The Ginseng Genome 3030303462, 9783030303464

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Compendium of Plant Genomes

Jiang Xu Tae-Jin Yang Hao-yu Hu   Editors

The Ginseng Genome

Compendium of Plant Genomes Series Editor Chittaranjan Kole, Raja Ramanna Fellow, Government of India, ICAR-National Research Center on Plant Biotechnology, Pusa, New Delhi, India

Whole-genome sequencing is at the cutting edge of life sciences in the new millennium. Since the first genome sequencing of the model plant Arabidopsis thaliana in 2000, whole genomes of about 100 plant species have been sequenced and genome sequences of several other plants are in the pipeline. Research publications on these genome initiatives are scattered on dedicated web sites and in journals with all too brief descriptions. The individual volumes elucidate the background history of the national and international genome initiatives; public and private partners involved; strategies and genomic resources and tools utilized; enumeration on the sequences and their assembly; repetitive sequences; gene annotation and genome duplication. In addition, synteny with other sequences, comparison of gene families and most importantly potential of the genome sequence information for gene pool characterization and genetic improvement of crop plants are described. Interested in editing a volume on a crop or model plant? Please contact Prof. C. Kole, Series Editor, at [email protected]

More information about this series at http://www.springer.com/series/11805

Jiang Xu • Tae-Jin Yang Hao-yu Hu

•

Editors

The Ginseng Genome

123

Editors Jiang Xu Institution of Chinese Materia Medica China Academy of Chinese Medical Sciences Beijing, China

Tae-Jin Yang Department of Plant Science, CALS Seoul National University Seoul, Korea (Republic of)

Hao-yu Hu Institution of Chinese Materia Medica China Academy of Chinese Medical Sciences Beijing, China

ISSN 2199-4781 ISSN 2199-479X (electronic) Compendium of Plant Genomes ISBN 978-3-030-30346-4 ISBN 978-3-030-30347-1 (eBook) https://doi.org/10.1007/978-3-030-30347-1 © Springer Nature Switzerland AG 2021 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Switzerland AG The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland

Preface to the Series

Genome sequencing has emerged as the leading discipline in the plant sciences coinciding with the start of the new century. For much of the twentieth century, plant geneticists were only successful in delineating putative chromosomal location, function, and changes in genes indirectly through the use of a number of “markers” physically linked to them. These included visible or morphological, cytological, protein, and molecular or DNA markers. Among them, the first DNA marker, the RFLPs, introduced a revolutionary change in plant genetics and breeding in the mid-1980s, mainly because of their infinite number and thus potential to cover maximum chromosomal regions, phenotypic neutrality, absence of epistasis, and codominant nature. An array of other hybridization-based markers, PCR-based markers, and markers based on both facilitated construction of genetic linkage maps, mapping of genes controlling simply inherited traits, and even gene clusters (QTLs) controlling polygenic traits in a large number of model and crop plants. During this period, a number of new mapping populations beyond F2 were utilized and a number of computer programs were developed for map construction, mapping of genes, and for mapping of polygenic clusters or QTLs. Molecular markers were also used in the studies of evolution and phylogenetic relationship, genetic diversity, DNA fingerprinting, and map-based cloning. Markers tightly linked to the genes were used in crop improvement employing the so-called marker-assisted selection. These strategies of molecular genetic mapping and molecular breeding made a spectacular impact during the last one and a half decades of the twentieth century. But still they remained “indirect” approaches for elucidation and utilization of plant genomes since much of the chromosomes remained unknown and the complete chemical depiction of them was yet to be unraveled. Physical mapping of genomes was the obvious consequence that facilitated the development of the “genomic resources” including BAC and YAC libraries to develop physical maps in some plant genomes. Subsequently, integrated genetic–physical maps were also developed in many plants. This led to the concept of structural genomics. Later on, emphasis was laid on EST and transcriptome analysis to decipher the function of the active gene sequences leading to another concept defined as functional genomics. The advent of techniques of bacteriophage gene and DNA sequencing in the 1970s was extended to facilitate sequencing of these genomic resources in the last decade of the twentieth century.

As expected, sequencing of chromosomal regions would have led to too much data to store, characterize, and utilize with the-then available computer software could handle. But the development of information technology made the life of biologists easier by leading to a swift and sweet marriage of biology and informatics, and a new subject was born—bioinformatics. Thus, the evolution of the concepts, strategies, and tools of sequencing and bioinformatics reinforced the subject of genomics—structural and functional. Today, genome sequencing has traveled much beyond biology and involves biophysics, biochemistry, and bioinformatics! Thanks to the efforts of both public and private agencies, genome sequencing strategies are evolving very fast, leading to cheaper, quicker, and automated techniques right from clone-by-clone and whole-genome shotgun approaches to a succession of second-generation sequencing methods. The development of software of different generations facilitated this genome sequencing. At the same time, newer concepts and strategies were emerging to handle sequencing of the complex genomes, particularly the polyploids. It became a reality to chemically—and so directly—define plant genomes, popularly called whole-genome sequencing or simply genome sequencing. The history of plant genome sequencing will always cite the sequencing of the genome of the model plant Arabidopsis thaliana in 2000 that was followed by sequencing the genome of the crop and model plant rice in 2002. Since then, the number of sequenced genomes of higher plants has been increasing exponentially, mainly due to the development of cheaper and quicker genomic techniques and, most importantly, the development of collaborative platforms such as national and international consortia involving partners from public and/or private agencies. As I write this preface for the first volume of the new series “Compendium of Plant Genomes,” a net search tells me that complete or nearly complete whole-genome sequencing of 45 crop plants, eight crop and model plants, eight model plants, 15 crop progenitors and relatives, and three basal plants is accomplished, the majority of which are in the public domain. This means that we nowadays know many of our model and crop plants chemically, i.e., directly, and we may depict them and utilize them precisely better than ever. Genome sequencing has covered all groups of crop plants. Hence, information on the precise depiction of plant genomes and the scope of their utilization are growing rapidly every day. However, the information is scattered in research articles and review papers in journals and dedicated Web pages of the consortia and databases. There is no compilation of plant genomes and the opportunity of using the information in sequence-assisted breeding or further genomic studies. This is the underlying rationale for starting this book series, with each volume dedicated to a particular plant. Plant genome science has emerged as an important subject in academia, and the present compendium of plant genomes will be highly useful to both students and teaching faculties. Most importantly, research scientists involved in genomics research will have access to systematic deliberations on the plant genomes of their interest. Elucidation of plant genomes is of interest not only for the geneticists and breeders, but also for practitioners of an array of plant science disciplines, such as taxonomy, evolution, cytology,

physiology, pathology, entomology, nematology, crop production, biochemistry, and obviously bioinformatics. It must be mentioned that information regarding each plant genome is ever-growing. The contents of the volumes of this compendium are, therefore, focusing on the basic aspects of the genomes and their utility. They include information on the academic and/or economic importance of the plants, description of their genomes from a molecular genetic and cytogenetic point of view, and the genomic resources developed. Detailed deliberations focus on the background history of the national and international genome initiatives, public and private partners involved, strategies and genomic resources and tools utilized, enumeration on the sequences and their assembly, repetitive sequences, gene annotation, and genome duplication. In addition, synteny with other sequences, comparison of gene families, and, most importantly, the potential of the genome sequence information for gene pool characterization through genotyping by sequencing (GBS) and genetic improvement of crop plants have been described. As expected, there is a lot of variation of these topics in the volumes based on the information available on the crop, model, or reference plants. I must confess that as the series editor, it has been a daunting task for me to work on such a huge and broad knowledge base that spans so many diverse plant species. However, pioneering scientists with lifetime experience and expertise on the particular crops did excellent jobs editing the respective volumes. I myself have been a small science worker on plant genomes since the mid-1980s and that provided me the opportunity to personally know several stalwarts of plant genomics from all over the globe. Most, if not all, of the volume editors are my longtime friends and colleagues. It has been highly comfortable and enriching for me to work with them on this book series. To be honest, while working on this series I have been and will remain a student first, a science worker second, and a series editor last. And I must express my gratitude to the volume editors and the chapter authors for providing me the opportunity to work with them on this compendium. I also wish to mention here my thanks and gratitude to the Springer staff, particularly Dr. Christina Eckey and Dr. Jutta Lindenborn for the earlier set of volumes and presently Ing. Zuzana Bernhart for all their timely help and support. I always had to set aside additional hours to edit books beside my professional and personal commitments—hours I could and should have given to my wife, Phullara, and our kids, Sourav and Devleena. I must mention that they not only allowed me the freedom to take away those hours from them but also offered their support in the editing job itself. I am really not sure whether my dedication of this compendium to them will suffice to do justice to their sacrifices for the interest of science and the science community. New Delhi, India

Chittaranjan Kole

This book series is dedicated to my wife Phullara and our children Sourav and Devleena Chittaranjan Kole

Preface

Panax ginseng C. A. Mey, a deciduous perennial plant belonging to the Araliaceae family, has been clinically used as a precious herbal medicine for several millennia in East Asia. The name ginseng was translated from the pronunciation of the Chinese words “Ren shen.” Modern pharmacological research confirmed that ginsenosides, the major bioactive compound of P. ginseng, exhibit multiple therapeutic activities. These activities include antitumor, antihypertensive, antivirus, and immune modulatory activities [3]. Therefore, P. ginseng is used as a general tonic or adatogen to promote longevity, particularly in China, Korea, and Japan. Currently, two genomes as well as several transcriptome data have been released for this valuable crop. Here, we present this book for readers to review the current achievements of omics of ginseng. This book comprised with 14 chapters can be generally separated into three parts. The first parts briefs about the background of ginseng studies. In the first two chapters, the authors introduced Panax ginseng from its origin, distribution, germplasm, cultivation, and medicinal properties. As molecular identification is the most early molecular biological tools used in ginseng industry, we introduced some work in this field in Chap. 3. In Chap. 4, the author introduced the progress of ginseng breeding which includes the breeding methods and mainstream varieties characteristics. The second part focused on the genome-wide research of ginseng. Cytogenetics once provided the image base for ginseng genetic research. Chapter 5 not only reviewed the historical achievements of P. ginseng cytogenetics but also discussed its future perspectives in the post-genomics era. In Chaps. 6 and 7, the authors introduced ginseng genome from two aspects—the metabolic pathway and the evolution. These two chapters will give the readers a panorama view of the genome information of this valuable medicinal plant. And besides the nuclear genome, cytoplasm genome is an unignoring part of plant genome. Chapter 8 presented chloroplast genome variation among Panax species and individuals. The authors also introduced the application of chloroplast genome in ginseng authentication. In the ninth chapter, the book focused on the transcriptome sequences and their technological advancements for the Panax species. The history of ginseng transcriptome research is far more than genome itself. So in this chapter, more technologies will be presented to our readers, including ESTs, 454 and up-to-date long-read single-molecule real-time technique. Chapter 10 xi

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Preface

discussed the metabolite pathways and metabolic dynamics. This chapter will help readers to joint to the metabolics and genes together. Chapter 11 gave a comprehensive overview of ginseng genome database, tools, and its broad utilities. Tools provided in this chapter will help readers use current achievements of ginseng omics even without profound informatics skills. Panax notoginseng is another important medicinal plant in genus Panax; here, the authors also introduced its genome characters in Chap. 12. In the third part, we introduced the progress of ginsenosides biosynthesis and the microbiome for ginseng medicine. Chapter 13 traced the recent progress of synthetic biology for ginsenoside production, including the elucidation of biosynthetic pathways and construction of cell factories for both natural and non-natural ginsenosides. These works will promote the industrialization of ginsenosides. In the last chapter, the authors summarized the role of gut microbiota in mediating the metabolism and enhanced bioavailability of ginseng. This book covered the mainly achievements of ginseng research in omic times. We believe the information contained in this book will benefit the researchers, students, and producers of ginseng. We also sincerely appreciate the help of our editorial group from Springer Nature. Beijing, China Seoul, Korea (Republic of) Beijing, China

Jiang Xu Tae-Jin Yang Hao-yu Hu

Contents

1.

2.

3.

Introduction of Panax Ginseng (Origin, Distribution, Germplasm, Cultivation and Economics Importance) . . . . . . . Zhao Lizi and Xu Yonghua The Ginseng Genome-Traditional Uses, Medicinal Properties, Phytochemistry, and Pharmaceutical Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Xiangyan Li, Daqing Zhao, Wenzhi Yang, Tiantian Zuo, Naiwu Zhang, Kondou Kenji, Zubair Ahmed Ratan, and Jae Youl Cho Nucleotide Signature and SNP Double Peak Methods Detect Adulterants and Substitution in Panax Products . . . . . . . . . . . Yang Liu, Gang Wang, and Jianping Han

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15

31

4.

Breeding of Superior Ginseng Cultivars . . . . . . . . . . . . . . . . . . Jang-Uk Kim, Dong-Yun Hyun, Hyeonah Shim, Tae-Jin Yang, and Young-Chang Kim

45

5.

Molecular Cytogenetics of Panax Ginseng . . . . . . . . . . . . . . . . Nomar Espinosa Waminal, Tae-Jin Yang, and Hyun Hee Kim

55

6.

Ginseng Genome and Metabolic Regulation . . . . . . . . . . . . . . Xing Zhi-han, Hu Hao-yu, and Xu Jiang

71

7.

Ginseng Genome Structure and Evolution . . . . . . . . . . . . . . . . Nam-Hoon Kim, Murukarthick Jayakodi, and Tae-Jin Yang

85

8.

Chloroplast Genome Diversity in Panax Genus . . . . . . . . . . . . Vo Ngoc Linh Giang, Woojong Jang, Hyun-Seung Park, and Tae-Jin Yang

95

9.

An Update to the Transcriptome Sequencing for the Genus Panax . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107 Deok-Chun Yang

10. Metabolic Dynamics and Ginsenoside Biosynthesis . . . . . . . . . 121 Shadi Rahimi, Padmanaban Mohanan, Dabing Zhang, Ki-Hong Jung, Deok-Chun Yang, Ivan Mijakovic, and Yu-Jin Kim

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11. Genomic Resources for Ginseng Genome Studies . . . . . . . . . . 143 Murukarthick Jayakodi and Tae-Jin Yang 12. Genomes of Other Species in Panax Linn . . . . . . . . . . . . . . . . 149 Zhang Guang-hui and Yang Sheng-chao 13. Synthetic Biology of Ginsenosides . . . . . . . . . . . . . . . . . . . . . . . 159 Pingping Wang, Lu Yu, Chaojing Li, Chengshuai Yang, Zhihua Zhou, and Xing Yan 14. Gut Microbiome for Ginseng Medicine . . . . . . . . . . . . . . . . . . 171 Xiao Shuiming and Zhang Xiaoyan

Contents

Contributors

Jae Youl Cho Department of Integrative Biotechnology, Sungkyunkwan University, Suwon, Republic of Korea Vo Ngoc Linh Giang Department of Plant Science, Plant Genomics and Breeding Institute, Research Institute of Agriculture and Life Sciences, College of Agriculture and Life Sciences, Seoul National University, Seoul, Korea Zhang Guang-hui National-Local, Joint Engineering Research Center on Germplasm Utilization and Innovation of Chinese Medicinal Materials in Southwest China, Yunnan Agricultural University, Kunming, China Jianping Han Engineering Research Center of Tradition Chinese Medicine Resource, Ministry of Education, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China Hu Hao-yu Key Laboratory of Beijing for Identification and Safety Evaluation of Chinese Medicine, Institution of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, China Dong-Yun Hyun Division of Ginseng, Department of Herbal Crop Research, National Institute of Horticultural and Herbal Science, Rural Development Administration, Eumseong, Korea Woojong Jang Department of Plant Science, Plant Genomics and Breeding Institute, Research Institute of Agriculture and Life Sciences, College of Agriculture and Life Sciences, Seoul National University, Seoul, Korea Murukarthick Jayakodi Department of Agriculture, Forestry and Bioresources, Plant Genomics and Breeding Institute, College of Agriculture and Life Sciences, Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul, Korea; Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Gatersleben, Seeland, Germany Xu Jiang Key Laboratory of Beijing for Identification and Safety Evaluation of Chinese Medicine, Institution of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, China

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Ki-Hong Jung Graduate School of Biotechnology and Crop Biotech Institute, Kyung Hee University, Yongin, Korea Kondou Kenji TSUMURA & CO, Tokyo, Japan Hyun Hee Kim Department of Life Science, Chromosome Research Institute, Sahmyook University, Seoul, Republic of Korea Jang-Uk Kim Division of Ginseng, Department of Herbal Crop Research, National Institute of Horticultural and Herbal Science, Rural Development Administration, Eumseong, Korea Nam-Hoon Kim Department of Agriculture, Forestry and Bioresources, Plant Genomics and Breeding Institute, College of Agriculture and Life Sciences, Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul, Korea; PHYZEN Genomics Institute, Seongnam-si, Gyeonggi-do, Korea Young-Chang Kim Division of Ginseng, Department of Herbal Crop Research, National Institute of Horticultural and Herbal Science, Rural Development Administration, Eumseong, Korea Yu-Jin Kim Department of Life Science and Environmental Biochemistry, Pusan National University, Miryang, Republic of Korea Chaojing Li CAS-Key Laboratory of Synthetic Biology, CAS Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China; University of Chinese Academy of Sciences, Beijing, China Xiangyan Li Key Laboratory of Active Substances and Biological Mechanisms of Ginseng Efficacy, Ministry of Education, Jilin Provincial Key Laboratory of BioMacromolecules of Chinese Medicine, Jilin Ginseng Academy, Changchun University of Chinese Medicine, Changchun, Jilin, China Yang Liu Engineering Research Center of Tradition Chinese Medicine Resource, Ministry of Education, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China Zhao Lizi The Engineering Research Institute of Agriculture and Forestry, Ludong University, Yantai, Shandong, P.R. China Ivan Mijakovic Systems and Synthetic Biology, Chalmers University of Technology, Göteborg, Sweden; The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Lyngby, Denmark Padmanaban Mohanan Plant Genomics and Breeding Institute, Research Institute of Agriculture and Life Sciences, College of Agriculture and Life Sciences, Seoul National University, Seoul, Korea

Contributors

Contributors

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Hyun-Seung Park Department of Plant Science, Plant Genomics and Breeding Institute, Research Institute of Agriculture and Life Sciences, College of Agriculture and Life Sciences, Seoul National University, Seoul, Korea Shadi Rahimi Systems and Synthetic Biology, Chalmers University of Technology, Göteborg, Sweden Zubair Ahmed Ratan Department of Biomedical Engineering, Khulna University of Engineering and Technology, Khulna, Bangladesh Yang Sheng-chao National-Local, Joint Engineering Research Center on Germplasm Utilization and Innovation of Chinese Medicinal Materials in Southwest China, Yunnan Agricultural University, Kunming, China Hyeonah Shim Department of Agriculture, Forestry and Bioresources, Plant Genomics and Breeding Institute, Research Institute of Agriculture and Life Sciences, College of Agriculture and Life Sciences, Seoul National University, Seoul, Korea Xiao Shuiming Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, China Nomar Espinosa Waminal Department of Life Science, Chromosome Research Institute, Sahmyook University, Seoul, Republic of Korea Gang Wang Engineering Research Center of Tradition Chinese Medicine Resource, Ministry of Education, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China Pingping Wang CAS-Key Laboratory of Synthetic Biology, CAS Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China Zhang Xiaoyan Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, China Xing Yan CAS-Key Laboratory of Synthetic Biology, CAS Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China Chengshuai Yang CAS-Key Laboratory of Synthetic Biology, CAS Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China Deok-Chun Yang Graduate School of Biotechnology and Department of Oriental Medicinal Biotechnology, College of Life Sciences, Kyung Hee University, Yongin, Republic of Korea

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Tae-Jin Yang Department of Agriculture, Forestry and Bioresources, Plant Genomics and Breeding Institute, Research Institute of Agriculture and Life Sciences, College of Agriculture and Life Sciences, Seoul National University, Seoul, Korea Wenzhi Yang Tianjin State Key Laboratory of Modern Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin, China Xu Yonghua Jilin Agricultural University, Changchun City, Jilin Province, P.R. China Lu Yu CAS-Key Laboratory of Synthetic Biology, CAS Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China Dabing Zhang Joint International Research Laboratory of Metabolic and Developmental Sciences, Shanghai Jiao Tong University–University of Adelaide Joint Centre for Agriculture and Health, State Key Laboratory of Hybrid Rice, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China; Graduate School of Biotechnology and Crop Biotech Institute, Kyung Hee University, Yongin, Korea Naiwu Zhang China Medico Coperation, Tianjin, China Daqing Zhao Key Laboratory of Active Substances and Biological Mechanisms of Ginseng Efficacy, Ministry of Education, Jilin Provincial Key Laboratory of BioMacromolecules of Chinese Medicine, Jilin Ginseng Academy, Changchun University of Chinese Medicine, Changchun, Jilin, China Xing Zhi-han Key Laboratory of Beijing for Identification and Safety Evaluation of Chinese Medicine, Institution of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, China Zhihua Zhou CAS-Key Laboratory of Synthetic Biology, CAS Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China Tiantian Zuo Tianjin State Key Laboratory of Modern Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin, China

Contributors

Abbreviations

AACT Ach AchE AFLP ANS APX Ara Ab BUSCO CAS CAT cDNA CEGMA COX cpDNA dCAPS DD DDS DESI-MS DMAPP DPPH DPR. Korea EPM EST FAD FOS FPP FPS Fuc GABA Gal GFP Glc GluA

Acetyl-CoA C-acetyltransferase Acetylcholine Acetylcholinesterase Amplified fragment length polymorphism Autonomic nervous system Ascorbate peroxidase Arabinose b-amyloid Benchmarking universal single-copy ortholog Cycloartenol synthase Catalase Complementary DNA Core eukaryotic genes mapping approach Cyclooxygenase Chloroplast DNA Derived cleaved amplified polymorphic sequence markers Dammarenediol-II Dammarendiol synthase Desorption electrospray ionization mass spectrometry Dimethylallyl diphosphate 2, 2-diphenyl-1-picrylhydrazyl Democratic People’s Republic of Korea Elevated plus maze Expression sequences tag Fatty acid desaturase Fructooligosaccharide Farnesyl diphosphate Farnesyl diphosphate synthase Fucose Gamma-aminobutyrate Galactose Green fluorescent protein Glucose Glucuronic acid xix

xx

GMPGIS GOS GP GPC GPP GPX Grx GT HMG-CoA HMGR HMGS HPA HPGPC HPLC-MS ICTRP IDI IPP IR LAS LHCP LOX LSC LTR LTR-RT Man MeJA MVA MVD MVK OA OAS OSC PD PMK PPD PPT PPTS PR Prx PSPG PVK R.O. Korea RAPD RE RFLP

Abbreviations

Regionalization Information System of the Ecological Suitability for Medicinal Plants Galactooligosaccharide Genome proportion Glycerophosphorylcholine Geranyl pyrophosphate Glutathione peroxidases Glutaredoxin Glycosyltransferase 3-hydroxy-3-methylglutaryl-CoA 3-hydroxy-3-methylglutaryl-CoA reductase 3-hydroxy-3-methylglutaryl-CoA synthase Hypothalamic-pituitary-adrenal High-performance gel permeation chromatography High-performance liquid chromatography–mass spectrometry International Clinical Trails Registry Platform Isopentenyl-diphosphate delta isomerase Isopentenyl diphosphate Inverted repeat Lanosterol synthase Light-harvesting chlorophyll a-b binding proteins Lipoxygenase Large single copy Long tandem repeat Long terminal repeat retrotransposon Mannose Methyl jasmonate Mevalonic acid Mevalonate diphosphate decarboxylase Mevalonate kinase Oleanolic acid Oleanolic acid synthase Oxidosqualene cyclase Parkinson’s disease Phosphomevalonate kinase Protopanaxadiol Protopanaxatriol Protopanaxatriol synthase Pathogenesis-related protein Peroxiredoxin Plant secondary product glycosyltransferase Phophomevalonate diphosphate kinase Republic of Korea Random amplified polymorphic DNA Repetitive element Restriction fragment length polymorphism

Abbreviations

xxi

Rha RNS ROS SE SNP SQE SS SSC TE TR UDP UGT UPLC-QTOF-MS WGD Xyl

Rhamnose Reactive nitrogen species Reactive oxygen species Squalene epoxidase Single nucleotide polymorphism Squalene epoxidase Squalene synthase Small single copy Transposable element Tandem repeat Utilize uridine diphosphate UDP-glycosyltransferase Ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry Whole-genome duplications Xylose

Introduction of Panax Ginseng (Origin, Distribution, Germplasm, Cultivation and Economics Importance)

1

Zhao Lizi and Xu Yonghua

Abstract

Panax ginseng C. A. Mey. is a precious and important medicinal plant, the roots of which have been used medicinally in various Asian countries for centuries, especially in East Asia. It originated in the inland high grounds of the northern cathaysia block as early as the Cretaceous Period. It is the intercontinental disjunction distribution of Panax genus caused by “continental drift” after the completion of the Tertiary continental drift that led to two ginseng plant centers in eastern Asia and eastern North America separately. Studies show that the Panax genus of the two continental plates in the northern hemisphere, though having developed into different species, respectively, have very close kinship. In the eastern Asia center, there developed a central region of Panax genus distribution areas in northeastern China and a sub-variation center in southeastern China due to the special geological climate changes.

Z. Lizi The Engineering Research Institute of Agriculture and Forestry, Ludong University, 186 Hongqizhong Road, Yantai, Shandong 264025, P.R. China X. Yonghua (&) Jilin Agricultural University, No. 2888, Xincheng Avenue, Changchun City, Jilin Province, P.R. China e-mail: [email protected]

Now, the original Panax ginseng is extremely rare in the wild and has almost reached extinction through over-harvesting. To meet people’s needs, the artificial cultivation of ginseng has emerged and has now become the major production mode. Nowadays, cultivated ginseng domesticated from the wild has developed into different cultivars. In China, for example, there are mainly three kinds of Panax ginseng species in China—wild, under forest and garden. The major cultivation areas are located in three Northeastern Provinces of China —Heilongjiang, Jilin and Liaoning Provinces. But affected by continuous cropping obstacles, the land resources suitable for ginseng growing in Jilin has decreased, so the ginseng production areas have been gradually moving northward further to Heilongjiang and even to the areas adjacent to Russia. Over nearly 400 years, the artificial ginseng cultivation out of the wild growing went through domestication to cultivation and developed three cultivation modes: wild cultivation, deforestation cultivation and farmland cultivation. Compared with deforestation and wild cultivation, the farmland ginseng planting has significant advantages of sustainable development since more land resources can be used without destroying forest resources, and ginseng–grain rotation can be conducted to realize the sustainable utilization of land resources. As the increasing deterioration of ecological environment have brought about a big challenge to the safety and effectiveness of ginseng, the

© Springer Nature Switzerland AG 2021 J. Xu et al. (eds.), The Ginseng Genome, Compendium of Plant Genomes, https://doi.org/10.1007/978-3-030-30347-1_1

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Z. Lizi and X. Yonghua

Fig. 1.1 Taxonomic hierarchy of Panax ginseng C. A. Mey

pollution-free cultivation has become the inevitable adoption and trend in future for the farmland ginseng planting. Up to now, some progress has been made in the related research such as the Regionalization Information System of the Ecological Suitability for Medicinal Plants (GMPGIS-II) used to analyze the ecological suitability of ginseng farming areas and the whole genome sequence of Panax ginseng assembled by the second-generation sequencing technology providing a basis for the breeding of new varieties of Panax ginseng in farmland. As a valuable herbal medicine, ginseng and its products are widely sold in the world, especially in East Asia. China and R. O. Korea are major exporters, while Asia remains the world's largest ginseng consumption region. Meanwhile, ginseng, as an important raw material, has been gradually applied in food, health drinks, cosmetics, chemicals (extraction) and other fields. Therefore, there is great potential for market demanding. In regards of ginseng production capacity, China and R. O. Korea are the major producing countries, and the annual output of China itself accounts for about 70% of the world’s total output, and China’s ginseng market is expected to reach $5 billion by 2025. Accordingly, it can be predicted that the economic and the world trade volume of ginseng will be gradually increasing in future. Panax ginseng C. A. Mey. is a perennial herb of Panax genus of Acanthopanax family (Fig. 1.1). It grows in coniferous and broadleaf mixed forests dominated by coniferous forests or deciduous broadleaf forests, in the range of 33 * 48 degrees north latitude, hundreds of meters above sea level. It distributes mainly in northeast China, DPR. Korea, R. O. Korea,

Japan, eastern Russia and other regions. Ginseng is 30 * 70 cm tall, with the main root fleshy, multiple branches at the lower end, the rhizome (reed head) upright or oblique, the stems on the ground solitary, the leaves palmately compound, umbel, the berries flat spherical, flowering period from May to June and fruiting period from July to August (Xiao et al. 1987) (Fig. 1.2) The fleshy root of ginseng is a famous sthenia nourishing tonic, which are good for adjusting blood pressure, restoring cardiac function, neurasthenia and physical weakness, and also has effects on expectoration, stomach health, diuresis, excitement and so on (Goldstein 1975; Liu and Xiao 1992; Gillis 1997; Coon and Ernst 2002). In China, ginseng has long been regarded as the king of herbs, and in the West, it is called Panax ginseng, of which, “PANAX” from Greek means “cure-all.” The pronunciation of “gin” stands for the Chinese character for “man” and “seng” is the equivalent of “essence” (Hu 1997). Therefore, in both modern or ancient times, ginseng has been held as a precious and important medicinal plant both in the east and west, especially in East Asia (Fig. 1.2).

1.1

Origin and Evolution of Panax Ginseng

1.1.1 Geological Age of Panax Ginseng Origin The transformation of ancient plants occurred in the mid-Cretaceous period, when prosperous ferns and gymnosperms drastically decreased, while

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Introduction of Panax Ginseng …

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Fig. 1.2 Pattern diagram of Panax ginseng C. A. Mey

angiosperms explosively developed. It was during this period that Araliaceae family (Panax genus under Araliaceae family) appeared. The fossils of Araliaceae family plants such as Hedera genus, Oplopanax genus, the Acanthopanax genus and Aralia genus of deciduous species have been found in eastern Siberia and North America as well as leaf fossils of Kalopanax genus in China. What is more, the ginseng fossils have been found in the Tertiary strata, and therefore, it can be inferred that Panax ginseng has already existed on Earth in the Cretaceous Period, and hence, it has obtained the name of living fossil.

1.1.2 Primitive Area of Panax Ginseng Origin According to the angiosperm origin theory by Kazuo Asami, a Japanese paleontologist, and the angiosperm fossils found in a coalfield of Taiyuan in Shanxi, China, Panax ginseng like other angiosperms originated in the inland-high grouds of the northern cathaysia block, now the Taihang Mountains in North China and southern Liaoning (Asami 1988). Famous Medical Records, an ancient Chinese medical book compiled around the Chinese Southern and Northern Dynasties

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(456 * 536 AD), also has records of ginseng distribution in this area (Tao 1986). From the late Cretaceous to the Paleogene, as the climate became warmer and wetter, the primitive species of the Panax genus had to migrate to higher latitudes along with other angiosperms from their birthplace, and extended to the Changbai Mountains in China, the high-latitude area of DPR. Korea and the far East of Russia. Then, after several thousand years of evolution, the ancient Panax ginseng species were formed.

1.1.3 Intercontinental Disjunction Distribution of Panax Genus From the mid-Mesozoic, the whole continent began to split, the North American plate drifting westward while the eastern Eurasian plate moving southward, and until Tertiary from the Cretaceous the whole continent was divided into two parts: the North American plate and the Eurasian plate. Therefrom came about the intercontinental disjunction distribution of Panax genus and finally formed the two ginseng distribution centers in eastern Asia and eastern North America separately. The ancient Panax ginseng, isolated geographically for tens of thousands of years, developed into different species of Panax genus. Studies reveal that when the plant of Panax species from North America are crossed with one from Asia, the hybrid seed set rate is high, and moreover, when the hybrid F1 pollen mother cells are meiotic, most of the chromosomes are paired with only a few univalent units. In addition, the isoenzyme spectrum of the two are also very similar. Moreover, the genome resequencing data of Panax ginseng indicates that Panax quinquefolius Linn and Panax trifolius Linn in North America evolved through intercontinental migration (Kim et al. 2018). Hence, it can be concluded that the Panax genus of the two continental plates in the northern hemisphere have very close kinship and they are very likely to have a common ancestor.

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1.1.4 Biological Evolution of Panax Genus After the formation of intercontinental disjunction distribution of ancient Panax ginseng, the Tertiary climate gradually changed from cool and semi-arid to warm and humid, and then, the temperature decreased and the air became dry. In the Quaternary, the climate was constantly warming due to the alternation of the glacial and interglacial ages, and the cold zone, temperate zone, subtropical and tropical flora today were thus gradually formed. Geographically, from the Tertiary Eocene to the Pliocene, the Rocky Mountains, the Atlas Mountains, the Andes, the Alps, and the Himalayas went rising successively. The crustal uplifting movements extended the earth surface, and forests areas reached the maximum in the Tertiary, after then reduced and appeared decline. By the Quaternary, the continents underwent rising and expanding, thus bringing high prosperity to herbs. All these changes in climate, geography and surface environment led to the gradual reproduction and evolution of ancient Panax ginseng, having developed a series of relatively new species and varieties, and finally forming Panax genus.

1.1.4.1 Evolution of Panax Genus in Eastern Asia As mentioned above, the ancient Panax ginseng formed in the early Cretaceous and gradually advanced northward to the Changbai Mountains in China, the high-latitude area of DPR. Korea and the Far East of Russia due to warm and humid climate in the late Cretaceous. After the completion of the Tertiary continental drift, the intercontinental disjunction distributions of Panax ginseng because of geographical migration of the population brought about two distribution centers in eastern Asia and the eastern North America, respectively. Since the middle Tertiary, there came a global temperature drop that restricted the migration of ginseng populations in Asia to the high-latitude area. As the Quaternary glacial age came, the climate became even colder, so the growth period

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Introduction of Panax Ginseng …

lasted only about 4 months. Consequently, the growth period of the original species of Panax genus was shortened, and to survive the cold climate of original forests, it had to develop out the cold-tolerant fleshy roots, but the distribution area was sharply reduced. As a result, this special geological climate changes narrowed down the geographical area of the birth center of Asian Panax genus, having left the Taihang Mountains and Liaodong region of China as the central regions of Panax genus origin, and the Changbai Mountains of China, the high-latitude area of North Korea and Far East of Russia as further extended high-latitude Panax genus distribution areas in order of geological chronology, which are all in the geographic region of the Panax genus distribution center of eastern Asia formed in the Tertiary. In the middle Tertiary, the distribution area of the ancient Panax ginseng decreased on the one hand as its expansion to high latitudes in eastern Asia was restricted by cold climate, and began to move southward on the other, where in the low latitudes of warm climate, the variations of the ancient Panax genus were evolving into new species over several thousand years, which propagated and grew well there in Yunnan, for the vegetation in southwestern China did not suffer destruction by the Quaternary continental ice sheet, only affected by the glacial age and subsequent fluctuation of the interglacial age. Besides, as the habitats there was more complicated and warmer, the Panax genus in the region was constantly undergoing convergence or divergence evolution with different new species and varieties evolved, and eventually, southwestern China turned into the secondary variation center of Panax genus (Kim et al. 2018). For example, Panax pseudoginseng Wall., spreading only in the middle Himalayas and southern Tibet of China, has similar chemical composition characteristics to ancient Panax ginseng, so is taken to be an intermediate type of the ancient species evolving to more evolutionary groups during long systematic development. Panax japonicus (T. Nees) C. A. Mey. is the most widely distributed species of Panax genus from southeastern China to Japan, but its main

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chemical composition and chromosome number have some changes reflecting the geographical transition nature of Panax japonicus. It is a taxa that has been constantly differentiating and developing to adapt to the present ecological environment. There are also some other different varieties such as Panax pseudoginseng Wall. var. elegantior (Burkill) Hoo & Tseng, Panax pseudoginseng Wall. var. japonicus (C. A. Mey.) Hoo & Tseng and Panax pseudoginseng Wall. var. bipinnatifidus (Seem.) Li.

1.1.4.2 Evolution of Panax Genus in Eastern North America In the early Tertiary, the North American continent plate completely separated from the Eurasian plate, geographically isolating the ancient Panax genus, and through several thousand years of evolution, there formed the population segregation. This segregation prevented the genus in North America from randomly crossing and communicating with the original population, thus minifying the population, causing easy interference of the gene pool there by accidental factors, consequently followed by genetic ratio deviation, random genetic drifting and furthermore divergence evolution, and eventually evolved unique species of Panax quinquefolius Linn and Panax trifolius Linn in eastern North America. However, North American Panax genus has retained some ancient features owing to the geographical isolation. It was found that Panax quinquefolius is rich in ginseng diol saponin Rb1, which is similar to the chemical properties of ancient ginseng.

1.1.5 Polyploidization of Panax Genus The polyploidy occurrence of angiosperms in evolution, widely accepted by the scientific community, has long been considered to be an important evolutionary dynamic of plant species as it causes a large-scale replication of genomes in the evolution. Studies show that Panax genus experienced polyploidization twice in its evolutionary history (Choi et al. 2013). The first time

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occurred before the formation of Panax genus, which formed the geographically isolated tetraploid species (2n = 4x = 48), mainly Panax ginseng, Panax quinquefolius and Panax japonicus, which is also one of the reasons why ginseng has a higher wintering ability (Kim et al. 2018). The second polyploidization occurs after the differentiation of the diploid Panax pseudoginseng species, which occurred inside, causing concurrence of diploid (2n = 2x = 24) and tetraploid (2n = 4x = 48). According to the geographical distribution, it is inferred that Panax ginseng is a more evolved species after polyploidy (Yang 1981). As far as the classification of the species of Panax genus concerned, it is rather confusing because of the similar external morphology and many intermediate variation types among the species of Panax ginseng. With the emergence and development of molecular systematics and genomics, the classification of Panax genus is also constantly changing. For example, Lee reconstructed the phylogenetic tree of Panax genus and confirmed that Panax genus and Aralia genus are the two closest genus in Araliaceae based on previous studies, although they were classified as the same genus in early morphological classification, but combined with molecular data, it was proved that Panax genus was a monophytic origin (Lee and Wen 2004). In the Panax genus, the morphology of Panax trifolius listed at the base of the phylogenetic tree is not only significantly different from that of other species of Panax genus, but also its phylogenetic tree position remains stable and is distinct from other species within the genus, but the phylogenetic relationship of Panax notoginseng, Panax pseudoginseng and Panax japonicus has always been controversial. The latest research shows that although the geographical location of Panax quinquefolius and Panax trifolius is in the eastern part of North America, their genetic relationship is closer to that of Panax ginseng and Panax japonicus, and it is only 0.8 * 1.2 million years ago that they were separated from each other (Fig. 1.4). This information suggests that the combination of polyploidy and geographical isolation promotes have contributed to the

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complex evolutionary process of the existing Panax genus (Shi et al. 2015).

1.2

Distribution of Panax Ginseng

Panax ginseng spreads mainly in eastern Asia, including China, DPR. Korea, R. O. Korea, Japan and Russia, with the growth areas largely in various mountains within 117 * 137 degrees east longitude and 33 * 48 degrees north latitude, that is, Northeast China, DPR. Korea and R. O. Korea, Honshu and Hokkaido of Japan and the Far East of Russia (Wang and Zhang 1984). Panax ginseng, originally wild, spreads in a very narrow area, mainly in the Changbai mountains of China, the border area between DPR. Korea and China as well as the coastal area of the Far East of Russia, but the region now has gradually shrunk through human over-collecting. To meet markets demands, the artificial cultivation of ginseng was gradually emerging, first in China, and then was introduced to DPR. Korea, R. O. Korea, Japan and Russia. Nowadays, artificial cultivation has become the major production mode of ginseng.

1.2.1 Panax Ginseng Distribution in China The types of Panax ginseng in China can be classified into three kinds—wild, under forest and garden, which are widely distributed in the region of 117 * 134 degrees east longitude and 40 * 44 degrees north latitude. The major cultivation areas are located in Fusong, Jingyu, Ji’an and other places of Jilin Province; Linkou, Dongning, Jiamusi, Yichun and other places of Heilongjiang Province; Kuandian, Huanren, Xinbin, Qingyuan and other places of Liaoning Province. Hebei, Shandong, Yunnan and other provinces also have a small amount of cultivation. Among them, Jilin Province was once the main producing area, reaching 6000 hectares at its best. Chinese ginseng production, with a large yield, covers 60 * 70% of the world's annual output, of which the yield of Jilin itself accounts

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for 85% of the total. But affected by continuous cropping obstacles, the land resources suitable for ginseng growing in Jilin has decreased, so the ginseng production areas have been gradually moving northward further. By 2017, about 70% of the major producing areas of ginseng in China have moved northward to Heilongjiang and even to the areas adjacent to Russia.

longitude and 43 * 48 degree north latitude, with the Ursuli River area in the Far East and the eastern coastal areas as its major producing areas, which later extended to the region of Moscow and the Caucasus (Zhuravlev et al. 2008).

1.2.2 Panax Ginseng Distribution in Korean Peninsula

The germplasm resources of Panax ginseng mainly include wild mountain ginseng and cultivated ginseng.

Panax ginseng in the Korean Peninsula has two kinds—wild ginseng and garden ginseng, accounting for 27% of the world’s annual production yield. It is mainly distributed in the area of 124 * 130 degrees east longitude and 33 * 43 degrees north latitude. The major cultivating areas of DPR. Korea are Kaesong, Jeollanam-do, Yanggang-do, Chagang-do, Chungcheongbuk-do and P'yǒnganbuk-do and so on, generally with artificial cultivation and only a small amount of wild plants, and Cam Son, Buyeo, Chungcheongnam-do, Chungcheongbukdo, Jeollabuk-do and other regions in R. O. Korea with no other but artificial cultivation.

1.2.3 Panax Ginseng Distribution in Japan Panax ginseng is all cultivated artificially in Japan, and the annual output takes less than 3% of the world's total production. It is mainly distributed in the area of 132 * 140 degrees east longitude and 35 * 37 degrees north latitude with the three counties of Nagano, Fukushima and Shimane in Honshu and the Hokkaido area as its major producing areas.

1.2.4 Panax Ginseng Distribution in Russia Panax ginseng in Russia is of two kinds—wild and garden, with less annual output. It is largely distributed in the area of 132 * 135 degrees east

1.3

Germplasm Resources of Panax Ginseng

1.3.1 Wild Panax Ginseng Resources Historically, wild ginsengs widely scattered in the Shangdang area of the Taihang Mountains, the Daxing’an and Xiaoxing’an Mountains in China as well as the Changbai Mountains at the junction of China and DPR. Korea. Nevertheless, since the 1980s, the natural resources of wild ginseng have been severely damaged and have almost reached extinction from over-harvesting and the destruction of the forest habitat, and eventually, most of the original distribution areas have disappeared. At present, having been in the dilemma of the ever-shrinking and endangered germplasm resources of wild ginseng, slow henogenesis, low self-breeding ability and the like, the natural ginseng population distribution regions have become very narrow (Reunova et al. 2010; Zhuravlev et al. 2010), limited to the area of 117.6 * 134 degrees east longitude and 40 * 48 degrees north latitude (Ma et al. 2000) ranging in the southeastern part of China’s Xiaoxing’an Mountains, the Changbai Mountains on the border between China and DPR. Korea, and the Ursuli River Basin at the junction of DPR. Korea and Russia. Statistics show that in Jilin, the major growing area in China, the collecting quantity of the Chinese wild ginseng was 750 kg in 1927, 362 kg in 1951, 128 kg in 1981 and only 10 kg in 1997, and thereafter, no official records of it at all. Now, in China, the natural resources of wild ginseng are still under

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declining, and disappeared in most of the original distribution areas, only sporadically scattering in the Changbai Mountains and the southern foot of Xiaoxing’an Mountains. In 1992, the ginseng has been listed as a rare and endangered plant in the Red Book of Chinese Plants (Fu 1992), and in 2005 it had been delisted from the Chinese Pharmacopoeia. Currently, the relatively complete wild population, very small in number, is only confined to the coastal areas to south Boli, Russia, and has long been listed as an endangered species in the Red Book (1988) by the Soviet Union (Grushwitsky 1961; Zhuravlev et al. 2008, 2010). Wild ginseng can be classified by growth process into pure wild ginseng and ginseng with regenerated roots (from sub-roots). Different wild ginsengs can be identified from four aspects: head and sub-roots, body and leg, skin and texture as well as fibrous roots and spots. Wild ginsengs are different in morphology owing to free from external interference in the growth process. Their typical features are as follows: the underground stem with a long head; the main root with thin and deep annular lines, longitudinally short and thick, having two branches transversely extended in Y-Shape; fibrous roots sparse and slender with pearl spots on. Various wild ginsengs may well display different morphological features influenced by diverse growing environments and geographical climates (Li and Wang 2008).

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In China, the principal varieties are as follows: Damaya (a big horse-teeth shape), Ermaya (a small-horse-teeth shape), roundhead with roundshoulder, long-neck and the like named by shapes of roots and the rhizomes; red fruit, yellow fruit and orange-yellow fruit by fruit colors; purple stem, green stem and indigo stem by stem colors; tight-ear and scattered-ear by ear tightness (Zhao et al. 2007). What needs to be added is that most of these varieties, named just by their morphological and cultivation features, not standardized, are local cultivars for daily production. With more attention given to the standardization of ginseng varieties, many new varieties have been selected in recent years in China, including Jilin Huangguo, Kangmei No.1, Xinkaihe No.1, Fuxing No. 1, Fuxing No.2, etc. The history of ginseng planting on the Korean Peninsula can be dated back to the sixteenth century. It is reported that its source was from the eastern part of Liaoning Province, China. R. O. Korea has a large planting area and has made great progress in developing the ginseng germplasm resources. Its breeding is generally for varieties suitable for farmland cultivation. So far, the new bred varieties are Gumpoong, Sunun, Sunpoong, Sunone, Cheongsun and Sunhyang. The varieties cultivated by DPR. Korea is mainly “Purple-stem No.1” and the like. Ginseng cultivation in Japan began in the eighteenth century. Its new bred varieties are “Yu Mu” and “Mimaki,” of which “Yu Mu” has better appearance but lower yield.

1.3.2 Cultivated Ginseng Resources

1.4 Cultivated ginseng, a mixed population domesticated from wild ginseng (Wen and Zimmer 1996), originated in China about 1600 years ago, and the scale cultivation has a history of more than 400 years (Yang and Tian 2004; Xiao et al. 1987). Under thousands of years of ecological environment motivation and a long-term selection and domestication, some variations have been gradually isolated from cultivated ginsengs of a hybrid population, and finally developed into different cultivars.

Cultivation of Panax Ginseng

Ginseng has been cultivated for more than 1600 years in the history. Studies show that the earliest documents for ginseng cultivation of China was seen in Shile Biography written in the late Western Jin Dynasty of China. It records the process of transplanting young wild ginseng into home gardens and selling it when it grows up. Over nearly 400 years, wild ginseng went through domestication to cultivation. The cultivation was carried out first by planting “ginseng

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shoot,” that is, transplanting wild ginseng seedling collected in the wild which was actually a semi-artificial cultivation, later followed by the “seed ginseng” cultivation, that is, propagating seeds of wild ginseng fruits collected, which was a complete artificial cultivation. At present, the ginseng cultivation methods fall into three modes: wild cultivation, logged land planting and farmland planting. And the existing cultivated varieties are the mixed population domesticated from wild ginseng.

1.4.1 Wild Cultivation Mode Wild cultivation mode of ginseng first appeared, also known as planting ginseng under forest. In this mode, ginseng is planted in the natural growth environment with “young plants” or “seeds” collected in the wild, growing usually 10 to 20 years with the products of some wild ginseng properties. The wild cultivation owns some advantages: It does not need to cut down trees and cover the shade, which not only protects forest resources and ecological environment, but also avoids soil erosion caused by artificial planting, and thus alleviates the land competition between ginseng and forests, and also saves manpower and shading materials. Usually, the most suitable places for the wild cultivation are the forest lands of tussah trees, linden trees and other broadleaf forests lands with the canopy density between 0.5 and 0.9, and so are the lands of mixed coniferous and broad-leaved forests. The slope direction is better to southwest or to southeast, and the soil is loose and fertile with the soil layer above 10 cm. Depending on the topography of the mountains and the distribution states of the trees, planting ginseng under forest may have two methods: semi-wild planting and furrow planting. The semi-wild planting is suitable for forest land with larger slope but smaller space; hence, it is difficult to manage; moreover, with slow growth and low yield; comparatively, the furrow planting is more convenient to manage and the yield is higher as well, but the manpower and material cost is relatively high (Zhao et al. 2013; Wang et al. 2016; Park 2001).

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1.4.2 Deforestation Model Deforestation model refers to the cultivation of ginseng by cutting down virgin forests or artificial forests, which is the major ginseng planting approach in China in the twentieth century. Generally, the best forestlands selected for planting ginseng are the lands with tussah trees, hazelnut trees and linden trees, followed by broad-leaved forests such as birch, for the broad leaves and thick deciduous layer of the above tree species make the soil rich in humus, loose and breathable and therefore good for ginseng growth. The forestlands to be used is better of humus-rich forest ash soil and live loess with good water retention and fertility, and the slope not too large, preferably 5 * 15 degrees. In general, this cultivation model adopts direct seeding or “three-three-year system” or “twofour-year system” and so on with growth period of 4 * 6 years. The advantages of deforestation model are as follows: fertile soil, less diseases and insect pests, mature technology and high yield. However, deforestation has destructive impact on the forests and the environment, not only destroying the vegetation, but also easily causing soil erosion. Therefore, in 1998 Chinese government implemented an industrial policy of “returning farmland to forests” for planting. New forestland is no longer approved for ginseng planting; planting ginseng by random cutting is forbidden; sloping land above 25 degrees or more must be returned to the forest. Hence, the traditional deforestation model has been gradually eliminated.

1.4.3 Farmland Planting Mode Farmland ginseng planting mode is carried out in farmland through appropriate soil improvement, with advantages of alleviating the land competition between ginseng and forests, protecting forest ecology, preventing soil erosion and facilitating intensive management. It generally adopts “three-three-year system” or “two-fouryear system” with 4 * 6 years growth period. This model was first adopted by R. O. Korea and

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gradually introduced to China, Japan and DPR. Korea, all of which now have perfect supporting technologies for farmland ginseng planting and ginseng–grain rotation. At present, R. O. Korea usually uses soil regulator and organic fertilizer for soil improvement and grows ginseng in the way of ginseng–corn rotation and ginseng–rice rotation, with 5 * 6 years growth periods normally. China’s farmland ginseng plantings started late, about 40 years ago, and is conducted in two ways: direct seeding and seedling transplantation. The transplanting can be carried out in the spring or autumn after one or two years of seedling cultivation. The transplanting bed is available in both horizontal and inclined styles. In Japan, the soil needs leisure for one year before planting ginseng. In leisure periods, the soil is improved by applying green manure and organic fertilizer and adding pentachloronitrobenzene powder and chloropicrin to speed up soil improvement. The two modes taken in Japan are direct sowing and transplanting, with fouryear or six-year growth period for direct sowing, and 1-year-old or 2-year-old seedling transplanting for the transplanting method, respectively. In DPR Korea, ginseng is mainly cultivated in farmland, for its soil contains granite parent material with loose texture, permeable water and good nutrient preservation. “One-five-year system” is widely used in production, that is, one-year seedling cultivation and five-year growing after transplanting.

1.4.4 Comparison of Different Planting Models The deforestation model, the traditional method of Chinese ginseng production, is based on cutting forests for ginseng cultivation. But the existing forest resources available for planting are no longer enough to support the demand. Moreover, the continuous cropping obstacles has not been overcame yet, so no substantial breakthrough in the land-reuse technology. Therefore, with more emphasis on ecological environment protection, the deforestation model has been prohibited. Wild breeding, as it imitates wild

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planting without cutting forests, has become an ecological industry model currently to be advocated, but difficult to meet the market demand for its low yield. Compared with deforestation and wild cultivation, the farmland ginseng planting has significant advantages of sustainable development since more land resources can be used without destroying forest resources, and ginseng–grain rotation can be conducted to realize the sustainable utilization of land resources.

1.4.5 Pollution-Free Ginseng Planting Mode As discussed above, farmland ginseng planting taking the advantages of high yield and more land resources, is the major approach of ginseng planting in future. However, with the increasing deterioration of ecological environment, improper site selection, random planting, unreasonable use of pesticides and chemical fertilizers, nonstandard cultivation and processing, the problems of yield decrease, quality decline, agricultural residues and heavy metals exceeding the standard are becoming increasingly serious, which have seriously affected the safety and effectiveness of ginseng (Shen et al. 2015). Therefore, the pollution-free cultivation is the inevitable adoption and future development trend of ginseng planting in farmland (Chen et al. 2018). Up to now, some progress has been made in the research of pollution-free cultivation of ginseng. The Regionalization Information System of the Ecological Suitability for Medicinal Plants (GMPGIS-II) has been used to analyze the ecological suitability of ginseng farming areas. The data indicates that the soil types suitable for farmland ginseng planting mainly are white pulp soil, strong leached soil, dark soil, cambisol soil, alluvial soil, latent soil, dwarf soil, podzolic soil, black soil and so on. The largest ecological similarity region (Ecological similarity degree between 99.9 and 100%) in the world is largely distributed in eastern Asia, central and eastern North America, central and southern Europe and the eastern coast of Oceania, with the USA, Canada, China, Russia, Japan, DPR. Korea,

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Introduction of Panax Ginseng …

France, Italy, R. O. Korea and other regions (Meng et al. 2018) on the top. What is more, the whole genome sequence of Panax ginseng has been assembled by the second-generation sequencing technology. The sequence contains 3.5 GB nucleotides, more than 60% of which are repetitive sequences, and the ginseng gene region encodes 42,006 kinds of predictive proteins. Besides, by the comparative analysis of the whole genome, 31 key enzyme coding genes of mevalonic acid pathway and 225 udpglycosyltransferase (UGT) have been identified. UGTs are one of the largest gene families of Panax ginseng. The sequencing and the analysis of Panax ginseng genome can reveal molecular mechanism of the ginsenoside biosynthesis, the evolution and the disease resistance of Panax ginseng, and thus can provide a basis for the breeding of new varieties of ginseng in farmland (Xu et al. 2017a; Sun et al. 2010; Chen et al. 2011). In addition, it was found that the soil physical structure and suitable pH value could be adjusted using green manure obtained by breaking the perilla, soybean, corn and other crops growing in the land (Xu et al. 2017b). At present, a new variety of “green manure perilla,” specially used for soil improvement in ginseng field, has been developed in China, which can also effectively inhibit the incidence of root rot of ginseng (Wang et al. 2016). In the meantime, Xujiang et al. on the basis of farmland ginseng planting research for many years, put forward the technical regulation and standard of pollution-free ginseng farmland cultivation in the aspects of cultivation land selection, soil improvement, high quality seed seedling production, field management, pest control, quality control and origin traceability etc. (Xu et al. 2018).

1.5

Economy and World Trade of Panax Ginseng

As a valuable herbal medicine, ginseng ranks the top in the field of traditional Chinese medicine, it has long been sought after as a top tonic in East Asia especially and its products are sold in many

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countries and regions around the world. Meanwhile, ginseng, as an important raw material, has been gradually applied in food, health drinks, cosmetics, chemicals (extraction) and other fields, and the demand has been growing steadily in recent years. China and R. O. Korea are the major producing countries with the annual output of China accounting for about 70% of the world’s total output, while that of R. O. Korea about 17%, respectively. The statistics of 2017 show that China’s ginseng production exceeded 10,000 tons (dry ginseng) with an output value over $3 billion, while the R. O. Korea ginseng market output value exceeded $700 million. As the consumption capacity increases, China’s ginseng market is expected to reach $5 billion by 2025. Therefore, there is great potential for market demanding. As a traditional bulk trade medicine, the volume of ginseng trade is not very big compared with other bulk herbs, but the total trade volume is on the top list. In terms of the export, China and R. O. Korea are major exporters. In 2016, China’s total export volume of ginseng products was 2076 tons with a total export value of $147 million, mainly exporting to Japan, R. O. Korea, Europe, the USA, Hong Kong (China) and Taiwan (China), R. O. Korea ginseng exports totaled $158 million (2017), mainly exporting to China, Hong Kong (China), Taiwan (China), Southeast Asia and North America. In terms of imports, Asia remains the world’s largest ginseng consumption region. Ginseng exports to Asia in 2016 totaled $122 million, reaching 83% of global ginseng exports, and the volume to Europe amounted to $22 million, covering 15% of global ginseng exports. Among them, the main importing countries and regions in Asia are Japan, Hong Kong (China) and Taiwan (China), while the main importers in Europe are Germany and Italy. On the whole, in the development of ginseng over the last decade, its yield and trade volume experienced short-term shocks and fluctuations, but they both remained in a relatively stable and increased steadily. In recent years, as the medicinal and tonic efficacy of ginseng has been

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globally recognized and applied, the demand for ginseng is increasing year by year. In 2012, the Ministry of Health of China issued Document No.17 The Announcement on Approving Ginseng (Artificial Planting) as a New Food Resource, allowing ginseng to enter the food production, thus expanding the application fields, hence further stimulating the demand of the ginseng. It can be predicted that the economic and trade volume of ginseng will be gradually increasing in the future.

1.6

Conclusion

Panax ginseng, as an important valuable Chinese herbal medicine, has been paid much attention by the researchers. In the past decades, much progress has been made in the research of ginseng breeding, cultivation, disease and insect pests control and medicinal components. However, due to the large and complex genome of ginseng, little is known about its genetic background and most of the related studies are therefore limited to traditional methods which have seriously restricted the development of ginseng industry. In recent years, owning to the advancement of genomics technology, the researchers have successfully assembled the whole genome of Panax ginseng, filling the gap of the research in the genetic background of ginseng. Thereby, a series of new molecular markers of ginseng breeding have been discovered, the genetic network of some functions clarified, and the key genes of ginsenoside synthesis pathway analyzed (Xu et al. 2017a), which have greatly promoted the research of ginseng breeding, cultivation and synthetic biology. In addition, with the discovery of the subsequent ginseng chloroplast genome, transcriptome, proteomics and rhizospheric metagenomics, the theoretical basis will be laid for the research of the evolution of the ginseng and the genetic polymorphism, the metabolism regulation, the disease resistance and the like (Dong et al. 2018; Kim et al. 2015).

References Asami K (1988) Origin of angiosperms. China Ocean Press, Beijing, pp 161–162. In Chinese Chen SL, Dong LL, Guo QS, Wei JH, Li XW et al (2018) Research of pollution-free and precision cultivation system of Chinese herbal medicines. China J Chin Materia Med 8:1517–1528 Coon JT, Ernst E (2002) Panax ginseng: a systematic review of adverse effects and drug interactions. Drug Saf 25(5):323–344 Choi H, Kim NH, Lee J, Choi BS, Kim KD, Park JY et al (2013) Evolutionary relationship of Panax ginseng and P. quinquefolius inferred from sequencing and comparative analysis of expressed sequence tags. Genet Resour Crop Evol 60(4):1377–1387 Chen S, Luo H, Li Y, Sun Y, Wu Q, Niu Y et al (2011) 454 EST analysis detects genes putatively involved in ginsenoside biosynthesis in Panax ginseng. Plant Cell Rep 30(9):1593–1601 Dong L, Xu J, Zhang L, Cheng R, Wei G, Su H et al (2018) Rhizospheric microbial communities are driven by Panax ginseng at different growth stages and biocontrol bacteria alleviates replanting mortality. Acta Pharmaceutica Sinica B 8(2):272–282 Fu LG (1992) Chinese plant red book vol 1. Science Press, Beijing, p 176. In Chinese Goldstein B (1975) Ginseng: its history, dispersion, and folk tradition. Am J Chin Med 3(3):223–234 Grushwitsky I (1961) Ginseng: the aspects of biology. Nauka, Leningrad Gillis CN (1997) Panax ginseng pharmacology: a nitric oxide link? ☆. Biochem Pharmacol 54(1):1–8 Kim K, Lee S, Lee J, Lee HO, Joh HJ, Kim NH et al (2015) Comprehensive survey of genetic diversity in chloroplast genomes and 45S nrDNAs within Panax ginseng Species. PLOS ONE, 10(6) Kim NH, Jayakodi M, Lee SC, Choi BS, Jang W, Lee J et al (2018) Genome and evolution of the shaderequiring medicinal herb Panax ginseng. Plant Biotechnol J 16(11):1904–1917 Li SY, Wang S (2008) Brief introduction to the origin, shape, production area, sort and differentiating of wild ginseng. Asia-Pacific Tradit Med 4(1):37–39 Lee C, Wen J (2004) Phylogeny of Panax using chloroplast trnC-trnD intergenic region and the utility of trnC-trnD in interspecific studies of plants. Mol Phylogenet Evol 31(3):894–903 Liu C, Xiao P (1992) Recent advances on ginseng research in China. J Ethnopharmacol 36(1):27–38 Meng XX, Shen L, Huang LF, Xiong C, Chen SL (2018) Exploring on environmental standard of high quality pollution-free Chinese herbal medicines. Chin J Exp Tradit Med Formulae 23:1–7 Ma XJ, Wang XQ, Xiao PG, Hong DY (2000) Research progress on domestic ginseng germplasm resources. Chin Pharm J 35(5):289–292

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Park KJ (2001) Fitness analysis of the forecasting model for the root rot progress of ginseng based on bioassay and soil environmental factors. Res Plant Dis 7:20–24 Reunova GD, Kats IL, Muzarok TI, Zhuravlev YN (2010) Polymorphism of RAPD, ISSR and AFLP markers of the Panax ginseng C. A. Meyer (Araliaceae) genome. Russ J Genet 46(8):938–947 Shi F, Li M, Li Y, Jiang P, Zhang C, Pan Y et al (2015) The impacts of polyploidy, geographic and ecological isolations on the diversification of Panax (Araliaceae). BMC Plant Biol 15(1):297–297 Sun C, Li Y, Wu Q, Luo H, Sun Y, Song J et al (2010) De novo sequencing and analysis of the American ginseng root transcriptome using a GS FLX Titanium platform to discover putative genes involved in ginsenoside biosynthesis. BMC Genomics 11 (1):262–262 Shen L, Xu J, Dong LL, Li XW, Chen SL (2015) Cropping system and research strategies in Panax ginseng. China J Chin Materia Med 40(17):3367– 3373 Tao HJ (1986) Famous medical records. People’s Medical Publishing House, Beijing In Chinese Wang R, Dong LL, Xu J, Chen WJ, Li XW, Chen SL (2016) Progress in improvement of continuous monoculture cropping problem in Panax ginseng by controlling soil-borne disease management. China J Chin Materia Med 41(21):3890–3896 Wen J, Zimmer EA (1996) Phylogeny and Biogeography of Panax L. (the Ginseng Genus, Araliaceae): inferences from ITS sequences of nuclear ribosomal DNA. Mol Phylogenet Evol 6(2):167–177 Wang S, Zhang QS (1984) Research on the environment of ecological climate and adaptability to cultivated region in Ginseng. Chin J Plant Ecol 8(2) Xu J, Chu Y, Liao B, Xiao S, Yin Q, Bai R et al (2017a) Panax ginseng genome examination for ginsenoside biosynthesis. Giga Sci 6(11):1–15

13 Xu J, Dong LL, Wang R, Niu WH, Zhang NW, Naoki F et al (2017b) Soil improvement promoted microecology of farmlands for ginseng cultivation. China J Chin Materia Med 42(5):875–881 Xu J, Shen L, Chen SL, Li G, Li XW, Dong LL, Kondo K (2018) Pollution-free criterion and technical regulations of radix ginseng cultivation in the farmland. Modernization Tradit Chin Med Materia MateriaWorld Sci Technol 20(07):102–111 Xiao PG, Zhu ZY, Zhang FQ, Zhu WH (1987) Studies of Panax Ginseng and its cultivation. China Agricultural Press, Beijing In Chinese Yang DQ (1981) The cyto-taxonomic studies on some species of Panax L. J Univ Chin Acad Sci 19(3):298– 303 Yang JX, Tian YX (2004) Medicinal plant cultivation. China Agriculture Press, Beijing In Chinese Zhao YH, Gu XH, Wu LJ, You W (2007) Researches on categories, characteristics, and utilization value of cultivated ginseng germplasm resources. Chin Tradit Drug 38(2):294–296 Zhuravlev YN, Koren OG, Reunova GD, Muzarok TI, Gorpenchenko TY, Kats IL, Khrolenko Y (2008) Panax ginseng natural populations: their past, current state and perspectives. Acta Pharmacol Sin 29 (9):1127–1136 Zhao DY, Li Y, Ding WL (2013) Isolation and characteristics of Panax ginseng autotoxin-degrading bacterial strains. China J Chin Materia Med 38(11):1703– 1706 Zhuravlev YN, Reunova GD, Kats IL, Muzarok TI, Bondar AA (2010) Genetic variability and population structure of endangered Panax ginseng in the Russian Primorye. Chin Med 5(1):21–21

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The Ginseng Genome-Traditional Uses, Medicinal Properties, Phytochemistry, and Pharmaceutical Applications Xiangyan Li, Daqing Zhao, Wenzhi Yang, Tiantian Zuo, Naiwu Zhang, Kondou Kenji, Zubair Ahmed Ratan, and Jae Youl Cho

Abstract

Panax ginseng C.A. Meyer is a valuable plant in the Araliaceae family, which has been used in clinical practices for over 2000 years and recorded in a series of literatures for the treatment of all deficiency syndromes. Recently, the safety and efficacies of ginseng as drug or

X. Li
D. Zhao (&) Key Laboratory of Active Substances and Biological Mechanisms of Ginseng Efficacy, Ministry of Education, Jilin Provincial Key Laboratory of BioMacromolecules of Chinese Medicine, Jilin Ginseng Academy, Changchun University of Chinese Medicine, Changchun, Jilin, China e-mail: [email protected] W. Yang (&)
T. Zuo Tianjin State Key Laboratory of Modern Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin, China e-mail: [email protected] N. Zhang China Medico Coperation, Tianjin, China K. Kenji TSUMURA & CO, Tokyo, Japan Z. A. Ratan Department of Biomedical Engineering, Khulna University of Engineering and Technology, Khulna, Bangladesh J. Y. Cho (&) Department of Integrative Biotechnology, Sungkyunkwan University, Suwon 16419, Republic of Korea e-mail: [email protected]

dietary supplement against multiple diseases were studied in ongoing and completed ginseng clinical trials. Different active components in ginseng including ginsenosides, polyacetylenes, and polysaccharides for different functions were identified and studied. Importantly, the pharmaceutical application of ginseng for memory, energy, stress, antioxidation, and immune system were extensively studied. Current findings for traditional uses, medicinal properties, phytochemistry, and pharmaceutical applications of ginseng are summarized.

2.1

Ginseng in Traditional Medicine and Clinical Uses

2.1.1 Ginseng in Traditional Medicine Concepts and Medicinal Uses 2.1.1.1 Traditional Literatures for Ginseng Panax ginseng C. A. Meyer is a valuable plant in the Araliaceae family, which has been used in clinical practices for over 2000 years (Yun 2001). As a medicinal herb, Ginseng was first recorded in the book, the Shennong Bencao Jing (Shennong’s Herbal Classic) edited in the first century AD, which depicted as having many pharmacological functions, including mainly supplementing the five viscera, quieting the essence spirit, settling the ethereal and corporeal

© Springer Nature Switzerland AG 2021 J. Xu et al. (eds.), The Ginseng Genome, Compendium of Plant Genomes, https://doi.org/10.1007/978-3-030-30347-1_2

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souls, checking fright palpitations, eliminating evil qi, brightening the eyes, opening the heart, sharpening the wits, making body light, and prolonging the life after long-term administration (Park et al. 2012). In the Shanghan Lun (Treatise on Treatment of Diseases Induced by Cold), Zhongjing Zhang from the end of Han Dynasty collected the use of ginseng in 21 formulas of 107 herbal prescriptions and described as mildly reinforcing, moistening, strengthening, enhancing sperm function, keeping warm, enhancing vision, and stabilizing spirit (Park et al. 2012). In the Jin Dynasty, Hongjing Tao wrote two medical books, Mingyi Bielu and Bencaojing Jizhu, and described the properties of ginseng as curing internal coldness and chest or abdomen pain, crushing chest fullness, relieving thirst, fixing feelings, enhancing cognitive function, and increasing blood circulation (Park et al. 2012). Renshen San from Zhouhou Beiji Fang (Handbook of Prescription for Emergency) by Hong Ge was first used in emergency. Ginseng was one of main herbs in Dingzhi Pill, which was used to stabilize the mind and described in Beiji Qianjin Yaofang (Essential Recipes for Emergent Use Worth A Thousand Gold) by Simiao Sun from the Tang Dynasty (Park et al. 2012). In the Song dynastic period, popular and effective formulas were collected in Taiping Huimin Heji Ju Fang, which was written by the Bureau of Taiping People’s Welfare Pharmacy and used in the public dispensaries. Pharmacological properties of ginseng mentioned above were also written in Bencao Gangmu by Shizhen Li from the Ming Dynasty, which is the most important pre-modern medical book (Park et al. 2012). In the Bencao Gangmu, ginseng was used to treat general weakness, spontaneous sweating and fever, vertigo and headache, alternating fever and chills, chronic diarrhea, fatigue, hematemesis, rectum bleeding, bloody urinary leakage, abnormal uterine bleeding, and discomfort before or after parturition (Park et al. 2012). With the development of history, ginseng was mainly discussed to treat multiple diseases in the ancient literatures, based on the medicinal essence of ginseng from Shennong Bencao Jing. In addition, many historical documents after Ming

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Dynasty have described similar activities. Taken together, ginseng was generally considered as the king of herb for the treatment of all deficiency syndromes, such as gastrointestinal problems, anxiety or mental symptoms, and prenatal or postpartum-related diseases (Fig. 2.1).

2.1.1.2 Medicinal Properties of Ginseng Ginseng is sweet and slightly bitter and reflects its actions, including supplement spleen, calming irritation, and nourishing the body. Moreover, the action of ginseng is relatively mild and a little cold, which means that ginseng can alleviate heat syndromes. Ginseng has good benefits for five viscera, which can nourish vital organs of the body and improve their abilities by significantly converting the nutrients into the Qi energy. In addition, ginseng can restore a normal pulse to stop palpitations, dispel the invasion of pathogenic factors, improve the activities of visual acuity and mentality, and enhance life expectancy after long-term intake. 2.1.1.3 Medicinal Use of Ginseng in Ancient Asian Medicine Based on the descriptions in the Shanghan Lun, 21 ginseng-containing prescriptions included Banxia Xiexin Tang, Xiao Chaihu Tang, and Baihu Jia Renshen Tang. Ginseng was prepared and used in different prescriptions, based on its properties of restorative effects, anti-aging function, or strengthening immune system. Sijunzi Tang was composed of four herbs, including Ginseng Radix, Atractylodis Macrocephalae Rhizoma, Poria, and Glycyrrhizae Radix et Rhizoma for treating hypodynamia, lassitude, and anorexia. In Sijunzi Tang, ginseng as a main ingredient exerts organ tonification and restorative activities. Sijunzi Tang and Siwu Tang consisting of Angelicae Gigantis Radix, Cnidii Rhizoma, Paeoniae Radix, and Rehmanniae Radix Preparata were prescribed as Shi-Quan-Da-Bu Tang for the patients with weakness or spontaneous sweating. In particular, the prescriptions containing ginseng relieves thirst caused internal fever as stated by the Minyi Bielu and the Bencao Gangmu, such as Baihu Jia Renshen Tang. Renshen San was first used for seriously ill patients in

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Fig. 2.1 Traditional literatures for ginseng

the Eastern Jin dynastic period. Dushen Tang was only composed of ginseng to tonify Qi and prevent exhaustion, which was described in Shi Yao Shen Shu by Kejiu Ge in 1348 during the Yuan Dynasty. Additionally, Dingzhi Wan and Guipi Tang were prescribed for treating anxiety, amnesia, anemia, and depression, in which ginseng as the main ingredient is used for the improvement of brain function. Now, these classic prescriptions, such as Sijunzi Tang, Renshen Yang Rong Tang, are studied in 11 different the clinical trial registries in the world. Overall, the medicinal use of ginseng in the ancient time was summarized as

tonifying Qi, ameliorating serious illness, improving brain function, and relieving thirst (Ogawa-Ochiai and Kawasaki 2018).

2.1.2 Clinical Studies of Ginseng as Drug or Dietary Supplement 2.1.2.1 The Registered Ginseng Clinical Trials In recent years, clinical trials about ginseng have been conducted and are steadily increasing in the

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Fig. 2.2 Sapogenins, sugars, and acyl groups in ginsenosides

globe, not only in Asian countries like Korea, China, and Japan, but also in Western countries, like USA and Canada (He et al. 2018). In 2005, the International Clinical Trials Registry Platform (ICTRP) was established by The World Health Organization (WHO). After a survey through the ICTRP Search Portal, 222 registration records were obtained from the 17 databases by advanced search for “Panax” MeSH term until June 2019. 169 trials were included from further analysis, and 53 trials were excluded because of not ginseng intervention. 11 registries for clinical trials of ginseng were found as following: Australian New Zealand Clinical Trials Registry (ANZCTR, 16 trials), Chinese Clinical Trial Register (ChiCTR, 9 trials), Clinical Research Information Service-Republic of Korea (CRiS, 31 trials), Clinical Trials.gov (68 trials), Clinical Trials Registry-India (CTRI, 2 trials), EU Clinical Trials Register (EU-CTR, 2 trials), German Clinical Trials Register (DRKS, 1 trial), Iranian Registry of Clinical Trials (IRCT, 19 trials),

ISRCTN (2 trials), Japan Primary Registries Network (JPRN, 18 trials), and Thai Clinical Trials Registry (TCTR, 1 trial). With respect to recruitment status, 118/169 of the trials (69.8%) have not yet begun to enroll participants. Of the 169 trials analyzed, the interventions of ginseng extracts and ginsenosides (139 trials) or prescriptions (39 trials) containing ginseng were conducted. The subjects were either healthy volunteers or patients with different pathological conditions, such as diabetes, fatigue, cognitive disorders, metabolic syndrome, hypertension, erectile dysfunction, mental disorders, and cancer-related symptoms, who were studied to achieve the primary purposes of the treatment, prevention, and safety for ginseng administration (Xiang et al. 2008). The duration of ginseng clinical trials was maintained from 4 to 24 weeks, and the dose of ginseng was ranging from 0.3 to 15 g each day, which made it difficult to compare the dose and duration of ginseng clinical trials.

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2.1.2.2 The Completed/Published Ginseng Clinical Trials (1) Safety Most clinical studies were to evaluate the efficacy of ginseng against different diseases, including cognitive and cardiovascular disorders, fatigue, sexual dysfunction, and diabetes (Kim et al. 2015). Some studies reported adverse events, including hot flushes, insomnia and dyspepsia, which were not significantly different in the frequency and symptom of ginseng administration, compared with placebo group (Lee et al. 2012). Meanwhile, some studies showed no adverse events or had no mention for any adverse events. (2) Alzheimer’s Disease In recent years, the studies indicated that ginseng plays protective effects in the prevention and treatment of neurological diseases. After assessing by the tests of the mini-mental state examination (MMSE) and Alzheimer’s disease (AD) Assessment Scale-Cognitive Subscale (ADAS-Cog), cognitive function was gradually improved by the long-term intake of Korean red ginseng (KRG) extract (Kim et al. 2018; Heo et al. 2011). In elderly patients with AD, frontal cortical activity was improved after the administration of relatively high amounts of KRG for more than 12 weeks (Heo et al. 2016). Several reports indicated that active components from ginseng extracts may prevent amyloid plaque accumulation-induced cognitive dysfunction (Kim et al. 2018; Wang et al. 2016; Heo et al. 2008). However, the protective effect of ginseng for the patients with AD is keeping inconclusive. In the future, larger and well-designed clinical studies in AD patients need to evaluate protective effect of ginseng to avoid current main limitations, such as small sample sizes and poor methodological qualities. (3) Fatigue Several studies showed that the intervention group had significantly improved fatigue outcome compared with the placebo group. In one prospective, open-label study, ginseng (800 mg,

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29 days) was safe and improved overall quality of life, appetite, and sleep in 30 patients with cancerrelated fatigue (Yennurajalingam et al. 2015). In a double-blind study of 60 patients with multiple sclerosis (MS), fatigue and quality of life were improved by ginseng treatment (250 mg, over 3 months) without side effects (Etemadifar et al. 2013). 20% ethanol extract of ginseng (1 g or 2 g/day) were administered to 90 subjects with idiopathic chronic fatigue (ICF) for 4 weeks can decrease the scores of total self-rating numeric scale (NRS) and the visual analogue scale (VAS), which is partly related with the antioxidant properties of ginseng (Kim et al. 2013). These present studies are not insufficient and limited; it suggests that larger and more diverse samples are needed to increase the generalizability and statistical power in the future (Arring et al. 2018). (4) Diabetes Mellitus Currently, patients with type 2 diabetes mellitus (T2DM) or impaired glucose tolerance (IGT) were studied to observe the efficacy of ginseng by several randomized clinical trials. Ginseng supplementation can improve glucose control and insulin sensitivity in patients with T2DM or IGT (Gui et al. 2016). A 4-week long, randomized, double-blind, and placebocontrolled trial of fermented red ginseng (FRG) in 42 subjects showed that FRG supplementation significantly reduced postprandial glucose level and also increased postprandial insulin level, compared to the placebo group, which had no effect on fasting glucose, insulin, and lipid profiles (Oh et al. 2014; Shishtar et al. 2014a). After receiving 1, 3, or 6 g of Korean white ginseng, the augmentation index and central blood pressure were investigated in 25 patients with well-controlled T2DM. This acute, randomized, placebo-controlled, double-blind, and crossover study indicated that Korean white ginseng showed a significant beneficial effect of arterial health and had no effect on any other parameters. Overall, long-term investigation of accountable components from ginseng in subjects with diabetes needs to be conducted and highlighted (Shishtar et al. 2014b).

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(5) Hypertension Currently, several randomized clinical trials for ginseng alone or as an adjunct therapy to other treatment in patients with pre-hypertension or hypertension were reported. Some studies reported positive effects of Korean red ginseng on reducing systolic and diastolic blood pressures, compared with placebo (Lee et al. 2017). Compared with control group, central and peripheral arterial pressures of healthy volunteers were acutely reduced by ginsenoside Rg3 (400 mg)-enriched KRG extract at 3 h after intervention (Jovanovski et al. 2014). However, one report showed that the drink with 800 mg of ginseng did not have a significant impact on blood pressure in 27 healthy individuals through assessing systolic blood pressure at baseline, 1, 2, 3.5, and 5.5 h (Shah et al. 2016). Based on the findings above, long-term clinical evaluation in higher-risk individuals should be done to evaluate the efficacy of ginseng on blood pressure. (6) Others The efficacies of the main components of ginseng, ginseng polysaccharides on the immune function were investigated in 72 healthy volunteers (Cho et al. 2014) or patients with non-small cell lung cancer (Ma et al. 2014) using randomized, placebo-controlled, and double-blind clinical trials. In 2019, the first international, multicenter, randomized controlled trial of ginseng for chronic obstruction pulmonary disease (COPD) was reported that 168 participants with moderate COPD were administrated ginseng capsules (100 mg, twice daily) and followed up for a further 24 weeks. Compared with placebo group, ginseng had no significant function on the health-related quality of life, and seemed safe and well-tolerated for the patients with COPD (Shergis et al. 2019). In addition, the ginseng prescriptions such as Sailuotong capsule, Shenfu decoction, or Shenmai injection (SMI) as adjuvant therapy were administrated to healthy adults or patients with chronic heart failure (CHF) to investigate their protective effects on cardiovascular function (Steiner et al. 2016; Wei et al.

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2015; Xian et al. 2016). In addition to standard therapies for CHF, 240 eligible patients received SMI (Panax ginseng and Ophiopogon japonicus) or placebo (100 ml/day) for 1 week. This double-blind, multicenter, and randomized study indicated that the New York Heart Association functional classification, 6-min walking distance, short-form 36 health survey score, and traditional Chinese medicines syndrome score were significantly improved by SMI without no apparent safety concerns, compared with the placebo group (Xian et al. 2016). Collectively, cognitive disorders, fatigue, diabetes, cancer-related symptoms, and CHF were the most common conditions in clinical trials of ginseng which were summarized (Table 2.1). However, present research for ginseng in healthy subjects or patients with various disorders is limited and small-scale clinical trial. Therefore, it is necessary to increase the number of the subjects and keep stricter regulation to evaluate the efficacy and safety of ginseng as complementary and alternative medicine in clinical studies.

2.2

Active Components/Chemistry from White and Red Ginseng

According to reports, Panax. Ginseng (P. ginseng) contains many chemical substances, which are the primary and secondary metabolites of plants. Multiple types of compounds have been isolated from different parts (rhizome/root, stem leaf, flower bud, fruit, and seed) of this medicinal herb, as well as red ginseng, sun ginseng, and the other steamed products (steamed leaf and steamed flower bud) (Yun 2001), including the saponins, organic acids/esters, polysaccharides, polyacetylenes, amino acids, sterols, flavonoids, and carbenes.

2.2.1 Ginsenosides The ginsenosides isolated from ginseng is an oligo-saccharide glycoside composed of sapogenin and sugars in structure. The sapogenin contained in ginsenosides is mainly composed of the tetracyclic triterpenoid dammarane, and

Intervention/Treatment

Korean Red Ginseng

Korean red ginseng

Panax ginseng

Korean ginseng

Location

Korean

Korean

United States

Iran

500 mg/3 months

800 mg/29 days

4.5 g/day, 12 weeks

4.5 g, 9 g/day, 24 weeks

Dose/Duration

52

30

14

61

Subject #

Table 2.1 Summary for the published clinical trials of ginseng

Multiple sclerosis

CRF

Alzheimer’s disease

Alzheimer’s disease

Diagnosis

Doubleblind placebocontrolled RCT

Single arm pre/post

Doubleblind placebocontrolled RCT

Doubleblind placebocontrolled RCT

Study design

Modified fatigue Impact Scale

Functional assessment of chronic illness therapy fatigue

Korean minimental state examination (KMMSE) and the frontal assessment battery

The Alzheimer’s disease assessment scale (ADAS), Korean version of the mini-mental status examination (K-MMSE)

Assessment

Total fatigue was significantly different in favor of ginseng. Physical dimension on the fatigue scale was significantly different for the ginseng group

Significantly improved fatigue scores at days 15 and 29 from baseline

Significant improvement on the frontal lobe function in AD after 12 weeks of KRG therapy

Feasible efficacies of long-term treatment of KRG for AD diseases

Main results

Reported no serious AEs. Did not report AEs in detail. Only reported one patient who had constipation, which resolved (continued)

No AEs attributed to the study. Grade >3, 6%. These were not attributed to the study. Most reported AEs: pain, nausea

No significant adverse events

Adverse events withdrew from the study

Reported AEs

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Intervention/Treatment

Panax ginseng

Fermented red ginseng

Korean white ginseng

Energy drink with ginseng

Location

Korea

Korean

Canada

United States

Table 2.1 (continued)

800 mg/5.5 h

1, 3, or 6 g/4 h

3 times/day, 4 weeks

1000, 2000 mg/4 weeks

Dose/Duration

27

25

42

90

Subject #

Healthy volunteer

type 2 diabetes

Impaired fasting glucose or type 2 diabetes

Idiopathic chronic fatigue

Diagnosis

Doubleblind placebocontrolled RCT

Doubleblind placebocontrolled RCT

Doubleblind placebocontrolled RCT

Doubleblind placebocontrolled RCT

Study design

QTc interval and systolic blood pressure, QT, PR intervals, QRS duration, heart rate, and diastolic blood pressure

Augmentation index and central blood pressure, ambulatory blood pressure, a symptoms questionnaire

Fasting and postprandial glucose profiles, insulin and lipid profiles

Fatigue numeric rating scale (NRS) and VAS

Assessment

No significant impact on blood pressure parameters

No effect for postprandial, vascular or glycemic parameters. A beneficial effect on arterial health

Lowered postprandial glucose levels in subjects with impaired fasting glucose or type 2 diabetes

NRS questions decreased for all three groups, with no significance between groups. cVAS scores declined for all groups, with P. ginseng(2 g) arm scores significantly lower than control

Main results

(continued)

Not to be reported

Be safe

No severe side effects, only one subject withdrawing due to hypoglycemia

AEs not reported

Reported AEs

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Intervention/Treatment

Rg3-enriched Korean ginseng

Ginseng capsules

Shenfu granules

Shenmai injection (SMI)

Location

Canada

Australia and China

China

China

Table 2.1 (continued)

100 ml/day

Ginseng 10 g, Heishunpaian, 10 g/day, 2 weeks

100 mg, twice daily/ 24 weeks

400 mg/1 week

Dose/Duration

240

40

168

23

Subject #

CHF

Symptomatic CHF

COPD

Healthy volunteer

Diagnosis

Doubleblind placebocontrolled RCT

Doubleblind placebocontrolled RCT

Doubleblind placebocontrolled RCT

Doubleblind placebocontrolled RCT

Study design

The primary endpoint of the New York Heart Association (NYHA) functional classification, 6min walking distance (6MWD), short-form 36 (SF36) heart survey score, traditional Chinese medicine (TCM) syndrome score, left ventricular ejection fractions (LVEF), B-type natriuretic peptide (BNP) level

Quality of life (QOL), cardiac function, left ventricular ejection fraction (LVEF)

St George’s respiratory questionnaire, COPD assessment test, and the Short Form Health Survey

Aortic augmentation index, central, and peripheral blood pressures

Assessment

The significant improvements of NYHA functional classification, 6MWD, SF-36 score, TCM syndrome score after 1 week treatment of SMI

Improved QOL and Increased the grading of cardiac function and LVEF

No significant differences between ginseng and placebo with overall results improving

Acutely lowered central and peripheral arterial pressures

Main results

Welltolerated with no apparent safety concerns

No significant adverse events

seemed safe and welltolerated

No selfreported adverse effects

Reported AEs

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protopanaxtriol (20S/20R; PPT), protopanaxadiol (20S/20R; PPD), the pentacyclic triterpenoid oleanolic acid (OA), and PPD and PPT are composed of general derivatives of H2Oaddition, hydroxylation, dehydration, carbonylation, dehydrogenation, cyclization, and oxidation together with versatile derivatives of PPD and PPT via H2O-addition, hydroxylation, dehydration, carbonylation, dehydrogenation, cyclization, and oxidation. These sugars are composed of glucose (Glc), glucuronic acid (GluA), rhamnose (Rha), arabinose (in the form of pyran or furan), and xylose (Xyl). The sugar groups sometimes have acyl substituents (such as acetyl, crotonyl, octenyl, and malonyl (Fig. 2.2). According to the structure differences of ginsenosides, the known ginsenosides can be reasonably divided into PPD type, PPT type, OAtype, C17 side chain variant type, etc. Among them, the PPD, PPT, and OA types of ginsenosides are the most common for P. ginseng, the OT-type ginsenosides are rare in P. ginseng, which are diagnostic for P. quinquefolius (American ginseng). According to our statistics, up to 2018, at least 247 ginsenosides compounds have been isolated from P. ginseng and its products, which include 57 of the PPD type, 45 of the PPT type, 13 of the OA type, 106 of the C17-side chain varied, and 26 miscellaneous. Among them, C17 side chain varied accounted for the most. Detailed information of these 247 ginsenosides is listed in Table 2.1 (Park et al. 2012; Ogawa-Ochiai and Kawasaki 2018; He et al. 2018; Xiang et al. 2008; Kim et al. 2015, 2018; Lee et al. 2012; Heo et al. 2011). PPD-type and PPT-type ginsenosides are the most common saponins for various Panax species. PPD (C-3/C-12/C-20) and PPT (C-3/C-6/C12/C-20) sapogenins have three and four hydroxyl groups, respectively. The glycosylation sites of PPT-type ginsenosides preferably include 6OH and 20-OH, while 3-OH and 20-OH for PPD-type ginsenosides. In addition, the C-20 a chiral center usually appears in the Sconfiguration, while the R-configuration isomer also exists in P. ginseng, such as 20(S)-/20(R)ginsenoside Rh1 (PPT type) and 20(S)-/20(R)ginsenoside Rg3 (PPD type).

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PPD and PPT ginsenosides are prone to malonylation, and the malonyl form is accompanied by most of the main saponins in P. ginseng (such as malonyl-ginsenoside Re, malonyl-ginsenoside Rb1, malonyl-ginsenoside Rc, malonyl-ginsenoside Rb2, and malonylginsenoside Rd). Malonyl ginsenosides are rich in flower buds of P. ginseng, but during the steaming process, malonyl ginsenosides can be converted into rare ginsenosides due to acidic hydrolysis and dehydration. What is more, the steaming process can also convert neutral ginsenosides into rare ginsenosides (Heo et al. 2016). OA-type ginsenosides have typical structure features: (1) glycosylation at 3-OH and 28COOH; (2) GluA as the inner sugar directed attached to 3-OH. OA-type ginsenoside Ro is a kind of abundant compound although its distribution is not as diverse as ginseng PPP type and PPT type (Wang et al. 2016). C-17 side chain ginsenosides are the most abundant ginsenosides in P. ginseng and the main source of novel ginsenosides. C-17 side chain changes mainly include H2O addition, oxidation (at the double bond), methoxylation, carbonylation, hydroxylation, peroxide, dehydrogenation, cyclization, degradation, rearrangement or integration of two or more reactions. It should be noted that the content of C-17 side chain ginsenoside in stems, leaves, buds, and processed products was higher than that in P. ginseng roots (Heo et al. 2016). Apart from the above five subtypes, according to reports, other saponins with abnormal changes or modifications in the sapogenin part have also been isolated from P. ginseng species. The changes include carbonylation at C-3 or C-19, 5H and 6 dehydration or further 7-hydroxylation, and even degradation products (Yun 2001).

2.2.2 Polyacetylenes Polyacetylenes are a class of biologically active natural molecules widely distributed in plants. It has a distribution in P. ginseng, such as the C17polyacetylene of P. ginseng. According to

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reports, panaxynol was first isolated from the root of P. ginseng in 1964. Among them, panaxydol is another polyacetylene compound. Their content in ginseng fiber roots is about 0.04% and 0.03%, respectively. Although they are low in content, they can still effectively anticancer and anti-inflammation, protect and nourish nerves (Heo et al. 2008). The current research polyacetylenes in P. ginseng is not thorough enough, the medicinal potential of these polyacetylenes in P. ginseng needs more study.

2.2.3 Polysaccharides Polysaccharide in P. ginseng has a variety of pharmacological properties, such as anti-cancer, anti-oxidation, anti-aging, reducing blood sugar and blood lipid, immune regulation, anti radiation. (Yennurajalingam et al. 2015). According to the review, at least 79 polysaccharides were isolated from the roots of Panax ginseng. The main components of polysaccharides isolated from Panax ginseng are starch-like glucans and pectin. Pectin of P. ginseng is composed of glucose (Glc), mannose (Man), rhamnose (Rha), galacturonic acid (GalA), galactose (Gal), arabinose (Ara), glucuronic acid (GlcA), fucose (Fuc), and xylose (Xyl). Those starch-like polysaccharides in P. ginseng contains side chain-free a-D(1,6)-glucans 6-branched a-D-(1,4)-glucans, aD-(1,4)-glucans, and 3-branched a-D-(1,6)-glucans. In future research, efforts should be made to develop novel analytical techniques capable of high-resolution and overall analysis of polysaccharides (polysaccharide fingerprints) to better control the quality of ginseng (Etemadifar et al. 2013).

2.3

Pharmaceutical Applications of Ginseng

2.3.1 Memory Memory is a natural brain process that requires continued attention and recording by parts of the brain, and different pathophysiological processes

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can impair memory. Our brain is an extensive network of neurons and memories generate when specific sets of neurons become reactivated. Stress, smoking, drug abuse, trauma, neurodegenerative disorders, and infections are the major causes of memory loss (Zhao et al. 2011; Maalouf et al. 2011). Zhang Jing-Yue, a famous physician from Ming Dynasty used “Qi Fu Yin” a special medicine for dementia, however, it was comprised of seven traditional herbs, and Panax ginseng was one of the most important ingredients of that mixture (Berke and Hyman 2000). Interestingly, ginseng is still widely used for the improvement of cognitive performance. Dementia became of the major causes of disability around the globe, especially for aged people (Ong et al. 2018). Different studies have revealed that ginsenosides can ameliorate the cognitive function in different-aged animal models with dementia (Flier and Scheltens 2005; Han et al. 2018). The cholinergic system in the brain plays a vital role in memory formation. Acetylcholine (Ach), the neurotransmitter is synthesized from choline and acetyl-CoA by acetyl-transferase, and it is widely distributed in the nervous system (Wang et al. 2009). Memory loss can be prevented by any substance that can inhibit acetylcholinesterase (AchE) activity or can increase the Ach level. One study revealed that ginsenoside Rb1 can enhance the uptake of choline in the cerebral cholinergic nerve ending as well as this can modulate the Ach release and uptake (Blokland 1995). Another neurotransmitter, glutamate has a significant role in learning, memory, and cognitive function (Benishin 1992). Yi Chang and his team identified that ginsenosides Rb1 and Rg1 can increase glutamate exocytosis from the cortical nerve terminals of rats, help to release glutamate by affecting mobilization of vesicles through activation of protein kinase C (Aigner 1995). Different studies have suggested that accumulation of amyloid b-peptide into oligomers or fibrils plays a significant role in the progression of Alzheimer's disease (AD). The pathological features of AD are mainly caused by deposition of b-amyloid (Ab) around the small vessel walls of the brain. One finding suggested that Panax notoginseng can inhibit the

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aggregation of Ab in a dose-dependent manner (Chang and Wang 2008). Another study revealed that administration of ginseng and its components can inhibit Ab aggregation in cultured neurons, and thus, it can improve the spatial learning abilities and memory (Choi et al. 2010). Oxidative stress plays a critical role in neuronal degeneration and nigral cell death in Parkinson’s disease (PD). Mitochondrial dysfunction and increased dopamine metabolism are the underlying causes of oxidative stress (Fang et al. 2012). Reactive oxygen species (ROS) and an impaired antioxidant defense system can deteriorate the neural degeneration (González-Burgos et al. 2015), interestingly, study enumerated that ginsenosides Rb1, Rg1, and Rd can inhibit the oxidative stress and neuroinflammation which ultimately reduce the progression of PD (Fang et al. 2012). Besides, ginsenoside Rg1 showed protective effects against apoptosis in substantia nigra neurons by decreasing the levels of cleaved caspase-3, Bax, and iNOS (Jenner 2003; Chen et al. 2002). One clinical study from the Clinical Research Institute, South Korea, demonstrated that intake of ginseng root for 12 weeks improved the mental performance in AD patients in a significant manner (Zhou et al. 2016).

2.3.2 Energy According to Grand View Research, Inc., it is expected that by the year 2024, the global dietary supplements market will reach USD 278.02 billion (Lee et al. 2008). However, ginseng has been recognized as one of the popular herbal dietary supplements, and some believe that it can increase endurance, energy, and strength. Ginsenosides, the major pharmacologically active components of ginseng which are predominantly composed of a dammarane skeleton with various sugar moieties, such as xylose, glucose, arabinose, and rhamnose. However, the levels of ginsenosides may vary from plant due to age or harvesting season (Radimer et al. 2004). Zhen Yu and his team evaluated the pharmacological activities of ginsenoside Rb1 to combat fatigue, interestingly, ginsenoside Rb1 showed a

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potential anti-fatigue effect by the repression of oxidative stress in skeletal muscles (Smith et al. 1996). Furthermore, ginseng can facilitate the mitochondrial activity during exercise, as well as it can inhibit the hydroxyl radical and lipid peroxidation (Tan et al. 2013). However, another study revealed that long-term use of ginseng can improve the cardiorespiratory function and decrease blood lactate concentration (Zhong and Jiang 1997). Thus, ginseng can improve physical condition. Different clinical studies have suggested that ginseng can improve running time, aerobic capacity, physical performance, etc. (Engels and Wirth 1997; Schepdael 1993). Caffeine, another source of energy, can alter sleep pattern, but ginseng does not show this side effects (Forgo and Kirchdorfer 1981). Therefore, ginseng could be a good source of energy with fewer side effects.

2.3.3 Stress Stress is defined as a state of threatened homeostasis that plays a vital role in the initiation of adaptive responses. The adaptive stress response mainly relies on an immensely interconnected neuroendocrine, cellular, and molecular infrastructure, and the hypothalamic–pituitary–adrenal (HPA) axis (Gyllenhaal et al. 2000). Stress is divided into four subtypes: acute distress (or too much stress), and chronic stress (or burnout stress), deficient stress, and optimum stress; however, when stress exceeds the optimum level, it can anticipate serious health problems, such as depression and anxiety (Tsigos and Chrousos 2002). Cortisol is closely linked to stress and is secreted by the adrenal glands for mitigating stress to maintain homeostasis. However, prolonged cortisol secretion can lead to immunosuppression, and the cortisol is regulated by the hypothalamic–pituitary–adrenal (HPA) axis (Fevre et al. 2003). Eiichi Tachikawa and his team found that components of ginseng can directly influence the adrenal glands, where the stress hormones adrenaline and cortisone are being produced, and they also revealed that ginsenosides and their metabolites have the

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potential to reduce stress (Park et al. 2005). The elevated plus maze (EPM) has been introduced to measure anxiety in laboratory animals as a screening test for putative anxiolytic or antigenic compounds. One study used the EPM in mice to suggest that ginseng can induce anxiolytic-like effects (Fevre et al. 2003). Therefore, ginseng is a potential agent for relieving stress, although more scientific studies are required to discover the exact mechanism.

2.3.4 Antioxidant In recent years, the increasing knowledge of free radicals and ROS opens a new era in the progression and prevention of diseases. The free radicals, including the ROS and reactive nitrogen species (RNS), are derived from endogenous and exogenous sources. Here, peroxisomes, endoplasmic reticulum, phagocytic cells are the endogenous sources, whereas different pollution, pesticides, smoking, heavy metals, certain drugs, radiation act as the exogenous source of free radicals (Hasegawa 2004). Different studies have revealed that cancer, different inflammatory disorders and degenerative disease are closely associated with free radicals (Phaniendra et al. 2015). Antioxidants act as radical scavengers, and these can detoxify ROS (Pham-Huy et al. 2008). Scientists are always looking for good antioxidants, and spices and herbs could be a potential source of antioxidants. Studies suggested that the ethanol and methanol extracts of ginseng leaves can potentially scavenge the free radicals, interestingly, the ethanol extract showed the highest 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical and ferrous ion chelating activities and hydroxyl radical scavenging activities (Lobo et al. 2010). In addition, the antioxidant enzymes such as glutathione peroxidase and superoxide dismutase have been increased by ginseng treatment (Lee et al. 2016). Besides, the antioxidant effect of ginseng has been identified through clinical trials, Son CG and his team investigated the role of Panax ginseng on healthy volunteers, and their result suggested that ginseng can significantly

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reduce the levels of serum ROS and methane dicarboxylic aldehyde (MDA) activity (Chen 1996).

2.3.5 Boost the Immune System Inflammation involves both the innate and adaptive immune systems, and heat, pain, redness, swelling, and loss of function are the five classical features of inflammation (Yang et al. 2017). Plant extracts have been used from ancient times for reducing pain and inflammation. Interestingly, Felix Hoffman, a famous scientist discovered aspirin, an inhibitor of the cyclooxygenase (COX) enzymes (FerreroMiliani et al. 2007). Different in vitro, in vivo, and clinical studies have suggested that ginseng can exhibit anti-inflammatory activity (Dinarello 2010; Lee et al. 2011; Jung et al. 2013). One study revealed that ginsan, a polysaccharide extract from P. ginseng, can inhibit the p38 MAP kinase pathway in in vitro; moreover, it can also inhibit the proinflammatory cytokines in in vivo experiments (Lee et al. 2012). Another study revealed that ginsenoside Rg3 inhibited phorbol ester-induced COX-2 and NF-jB induction (Ahn et al. 2006). In 2006, David M. Shepherd and his team examined the immunomodulatory effects of the herbal extract Panax notoginseng and they found that it can inhibit the LPS-induced production of TNF-a and IL-6 in a concentration-dependent manner (Friedl et al. 2001). A group of scientists from Seoul National University, Korea, examined the impact of ginseng on 39 patients who were recovering from surgery due to stomach cancer. Interestingly, these persons showed significant improvement in immune functions (Rhule et al. 2006). In conclusion, multiple protective effect of ginseng was recorded based on the traditional medicine theory and clinical practices since ancient times. Now, active components of ginseng and its medical properties and clinical trials against a series of diseases are being studied. However, three challenges we meet still need to be investigated as following: (1) clinical studies

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for ginseng or its prescription against various disorders should be further studied in large and multicenter patients, according to standard clinical trial protocols. (2) Except for ginsenosides, other active components of ginseng should be clarified and studied for exploring their efficacies. (3) Molecular mechanism and target network of active ingredients from ginseng for good benefits need to be deeply clarified. We believe that continued and extensive studies of ginseng could bring a good perspective for ginseng industry.

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30 placebo-controlled, multiple-crossover, acute dose escalation trial. Clin Nutr Res 3(2):89–97. Shishtar E, Sievenpiper JL, Djedovic V, Cozma AI, Ha V, Jayalath VH et al (2014a) The effect of ginseng (the genus panax) on glycemic control: a systematic review and meta-analysis of randomized controlled clinical trials. PLoS ONE 9(9):e107391 Smith RG, Caswell D, Carriere A, Zielke B (1996) Variation in the ginsenoside content of American ginseng, Panax quinquefolius L., roots. Can J Bot 74 (10):1616–20. Steiner GZ, Yeung A, Liu JX, Camfield DA, Blasio FM, Pipingas A et al (2016) The effect of Sailuotong (SLT) on neurocognitive and cardiovascular function in healthy adults: a randomised, double-blind, placebo controlled crossover pilot trial. BMC Complement Altern Med 16:15 Tan S, Zhou F, Li N, Dong Q, Zhang X, Ye X, et al (2013) Anti-fatigue effect of ginsenoside Rb1 on postoperative fatigue syndrome induced by major small intestinal resection in rat. Biol Pharm Bull b13– 00522 Tsigos C, Chrousos GP (2002) Hypothalamic–pituitary– adrenal axis, neuroendocrine factors and stress. J Psychosom Res 53(4):865–871 van der Flier WM, Scheltens P (2005) Epidemiology and risk factors of dementia. J Neurol Neurosurg Psychiatry 76(suppl 5):v2–v7 van Schepdael P (1993) The effects of ginseng G115 on the physical capacity of endurance sports. Acta Ther 19:337–347 Wang Y-Z, Chen J, Chu S-F, Wang Y-S, Wang X-Y, Chen N-H et al (2009) Improvement of memory in mice and increase of hippocampal excitability in rats by ginsenoside Rg1’s metabolites ginsenoside Rh1 and protopanaxatriol. J Pharmacol Sci 109(4):504– 510 Wang Y, Yang G, Gong J, Lu F, Diao Q, Sun J et al (2016) Ginseng for Alzheimer’s disease: a systematic

X. Li et al. review and meta-analysis of randomized controlled trials. Curr Top Med Chem 16(5):529–536 Wei H, Wu H, Yu W, Yan X, Zhang X (2015) Shenfu decoction as adjuvant therapy for improving quality of life and hepatic dysfunction in patients with symptomatic chronic heart failure. J Ethnopharmacol 169:347–355 Xian S, Yang Z, Lee J, Jiang Z, Ye X, Luo L et al (2016) A randomized, double-blind, multicenter, placebocontrolled clinical study on the efficacy and safety of Shenmai injection in patients with chronic heart failure. J Ethnopharmacol 186:136–142 Xiang YZ, Shang HC, Gao XM, Zhang BL (2008) A comparison of the ancient use of ginseng in traditional Chinese medicine with modern pharmacological experiments and clinical trials. Phytother Res 22 (7):851–858 Yang Y, Ren C, Zhang Y, Wu X (2017) Ginseng: an nonnegligible natural remedy for healthy aging. Aging Dis 8(6):708 Yennurajalingam S, Reddy A, Tannir NM, Chisholm GB, Lee RT, Lopez G et al (2015) High-dose asian ginseng (Panax ginseng) for cancer-related fatigue: a preliminary report. Integr Cancer Ther 14(5):419–427 Yun TK (2001) Brief introduction of Panax ginseng C. A. Meyer. J Korean Med Sci 16(Suppl):S3-5 Zhao B, Lv C, Lu J (2019) Natural occurring polysaccharides from Panax ginseng C. A. Meyer: a review of isolation, structures, and bioactivities. Int J Biol Macromol 133:324–336 Zhong G, Jiang Y (1997) Calcium channel blockage and anti-free-radical actions of ginsenosides. Chin Med J 110(1):28 Zhou Q-l, Xu W, Yang X-W (2016) Chemical constituents of Chinese red ginseng. Chin J Tradit Chin Med 41(02):233–249

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Nucleotide Signature and SNP Double Peak Methods Detect Adulterants and Substitution in Panax Products Yang Liu, Gang Wang, and Jianping Han

Abstract

There is a big international market for medicinal Panax medicines and their products, such as Panax ginseng, Panax quinquefolium, Panax notoginseng and so on. Panax species share similar morphological characteristics, however possess obvious difference in pharmacological effects and price. Thus, the adulterants are of widespread in ginseng market. Commonly used identification methods, morphological identification and chemistry analysis for example, are sometimes neither efficient nor accurate dealing with ginseng adulterations. DNA barcoding and mini-barcoding technique give us new insights into identification of ginseng materials and products. DNA barcoding technique could succeed in distinguishing species by obvious differentials between interspecific distances and intraspecific distances, however fail in distinguishing closely related species among which the difference between interspecific distances and intraspecific distances is ambiguous. According to the alignment of

Y. Liu
G. Wang
J. Han (&) Engineering Research Center of Tradition Chinese Medicine Resource, Ministry of Education, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100193, China e-mail: [email protected]

all available DNA barcoding sequences of Panax species, the species-specific SNPs are excavated and then nucleotide signature method and double-peak detection method are established based on the species-specific SNPs. Nucleotide signature of Panax ginseng, Panax quinquefolium and Panax notoginseng has been exploited. The nucleotide signature combined with double-peak detection method or NJ tree analysis could accurately identify closely related Panax species and deal with the DNA degradation occurred in ginseng products with high efficiency.

3.1

Panax ginseng and its Closely Related Species

Panax ginseng is a precious traditional medicine with a long application history in the Asia, which could date back to prehistory. Its high medicinal values can be indicated by its genus name Panax of which the meaning is ‘cure all’ in Greek. This medicine is regarded as the lord or king of herbs (Hu 1976). According to the Compendium of Materia Medica, ginseng can be used as a curative medicine for 23 diseases (Yun 2001). According to the Dongeui Bogam (Korean Clinical Pharmacopoeia), ginseng is included in up to 653 prescriptions (Kim et al. 2014). Also, in daily life, ginseng is mainly eaten as a tonic to invigorate weak bodies.

© Springer Nature Switzerland AG 2021 J. Xu et al. (eds.), The Ginseng Genome, Compendium of Plant Genomes, https://doi.org/10.1007/978-3-030-30347-1_3

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P. ginseng belongs to the Family Araliaceae. And, totally thirteen species are named as ginseng. Different species mainly distributes to different areas. P. ginseng is cultivated in Korea, Japan, China, Russia and Germany; Panax quinquefolius, so-called American ginseng, is native to Southern Canada and the USA; Panax trifolius, so-called Dwarf ginseng, also grows in America, mainly from Nova Scotia to Wisconsin. Panax japonicas, so-called Japanese ginseng, mainly grows in Japan; Panax notoginseng is mainly distributed in Yunnan province of China; Panax major, Panax omeiensis and Panax pseudoginseng are mainly distributed in Nepal and the eastern Himalayas. The rest includes Panax sinensis, Panax stipuleanatus, Panax wangianus, Panax zingiberensis (Wen and Zimmer 1996) and Panax vietnamensis (Duc et al. 1994). And, they all have their own major agricultural regions. Among all these ginseng species, P. ginseng, P. notoginseng, P. quinquefolius and P. japonicas are commonly used traditional medicines with different clinical efficacy. P. ginseng can enhance stamina and capacity to cope with fatigue and physical stress. P. quinquefolius is used to treat Qi deficiency anemia, cough and asthma (Chen et al. 2008a). P. notoginseng, also named Sanqi, is commonly used to relieve swelling and pain; and it also exhibits cardiovascular benefits due to its hemostatic activity. (Ng 2006), effectively treating hemoptysis, epistaxis, hematochezia, traumatic bleeding, etc. (Chinese pharmacopoeia 2015). P. notoginseng contains a variety of active ingredients including saponins and dencichine, performing well in regulating cardiovascular system, nervous system and immune system (Liu et al. 2014; Su et al. 2014; Wang et al. 2008; Xia et al. 2014). Due to its unique hemostatic effect, P. notoginseng’s annual consumption is nearly 8000 tons and the market demand is still rising, whose consumers contain Yunnan Baiyao Group, Tianjin Tianshili, Guangzhou Baiyunshan and other enterprises (Cui et al. 2014). P. notoginseng mostly comes from artificial cultivation, which planted in Yunnan is considered to be of the best quality (Cui et al. 2005). P. notoginseng has special

Y. Liu et al.

requirements for growth environment, which is suitable for dark and humid environment but not cold and heat. Due to the differences in cultivation environment like light, temperature, soil, humidity, diseases and insect pests, there are great variations in the characters of P. notoginseng, such as individual size, root shape, skin color, etc., which make species identification more difficult (Yao et al. 2011). The commodity price of P. notoginseng is relatively high, which leads to an endless supply of confused and counterfeit drugs on markets, bringing potential threats to the drug security of patients (Han 2013). These medicinal Panax species have very similar morphological characteristics and chemical profiles but exhibit different properties in pharmacology, so it is difficult to identify Panax and its related species by the traditional method of character authentication because of their similar appearance. In addition, the existence of flowers and powders in herbal medicine markets also brings great challenges to the identification of the authenticity of medicinal materials. Meanwhile, in China, some species like Gynura segetum, Phedimus aizoon and Anredera cordifolia with ‘Sanqi’ in their Chinese names also may cause confusion in clinical use. As a result, they are usually adulterated by each other intentionally or otherwise, which maybe harm health and safety of consumers. Thus, it is very important to accurately identify these species for the safety of clinical medication.

3.2

Panax ginseng Products and Chinese Patent Medicines

The medicinal Panax medicines and their products have a very large market share each year. Based on the growing years and preliminary processing methods, P. ginseng are commonly sold in three types: (a) fresh ginseng (less than 4 years old; can be consumed in its fresh state); (b) white ginseng (4–6 years old; dried after peeling); and (c) red ginseng (harvested when 6 years old, and then steamed and dried). After the preliminary process, these materials can be further processed into different types of products

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Nucleotide Signature and SNP Double Peak Methods Detect …

patent medicine market urgently needs to be completed with effective standardization methods. P. ginseng Chinese patent medicines play a very important role in clinical medication all over the world; however, the quality of this kind of drugs is sometimes uninsured because of the identification difficulty of Panax species used as raw materials. Asian ginseng and American ginseng, for example, are extremely difficult to be distinguished using traditional taxonomic methods. And this case become much more difficult when it comes to the Chinese patent medicines or decoctions. Because of the high price and identification difficulty of medicinal Panax species, Chinese patent medicines or some other products are intentionally or unintentionally adulterated, which pose a threat to consumers’ health.

including medicinal slices, decoction, extract, tea, pills, powder and so on. Fresh ginseng with two years of growing ages can also be directed eaten as daily supplementary. As food therapy concept with herbal medicine has enjoyed popular support, the market demand for ginseng products maintain a steady growth. The supply of ginseng is far behind requirement even these herbals have been cultivated over one hundred years (Harding 1908). There is another type of P. Ginseng product that is greatly consumed, Chinese patent medicines. This kind of products mainly refers to drugs made of traditional Chinese herbal medicines. Chinese patent medicines include different dosage forms: pills, powders, paste, particles, oral liquids, injections and so on. Chinese pharmacopoeia (2015) has totally recorded 86 prescriptions of P. ginseng Chinese patent medicines (Table 3.1.). Chinese patent medicines containing P. ginseng have been broadly used in many diseases, like coronary heart diseases, angina, arteriosclerosis, gastropathy and diabetes. To achieve the best therapeutic effect, different prescriptions need different processing technology, called “PaoZhi” in Chinese. The commonly used processes of P. ginseng Chinese patent medicines mainly include powdering, stewing and extracting (Table 3.1). Chinese patent medicines can be easily used and be purchased all over the world. However, the quality of Chinese patent medicines cannot be fully guaranteed since the regulation mechanism of Chinese patent medicines is very imperfect (Wang et al. 2011). The supervision of the Chinese Table 3.1 Dosage form and processing tech statistics of Panax ginseng Chinese patent medicines

Dosage form

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3.3

Identification Methods of Panax Species

In early studies, morphological identification and chemistry analysis are commonly used for the identification of Panax species; however, these methods are neither efficient nor accurate enough. The morphological characteristics of medicinal Panax species are very similar, and they cannot be easily observed after the raw materials have been shredded slice and powdered. As for chemistry analysis, highperformance liquid chromatography-mass spectrometry (HPLC–MS) is often applied using

Number

Processing tech

Number

Capsule

21

Powdering

52

Particle

11

Stewing

17

Pill

25

Ethanol reflux extract

8

Tablet

18

Water immersion

1

Ethanol immersion

1

Oral liquid

7

Mixture

1

Others

7

Suppository

1

Total

86

Powder

2

34

Y. Liu et al.

ginsenosides as detective markers (Chen et al. 2008b). So far more than 30 different ginsenosides have been reported to be extracted from American ginseng (Assinewe et al. 2003), and different Panax species share similar profiles of the ginsenosides. Thus, the species-specific chemical marker is not easy to be detected. Nowadays, molecular biotechnology has gained more and more attention taxonomical studies. DNA molecular markers such as random amplified polymorphic DNA (RAPD) analysis (Shaw and But 1995), amplified fragment length polymorphism (AFLP) (Ha et al. 2002; Hyekyoung and Yongeui 2009), EST-SSR Markers (Choi et al. 2011) and gradient PCR method (Shim et al. 2005) have been successfully applied. In addition, many other PCR-free technology, e.g., restriction fragment length polymorphism (RFLP) and DNA fingerprinting, are included in some cases as well (Fushimi et al. 1997; Ho and Leung 2002). However, these markers have their own deficiencies.

3.4

DNA Barcoding and MiniBarcoding

DNA barcoding technique uses one wellrecognized and short DNA sequence to realize the fast and accurate species identification (Hollingsworth et al. 2011), DNA barcoding was developed in 2003 by Paul Hebert et al., at the University of Guelph in Canada (Hebert et al. 2003). Based on the comparative analysis of gene sequences of mitochondria Cytochrome c oxidase I (COI) in 13,320 species, 11 phyla, animal kingdom, CO I was found to be used for animal identification. They suggested that a single short gene fragment could be applied for quick and precise species identification. According to the study of more than 6600 samples of over 400 medicinal plant species, Chen et al. proposed ITS2 region as DNA barcoding sequence, which possessed the advantages of high amplification efficiency, small intraspecific variation and large interspecific variation (Chen et al. 2010; Han et al. 2013). In another study, Yao et al. (Hui et al. 2010) proposed to use ITS2

sequence as standard markers for the identification of plant species and developed a public database for the identification of TCMs (https:// www.tcmbarcode.cn/). ITS2 has been successfully used in the identification of many medicinal plants (Chen et al. 2013; Han et al. 2011; Hou et al. 2015; Xin et al. 2015; Zhao et al. 2014). The rapid development of molecular biology and bioinformatics has laid a foundation for the advent of DNA barcoding, and the requirement for rapid and accurate identification of species has promoted this technology. Compared with other methods, DNA barcoding technique combined with DNA sequencing can always bring us more specific information for identification. And it is very convenient especially for nontaxonomist scientists due to its simple operating steps. This technique shows high operation convenience, identification efficiency and accuracy, which therefore can be considered as powerful tool for the identification of medicinal herbals (Hao et al. 2010). As for the DNA barcoding research on Panax species, ITS sequence showed the highest distinguishability among ten candidate sequences (Zuo et al. 2010). DNA barcoding technique has been proved to be a useful identification tool and has been included in the Chinese Pharmacopoeia as standard method. DNA barcoding technique can identify the raw materials and preliminarily processed products but difficultly deal with heavily processed products, e.g. decoctions and Chinese patent medicines, due to the serious DNA degradation during the manufacturing processes. DNA degradation can cause the failure of PCR of sequences over 200 bp in length (Goldstein and Desalle 2003; Hajibabaei et al. 2006; Wandeler et al. 2007). Meusnier et al. proposed to use “mini-barcode” in order to break this bottleneck. In their study, the whole-length CO1 sequence was cut into different lengths to evaluate the identification success rate. As a result, the short length (150 bp and 100 bp) of CO1 sequence could still obtain a high identification rate, 95% and 90%, respectively, (Meusnier et al. 2008). Shaw et al. (Lo et al. 2015) evaluated the DNA degradation degree in TCM decoction and found

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Nucleotide Signature and SNP Double Peak Methods Detect …

that an 88 bp sequence fragment could be amplified from TCM material after boiling for 120 min while the amplification of a 121 bp sequence could not be obtained, which indicates that under the circumstances of DNA degradation shorter DNA sequences might get more chances to be amplified. Hence, conclusion can be easily drawn that within certain limits, shorter sequences has higher amplification efficiency. And, as a result, to shorten the amplification sequence can help overcome the identification obstacle caused by DNA degradation occurred in Chinese patent medicines. Considering the complex ingredients and conditions of DNA degradation within Chinese patent medicines, an efficient approach to accurately detect ingredients of processed ginseng products is urgently needed.

3.5

Intraspecific Genetic Distances of Medicinal Panax Species

Chen et al. systematically studied the intraspecific genetic distance and variation among of three DNA barcode sequences (totally 377 sequences), ITS2, matK and psbA-trnH, of P. ginseng, P. quinquefolius and P. notoginseng (Chen et al. 2013). In the results, ITS2 sequences showed unexpected low intraspecific divergence among these three species. And no SNP sites was observed in 54 samples of P. ginseng or 18 samples of P. quinquefolius. Same results were also obtained from other research. 17 ITS2 sequences of ginseng species submitted by a Korean institution (GenBank accession nos. AB043871, AB043872, HM446498-504, AY548192, AF274532-534, DQ284918 and DQ339097099), including five different cultivars with considerable morphological variation, showed little variation. Also, no intraspecific variation was observed. ITS2 sequences of P. notoginseng have a higher diversity, 19 of 24 (79.2%) sequences were identical. ITS2 sequences of P. japonicus have the highest level of sequence diversity, only 8 of 18 (44.4%) sequences were identical.

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The variation rate of matK sequences is also very low among these three species. P. ginseng or P. quinquefolius showed no variation within matK sequences (51 sequences and 31 sequences, respectively) of their own species. In contrast, the intraspecific divergence in P. notoginseng and P. japonicus was higher. Twelve of 16 (75.0%) matK sequences from P. notoginseng were identical. However, matK sequences of P. notoginseng from GenBank are more consistent; only 1 of the 8 matK sequences contained variations. Similarly, 13 of 14 matK sequences of P. japonicas from GenBank are completely identical. As for psbA-trnH sequences, no SNP was exhibited in 39 sequences of P. ginseng. Four of 25 sequences of P. quinquefolius from GenBank (HQ112888, SN03MT02, SN03MT12, and SN03MT26) had similar variability, with 9 indels, and no other variations were found. Within P. notoginseng species, only 1 of the 29 sequences includes one SNP site, which demonstrated that the psbA-trnH sequences also have a very low intraspecific divergence. In P. japonicas species, 6 of 12 psbA-trnH sequences from GenBank exhibit only one variation site.

3.6

SNPs Analysis and Double Peak Method Development

Single Nucleotide Polymorphism (SNP) has contributed a lot to taxonomical studies, particularly to researches of very closely related species (Arif et al. 2010). Based on the investigation of DNA barcoding sequences of four commonly used Panax species, Chen et al. established an effective method to identify Panax species based on the SNP within ITS2 sequences. All the ITS2 sequences of the Panax species were analyzed to screen out SNPs at the interspecies level. As a result, totally five stable SNPs were found. And, based on these SNPs, a unique marker was found to be able to accurately distinguish these Panax species. Among Panax species, P. ginseng and P. quinquefolius share the most similar morphological characteristics.

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Y. Liu et al.

Fig. 3.1 Sequencing result of mixed powder of different ratio. The ratio of P. ginseng and P. quinquefolius is a 13:7; b 1:1; c 6:14; and d 1:19. Note: X. Chen et al./Gene 530(2013)39–43

Therefore, they are often misidentified as each other. SNPs can directly show the difference of these two species. Two stable species-specific SNP sites was found based the investigation of all the experimental and the published GenBank ITS2 sequences of P. ginseng and P. quinquefolius. A combination of base C and T at positions 32 and 43, respectively, specifically belongs to P. ginseng while a combination of T and C specifically belongs to P. quinquefolius. In addition, there is a base T at position 32 in the ITS2 sequences of P. quinquefolius while a base C in the same position of all the other speices. Thus, P. quinquefolius could be simply distinguished from the all its closely related Panax species by the SNP site. Similarly, species-specific SNPs were also found in P. notoginseng. At positions 28,140 and 207, the nucleotide base is T in P. notoginseng while it is A or C in all the other Panax species. Then, the two SNPs of P. ginseng and P. quinquefolius were used to identify these two species. Powder of P. ginseng and P. quinquefolius were artificially mixed by different ratios (P. ginseng–P. quinquefolius 13:7, 10:10, 6:14

and 1:19) to stimulate the contamination and adulteration in the market. DNA of the mixed powder was extracted. The sequencing result of PCR amplification of sequences containing the two SNPs showed that as low as 5% P. ginseng ingredient can be detected from 1:19 powder (Fig. 3.1). Besides, the height of the double peaks in the SNPs in the sequencing trace data could roughly reflect the mix ratio; e.g., at the ratio of 1:19, the main peaks G/A from reverse sequencing (equals to T/C in forward sequencing) attributed to P. quinquefolius were much higher than the lower peaks A/G (equals to C/T in forward sequencing) attributed to P. ginseng.

3.7

Nucleotide Signature Combined with Double Peak Method Identify Ginseng Chinese Patent Medicines

The term “nucleotide signature” means an extremely short DNA sequences unique to one taxon. Liu et al. developed a nucleotide signature sequence for the differentiation of P. quinquefolius

3

Nucleotide Signature and SNP Double Peak Methods Detect …

and its Chinese patent medicines (Liu et al. 2016). The sequence is 27 bp in length and is completely conserved within the P. quinquefolius species. Using this sequence, 24 batches of herbal products containing American ginseng were successfully detected. In this research, totally 54 ITS2 sequences of P. quinquefolius were analyzed. Two SNPs in ITS2 stably existed between American ginseng and Asian ginseng was included in the 27-bp nucleotide signature. To further confirmed the stability of these two SNPs, 283 ITS2 sequences of closely related Panax species were analyzed. All the haplotypes of the same position of SNP site is aligned in Fig. 3.2. It is clearly shown that at least one variation site exists in the sequences of all the other species, which indicates that the 27 bp is completely unique to P. quinquefolius and thus can be defined as the nucleotide signature. To figure out whether the nucleotide signature method can deal with processed medicinal materials, experiment was conducted upon the boiled American ginseng. Five primers with different size of sequences containing the nucleotide signature were designed. The wholelength ITS2 sequences could not be amplified from the decoction but the shorter sequences could be amplified using the specially designed primer pares. The length of these amplification products from short to long was 49 bp, 72 bp, 95 bp, 149 bp and 168 bp. This result indicated that shorter length may get better amplification result in a specific range but not the shortest the best. In order to include both SNPs and obtain an optimum sequencing result, the primer 4F/4R was chosen to amplify the nucleotide signature sequence. And, the nucleotide signature sequence could be successfully obtained from American ginseng decoction. Chinese patent medicines might suffer more complicated manufacturing process than decoction which may cause more serious DNA degradation. Taking Yangshen Baofei Wan as an example, a representative Chinese patent medicine that is made from American ginseng and 11 other ingredients. The whole-length ITS2 sequence was

37

unable to be amplified from DNA extracted from Yangshen Baofei Wan. However, short sequences containing the nucleotide signature could be amplified using the 4F/4R primer pair to from the same DNA. Besides, other commonly purchased Chinese patent medicine containing American ginseng were also successfully detected. Twenty-two batches of other Chinese patent medicines containing American ginseng, Asia ginseng and notoginseng were detected as well. The PCR and sequencing success rate was 100%. In the result, 5 of 21 Chinese patent medicines labeled as containing Asian ginseng were found to contain American ginseng instead. And, no Asian ginseng were detected in these five batches. By analyzing the double peak at the SNP positions (Fig. 3.3) 2 batches were adulterated with American ginseng. The other 17 Chinese patent medicines were found to match their labels (Table 3.2.). According to the traditional Chinese medicine theory, American ginseng and Asian ginseng have different medicinal properties. American ginseng can tonify “qi” and nourish “yin” while Asian ginseng can reinforce “qi” and benefit the spleen and the lungs. So, they must be clearly distinguished in clinical uses, especially in Chinese patent medicines which is usually made from complex ingredients. As shown in Table 3.3, the percentage of counterfeit samples identified out of the 24 commercial Chinese patent medicine samples was 21%, including 2/2 of the Shenwu Jiannan capsules, 1/3 of the Renshen Guipi pills, and 2/7 of the Qipi pills. In addition, two batches of Chinese patent medicine were detected to be adulterated with American ginseng. The specific primer pair 4F/4R could be used to amplify the nucleotide signature region and to identify the ginseng product used in Chinese patent medicines. However, to distinguish all the ingredients in complex Chinese patent medicines, the high-throughput sequencing method are recommended. For the further study of Chinese patent medicine ingredients, a promising direction would be to combine the short nucleotide signature method with the highthroughput sequencing method.

38 Fig. 3.2 Alinement SNP site of Panax quinquefolium Note Front. Plant Sci. 7:319. https://doi.org/10.3389/fpls. 2016.00319

Y. Liu et al.

3

Nucleotide Signature and SNP Double Peak Methods Detect …

Fig. 3.3 Peak profile of four representing batches of Chinese patent medicine labeled containing Panax species. A-YSBF01 is Yangsheng baofei Pills. BHG04MT02 is Renshen Guipi Pills. C-HG02MT06 is

Table 3.2 Identification results of ginseng chinese patent medicines (CPM)

39

Qipi Pills. D-HG05MT02 is Shenlin Baizhu capsules. And the base in frame is SNP site. Note: Front. Plant Sci. 7:319. https://doi.org/10.3389/fpls.2016.00319

Name of CPM

Labelled species

Identification results

Yangshen Baofei Pills01

P. quinquefolius

P. quinquefolius

Yangshen Baofei Pills02

P. quinquefolius

P. quinquefolius

Xiyangshen Pills01

P. quinquefolius

P. quinquefolius

Xiyangshen tablets02

P. quinquefolius

P. quinquefolius

Xiyangshen capsules09

P. quinquefolius

P. quinquefolius

Xiyangshen tablets13

P. quinquefolius

P. quinquefolius

Xiyangshen capsules16

P. quinquefolius

P. quinquefolius

Qipi Pills01

P. ginseng

P. ginseng

Qipi Pills02

P. ginseng

P. ginseng

Qipi Pills03

P. ginseng

P. quinquefolius

Qipi Pills04

P. ginseng

P. ginseng

Qipi Pills05

P. ginseng

P. quinquefolius

Qipi Pills06

P. ginseng

P. ginseng

Qipi Pills07

P. ginseng

P. ginseng

Renshen Guipi Pills02

P. ginseng

P. ginseng

Renshen Guipi Pills01

P. ginseng

P. quinquefolius (continued)

40

Y. Liu et al.

Table 3.2 (continued)

Name of CPM

Labelled species

Identification results

Renshen Guipi Pills02

P. ginseng

P. ginseng

Shenlin Baizhu capsules01

P. ginseng

P. ginseng

Shenlin Baizhu capsules02

P. ginseng

P. ginseng

Shenlin Baizhu Pills01

P. ginseng

P. ginseng

Shenlin Baizhu Pills04

P. ginseng

P. ginseng

Shenwu Jiannan capsules01

P. ginseng

P. quinquefolius

Shenwu Jiannan capsules02

P. ginseng

P. quinquefolius

Dieda Pills02

P. notoginseng

P. notoginseng

Note: Front. Plant Sci. 7:319. 10.3389/fpls.2016.00319

3.8

A Fast Identification Method of Panax notoginseng Based on Nucleotide Signature and NJ Tree

A total of 46 samples of P. notoginseng (Sanqi) and its adulterant Gynura segetum (Ju sanqi) were purchased from different crude drug markets and genuine producing areas. The sample information is shown in Table 3.3. The sequences obtained from the experiment and downloaded from GenBank were screened, and only one identical sequence of the same species was retained to obtain the haplotype sequences of all samples, which were then aligned by MEGA 5.0 software via the “MUSCLE” method (Edgar 2004; Tamura et al. 2013). Fourty-six ITS2 sequences of P. notoginseng were obtained from the experimental materials, and the aligned length was 230 bp with 63.35% of the bases GC. According to the alignments with downloaded sequences from GenBank, three haplotype sequences with total four variable sites were obtained. There were 36 dominant haplotype sequences with an average intraspecific distance of 0.002. Two hundred forty-eight ITS2 sequences were downloaded from GenBank and the length of aligned haplotype sequences was 287 bp. A unique nucleotide signature region of P. notoginseng was obtained: 20-54 bp: 5′-AACCCATCATTCCCTCGCGG GAGTCGATGCGGAGG-3′, which was conserved within species and species specific. In this

region, there were no variable sites in the there haplotypes of P. notoginseng, four variable sites with P. ginseng, five variable sites with P. quinquefolium, one to four variable sites with P. japonicus, three variable sites with P. japonicus var. Major, two to five variable sites with P. japonicus var. Bipinnatifidus, three variable sites with P. zingiberensis and six to seven variable sites with P. stipuleanatus. P. notoginseng and its closely related species could be distinguished by the specific nucleotide fragments. This sequence was searched by BLAST tool in NCBI website. For the first 1000 results (sorted from high to low similarity), it was found that the similarity between this sequence and P. notoginseng was 100%, while the similarity with other non-P. notoginseng species was lower than 100%, indicating that the sequence was highly conservative in P. notoginseng.

3.9

Identification of P. notoginseng and Its Related Species and Adulterants

The identification of P. notoginseng and its closely related species can be realized by nucleotide signature sequences. For the species with a greater genetic distance from P. notoginseng like Gynura segetum, Phedimus aizoon and Anredera cordifolia, their sequence fragments were quite different and many insertions and deletions appeared in the alignments, so NJ tree was adopted for the identification (Fig. 3.4). Five

3

Nucleotide Signature and SNP Double Peak Methods Detect …

Table 3.3 Detail information of samples

41

Sample No

Species

Source

Sample type

SN02MT01

P. notoginseng

Hebei Anguo TCM market

Rhizome

SN02MT02

P. notoginseng

Hebei Anguo TCM market

Rhizome

SN02MT03

P. notoginseng

Anhui Bozhou TCM market

Rhizome

SN02MT04

P. notoginseng

Hebei Anguo TCM market

Rhizome

SN02MT05

P. notoginseng

Hebei Anguo TCM market

Rhizome

SN02MT06

P. notoginseng

Wenshan, Yunnan

Rhizome

SN02MT07

P. notoginseng

Wenshan, Yunnan

Rhizome

SN02MT08

P. notoginseng

Wenshan, Yunnan

Rhizome

SN02MT09

P. notoginseng

Wenshan, Yunnan

Rhizome

SN02MT10

P. notoginseng

Wenshan, Yunnan

Rhizome

SN02MT11

P. notoginseng

Wenshan, Yunnan

Rhizome

SN02MT12

P. notoginseng

Wenshan, Yunnan

Rhizome

SN02MT13

P. notoginseng

Wenshan, Yunnan

Rhizome

SN02MT14

P. notoginseng

Wenshan, Yunnan

Rhizome

SN02MT15

P. notoginseng

Wenshan, Yunnan

Rhizome

SN02MT16

P. notoginseng

Wenshan, Yunnan

Rhizome

SN02MT17

P. notoginseng

Wenshan, Yunnan

Rhizome

SN02MT18

P. notoginseng

Wenshan, Yunnan

Rhizome

SN02MT19

P. notoginseng

Wenshan, Yunnan

Rhizome

SN02MT20

P. notoginseng

Wenshan, Yunnan

Rhizome

SN02MT21

P. notoginseng

Wenshan, Yunnan

Rhizome

SN02MT22

P. notoginseng

Wenshan, Yunnan

Rhizome

SN02MT23

P. notoginseng

Wenshan, Yunnan

Rhizome

SN02MT24

P. notoginseng

Wenshan, Yunnan

Rhizome

SN02MT25

P. notoginseng

Wenshan, Yunnan

Rhizome

GZBJ094

P. notoginseng

Bijie, Guizhou

Leaf

GZBJ096

P. notoginseng

Bijie, Guizhou

Leaf

GZBJ097

P. notoginseng

Bijie, Guizhou

Leaf

Y1301030

P. notoginseng

A TCM company

Powder

SQ26

P. notoginseng

Wenshan, Yunnan

Leaf

FHAG403

P. notoginseng

Hebei Anguo TCM market

Rhizome

FDC095

P. notoginseng

NIFDC

Rhizome

RC-SN02MT01

P. notoginseng

Hebei Anguo TCM market

Rhizome

RC-SN02MT03

P. notoginseng

Anhui Bozhou TCM market

Rhizome

RC-SN02MT04

P. notoginseng

Hebei Anguo TCM market

Rhizome

RC-SN02MT06

P. notoginseng

Weshan, Yunnan

Rhizome

RC-SN02MT10

P. notoginseng

Weshan, Yunnan

Rhizome

RC-SN02MT11

P. notoginseng

Weshan, Yunnan

Rhizome

RC-SN02MT13

P. notoginseng

Weshan, Yunnan

Rhizome

RC-SN02MT14

P. notoginseng

Weshan, Yunnan

Rhizome (continued)

42 Table 3.3 (continued)

Y. Liu et al. Sample No

Species

Source

Sample type

RC-SN02MT16

P. notoginseng

Weshan, Yunnan

Rhizome

RC-SN02MT17

P. notoginseng

Weshan, Yunnan

Rhizome

RC-SN02MT18

P. notoginseng

Weshan, Yunnan

Rhizome

RC-SN02MT19

P. notoginseng

Weshan, Yunnan

Rhizome

RC-SN02MT23

P. notoginseng

Weshan, Yunnan

Rhizome

SH-SN02MT17

P. notoginseng

Weshan, Yunnan

Rhizome

H4

G. segetum

A pharmacy from H.K

Powder

H10

G. segetum

A pharmacy from H.K

Powder

H11

G. segetum

A pharmacy from H.K

Powder

H12

G. segetum

A pharmacy from H.K

Powder

NH5

G. segetum

A pharmacy from H.K

Powder

NH6

G. segetum

A pharmacy from H.K

Powder

NH29

G. segetum

A pharmacy from H.K

Powder

Note: Chin Pharm J, 2015, 50(22):1954–1959

Fig. 3.4 Neighbor-joining phylogenetic tree based on ITS2 haplotype sequences from P. notoginseng and its adulterants

sequences of Phedimus aizoon and two sequences of Anredera cordifolia were downloaded from GenBank. The result indicated that P. notoginseng and its adulterants were successfully distinguished according to the NJ tree. The purpose of this study is to overcome the defects in traditional identification strategies and provide a simple and accurate identification method for P. notoginseng. Based on the genus, a nucleotide signature ITS2 sequences of P. notoginseng and other species of Panax containing the unique fragment of 5′-AACCC

ATCAT TCCCT CGCGG GAGTC GATGC GGAGG-3′ was developed, which was characterized by interspecific specificity and intraspecific conservation. An unknown species can be identified as P. notoginseng when the sequence is obtained from the species. DNA barcoding is usually based on the similarity of BLAST results to identify species. The higher the similarity was, the more likely it is that the sample is the corresponding species in GenBank. Another authentication method is according to the genetic distance within and between species.

3

Nucleotide Signature and SNP Double Peak Methods Detect …

However, for different species, the intraspecific threshold is different, so the identification of species based on the genetic distance or barcoding gap has some ambiguity and uncertainty. By contrast, the identification results obtained by unique nucleotide signature are straightforward. For patent medicines and high temperature treated herbs, DNA degradation is serious and long DNA barcode sequences are difficult to obtain. However, the length of P. notoginseng nucleotide signature is only 35 bp, which is very suitable for samples with serious DNA degradation. Therefore, the nucleotide signature can be used to identify P. notoginseng quickly and accurately. The discovery of specific sequences is representative only if it is based on a large enough sample size. In this study, the analysis results of all experimental sequences of P. notoginseng and sequences of Panax genus in GenBank showed that the unique nucleotide fragment of P. notoginseng are representative enough. With the decrease of sequencing cost, high-throughput sequencing of short fragments will lead the whole molecular identification industry, and species authentication based on the specific nucleotide signature will have great application prospects.

References Arif IA, Bakir MA, Khan HA, Farhan AHA, Homaidan AAA, Bahkali AH, Sadoon MA, Shobrak M (2010) A brief review of molecular techniques to assess plant diversity. Int J Mol Sci 11:2079 Assinewe VA, Baum BR, Gagnon D, Arnason JT (2003) Phytochemistry of wild populations of Panax quinquefolius L. (North American ginseng). J Agric Food Chem 51:4549–4553 Chen C, Chiou W, Zhang J (2008) Comparison of the pharmacological effects of Panax ginseng and Panax quinquefolium. Acta Pharmacol Sin 29:1103–1108 Chen FD, Wu MC, Wang HE, Hwang JJ, Hong CY, Huang YT, Yen SH, Ou YH (2008) Sensitization of a tumor, but not normal tissue, to the cytotoxic effect of ionizing radiation using Panax notoginseng extract. Am J Chin Med 29:517–524 Chen S, Yao H, Han J, Liu C, Song J, Shi L, Zhu Y, Ma X, Gao T, Pang X (2010) Validation of the ITS2 region as a novel DNA barcode for identifying medicinal plant species. PLoS ONE 5:e8613

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Chen X, Liao B, Song J, Pang X, Han J, Chen S (2013) A fast SNP identification and analysis of intraspecific variation in the medicinal Panax species based on DNA barcoding. Gene 530:39–43 Chinese Pharmacopoeia Commision (2015) Pharmacopoeia of the People’s Republic of China 2015. China Medical Science Press, Beijing Choi HI, Kim NH, Kim JH, Choi BS, Ahn IO, Lee JS, Yang TJ (2011) Development of reproducible ESTderived SSR markers and assessment of genetic diversity in Panax ginseng cultivars and related species. J Ginseng Res 35:399–412 Cui X, Xu L, Wang Q, Chen Z (2005) Analysis on the geologic background and physicochemical properties of soil for the cultivation of Panax notoginseng in Yunnan province. China J Chin Mater Med 30 (05):332–335 Cui XM, Huang LQ, Guo LP, Liu DH (2014) Chinese Sanqi industry status and development countermeasures. China J Chin Mater Med 39(04):553–557 Duc NM, Kasai R, Ohtani K, Ito A, Nham NT, Yamasaki K, Tanaka O (1994) Saponins from vietnamese ginseng, Panax vietnamensis ha et grushv. collected in central Vietnam III. Chem Pharm Bull 42:634–640 Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32(5):1792–1797 Fushimi H, Komatsu K, Isobe M, Namba T (1997) Application of PCR-RFLP and MASA analyses on 18S ribosomal RNA gene sequence for the identification of three ginseng drugs. Bio Pharm Bull 20:765– 769 Goldstein PZ, Desalle R (2003) Calibrating phylogenetic species formation in a threatened insect using DNA from historical specimens. Mol Ecol 12:1993 Ha WY, Shaw PC, Liu J, Yau FC, Wang J (2002) Authentication of Panax ginseng and Panax quinquefolius using amplified fragment length polymorphism (AFLP) and directed amplification of minisatellite region DNA (DAMD). J Agric Food Chem 50:1871– 1875 Hajibabaei M, Smith MA, Janzen DH, Rodriguez JJ, Whitfield JB, Hebert PDN (2006) A minimalist barcode can identify a specimen whose DNA is degraded. Mol Ecol Resour 6:959–964 Han J, Li M, Luo K, Liu M, Chen X, Chen S (2011) Identification of Daturae Flos and its adulterants based on DNA barcoding technique. Acta Pharm Sin B 46(11):1408–1412 Han QQ (2013) Methods and analysis for identifying authenticity of Sanqi. Clin J Chin Med 5(12):49–50 Han J, Zhu Y, Chen X, Liao B, Yao H, Song J, Chen S, Meng F (2013) The short ITS2 sequence serves as an efficient taxonomic sequence tag in comparison with the full-length ITS. Biomed Res Int 2013:741476 Hao DC, Chen SL, Xiao PG, Peng Y (2010) Authentication of medicinal plants by DNA-based markers and genomics. Chin Herbal Med 02:250–261

44 Harding AR (1908) Ginseng and other medicinal plants. A R Harding Publishing Co Hebert PD, Cywinska A, Ball SL, deWaard JR (2003) Biological identifications through DNA barcodes. Proc Biol Sci 270(1512):313–321 Ho IS, Leung FC (2002) Isolation and characterization of repetitive DNA sequences from Panax ginseng. Mol Genet Genomics 266:951–961 Hollingsworth PM, Graham SW, Little DP (2011) Choosing and using a plant DNA barcode. PLoS ONE 6:e19254 Hou D, Zhou H, Song J (2015) Molecular identification of Dallbergiae Dderiferae and its adulterants using the ITS sequence. Chin Pharm J 50(15):1273–1276 Hu SY (1976) The genus Panax (Ginseng) in Chinese medicine. Econ Bot 30:11–28 Hui Y, Song J, Chang L, Luo K, Han J, Ying L, Pang X, Xu H, Zhu Y, Xiao P (2010) Use of ITS2 region as the universal DNA barcode for plants and animals. PLoS ONE 5(10):e13102 Hyekyoung K, Yongeui C (2009) Molecular authentication of Panax notoginseng by specific AFLP-derived SCAR marker. J Med Plant Res 3:957–966 Kim YH, Ahn IO, Khan AL, Kamran M, Waqas M, Lee JS, Kim DH, Jang SW, Lee IJ (2014) Regulation of endogenous gibberellins and abscisic acid levels during different seed collection periods in Panax ginseng. Hortic Environ Biotechnol 55:166–174 Liu J, Wang Y, Qiu L, Yu Y, Wang C (2014) Saponins of Panax notoginseng: chemistry, cellular targets and therapeutic opportunities in cardiovascular diseases. Expert Opin Investig Drugs 23(4):523–539 Liu Y, Wang X, Wang L, Chen X, Pang X, Han J (2016) A nucleotide signature for the identification of American ginseng and its products. Front Plant Sci 7:34940 Lo YT, Li M, Shaw PC (2015) Identification of constituent herbs in ginseng decoctions by DNA markers. Chin Med 10:1 Meusnier I, Singer GA, Landry JF, Hickey DA, Hebert PD, Hajibabaei M (2008) A universal DNA mini-barcode for biodiversity analysis. Bmc Genomics 9:214 Ng TB (2006) Pharmacological activity of sanchi ginseng (Panax notoginseng). J Pharm Pharmacol 58(8):1007– 1019 Shaw PC, But PP (1995) Authentication of Panax species and their adulterants by random-primed polymerase chain reaction. Planta Med 61:466–469

Y. Liu et al. Shim YH, Park CD, Kim DH, Cho JH, Cho MH, Kim HJ (2005) Identification of Panax species in the herbal medicine preparations using gradient PCR method. Biol Pharm Bull 28:671–676 Su P, Wang L, Du SJ, Xin WF (2014) Advance in studies of Panax notoginseng saponins on pharmacological mechanism of nervous system disease. China J Chin Mater Med 39(23):4516–4521 Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: molecular evolutionary genetics analysis version 6.0 [J]. Mol Biol Evol 30(12):2725– 2729 Wandeler P, Hoeck PE, Keller LF (2007) Back to the future: museum specimens in population genetics. Trends Ecol Evol 22:634–642 Wang J, Zhang GF, Hong LU, Jin D (2011) Investigation and analysis of 240 Package inserts of chinese patent medicine. Eval Anal Drug-Use Hosp China Wang N, Wan JB, Li MY, Wang YT (2008) Advances in studies on Panax notoginseng against atherosclerosis. Chin Tradit Herbal Drugs 39(05):787–790 Wen J, Zimmer EA (1996) Phylogeny and biogeography of Panax L. (the ginseng genus, araliaceae): inferences from ITS sequences of nuclear ribosomal DNA. Mol Phylogenet Evol 6:167–177 Xia PG, Zhang SC, Liang ZS, Qi ZH (2014) Research history and overview of chemical constituents of Panax notoginseng. Chin Tradit Herbal Drugs 45 (17):2564–2570 Xin T, Li X, Yao H, Lin Y, Ma X, Cheng R, Song J, Ni L, Fan C, Chen S (2015) Survey of commercial Rhodiola products revealed species diversity and potential safety issues. Sci Rep 5:8337 Yao J, Yang KH, Peng ER (2011) The analysis and control of influencing factors on Panax notoginseng. Guangdong Agric Sci 2(24):22–24 Yun TK (2001) Brief introduction of Panax ginseng C. A. Meyer. J Korean Med Sci 16(Suppl):S3 Zhao S, Pang X, Song J, Chen S (2014) Identification of cortex acanthopanacis and its adulterants using the DNA barcode ITS2. The 14th national symposium on traditional chinese medicine and natural medicine Zuo Y, Chen Z, Kondo K, Funamoto T, Wen J, Zhou S (2010) DNA barcoding of Panax species. Planta Med 77:182–187

4

Breeding of Superior Ginseng Cultivars Jang-Uk Kim, Dong-Yun Hyun, Hyeonah Shim, Tae-Jin Yang, and Young-Chang Kim

Abstract

There is a total of 32 ginseng cultivating varieties (cultivars) in Korea which have unique agronomic and morphological traits. Each variety was registered as an official cultivar and protected for breeder’s rights permitted by the Korean government. All 32 elite cultivars have been developed by pure-line selection from local landraces and show diverse agricultural performances and characteristics such as growth, floral and fruit shape, root growth, shapes and processing traits, characteristics for biotic or abiotic stress, and ginsenoside compositions. This chapter will discuss the breeding method and characteristics, breeding history in Korea, and characteristics of officially registered ginseng varieties.

J.-U. Kim
D.-Y. Hyun
Y.-C. Kim (&) Division of Ginseng, Department of Herbal Crop Research, National Institute of Horticultural and Herbal Science, Rural Development Administration, Eumseong, Korea e-mail: [email protected] H. Shim
T.-J. Yang Department of Agriculture, Forestry and Bioresources, Plant Genomics and Breeding Institute, Research Institute of Agriculture and Life Sciences, College of Agriculture and Life Sciences, Seoul National University, Seoul, Korea

4.1

Introduction

‘Panax ginseng C.A. Meyer’ is the scientific name for ginseng that was assigned by ‘Carl Anton von Meyer’ of the Soviet Union in 1843 (Brekhman II 1957). Plant taxonomical pedigree of ginseng varies according to the development of plant taxonomy, but ginseng is generally classified according to Engler’s classification system: vascular plants/ Angiospermae/ Dicotyledoneae/Archichlamydeae/Apiales/Araliaceae/Panax. The genus name ‘Panax’ is the combination of ‘Pan’ and ‘Axos,’ which means ‘all’ and ‘cure’ in Greek, meaning the ultimate elixir. There are 17 Panax species reported, although some confusion for classification remains (Hu 1976; Yun 2001; Duc et al. 1993). Ginseng has long been used for medicinal purposes in Korea and China. Four other species, such as the North American ginseng (Panax quinquefolius L.), the bamboo ginseng (Panax japonicus C. A. Meyer), the sanchi ginseng (Panax notoginseng Burkill), and the Vietnamese ginseng (Panax vietnamensis Ha et Grushv), are also cultivated as medicinal plants in North America, Southwestern China, and Vietnam, respectively (Duy et al. 2016; Kim et al. 2016, 2018; Nguyen et al. 2017, 2018). Plants in the Panax genus can naturally grow in cool and humid deciduous forests with consistently low temperatures in the winter and adequate rainfall in the summer. The East Asian

© Springer Nature Switzerland AG 2021 J. Xu et al. (eds.), The Ginseng Genome, Compendium of Plant Genomes, https://doi.org/10.1007/978-3-030-30347-1_4

45

46

J.-U. Kim et al.

region between longitudes 85°E (Nepal) and 140°E (Japan) provides a natural habitat for P. ginseng throughout the Korean Peninsula, a part of Primorsky Krai, Russia, Manchuria, and China. In North America, P. quinquefolius naturally grows in the northeastern part of the USA and the southeastern part of Canada along the Appalachia Mountains between longitudes 70°W and 90°W. The latitudinal range is between latitudes 22°N and 48°N in East Asia and latitudes 34°N and 47°N in North America.

4.2

Reproduction of Ginseng

4.2.1 Flowering and Reproduction Ginseng flowers are bell-shaped and have florets that form an umbel around the stalk. If grown vigorously (Chung et al. 1989), ginseng can flower in its second year, though it is more common for ginseng to flower in its third year (Cha et al. 2003). The flowers bloom light green and have five yellow-green petals and green calyxes, with the number of flowers varying with the ginseng’s age. The flowers are hermaphrodite (both pistil and stamens are in a single flower) with five stamens and one pistil, which is divided into two stigmas (KT&G 1996). Ginseng also has an inferior ovary (ovary placed below the corolla) divided into two parts where seeds are generated in each ovule. The flowering period is between 8 and 10 in the morning, though it can be 1 h late on a cloudy day. Flowering takes less than an hour on a clear day, but in some cases, flowering can take place within a few minutes. The anther burst

Flowering

begins between 11 AM and 12 PM, which is 2– 3 h after flowering, and the bursting process for all five anthers finishes within 3–5 h; however, the pollen remains in the anthers for a day and disperses in the following morning. Ginseng flowers are protogynous, which means they have pistils that mature earlier than stamens. Ginseng berry fruiting and seed formation can be achieved through natural pollination mediated by wind or insects, as well as through artificial fertilization (KT&G 1996). The natural crosspollination rate is less than 4%. When pollination occurs, the ovule tissues begin to grow and differentiate, and the ovary enlarges to bear fruit. Since ginseng has two ovules, each ginseng berry bears two seeds. The number of fruits varies with plant age, vegetative growth stages, and weather conditions. Normally, about 10 fruits are borne in the third year, 30 in the fourth, 40 in the fifth, and about 50 in the sixth year (Fig. 4.1).

4.2.2 Seed Harvest and Stratification In late July, the fruits turn orange-red, signaling that the fruits are ripe and ready for harvest. Although the berries and seeds may seem mature during the harvesting stage, the embryo inside is at an immature state not ready for germination. To ensure stratification, which refers to the opening of seed coats, it is important to maintain the seeds in an appropriate environment and allow for growth of the embryo. Stratification is the artificial maturation process used to prepare seeds for germination. In Korea, the seeds are harvested in late July. If the immature seeds are sowed and watered

Fruiting

Fig. 4.1 Flowering, fruiting, and fruit maturation of ginseng

Early maturation

Maturation

4

Breeding of Superior Ginseng Cultivars

properly in early August, 55–60% of seeds germinate in April of the following year. Ungerminated seeds then become dormant and start growing a year later. Majority of the seeds germinate 21 months after being sowed, under the condition that the immature seeds have been sowed directly in the soil. Consequently, it is crucial that seeds are stored in a specific environment for 3–4 months after being harvested. In order to do so, a proper environment for seed storage is created with cool temperatures around 18 °C and enough moisture that can promote maturation of the immature embryos, ultimately reducing the time required for germination and enhancing the rate of germination (Won et al. 1988).

4.3

Characteristics of Ginseng Breeding

Ginseng breeding is challenging because it is a lengthier process compared to breeding of other crops that normally takes one or two years for one generation. In general, ginseng requires four years for one generation because a favorable number of seeds can be obtained from four-yearold ginseng plants (Kwon et al. 1998b). Ginseng has been cultivated for several hundred years as local landraces, with its cultivation areas having mixed lines. Compared to regular crops, ginseng has low genetic and phenotypic variation, although numerous variations have been identified in ginseng DNA (Choi et al. 2013, 2014; Jang et al. 2017). Local landraces that contain diverse genotypes are good resources for breeding elite cultivars; however, ginseng cultivation is much more difficult due to its lengthy generation time and usage of pure-line selection method. Breeding efforts include selection of superior individuals, self-pollination to develop pure inbred lines, multiplication of seeds, and regional adaptation and productivity tests. Each ginseng individual will produce fewer than 120 seeds, even if the seeds are harvested once every two years for six consecutive years. As such, more than 12 years would be required to propagate the seeds to sufficiently supply new

47

cultivars. In the case that cross-hybridization breeding is applied, it will take more than 60 years to develop pure inbred line cultivars with conventional breeding methods (Cho 1998).

4.3.1 The Breeding History of Korean Ginseng Research on Korean ginseng breeding started with the discovery of the yellow-berry variants and green-stem variants from local landraces in 1926, but research was limited to ginseng’s morphological characteristics. In the 1960s, systematic research on the development of new cultivars started with comparative research between yellow-berry variants and violet-stem variants in 1962, and resource collection and trait assessment began in 1966. Yunpoong and Gopoong cultivars were collected and selected in 1968 (Kwon et al. 2000), Chunpoong and Sunpoong in 1972, Gumpoong in 1979 (Kwon et al. 1998a), Chungsun in 1982, and Sunun in 1985, resulting in the cultivars they are today (Wang et al. 2009). Ginseng breeding really took place in the 1970s, with systematic investigation of selected lines, mainly dealing with morphological variations (Choi et al. 1980) such as multi-stem shoots (Park et al. 1980) and shortening of breeding time via tissue culture methods (Cho 1979) and development of mutants (Choi et al. 1981). There was an expansion of ginseng breeding research in the 1980s, focusing on local adaptation tests of collection lines (Kwon et al. 1991), ginsenoside contents (Ahn and Choi 1984b), red ginseng quality (Kwon et al. 1994), interspecific crossing with foreign ginseng (Choi et al. 1983), and general characteristics and components (Ahn and Choi 1984a). P. quinquefolius and other Panax species were introduced into Korea in the 1990s, and interspecific crossing was applied for research and breeding (Cheon et al. 1985). Accessions were bred to increase rusty-root tolerance, high saponin levels, and root rot resistance (Kim et al. 1991; Kwon et al. 1991). Nine cultivars had been registered from the early 2000s up until 2010 (KSVS 2010). By then, ginseng research was done not only in national

48

institutions, but also in local government research institutes. Active research was done on areas such as collection of genetic resources, variation expansion, cultivation of superior accessions, and cultivar-specific DNA marker development (Wang et al. 2009, 2010a; b; Lee et al. 2011). Recently, diverse molecular markers are also developed and applied for the authentication of cultivars based on ginseng genome sequences (Choi et al. 2013, 2014, Jang et al. 2017, Kim et al. 2014, 2015, 2018). Furthermore, tissue culture and genetic transformation are established for mass propagation, shortening of generation time, and in vitro metabolite production (Choi et al. 1999; Lee et al. 2017).

4.3.2 Ginseng Breeding Method 4.3.2.1 Pure-Line Selection The self-pollinating crops become homozygous by increasing generations. Each individual is highly homozygous even though the population contains heterogeneous mixed lines. If rare natural outcrossing events occur, they become homozygous after several generations of selfpollination, and each individual becomes a genetically fixed homozygous line. Pure-line selection is a breeding method in which superior individuals are selected within a population to be multiplied as an inbred line. The basis of pure-line selection method is selecting the most elite individuals from the genetically diverse heterogeneous population. The seeds of individual ginseng plants can be collected from ginseng farms that cultivate mixed populations as local landraces. The progenies are evaluated for agricultural characteristics and homogeneity. Seeds collected from the selected progenies are used to evaluate the second generation and nurture for genetic homogenization. Once an outstanding line has superior agronomic traits and homogeneity after the third generation, seeds are pooled and planted for mass production and evaluated for productivity and regional

J.-U. Kim et al.

adaptation before being registered as an official cultivar.

4.3.2.2 Crossbreeding Pure-line selection is based on natural pollination without artificial cross-hybridization. Crossbreeding is based on artificial crossing between two parental lines, selecting superior inbreds after several rounds of self-pollination for genetic fixation. Selection of a superior line can be achieved by pedigree selection and bulk selection methods. The pedigree selection method is recommended for ginseng because otherwise it takes a long time to pass one generation. Successful crossbreeding depends on the selection of ideal parental lines. The genetic variation is narrow, and the diversity of genetic resources is limited compared to that of other annual crops. However, breeding collections contain abundant alleles in the population (Jang et al. 2019). The seed parent (female parent) should carry elite agronomic traits. In order to improve genetic recombination and replace the poor traits of the female parent, the male parent should have other superior traits. 4.3.2.3 Artificial Crossing Artificial crossing should take place prior to flowering to eliminate potential self-pollination events. The female parent’s petals and five stamens are completely removed when the flowers are in early bud stages in May, and artificial crossing is performed using pollen from the male flower. It takes about two weeks for a flower to bloom from the edges to the center, so crossbreeding should take place every day for two weeks. The envelope should be replaced after crossing to prevent natural pollination. The fruit begins to ripen 40 days after fertilization, and the seeds can be harvested 50–60 days later (Fig. 4.2). Self-pollinated plants should be identified by comparing with the maternal parents and removed, obtaining genuine F1 plants for succeeding generations.

4

Breeding of Superior Ginseng Cultivars

emasculation

49

pollen

fertilization

fruiting

Fig. 4.2 Process of cross-hybridization for ginseng

4.3.2.4 Breeding and Selection of Superior Inbred Lines After cross-hybridization, the F2 population and individuals in the F2 population are highly heterozygous. Inbred lines can be developed by several rounds of self-pollination and selection, consequently increasing homozygosity. In pedigree selection, individuals should be selected from the 4-year-old plants of the F2 population. From the F3 population, selection of individuals and pedigrees should be repeated until fixed pedigree inbred lines are selected. Bulk selection is achieved by harvesting seed pools from all the F2 plants after removing defective individuals. The harvest of seed pools and bulk cultivation should be repeated for four generations. Breeding time can be shortened when the seeds of three-year-old plants are harvested from each hybrid generation. By proceeding up to the F6 generation, the seeds are genetically fixed to a certain degree, with over 95% homogeneity, so the selected lines can be evaluated as a cultivar.

4.3.3 Registration of Novel Ginseng Cultivars The breeding lines should be evaluated for agronomic characteristics, productivity, and genetic uniformity. The most elite breeding lines can be registered as a novel cultivar by evaluating four requirements: novelty, distinctness, uniformity, and stability. Novelty is the newness of the cultivar in which seeds or harvests of the breeding lines have not been released or transferred for at least 1 year in Korea and 4 years in

foreign countries before registration. Distinctness requires at least one unique trait that can be distinctive from previously developed cultivars. Uniformity refers to the case where the inherent characteristics of a variety are sufficiently uniform considering the expected variation in the breeding method of the variety, and the number of heterologous individuals in the group is within an acceptable range. Stability is long-term maintenance of the elite traits. Continuous propagation should not increase the variation or changes essential characteristics in the cultivar. Finally, a new cultivar must have a unique name to meet plant variety protection requirements (KSVS 2019).

4.4

Characteristics of Major Ginseng Cultivars

A total of 32 ginseng cultivating varieties have been developed in Korea, and all of them have been developed from landraces using the pureline selection method (KSVS 2019). In China, 13 cultivars are known to be registered recently. There are no officially registered cultivars in other countries other than Korea and China. The characteristics of major Korean varieties are as shown in Fig. 4.3.

4.4.1 Chunpoong Chunpoong is the first registered cultivar in Korea. The stems of young and 1 * 2-year-old plants are light purple, while they are mostly green in 4-year-olds and older except for the light

50 Fig. 4.3 Characteristics of leaf, stem, berry, and root in major Korean ginseng cultivars

J.-U. Kim et al. Variety (registration year) Chunpoong (2002)

Yunpoong (2002)

Gopoong (2003)

Sunpoong (2003)

Gumpoong (2003)

Sunun (2006)

Sunwon (2006)

Chungsun (2007)

Sunhyang (2009)

K-1(2012)

Geumsun (2013)

Cheonryang (2013)

Kowon (2015)

Cheonmyeong (2019)

Jinwon (2019)

Leaf

Stem

Fruit

Root

4

Breeding of Superior Ginseng Cultivars

purple areas between the leaf base and petiole. Leaves are slightly rolled up and yellow with a hint of red in the fall. Ripe fruits are light orange. The main root is cylindrical, and the quality of fresh ginseng is excellent. Due to its low frequency of developing rusty roots, Chunpoong can be cultivated in paddy fields and has moderate resistance to spotting disease (Kwon et al. 1998a).

51

4.4.5 Gumpoong Gumpoong stems are green, and ripe fruits are yellow or gold. Roots are long and creamy white. Native yellow-berry variants are rusty-root susceptible, but Gumpoong is rusty-root tolerant. Germination is relatively fast. Gumpoong has big roots and is also strong against diseases (Lee et al. 2015).

4.4.2 Yunpoong 4.4.6 Sunun Yunpoong has short stems in light purple. There are many small leaves with high occurrences of stipules from seedling stages. Leaves turn red in the fall, and ripe fruits are red. There are more than 2 stems in individuals that are 4 years and older. It has a main root that is short and thick in a cylindrical shape and is a high-yielding cultivar. Depending on cultivation conditions, leaf edges of 2-year-olds may dry up or turn slightly yellow commonly seen in high temperature conditions (Kwon et al. 2000).

4.4.3 Gopoong All petioles and stems of Gopoong are dark purple. Leaves are red in the fall with dark red fruits. The petiole is horizontal, peduncle length is average, and berry clusters are in inverted triangle shapes. The main root is long and creamy white. Gopoong is high yielding with an average rate of germination. Gopoong roots contain higher levels of saponin compared to other cultivars. Gopoong individuals generally have big roots (Kwon et al. 2003).

All stems of Sunun are purple. Small leaf surfaces are very wrinkly. The cross section of the leaf is flat, and leaves turn red in the fall. Fruits are red, and berry clusters are fan-shaped. Germination is relatively late compared to other cultivars (Lee et al. 2015).

4.4.7 Sunwon Sunwon stems are long and purple. Fruits are red, and berry clusters are fan-shaped. Leaves turn red in the fall. Petioles of the second lateral leaflet are short, which causes the palmately compound leaf to look round and clustered (Lee et al. 2015).

4.4.8 Chungsun Chungsun stems are green like those of Gumpoong, but fruits and leaves in the fall are red, a trait that is different from Gumpoong’s morphology. Germination is relatively fast (Lee et al. 2015).

4.4.4 Sunpoong 4.4.9 Sunhyang Sunpoong stems are purple. Leaves in the fall are red, and peduncles are long. Fruits are red, and berry clusters are fan-shaped. Roots are creamy white, and germination occurs earlier than other cultivars which makes this Sunpoong’s unique trait. Seedlings are long, and big roots are produced (Lee et al. 2015).

Sunhyang has one long stem with a purple stem base. There are many small leaves per leaf stem, with relatively long petioles. There are many stipules, and leaves are wide and oval. Pedicels droop, and fruits are red. Roots are creamy white and have fibrous roots (Lee et al. 2015).

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4.4.10 Cheonryang

References

Cheonryang leaves are convexed, and most of the stems are light green except for light purple areas between the base and petiole. Ripe fruits are red. The main and lateral roots are well developed with relatively high yield. Frequency of fruiting is relatively low (Kim et al. 2013).

Ahn SD, Choi KT (1984) Characteristics of flower organ, inflorescence and flowering in Panax ginseng and Panax quinquefolium. J Ginseng Res 8:45–56 Ahn SD, Choi KT (1984) Saponin contents of root and aerial parts in Panax ginseng and Panax quinquefolium. Korean Soc Crop Sci 29:342–349 Bevan MW, Uauy C, Wulff BBH, Zhou J, Krasileva K, Clark MD (2017) Genomic innovation for crop improvement. Nature 543:346–354 Brekhman II (1957) Ginseng. Government publisher of medicine literature. Medgiz, Saint Petersburg, pp 3– 163 Cha SK, Kim YC, Choi JE, Choi JS, Kang KK (2003) Genetic variation in among cultivated field populations of Korean ginseng (Panax ginseng C. A. Meyer) using RAPD. Korean J Plant Resour 16:251–256 Chung CM, Nam KY, Kim YT (1989) Growth characteristics of early peduncle developing plant in Korean ginseng (Panax ginseng C. A. Meyer). Korean Journal of Crop Science 34(1):92–97 Cheon SR, Ahn SD, Choi KT, Kwon WS (1985) Characters and inheritance of stem color in F1 and F2 of violet-stem variant x yellow-berry variant in Panax ginseng C. A. Meyer. J Ginseng Res 9:264–269 Cho JS (1979) Studies on the tissue culture of Korean ginseng(I)—Effect of temperature on the growth of ginseng plant and ginseng callus. Korean J Crop Sci 24:72–77 Cho JS (1998) Ginseng culture. Seonjin Press Choi HI, Kim NH, Lee J, Choi BS, Kim KD, Park JY, Lee SC, Yang TJ (2013) Evolutionary relationship of Panax ginseng and P. quinquefolius inferred from sequencing and comparative analysis of expressed sequence tags. Genet Resour Crop Evol 60:1377– 1387 Choi HI, Waminal NE, Park HM, Kim NH, Choi BS, Park M, Choi D, Lim YP, Kwon SJ, Park BS, Kim HH, Yang TJ (2014) Major repeat components covering one-third of the ginseng (Panax ginseng C. A. Meyer) genome and evidence for allotetraploidy. Plant J 77:906–916 Choi KT, Ahn SD, Shin HS (1980) Correlations among agronomic characters of ginseng plants. Korean J Crop Sci 25:63–67 Choi KT, Ahn SD, Park KJ (1981) Effects of ethyl methane sulphonate treatment on ginseng seeds. J Ginseng Res 5:163–169 Choi KT, Ahn SD, Park KJ, Yang DJ (1983) The characteristics and correlation coefficients of characters in Panax ginseng violet stem variant and yellow berry variant and Panax quinquefolium. J Ginseng Res 7:133–147

4.4.11 Gowon Gowon has flat leaves, and most of the stems are light purple with the base and petiole areas in darker purple. Ripe fruits are red. The main root is well developed and is high yielding. It has resistance to spotting disease and has tolerance to salinity and high temperatures.

4.5

Conclusion and Perspective

Pure-line selection method is widely used for ginseng breeding because superior homozygous individuals can be selected from the heterogeneous landrace population. On the other hand, it takes a long time of over 60 years to develop a cultivar via crossbreeding, so no cultivars have been developed using this method. However, this method is valuable to make diverse and superior genetic combinations by combining different genotypes that could not be detected in the natural population. The superior varieties can be more effectively developed if the genome sequence information and genomics-based selection are applied to promote the selection efficiency in early generations (Kim et al. 2018). Once advanced breeding technologies related to molecular and genomics tools, haploid breeding, mutation breeding, tissue culture, and genome editing technology are further developed and applied in the future, new elite cultivars can be developed in an accurate and fast digital breeding manner (Bevan et al. 2017).

4

Breeding of Superior Ginseng Cultivars

Choi YE, Yang DC, Yoon ES, Choi KT (1999) High efficiency plant production via direct somatic single embryogenesis from preplasmolysed cotyledons of Panax ginseng and possible dormancy of somatic embryos. Plant Cell Report 18:493–499 Duc NM, Nham NT, Kasai R, Ito A, Yamasaki K, Tanaka O (1993) Saponins from vietnamese ginseng, Panax vietnamensis ha et grushv. collected in central vietnam. 1. Chem Pharm Bull 41(11):2010–2014 Duy NV, Triue LN, Chinh ND, Tran VT (2016) A new variety of Panax (Araliaceae) from lam vien plateau, vietnam and its molecular evidence. Phytotaxa 277:47–58 Hu SY (1976) The genus Panax (ginseng) in Chinese medicine. Econ. Bot. 30:11–28 Jang W, Kim NH, Lee J, Waminal NE, Lee SC, Jayakodi M, Choi HI, Park JY, Lee JE, Yang TJ (2017) A Glimpse of Panax ginseng genome structure revealed from ten BAC clone sequences obtained by SMRT sequencing platform. Plant Breed Biotech 5 (1):25–35 Jang W, Jang Y, Kim N-H, Waminal NE, Chang Kim Y, Lee JW, Yang T-J, Genetic diversity among cultivated and wild Panax ginseng populations revealed by highresolution microsatellite markers. J Ginseng Res https://doi.org/10.1016/j.jgr.2019.05.008 Joh HJ, Kim NH, Jayakodi M, Jang W, Park JY, Kim YC, In JG, Yang TJ (March 2017) Authentication of golden-berry P. ginseng cultivar ‘Gumpoong’ from a landrace ‘Hwangsook’ based on pooling method using chloroplast-derived markers. Plant Breed Biotech 5 (1):16–24 Kim KS, Schuster W, Brennicke A, Choi KT (1991) Korean ginseng mitochondrial DNA encoded an intact rps12 gene downstream of the nad3 gene. Plant Physiol 97:1602–1603 Kim YC, Kim DH, Bang KH, Kim JU, Hyun DY, Lee SW, Kang SW, Cha SW, Kim KH, Choi JK, Han SH, An YN, Jeong HN (2013) High yielding and salt resistance ginseng variety ‘cheonryang.’ Korean J Breed Sci 45(4):434–439 Kim NH, Choi HI, Ahn IO, Yang TJ (2014) Evidence of genome duplication revealed by sequence analysis of multi-loci expressed sequence tag-simple sequence repeat bands in Panax ginseng Meyer. J Ginseng Res 38:130–135 Kim K, Lee SC, Lee J, Lee HO, Joh HJ, Kim NH, Park HS, Yang TJ (2015) Comprehensive survey of genetic diversity in chloroplast genomes and 45S nrDNAs within Panax ginseng species. PLoS ONE 10 (6):e0117159 Kim K, Lee SC, Lee J, Kim NH, Jang W, Yang TJ* (2016) The complete chloroplast genome sequence of Panax quinquefolius (L.). Mitochondrial DNA Part A 27(4):3033–3034 Kim K, Nguyen VB, Dong J, Wang Y, Park JY, Lee SC, Yang TJ (2017a) Evolution of the araliaceae family inferred from complete chloroplast genomes and 45S nrDNAs of 10 Panax-related species. Sci Rep 7:4917 Kim YC, Kim KJU, Lee JW, Hong CE, Bang KH, Kim DH, Hyun DY, Choi JK, Seong BJ, An YN,

53 Jeong HN, Jo IH (2017b) ‘Kowon’, a new Korean ginseng cultivars with high yield and Alternaria blight resistance. Korean J Hortic Sci Technol 35(4):499– 509 Kim NH, Jayakodi M, Lee SC, Choi BS, Jang W, Lee J, Kim HH, Waminal NE, Lakshmanan M, Van Nguyen B, Lee YS, Park HS, Koo HJ, Park JY, Perumal S, Joh HJ, Lee H, Kim J, Kim IS, Kim K, Koduru L, Kang KB, Sung SH, Yu Y, Park DS, Choi D, Seo E, Kim S, Kim YC, Hyun DY, Park YI, Kim C, Lee TH, Kim HU, Soh MS, Lee Y, In JG, Kim HS, Kim YM, Yang DC, Wing RA, Lee DY, Paterson AH, Yang TJ (2018) Genome and evolution of the shade-requiring medicinal herb Panax ginseng. Plant Biotechnol J 16:1904–1917 KSVS (Korea Seed and Variety Service, 2010) UPOV test guideline for ginseng Korea seed and variety service (KSVS) (2013) https:// www.seed.go.kr KT&G (1972) Annual report of ginseng-culture part KT&G (1996) The latest Korean ginseng-culture part. pp 42–44 Kwon WS, Chung CM, Kim YT, Choi KT (1991) Comparisons of growth, crude saponin, ginsenosides, and anthocyanins in superior lines of Panax ginseng C. A. Meyer. Korean J Breed 23:219–228 Kwon WS, Lee JH, Kang JY, Kim YT, Choi KT (1994) Red ginseng quality and characteristics of jakyungjong and hwangsook-jong in Panax ginseng C. A. Meyer. Korean J Breed 26:400–404 Kwon WS, Chung CM, Kim YT, Lee MG, Choi KT (1998a) Breeding process and characteristics of KG101, a superior line of Panax ginseng C. A. Meyer. J Ginseng Res 22:11–17 Kwon WS, Kang JY, Lee JH, Lee MG, Choi KT (1998b) Red ginseng quality and characteristics of KG101 a promising line of Panax ginseng C. A. Meyer. J Ginseng Res 22:244–251 Kwon WS, Lee MG, Choi KT (2000) Breeding process and characteristics of Yunpoong, a new variety of Panax ginseng C. A. Meyer. J Ginseng Res 24:1–7 Kwon WS, Lee JH, Park CS, Yang DC (2003) Breeding process and characteristics of gopoong, a new variety of Panax ginseng C. A. Meyer. J Ginseng Res 27 (2):86–91 Lee JW, Kim YC, Jo IH, Seo AY, Lee JH, Kim OT, Hyun DY, Cha SW, Bang KH, Cho JH (2011) Development of an ISSR-derived SCAR marker in Korean ginseng cultivar (Panax ginseng C. A. Meyer). J Ginseng Res 35:52–59 Lee JH, Lee JS, Kwon WS, Kang JY, Lee DY, In JG, Kim YS, Seo JH, Baeg IH, Chang IM, Grainger K (2015) Characteristics of Korean ginseng varieties of gumpoong, sunun, sunpoong, sunone, cheongsun, and sunhyang. J Ginseng Res KR 39(2):94–104 Lee YS, Park HS, Lee DK, Jayakodi M, Kim NH, Lee SC, Kundu A, Lee DY, Kwon SW, Yang TJ (2017) Comparative analysis of the transcriptomes and primary metabolite profiles of in vitro cultured

54 adventitious roots of five P. ginseng cultivars. J Ginseng Res 41:60–68 Nguyen B, Kim K, Kim YC, Lee SC, Shin JE, Lee J, Kim NH, Jang W, Choi HI, Yang TJ (2017) The complete chloroplast genome sequence of Panax vietnamensis Ha et Grushv (Araliaceae). Mitochondrial DNA Part A 28(1):85–86 Park H, Ohh SW, Kwon SC (1980) A case of ten-stem plant in Panax ginseng. J Ginseng Res 4:194–196 Van Binh N, Linh Giang VN, Waminal NE, Park H-S, Kim N-H, Jang W, Lee J, Yang TJ (2018) Comprehensive comparative analysis of chloroplast genomes from seven Panax species and development of an authentication system based on species-unique SNP markers. J Ginseng Res https://doi.org/10.1016/j.jgr.2018.06.003 Wang H, Sun H, Kwon WS, Jin H, Yang DC (2009) Molecular identification of the Korean ginseng

J.-U. Kim et al. cultivar “Chunpoong” using the mitochondrial nad7 intron 4 region. Mitochondrial DNA 20:41–45 Wang H, Sun H, Kwon WS, Jin H, Yang DC (2010a) A PCR-based SNP maker for specific authentication of Korean ginseng (Panax ginseng) cultivar “Chunpoong.” Mol Biol Rep 37:1053–1057 Wang H, Sun H, Kwon WS, Jin H, Yang DC (2010b) A simplified method for identifying the Panax ginseng cultivar Gumpoong based on 26S rDNA. Planta Med 76:399–401 Won JY, Jo JS, Kim HH (1988) Studies on the germination of Korean ginseng (Panax ginseng C. A. Meyer) seed; II. Influences of temperature and seed treatment on embryo growth and germination. Korean J Crop Sci 33(1):59–63 Yun TK (2001) Brief introduction of Panax ginseng C. A. Meyer. J Korean Med Sci 16(suppl):S3-5

Molecular Cytogenetics of Panax Ginseng

5

Nomar Espinosa Waminal, Tae-Jin Yang, and Hyun Hee Kim

Abstract

While the medicinal importance of Panax ginseng has long been established, its molecular cytogenetic data have not been available until the recent decade. Slow growth, low seed yield, long seed dormancy, sensitivity to light and high temperature, and low mitotic index are just some of the biological factors that hinder speedy progress in P. ginseng cytogenetics. Past reports were simple chromosome counting using chromogenic dyes, but the development of fluorescence in situ hybridization (FISH) and next-generation sequencing (NGS) technologies have sparked a renewed interest in P. ginseng cytogenetics in the context of genomics. Different classes of repetitive elements (REs) including transposable elements (TEs) and tandem repeats (TRs) representing about 80% of the genome have been identified from whole-genome screening of NGS reads and the

N. E. Waminal
H. H. Kim (&) Department of Life Science, Chromosome Research Institute, Sahmyook University, Seoul 01795, Republic of Korea e-mail: [email protected] T.-J. Yang Department of Agriculture, Forestry and Bioresources, Plant Genomics and Breeding Institute, Research Institute of Agriculture and Life Sciences, College of Agriculture and Life Sciences, Seoul National University, Seoul 08826, Republic of Korea

draft P. ginseng genome assembly. Chromosomal mapping of these repetitive elements through FISH was instrumental in establishing a high-resolution P. ginseng karyotype (2n = 4x = 48), in elucidating its allopolyploid nuclear genome, and in providing insights into its genome history. Chromosome data have contributed valuable information in understanding the P. ginseng genome structure and continued integration of new innovative approaches like CRISPR/dCas9 chromosome tagging and chromosome flow sorting will continue to highlight the importance of cytogenetic works in P. ginseng. In this chapter, we review the history, challenges, achievements, and future directions of P. ginseng cytogenetics in the post-genomics era.

5.1

Introduction

Panax ginseng C. A. Meyer (Asian ginseng) is a slow-growing shady perennial crop in the family Araliaceae, highly priced for the varied health benefits it offers owing to its different types of ginsenosides (Chen et al. 2016; Park et al. 2018). There are 12 accepted species in the genus Panax (www.theplantlist.org, Table 5.1), which originate in eastern Asia. Although ginsenosides and other health-promoting secondary metabolites are present in different Panax species (Chan et al. 2011; Wu et al. 2010; Zhang and Li 2017; Zhang

© Springer Nature Switzerland AG 2021 J. Xu et al. (eds.), The Ginseng Genome, Compendium of Plant Genomes, https://doi.org/10.1007/978-3-030-30347-1_5

55

56

N. E. Waminal et al.

Table 5.1 Chromosome numbers of some Panax and related araliaceae species

a

Species name

Chr. No. (2n)

Reference

Aralia cordata thunb

24

IPCN

Aralia cordata thunb

48

IPCN

Aralia elata (Miq.) seem

24

IPCN

Dendropanax morbiferus

48

Zhou et al. (2019)

Eleutherococcus koreanus nakai

48

IPCN

Eleutherococcus senticosus (Rupr. ex Maxim.) maxim

48

IPCN

Eleutherococcus sessiliflorus (Rupr. & Maxim.) S.Y. Hu

48

IPCN

Eleutherococcus sieboldianus (Makino) koidz

48

IPCN

Fatsia japonica (Thunb.) decne. & planch

24

IPCN

Fatsia oligocarpella koidz

48

IPCN

Hedera hibernica (Kirchn.) carrière

96

IPCN

Hedera nepalensis K. koch

24

IPCN

Hedera rhombea (Miq.) bean

48

IPCN

Hydrocotyle asiatica L

24

IPCN

Kalopanax septemlobus (Thunb.) koidz

48

IPCN

Macropanax dispermus (Blume) kuntze

48

IPCN

Macropanax oreophilus Miq

48

IPCN

Metapanax davidii (Franch.) J. Wen & Frodin

48

IPCN

Metapanax delavayi (Franch.) J. Wen & Frodin

48

IPCN

Panax bipinnatifidus Seem

48

IPCN

Panax ginseng C. A. Mey

48

IPCN

Panax japonicus (T.Nees) C. A. Mey

24

Darlington and Wylie (1956)

Panax notoginseng (Burkill) F.H. Chen ex C.H. Chow

24

IPCN

Panax pseudoginseng wall

24

Hara (1970)

Panax quinquefolius L

48

IPCN

Panax sokpayensis Shiva K. Sharma & Pandit

–

Panax stipuleanatus Tsai & K. M. Feng

24

IPCN

Panax trifolius L

24

Hu et al. (1980)

Panax vietnamensis Ha & Grushv

24

Zhu et al. (2003)

Panax wangianus S. C. Sun

–

Panax zingiberensis C. Y. Wu & Feng

–

Pentapanax leschenaultii var. umbellatus (Seem.) C. B. Clarke

24

IPCN

Pseudopanax arboreus var. arboreus

48

IPCN

Tetrapanax papyrifer (Hook.) K. Koch

48

IPCN

Tetraplasandra meiandra (Hillebr.) Harms

48

IPCN

Index to plant chromosome numbers. 1979. P. Goldblatt & D. E. Johnson, eds. Missouri Botanical Garden, St. Louis

5

Molecular Cytogenetics of Panax Ginseng

et al. 2015b), P. ginseng is the most important commercially (Court 2000). The accepted base chromosome number of the family Araliaceae is x = 12 (Yi et al. 2004), although taxonomic reclassification within the family and molecular data from P. ginseng suggest that an ancient base chromosome number of x = 6 cannot be ruled out (Choi et al. 2011; Yi et al. 2004). The latter hypothesis, although not invalid, may need further detailed cytogenomic analyses to be established. So, based on the current convention of x = 12, species with 2n = 24 are considered diploids, and those with 2n = 48 are tetraploids (Yi et al. 2004). Species in the genus Panax have undergone lineage-specific rounds of whole-genome duplications (WGDs), a more ancient Pg-b WGD which occurred about 28 million years ago (MYA) is shared by all species in Panax and related genera, and a more recent Pg-a WGD which occurred about 2.2 MYA is shared by P. ginseng and P. quinquefolius (Kim et al. 2018; Shi et al. 2015). Consequently, most Panax species are diploids (Yi et al. 2004). However, it is evident that a comprehensive comparative cytogenetic analysis is needed in the genus Panax using taxonomically defined taxa, taking into consideration inconsistencies and relatively old chromosomal data reported by different groups (Graham 1966; Ko et al. 1993; Waminal et al. 2012; Yang 1981), and also to make use of more recent technology advances in molecular cytogenetics for gathering of more accurate information. Establishing a cytogenetic reference for these comparative studies would be essential, and what is more relevant than starting with the characterization of the karyotype of the most commercially important Panax crop, P. ginseng. The establishment of cytogenetic resources in P. ginseng will translate to understanding its cytogenomic relationship with other species in the genus or beyond.

57

5.2

History of P. ginseng Cytogenetics

Although chromosome counts of P. ginseng have been available since the 1920s (Oehm 1924; Yi et al. 2004), cytogenetic progress has been rather slow. Many of the chromosomal data in P. ginseng have been reported in the recent decade (Choi et al. 2014, 2009; Waminal et al. 2012, 2017, 2018). There might have been less interest in its chromosomes then than now. Nevertheless, aside from the difficulties in preparing P. ginseng sporophytic metaphase chromosomes (see below), there were limited applications of cytological data aside from chromosome counting and simple karyotyping. Some earlier chromosome counts like those of Graham (1966) and Yang (1981) showing 2n = 44 were inconsistent with later reports of Ko et al. (1993), Choi et al. (2009), and Waminal et al. (2012) showing 2n = 48. This discrepancy may be due to the small and numerous P. ginseng chromosomes which may have been difficult to resolve using older less sophisticated chromosome preparation and microscopy techniques. With the discovery of the fluorescence in situ hybridization (FISH) technique in the Pinkel et al. (1986), molecular cytogenetics became more efficient and less hazardous, attracting more plant scientists to use it in analyzing the location of ribosomal RNA gene families (5S and 45S rDNA) not only to count the number of chromosomal loci in their plant of interest but also to exploit the number and distribution of these genes as cytotaxonomic markers (Belandres et al. 2015; Cai et al. 2006; Guerra 2012; Pellerin et al. 2019; Remnyl Joyce et al. 2018). The first molecular cytogenetics-based P. ginseng karyotype was reported by Waminal et al. (2012). Despite the limited number of chromosomes bearing the 5S and 45S rDNA loci, numerous DAPI bands inherent in the P. ginseng genome were instrumental in producing the first

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N. E. Waminal et al.

P. ginseng FISH karyotype. This karyotype was later improved after the identification of a 167-bp highly abundant satellite DNA, Pg167TR (Choi et al. 2014; Waminal et al. 2017). With the availability of high-throughput sequencing technologies, several repeat-rich bacterial artificial chromosomes (BACs) from a P. ginseng BAC library were sequenced, and several transposable elements (TEs) were identified like the Ty3/Gypsy PgDel1 and PgDel2. FISH localization of these representative TEs provided a hint of the origin of P. ginseng ploidy (Choi et al. 2014) and also further improved the karyotype to what we currently know by grouping the chromosomes according to the abundance/intensity of PgDel2 signals. While cytogenetics is a broad field which includes understanding centromere structure and function, chromosome organization, meiosis, and integration of genetic and physical maps among others, and that most of the recent reports are based on chromosomal mapping of DNA sequences on sporophytic chromosomal preparations, it is evident that P. ginseng cytogenetics has still a wide area of unexplored opportunities and questions to address essential issues of P. ginseng biology.

5.3

Challenges and Alternative Approaches in Chromosome Preparations

While sporophytic metaphase chromosomes could be obtained from any dividing cells, roots are often the primary source for these chromosomes because of their generally higher rate of cell division and established easy preparation methods for many plant species that need only few modifications to suit specific plants (Kato et al. 2004; Kirov et al. 2014; Mirzaghaderi 2010). Moreover, roots are often harvested from germinated seeds, although mature plants or shoot meristem can also provide right metaphase

chromosomes, especially for species with recalcitrant seeds or difficulty in root sampling (Anamthawat-Jónsson 2004). In P. ginseng, harvesting roots from seeds is more practical than harvesting from mature plants as the latter could be impractically laborious, detrimental to commercially propagated plants, extremely low throughput, and mature plants are tough to manage for this purpose. However, harvesting roots from P. ginseng seeds also pose many challenges. It is essential to understand the growth cycle of P. ginseng to understand these challenges. Cultivating P. ginseng is laborious and expensive due to its temperature and light sensitivity and slow-growing nature (Court 2000; Kim et al. 2018). It takes about four years for P. ginseng to flower and bear fruit from the time of planting and sets an average of only 40 seeds per year (Kim et al. 2018). Consequently, unlike cereals and other easily germinating plants, P. ginseng seeds are not available all year round and are not amenable to prolonged storage in dry conditions for use in the future cytogenetic investigations. Additionally, P. ginseng seeds are quite expensive and require extensive care. The zygotic embryo of a ripe P. ginseng berry is still immature at the time of harvest and takes about 21 months to mature in natural environments (Choi et al. 1998; Court 2000). Although the maturity process has been shown to shorten significantly after seed stratification, this process generally lasts up to three or more months (Court 2000; Son and Reuther 1977). Besides, stratified ginseng seeds need controlled temperature (10° C) to germinate, are prone to fungal contamination, and often have low germination rate (Court 2000; Kim et al. 2017; Park et al. 2012). Since seeds are not available all year round, it is essential to harvest as many roots as possible to be fixed and analyzed during which seeds are not available. Very often, these roots have very low mitotic index, and because of the numerous P. ginseng chromosomes, and because there is no

5

Molecular Cytogenetics of Panax Ginseng

59

5.4

established cell synchronization protocol like those of cereals and other crops that augment the mitotic index (Dolezel et al. 1999), it is complicated to obtain well-spread P. ginseng chromosomes suitable for karyotyping. Nevertheless, the first FISH karyotype of P. ginseng was generated using roots from stratified P. ginseng seeds (Waminal et al. 2012). Moreover, it is evident that more efficient methods in breaking seed dormancy or cell synchronization are needed for high-throughput harvest of P. ginseng roots suitable for rapid cytogenetic and other downstream analyses.

Repeats in the P. ginseng Genome

Like many plant species, repetitive elements (REs) are abundant in the P. ginseng genome. A vast majority of these REs are Class I transposable elements (TEs), while tandem repeats (TRs) represent only a little fraction (Table 5.2) (Choi et al. 2014; Kim et al. 2018; Lee et al. 2017). REs comprise about 80%, with LTR elements alone comprising about 57% of the ginseng genome (Kim et al. 2018). Of these LTR elements, four families (PgDel, PgTat, PgAthila,

Table 5.2 Summary of repetitive elements identified in the P. ginseng genome Repeat type

Name

Length (bp)

GPa (%)

Chromosomal distribution

References

TE-Ty3/Gypsy

PgDel1

10,039

24.28

Dispersed in all chromosomes

Choi et al. (2014)

PgDel2

12,515

1.62

Intense in 12 (13–24) out of 24 chromosomes

Choi et al. (2014)

PgDel3

11,809

2.48

–

Choi et al. (2014)

TE-Ty1/Copia

PgDel4

11,050

0.80

–

Jang et al. (2017)

PgDel5

12,860

0.92

Intense in 12 (1–12) out of 24 chromosomes

Jang et al. (2017)

PgDel6

12,252

1.95

–

Jang et al. (2017)

PgTat1

22,881

6.78

In subtelomeric regions of all chromosomes

Choi et al. (2014), Kim et al. (2018)

PgTat2

10,965

0.76

–

Choi et al. (2014)

PgAthila

9,893

1.62

–

Choi et al. (2014)

PgTork

9,707

1.19

Pericentromeric regions

Choi et al. (2014)

PgOryco

7,772

0.10

–

Choi et al. (2014)

TE-TIR

PgCACTA

10,964

–

Kim et al. (2018)

TR satellite

Pg167TRb

1,577

1.33

Distinct loci in all chromosomes

Choi et al. (2014), Waminal et al. (2017)

45S rDNAc

5,877

0.45

Short arm of chromosome 16

Kim et al. (2018), Waminal et al. (2017)

5S rDNA

898

0.40

Short arm of chromosomes 2, 14, and 22

Kim et al. (2018), Waminal et al. (2017)

TR minisatellite

Pgms1

11

0.02

Long arm of chromosome 1

Waminal et al. (2018)

TR microsatellite

PgGA15

2

0.01

Long arm of chromosome 20

Waminal et al. (2018)

a

Genome proportion = (total bases or repeats/P. ginseng genome size) * 100% P. ginseng tandem repeats (PgTR) were composed of 9.4 copy number units of 167 bp consensus sequences c The 45S rDNA sequence includes only transcriptional unit sequences (18S-ITS1-5.8S-ITS2-26S) b

60

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and PgTork) that were identified from full sequencing of repeat-rich BACs represent 39% (*1.4 Gbp) of the P. ginseng genome (Choi et al. 2014; Lee et al. 2017). The PgDel family is the most abundant TE, and its dynamics is considered to have caused a significant impact on the species divergence in the genus Panax (Kim et al. 2018; Lee et al. 2017). Different PgDel subfamilies vary in abundance and chromosomal distribution. PgDel1 is the most abundant subfamily and is distributed along the entire lengths of all P. ginseng chromosomes (Table 5.2, Fig. 5.1). One cytogenetically important PgDel subfamily is PgDel2. Its genome proportion (GP) is much lower than that of PgDel1, and it is abundantly distributed in 12 of 24 P. ginseng chromosomes (Choi et al. 2014; Lee et al. 2017). This biased distribution to a P. ginseng subgenome representing half of the total chromosomes was instrumental in reorganizing and simplifying the P. ginseng karyotype into either PgDel2-poor or PgDel2-rich chromosomes (Fig. 5.1) (Kim et al. 2018). More importantly, PgDel2 distribution is good cytogenetic evidence of an allotetraploidization event in the P. ginseng genome history, supporting in silico data (Kim et al.

2018). Another PgDel subfamily, PgDel5, showed more intense FISH signals to PgDel2poor chromosomes (Jang et al. 2017). The PgTat family, although not as abundant as the PgDel family, also impacted the genome expansion of allotetraploids P. ginseng and P. quinquefolius (Kim et al. 2018). FISH analysis of the major PgTat subfamily, PgTat1, revealed its preferential distribution at the subtelomeric regions of P. ginseng chromosomes (Fig. 5.1). Three other LTR families PgAthila, PgTork, and PgOryco have been characterized in the P. ginseng genome (Table 5.2). While there are no chromosomal localization data for PgAthila and PgOryco, FISH with PgTork showed hybridization mostly at pericentromeric regions (Fig. 5.1). Class II TEs represent less than 5% of the P. ginseng genome (Kim et al. 2018). One Class II terminal inverted repeat (TIR) element, PgCACTA1 (Table 5.2), was characterized showing greatest expansion in P. ginseng than in other diploid or tetraploid species (Kim et al. 2018). One interesting feature of this element is the presence of a 167-bp TR (Pg167TR) in the subterminal region of the 3′ TIR. A comparative FISH mapping of a PgCACTA1 transposase and the Pg167TR repeat between P. notoginseng (diploid) and

Fig. 5.1 Karyogram of P. ginseng with a chromosomal distribution of major ginseng REs. a rDNA families, b– e LTR retrotransposon probes (b: PgDel1, c: PgDel2, d: PgTat1, and e: PgTork), and f Pg167TR tandem repeat. Gray chromosomes in c–e are raw images of RE signals. PgDel1 (green) is shown to localize along the entire chromosomes (Choi et al. 2014). PgDel2 localized mostly

at the intercalary and the pericentric area (b, pink arrows) and more intensely in chromosomes 13–24 (red arrows). PgTat1 localized mostly at the subtelomeric regions (green arrows) (Kim et al. 2018). PgTork is shown to hybridize at the centromeric regions in addition to the euchromatic regions. Scale bar = 5 lm

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P. ginseng showed a dispersed distribution of the transposase along both P. notoginseng and P. ginseng chromosomes. However, the Pg167TR showed a distinct distribution pattern along P. ginseng chromosomes while very weak signals in P. notoginseng (Kim et al. 2018). Moreover, the chromosomal distribution of Pg167TR was crucial

in fine-tuning the P. ginseng karyotype (Tables 5.1 and 5.2, Fig. 5.2) (Waminal et al. 2017), and different Pg167TR repeat variants, Pg167TRa and Pg167TRb, show differential distribution in P. ginseng chromosomes (Fig. 5.3). Other P. ginseng satellite repeats that have characterized are the 45S and 5S nrDNA families

Fig. 5.2 Identification of individual P. ginseng chromosomes using the distinct chromosomal distribution of Pg167TR. a Several sets of P. ginseng chromosomes show consistent Pg167TR signals. b Chromosome 16 shows the 45S rDNA locus at the short arm and two Pg167TR signals at the intercalary region of the long arm.

c Two images of P. ginseng root metaphase chromosome spread after FISH with Pg167TR. Bars = 10 µm. Note: This image is a modified version of that presented in Waminal et al. (2017) after rearranging the chromosomes based on PgDel2 signals (Kim et al. 2018)

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Fig. 5.3 FISH idiogram of the P. ginseng karyotype. Gray and blue chromosomes represent PgDel2-poor and PgDel2rich chromosomes

(Kim et al. 2015). The 45S nrDNA family localized on only one chromosome (short arm of chr. 16), while the 5S nrDNA localized to three chromosomes (short arms of chrs. 2, 14, and 23). The locus on chromosome 14 has the most intense FISH signal and is considered the major 5S rDNA locus, while those of the two other chromosomes are minor loci (Fig. 5.3) (Waminal et al. 2017). One 11-bp minisatellite (Pgms1) was identified by screening a P. ginseng whole-genome NGS dataset for abundant micro- (2–5 bp) and minisatellite (6–100 bp) repeats (Mehrotra and Goyal 2014; Waminal et al. 2018). Several microsatellites were also identified, but so far only one (PgGA15) has been confirmed to be chromosome-specific (Waminal et al. 2018). FISH mapping of Pgms1 and PgGA15 showed localization at the long arms of chromosomes 1 and 20, respectively (Table 5.2) (Waminal et al. 2018). Although centromere-specific satellite repeats are often abundant in many plant species like Brassica (Lim et al. 2007), Zea mays (Jin et al. 2005; Lamb et al. 2007), Glycine max (Findley et al. 2016, 2010), Oryza sativa (Cheng et al. 2002), and others, to date, centromere-specific tandem repeat in P. ginseng has yet to be identified.

5.5

The P. ginseng Karyotype and Chromosome Counts of Related Species

The characterization of the major P. ginseng REs was crucial in establishing a refined P. ginseng karyotype. As mentioned above, the different

subgenome or chromosome-specific distributions of different TE and TR repeat families enabled discrimination of relatively small and numerous ginseng chromosomes (Waminal et al. 2012, 2017). The current karyotype has the P. ginseng chromosomes grouped into two subgenomes based on the abundance of PgDel2 (Figs. 5.1 and 5.3). The rest of the chromosome features are based on the distribution of inherent DAPI bands (Waminal et al. 2012), nrDNA families, and Pg167TR (Table 5.3, Fig. 5.3) (Waminal et al. 2017). The Pg167TR repeat alone can be used to discriminate individual chromosomes. This karyotype will continue to be updated as new cytogenetic markers are identified. Cytogenetic markers identified in Panax ginseng will be useful in comparative studies with related species. However, comparative cytogenetics in Panax and related species is not straightforward owing to the difficulties in obtaining root samples. Moreover, although chromosome counts for these species have been reported (Table 5.1), comprehensive comparative analyses have not yet been conducted. In order to carry out these analyses in the future, it is important to obtain a collection of species for chromosome counting and FISH screening. Chromosome count and rDNA FISH data of the diploid Panax notoginseng and tetraploid P. ginseng and P. quinquefolius along with related medicinal plants, Aralia elata, Dendropanax morbiferus, Eleutherococcus sessiliflorus, and Kalopanax septemlobus are presented in Fig. 5.4. FISH karyotype of these four related species was recently published (Zhou et al. 2019).

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Table 5.3 Summary of cytogenetic landmarks in 24 P. ginseng chromosomes Chr. No

Chromosome features

1

Paracentromeric DAPI band on the short arm (S), more intense intercalary on the long arm (L). Two medium-intense Pg167TR loci on 1S, one being paracentromeric and the other intercalary. Another two weak Pg167TR loci on 1L flanking the 1L DAPI band. Pgms1 along the 1L DAPI band

2

Minor 5S rDNA locus on the paracentromeric region of 2S, proximal to the Pg167TR locus. Weak subtelomeric DAPI band on 2L. One paracentromeric Pg167TR locus on 2S

3

Subtelomeric and average intensity DAPI band on 3L. Large and intense Pg167TR on intercalary 3L, with small and weak loci on paracentromeric 3L and intercalary 3S

4

Three Pg167TR loci on 4L, pericentromeric, intercalary (about 50% from centromere and telomere), and telomeric, with the pericentromeric signal being the most intense

5

This chromosome is easily distinguishable owing to its intense Pg167TR signals localized in the pericentromeric and intercalary regions of 5L and subtelomeric region of 5S. Another weak Pg167TR signal can be seen just proximal to the 5S intense signal

6

One intense paracentromeric DAPI band, two intercalary with weaker proximal. Weak Pg167TR signal is proximal to the intercalary DAPI band on 6L. Another two weak Pg167TR signals on 6S, one at the intercalary region and one at the paracentromeric area

7

Average intensity DAPI bands on 7L. This is easily distinguishable for its large and intense Pg167TR signal at the intercalary region of 7L, one of the most intense signals in the genome. Additional Pg167TR signal is localized at the intercalary region of 7S

8

Intercalary 8S and 8L Pg167TR signals

9

Weak intercalary DAPI band in 9L. Pg167TR signals localized at the paracentromeric regions of 9S and 9L

10

Weak paracentromeric DAPI band on 10S. One intercalary Pg167TR signal on 10L, proximal to the DAPI band

11

Intercalary 11L and weak paracentromeric 11S Pg167TR signals

12

Two Pg167TR loci, one at the pericentromeric region of each arm that can overlap to be seen as one signal. This looks similar to chromosome 23 except for the DAPI band that is absent here

13

Weak paracentromeric DAPI band, two intercalaries with a very intense middle signal on 13L and weak at intercalary on 13S. Only one Pg167TR signal at the paracentromeric region

14

Major 5S rDNA locus. Two moderate DAPI band intensity flanking 5S rDNA on 14S and one weak subtelomeric on 14L. One intercalary Pg167TR signal proximal to the subtelomeric DAPI band on 14L

15

Intercalary moderate intensity DAPI band on 15L. Weak paracentromeric 15S and intercalary 15L Pg167TR signals

16

Weak DAPI band on the satellited chromosome. Two intense Pg167TR loci can be found in the intercalary region of 16L, which sometimes overlap and can be seen as one large signal in some spreads

17

Weak intercalary Pg167TR signal on 17S and 17L

18

Weak subtelomeric DAPI band on 18S, more intense intercalary on 18L. One intense intercalary Pg167TR signal on 18S paracentromeric region, and another one proximal to the DAPI band on 18L

19

Weak paracentromeric and weak intercalary DAPI bands. Intense Pg167TR signals that correspond to the DAPI bands on both arms. This chromosome is easily distinguishable owing to the intense Pg167TR signals

20

Intercalary DAPI bands on both arms, more intense on 20L which overlap with the PgGA15 signal. Two closely localized intercalary 20L Pg167TR signals that sometimes overlap to be seen as one signal. Another weak intercalary Pg167TR signal seems to colocalize with the 20S DAPI band

21

Intercalary DAPI bands on both arms, more intense on 21L. One weak 21L Pg167TR signal is just distal to the 21L DAPI band (continued)

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Table 5.3 (continued) Chr. No

Chromosome features

22

Two intercalary DAPI bands on 22L, proximal more intense than distal. Intense paracentromeric Pg167TR locus is observable on 22S, plus two other weaker loci on 22L, one in between the DAPI bands and one at the subtelomeric area

23

Minor 5S rDNA locus at the paracentromeric region of 23S. Intercalary DAPI bands on both arms, more intense on 23L than 23S and that on chromosome 20L. Two Pg167TR loci, one at the pericentromeric region of each arm that can overlap to be seen as one signal

24

Weak subtelomeric DAPI band on 24S, more intense intercalary on 24L. Two closely localized Pg167TR at the paracentromeric area of 24L

Fig. 5.4 Chromosome count and rDNA FISH of representative Panax and related species. a Panax notoginseng, b P. ginseng, c P. quinquefolius, d Aralia elata, e Dendropanax morbiferus, f Eleutherococcus

5.6

Repeats Are Drivers for Genome Diversification in Panax Species

An abundance of REs in a genome poses a significant challenge in genome assembly projects even with the advent of third-generation sequencing technologies (Jiao and Schneeberger 2017; Liu et al. 2014; Michael and Jackson 2013; Schatz et al. 2012). Nevertheless, the growing report about their numerous crucial functions and applications warrant attention (Chuong et al. 2017; Lai 1994; Plohl et al. 2012; Ugarkovic 2005).

sessiliflorus, g Kalopanax septemlobus. Numbers in each panel represent chromosome counts. Red and green FISH signals represent 45S and 5S rDNA, respectively. Bar = 10 µm

Once regarded as ‘junk DNA’ because of their abundance in a genome without known direct function (Maumus and Quesneville 2016), REs have now been shown to play crucial roles in cellular biology. Although many REs do not code for proteins (Jagannathan et al. 2018; Sultana et al. 2017), their functions are essential which include regulation of cellular processes like responses to biotic and abiotic stresses (Bartlett and Hunter 2018; Capy et al. 2000; Garrido-Ramos 2015; Kalendar et al. 2000; Lanciano and Mirouze 2018; Makarevitch et al. 2015; Voinnet 2009) and genome size plasticity (Schubert and Vu 2016) via genetic (e.g.,

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Molecular Cytogenetics of Panax Ginseng

unequal crossovers, gene conversion, and transposition) and epigenetic (e.g., RNA interference) mechanisms (Fedoroff 2012; Fedoroff and Bennetzen 2013; Hall et al. 2005; Talbert and Henikoff 2010). Moreover, they have been used in different applications in genetic and biomedical research (Lai 1994; Schumann et al. 2019). REs are also known to influence genome evolution and species diversification (Biémont and Vieira 2006; Hall et al. 2005; Nowak 1994; Pardue and DeBaryshe 2003; Shapiro and von Sternberg 2005; Tank et al. 2015; Wei et al. 2013). Their insertions to new genomic loci may affect certain biosynthetic pathways such as pigment production (Buckner et al. 1996; Yan et al. 2015; Zabala and Vodkin 2014; Zhang et al. 2015a). Differential abundance and expression may also confer differential adaptation (Kalendar et al. 2000; Lanciano and Mirouze 2018). Fixation of these variations in different populations followed by reproductive isolation may promote speciation (Ricci et al. 2018; Serrato-Capuchina and Matute 2018). While REs may drive change in the genome and phenome, the same REs may also be utilized to trace the evolutionary history of a genome through comparative analyses. In the genus Panax, the Ty3/Gypsy LTR retrotransposons are the major players in genome size diversification and speciation (Kim et al. 2018; Zhang and Li 2017). While the genome proportion (GP) of PgDel2 is much lower than that of PgDel1, the observation that diploid relatives of P. ginseng have about twice the abundance of PgDel2 than in tetraploids suggests an allotetraploid origin of both P. ginseng and P. quinquefolius. Comparative FISH analysis of PgDel2 between P. ginseng and P. quinquefolius indicates that at least one of the progenitor genomes of the two allotetraploids is common (Lee et al. 2017). Although developing data are providing clues to the closest related species of the allotetraploid progenitors, further analyses are needed to decipher the exact route of the P. ginseng evolution unequivocally. Moreover, even though P. ginseng is generally considered a tetraploid, molecular data obtained from P. ginseng (Choi et al. 2011; Kim et al. 2014), the prevalent

65

reticulated evolution in Araliaceae (Yi et al. 2004), the chromosome number, and the chromosomal distribution of Pg167TR variants (Kim et al. 2018) open the table for discussion on an ancient octopolyploidy of the P. ginseng genome.

5.7

Future Directions in P. ginseng Cytogenetics

P. ginseng molecular cytogenetics is relatively young, and despite the several challenges in working with P. ginseng, cytogenetic data have made considerable contributions in elucidating the chromosomal-level organization and visualizing the allotetraploidization relics of the P. ginseng genome. Nonetheless, more critical questions about P. ginseng biology are yet to be answered, and the use of chromosome-based data may be useful in addressing some of these questions. Centromere function is at the core of cytogenetics (Kalinowska et al. 2019; Lermontova and Sandmann 2017; Lermontova and Schubert 2013). Disruptions of this function are primary drivers of aneuploidy, dysploidy, and even speciation (Cano-Roldán et al. 2016; Gérard et al. 2017; Winterfeld et al. 2018). New breeding methods target the centromere to generate haploid and doubled haploid lines bypassing issues of heterozygosity and can be used for many purposes such as genome sequencing and breeding (Ishii et al. 2016; Kalinowska et al. 2019; Lermontova and Sandmann 2017; Ravi and Chan 2013). Similar approaches can be used in P. ginseng breeding. To follow centromere dynamics, developing centromeric repeat probes and PgCENH3 antibody (Dunemann 2016; Dunemann et al. 2014) is necessary. However, centromeric-specific repeats are not yet identified in P. ginseng to date. Future research in this field, although may seem quite challenging, may be beneficial in the long term. A hairy root system is an excellent alternative to a seed-based sporophytic metaphase chromosome preparation in P. ginseng. Such a system can address issues in uniformity of source

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material and availability of metaphase chromosomes for cytogenetic analysis. Studies have shown that chromosome number remains stable from hairy root cultures (Aird et al. 1988; Hänisch ten Cate et al. 1987). Moreover, establishing a cell-cycle synchronization protocol for P. ginseng seedlings or hairy root cultures may also address the low mitotic index. Our preliminary data also show a promising future for this system in P. ginseng. Metaphase chromosomes harvested from synchronized cells can be used for singlechromosome isolation through flow sorting (Dolezel et al. 2014), by labeling chromosomespecific repeats as chromosome tags (Giorgi et al. 2013). Recently, a CRISPR/dCas9 fluorescence labeling system can be used to target specific chromosomal area (Deng et al. 2015; Ishii et al. 2019). Isolated chromosomes can be used for chromosome-specific sequencing and assembly using third-generation sequencing platforms and assembly pipelines and for other downstream chromosomal genomics analyses (Šimková et al. 2018). The sequence characterization and chromosomal mapping of several P. ginseng REs though useful in the general description of the genome do not provide direct information on how they affect P. ginseng biology. Analyses on the evolution, insertion, and expression dynamics of these REs may reveal significant functional associations with pathway genes responsible for stress and pathogen resistances, seed dormancy, and ginsenoside biosynthesis among others. Moreover, integration of new relevant scientific innovations with cytogenetics will provide a more holistic approach in further analyzing P. ginseng genome evolution and function.

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Molecular Cytogenetics of Panax Ginseng

Talbert PB, Henikoff S (2010) Centromeres convert but don’t cross. PLoS Biol 8:e1000326 Tank DC, Eastman JM, Pennell MW, Soltis PS, Soltis DE, Hinchliff CE, Brown JW, Sessa EB, Harmon LJ (2015) Nested radiations and the pulse of angiosperm diversification: increased diversification rates often follow whole genome duplications. New Phytol 207:454–467 Ugarkovic D (2005) Functional elements residing within satellite DNAs. EMBO Rep 6:1035–1039 Voinnet O (2009) Origin, biogenesis, and activity of plant microRNAs. Cell 136:669–687 Waminal N, Park HM, Ryu KB, Kim JH, Yang TJ, Kim HH (2012) Karyotype analysis of Panax ginseng C. A. Meyer, 1843 (Araliaceae) based on rDNA loci and DAPI band distribution. Comp Cytogenet 6:425–441 Waminal NE, Choi HI, Kim NH, Jang W, Lee J, Park JY, Kim HH, Yang TJ (2017) A refined Panax ginseng karyotype based on an ultra-high copy 167-bp tandem repeat and ribosomal DNAs. J Ginseng Res 41:469– 476 Waminal NE, Pellerin RJ, Jang W, Kim HH, Yang T-J (2018) Characterization of chromosome-specific microsatellite repeats and telomere repeats based on low coverage whole genome sequence reads in Panax ginseng. Plant Breed Biotechnol 6:74–81 Wei L, Xiao M, An Z, Ma B, Mason AS, Qian W, Li J, Fu D (2013) New insights into nested long terminal repeat retrotransposons in Brassica species. Mol Plant 6:470–482 Winterfeld G, Becher H, Voshell S, Hilu K, Röser M (2018) Karyotype evolution in phalaris (poaceae): the role of reductional dysploidy, polyploidy and chromosome alteration in a wide-spread and diverse genus. PLoS ONE 13:e0192869 Wu Q, Song JY, Sun YQ, Suo FM, Li CJ, Luo HM, Liu Y, Li Y, Zhang XW, Yao H et al (2010) Transcript profiles of Panax quinquefolius from flower, leaf and root bring new insights into genes

69 related to ginsenosides biosynthesis and transcriptional regulation. Physiol Plant 138:134–149 Yan F, Di S, Takahashi R (2015) CACTA-superfamily transposable element is inserted in MYB transcription factor gene of soybean line producing variegated seeds. Genome 58:365–374 Yang D-Q (1981) The cyto-taxonomic studies on some species of Panax L. Acta Phytotaxonomica Sinica 19:298–303 Yi T, Lowry PP, Plunkett GM, Wen J (2004) Chromosomal evolution in araliaceae and close relatives. Taxon 53:987–1005 Zabala G, Vodkin LO (2014) Methylation affects transposition and splicing of a large CACTA transposon from a MYB transcription factor regulating anthocyanin synthase genes in soybean seed coats. PLoS ONE 9:e111959 Zhang B, Liu C, Wang Y, Yao X, Wang F, Wu J, King GJ, Liu K (2015a) Disruption of a carotenoid cleavage dioxygenase 4 gene converts flower colour from white to yellow in Brassica species. New Phytol 206:1513–1526 Zhang G-H, Ma C-H, Zhang J-J, Chen J-W, Tang Q-Y, He M-H, Xu X-Z, Jiang N-H, Yang S-C (2015b) Transcriptome analysis of Panax vietnamensis var. Fuscidicus discovers putative ocotillol-type ginsenosides biosynthesis genes and genetic markers. BMC genomics 16:159 Zhang D, Li W, Xia E-h, Zhang Q-j, Liu Y, Zhang Y, Tong Y, Zhao Y, Niu Y-C, Xu J-HJMP (2017) The medicinal herb Panax notoginseng genome provides insights into ginsenoside biosynthesis and genome evolution. 10:903–907 Zhou HC, Pellerin RJ, Waminal NE, Yang T-J, Kim HH (2019) Pre-labelled oligo probe-FISH karyotype analyses of four araliaceae species using rDNA and telomeric repeat. Genes Genomics

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Ginseng Genome and Metabolic Regulation Xing Zhi-han, Hu Hao-yu, and Xu Jiang

Abstract

Here, we reviewed the genome of P. ginseng. Its 3.5 Gb nucleotide sequence represents one of the most complex plant genomes which contain more than 60% repeats. More than 40,000 protein-encoding genes were predicted and annotated, those genes include 488 cytochrome P450s, 2556 transcription factors, and 3745 transporter genes. The mevalonic acid pathway includes thirty-one genes. Eight of the 31 genes were labeled as 3-hydroxy-3-methylglutarylCoA reductases. They showed various expression patterns among ginseng tissues. UDP-glycosyltransferase (UGT) is a major gene family in P. ginseng species. Also, it was inferred to be responsible for the diversification of ginsenosides. In ginseng genome, at least 200 UGTs were annotated, and genome analysis showed that tandem repeats lead to UGTs’ divergence and duplication. The UGTs molecular modeling of 71, 74, and 94 families shows that there is a regionally specific conservative motif located at the N terminus. A prediction is made by molecular docking that captured ginsenoside precursors subsequently.

X. Zhi-han
H. Hao-yu
X. Jiang (&) Key Laboratory of Beijing for Identification and Safety Evaluation of Chinese Medicine, Institution of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China e-mail: [email protected]

6.1

Introduction

Recent pharmacological reports indicate that ginsenoside is the primary bioactive compound of P. ginseng and plays different therapeutic roles including antitumor, antihypertensive, antivirus, immunomodulation activities etc. (Leung and Wong 2010). Different parts of ginseng tissues, for example, the leaves, the root, or the rhizome, show remarkable variations in clinical efficacy, commercial uses, and quality evaluation, partly because of their ginsenoside variations (Zhang et al. 2014). As defense metabolites for ginseng itself, ginsenosides are regularly distributed and concentrated in particular organizations through transporting systems. Chemical analysis demonstrated that ginsenosides distributed unevenly among ginseng tissues, such as periderm and cortex, include higher quantity of protopanaxatriol (PPT)-type ginsenosides and protopanaxadiol (PPD)-type ginsenosides than ginsenosides in the root medulla (Fukuda et al. 2005; Taira et al. 2010; Yokota et al. 2011). Histochemical staining indicated that most of the ginsenosides reside on the periderm oil canals and roots’ outer cortex area (Christensen et al. 2006; Tani et al. 1981). Considering the potential physiological mission (Augustin et al. 2011), plenty of the ginsenoside in the periderm is corresponding to the P. ginseng’s biological function as phytoanticipin that helps P. ginseng defense pathogens.

© Springer Nature Switzerland AG 2021 J. Xu et al. (eds.), The Ginseng Genome, Compendium of Plant Genomes, https://doi.org/10.1007/978-3-030-30347-1_6

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Though the significance of ginsenosides in pharmacology has been extensively researched, the regulatory patterns and biosynthetic enzymes of ginseng saponins are still a mystery (Haralampidis et al. 2002; Jenner et al. 2005; Liang and Zhao 2008; Osbourn et al. 2011; Sawai and Saito 2011; Thimmappa et al. 2014). Via the cytosolic mevalonic acid (MVA) pathway, precursors of ginsenosides are biosynthesized. It is originated by acetyl coenzyme A and completed with isopentenyl diphosphate (IPP). Monoterpene molecules endured a series of condensation reactions finally turned into a linear C30 molecule–squalene (Lee et al. 2004) and transformed to (S)-2,3-oxidosqualene (Han et al. 2009) via cyclization process(Phillips et al. 2006). Then, a variety of events (e.g., mediated by cytochrome P450-dependent monooxygenases) (Han et al. 2011, 2012, 2013) generated multiple types of ginsenoside precursors, containing PPD/PPT and oleanolic. Those precursors are then modified by glycosylation reactions. Glycosylation transfers the glycosyl group to the target acceptor. This reaction is conducted by groups of multigene superfamilies, glycosyltransferases (GTs). UDP-glycosyltransferases (UGTs) are GTs that use sugar molecules activated by uridine diphosphate (UDP) as a supply. The UGTs diversity has been confirmed by matching genomic, transcriptome, or proteome sequences. In ginseng, Sun et al. found 129 potential UGT sequence bases on annotation results from the transcriptome data of P. ginseng roots, stems, leaves, and flowers. A number of the items encode enzymes that is in charge of ginsenoside backbone decoration (Li et al. 2013). Based on this dataset, Yan et al. (2014) reported a UGT molecule (UGTPg1) that glycosylated the C20–OH of PPD. Another report identified two UGTs (PgUGT94Q2 and PgUGT74AE2) catalyzed the glycosylation of the C3–OH of PPD to generate ginsenoside Rh2 and elongate the glucose moiety of Rh2 to obtain ginsenoside Rg3 (Jung et al. 2014). References (Wei et al. 2015; Wang et al. 2015a) discovered that UGT1 and the homologous genes had the function to glycosylate PPT

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and then produced PPT-derived ginsenosides. In fact, there are a limited number of reports of plantbased UGTs of glycosylate triterpenoid aglycones, such as UGTs from Medicago truncatula (Achnine et al. 2005), Saponaria vaccaria (Meesapyodsuk et al. 2007), Barbarea vulgaris (Augustin et al. 2012), Glycine max (Shibuya et al. 2010), and P. ginseng (Yan et al. 2014; Jung et al. 2014; Wei et al. 2015; Wang et al. 2015a). Therefore, both evolutionary analysis and bioengineering industry require whole-genome analysis of UGTs in plant community. Recently, two maps of ginseng genome have been released (Xu et al. 2017; Kim et al. 2018), while a large set of ginseng transcriptomes and proteomics work can also be achieved publicly. In this chapter, we took the genome map of the institute of Chinese materia medica as an example and combined other peer-reviewed data together to elucidate the synthesis and regulation of ginsenosides. We hope this work will help our reader to establish a panorama view for natural generation process of those important bioactive compounds.

6.2

Characteristics of P. ginseng Genome

According to the k-mer simulation and flow cytometry analysis, P. ginseng’s genome size is approximated 3.5 Gb. The next-generation sequencing data confirmed the prediction. Using MAKER pipeline, which combining the ab initio and comparison methods together. The total predicted number of protein-coding gene models is 42 006. Approximately, 90% predicted models were backed by the assembled RNA-Seq transcripts. There are over ninety-five percent of predicted genes in the NCBI GenBank nonredundant database, which include homologs. Nearly, seventy-five percent predicted genes could be assigned to gene ontology (GO) catalogs. Over sixty-eight percent genes could be assigned to Genomes (KEGG) pathways and Kyoto Encyclopedia of Genes. Several appealing gene families were detected, those included 2556

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transcription factors, 3745 transporters, and 488 cytochrome P450 genes. Key enzymes for ginsenoside cyclization—the PPD ginsenoside synthase (PPDS) CYP716A47, PPT ginsenoside synthase (PPTS) CYP716A53, and oleanolic acid synthase CYP716A52—were all detected. Repeat elements were taken the most part of ginseng genome (about 62%). In those sequences, more than eighty-three percent elements were labeled as long terminal repeats (LTRs). The most abundant retro-element superfamily is Ty3/Gypsy which accounts for nearly forty-three percent of the genome. Approximately 8% of the entire genome was comprised by the Ty1/Copia family. In the DNA transposon class, the most common repeat type was CMC, comprised 43 Mb sequence of the genome. Ortholog analysis (with a set containing 14 plants) showed that more than 30,000 gene models in P. ginseng could be classified into 12 231 gene families. Among these families, 1648 gene families are unique for P. ginseng species (Fig. 6.1c). There

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are average 2.59 genes per gene family, and it was the highest in all tested plants. This shows that duplication events happen in P. ginseng’s evolution. A phylogenetic tree was constructed using the maximum likelihood method based on the 383 single copy gene indexes by the ortholog analysis. Daucus carota diverging about sixtysix Myr ago (Fig. 6.1b) from Umbelliferae was discovered to be the most related species of P. ginseng in all the compared plant species.

6.3

Metabolism and Transcription Characteristics of Ginseng Root

For visualizing the spatial distribution of ginsenosides straightforwardly, desorption electrospray ionization–mass spectrometry (DESI–MS) imaging was employed on ginseng root sections. The identified and summarized ginsenosides included ginsenoside Rg1/Rf, Rd/Re, Rs1/Rs2, Ra1/Ra2, Ra3, and pseudo-Rc1. Some

Fig. 6.1 Brief account of P. ginseng genome. a Statistical analysis of the P. ginseng draft genome. b Phylogenetic tree of P. ginseng. c Orthologous analysis

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Fig. 6.2 Mass spectrometry based on the desorption electrospray ionization–mass spectrometry (DESI–MS) studied the distribution of ginsenoside in P. ginseng root cross sections. a Optical image of main root. b TMS

image spectrum. c DESI–MS image of metabolites and ginsenosides: a maltose, b citbismine C, c Rg1/Rf, d pseudo-Rc1, e Ra1/Ra2, f Rd/Re, g Rs1/Rs2, and h Ra3. Scale bar = 2 mm (Xu et al. 2017)

ginsenosides such as Rg1/Rf were highly enriched in the inner core areas and the outer bark of the rhizome cross section. Some other ginsenosides, Rd/Re Rs1/Rs2, Ra1/Ra2, and pseudoginsenoside Rc1, for example, have low concentrations in the inner core areas and high concentrations in the bark (Fig. 6.2c–g). The ginsenoside Ra3 showed a spread distribution in the core section and a high enriched distribution around the bark (Fig. 6.2c, h). The DESI–tandem mass spectrometry (MS/MS) was further distinguished for isomers. For Rf/Rg1, fragmentation

of the disaccharide group C12H22O11 (342.12 Da) and monosaccharide group C6H10O5 (162.05 Da) produced fragments at m/z 457.15 and 637.46, which consist of various spatial distributions. The characteristic MS/MS transitions were m/z 799.52 for Re and m/z 603.08 for Rd. High-performance liquid chromatography analysis confirmed the concentration of ginsenosides Rg1, Rg2, Re, Rc, Rf, Rb, Rb1, and Rd2 was remarkably lower in the cortex and stele parts(P < 0.001) than in the periderm of ginseng root (Fig. 6.3a). Centralize among the periderm, cortex, and stele organs were

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distinct when using the PCA and PLS-DA plots algorithm (Fig. 6.3b–c). The detections indicate different distributions of ginsenosides. So, transcriptome analysis for ginseng root should go deep into the tissue level. Tissue-level transcriptome showed that 27 450 genes were expressed in the periderm, cortex, and stele of ginseng root. Samples can be well grouped by expression profile. The distance between the stele and cortex is closer than any tissue to periderm (Fig. 6.3d). Between the stele and periderm, cortex and periderm, and the stele and cortex, 2688, 2530, and 711 differentially expressed genes were detected, respectively. The genes that differ between the stele and periderm, as well as most of the cortex and periderm were enriched in genes related to metabolic approach and stimulation reactions. Three of the sixty-four weighted gene coexpression network analyses (WGCNAs) and grouped 64 modules were positively correlated with total ginsenoside content. The closest related module included more than 15,000 genes, signifying the detailed mechanisms under ginsenoside regulation and synthesis (Fig. 6.3e).

6.4

Conserved Biosynthesis Pathway of Ginsenosides

MVA pathway is conserved in eukaryotes. In ginseng genome, by BLAST search and motif finding, 31 genes encoding for 10 MVA enzymes were identified (Fig. 6.4a). Except for acetylCoA C-acetyltransferase (AACT), most of the 10 enzymes contain isoforms and multiple copies. The mevalonate kinase [MVK], isopentenyldiphosphate delta-isomerase [IDI], mevalonate diphosphate decarboxylase [MVD], and farnesyl diphosphate synthase [FPS]) regulated two duplicates of encoding genes each. For other 5 enzymes, 3-hydroxy-3-methylglutaryl-CoA reductase [HMGR] has 8 copies, squalene synthase [SS] and squalene epoxidase [SE] have 4 copies, and phosphomevalonate kinase [PMK] and 3-hydroxy-3-methylglutaryl-CoA synthase [HMGS] have 3 copies each. One of the PMKs might be a potential pseudogene and divides the encoding region with several termination codons.

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Such a common multicopy phenomenon in MVA enzymes of ginseng signified the varied regulatory of triterpenoid/steroid biosynthesis on this plant. In addition, more than 100 terpenoid synthases were labeled, containing a cycloartenol synthase (CAS), a lanosterol synthase (LAS), 5 beta-amyrin synthases (beta-ASs), 3 PPTSs, 3 PPDSs, 3 oleanolic acid synthases (OASs), and 3 dammarenediol synthases (DDSs). Transcriptome data, including root periderm, root cortex, root stele, stem, leaf blade, leaflet pedicel, leaflet peduncle, fruit flesh, and fruit pedicel, showed interesting pattern when used for representing analysis of the upstream genes of ginsenoside (Wang et al. 2015b). Three tissues originate ginseng root that was gathered into a same clan. The aerial parts, including the stem, leaf blade, leaflet pedicel, leaflet peduncle, and fruit pedicel but not fruit flesh, were gathered into another clan (Fig. 6.4b). Data of fruit flesh formed a single branch itself maybe because it is the only reproductive organ in this comparison. Certain correlations were exhibited among those genes. For instance, PG03014 from MVK, PG07131 and PG03840 from HMGR, PG30418 from SS, and PG00849 from beta-AS were mainly represented in the fruit flesh dataset instead of in other samples. PG20228 from MVK, PG37213 from MVD, and two FPS isoforms (PG34283 and PG21761) were particularly co-expressed in stem. The upstream genes were classified into several groups, according to hierarchical cluster analysis. HMGRs are in charge of the conversion of HMG-COA into MVA. This process has been identified to be the primary step in the synthesis of ginsenoside. As illustrated, 8 HMGR-encoding genes were detected. Based on the primary structure of putative proteins, we produced four subfamilies of the 8 PgHMGRs, or PgHMGR1.1 (PG16235, PG37498), PgHMGR1.2 (PG00233, PG15732), PgHMGR2.1 (PG03840, PG07131), and PgHMGR2.2 (PG38245, PG02251) (Fig. 6.5a). PgHMGR1 group is compared to be short, with 573 amino acids (aa) for HMGR1.1 and 565 aa for HMGR1.2. The coding area of the PgHMGR2 family is relatively long, with the coding area aa of HMGR 2.1 at 594 and the

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Fig. 6.3 Transcriptome and metabolism analysis of P. ginseng root. a HPLC chromatograms of the ginsenosides Rg1, Re, Rf, Rg2, Rb1, Rc, Rb2, and Rd standards. b PCA score plots based on the HPLC dataset (● periderm, ● cortex, and ● stele). c PLS-DA score plots

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based on the HPLC dataset ( ● periderm, ● cortex, and ● stele). d Cluster tree of the ginseng samples based on the expression pattern of 42,006 genes. e Hierarchical cluster tree of ginseng genes (Xu et al. 2017)

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Fig. 6.4 MVA pathway in P. ginseng a and expression b

coding area aa of HMGR2.2 at 589 (Fig. 6.5d). Most of PgHMGR-encoding genes have the alike exon phase pattern with the makeup “0–2-1–0”. The fluctuation of the size of the intron in the PgHMGR-coding genes is greater than that of the exon. In the first exon region, the PgHMGR1 family was 63 bp shorter than PgHMGR2 family. The second intron changed the most and the standard variation of 187 bp (Fig. 6.5c). In the coding area, PgHMGRs were heavily conservative at the C-terminal (which was identified for MVA catalysis). However, they were differentiated at the N-terminal (which was identified for membrane anchoring). It means maybe those isoforms varied in subcellular location. Similar to most identified plants HMGRs, PgHMGRs have a membrane anchor domain with a characteristic helix–loop–helix structure, connection linker area, 2 HMG-COAbinding motifs (MP(I/V)GY(I/V)QIP and TTEGCLVA), and 2 NADPH-binding motifs (GTVGGGT and DAMGMNM) (Fig. 6.5d). Then, the amino acids in the functional sites were conserved of all HMGRs peculiarly in the catalytic domains containing core region. Except PG00233 and PG15732 in HMGR1.2, all other isoforms contained a triple consecutive arginine

region. The identity obsessed the difference of HMGR1.2 and other isoforms in endoplasmic reticulum retention. The expression patterns of HMGRs were also varied in types (Figs. 6.4b and 6.5b). The 2 insiders of the HMGR2.2 family were expressed mainly in the blades and high in the plant roots. Different HMGRs expression patterns indicate that they may play different tasks during ginseng development.

6.5

Microbial Resistance Genes and HMGR Expression Pattern During Cylindrocarpon Destructans Infection

Ginsenosides comprise a group of defense metabolites. Hence, defense microbial infection is essential for biosynthesis of ginsenoside. Resistance genes are involved in the plants’ defense mechanism and generally play an important role in the recognition step in immune response. On the basis of the plant resistance gene database, 1652 resistance genes were annotated. Given the structural characteristics, these genes were divided into seven groups as follows: 50 CNLs (proteins with at least a

78

X. Zhi-han et al.

Fig. 6.5 Comparative analysis of the HMGR gene family. a Classification of PgHMGRs. b Tissue-specific PgHMGR expression patterns in 4-year-old roots. The data represents the mean ± SD of the three independent samples. c Genomic DNA structure of PgHMGRs. The exons are represented by the green-filled square boxes. The lines among the boxes correspond to the introns. The numbers above the exons indicate the length in bp. d Multiple alignments of the amino acid sequences of

PgHMGRs with homologous HMGRs from Arabidopsis. The black boxes indicate identical residues; the gray boxes represent identical residues for at least two of the sequences. Functional domains are highlighted in the colored boxes (red, membrane domain; green, linker domain; and blue, catalytic domain). The two putative HMGR-CoA-binding sites, two NADP(H)-binding sites, and ER retention motifs are denoted by square boxes

nucleotide binding site, a coiled-coil domain, and a leucine-rich repeat), 21 TNLs (proteins with a nucleotide binding site, a Toll–interleukin receptor-like domain, and a leucine-rich repeat), two NLs (proteins with a leucine-rich copy and a nucleotide binding site, excluding CNL and TNL), 130 RLPs (proteins with an extracellular leucine-rich repeat, and a receptor serine–threonine kinase-like domain), 877 RLKs (proteins with a kinase domain and an extracellular leucinerich repeat), 139 kinase-type enzymes, and 433 other resistance-type genes (Fig. 6.6a). In

comparison with eight plants, ginseng possessed the highest number of RLK and kinase-type resistance genes and the lowest number of TNLtype genes (Fig. 6.6a). The expression analysis showed that 160 resistance genes were expressed lower in the cortex than in the periderm; the 156 resistance genes were expressed higher in the than in the stele. The 134 expressions of resistance genes in the periderm were higher than those in the stele and cortex. Half of these genes belong to the RLK/RLP/kinase type, whereas none of the genes belong to the TNL type.

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Fig. 6.6 Microbe resistance and HMGR expression pattern during the C. destructans infection. a Distribution of resistance genes in P. ginseng and eight other plants. b HMGR coexpression pattern in the P. ginseng root stele, cortex, and periderm. Module visualization of the network connections for HMGRs. The HMGR genes were manually selected from the WGCNA, and only the first neighbor genes for each HMGR were presented in the graph. c HMGR coexpression pattern in P. ginseng root during the process of C. destructans infection

The ginseng transcriptomes consisted by C. destructans, a major pathogen that causes ginseng root-rot disease, were recalculated for immune response analysis. The datasets involved seven time points, including the control and 0.25, 0.5, 1, 4, 7, and 12 days post-inoculation (DPI) of C. destructans. A total of 35 008 genes were predicted. Among the predicted genes, 28 481 were expressed at a higher level at any post-inoculation time point than the initial point. One hundred and four resistance genes were highly expressed at 0.25 DPI, and they might be involved in C. destructans recognition; 41 of these genes were RLKs. GO enrichment analysis showed that at all stages, the family “defense response to fungus” has increased significantly. The “response to

ethylene stimulus” and “jasmonic acid mediated signaling pathway” were also enriched, indicating the coordination between secondary metabolism and microbial resistance. A total of 151 transcriptional factors were also highly expressed at 0.25 DPI; these factors included 21 basic helix– loop–helix (bHLHs), 12 ethylene responsive factors (ERFs), and 24 MYBs. These findings were consistent with the GO enrichment data. We also evaluated the relationship of HMGRs and resistance genes. The co-expression patterns showed that PG16235 and PG02251 were mostly co-expressed with resistance genes during C. destructans infection. Hence, these two genes belong to inducible-type HMGRs during microbial infection (Fig. 6.6b–c).

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6.6

X. Zhi-han et al.

The MEP Pathway

Ginsenoside biosynthetic IPP biosynthesis is normally produced through the MVA route. Methylerythritol phosphate (MEP) is another way to make up for IPP production when MVA was blocked. The MEP pathway was started with the compression between pyruvate and Dglyceraldehyde-3-phosphate by 1-deoxy-Dxylulose 5-phosphate synthase (DXS, DXP synthase), and eventually converts 4-hydroxy-3methyl-butenyl 1-diphosphate (HMBPP) into dimethylallyl diphosphate (DMAPP) or IPP by isoprenoid synthase-containing protein H (IspH). A total of 33 putative genes relevant to the MEP pathway were detected, which include 9 DXSs, 4 DXRs (DXP reductoisomerase), 2 IspDs, 4 IspEs, 5 IspFs, 4 IspGs, and 5 IspHs. That means the multicopy phenomenon existed in MEP pathway like in the MVA route. The existence of multiple isoforms in the MVA/MEP pathway can facilitate elastic invoking or regulation of triterpene biosynthesis in ginseng.

6.7

UGTs of P. ginseng

UGTs are responsible for the glycosyl moiety transfer to target molecules, containing ginsenoside precursors. The ginseng genome encodes a large number of different UGTs arrays. In Xu et al. version, a total of 225 UGTs were annotated, a major ginseng gene family. The predications ranged in length from 74 to 575 aa. A few short, fragmented remains may due to the complexity of the genome or the pseudogene during evolution. The predicted isoelectric points fluctuated between 4.45 and 9.54. Pursuant to the standardization of the UGT Nomenclature Committee, all the labeled UGTs were classified into 24 subfamilies (Fig. 6.7a). The largest group was UGT73 with 30 members. The second is the UGT74 and UGT94 families, with 25 and 24 members each. Contrast to D. carota, the UGT71

and UGT74 families expanded significantly, while the family UGT93 significantly shrank. Based on genomic physical arrangement, a total of 78 UGTs were detected and divided into thirty groups. The largest group has five UGT members. PgUGTs in the same group were belonged to one subfamily, arranged in array. Like the largest group, all the group members are from the same ancestral UGT73, with similarity between 48 and 92%. This highly consistent suggests that the genes possibly have evolved since recent genome repeats or recent unequal recombination events. UGTs expression modules have high tissue specificity as well. Alike those gene groups, the UGTs expression patterns vary widely, although they all originated in the same gene family (UGT73) (Fig. 6.7b). PG22765-1 as the highest expressed member of the root, average FKPM of 3089, the only highly expressed gene in the root, followed by average FKPM of 1957 PG22765-2. At the same time, PG22765-5 with a CV of 186.72 was the most volatile gene. This UGT was seldom expressed in the roots, stems, or leaves, but was highly expressed in plants’ fruit. Therefore, UGTs belong to one family or located intimately, and exhibit significant gene expression regulation. Eighteen UGT71, UGT74, or UGT94 genes were selected for molecular modeling and docking function analysis. The Uniprot models of PPD and PPT were selected as the docking substrates, and UGT-Glc was chosen as sugar supply. The C-terminal W-N-S-X-L-E motif, the N-terminal I/V-G/S-H motif, and the C-terminal Y-G/A-E-Q motif of UGT71 family; the C-terminal H-C/S-G-W-N-S-T-X-E motif and the N-terminal motif Q-G-H-X-N/S of UGT74 family; and the C-terminal D-Q motif and the N-terminal H/Q/Y-G-H motif of UGT94 family were forecast to bind particularly to acceptors of sugar. The results implicated that during evolution for a specific substrate binding, indispensable residues in the N-terminal might have been subject to selection pressure.

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Fig. 6.7 P. ginseng’s UGTs. a Phylogenetics of ginseng UGTs. b An example of ginseng UGTs cluster

6.8

Transcriptional Factors and Transporters

The bHLH transcription factor family is a wellknown transcription factor family in plants. This family has been proved to be involved in numerous regulatory processes. Chu et al. (2018) identified putative 169 bHLH transcription factor genes in the P. ginseng genome. The phylogenetic analysis assigned those PGbHLHs to 24 subfamilies. Six PGbHLHs from four subfamilies were chosen as potential regulators of ginsenoside biosynthesis based on a combinatorial analysis of gene expression and ginsenoside content. The target genes of these six PGbHLHs were also predicted to be different. WRKY family is another unignored transcription factor of plants. Guo et al.

(2019) analyzed the WRKY gene in ginseng and five other plants from the superorder of Asteranae. They figured out that segmental duplication may play an indispensable part in the evolution of WRKY. For compound transportation, plants also encode a large set of transporters in genome. In ginseng, Su et al. picked out 37 oligopeptide transporter (OPT) family members. These OPT genes can be grouped into two separate clades which conserved in plants. Su suggested that segmental duplication and subsequent structural variation may contribute to the abundance of OPT paralogs in ginseng genome. Organspecific or tissue-specific expression motifs were also observed in some OPT genes when using RNA-Seq data for expression analysis (Su et al. 2018).

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References Achnine L, Huhman DV, Farag MA, Sumner LW, Blount JW, Dixon RA (2005) Genomics-based selection and functional characterization of triterpene glycosyltransferases from the model legume Medicago truncatula. Plant J 41:875–887 Augustin JM, Kuzina V, Andersen SB, Bak S (2011) Molecular activities, biosynthesis and evolution of triterpenoid saponins. Cheminform 72:435–457 Augustin JM, Drok S, Shinoda T et al (2012) UDPglycosyltransferases from the UGT73C subfamily in Barbarea vulgaris catalyze sapogenin 3-Oglucosylation in saponin-mediated insect resistance. Plant Physiol 160:1881–1895 Christensen LP, Jensen M, Kidmose U (2006) Simultaneous determination of ginsenosides and polyacetylenes in American ginseng root (Panax quinquefolium L.) by high-performance liquid chromatography. J Agric Food Chem 54:8995–9003 Chu Y, Xiao SM, Su H et al (2018) Genome-wide characterization and analysis of bHLH transcription factors in Panax ginseng. Acta Pharm Sin B 8:178– 189 Fukuda N, Shan S, Tanaka H, Shoyama Y (2005) New staining methodology: eastern blotting for glycosides in the field of Kampo medicines. J Nat Med 60:21–27 Guo H, Zhang Y, Wang Z et al (2019) Genome-wide identification of WRKY transcription factors in the Asteranae. Plants 8:393 Haralampidis K, Trojanowska M, Osbourn AE (2002) Biosynthesis of triterpenoid saponins in plants. Adv Biochem Eng Biotechnol 75:31–49 Han J-Y, In J-G, Kwon Y-S, Choi YE (2009) Regulation of ginsenoside and phytosterol biosynthesis by RNA interferences of squalene epoxidase gene in Panax ginseng. Phytochemistry 71:36–46 Han J-Y, Kim H-J, Kwon Y-S, Choi Y-E (2011) The Cyt P450 enzyme CYP716A47 catalyzes the formation of protopanaxadiol from dammarenediol-II during ginsenoside biosynthesis in Panax ginseng. Plant Cell Physiol 52:2062–2073 Han JY, Hwang HS, Choi SW, Kim HJ, Choi YE (2012) Cytochrome P450 CYP716A53v2 catalyzes the formation of protopanaxatriol from protopanaxadiol during ginsenoside biosynthesis in Panax ginseng. Plant Cell Physiol 53:1535–1545 Han J-Y, Kim M-J, Ban Y-W, Hwang H-S, Choi Y-E (2013) The involvement of b-amyrin 28-oxidase (CYP716A52v2) in oleanane-type ginsenoside biosynthesis in Panax ginseng. Plant Cell Physiol 54:2034–2046 Jenner H, Townsend BJ, Osbourn A (2005) Unravelling triterpene glycoside synthesis in plants: phytochemistry and functional genomics join forces. Planta 220:503–506

X. Zhi-han et al. Jung S-C, Kim W, Park SC et al (2014) Two ginseng UDP-glycosyltransferases synthesize ginsenoside Rg3 and Rd. Plant Cell Physiol 55:2177–2188 Kim NH, Jayakodij M, Lee SC et al (2018) Genome and evolution of the shade-requiring medicinal herb Panax ginseng. Plant Biotechnol J 16:1904–1917 Lee M-H, Jeong J-H, Seo J-W et al (2004) Enhanced triterpene and phytosterol biosynthesis in Panax ginseng overexpressing squalene synthase gene. Plant Cell Physiol 45:976–984 Leung KW, Wong ST (2010) Pharmacology of ginsenosides: a literature review. Chin Med 5:20 Li C, Zhu Y, Xu G et al (2013) Transcriptome analysis reveals ginsenosides biosynthetic genes, microRNAs and simple sequence repeats in Panax ginseng C. A. Meyer. BMC Genomics 14:245. https://doi.org/10. 1186/1471-2164-14-245 Liang Y, Zhao S (2008) Progress in understanding of ginsenoside biosynthesis. Plant Biol 10:415–421 Meesapyodsuk D, Balsevich J, Reed DW, Covello PS (2007) Saponin biosynthesis in Saponaria vaccaria. cDNAs encoding beta-amyrin synthase and a triterpene carboxylic acid glucosyltransferase. Plant Physiol 143:959–969 Osbourn A, Goss RJM, Field RA (2011) The saponins: polar isoprenoids with important and diverse biological activities. Nat Prod Rep 28:1261–1268 Phillips DR, Rasbery JM, Bartel B, Matsuda SP (2006) Biosynthetic diversity in plant triterpene cyclization. Curr Opin Plant Biol 9:305–314 Sawai S, Saito K (2011) Triterpenoid biosynthesis and engineering in plants. Front Plant Sci 2:25. https://doi. org/10.3389/fpls.2011.00025 Shibuya M, Nishimura K, Yasuyama N, Ebizuka Y (2010) Identification and characterization of glycosyltransferases involved in the biosynthesis of soyasaponin I in Glycine max. FEBS Lett 584:2258–2264 Su H, Chu Y, Bai J, et al (2018) Genome-wide identification and comparative analysis for OPT family genes in Panax ginseng and eleven flowering plants. Molecules 24(1):15. Published 20 Dec 2018. https://doi.org/10.3390/molecules24010015 Tani T, Kubo M, Katsuki T, Higashino M, Hayashi T, Arichi S (1981) Histochemistry II. Ginsenosides in ginseng (Panax ginseng, Root). J Nat Prod 44:401– 407 Taira S, Ikeda R, Yokota N et al (2010) Mass spectrometric imaging of ginsenosides localization in Panax ginseng root. Am J Chin Med 38:485–493 Thimmappa R, Geisler K, Louveau T, O’Maille P, Osbourn A (2014) Triterpene biosynthesis in plants. Annu Rev Plant Biol 65:225–257 Wang P, Wei Y, Fan Y et al (2015a) Production of bioactive ginsenosides Rh2 and Rg3 by metabolically engineered yeasts. Metab Eng 29:97–105 Wang K, Jiang S, Sun C et al (2015b) The spatial and temporal transcriptomic landscapes of ginseng, Panax

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ginseng C. A. Meyer. Sci Rep 5:18283. https://doi.org/ 10.1038/srep18283 Wei W, Wang P, Wei Y et al (2015) Characterization of Panax ginseng UDP-glycosyltransferases catalyzing protopanaxatriol and biosyntheses of bioactive ginsenosides F1 and Rh1 in metabolically engineered yeasts. Mol Plant 8:1412–1424 Xu J, Chu Y, Liao B et al (2017) Panax ginseng genome examination for ginsenoside biosynthesis. GigaScience 6(gix093):1–15 Yan X, Fan Y, Wei W et al (2014) Production of bioactive ginsenoside compound K in metabolically engineered yeast. Cell Res 24:770–773

83 Yokota S, Onohara Y, Shoyama Y (2011) Immunofluorescence and immunoelectron microscopic localization of medicinal substance, Rb1, in several plant parts of Panax ginseng. Curr Drug Disc Technol 8:51–59 Zhang YC, Li G, Jiang C et al (2014) Tissue-specific distribution of ginsenosides in different aged ginseng and antioxidant activity of ginseng leaf. Molecules 19:17381–17399

7

Ginseng Genome Structure and Evolution Nam-Hoon Kim, Murukarthick Jayakodi, and Tae-Jin Yang

Abstract

Ginseng (Panax ginseng) genome has been difficult to unlock its genomics due to its size, high repeat content, and polyploidy nature. Although chromosome-scale genome assembly for ginseng holds numerous challenges, a draft gene space assembly can open many avenues for ginseng crop improvements. This chapter provides an overview of the current ginseng genomic status, evolution, gene annotation of novel genes associated with repeat evolution, and environmental adaptations.

N.-H. Kim
M. Jayakodi
T.-J. Yang (&) Department of Agriculture, Forestry and Bioresources, Plant Genomics and Breeding Institute, College of Agriculture and Life Sciences, Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul 08826, Korea e-mail: [email protected] N.-H. Kim PHYZEN Genomics Institute, Seongnam-si, Gyeonggi-do 13558, Korea M. Jayakodi Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), 06466 Gatersleben, Seeland, Germany

7.1

Introduction

Panax ginseng C.A. Meyer (Ginseng) is an important and popular herbal plant in Asia and worldwide. The genus Panax is comprised of 15 species within Araliaceae family. Panax species can be classified into diploid and tetraploid species according to its chromosome number. The tetraploid species, ginseng and P. quinquefolius, have characteristics of 2n = 48 (Obae and West 2012), and the other diploid Panax species have half of chromosome set, 2n = 24. Most diploid ginseng species grow at the freeze-free region at Eastern Himalayas to Southern China, while tetraploid Panax species overwinter and grow at higher latitudes in Northeast Asia and North America. Roots of ginseng have been used for thousands of years to strengthen the body and mental well-being of humans. The ginseng has therapeutic effects on notable serious diseases such as neurodegenerative (Cho 2012) and cardiovascular diseases (Zheng et al. 2012), diabetes (Xie et al. 2005), and cancer (Wong et al. 2015). The major medicinal efficacy of ginseng is attributed to its various unique saponins called ginsenosides, classified as dammarane- or oleanane-type. Particularly, dammarane-type ginsenosides are exclusively biosynthesized in the plants that belong to the genus Panax (Kim et al. 2018). Though limited genomic information and difficulties of breeding had been hindered

© Springer Nature Switzerland AG 2021 J. Xu et al. (eds.), The Ginseng Genome, Compendium of Plant Genomes, https://doi.org/10.1007/978-3-030-30347-1_7

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genomic and genetic study of ginseng, two draft genome sequences of ginseng were completed and opened to researchers in 2017 and 2018 (Xu et al. 2017; Kim et al. 2018). The first one is comprised with 83,064 scaffolds, and the second one contained 9,845 scaffolds. In this chapter, we give you an overview of ginseng genome structure and its evolution based on better assembly, Kim et al. 2018. Even though the draft genome sequences have not been completed as pseudomolecule, the length of scaffolds is long-enough to cover gene space and elucidate its genome characteristics.

7.2

Genome Size

Genome size could be estimated by various methods, of which flow cytometry and k-merbased analysis using next-generation sequencing (NGS) reads are the most popular methods owing to its fastness and accuracy. Flow cytometry calculated C-value by florescent signal of nuclei comparison to control sample. In k-mer analysis, Table 7.1 Characterized repeat elements in P. ginseng BAC clone sequences

the NGS reads are split into small size of nucleotide (k-mer) around 17 bp, and its frequency and distribution pattern are calculated. The first ginseng genome size was estimated in 2004 by flow cytometry, resulting in 3.12 Gbp (Hong et al. 2004). The newly estimated genome size of ginseng revealed 3.3 to 3.6 Gbp, slightly bigger than previous report, using both flow cytometry and k-mer analysis methods.

7.3

Genome Assembly and Annotation

The draft genome sequence of ginseng comprised of 2.98 Gbp, 9,845 scaffolds with 569 kb of N50 value. The genome size variation among organism is often associated with the content of repetitive DNA sequences. Using 20 reported repeat elements (Table 7.1) in BAC clone sequences (Choi et al. 2014; Jang et al. 2017) and de novo repeat annotation revealed that 80% of ginseng genome is comprised with repeat elements, of which Ty3/gypsy long terminal repeat

Type

Name

Length (bp)

References

Ty3/Gypsy

PgDel1_1

10,039

Choi et al. (2014)

PgDel1_2

10,120

Choi et al. (2014)

PgDl1_3

9,477

Choi et al. (2014)

PgDel1_4

8,004

Choi et al. (2014)

PgDel1_5

7,714

Choi et al. (2014)

PgDel2

12,515

Choi et al. (2014)

PgDel3

11,809

Choi et al. (2014)

PgDel4

11,050

Jang et al. (2017)

PgDel5

12,860

Jang et al. (2017)

PgDel6

12,252

Jang et al. (2017)

PgTat1_1

22,881

Choi et al. (2014)

PgTat1_2

12,289

Choi et al. (2014)

PgTat2

10,965

Choi et al. (2014)

PgAthila

9,893

Choi et al. (2014)

Ty1/Copia

PgTork

9,707

Choi et al. (2014)

PgOryco

7,772

Choi et al. (2014)

Tandem unit

PgTR

1,577

Choi et al. (2014)

45S rDNA

5,877

Kim et al. (2015)

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Ginseng Genome Structure and Evolution

retrotransposon (LTR-RT) family occupied almost half of ginseng genome (Table 7.2). 59,352 genes were predicted in ginseng, using transcriptome data by NGS platform (Jayakodi et al. 2014, 2015) and third-generation sequencing (TGS) as well as protein evidence and ab initio prediction. The Iso-Seq sequences from TGS were enabled to annotate expanded gene length of 93 kb and were helpful for validation gene structure. Annotation by known protein databases such as InterPro, NCBI Nr, arabidopsis, and tomato revealed that 97% of the ginseng genes have attained functional descriptions. Additionally, 22,384 of alternative splicing transcripts, 19,496 long noncoding RNAs (lncRNAs), and 451 conserved microRNAs (miRNAs) were characterized. The genome assembly, annotations, and other multi-omics

87

data are available at ginseng genome database (https://ginsengdb.snu.ac.kr/) (Jayakodi et al. 2018).

7.4

Genome Duplication

Polyploids are generated with genome doubling or triplication of same or different genome sets (Leitch and Bennett 1997). This phenomenon is an important driving force of genome evolution and speciation in angiosperm (Hegarty and Hiscock 2008; Soltis 2005). The chromosome number is the clear evidence for polyploidization; however, diploidization and chromosomal rearrangement hindered whole-genome duplication (WGD) event occurred long ago. Comparative analysis of the number of synonymous

Table 7.2 Annotation of repetitive sequence in P. ginseng draft sequences Number of elements

Length occupied (bp)

% of sequences

11,214

5,670,094

0.21

ALU-like

5,864

4,036,665

0.15

MIRs

5,350

1,633,429

0.06

LINEs:

46,273

30,551,064

1.11

LINE1

30,168

25,501,454

0.93

SINEs:

Tad1

13,895

4,720,262

0.17

LTR elements:

1,041,922

1,561,608,460

56.94

LTR/Gypsy

855,522

1,346,645,416

49.10

LTR/Copia

134,616

166,958,332

6.09

LTR/unknown

39,414

35,454,270

1.29

LTR/Caulimovirus

7,760

10,961,815

0.40

DNA elements:

282,863

127,715,344

4.67

CMC-EnSpm

46,937

28,535,729

1.04

TcMar-Stowaway

35,332

5,882,125

0.21

hAT-Ac

29,638

8,073,451

0.29

Unclassified:

829,017

454,133,147

16.56

Total interspersed repeats

2,215,834

2,181,212,651

79.52 (of 2.7 Gb)

Satellites:

28,615

21,084,254

0.77

Simple repeats:

297,483

19,750,362

0.72

Low complexity:

53,676

2,742,099

0.10

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substitutions per synonymous site (Ks) between two homologous gene sequences indicates the divergence scale (Sterck et al. 2005). Because synonymous substitutions did not change the amino acid, nucleotide change was accumulated between two genes. The calculated divergence time via Ks analysis revealed WGD event between paralogous genes and diverged time between orthologous genes. A comparative analysis of annotated ginseng paralog genes revealed two rounds of WGD, one at 2.2 million years ago (MYA), represented as Pg-a, and the other at 28 MYA (Pg-b). Many ginseng researchers had problems with marker development or gene identification (Kim et al. 2014), because paralogous genes resulted by Pg-a WGD showed 99% of similarity. Moreover, the flanking regions were highly conserved between paralogous scaffolds that resulted from Pg-a WGD (Fig. 7.1). A detailed sequence analysis revealed that paralogous scaffolds from Pg-a WGD have 95% of sequence similarity except repeatmediated regions, whereas only genic region showed synteny relationship between Pg-b WGD-related paralogous scaffolds.

7.5

Phylogenomics of Panax Species

Repetitive DNA proportion could be estimated in genome through mapping of low-coverage NGS reads (Lee et al. 2017). The PgDel LTR-RT family is major repeat element in ginseng, of which

PgDel1 occupied 1/3 of whole genome. Particularly, PgDel2 accumulated in only half of chromosomes of tetraploid Panax species, supporting allotetraploidization event which are combination of two distinct subgenomes. The genome proportion of five major LTR-RT families (PgDel, PgTat, PgAthila, PgTork, and PgSire) in seven Panaxrelated species revealed that PgDel has mainly contributed to genome size variation (Lee et al. 2017) in Panax species. Especially, P. quinquefolius have larger genome size than ginseng, 4.9 Gb, PgDel family illuminated genome size difference of two species. An En/Spm-like CACTA transposon (PgCACTA1) has diverse copy number of 167 bp of tandem repeat (Pg167TR) (Waminal et al. 2017). The genome proportions of PgCACTA1 and Pg167TR are distinctly diverse between diploid and tetraploid Panax species. Accumulated Pg167TR in ginseng was verified with comparative fluorescence in situ hybridization (FISH) analysis with P. notoginseng (Kim et al. 2018). The chloroplast and ribosomal DNA are conserved in plants; thus, molecular phylogenetic analysis has widely been conducted based on them as a target. The integrated phylogenomic analysis of complete chloroplast genome and ribosomal DNA of ten Panax-related species revealed that Panax–Aralia lineage diverged approximately 7.97–7.50 (Kim et al. 2017; Nguyen et al. 2018). Two Panax tetraploids diverged from diploid species *2.59 MYA, and ginseng and P. quinquefolius diverged at *0.77 MYA.

Fig. 7.1 Four homoeologous sequences resulted by two WGDs (adopted in Kim et al. 2018). Two green and blue scaffolds represent paralogous scaffolds by Pg-a, respectively, and green and blue scaffolds are related to Pg-b WGD

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Ginseng Genome Structure and Evolution

7.6

Evolution History of Ginseng

Postulated evolutionary history of ginseng was constructed by molecular clock with Ks analysis and chloroplast sequence comparison with other known species. Approximately, the Araliaceae– Apiaceae was diverged in 51 MYA, estimating divergent time with carrot (Fig. 7.2a), corresponded to the global warming of the Eocene era (56–34 MYA). The WGD was followed in Apiaceae and Araliaceae family at 45–28 MYA (Fig. 7.2a). The common ancestor of Panax and Aralia species was distributed at Qinghai–Tibetan Plateau and southwestern part of Asia before * 10 MYA, and Eleuthercoccus– Dedropanax–Schefflera group were diverged with Aralia–Polyscias–Pseudopanax group (Fig. 7.2b) (Wen et al. 2001). Around 7.5 MYA, Aralia and Panax genuses were divided and major repeat elements including PgDel were proliferated in Panax-specific. In addition, one of the diploid ginseng species crossed into North America and diverged to P. trifolius (Fig. 7.3). At south-central China, the speciation of diploid Panax species and WGD (allotetraploidization) in the ancestor of ginseng and P. quinquefolius occurred sequentially at 3–2 MYA, and tetraploid Panax gained overwintering and dispersed in Northeast Asia. Genome shock such as drastic climate change or polyploidization caused explosive repeat element amplification (McClintock 1978). Comparison of two LTRs of intact LTR-RT and transposase domain lead when repeat elements were inserted or diverged. Tetraploid Panax species have double to ten of PgCACTA1 and Pg167TR than diploid Panax and the estimated insertion time by intact LTR sequences was 1.8–1.4 MYA indicated various repeat elements were proliferated following Pg-a WGD and affected the genome size in Panax species. Pleistocene (*2.58 MYA) glaciation may influence isolation of cold-susceptible diploid Panax species in warm Southern Asia and migration of P. quinquefolius to North America around 1.2–0.8 MYA (Fig. 7.3) (Choi et al. 2013; Kim et al. 2017).

89

7.7

Environmental Adaptive Gene Families

Fatty acid desaturases (FADs) are enzymes that synthesize mono- and poly-unsaturated fatty acids such as oleic acid and linoleic acid. In ginseng, a large number of (85) FAD gene were found, which is three times higher than model annual plants of A. thaliana, S. lycopersicum, and O. sativa. Notably, the many members of this gene family have arisen through tandem duplication. The digital gene expression of FADs between abiotic stress RNA-Seq samples in P. ginseng showed a higher expression pattern under cold stress as compared with other stresses. Further, a phylogenetic analysis notably formed two separate groups specific to P. ginseng, containing twenty-six FAD genes related to putative acetylene and a group of nine genes identified as newly evolved delta-12-FADS-like genes (Kim et al. 2018). In addition, the diversification of FAD structures was happened in the evolution of P. ginseng. Gene presence/absence analysis showed specific expansion of FADs in P. ginseng with strong expression against cold stress. These evidences postulated that expansion of FAD genes with diverse FAD structures might be associated with survival of ginseng at low temperatures. Ginseng has been grown under canopy or artificial shade; however, the reason behind this process is largely unexplored. It is obvious that the ginseng plant should have acquired a novel mechanism to ensure an efficient photosynthesis under low light conditions. The light-harvesting chlorophyll a-b binding proteins (LHCPs or CAB) are the key components of the photosynthesis antennae complexes which transfer the light energy to the reaction centers of photosystem I (PS I) and photosystem II (PS II) where the light energy is converted to form chemical bond energy (i.e., NADPH and ATP). Ginseng genome contains more CAB genes (49 genes) than any plant species to date. A phylogenetic analysis of CAB genes between P. ginseng, P. notoginseng, A. thaliana, S. lycopersicum, O. sativa,

90

N.-H. Kim et al.

C

D

Fig. 7.2 Evolutionary history of P. ginseng with comparative sequence analysis. a Reconstructed evolution model via Ks analysis with carrot (adopted in Kim et al.

2018). b Evolution diagram within Araliaceae through comparison of chloroplast genomes

7

Ginseng Genome Structure and Evolution

91

Fig. 7.3 Postulated evolution model of seven Panax species; P. stipuleanatus (Ps), P. notoginseng (Pn), P. vietnamensis (Pv), P. japonicas (Pj), P. trifolius (Pt), P. ginseng (Pg), and P. quinquefolius (Pq) (adopted in Kim et al. 2018). Blue and yellow circles indicate current

habitats of the Panax species, whereas gray circle indicates putative habitats of common ancestor. The 1st migration of diploid Panax marked as blue line and 2nd migration of tetraploid Panax marked as yellow line

and D. carota revealed that a member of CAB gene in P. ginseng have 4:1 (four copies in P. ginseng: one copy in other species), and 4:2 and 4:3 relationship with other species (Kim et al. 2018). Additionally, estimation of presence/absence of orthologous gene copies in P. vietnamensis revealed the abundance of CAB genes, both shade plants, tetraploid and diploid ginseng species. Equivalently, a total of 53 genes (including pseudogenes) were also identified in the genome of brown algae (Cock et al. 2010) (Ectocarpus siliculosus) and that expansion was attributed to adapt to variable or dim light conditions. Similarly, it was speculated that the large number of CAB genes in P. ginseng might

enable the ginseng plant to adapt to an environment with low light conditions.

7.8

Conclusion and Perspective

The genome sequence of P. ginseng provides its own duplicated genome feature, evolution, and characterization of functional genes. Two rounds of WGD might have shaped ginseng plant for surviving and adopting to environmental stress with expansion of freezing and shade-related genes. Constructed metabolic network of ginsenosides provides essential targets to increase the production of ginsenosides through latest

92

biotechnological approaches. The ginseng genome sequence and annotated genes will enable ginseng researchers to develop new cultivars carrying resistant to biotic/abiotic stresses, tolerant to direct sunlight, and enhanced ginsenoside production through advanced breeding technologies and metabolic engineering.

References Cock JM, Sterck L, Rouzé P, Scornet D, Allen AE, Amoutzias G, Anthouard V, Artiguenave F, Aury JM, Badger JH, Beszteri B, Billiau K, Bonnet E, Bothwell JH, Bowler C, Boyen C, Brownlee C, Carrano CJ, Charrier B, Cho GY, Coelho SM, Collén J, Corre E, Da Silva C, Delage L, Delaroque N, Dittami SM, Doulbeau S, Elias M, Farnham G, Gachon CM, Gschloessl B, Heesch S, Jabbari K, Jubin C, Kawai H, Kimura K, Kloareg B, Küpper FC, Lang D, Le Bail A, Leblanc C, Lerouge P, Lohr M, Lopez PJ, Martens C, Maumus F, Michel G, Miranda-Saavedra D, Morales J, Moreau H, Motomura T, Nagasato C, Napoli CA, Nelson DR, Nyvall-Collén P, Peters AF, Pommier C, Potin P, Poulain J, Quesneville H, Read B, Rensing SA, Ritter A, Rousvoal S, Samanta M, Samson G, Schroeder DC, Ségurens B, Strittmatter M, Tonon T, Tregear JW, Valentin K, von Dassow P, Yamagishi T, Van de Peer Y, Wincker P (2010) The Ectocarpus genome and the independent evolution of multicellularity in brown algae. Nature 465(7298):617–621 Cho IH (2012) Effects of Panax ginseng in neurodegenerative diseases. J Ginseng Res 36:342 Choi HI, Kim NH, Lee J, Choi BS, Kim KD, Park JY, Lee SC, Yang TJ (2013) Evolutionary relationship of Panax ginseng and P. quinquefolius inferred from sequencing and comparative analysis of expressed sequence tags. Genet Resour Crop Evol 60:1377– 1387 Choi HI, Waminal NE, Park HM, Kim NH, Choi BS, Park M, Choi D, Lim YP, Kwon SJ, Park BS, Kim HH, Yang TJ (2014) Major repeat components covering one-third of the ginseng (Panax ginseng C. A. Meyer) genome and evidence for allotetraploidy. Plant J 77:906–916 Hegarty MJ, Hiscock SJ (2008) Genomic clues to the evolutionary success of polyploid plants. Curr Biol 18: R435-444 Hong C, Lee S, Park J, Plaha P, Park Y, Lee Y, Choi J, Kim K, Lee J, Lee J (2004) Construction of a BAC library of Korean ginseng and initial analysis of BACend sequences. Mol Genet Genomics 271:709–716 Jang W, Kim NH, Lee J, Waminal NE, Lee SC, Jayakodi M, Choi HI, Park JY, Lee JE, Yang TJ (2017) A glimpse of Panax ginseng genome structure revealed from ten BAC clone sequences obtained by

N.-H. Kim et al. SMRT sequencing platform. Plant Breed Biotechnol 5:25–35 Jayakodi M, Lee SC, Park HS, Jang W, Lee YS, Choi BS, Nah GJ, Kim DS, Natesan S, Sun C, Yang TJ (2014) Transcriptome profiling and comparative analysis of Panax ginseng adventitious roots. J Ginseng Res 38 (4):278–288 Jayakodi M, Lee SC, Lee YS, Park HS, Kim NH, Jang W, Lee HO, Joh HJ, Yang TJ (2015) Comprehensive analysis of Panax ginseng root transcriptomes. BMC Plant Biol 15:138 Jayakodi M, Choi BS, Lee SC, Kim NH, Park JY, Jang W, Lakshmanan M, Mohan SVG, Lee DY, Yang TJ (2018) Ginseng Genome Database: an openaccess platform for genomics of Panax ginseng. BMC Plant Biol 18:62 Kim K, Lee SC, Lee J, Lee HO, Joh HJ, Kim NH, Park HS, Yang TJ (2015) Comprehensive survey of genetic diversity in chloroplast genomes and 45S nrDNAs within Panax ginseng species. PLoS ONE 10 (6):e0117159 Kim K, Nguyen VB, Dong J, Wang Y, Park JY, Lee SC, Yang TJ (2017) Evolution of the Araliaceae family inferred from complete chloroplast genomes and 45S nrDNAs of 10 Panax-related species. Sci Rep 7:4917 Kim NH, Choi HI, Ahn IO, Yang TJ (2014) Evidence of genome duplication revealed by sequence analysis of multi-loci expressed sequence tag-simple sequence repeat bands in Panax ginseng Meyer. J Ginseng Res 38:130–135 Kim NH, Jayakodi M, Lee SC, Choi BS, Jang W, Lee J, Kim HH, Waminal NE, Lakshmanan M, Van Nguyen B, Lee YS, Park HS, Koo HJ, Park JY, Perumal S, Joh HJ, Lee H, Kim J, Kim IS, Kim K, Koduru L, Kang KB, Sung SH, Yu Y, Park DS, Choi D, Seo E, Kim S, Kim YC, Hyun DY, Park YI, Kim C, Lee TH, Kim HU, Soh MS, Lee Y, In JG, Kim HS, Kim YM, Yang DC, Wing RA, Lee DY, Paterson AH, Yang TJ (2018) Genome and evolution of the shade-requiring medicinal herb Panax ginseng. Plant Biotechnol J 16:1904–1917 Lee J, Waminal NE, Choi HI, Perumal S, Lee SC, Nguyen VB, Jang W, Kim NH, Gao LZ, Yang TJ (2017) Rapid amplification of four retrotransposon families promoted speciation and genome size expansion in the genus Panax. Sci Rep 7:9045 Leitch IJ, Bennett MD (1997) Polyploidy in angiosperms. Trends Plant Sci 2:470–476 McClintock B (1978) Mechanisms that rapidly reorganize the genome. Stadler Genet Symp 10:25–48 Nguyen VB, Giang VNL, Waminal NE, Park HS, Kim NH, Jang W, Lee J, Yang TJ (2018) Comprehensive comparative analysis of chloroplast genomes from seven Panax species and development of an authentication system based on species-unique SNP markers. J Ginseng Res https://doi.org/10.1016/j.jgr. 2018.06.003 Obae SG, West TP (2012) Nuclear DNA content and genome size of American ginseng. J Med Plants Res 6:4719–4723

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Ginseng Genome Structure and Evolution

Soltis DE (2005) Ancient and recent polyploidy in angiosperms. New Phytol 166:5–8 Sterck L, Rombauts S, Jansson S, Sterky F, Rouze P, Van de Peer Y (2005) EST data suggest that poplar is an ancient polyploid. New Phytol 167:165–170 Waminal NE, Choi HI, Kim NH, Jang WJ, Lee J, Park JY, Kim HH, Yang TJ (2017) A refined Panax ginseng karyotype based on an ultra-high copy 167-bp tandem repeat and ribosomal DNAs. J Ginseng Res 41:469–476 Wen J, Plunkett GM, Mitch AD, Wagstaff SJ (2001) The evolution of araliaceae: a phylogenetic analysis based on ITS sequences of nuclear ribosomal DNA. Syst Bot 26:144–167 Wong AS, Che CM, Leung KW (2015) Recent advances in ginseng as cancer therapeutics: a functional and mechanistic overview. Nat Prod Rep 32:256–272

93 Xie JT, Mehendale SR, Li X, Quigg R, Wang X, Wang CZ, Wu JA, Aung HH, Bell GI, Yuan CS (2005) Anti-diabetic effect of ginsenoside Re in ob/ob mice. Biochem Biophys Acta 1740:319–325 Xu J, Chu Y, Liao B, Xiao S, Yin Q, Bai R, Su H, Dong L, Li X, Qian J, Zhang J, Zhang Y, Zhang X, Wu M, Zhang J, Li G, Zhang L, Chang Z, Zhang Y, Jia Z, Liu Z, Afreh D, Nahurira R, Zhang L, Cheng R, Zhu Y, Zhu G, Rao W, Zhou C, Qiao L, Huang Z, Cheng YC, Chen S (2017) Panax ginseng genome examination for ginsenoside biosynthesis. Gigascience 6:1–15 Zheng Sd, Wu HJ, Dl Wu (2012) Roles and mechanisms of ginseng in protecting heart. Chin J Integr Med 18:548–555

8

Chloroplast Genome Diversity in Panax Genus Vo Ngoc Linh Giang, Woojong Jang, Hyun-Seung Park, and Tae-Jin Yang

Abstract

The chloroplast genome variation between plant species and individuals is especially valuable for studying plant genome diversity. Recently, 45S rDNA and the complete chloroplast genome sequences from seven Panax and relative species have enhanced our understanding of the genetic and molecular basis that can exploit genome evolution, diversity and conservation in the Araliaceae family. In this chapter, we characterize the genetic diversity and present the phylogenetic relationship of Panax and relative species. We also show the 60kbp of chloroplast genome segments which transferred into mitochondrial genomes and remained conserved as extra copies in the mitochondrial genomes that can cause false authentication or confusion. The study provides genomic resources for understanding of evolution in the Panax genus and practical DNA markers suitable for authentication and barcoding of each species.

V. N. L. Giang
W. Jang
H.-S. Park
T.-J. Yang (&) Department of Plant Science, Plant Genomics and Breeding Institute, Research Institute of Agriculture and Life Sciences, College of Agriculture and Life Sciences, Seoul National University, Seoul, Korea e-mail: [email protected]

8.1

Introduction

Chloroplast are photosynthesis organelles playing vital roles in plant physiology, such as the major synthesis and a plethora of metabolites that required for plant development (e.g., vitamins, fatty acids, amino acids, nucleotides and phytohormones) (Bobik and Burch-Smith 2015). They contain independent genomes (chloroplast DNA, cpDNA), which are highly conserved, not subject to Mendelian inheritance and uni-parentally inherited (Birky 1995). In angiosperms, cpDNA occurs in circular form with length ranging from 120 to 160 kb, the cpDNA sequence is arranged in a quadripartite fashion consisting of an 80–90 kb large single copy (LSC), two copies of 20–28 kb inverted repeats (IRs) and a 16–27 kb small single copy (SSC) (Jansen et al. 2005). As technology advances, the next-generation sequencing (NGS) technologies have increased the ability to accomplish chloroplast genetics and genomics astonishingly. Insights gained from the development and application of NGS to chloroplast genome sequences are improved our scientific knowledge for plant genetic structure, genome diversity, phylogenetic and systematic evolutionary analyses (Daniell et al. 2016). Ginseng, Panax species (Araliaceae), is a slow growing perennial herbal medicine plant, containing triterpene glycosides commonly referred to as ginsenosides. Eastern societies widely used ginseng for the treatment or

© Springer Nature Switzerland AG 2021 J. Xu et al. (eds.), The Ginseng Genome, Compendium of Plant Genomes, https://doi.org/10.1007/978-3-030-30347-1_8

95

96

prevention of diseases from thousands of years ago, and over the last three decades, it has gained popularity in western societies like Americas, Canada, and Europe (FM 2009). According to the recent classification, 16–18 species are defined in the Panax genus (Sharma and Pandit 2009), and most species are considered seriously endangered medicinal plants. Among them, five species (Panax ginseng, P. notoginseng, P. vietnamensis P. japonicus and P. quinquefolius) have been cultivated for medicinal purposes in Korea, China, Vietnam, Japan and USA. However, illegal trades happen occasionally that cause economic loss and confusion to consumers. For enforcement of international treaties and national laws in deterring the illegal acquisition of wild endangered species, established approaches for ginseng identification is highly essential. Morphological and histological assays could not authenticate the origin of processed ginseng products such as powders or decoctions. The applications of ginsenosides (secondary metabolites) profiling (Yang et al. 2016) have limited practical use. Ginsenosides accumulation may be variable when derived from different tissues (such as flower buds, berries, leaves, stems and roots) (Shi et al. 2007), cultivars (Lee et al. 2017), environmental conditions (Jiang et al. 2016), storage conditions and manufacturing processes (Nguyen et al. 2017). In general, the cp genome sequences and genes order are highly conserved (Gao et al. 2011). Therefore, variations among related species in cp genomes provide sources of information for systematic study, including genetic diversity and phylogenetic analysis in diverse plants. Recently, the nuclear ribosomal DNA and the whole chloroplast genome sequences were obtained from Panax species and relative genus by our group and also from the global plant genomic research community (Kim et al. 2015, 2017, 2018; Nguyen et al. 2018; Liu et al. 2018). Here, we added some more cp genome sequences and reviewed the genetic diversity intra- and inter-species level in Panax genus. We also show practical DNA markers derived from cp genome which can be applied for species authentication and cultivar identification.

V. N. L. Giang et al.

8.2

Chloroplast Genome Diversity Among P. Ginseng Accessions

To investigate intraspecific cp genome diversity in P. ginseng, a study was undertaken to compare the cp genome sequences of 11 genetic resources (two Korean local landraces and nine registered inbred cultivars in Korea) (Kim et al. 2015). Typically, comparative genome studies for intraspecies variation indicate limited polymorphism in cp genome; however, six SNPs and six InDels were identified from P. ginseng cp genomes (Table 8.2). Among these variations, five were in intergenic regions and seven were derived from genic regions. Six molecular markers were successfully developed based on those variations in cp genome. Some cultivars including cv. ChP, cv. SH, cv. HS and cv. GU were authenticated by combination of developed markers (Kim et al. 2015). The availability of the polymorphisms found in this study are suitable for studying evolutionary genomics, population structure analysis, identification of ginseng cultivars and to improve the ginseng breeding and industry through maintaining the purity of each cultivar. Moreover, an additional genetic diversity in cp genome of P. ginseng could be found through comparative analysis using diverse wild collections (Table 8.1).

8.3

Phylogenetic Relationship of Panax and Relative Species

The cp genomes of twelve species: P. ginseng cv. ChP, P. notoginseng, P. vietnamensis, P. stipuleanatus, P. japonicus, P. quinquefolius, P. trifolius, A. elata, A. undulata, D. morbifera, E. senticosus and E. sessiliflorus were obtained for comparative analysis Panax and relative species genetic diversity (Kim et al. 2017, 2018) (Fig. 8.1). The measure of divergence time and Ks values (Kim et al. 2017) provided the substitution occurring among their cp genomes was approximately 1.0  10−9 per year. The study shows the average performance of Ks values among the Panax population (seven species) was lower (0.0056) than those between Aralia-Panax

Dendropanax

Eleutherococcus

Aralia

86,128

Hwangsook (HS)

18,013

D. morbifera (DM)

86,475

86,603 86,755

E. sessiliflorus (ES)

E. senticosus (ESen)

86,263

P. vietnamensis (PV) 86,029

86,178

P. trifolius (PT)

A. elata (AE)

86,322

P. stipuleanatus (PS)

A. undulata (AU)

86,095 86,116

P. quinquefolius (PQ)

86,199

86,128

Yunpoong (YP) 86,190

18,077

86,128

Sunun (SU)

18,077

18,125

18,153

18,213

18,092

18,111

17,935

18,047

18,174

17,993

18,004

18,077

18,077

86,128 86,128

Sunpoong (SP)

18,077

18,077

18,077

18,077

18,077

18,084

18,077

SSC

Sunone (SO)

86,128

86,129

Gumpoong (GU)

86,200

86,128

Gopoong (GO)

Sunhyang (SH)

86,128

Chunpoong (ChP)

Jakyung (JK)

86,129

Cheongsun (CS)

LSC

25,883

25,930

25,957

26,106

25,923

25,940

25,894

25,887

26,000

26,136

25,988

26,075

26,075

26,075

26,075

26,074

26,075

26,018

26,075

26,075

26,018

26,075

IR

Chloroplast genome

P. japonicus (PJ)

P. ginseng (PG)

Panax

Cultivar

P. notoginseng (PN)

Species (Abbreviated name)

Genus

156,366

156,768

156,730

156,333

156,220

155,993

156,157

156,064

156,088

156,466

156,188

156,355

156,355

156,355

156,355

156,425

156,355

156,241

156,356

156,355

156,248

156,356

Entire

Table 8.1 Summary statistics of Panax and relative species and chloroplast genome assembly

37.99

37.95

37.95

38.1

38.08

38.05

38.08

38.03

38.07

38.07

38.07

38.08

38.08

38.08

38.08

38.06

38.08

38.07

38.08

38.08

38.07

38.08

GC (%)

KR136270

NC_016430

KT153019

NC_022810

KT153023

KP036470

MF100782

KX247147

KM088018

KP036468

KP036469

KM088020

KM067392

KM067391

KM067390

KM067393

KM067389

KM067394

KM067388

KM067387

KM088019

KM067386

GenBank No.

Kim et al. (2017)

Kim et al. (2017), Van Binh Nguyen et al. (2018), Liu et al. (2018)

Kim et al. (2015)

References

8 Chloroplast Genome Diversity in Panax Genus 97

ndhF-rpl32

SNP

rps16 intron

rps16-trnUUG

trnUUC-trnGGU

trnUGC intron

ycf1

rpl32-trnUAG

InDel

InDel

InDel

InDel

InDel

InDel

ccsA

ycf1c

SNP

SNP

c

rpoC1

SNP

115,833

111,304/130,897

105,431/136,936

32,850

7,189

5,473

127,069

117,376

115,466

22,287

21,344

c

7,159

rps16-trnUUG

rpoC2b

SNP

SNP

Nucleotide positiona

Position

Type

(G)11 57  4 72

(G)11 57  3 73

(C)8 13  1

(C)8

A

A

G

G

C

G

YP

13  1

A

A

G

T

C

G

ChP

72

57  4

(G)11

13  1

(C)9

A

G

G

G

T

G

GU

72

57  4

(G)11

13  1

(C)8

A

A

G

G

C

G

GO

72

57  4

(G)11

13  1

(C)8

A

A

G

G

C

G

SO

72

57  4

(G)11

13  1

(C)8

A

A

G

G

C

G

SU

72

57  4

(G)11

13  1

(C)8

A

A

G

G

C

G

SP

Table 8.2 Summary of nucleotide polymorphisms in cp genomes of 11 P. ginseng accessions, after (Kim et al. 2015)

72

57  4

(G)10

59

13  2

(C)8

A

A

T

G

C

T

SH

72

57  4

(G)11

13  1

(C)9

A

A

G

G

C

G

CS

72

57  4

(G)11

13  1

(C)8

A

A

G

G

C

G

JK

72

57  3

(G)11

13  1

(C)8

T

A

G

G

C

G

HS

98 V. N. L. Giang et al.

8

Chloroplast Genome Diversity in Panax Genus

(0.0110) and Eleutherococcus-Dendropanax (0.0080). In addition, KaKs calculator was applied to calculate Ks values for 79 conserved protein-coding sequences. The Ks values of genes in LSC and SSC regions were higher than in IR regions. The average Ka and Ks values in IRs, LSC and SSC regions were low among the Panax relatives and ranged from 0.0011 to 0.0046 and from 0.0022 to 0.0236, respectively. Among the protein-coding genes, atpF, atpE, ycf2 and rps15 are hotspot regions to be explored for development of selection markers in the ten Araliaceae species. The groups of conserved cp genes were identified in the Araliaceae family; one (rpl23) was related to the ribosomal large subunit; and four (psbN, psbF, psaJ and petN) were related to photosynthesis. Understanding the diversity in the cp gene pool will be key to determine the species classification and gain knowledge of evolution in the Apiales order. Apiaceae and Araliaceae have evolved over time from a common ancestor in the Apiales order approximately 60.2 MYA (Shi et al. 2015; Magallón et al. 2015; Court 2000; Tank et al. 2015). Evolutionary and phylogenetic analysis of cp genomes indicated that the Araliaceae family diverged into two monophyletic lineages approximately 8.81–10.59 MYA. Aralia and Panax, two closest-related genera, diverged around 7.97–8.46 MYA, and Dendropanax and Eleutherococcus diverged after that (4.48–5.60 MYA) (Fig. 8.2). The Panax genus had speciation events more recently around 2.89–3.20 MYA which was similar to common evolutionary history of other plants affected by an Asian temperate climate change. This speciation was the important consequence of the Tibetan Plateau uplift; the strong influence caused Asian temperate climate change in the late Cenozoic era (Thompson and Lumaret 1992; Yamane et al. 2003; Chen et al. 2007). The center of origin of Panax was suggested to be in the southwestern China and Himalayas (Wen and Zimmer 1996), and these regions were also reported to have a high rate of polyploidy because of the appearance of diverse species in the widespread alpine environment (Nie et al. 2005). The magnitude of molecular clock

99

estimation depends on the complete cp coding sequences supported the previous studies that P. ginseng arising spontaneously in nature as a result of a recent whole-genome duplication 2.3 MYA (Choi et al. 2014), an allotetraploidization event merging different diploid ancestors triggered by a large Asian temperate climate change. The divergence time between P. ginseng and P. quinquefolius is approximately 0.72–0.87 MYA. In North America, the settlement of P. quinquefolius was suggested by the entailment of P. ginseng seeds transported on drifting sea ice via the Beringia (Bering land bridge) that once connected Siberia and Alaska and disjunction by the shallow Bering Sea 0.9– 2.3 MYA (Choi et al. 2013).

8.4

Distribution of Mitochondrial Plastid DNAs (MTPTs) Across the P. Ginseng Chloroplast Genome

The integration of “alien” intracellular DNA from the chloroplast and nucleus has played a critical role in the increasing size of the mitochondrial genomes (Wang et al. 2007). Recently, the prevalence and importance of plastid-tomitochondrion DNA transfer phenomena in the evolution mechanism of plant is being increasingly recognized. In general, the mitochondrial plastid DNAs (MTPTs) are non-functional based on consistency of a number of frameshift mutations and indels (Sloan and Wu 2014). However, these noncoding sequences may be artifacts coamplified with the target cpDNA sequences in polymerase chain reaction (PCR), leading to erroneous declaring like nuclear mitochondrial DNAs in animal (Park et al. 2020; Song et al. 2008). High throughput and sensitive variation detection techniques like fluorescence based SNP markers are useful for Panax species authentication, but might be susceptible to interference from co-amplified of MTPTs during PCR. For SNP analyses, careful selection of target sequences must be carried out to escape from confusion by the disadvantageous MTPTs. The simple sequence alignment of chloroplast

100

V. N. L. Giang et al.

Fig. 8.1 Gene map of Panax-related species chloroplast genome sequence was created using OGDRAW. Genes transcribed clockwise and counterclockwise are indicated on the outside and inside of the large circle, respectively.

Genes associated with different functional groups are color coded. Four parts of chloroplast genome and GC content are indicated on the middle circle (Kim et al. 2015)

and mitochondrial genomes using the BLAST program can find the MTPT regions easily. Twelve large MTPTs ranging from 2,297 to 8,250 bp had been observed in P. ginseng mitochondrial genome (Nguyen et al. 2018) (Fig. 8.3). In total, 60,331 bp of MTPTs were detected in P. ginseng mitochondrial genome, which corresponds to approximately 38.6% of

chloroplast genome. The SNPs in pure chloroplast genome sequences without homology with the 12 obvious MTPT regions can be considered as possible candidates for species identification. In other words, the chloroplast genomes escaped from the MTPTs considerably expands the resolution to achieve the goal of species unique SNP in seven Panax species.

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Chloroplast Genome Diversity in Panax Genus

101

Fig. 8.2 Phylogenetic tree and divergence time of 12 Araliaceae species. Phylogenetic trees were generated based on cp protein-coding sequences. Numbers indicate average divergence time based on Ks values, and median

divergence time using the BEAST program (Suchard et al. 2018) with 95% highest posterior density (adopted and modified from Kim et al. 2015, 2018)

Fig. 8.3 Schematic representation of twelve large mitochondrial plastid DNAs (MTPTs) distribution throughout the chloroplast and mitochondrial genomes in P. ginseng. Gray quadrilaterals are connected the start and end positions of high similarity sequences between chloroplast

and mitochondrial genomes. Colored boxes show conserved chloroplast and mitochondrial genes, classified based on product function (https://chlorobox.mpimpgolm.mpg.de/OGDraw.html) (adpoped and modified from Nguyen et al. 2018)

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V. N. L. Giang et al.

Fig. 8.4 Circos plot distribution of 1913 unique SNPs. (A–G) Distribution of unique SNPs detected on P. trifolius (PT), P. stipuleanatus (PS), P. vietnamensis (PV), P. japonicus (PJ), P. notoginseng (PN), P. quinquefolius (PQ) and P. ginseng (PG) chloroplast genomes,

8.5

Development of Cp Genome DNA Markers to Authenticate Panax Species

Five Panax species chloroplast genome sequences (P.ginseng cv. ChP and cv. YP, P. notoginseng, P. vietnamensis, P. japonicus and P. quinquefolius) were compared by using MAFFT (http://mafft.cbrc.jp/alignment/server/),

respectively. The unique SNPs are represented by lines in different colors for each species. (H) Plot of coding sequences (CDS). (J) Pie charts representing the relative number of unique SNPs resulting from non mitochondrial CDS (54)/total unique SNPs for each Panax species

MEGA6 (Tamura et al. 2013) and mVISTA (http://genome.lbl.gov/vista/mvista/submit.shtml) programs. Based on comparative analysis, the 14 InDel markers were developed and validated to authenticate these Panax species (Nguyen et al. 2017). These markers effectively classify the five Panax species, either in combination or independently. Up to date, the SNPs are readily amenable to use as the polymorphic markers to validate the

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Fig. 8.5 Validation of 18 dCAPS markers derived from CDS SNP regions of seven Panax chloroplast genomes. The 18 denoted dCAPS markers, Pgdm1-3, Pqdm4-6, Pndm7-9, Pjdm10-11, Pvdm12-13, Psdm14-16, and Ptdm17 and 18 are unique for P. ginseng (Pg),

P. quinquefolius (Pq), P. notoginseng (Pn), P. japonicus (Pj), P. vietnamensis (Pv), P. stipuleanatus (Ps), and P. trifolius (Pt), respectively. M, 100-bp DNA ladder (adopted and modified from Nguyen et al. 2018)

variation between individuals. The complete cp genome sequencing is a more comprehensive approach to identify genome wide SNPs. SNPderived markers were developed from the complete cp genomes of seven Panax species for authentication of each species (P.ginseng, P. notoginseng, P. vietnamensis, P. stipuleanatus, P. japonicus, P. quinquefolius and P. trifolius). Prior to use, 1,783 SNP sites in the whole chloroplast genome sequences and 1,128 sites in protein-coding regions were identified and validated (Nguyen et al. 2018). P. ginseng and P. quinquefolius (two Panax tetraploid species) had a lower number of SNPs, and P. trifolius had the highest SNPs from other species in both CDSs and whole chloroplast genome (Fig. 8.4). The MTPT regions were not included for marker development to avoid co-amplification with the original chloroplast genome targets. The target regions were mainly focused on species-specific SNPs in the chloroplast CDSs, which can

distinguish the one from other six species. In total, 18 derived cleaved amplified polymorphic sequence markers (dCAPS) were developed to discriminate seven Panax species, each of which had least two unique markers (Fig. 8.5). They showed clear authentication results from multiple accessions of the seven Panax species.

8.6

Conclusion and Perspective

Various molecular authentication of Panax and the relatives were subjected to many adversity, including the experimental difficulty, multiple rounds of PCR reactions (Ha et al. 2002; Shim et al. 2003; Choi et al. 2008, 2011; Lee et al. 2011), and the risk of contamination from MTPTs that were not associated with the target genome but are artifacts of the PCR condition. However, the current advances of high-throughput sequencing methods provide high-quality datasets

104

of complete chloroplast genomes. The information of complete chloroplast genome has contributed to improve our knowledge in the field of genetic diversity and evolution of Panax and their relative species. In intraspecific level, the highly conserved chloroplast genomes of Panax ginseng showed a few variations among ginseng accessions. Abundant variations were identified at inter-specific level in Panax genus and useful DNA markers were successfully developed based on comparative analysis. Furthermore, the phylogenetic relationship using Panax and relative species (Kim et al. 2015, 2018) provides strong evidence of the evolutionary hypotheses which suggest that climate change in ice age might lead to allotetraploidization (a whole-genome duplication via inter-species hybridization between different diploid ancestors) in P. ginseng. The species-specific DNA markers derived from cp genome diversity analysis will be the useful tools for authentication of Panax species and the cultivars, in order to promote and protect the ginseng industry.

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V. N. L. Giang et al. Kim HH, Yang T-J (2014) Major repeat components covering one-third of the ginseng (Panax ginseng C. A. Meyer) genome and evidence for allotetraploidy. The Plant J 77(6):906–916. https://doi.org/10.1111/ tpj.12441 Choi Y-E, Ahn CH, Kim B-B, Yoon E-S (2008) Development of Species Specific AFLP-Derived SCAR marker for authentication of panax japonicus C.A. Meyer. Biol Pharm Bull 31(1):135–138. https:// doi.org/10.1248/bpb.31.135 Court WE (2000) Ginseng: the genus Panax. CRC Press Daniell H, Lin C-S, Yu M, Chang W-J (2016) Chloroplast genomes: diversity, evolution, and applications in genetic engineering. Genome Biol 17(1):134. https:// doi.org/10.1186/s13059-016-1004-2 FM P (2009) Panax ginseng. Monograph. Altern Med Rev 14 (2):172–176 Gao L, Zhou Y, Wang Z-W, Su Y-J, Wang T (2011) Evolution of the rpoB-psbZ region in fern plastid genomes: notable structural rearrangements and highly variable intergenic spacers. BMC Plant Biol 11:64 Ha W, Shaw P, Liu J, Yau FC, Wang J (2002) Authentication of panax ginseng and panax quinquefolius using amplified fragment length polymorphism (AFLP) and directed amplification of minisatellite region DNA (DAMD). J Agri Food Chem 20 (7):1871–1875. https://doi.org/10.1021/jf011365l Jansen RK, Raubeson LA, Boore JL, Depamphilis CW, Chumley TW, Haberle RC, Wyman SK, Alverson AJ, Peery R, Herman SJ (2005) Methods for obtaining and analyzing whole chloroplast genome sequences. In: Methods in enzymology, vol 395. Elsevier, pp 348– 384. doi:https://doi.org/10.1016/S0076-6879(05) 95020-9 Jiang M, Liu J, Quan X, Quan L, Wu S (2016) Different chilling stresses stimulated the accumulation of different types of ginsenosides in Panax ginseng cells. Acta Physiol Plant 38(8):210. https://doi.org/10.1007/ s11738-016-2210-y Kim K, Lee S-C, Lee J, Lee HO, Joh HJ, Kim N-H, Park H-S, Yang T-J (2015) Comprehensive survey of genetic diversity in chloroplast genomes and 45S nrDNAs within Panax ginseng species. PLoS ONE 10 (6):e0117159. https://doi.org/10.1371/journal.pone. 0117159 Kim K, Nguyen VB, Dong J, Wang Y, Park JY, Lee S-C, Yang T-J (2017) Evolution of the Araliaceae family inferred from complete chloroplast genomes and 45S nrDNAs of 10 Panax-related species. Sci Rep 7 (1):4917. https://doi.org/10.1038/s41598-017-05218-y Kim N-H, Jayakodi M, Lee S-C, Choi B-S, Jang W, Lee J, Kim HH, Waminal NE, Lakshmanan M, van Nguyen B, Lee YS, Park H-S, Koo HJ, Park JY, Perumal S, Joh HJ, Lee H, Kim J, Kim IS, Kim K, Koduru L, Kang KB, Sung SH, Yu Y, Park DS, Choi D, Seo E, Kim S, Kim Y-C, Hyun DY, Park Y-I, Kim C, Lee T-H, Kim HU, Soh MS, Lee Y, In JG, Kim H-S, Kim Y-M, Yang D-C, Wing RA, Lee D-Y, Paterson AH, Yang T-J (2018) Genome and evolution of the shade-requiring medicinal herb Panax ginseng.

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Plant Biotechnol J 16(11):1904–1917. https://doi.org/ 10.1111/pbi.12926 Lee J-W, Kim Y-C, Jo I-H, Seo A, Lee J-H, Kim O-T, Hyun D-Y, Cha S-W, Bang K-H, Cho J-H (2011) Development of an ISSR-derived SCAR marker in Korean ginseng cultivars (Panax ginseng C. A. Meyer). J Ginseng Res 35(1):52–59 Lee YS, Park H-S, Lee D-K, Jayakodi M, Kim N-H, Koo HJ, Lee S-C, Kim YJ, Kwon SW, Yang T-J (2017) Integrated transcriptomic and metabolomic analysis of five panax ginseng cultivars reveals the dynamics of ginsenoside biosynthesis. Frontiers Plant Sci 8(1048). https://doi.org/10.3389/fpls.2017.01048 Liu C, Yang Z, Yang L, Yang J, Ji Y (2018) The complete plastome of Panax stipuleanatus: Comparative and phylogenetic analyses of the genus Panax (Araliaceae). Plant Divers 40(6):265–276. https://doi.org/10. 1016/j.pld.2018.11.001 Magallón S, Gómez-Acevedo S, Sánchez-Reyes LL, Hernández-Hernández T (2015) A metacalibrated time-tree documents the early rise of flowering plant phylogenetic diversity. New Phytol 207(2):437–453. https://doi.org/10.1111/nph.13264 Nguyen VB, Giang VNL, Waminal NE, Park H-S, Kim N-H, Jang W, Lee J, Yang T-J (2018) Comprehensive comparative analysis of chloroplast genomes from seven Panax species and development of an authentication system based on species-unique single nucleotide polymorphism markers. J Ginseng Res. doi:https://doi.org/10.1016/j.jgr.2018.06.003 Nguyen VB, Park H-S, Lee S-C, Lee J, Park JY, Yang T-J (2017) Authentication markers for five major Panax species developed via comparative analysis of complete chloroplast genome sequences. J Agric Food Chem 65(30):6298–6306. https://doi.org/10.1021/acs. jafc.7b00925 Nie Z-L, Wen J, Gu Z-J, Boufford DE, Sun H (2005) Polyploidy in the flora of the hengduan mountains hotspot, southwestern China. Ann Mo Bot Gard, 275–306 Park HS, Jayakodi M, Lee SH, Jeon JH, Lee HO, Park JY, Moon BC, Kim CK, Wing RA, Newmaster SG, Kim JY, Yang T-J (2020) Mitochondrial plastid DNA can cause DNA barcoding paradox in plants. Scientific reports 10, 6112 Sharma SK, Pandit MK (2009) A new species of Panax L. (Araliaceae) from Sikkim Himalaya, India. Syst Bot 34:434–438. https://doi.org/10.1600/ 036364409788606235 Shi F-X, Li M-R, Li Y-L, Jiang P, Zhang C, Pan Y-Z, Liu B, Xiao H-X, Li L-F (2015) The impacts of polyploidy, geographic and ecological isolations on the diversification of Panax (Araliaceae). BMC Plant Biol 15(1):297. https://doi.org/10.1186/s12870-015-0669-0 Shi W, Wang Y, Li J, Zhang H, Ding L (2007) Investigation of ginsenosides in different parts and ages of Panax ginseng. Food Chem 102(3):664–668

105 Shim Y-H, Choi J-H, Park C-D, Lim C-J, Cho J-H, Kim H-J (2003) Molecular differentiation of Panax species by RAPD analysis. Arch Pharmacal Res 26(8):601. https://doi.org/10.1007/bf02976708 Sloan DB, Wu Z (2014) History of plastid DNA insertions reveals weak deletion and at mutation biases in angiosperm mitochondrial genomes. Genome Biol Evol 6(12):3210–3221. https://doi.org/10.1093/ gbe/evu253 Song H, Buhay JE, Whiting MF, Crandall KA (2008) Many species in one: DNA barcoding overestimates the number of species when nuclear mitochondrial pseudogenes are coamplified. Proc Natl Acad Sci 105 (36):13486–13491. https://doi.org/10.1073/pnas. 0803076105 Suchard MA, Baele G, Lemey P, Ayres DL, Drummond AJ, Rambaut A (2018) Bayesian phylogenetic and phylodynamic data integration using BEAST 1.10. Virus Evol 4(1). https://doi.org/10.1093/ve/ vey016 Tamura K, Filipski A, Peterson D, Stecher G, Kumar S (2013) MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30(12):2725– 2729. https://doi.org/10.1093/molbev/mst197 Tank DC, Eastman JM, Pennell MW, Soltis PS, Soltis DE, Hinchliff CE, Brown JW, Sessa EB, Harmon LJ (2015) Nested radiations and the pulse of angiosperm diversification: increased diversification rates often follow whole genome duplications. New Phytol 207(2):454–467. https://doi.org/10.1111/ nph.13491 Thompson JD, Lumaret R (1992) The evolutionary dynamics of polyploid plants: origins, establishment and persistence. Trends Ecol Evol 7(9):302–307. https://doi.org/10.1016/0169-5347(92)90228-4 Wang D, Chaw S-M, Wu C-S, Shih AC-C, Wu Y-W, Wang Y-N (2007) Transfer of chloroplast genomic DNA to mitochondrial genome occurred at least 300 MYA. Mol Biol Evol 24(9):2040–2048. https://doi. org/10.1093/molbev/msm133 Wen J, Zimmer EA (1996) Phylogeny and biogeography of Panax L. (the Ginseng Genus, Araliaceae): inferences from ITS sequences of nuclear ribosomal DNA. Mol Phylogenet Evol 6(2):167–177. doi:https://doi. org/10.1006/mpev.1996.0069 Yamane K, Yasui Y, Ohnishi O (2003) Intraspecific cpDNA variations of diploid and tetraploid perennial buckwheat, Fagopyrum cymosum (Polygonaceae). Am J Bot 90(3):339–346. https://doi.org/10.3732/ajb. 90.3.339 Yang W, Qiao X, Li K, Fan J, Bo T, Guo D-a, Ye M (2016) Identification and differentiation of Panax ginseng, Panax quinquefolium, and Panax notoginseng by monitoring multiple diagnostic chemical markers. Acta Pharm Sinica B 6(6):568–575

9

An Update to the Transcriptome Sequencing for the Genus Panax Deok-Chun Yang

Abstract

Transcriptome sequencing for the non-model plants is currently becoming a preliminary data that assists the researchers in getting an initial glimpse of the secondary metabolites in plants. At present, it is becoming feasible for an extensive ginseng research community because of the advancements in nextgeneration sequencing technologies. In particular, the plants belonging to genus Panax are remarkably treated as superior (king) to other herbal plants in ancient medicines (Oriental and Chinese), since it is believed to be a cure-for-all the diseases. Besides, the secondary metabolite “ginsenoside” is considered as a major therapeutic component consisting of * 150(approx.) isoforms. Initially, the transcriptomes were sequenced using the Sanger method in 2003 to mainly characterize genes involved in ginsenoside biosynthesis pathway. Later, the researchers have focused on different issues of the ginseng plant

D.-C. Yang (&) Graduate School of Biotechnology, College of Life Science, Kyung Hee University, Yongin-Si, Gyeonggi-Do 17104, Republic of Korea e-mail: [email protected] Department of Oriental Medicinal Biotechnology, College of Life Science, Kyung Hee University, Yongin-Si, Gyeonggi-Do 17104, Republic of Korea

development, problems related to its agriculture and physiological process more broadly using high-throughput sequencing technologies for more than a decade now. This chapter has mainly focused on the transcriptome sequences and their technological advancements for the species Panax, including on how it has helped the researchers to annotate the ginseng genome, respectively.

9.1

Introduction

It is more remarkable to decode the genetics of medicinal plants to acknowledge their phytochemical constituents. The knowledge acquired from it is more beneficial for the pharmaceutical industries to develop a standard natural drug. Till date, the knowledge acquired from the past medicinal history has educated us the significance of medicinal plants and how they can facilitate humans in retaining their health from several diseases (Petrovska 2012). The contemporary science has acknowledged the derived knowledge and impelled us from a logical search to an empirical search. Hence, the term “phytochemical genomics,” that integrates multi-omics systematically such as genomics, transcriptomics, proteomics and metabolomics, has been originated from those highly emerging empirical searches (Saito 2013; Abbai et al. 2017). This systematic integration helps researchers to discern the understanding of the biosynthesis of

© Springer Nature Switzerland AG 2021 J. Xu et al. (eds.), The Ginseng Genome, Compendium of Plant Genomes, https://doi.org/10.1007/978-3-030-30347-1_9

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plant-specific phytochemicals. The nextgeneration sequencing technologies (NGS) and LC/GC-MS/MS-based metabolomics are made more accessible to larger plant communities as a consequence of these omics sequencing technologies. Hence, it was more beneficial in implementing the concept of “gene-tometabolite” (Yin and Xu 2014; Goodwin et al. 2016). On the other hand, the statistical and computational models have also helped the scientific community to integrate multiple datasets for deriving a testable hypothesis (Krumsiek et al. 2016). Ginseng is a non-model plant. It is widely known as an adaptogen that belongs to the genus Panax from the family Araliaceae, and it consists of fifteen species and seven sub-species. The word “Panax” is derived from the Greek word “Panacea,” which means a heal for all the diseases. Moreover, the unique life cycle (Fig. 9.1) and human-shaped root of the Panax ginseng (Fig. 9.2) are more striking toward its identified medicinal properties. In addition, it has a unique correlative factor that its genome size is homologous to the human genome. Among all the fifteen species of the genus Panax, only four species, namely P. ginseng (Korean ginseng), P. notoginseng (Chinese ginseng), P. quinquefolius (American ginseng), and P. japonicus (Japanese ginseng), have been studied widely (Fig. 9.2) for its biologically active ingredients (Yang et al. 2014) as well as their pharmacological properties. “Ginsenoside” is the most valuable active bio-ingredient that is present in the ginseng, which has numerous benefits on various human diseases and its activities. Also, ginsenosides have more isoforms, and hence, it is used disparately to treat multiple conditions, and these individual specifications are reviewed by various people as follows, for instance, cancer (Ahuja et al. 2018), obesity (Zhang et al. 2017b), cardiovascular (Kim 2018), inflammation (Kim et al. 2017), neurodegenerative diseases (Kim et al. 2018a), diabetes (Bai et al. 2018), and ginsenosides-signaling molecular interactions (Mohanan et al. 2018). To emphasize, considering its botanical nature ginseng has an exceptional complexity and different types of roots

D.-C. Yang

which possess a great interest in the market for its age and the cultivation type (wild roots and cultivated roots). Thus, the age and the type of root have a direct impact on its price on the market. In the genome of an organism, the transcriptome is a total representation of its RNAs. The basic goal of transcriptomics is to understand the functions of proteins/genes of an organism. It is mainly to know which group of genes is highly responsible for making them unique from one another. Currently, the RNA-sequencing (RNASeq) is the cutting edge technology used in the field of transcriptomics to understand how they are expressed in an organism. Also, RNA-Seq technology has led a way to acquire basic knowledge on the information about a gene or protein for all non-model plants. Furthermore, the long-read sequencing technologies have also helped in discovering the genome-wide fulllength transcriptome along with their isoforms for any non-model plants (An et al. 2018). Simultaneously, to analyze and integrate multiple technologies, computational methods were developed as it have also helped the plant research community to outreach their goal remarkably using the transcriptomic data(Conesa et al. 2016). In medicinal plants, RNA-Seq plays an important role in understanding the biosynthesis of their distinctive metabolites. Similarly, the RNA-Sequencing projects in the genus Panax were primarily applied to acknowledge the ginsenoside biosynthesis mechanism from different plant organs and stages (Fig. 9.3). However, RNA-seq is not only available to specifically investigate on how ginsenosides are biosynthesized, but also used highly for various other applications in the Panax species according to other studies reported in the literature.

9.1.1 Panax Ginseng In 2003, the transcriptome sequencing for P. ginseng was initiated using Sanger sequencing by constructing the expressed sequence tags (ESTs) from various parts of the plant such as the roots (i.e. different age and types), leaves, and

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An Update to the Transcriptome Sequencing for the Genus Panax

109

Fig. 9.1 Illustration of P. ginseng developmental life cycle. Adapted from (Boopathi et al. 2019)

Fig. 9.2 Roots of various Panax species. Adapted from (Boopathi et al. 2019)

flower buds. Later, the same project was extended, in which the roots were sequenced after the treatment of methyl jasmonic acid (MeJA). Further, it was also assessed for differential expression patterns to observe the terpenoid backbone gene biosynthesis (Jung et al. 2003; Choi et al. 2005; Kim et al. 2006; Sathiyamoorthy et al. 2010a, b; Sathiyamoorthy et al. 2011). The 454pyrosequencing technology was used for tissuespecific and hormone treatments to enhance the throughput of the P. ginseng transcriptome from the year 2011 to 2014. The coverage of genes was still insufficient even though those transcriptomic datasets from 454-pyrosequencing technology were similar to the datasets prepared from Sanger method. (Chen et al. 2011; Li et al. 2013; Subramaniyam et al. 2014). Hence, from the year 2014 to 2016, a different nextgeneration short-read sequencing technology (Illumina) was employed to sequence a largescale transcriptome to improve the throughput and coverage of the genes in a cost-effective basis (Jayakodi et al. 2014; Cao et al. 2015;

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D.-C. Yang

Fig. 9.3 Illustration of the various sequencing technologies that were used to achieve the different levels of the transcriptome

Jayakodi et al. 2015; Wang et al. 2015; Wu et al. 2015; Gao et al. 2016; Liu et al. 2016a, b). The short-read sequencing technology was a wellstructured method for P. ginseng, but still this method was not enough to obtain their full-length transcripts, because the genome of P. ginseng is composed of *75% of repeats. For this reason, a long-read single-molecule real-time (SMRT) method was utilized for various tissues to defeat those difficulties consisting of higher repeats (Jo et al. 2017). Thus, so far, totally sixteen transcriptome projects were performed from the year 2003 to 2018 using the denovo analysis (Table 9.1). From all the available studies regarding transcriptomics so far, it is concluded that they were conducted with just the three following strategies: (1) to analyze the content of the ginsenosides that vary upon different age and cultivation types of Panax roots; (2) to study the tissue-specific and cultivar-specific transcripts/ genes; (3) to learn plant responses with different environmental stresses (Table 9.1). Discrete and continuous are the two types of bioinformatics analytical strategies used to prepare every single transcriptomic datasets (Table 9.1). Out of all the transcriptomic studies, only two of them were produced as continuous series. It is to acknowledge the mechanisms of environmental stress in P. ginseng. For instance, it is important to know the reaction of P. ginseng

root to benzoic acid while it is subjected to autotoxicity (Wu et al. 2015). Additionally, it is significant to understand its responses to root-rot diseases caused from Cylindrocarpon destructants (Gao et al. 2016), whereas all other transcriptomic studies were developed into a discrete model. Transcriptomic projects were highly aimed toward the denovo method as more perceptions of it are based on the final-result explications. Major concept on almost all the studies was highly focusing on the understanding of ginsenoside biosynthesis enzymes. The expression patterns among various tissues types/age groups of roots are also highly concentrated. Recently, due to two distinct versions of the draft genome (chunpoong and yunpoong), there will be a reduction on those perceptions for this specific species. Chunpoong draft is reproduced more exactly with co-expression networks, out of the two drafts. Eventually, an orderly and userfriendly database with the largest number of transcriptome libraries has been donated to the public (Jayakodi et al. 2018a). Concluding the era of the P. ginseng denovo transcriptome, its next aspect will be on the reference transcriptome. It is expected to mine new genes along with the functional annotations of the foresee genes. Hence, focusing that, out of those two cultivars (chunpoong and yunpoong) the initial study

Type of sequencing

ESTSanger

ESTSanger

ESTSanger

ESTSanger

ESTSanger

454-PyroSeq

454-PyroSeq

454-PyroSeq

Illumina

Illumina

Illumina

Illumina

S. No

1

2

3

4

5

6

7

8

9

10

11

12

Pg

Pg

Pg

Pg

Pg

Pg

Pg

Pg

Pg

Pg

Pg

Pg

Species

Damaya

Damaya

Damaya

Chunpoong & Cheongsun

NA

NA

NA

NA

NA

NA

NA

NA

Cultivars

19

18

2

2

5

4

1

2

3

1

1

5

No. of. Samples

Table 9.1 Transcriptome, adapted from: (Boopathi et al. 2019)

R (Fiber, Leg, Epiderm, Cortex, Arm), Rh, S, L (Peduncle, Blade, Pedicel), F (Peduncle,

R, S, L (Benzoic acid stress)

MeJA treated AR (Control, Treated)

AR

MeJA treated AR (Control, 2 h,6 h,12 h, and 24 h)

R, S, L, F (4 Y)

R (11 Y)

Embryonic callus, F. buds

HR, R (14 Y, 4 Y)

L (4Y)

HR treated with 10 uM MeJA

Rh(4Y), Sh (In vitro culture), R(4Y), Sh (3 weeks old seedling), S (Green color stage)

Samples

D

C

C

D

C

D

D

D

D

D

D

D

Type

DE

DE

DE

DE

DE

DE

DE

DE

DE

DE

DE

DE

Project Type

248,993

72,732

71,095

63,243

534,324

2,423,076

217,529

6,226

6,757

2,896

3,134

11,636

No. of. Transcripts

2015

2015

2015

2014

2014

2013

2011

2011

2010

2006

2005

2003

Year

An Update to the Transcriptome Sequencing for the Genus Panax (continued)

SRP066368

SRP049125

NA

SRA061905

SRP039367

SRP015263

SRX017443

NA

NA

NA

NA

NA

GenBank Accessions

9 111

Type of sequencing

Illumina

Illumina

Illumina

PacBio

454-PyroSeq

Illumina

Illumina

Illumina

454 Seq

454-PyroSeq

ESTSanger

S. No

13

14

15

16

17

18

19

20

21

22

23

Table 9.1 (continued)

Pq

Pq

Pq

Pq

Pn

Pn

Pn

Pg

Pg

Pg

Pg

Species

NA

NA

NA

NA

NA

NA

NA

NA

Damaya

NA

Chunpoong

Cultivars

4

2

1

7

4

3

1

4

6

1

4 (3 Replicates)

No. of. Samples

F, L, R, S-4Y

R, L

R- 3Y & 4Y

F(Ovule), S, E, endosperm, R, S, and L

Seedlings with Arsenic treatment

L, R, F (3-Y Plant)

R (4-Y)

F, L, S and R (4-Y)

3-Y R inoculated with Cylindrocarpon destructans (0 to 12 days)

R (Leaf Expansion Period)

Whole R (1Y), R- 6Y (Main body, Lateral rhizome)

Pedicel, Flesh), S and R (5Y, 12Y, 18 Y, & 25 Y)

Samples

D

D

C

C

D

D

D

D

C

D

D

Type

DE

DE

DE /TR

DE /TR

DE

DE

DE

DE

DE

DE

DE

Project Type

6,678

209,747

41,623

54,292

81,575

107,340

30,852

135,317

73,335

161,176

55,949

No. of. Transcripts

2010

2010

2013

2015

2016

2015

2011

2017

2016

2016

2015

Year

GR870658GR875376 (continued)

NA

SRX247045, SRX247043, SRX247042, SRX247040, and SRX247037- 39,

SRR1586196

NA

PRJNA228978

SRX017444

SUB2796783

SRR1639601

E–MTAB–974

SRR1648321 * 380

GenBank Accessions

112 D.-C. Yang

454-PyroSeq

Illumina

Illumina

Illumina

ESTSanger

24

25

26

27

28

Ps

Pj

Pj

Pv

Pq

Species

NA

NA

NA

NA

Cheonryang/Yunpoong

Cultivars

3

1

5

1

1

No. of. Samples

L, S, and Rh

Rh

F, L, sec R, Rh_Y, Rh_O

R

R

Samples

D

D

D

D

D

Type

TR

DE/TR

TR

TR

DE/TR

Project Type

234

553,294

135,235

14,703,210

308,313

No. of. Transcripts

2016

2015

2016

2015

2014

Year

JZ822732 to JZ822971

SRP06294

GSE78893/E453242-0003-3-C4

NA

GenBank Accessions

Note Pg: P. ginseng, Pn: P. notoginseng, Pq: P. quinquefolium, Pv: P. vietnamensis, Pj: P. japonicus, Ps: P. sokpayensis L: Leaf, S: Stem, Sh: Shoot, R: Root, Rh: Rhizome, E: Embryo, F: Flower, Adventitious Roots: AR, Hairy roots: HR. MeJA: Methyl jasmonic acid. D: Discrete, C: Continuous, Seq: Sequencing, Tech: Technology, Y: Year(s), DE: Denovo, TR: Transcriptome

Type of sequencing

S. No

Table 9.1 (continued)

9 An Update to the Transcriptome Sequencing for the Genus Panax 113

114

has used the chunpoong draft for predicting the heat stress responsive genes. As a result, it has given a hypotheses that, the CAB, FAD, and WRKY genes will be an authentic candidate for future characterizations (Jayakodi et al. 2018b). Some of the additional benefits of utilizing this reference genome is to help scientists arrange their experimental outputs in a genome centric manner and to thoroughly characterize various problems in P. ginseng.

9.1.2 Panax Notoginseng The 454-pyrosequencer was used to initiate the transcriptome of P. notoginseng in 2011. It is to find the genes behind ginsenoside biosynthesis as well as their simple sequence repeat (SSR) markers (Luo et al. 2011). So far, three transcriptome studies along with the previous one, i.e. the research that was performed using Illumina sequencer, have been conducted. It was reported that it was initiated mainly to study the alkaloid and ginsenoside biosynthesis from different plant organs (Liu et al. 2015). And another study has investigated the environmental toxicity of the plant, and it was to discover on how it regulates the ginsenosides and flavonoid responses from the arsenic heavy metal stress (Liu et al. 2016a, b). Besides, the organ-specific transcriptome for this Panax species was also generated from three different genomic studies, respectively (Chen et al. 2017; Zhang et al. 2017a; Fan et al. 2018). P. notoginseng is dissimilar to P. ginseng as it highly focusses on its different secondary metabolites, such as alkaloids and flavonoids; hence, it has fewer studies related to genome-wide expression analysis.

9.1.3 Panax Quinquefolius In 2010, the transcriptome for P. quinquefolius was started through establishing ESTs and then sequenced using the Sanger sequencer for their different organs (Wu et al. 2010). Simultaneously, to study its ginsenoside biosynthesis extensively by the elicitation of MeJA, the

D.-C. Yang

transcriptome of the root was also sequenced in the same year using with 454-pyrosequencer (Sun et al. 2010). Following this, three more work was prepared to know the different molecular mechanisms of P. quinquefolius, i.e. (1) to know the ginsenoside biosynthesis in different growth stages (Wu et al. 2013); (2) seed dormancy mechanism (Qi et al. 2015); and (3) ginsenosides biosynthesis transcripts expression after the MeJA treatment at different time points (Wang et al. 2016). In between, another study has assessed the single nucleotide polymorphism (SNP) between P. ginseng and P. quinquefolius. To create continuous datasets rather than the discrete samples, at last the hithroughput transcriptomic studies for P. quinquefolius was developed. To construct the coexpression networks for P. quinquefolius, those continuous datasets are an additional development and a vital dataset that could help in characterizing the other genes accountable for ginsenoside biosynthesis. Like any other species, the reference genome is not available for P. quinquefolius, but still, the transcriptome dataset generated so far may aid the researchers to conduct a structured bioinformatics analysis to reveal the numerous theories on the ginsenoside biosynthesis.

9.1.4 miRNAs Non-coding RNAs (21–24 bases) having various regulatory roles specifically in the posttranscriptional modifications (Borges and Martienssen 2015) are named as miRNAs. In plants, the miRNAs are a part of the of 3’-prime, 2’-Omethylation process, but, in animals the same action will take place in the 5’-prime. The molecular signatures that are from the machines of DICER and ARGONAUTE proteins are viewed as the features for establishing computational procedures of miRNA predictions in plants as well as animals (Bilichak et al. 2017; Morgado and Johannes 2017). These computational techniques combined with deep sequencing technology are utilized to profile miRNAs in the Panax species. The first profiling of miRNAs

9

An Update to the Transcriptome Sequencing for the Genus Panax

were from the 4-5 years old plant tissues of P. ginseng during the year 2012 and 2013 (Wu et al. 2012; Mathiyalagan et al. 2013). This study has very few miRNAs as a result (i.e. 73 conserved and 28 non-conserved and 69 conserved miRNAs) comparing to the other plants. The mature tissues are not good for profiling the miRNA, and hence, the younger tissues are mostly suggested. It is because the organogenesis takes place highly in the younger tissues comparing to the matured ones. Apart from the previous study, one more article has also been reported with very less miRNAs revealing that it varies among distinct age groups of P. ginseng roots (1–3 years old) (Wei et al. 2015). The role of an individual miRNA (i.e. miR156) was discovered being a vital regulator for different root sizes in P. notoginseng (Zheng et al. 2017). Further, the miR171 is crucial for pentatricopeptide repeats proteins as it has an important regulatory role. It also has a required function in achieving the degradome sequencing (Chen et al. 2018). The datasets from the study of Mathiyalagan et al. were finally employed to interpret the miRNAs in P. ginseng genome (Kim et al. 2018b).

9.1.5 Other Panax Species There are fifteen species and six sub-species in the genus Panax (as per the NCBI Taxonomy classifications). Only seven species among them have a transcriptome for acknowledging the theory behind ginsenoside biosynthesis, and the other four from the above-mentioned seven species are namely P. sokpayensis, P. vietnamensis, P. zingiberensis, and P. japonicas (Fig. 9.2). The P. sokpayensis has been reported recently from Sikkim located in the Indian Himalayan valley. Due to the suppression subtractive hybridization (SSH) and Sanger sequencing techniques, the minimal ESTs were generated for this species. Sanger sequencing and the SSH methods were used to assess the differentially expressed transcripts among the

115

rhizomes and the leaves of P. sokpayensis (Gurung et al. 2016). It was then included to the Panax family as an important dataset. In Japan, the P. japonicus is very famous since it is used as a substitute for P. ginseng from the ancient times. The oleanane-type saponins are highly present in this particular species comparing to other Panax species (Rai et al. 2016). For P. japonicus, a transcriptome data using Illumina was generated from five individual tissues. Also, the transcripts behind the ginsenoside biosynthesis, i.e. the cytochrome and glycosyltransferase, were also discovered for P. japonicus (Rai et al. 2016). Likewise, a different set of transcriptome for this species’ s rhizome was sequenced in China (Zhang et al. 2015b). The P. vietnamensis, known as Vietnam ginseng is abundant in ocotillol-type saponins such as majonoside R2. Illumina was used to generate the root transcriptome of this species, and further, it was also evaluated for the ginsenoside biosynthesis pathway (Zhang et al. 2015a). South China is native to one of the near extinct Panax species named P. zingiberensis which is rich in oleanane and dammarane-type ginsenosides. To analyze the ginsenoside biosynthesis pathway, transcriptomes from these plant tissues were generated and utilized (Tang et al. 2018). In general, comparing to other research components, almost every transcriptomic projects for all the above-mentioned species were carried particularly focusing on revealing and understanding the genes responsible for the biosynthesis of novel and species-specific saponins.

9.2

Conclusion and Perspective

In the future, the ginseng transcriptome can be utilized for overcoming the following issues: (a) As ginseng is a shade-requiring plant, it requires higher cultivation cost and, hence, it can be widely reduced by modifying it genetically to be cultivated in open sunlight. In addition, (b) it could also aid in producing a high-yield

116

ginsenoside variety through the genome assisted breeding schemas, (c) further it can also be used for transferring the ginsenoside biosynthesis system to the microbial system as a whole for the production of a specific type of ginsenoside (Quan et al. 2012), and d) another major problem with ginseng is its longer developmental life cycle (DLC-4 years), and it can be resolved by producing an adventitious root that can synthesis higher amount of ginsenosides in a shorter period of time, which may reduce the DLC of ginseng from four years to one year or lesser. Likewise, other various related complexities can also be resolved using the ginseng genetics with a constant effort. To achieve all the above-mentioned benefits, firstly, the complete ginsenoside pathway should be characterized from the available genome. Even though a larger transcriptome dataset is available currently, still, it is not sufficient enough to construct a significant mathematical network model to elucidate the various molecular mechanisms with respect to the ginseng growth and stress environments (Conesa et al. 2016). Furthermore, most of the transcriptome projects are highly focusing only on the genotypes and its expression profiles rather than the morphological or environmental metadata, which is consequently reducing the utilization of the RNA-seq dataset from conducting further analysis on the comprehensive transcriptome. On the other hand, the computational methods for the transcriptomic data were developed to analyze it into two data models, i.e. paired-datasets and continuous datasets. Further, in this regard, the ginseng transcriptome has to highly concentrate on these protocols and more specifically toward the continuous datasets, such as the time serious dataset by following suggestions from the network modeling experts (van Dam et al. 2018). Ultimately, to support the prospective researchers who are working for the empowerment of ginseng worldwide, it is highly recommended to establish an international genome consortium, which will support to improve the ginseng genome more effectively with the available collective knowledge.

D.-C. Yang

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D.-C. Yang Brief Bioinform 19(4):575–592. https://doi.org/10. 1093/bib/bbw139 Wang J, Li J, Li J, Liu S, Wu X, Li J et al (2016) Transcriptome profiling shows gene regulation patterns in ginsenoside pathway in response to methyl jasmonate in Panax Quinquefolium adventitious root. Sci Rep 6:37263. https://doi.org/10.1038/srep37263 Wang K, Jiang S, Sun C, Lin Y, Yin R, Wang Y, et al (2015) The spatial and temporal transcriptomic landscapes of ginseng, Panax ginseng C. A. Meyer. Sci Rep 5:18283. https://doi.org/10.1038/srep18283 https://www.nature.com/articles/ srep18283#supplementary-information Wei R, Qiu D, Wilson IW, Zhao H, Lu S, Miao J et al (2015) Identification of novel and conserved microRNAs in Panax notoginseng roots by high-throughput sequencing. BMC Genom 16(1):835. https://doi.org/ 10.1186/s12864-015-2010-6 Wu B, Long Q, Gao Y, Wang Z, Shao T, Liu Y et al (2015) Comprehensive characterization of a timecourse transcriptional response induced by autotoxins in Panax ginseng using RNA-Seq. BMC Genom 16 (1):1010. https://doi.org/10.1186/s12864-015-2151-7 Wu B, Wang M, Ma Y, Yuan L, Lu S (2012) HighThroughput Sequencing and Characterization of the Small RNA Transcriptome Reveal Features of Novel and Conserved MicroRNAs in Panax ginseng. PLoS ONE 7(9):e44385. https://doi.org/10.1371/ journal.pone.0044385 Wu D, Austin RS, Zhou S, Brown D (2013) The root transcriptome for North American ginseng assembled and profiled across seasonal development. BMC Genom 14(1):564. https://doi.org/10.1186/1471-2164-14-564 Wu Q, Song J, Sun Y, Suo F, Li C, Luo H et al (2010) Transcript profiles of Panax quinquefolius from flower, leaf and root bring new insights into genes related to ginsenosides biosynthesis and transcriptional regulation. Physiol Plant 138(2):134–149. https://doi.org/10.1111/j.1399-3054.2009.01309.x Yang W-Z, Hu Y, Wu W-Y, Ye M, Guo D-A (2014) Saponins in the genus Panax L. (Araliaceae): a systematic review of their chemical diversity. Phytochemistry 106:7–24. https://doi.org/10.1016/j. phytochem.2014.07.012 Yin P, Xu G (2014) Current state-of-the-art of nontargeted metabolomics based on liquid chromatography– mass spectrometry with special emphasis in clinical applications. J Chromatogr A 1374:1–13. https://doi. org/10.1016/j.chroma.2014.11.050 Zhang D, Li W, Xia EH, Zhang QJ, Liu Y, Zhang Y et al (2017a) The medicinal herb Panax notoginseng genome provides insights into ginsenoside biosynthesis and genome evolution. Mol Plant 10(6):903–907. https://doi.org/10.1016/j.molp.2017.02.011 Zhang G-H, Ma C-H, Zhang J-J, Chen J-W, Tang Q-Y, He M-H et al (2015a) Transcriptome analysis of Panax vietnamensis var. fuscidicus discovers putative ocotillol-type ginsenosides biosynthesis genes and genetic markers. BMC Genomics 16(1):159. https:// doi.org/10.1186/s12864-015-1332-8

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An Update to the Transcriptome Sequencing for the Genus Panax

Zhang L, Virgous C, Si H (2017b) Ginseng and obesity: observations and understanding in cultured cells, animals and humans. J Nutr Biochem 44:1–10. https://doi.org/10.1016/j.jnutbio.2016.11.010 Zhang S, Wu Y, Jin J, Hu B, Zeng W, Zhu W et al (2015b) De novo characterization of Panax japonicus C. A. Mey transcriptome and genes related to triterpenoid saponin biosynthesis. Biochem Biophys

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Metabolic Dynamics and Ginsenoside Biosynthesis

10

Shadi Rahimi, Padmanaban Mohanan, Dabing Zhang, Ki-Hong Jung, Deok-Chun Yang, Ivan Mijakovic, and Yu-Jin Kim

Abstract

Panax ginseng, a medicinally important perennial herb, has been widely used as the medicinal plant in South East Asian countries such as Korea, Japan, and China. Various pharmacological efficacies of ginseng are mainly correlated to its unique triterpenoid saponins ginsenosides. The backbone of ginsenosides are synthesized as triterpene saponins via the isoprenoid pathway, and the various ginsenosides are formed by glycosyl transferases. Ginseng plant has a long life cycle over 4–6 years, and it is frequently S. Rahimi
I. Mijakovic Systems and Synthetic Biology, Chalmers University of Technology, Göteborg, Sweden e-mail: [email protected] I. Mijakovic e-mail: [email protected] P. Mohanan Plant Genomics and Breeding Institute, Research Institute of Agriculture and Life Sciences, College of Agriculture and Life Sciences, Seoul National University, Seoul, Korea e-mail: [email protected] D. Zhang Joint International Research Laboratory of Metabolic and Developmental Sciences, Shanghai Jiao Tong University–University of Adelaide Joint Centre for Agriculture and Health, State Key Laboratory of Hybrid Rice, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China e-mail: [email protected]

exposed to environmental stresses during this long-term cultivation. Therefore, ginseng needs to activate an array of defense mechanisms controlled by defense-related genes to confer the enhanced resistance with minimal fitness cost. Infection, wound, irradiation, and other abiotic stresses induce several defense-related genes. Such responses are caused by salicylates and jasmonates, although their roles and interactions in development and signaling are not yet fully studied in ginseng. This chapter summarizes the genes involved in the synthesis of ginseng saponins D. Zhang
K.-H. Jung Graduate School of Biotechnology and Crop Biotech Institute, Kyung Hee University, Yongin 446-701, Korea e-mail: [email protected] D.-C. Yang Graduate School of Biotechnology and Department of Oriental Medicinal Biotechnology, College of Life Sciences, Kyung Hee University, Yongin 446-701, Republic of Korea e-mail: [email protected] I. Mijakovic The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Lyngby, Denmark Y.-J. Kim (&) Department of Life Science and Environmental Biochemistry, Pusan National University, Miryang 50463, Republic of Korea e-mail: [email protected]

© Springer Nature Switzerland AG 2021 J. Xu et al. (eds.), The Ginseng Genome, Compendium of Plant Genomes, https://doi.org/10.1007/978-3-030-30347-1_10

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and defense-related genes in ginseng in response to biotic and abiotic stresses. Moreover, this chapter discusses the kinetics of metabolic pathways throughout the ginseng development supporting the therapeutic significance and bioactive ingredients of ginseng during plant growth and development.

together with the metabolic dynamics corresponding to the aging in ginseng. This chapter may give an insight into the genetic and metabolic adaptations of ginseng plant to adverse growth conditions according to the age.

10.2 10.1

Introduction

Panax ginseng Meyer from Araliaceae family has highly valued root used for medicinal purposes. P. ginseng with long history since ancient times is considered as the health tonic and healing drug in Japan, Korea, China, and other Asian countries. The principle active components isolated from ginseng are saponins. Ginseng saponins, generally called as ginsenosides, are glycosides consisting of a sapogenin moiety, which may be a steroid or a triterpene, attached to one or more sugar moieties. Most of the ginsenosides have basic structure having 17 carbon dammarane steroidal nucleus arranged in four carbon rings. Ginsenosides are classified into protopanaxadiol, protopanaxatriol, oleanolic acid, occolitol based on the steroidal nucleus, and number of hydroxyl groups and sugar moieties attached (Nag et al. 2012; Kim et al. 2015b). In Panax species, around 150 natural ginsenosides have been discovered (Kim et al. 2015a). The ginsenosides have numerous medical effects such as adaptogen, anticancer, antiaging, and antioxidative activities. (Kim et al. 2015b). The current supply of ginseng mainly depends on field cultivation. P. ginseng growth can be affected by many environmental factors including sunlight, soil, temperature, pests, and pathogens. Due to the long cultivation period (generally 4–6 years), this highly valued medicinal plant is more susceptible to ecological stresses. However, ginseng activates distinct defense mechanisms mediated by a complex hormonal network to adapt to unfavorable conditions during growth and development. In this chapter, we provide an overview of ginsenoside biosynthetic genes and defenserelated genes and their active roles in ginseng,

Ginsenoside BiosynthesisRelated Genes

Ginsenoside saponins known as the main bioactive compounds in ginseng are glycosylated triterpenes in Panax species. Isopentenyl diphosphate (IPP) is the precursor of triterpene ginsenosides, biosynthesized through the mevalonate pathway (MVA) by the key regulatory enzyme of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) (Fig. 10.1). In next steps, squalene is catalyzed from two molecules of farnesyl diphosphate (FPP) alongside squalene synthase (SS). Then, squalene epoxidase (SE) oxidizes squalene to give rise to 2, 3oxidosqualene. Ginsenosides are synthesized from 2, 3-oxidosqualene (a common precursor of sterols) by additional cyclization, hydroxylation, and glycosylation by dammarenediol synthase (DDS), cytochrome P450 and glycosyltransferase (GT) enzymes, respectively (Devi et al. 2011; Khorolragchaa et al. 2014; Kim et al. 2015b). Following sections briefly introduce how the ginseng biosynthetic genes involve in the production of ginseng saponins.

10.2.1 3-Hydroxy-3-MethylglutarylCoA Reductase (HMGR) Condensation of three units of acetyl-coA and 3hydroxy-3-methylglutaryl-CoA (HMG-CoA) formation is the beginning of MVA pathway in the cytosol. HMGR then converts HMG-CoA to mevalonate, and this conversion initiates the production of IPP by subsequent enzymatic reactions. There are two isoenzymes of HMGR in ginseng known as PgHMGR1 and PgHMGR2, PgHMGR1 expresses abundantly in the root tissue, while PgHMGR2 transcripts can be found as the ginseng aged. Although PgHMGR2 having

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Fig. 10.1 Possible biosynthetic pathway for the production of ginsenosides in major Panax species. Pg-Panax ginseng; Pq—Panax quinqefolius; Pj—Panax japonicus; Pn—Panax notoginseng Adapted from: Kim et al. 2015a. Ginsenosides produced from the triterpenoid nucleus form the precursor IPP synthesized via mevalonate pathway. Further cyclization by DDS leads to the production of dammarane nucleus ginsenosides. The presence of cytochrome P450 and UGTs adds glucose moieties to make diverse array of ginsenosides. HMG-CoA—3Hydroxy-3-methylglutaryl-CoA; HMGS—3-Hydroxy-3methylglutaryl-CoA synthase; HMGR—3-hydroxy-3methylglutaryl-CoA reductase; MVK—mevalonate kinase; MVPmevalonate phosphate; PMK-

phosphomevalonate kinase; MVPP—diphosphomevalonate; MVD—mevalonate diphosphate decarboxylase; FPP—farsenyldiphosphate; DMAPP—dimethylally diphosphate; IDI—isopentenyl diphosphate deltaisomerase; DXP—1- deoxy-D-xylulose-5-phosphate; MEP—methylerythritol phosphate; FPS—farnesyl diphosphate synthase; SS—squalene synthase; SE— squalene epoxidase; DDS—dammarenediol synthase; bAS—b-amyrin synthase; CAS—cycloartenol synthase; LAS—lanosterol synthase; PPDS—protopanaxadiol synthase; PPTS—protopanaxatriol synthase; CYP—cytochrome P450; GT—glycosyltransferase; UGT—UDPglycosyltransferase. Reused from Kim et al. (2015b) with permission

the same catalytic domain to that of Arabidopsis, it fails to complement athmg1, suggesting an evolutionary divergence of HMGR isoenzymes (Kim et al. 2014c). Moreover, continuous dark treatment enhanced PgHMGR1 transcription and production of total ginsenosides in 3-year-old ginseng, implying the regulation of saponins

biosynthesis by light and dark. Overexpression of PgHMGR1 in ginseng and Arabidopsis increases the production of triterpenes and phytosterol. PnHMGR1 and PnHMGR2 are also identified from P. notoginseng with the similar transcription profile as P. quinquefolius and P. ginseng. Both genes were expressed most in

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flowers followed by roots, stems, and least in leaves (Liu et al. 2016). Overall, HMGRs play a key role in production of triterpenes ginsenosides in ginseng (Kim et al. 2014c).

10.2.2 Mevalonate Diphosphate Decarboxylase (MVD) The conversion of mevalonate IPP by mevalonate kinase (MVK) and phophomevalonate diphosphate kinase (PVK) followed by mevalonate diphosphate decarboxylase (MVD) from diphospho mevalonate (MVPP) is uncertain. When PgMVD is overexpressed, it upregulates cycloartenol synthase (CAS) which subsequently produce campesterol, stigmasterol, and b-stiosterol, with no significant increase in ginsenoside content and b-amyrin. Thus, MVD was suggested as the last enzyme in mevalonate pathway, playing crucial role in biosynthesis of phytosterol rather than ginsenoside (Kim et al. 2014d).

10.2.3 Farnesyl Phosphate Synthase (FPS) The MVA-synthesized IPP along with dimethylallyl diphosphate (DMAPP) condensed to produce geranyl pyrophosphate (GPP), which along with one more molecule of IPP yields FPP by farnesyl diphosphate synthase (FPS). P. ginseng contains two copies of FPS gene, and when it was overexpressed in Escherichia coli, the purified protein contains FPS like activity. Moreover, 0.1 mM methyl jasmonate (MeJA) treatment enhances the transcript of FPS 12 h after treatment (Kim et al. 2010a), and overexpression of FPS gene in ginseng hairy root cultures increases the ginsenoside content in transgenic ginseng roots, confirming the importance of PgFPS in ginsenoside biosynthesis (Kim et al. 2014d). Interestingly, the longitudinal expression pattern of key genes in the saponin synthesis indicates that the synthesis and/or accumulation of the Rb1 resulted from the contribution of the HMGR and FPS in P. notoginseng stems. However, the total saponins synthesis might result firstly from the

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contribution of the FPS, next from those of the SS and DDS (Xu et al. 2018). Furthermore, overexpressing FPS in P. notoginseng cells positively affects saponin synthesis including Rh1, Rg1, and Rd with the most significant effect on Re content (Yang et al. 2015). A fusion protein was constructed to combine a ginseng FPS and a Centella asiatica SS via a short peptide (Gly-Ser-Gly) linker. Approximately 94% and 71% higher FPS and SS enzyme activities were found by heterologous expressed soluble fusion protein in E. coli compared with single enzyme activities, respectively (Jung et al. 2017). It can serve as the valuable tool for genetic engineering of efficient saponins production.

10.2.4 Squalene Synthase (SS) Triterpenes and phytosterols are derived from a common biosynthetic intermediate, squalene in P. ginseng. PgSS1 transcripts are ubiquitously present in whole plant but higher in shoot apex and roots (Lee et al. 2004), while PgSS2 and PgSS3 are only detected in specific organs. Functional complementation analysis was performed by the expression of all these genes in the yeast mutant erg9 lacking SS activity. The recombinant mutant yeast produced squalene, squalene epoxide, and ergosterol (Kim et al. 2011). P. notoginseng SS was also cloned and investigated followed by its recombinant expression in E. coli (Jiang et al. 2017a). MeJA treatment increases the expression of ginsenoside biosynthetic genes including SE, bamyrin synthase (b-AS) but not cycloartenol synthase (CAS), and especially, PgSS1 transcripts were increased 12–96 h after treatment. The proximal promoter of P. quinquefolium SS was identified in which potential cis elements mediating the response to MeJA, and other abiotic factors were localized (Kochan et al. 2018). When PgSS1 is overexpressed in the adventitious roots, it upregulates the expression of downstream genes SE, b-AS, and CAS (Lee et al. 2004). Co-overexpression of PnSS and PnHMGR cell line also accumulates six major saponins (Rb1, Re, Rg1, Rh2, F1, and Rh1), and

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phytosterols higher than those in control and the cell line overexpressed PnHMGR alone (Deng et al. 2017). Thus, the biochemical activities of ginseng SSs are necessary for producing phytosterols and triterpenes.

derived phytosterols (Kim et al. 2015b). PgDDS fused with the green fluorescent protein (GFP) showed membrane localization and improved DD production (Liang et al. 2016). The transgenic rice of PgDDS gene produces the dammarane-type sapogenins, creating a new germplasm called “ginseng rice” (Huang et al. 2015b). Furthermore, the interaction between PgMYB2 protein and DDS promoter may positively regulate the expression of PgDDS (Liu et al. 2019).

10.2.5 Squalene Epoxidase (SE) The production of 2,3-oxidosqualene from squalene was catalyzed by SE, which is a rate limiting oxygenation reaction in the triterpene saponin synthesis. In case of Arabidopsis, there are six isoenzymes of SE, which among them SE1, SE2, and SE3 were active. AtSE1 is responsible for the normal development and important for the root sterol biosynthesis. P. ginseng has two isoenzymes of SE, PgSE1 is expressed in all tissues, whereas PgSE2 is weekly expressed only in petioles and flower buds. MeJA treatment to the ginseng roots increases the expression of PgSE1 and downregulates PgSE2. RNA interference knockdown of PgSS1 significantly reduces the ginsenoside concentration in the transgenic plants, while it enhances PgSE2 and CAS transcription, eventually leading to the accumulation of phytosterol. Hence, PgSE1 is essentially needed for producing ginsenosides in ginseng (Lee et al. 2004).

10.2.6 Dammaranediol Synthase (DDS) Cyclization of 2, 3-oxidosqualene to dammarene diol-II (DD) is the first committed step in ginsenoside biosynthesis, which catalyzed by DDS, one of oxidosqualene cyclase (OSCs). DDS is highly expressed in flower buds compared to the petioles, leaves, and roots in ginseng, and MeJA treatment upregulates the expression of DDS. RNA interference knockdown of DDS resulted in 84.5% reduction in the production of ginsenosides in roots, suggesting that DDS is an important enzyme in the ginsenoside biosynthesis (Han et al. 2006). Transgenic ginseng roots overexpressing DDS enhances the production of ginsenosides by 50-100% than the control roots and suppresses CAS branch pathway-

10.2.7 b-Amyrin Synthase (b-AS) b-AS (also called as PNY) catalyzes the production of oleanane-type ginsenoside R0 from bamyrin in P. ginseng and P. quinqefolius. In P. ginseng, PNY1 shares 65% similarity with DDS and expresses ubiquitously in all tissues, but PNY2 expresses only in flowers. The DDS gene silencing led to the expression of PNY2 and PNZ, suggesting tight coordination of DDS with other OSCs due to the same precursor utilization. Phylogenetic analysis reveals divergence of bAS and DDS from the common ancestor lanosterol synthase (LAS) in higher plants which is mainly through the positive selection and tandem duplication (Kim et al. 2015b). Transformation of P. japonicus b-AS gene into rice successfully produced a new rice germplasm, producing oleanane-type sapogenin (Huang et al. 2015a). Interestingly, biosynthetic pathway of oleananetype saponins was constructed in P. notoginseng by introducing P. japonicus b-AS gene into P. notoginseng cells (Zhang et al. 2019).

10.2.8 Cytochrome P450s Among the cytochrome P450 genes identified in ginseng, CYP716A47 is known as protopanaxadiol synthase (PPDS), which involved in hydroxylating DD at C-12 thereby producing protopanaxadiol (PPD) (Devi et al. 2011). The production of PPD was improved in yeast, but also a genetic method was provided to improve the plant cytochrome P450 activities (Zhao et al. 2016). PPD and DD were produced in transgenic

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rice grains overexpressing PgDDS and PgPPDS driven by a rice endosperm-specific a-globulin promoter. The finding can be applied to the rice breeding for the reinforcement of new medicinal values (Han et al. 2019). In addition, CYP716A subfamily genes including CYP716A52v2 and CYP716A53v2 were also identified, and the gene product of CYP716A53v2, known as protopanaxatriol synthase (PPTS), catalyzes the formation of protopanaxatriol (PPT) from PPD in ginseng during ginsenoside biosynthesis (Han et al. 2012). Transgenic P. ginseng lines overexpressing the CYP716A53v2 gene lead to increase PPT-group ginsenosides (Rg1, Re, and Rf) and decreased accumulation of PPD-group ginsenosides (Rb1, Rc, Rb2, and Rd). By contrast, silencing the CYP716A53v2 by RNA interference results lower levels of PPT-group compounds and higher levels of PPD-group compounds in transgenic ginseng roots (Park et al. 2016). Transgenic tobacco co-overexpressing PgDDS, PgPPDS, and PgPPTS produces DD, PPD, and PPT in leaves. However, transgenic tobacco was not able to set seeds because of microspore degeneration in anthers. The phloem cells in the center of anther showed an abnormally degenerated mitochondria and condensed nuclei (Gwak et al. 2019). CYP716A52v2 transcript was found only in trichome, and it catalyzes the synthesis of oleanolic acid from b-AS, and it has been known as oleanolic acid synthase (OCS) (Han et al. 2013). MeJA treatment leads to the obvious accumulation of CYP716A47 in adventitious roots, not for CYP716A52v2 and CYP716A53v2 (Han et al. 2012). It is consistent with the higher expression of UGTs involve in PPD biosynthesis and PPD-type ginsenosdie production upon MeJA treatment (Rahimi et al. 2019). 30 transcription factors, 20 cytochromes, and 11 glycosyl transferases with potential function in the ginsenoside biosynthesis were identified by transcriptomic profiling of in vitro grown ginseng adventitious roots (Subramaniyam et al. 2014). The transcriptome resources of Panax could predict the gene networks in P. ginseng.

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10.2.9 Uridine Diphosphate (UDP)Glucosyltransferases (UGTs) Specific ginsenosides are synthesized from the triterpene aglycones moiety by addition of sugar molecules catalyzing by UGTs. Solubility, stability, bioactivity, or bioavailability of metabolites are increased by UGTs typically acting at the final step of their biosynthesis (Jung et al. 2014). Although, there are several UGTs with known activities on triterpene aglycones glycosylation (Rahimi et al. 2019), there are still some unknown functional UGTs for ginsenosides diversification (question marks in Fig. 10.2). Rahimi et al. (2019) found 416 putative P. ginseng UGTs in public databases. Genomewide search analysis predicted more than 100 unique UGT complementary DNA (cDNA) sequences in P. quinguefolium, P. ginseng, and P. notoginseng. These may be potential candidates functioning in diversification of ginsenosides (Rahimi et al. 2019). Plant secondary product glycosyltransferase (PSPG) motif with 44-amino acid consensus sequence is typical motif in UGTs involved in plant secondary metabolism. Grouping of triterpene-related UGTs was done on the basis of PSPG motif sequences (Rahimi et al. 2019). 12 UGTs were selected as the most likely candidates for biosynthesis of triterpenoid (Khorolragchaa et al. 2014). They were classified into eight families of UGT71, UGT72, UGT75, UGT79, UGT82, UGT93, UGT94, and UGT709, which grouped in A, E, G, L, N, and O groups.

10.2.9.1 C-3 Glycosylation PgUGT74AE2 (Group L) glycosylates hydroxyl groups at C-3 positions and thereby converting compound K and PPD into F2 and Rh2, respectively (Jung et al. 2014). Subsequently, PgUGT94Q2 from Group A glycosylated C-3, synthesizing Rd and Rg3 from F2 and Rh2, respectively (Jung et al. 2014). In P. quinquefolius, Pq3-O-UGT1 and Pq3-O-UGT2 were characterized (Lu et al. 2017a, b). Pq3-O-UGT2 sequence showed high similarity to PgUGT94Q2

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Fig. 10.2 Pathway for ginsenoside biosynthesis. Unknown enzymes are classified and marked with different colors. MeJA-induced biosynthetic pathway is indicated by red arrows. CK, compound K; CY,

compound Y; CO, compound O; DMG, 20S-O-b-(Dglucosyl)-dammarenediol II; PPD, protopanaxadiol; PPT, protopanaxatriol Adapted from: Rahimi et al. 2019

in P. ginseng. RNA interference of both Pq3-OUGT2 and PgUGT94Q2 showed reduced levels of total and PPD-type ginsenosides, alongside higher transcription level of PPDS and PPTS. Similar to PgUGT94Q2, Pq3-O-UGT2

glycosylates C-3 position of F2 and Rh2 and produces Rd and Rg3 (Lu et al. 2017a). In addition, Pq3-O-UGT1 shares high similarity with the deduced amino acid sequence of PgUGT74AE2. Also, both PgUGT74AE2 and

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Pq3-O-UGT1 showed same catalytic activities in glycosylation of C-3 and production of Rh2. Therefore, from the evolutionarily point of view, it can be proposed that UGT catalytic activities are conserved before diversification of species.

10.3

Physiological Changes of Ginsenoside Accumulation

10.2.9.2 C-6 and C-20 Glycosylation C-6 and/or C-20 in PPT-type ginsenosides and C-3 and/or C-20 in PPD-type ginsenosides is mainly glycosylated by P. ginseng UGTs. PgUGTPg100 (Group E) catalyzed glycosylation of C-6 in PPT and thereby synthesis of ginsenoside Rh1 (Wei et al. 2015). Moreover, P. notoginseng UGRdGT glycosylates Rd at C-20 and synthesizes ginsenoside Rb1 (Yue and Zhong 2005). Same group member (Group E), PgUGTPg1 catalyzes glycosylation of C-20 position in PPD, DD, Rg3, and Rh2 and synthesis of compound K, new compounds 20S-O-b-(Dglucosyl)-dammarenediol II (DMG), Rd, and F2, respectively (Yan et al. 2014). PgUGTPg101 (Group E) functions on multiple positions of C-6 and C-20 in PPT-type ginsenosides. A UGT protein (GpUGT23) in Gynostemma pentaphyllum was patented with glycosyltransferase activity at C-20 in PPD- or PPT-type ginsenosides (Kim et al. 2015a). F2, compounds K, F1, and Rd are converted by GpUGT23 (Group A) into gypenoside XVII, gypenoside LXXV, notoginsenoside U, and Rb1, respectively. Although it can use substrates with a broad range, but its regioselectivity is still conserved. Broad substrate specificity was found among the phylogenetic Group D UGTs members including UGT73. In Berberis vulgaris and soybean, 73 family is found as the best candidate group for saponin biosynthesis (Rahimi et al. 2019). In P. ginseng, we could also observe 73 family members, but none of them was functionally analyzed which can be due to that oleanane-type saponin exists only in minor quantities in P. ginseng. Overall, unknown UGTs involved in ginsenoside biosynthesis (question marks in Fig. 10.2) need to be investigated to define the different pathways involved in biosynthesis of ginsenoside.

MeJA treatment stimulated not only the expression of P. ginseng UGTs but also upregulated the upstream terpenoid biosynthetic genes, including HMGR, FPS, SS, SE, DS, and PPDS (Rahimi et al. 2019). Among 19 UGT contig groups induced by MeJA, PgUGT71A27 from Group E, PgUGT74AE2 from Group L, and PgUGT94Q2 from Group A were active for ginsenosides. Furthermore, early response of 6 h after MeJA treatment was shown by PgUGT1 and PgUGT2 (Group E members from family UGT71) (Khorolragchaa et al. 2014). Kang et al. (2018) also demonstrated similar results for UGT71 candidates. Remarkably, MeJA affected only PPD-type ginsenosides, but not for PPT- or oleane-type ginsenosides (Kang et al. 2018). It can be suggested that the PPD-type ginsenoside production and certain groups of UGTs involved in PPD-type ginsenosides biosynthesis are induced by MeJA. Interestingly, MeJA elicited production of PPD-type ginsenoside Rg3 in hairy root cultures, while Rg3 is not naturally occurring in P. ginseng (Rahimi et al. 2019). It was due to the upregulation of a Group A UGT catalyzing Rh2 conversion into Rg3 (Kim et al. 2013). Similarly, about 100-fold, increased level of Rg3 was observed in 120 h MeJA-treated adventitious roots. It was accompanied with upregulation of UGT74 from Group L and UGT94 from Group A candidates which are involved in biosynthesis of Rg3 (Kang et al. 2018). Thus, PPD precursor goes into the biosynthesis of Rg3 through group L, E, and A UGT activities using treatment with MeJA. Subsequently, higher activity of UDPG- ginsenoside Rd glucosyltransferase (UGRdGT) converted Rd into Rb1 ginsenoside after six days treatment (Kim et al. 2009a). Red arrows in Fig. 10.2 marked the MeJA-induced pathway in biosynthesis of ginsenoside.

10.3.1 MeJA Effect on Ginseng UGTs and Ginsenoside Accumulation

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Fig. 10.3 Ginsenoside metabolic pathways and the changes of ginsenoside metabolites in different years. Dashed arrows represent putative pathways. Green and yellow graphs represent metabolic changes in leaves and

roots, respectively. Data represent mean values of three samples for each growth stage (Y1, Y4, or Y6). Error bars represent SDs (n = 3). Adapted from: Kim et al. 2018

10.3.2 UGTs and Ginsenoside Accumulation Regarding Ginseng Development

tissues, and UGTs also differently expressed among plant tissues. UGT94 and UGT71 candidates are as the most putative triterpene-related PgUGT genes expressing in roots and leaves of P. ginseng (Khorolragchaa et al. 2014). 11 major ginsenosides were observed by Kim et al. (2018) as the ginseng develops (Fig. 10.3). It is consistent with an earlier report using terpenoids to discriminate ginseng ages (Cui et al. 2015). In case of the squalene precursor, there is an increase in 4- and 6-year-old leaves compared to 1-year-old leaves. Meanwhile, no significant

During the evolution, the triterpene UGTs are enriched in P. ginseng genome compared with other plant species. Specific saponins are generally produced during development of roots and fruit in P. ginseng and Siraitia grosvenorii, respectively (Kim et al. 2015b; Rahimi et al. 2019). Based on protection need, the saponins accumulate differently among plant organs and

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Fig. 10.4 Schematic function of defensive genes in ginseng during biotic and abiotic stress. Different environmental stresses alleviate ROS production from chloroplast and mitochondria. Ginseng cell nucleus activates its own defense proteins to negotiate the generated ROS and

protects the cell from free radicals. Likewise, the ginseng PR proteins protect from pathogen attack by rapidly counter attacking the pathogen invaders. CAT1—catalase, GPX—glutathione peroxidase, Grx—glutaredoxin, Prx— peroxiredoxin, PR—pathogenesis related

decrease was observed in squalene content of root during growth. It can suggest that the ginsenosides or their precursors can be actively biosynthesized in the leaves followed by their movement into the root. It was suggested that the glycosylation and ginsenosides synthesis or their transport into the roots was contributed by the derivatives of photosynthetic metabolites (glucose and fructose) (Schramek et al. 2014). The biosynthesis of bioactive compound in underground tissues (lateral root and main root) is regulated by photosynthesis and consumption of energy in aboveground tissues (stems, petioles, and leaves). However, the mechanism of ginsenoside synthesis and location, as well as the long-distance allocation of ginsenosides is not identified, and further investigations need to be performed. 40 different ginsenosides are identified and isolated from different tissues, Rd, Rb1, Rb2, and Rc, as the major PPD-type and and Rf, Rg1, and Re as PPT-type ginsenosides which composed of more than 80% of the total ginsenosides in roots and leaves of P. ginseng (Kim et al. 2015b).

Seven major ginsenosides and other PPD- and PPT-type ginsenosides such as Rg2, F2, F1, and Rh1 were identified by metabolomic analysis (Kim et al. 2018). Different studies reported various total ginsenoside content and ginsenoside profiles, but roots continuously accumulated ginsenosides over the time (Kim et al. 2015b, 2018). It is consistent with the medicinal significance of ginseng roots, as the plant grew ginsenosides dramatically accumulated up to 1.2—to sevenfold in roots (Kim et al. 2018). All detected ginsenosides significantly accumulated in 4-yearold roots. Meanwhile, Rb1 and Rg2 continued to increase in 6-year-old roots. Interestingly, during the roots growth, Rb1 content increases; however, it decreases during the leaves growth (Kim et al. 2018). Hence, Rb1 can be used as a marker for 6-year-old roots. Compared with the roots, leaves have a lower level of ginsenosides (Kim et al. 2018). 6-yearold leaves contain lower levels of Rb1, Rb2, Rg1, Rf, and Rh1 than 1-year-old leaves. Compared to 1-year-old leaves, two- and sixfold

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increases of Re and Rg2 contents were exceptionally found in 6-year-old leaves (Kim et al. 2018). Different biological activities of individual ginsenosides may describe the different patterns of their accumulation in leaves and roots during growth (Kim et al. 2015b), which needs to be elucidated. Altogether, in both leaves and roots of ginseng plants, a high degree of metabolic dynamics is found at different ages. Biosynthesis of ginsenosides in leaves can be followed by transport and accumulation in ginseng roots. It can give insight into the metabolites dynamics in the source (leaves) and sink (roots) tissues and the metabolic alterations due to the unfavorable growth conditions during ginseng plant development. Moreover, significant contents of major ginsenosides are found in the peridermal cells in roots compared to the stele and cortex (Jiang et al. 2017b). In roots cross sections, types of ginsenoside are also continuously altering. It can be suggested that the transport mode, physiological role in growth and development, defense system, and responses to environmental stresses can influence accumulation of saponins.

main role of ginsenosides in defense against pathogens, little is known about their physiological role in plants. Association with plant growth is revealed by different localizations of ginsenosides at xylem, periderm, and cortex at different root ages. However, the metabolomic analysis of ginseng indicates that a broad range of metabolic strategies are utilized by the ginseng plant to adapt to the adverse conditions. Whereas, ginsenoside contents are not definitely related with the ginseng age in 5–18-yearold ginseng roots. It is in conflict that older wild ginseng may result in better medicinal ginseng for herbal medicines. The samples collected from different cultivation regions, ginsenoside Rc, Rd, and Rb1 contents differed extensively with the ages. Harvesting season is the determinant factor for the changes in Re and Rg1 ratio, as well. Altogether it suggests that ginsenosides accumulation varies according to the physiological status (Kim et al. 2018).

10.3.3 Effect of Environmental Changes on Ginsenosides Accumulation Environmental stimuli (light, salt, JA, and other signaling molecules) significantly affect the ginsenosides concentrations (Oh et al. 2014; Rahimi et al. 2015a). The balance between primary and secondary metabolisms is differently affected by various environmental circumstances in plants. For example, plants grow slowly under nutrientlimiting or stressful environments. At this condition, they need to allocate more resources for constitutive defense. For example, the low nitrate concentrations in ginseng cell culture could induce ginsenoside production. However, high-N hydroponic ginseng root culture displayed faster growth but lower ginsenoside contents. Osmotic stress could also inhibit the growth but stimulate the ginsenosides accumulation. In addition to the

10.4

Defense Genes Involved in Biotic and Abiotic Stresses in Ginseng

In order to protect from environmental stresses caused by biotic and abiotic factors, plants evolve with protection mechanism which can vary from physical to molecular barrier (Fig. 10.4). A complex polymer from esterified fatty acids covered by waxes and lignin composed of the cuticle layer in plant cell wall, playing a protective function role in structural defense. The hypersensitive response associated with oxidative burst, secondary metabolites accumulation, signaling for increasing antioxidant enzymes activities, and inducing pathogenesis-related proteins is the second line of defense against biotic and abiotic stresses in plant (Van Loon 1999; Van Loon et al. 2006). Transcriptomic analysis of hairy roots, 4—and 14-year-old roots of ginseng have shown the genes involved in stress, secondary metabolism, and long survival. Among them, around 1,365 EST sequences are related to plant secondary metabolism, and 745 sequences are related to

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stresses (Sathiyamoorthy et al. 2010). The differential expression and upregulation of ginseng antioxidant genes under abiotic stresses may indicate response of these genes during the generation of reactive oxygen species (ROS). However, the upregulation of transcript level is merely based on the incompatible interactions under biotic stress condition (Sathiyaraj et al. 2011).

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ginseng tissues. However, both PgGPXs transcripts were strongly induced by salt and chilling stresses as well as MeJA in ginseng seedling and adventitious roots, respectively (Kim et al. 2014a; Rahimi et al. 2016a). But they have shown different responses to biotic stresses, and they sense oxidative damage differently, as well (Kim et al. 2014a). This suggests that PgGPXs are regulated differently, thus preventing ginseng from different stress states.

10.4.1 Catalase (CAT) 10.4.3 Glutaredoxin (Grx) Catalase (H2O2:H2O2 oxidoreductase, E.C. 1.11.1.6; CAT) is an iron porphyrin enzyme, catalyzing dismutation of H2O2:H2O2 to water and dioxygen. There are monofunctional and tetrameric catalases mostly localizing in glyoxysomes or peroxisomes in plants (Willekens et al. 1995). Multiple CAT isozymes are found in plants, functioning as the antioxidant defense genes and responding to environmental and physiological oxidative stresses (Scandalios 1990, 1994). A cDNA clone containing a catalase (PgCAT1) gene representing multi-gene family was isolated from P. ginseng. It was expressed relatively high in leaves and stems but at moderate levels in roots. Different stresses such as heavy metals, plant hormones, osmotic agents, MeJA, and high light irradiance, triggered a significant induction of PgCAT1 (Balusamy et al. 2013; Purev et al. 2010; Rahimi et al. 2016a). It suggests a potential role of PgCAT1 in ginseng protection against environmental stresses and ROS generation.

10.4.2 Glutathione Peroxidases (GPX) GPXs catalyze the reduction of H2O2 or organic hydro peroxides to water or alcohols by reduced glutathione. These are a group of enzymes that protecting cells against oxidative damage generated by ROS. Two GPX cDNAs (PgGPX1 and PgGPX2) were isolated and characterized from P. ginseng. PgGPX2 was constitutively expressed in all ginseng tissues, while PgGPX1 was expressed at a lower level than PgGPX2 in

Grxs are ubiquitous thiol-disulfide oxidoreductases present in most of living organisms from virus and bacteria to mammals (Fernandes and Holmgren 2004). This enzyme plays a role in active oxygen-scavenging systems in animals and virus (Wells et al. 1990; Terada et al. 1992). PgGrx ubiquitously expressed in root, stem, and leaves of ginseng. The abiotic stresses such as chilling, salt, ultraviolet (UV) exposure, and heavy metal treatments showed slight decrease in the transcription of this gene initially for a brief period after which their transcription has been increased. These results suggest their involvement in abiotic stresses (Kim et al. 2008).

10.4.4 Peroxiredoxin (Prx) Prxs are most recently identified as the H2O2decomposing antioxidant enzymes. They are thiol-based peroxidases existing in whole organisms. They can reduce various hydroperoxides into the corresponding alcohol or water (Dietz et al. 2006; Rouhier et al. 2001). Plants have four Prx subgroups which are classified according to the number and position of the conserved cysteine residues: 1-Cys Prx, 2-Cys Prx, type II Prx, and Prx Q (Dietz et al. 2006). A Prx cDNA (PgPrx) from P. ginseng is highly expressed in leaves, but it was expressed at a low level in the stem. PgPrx was upregulated by low temperature, UV irradiation, and salt, suggesting its essential role in ginseng defense mechanism against abiotic stresses (Kim et al. 2010b).

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10.4.5 Ascorbate Peroxidase (APX) APX enzyme (EC 1.11.1.1) plays a fundamental role in the plant antioxidative defense mechanism. APX combined with ascorbate–glutathione cycle acts preventing H2O2 toxicity in plants in response to multiple stress conditions (Shigeoka et al. 2002; Blokhina et al. 2003). Two PgAPX genes were isolated from P. ginseng. The most abundant PgAPXs transcripts were found in leaves. Arabidopsis lines expressing PgAPX1 showed higher germination rate and root length than wild-type (WT) when exposed to salt stress. It is consistent with higher expression of PgAPX1 in response to salt stress in ginseng. Higher total APX activity was along with higher content of relative water, proline, and chlorophyll, as well as lower accumulation of H2O2 in transgenic plants under salt stress. Similarly, salt stress increases expression of chlorophyll a/bbinding proteins (CAB) and geranylgeranyldiphosphate synthase (GGDPS) catalyzing formation of chlorophyll precursor (Silva et al. 2016; Rahimi et al. 2015b). Higher expression of PgAPX1 conferred salt tolerance to transgenic Arabidopsis lines by upregulating the ion homeostasis mechanism (Sukweenadhi et al. 2017).

10.4.6 Pathogenesis-Related Protein (PRs) The term of “PRs” is attributed to the proteins induced in the incompatible host-pathogen interactions, and they are classified base on their characteristics in pathological situations (Van Loon et al. 1994, 2006). PRs is a proteinbased defensive system in higher plants against biotic stresses, especially pathogen infections. PR proteins are grouped based on their mode of action. Although they show anti-microbial activity and their accumulations are indicated in plant resistance mechanism, but their direct functions in plant defense are not clearly understood (Sels et al. 2008). Several PR protein families are characterized in P. ginseng, and their role in biotic and abiotic stresses is as follows.

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10.4.6.1 PgPR2 (b-Glucanase) PR2 family of proteins is rapidly accumulated in response to hormonal responses, elicitor treatment, and pathogen attack (Leubner-Metzger et al. 1999). Pg-glu1 in ginseng with 46 kD molecular weight shares 60% identity with tomato, tobacco, and potato b-1,3-glucanases (Kiselev et al. 2006). Pg-glu1 expression was significantly increased in shoots upon wounding and after treatment with salicylic acid (SA), cytokines, ethylene, and fungal elicitors. The overexpressed Pg-glu1 ginseng calli showed an upregulated expression and activity of b-1, 3— glucanase. Phyto-pathogens overcome b-1,3glucanase activity within a short period of time. Thus, b-1,3—glucanases combined with chitinase is obviously more effective strategy of fungal cell walls degradation (Broekaert et al. 2000). 10.4.6.2 PgPR3 (Chitinase) Chitinase (EC3.2.1.14) hydrolyzes b-1,4-linked N-acetylglucosamine and N-acetylmuramic acid, which are different forms of lysozyme. The fungal cell wall can be degraded by chitinase, and the manipulating chitinase genes enhance plants to resist against fungal pathogen. Based on their primary structures, the chitinases are classified into the six different classes (Collinge et al. 1993). The chitinase gene (PgChi-1) has been identified and characterized in ginseng (Pulla et al. 2011). PgChi-1 was highly expressed in ginseng roots rather than stem or leaves. Especially, the transcription level of PgChi-1 was induced upon wounding and after treatment with oxidative stress, osmotic stress, SA, jasmonic acid (JA), heavy metals, and fungal and nematode infection (Pulla et al. 2011). 10.4.6.3 PgPR4 The PR4 family is a group of defensive proteins that consist of a Barwin domain at C-terminus. It was found that PgPR4 gene is induced by salt, wounding, pathogen infection, and hormone stresses. It can propose that the PgPR4 involves in the molecular response of ginseng defense against pathogen attack and abiotic stress (Kim et al. 2014b). Class II PgPR4 abundantly

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accumulated in the apoplast might protect against the imbalance. The improved tolerance against pathogen and salt stresses may be conferred by PgPR4 through the SA, JA, and abscisic acid (ABA) signaling pathways.

10.4.6.4 PgPR5 (Thaumatin-Like Protein) Thaumatin-like and osmotin-like proteins are classified in the PR5 family, playing role in plant defenses against infections (Vigers et al. 1991). A PR5 (PgPR5) was identified in ginseng leaves, sharing 87% similarity to that of Actinidia deliciosa. It was found that PgPR5 upregulated by salt, infection, heavy metals, and cold in ginseng (Kim et al. 2009b), while the functional role has not been revealed yet. 10.4.6.5 PgPR6 (Protease Inhibitor) PR6 acting as a protease inhibitor plays a crucial role in defense mechanism in plants (Van Loon 1999). Based on the active amino acid of the “reaction center” (Koiwa et al. 1997), they are classified into aspartic, cysteine, serine, and metallo-proteases. A cysteine protease inhibitor PgCPI with a multi-gene family was characterized in ginseng (Jung et al. 2010). PgCPI was moderately upregulated by ABA, osmotic stress, and JA, and meanwhile, it was significantly upregulated by UV radiation, light, wounding, and MeJA. In ginseng, infection with Botrytis cinerea, Colletotrichum gloeosporoides, or a nematode induces PgCPI transcription (Jung et al. 2010). Myagmarjava et al. (2017) isolated a PR6 from ginseng embryogenic callus. PgPR6 belongs to Kunitz-type PI family classification. High transcript level of PgPR6 was found in ginseng roots. PgPR6 was highly induced by signaling molecules, mechanical wounding, chilling, heavy metals, salt, mannitol, and sucrose stress, indicating an essential role of PgPR6 in defense mechanism against broad range of environmental stresses. 10.4.6.6 PgPR10 (Ribonuclease) PR10 proteins as a ubiquitous class of intracellular PRs belong to the family of small, homologous, and acidic proteins (Van Loon 1999). To

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date, three different groups of PR10 genes are investigated in ginseng (Moiseyev et al. 1997; Pulla et al. 2010; Lee et al. 2012a, b). Specific ribonuclease activity was shown for two PR10 proteins (Moiseyev et al. 1994, 1997; Pulla et al. 2010). Overexpressed PgPR10-1 (Lee et al. 2012a) and PgPR10-2 (Pulla et al. 2010) gave biotic stresses resistance in Arabidopsis and tobacco, respectively. Heterologous overexpressed PgPR10-4 in Arabidopsis showed resistance against bacterial and fungal stresses. Furthermore, full-length coding sequence of PR10-1 was identified in P. notoginseng and showed high similarity with that of P. ginseng. It was suggested that it might be involved in growth, development, and secondary metabolism. It was also significantly upregulated with the infection of Fusarium oxysporum in roots, suggesting that it might be involved in defense against many diseases including root rot in P. notoginseng (Tang et al. 2015).

10.4.7 Lipoxygenase (LOX) LOX enzyme (EC1.13.11.12) ubiquitously occurring in plants and mammals catalyzes hydroperoxidation of polyunsaturated fatty acids. LOX-derived oxylipins in plants such as JA play a role in wound healing and defense mechanism, while they are involved in asthma, heart disease, and inflammation in mammals (Andreou and Feussner 2009). Bae et al. 2016 characterized five LOX cDNA clones from P. ginseng. PgLOXs are classified into two diverse classes of 9-LOX (LOX1, LOX2, and LOX3) and 13-LOX (LOX4, LOX5). PgLOXs are highly expressed in aerial parts of the plant. PgLOXs were strongly induced by bacterial infection. However, ginseng 13-LOXs upregulated upon wounding that may involve in C6 volatiles and jasmonic acid production at the wounded sites. Rahimi et al. (2016b) also identified a JA biosynthetic 13-LOX (PgLOX6) in P. ginseng that promotes ginsenoside production. Overexpressing PgLOX6 in Arabidopsis increases amounts of JA signaling molecules and squalene content. Meanwhile, MeJA treatment of

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transgenic ginseng roots overexpressing PgLOX6 increases JA signaling molecules and ginsenoside content. Altogether, these results provide insights into the biological significance of PgLOXs in plant interaction with environmental stresses as well as JA role in biosynthesizing secondary metabolites and ginsenoside production (Balusamy et al. 2015; Rahimi et al. 2014, 2015a).

mannitol in older ginseng leaves (Kim et al. 2018) (Table 10.1). Compounds with strong osmotic properties (quaternary ammonium compounds, GPC, and sugar alcohol mannitol), as well as amino acidderived metabolites (polyamines and gammaaminobutyrate (GABA)) are accumulated alongside high salts concentrations in old ginseng leaves of the field cultivated 4–6-year-old ginseng. Thus, it can be proposed that these plants suffering from osmotic stress adapt metabolism to adverse growth condition and improve stress resistance with the action of multiple compounds.

10.5

Defensive Metabolites in Ginseng

10.5.1 Lipid Metabolites The free fatty acids including 16:0 palmitate and 18:0 stearate are decreased in 4-year-old leaves, whereas the oxidized fatty acid intermediates such as 2-hydroxypalmitate and 2-hydroxysterate are accumulated in 6-year-old leaves indicating that aboveground organs of ginseng plant may suffer from increased oxidative stress. The reduced level of linoleic acid phospholipids (the most abundant fatty acid in roots of ginseng) and increased level of free fatty acids and other intermediates (glycerophosphorylcholine (GPC)) propose an active defense mechanism in 6-year-old plant via reprogramming lipid metabolic pathways and membrane lipids remobilization (Zhang et al. 2013; Kim et al. 2018) (Table 10.1).

10.5.2 Soluble Sugars and Osmoprotectants The soluble sugar level changes link with the metabolism in plant with the environment, as both biotic/abiotic stresses and sink activities affect the sugar levels. Raffinose and galactinol accumulate in both 4-year-old leaves and roots which may act as the osmoprotectants in defenseresponse against drought and salt stresses. Sugar alcohols such as sorbitol and mannitol have been involved in tolerating drought and salinity stresses. The dramatic increase of mannitol and the decrease of sorbitol in 6-year-old leaves indicate that the preferred osmoprotectant may be

10.5.3 Antioxidants A variety of reactive molecules generates Met sulfoxide, an oxidized metabolite involved in plant oxidative stresses. The increased relative ratio of methionine sulfoxide to methionine in 6year-old leaves suggests that 6-year-old plant suffered from oxidative stress. Similarly, reduced level of ascorbate and increased level of dehydroascorbate in aged ginseng leaves are consistent with the age-dependent oxidative stress. Therefore, key characteristics of aging in ginseng are the higher level of oxidative stress as well as losing the membrane integrity. However, higher levels of antioxidants are produced against ROS production in 6-year-old plant (Kim et al. 2018). It can suggest the aged plants adaption to stress condition through the balance between ROS production and quenching. Production of endogenous antioxidants including ascorbate, glutathione, and vitamins is the ginseng strategies to avoid oxidative damages. Ascorbate, glutathione, and tocopherol pools also play a role as the redox buffers in plant cells. The vitamins B, C, and E as the antioxidant metabolites change in ginseng leaves as the plant aged. Pantothenate (vitamin B5) and alphatocopherol (vitamin E) accumulate in 4- and 6year-old leaves triggering antioxidant defense system in aged ginseng leaves (Table 10.1). Alpha-tocopherol in chloroplast membranes acts

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Table 10.1 Top 40 metabolites showing strong changes in 6-year-old leaves compared with 1-year-old leaves

Red and green shaded cells indicate that the mean values are significantly higher and lower, respectively a

indicates metabolites responsible for the separation by PLS-DA. P-value and FDR-value indicate the significance and false discovery rate of difference of the relative metabolite levels between Y1L and Y6L, respectively. Adapted from Kim et al. 2018

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in quenching ROS and protecting lipid from peroxidation under oxidative stress or photooxidation. Moreover, the increased level of these antioxidative metabolites could avoid accumulation of oxidized fatty acids in matured leaves.

Panax genus, so that they can thrive in the environment that they are in. The list of genes discussed here will be extended when the new genes involved in ginsenoside biosynthesis and genes responsible for stresses identified in future. In accordance with the recent findings, diverse array of genes and isoforms/isoenzymes found in ginseng are formed from the gene duplication as a result of gene expansion. However, the mechanisms driving the formation of ginsenosides and diversification of ginsenoside pathways in Panax species are yet to be elucidated. There were few attempts made to produce stress-tolerant crops/plants using ginseng stress-related genes having interesting future. Also, there are some efforts to produce ginsenosides by heterologously expressing ginseng genes in yeast, tobacco which might leads to the production of ginsenosides that benefits the society.

10.5.4 Secondary Metabolites The ginseng plant evolves to avoid oxidative damages by production of secondary metabolites. In correlation with the therapeutic significance of older ginseng roots, phenolics (five benzenoids and five phenylpropanoids) and alkaloid (tryptophan betaine) accumulate in 4- and 6-year-old roots as compared with 1-year-old roots, suggesting that mainly carbon (C)-based secondary metabolites are accumulated in ginseng roots (Kim et al. 2018). Flavonols including quercetin, kaempferols, and flavanone naringenin are identified in ginseng leaves. Kaempferol 7-O-glucoside and kaempferol highly increase in 4-year-old leaves compared with 1-year-old leaves, while quercetin 3-O-glucoside increases in 4-year-old leaves but highly decreases in 6-year-old leaves. The benzenoids, such as benzyl alcohol, vanillate, antioxidant polyphenol, and a type of phenolic acid also increase in 4- and 6year-old roots (Kim et al. 2018). All these are consistent with the high accumulation of antioxidants in old ginseng leaves inhibiting oxidative stress. Though, most phenylpropanoids do not change in the roots, except secoisolariciresinol accumulating in 6-year-old roots. Interestingly, heterologous expression of ginseng geraniol 10-hydroxylase P450 gene in Arabidopsis caused terpenoid indole alkaloid dihydrositsirikine production and also conferred enhanced resistance to bacterial infection (Balusamy et al. 2017).

10.6

Summary and Conclusions

A slow growing, perennial ginseng has adopted itself according to the conditions over the course of evolution. The ginsenoside gene cassettes were also evolved between different species of

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synthase genes associated with saponin biosynthesis in Panax ginseng. Plant Cell Physiol 52(1):125–137 Kim OT, Yoo NH, Kim GS, Kim YC, Bang KH, Hyun DY, Kim SH, Kim MY (2013) Stimulation of Rg3 ginsenoside biosynthesis in ginseng hairy roots elicited by methyl jasmonate. Plant Cell Tissue Organ Cult (PCTOC) 112(1):87–93 Kim YJ, Jang MG, Noh HY, Lee HJ, Sukweenadhi J, Kim JH et al (2014a) Molecular characterization of two glutathione peroxidase genes of Panax ginseng and their expression analysis against environmental stresses. Gene 535(1):33–41 Kim YJ, Lee HJ, Jang MG, Kwon WS, Kim SY, Yang DC (2014b) Cloning and characterization of pathogenesis-related protein 4 gene from Panax ginseng. Russ J Plant Physiol 61(5):664–671 Kim YJ, Lee OR, Oh JY, Jang MG, Yang DC (2014c) Functional analysis of 3-hydroxy-3-methylglutaryl coenzyme a reductase encoding genes in triterpene saponin-producing ginseng. Plant Physiol 165(1):373– 387 Kim YK, Kim YB, Uddin MR, Lee S, Kim SU, Park SU (2014d) Enhanced triterpene accumulation in Panax ginseng hairy roots overexpressing mevalonate-5pyrophosphate decarboxylase and farnesyl pyrophosphate synthase. ACS Synth Biol 3(10):773–779 Kim SC, Choi G, Jung SC, Kim W, Lim S, Im WT (2015a) U.S. Patent Application No. 14/857,703 Kim YJ, Zhang D, Yang DC (2015b) Biosynthesis and biotechnological production of ginsenosides. Biotechnol Adv 33(6):717–735 Kim YJ, Joo SC, Shi J, Hu C, Quan S, Hu J, Sukweenadhi J, Mohanan P, Yang DC, Zhang D (2018) Metabolic dynamics and physiological adaptation of Panax ginseng during development. Plant Cell Rep 37(3):393–410 Kiselev KV, Kusaykin MI, Dubrovina AS, Bezverbny DA, Zvyagintseva TN, Bulgakov VP (2006) The rolC gene induces expression of a pathogenesis-related beta-1,3-glucanase in transformed ginseng cells. Phytochemistry 67(20):2225– 2231 Kochan E, Balcerczak E, Lipert A, Szymańska G, Szymczyk P (2018) Methyl jasmonate as a control factor of the synthase squalene gene promoter and ginsenoside production in American ginseng hairy root cultured in shake flasks and a nutrient sprinkle bioreactor. Ind Crops Prod 115:182–193 Koiwa H, Bressan RA, Hasegawah PM (1997) Regulation of protease inhibitors and plant defense. Trends Plant Sci 2:379–384 Lee MH, Jeong JH, Seo JW, Shin CG, Kim YS, In JG, Yang DC, Yi JS, Choi YE (2004) Enhanced triterpene and phytoesterol biosynthesis in Panax ginseng overexpressing Squalene synthase gene. Plant Cell Physiol 45(8):976–984 Lee OR, Kim YJ, Balusamy SRD, Khorolragchaa A, Sathiyaraj G, Kim MK, Yang DC (2012a) Expression of the ginseng PgPR10-1 in Arabidopsis confers

resistance against fungal and bacterial infection. Gene 506(1):85–92 Lee OR, Pulla RK, Kim YJ, Balusamy SRD, Yang DC (2012b) Expression and stress tolerance of PR10 genes from Panax ginseng CA Meyer. Mol Biol Rep 39(3):2365–2374 Leubner-Metzger G, Meins F Jr (1999) Functions and regulation of plant b-1,3-glucanases (PR-2). In: Muthukrishnan S (ed) Datta SK. CRC Press, Boca Raton, pp 49–76 Liang HC, Gao LL, Hu ZF, Gong T, Chen JJ, Yang JL, Zhu P (2016) Expression, subcellular localization and characterization of dammarenediol-II synthase of Panax ginseng in Saccharomyces cerevisiae. Yao xue xue bao = Acta pharmaceutica Sinica 51(6):998–1003 Liu WJ, Lv HZ, He L, Song JY, Sun C, Luo HM, Chen SL (2016) Cloning and bioinformatic analysis of HMGS and HMGR genes from Panax notoginseng. Chin Herbal Med 8(4):344–351 Liu T, Luo T, Guo X, Zou X, Zhou D, Afrin S, Li G, Zhang Y, Zhang R, Luo Z (2019) PgMYB2, a MeJAresponsive transcription factor, positively regulates the dammarenediol synthase gene expression in Panax ginseng. Int J Mol Sci 20(9):2219 Lu C, Zhao S, Wei G, Zhao H, Qu Q (2017a) Functional regulation of ginsenoside biosynthesis by RNA interferences of a UDP-glycosyltransferase gene in Panax ginseng and Panax quinquefolius. Plant Physiol Biochem 111:67–76 Lu C, Zhao SJ, Wang XS (2017b) Functional regulation of a UDP-glucosyltransferase gene (Pq3-O-UGT1) by RNA interference and overexpression in Panax quinquefolius. Plant Cell Tissue Organ Cult (PCTOC) 129 (3):445–456 Moiseyev GP, Beintema JJ, Fedoreyeva LI, Yakovlev GI (1994) High sequence similarity between a ribonuclease from ginseng calluses and fungus-elicited proteins from parsley indicates that intracellular pathogenesisrelated proteins are ribonucleases. Planta 193(3):470– 472 Moiseyev GP, Fedoreyeva LI, Zhuravlev YN, Yasnetskaya E, Jekel PA, Beintema JJ (1997) Primary structures of two ribonucleases from ginseng calluses. New members of the PR-10 family of intracellular pathogenesis-related plant proteins. FEBS Lett 407:207–210 Myagmarjav D, Sukweenadhi J, Kim YJ, Jang MG, Rahimi S, Silva J, Choi JY, Mohanan P, Kwon WS, Kim CG, Yang DC (2017) Molecular characterization and expression analysis of pathogenesis related protein 6 from Panax ginseng. Russ J Genetics 53 (11):1211–1220 Nag SA, Qin JJ, Wang W, Wang MH, Wang H, Zhang R (2012) Ginsenosides as anticancer agents: invitro and invivo activities, structure activity relationships and molecular mechanisms of action. Frontiers Pharmacol 3(25):1–18 Oh JY, Kim YJ, Jang MG, Joo SC, Kwon WS, Kim SY, Jung SK, Yang DC (2014) Investigation of

140 ginsenosides in different tissues after elicitor treatment in Panax ginseng. J Ginseng Res 5:1–8 Park SB, Chun JH, Ban YW, Han JY, Choi YE (2016) Alteration of Panax ginseng saponin composition by overexpression and RNA interference of the protopanaxadiol 6-hydroxylase gene (CYP716A53v2). J Ginseng Res 40(1):47–54 Pulla RK, Lee OR, In JG, Kim YJ, Senthil K, Yang DC (2010) Expression and functional characterization of pathogenesis-related protein family 10 gene, PgPR102, from Panax ginseng CA Meyer. Physiol Mol Plant Pathol 74(5–6):323–329 Pulla RK, Lee OR, In JG, Parvin S, Kim YJ, Shim JS, Sun H, Kim YJ, Senthil K, Yang DC (2011) Identification and characterization of class I chitinase in Panax ginseng C. A. Meyer. Mol Biol Rep 38:95– 102 Purev M, Kim YJ, Kim MK, Pulla RK, Yang DC (2010) Isolation of a novel catalase (Cat1) gene from Panax ginseng and analysis of the response of this gene to various stresses. Plant Physiol Biochem 48(6):451– 460 Rahimi S, Devi BSR, Khorolragchaa A, Kim YJ, Kim JH, Jung SK, Yang DC (2014) Effect of salicylic acid and yeast extract on the accumulation of jasmonic acid and sesquiterpenoids in Panax ginseng adventitious roots. Russ J Plant Physiol 61(6):811–817 Rahimi S, Kim YJ, Devi BSR, Khorolragchaa A, Sukweenadhi J, Yang DC (2015a) Isolation and characterization of Panax ginseng geranylgeranyldiphosphate synthase genes responding to drought stress. Eur J Plant Pathol 142(4):747–758 Rahimi S, Kim YJ, Yang DC (2015b) Production of ginseng saponins: elicitation strategy and signal transductions. Appl Microbiol Biotechnol 99 (17):6987–6996 Rahimi S, Kim YJ, Devi BSR, Oh JY, Kim SY, Kwon WS, Yang DC (2016a) Sodium nitroprusside enhances the elicitation power of methyl jasmonate for ginsenoside production in Panax ginseng roots. Res Chem Intermed 42(4):2937–2951 Rahimi S, Kim YJ, Sukweenadhi J, Zhang D, Yang DC (2016b) PgLOX6 encoding a lipoxygenase contributes to jasmonic acid biosynthesis and ginsenoside production in Panax ginseng. J Experiment Bot 67 (21):6007–6019 Rahimi S, Kim J, Mijakovic I, Jung K, Choi G, Kim SC, Kim YJ (2019) Triterpenoid-biosynthetic UDPglycosyltransferases from plants. Biotechnol Adv Rouhier N, Gelhaye E, Sautiere PE, Brun A, Laurent P, Tagu D, Gerard J, de Fäy E, Meyer Y, Jacquot JP (2001) Isolation and characterization of a new peroxiredoxin from poplar sieve tubes that uses either glutaredoxin or thioredoxin as a proton donor. Plant Physiol 127:1299–1309 Sathiyamoorthy S, In JG, Gayathri S, Kim YJ, Yang DC (2010) Generation and gene ontology based analysis of expressed sequence tags (EST) from a Panax ginseng CA Meyer roots. Mol Biol Rep 37(7):3465– 3472

S. Rahimi et al. Sathiyaraj G, Lee OR, Parvin S, Khorolragchaa A, Kim YJ, Yang DC (2011) Transcript profiling of antioxidant genes during biotic and abiotic stresses in Panax ginseng CA Meyer. Mol Biol Rep 92(4):2761– 2769 Scandalios JG (1990) Response of plant antioxidant defense genes to environmental stress. Adv Genet 28:1–41 Scandalios JG (1994) Regulation and properties of plant catalases. In: Foyer CH, Mullineaux PM (eds) Causes of Photooxidative stress and amelioration of defense dystems in plants. CRC Press, Boca Raton, FL, ISBN 0-8493-5443-9, p 275e315 Schramek N, Huber C, Schmidt S, Dvorski SE, Kmispel N, Ostrozhenkova E, Pena-Rodriguez LM, Cusido RM, Wischmann G, Eisenreich W (2014) Biosynthesis of ginsenosides in field-grown Panax ginseng. JSM Biotechnol Biomed Eng 2:1033–1048 Sels J, Mathys J, De Coninck BMA, Cammue BPA, De Bolle MFC (2008) Plant pathogenesis-related (PR) proteins: a focus on PR peptides. Plant Physiol Biochem 46(11):941–950 Shigeoka S, Ishikawa T, Tamoi M, Miyagawa Y, Takeda T, Yabuta Y, Yoshimura K (2002) Regulation and function of ascorbate peroxidase isoenzymes. J Experiment Bot 53:1305–1319 Silva J, Kim YJ, Sukweenadhi J, Rahimi S, Kwon WS, Yang DC (2016) Molecular characterization of 5chlorophyll a/b-binding protein genes from Panax ginseng Meyer and their expression analysis during abiotic stresses. Photosynthetica 54(3):446–458 Subramaniyam S, Mathiyalagan R, Natarajan S, Kim YJ, Jang MG, Park JH, Yang DC (2014) Transcript expression profiling for adventitious roots of Panax ginseng Meyer. Gene 546(1):89–96 Sukweenadhi J, Kim YJ, Rahimi S, Silva J, Myagmarjav D, Kwon WS, Yang DC (2017) Overexpression of a cytosolic ascorbate peroxidase from Panax ginseng enhanced salt tolerance in Arabidopsis thaliana. Plant Cell Tissue Organ Cult (PCTOC) 129(2):337–350 Tang MQ, Min DD, Li G, Jiang N, Ye YF (2015) Cloning and functional characterization of pathogenesis-related PR10-1 gene in Panax notoginseng. Yao xue xue bao = Acta pharmaceutica Sinica 50(2):227–323 Terada T, Oshida T, Nishimura M, Maeda H, Hara T, Hosomi S, Mizoguchi T, Nishihara T (1992) Study on human erythrocyte thioltransferase: comparative characterization with bovine enzyme and its physiological role under oxidative stress. J Biochem 111:688–692 Van Loon LC (1999) Occurrence and properties of plant pathogenesis-related proteins. In: Datta SK, Muthukrishnan S (eds) Pathogenesis-related proteins in plants. CRC Press, Boca Raton, pp 1–19 Van Loon LC, Pierpoint WS, Boller T, Conejero V (1994) Recommendations for naming plant pathogenesisrelated proteins. Plant Mol Biol Reporter 12:245–264 Van Loon LC, Rep M, Pieterse CM (2006) Significance of inducible defense-related proteins in infected plants. Ann Rev Phytopathol 44:135–162

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Vigers AJ, Roberts WK, Selitrennikoff CP (1991) A new family of plant antifungal proteins. Mol Plant-Microbe Interact 4:315–323 Wei W, Wang P, Wei Y, Liu Q, Yang C, Zhao G, Yue J, Yan X, Zhou Z (2015) Characterization of Panax ginseng UDP-glycosyltransferases catalyzing protopanaxatriol and biosyntheses of bioactive ginsenosides F1 and Rh1 in metabolically engineered yeasts. Mol Plant 8(9):1412–1424 Wells WW, Xu DP, Yang Y, Rocque PA (1990) Mammalian thioltransferase (glutaredoxin) and protein disulfide isomerase have dehydroascorbate reductase activity. J Biol Chem 265:15361–15364 Willekens H, Inze D, Van Montagu M, Van Camp W (1995) Catalases in plants. Mol Breed 1:207–228 Xu S, Zhao C, Wen G, Zhang H, Zeng X, Liu Z, Xu S, Lin C (2018) Longitudinal expression patterns of HMGR, FPS, SS, SE and DS and their correlations with saponin contents in green-purple transitional aerial stems of Panax notoginseng. Ind Crops Prod 119:132–143 Yan X, Fan Y, Wei W, Wang P, Liu Q, Wei Y, Zhang L, Zhao G, Yue J, Zhou Z (2014) Production of bioactive ginsenoside compound K in metabolically engineered yeast. Cell Res 24(6):770–773

Yang Y, Liu DQ, Ge F, Chen CY (2015) Effect of overexpressing farnesyl pyrophosphate synthase (FPS) gene of Panax notoginseng cell on saponin synthesis. Modern Food Science & Technology 31:59–64 Yue CJ, Zhong JJ (2005) Purification and characterization of UDPG: ginsenoside Rd glucosyltransferase from suspended cells of Panax notoginseng. Process Biochem 40(12):3742–3748 Zhang XJ, Huang LL, Cai XJ, Li P, Wang YT, Wan JB (2013) Fatty acid variability in three medicinal herbs of Panax species. Chem Central J 7:12–21 Zhang X, Yu Y, Jiang S, Yu H, Xiang Y, Liu D, Qu Y, Cui X, Ge F (2019) Oleanane-Type Saponins Biosynthesis in Panax notoginseng via Transformation of bAmyrin Synthase Gene from Panax japonicus. J Agric Food Chem Zhao F, Bai P, Liu T, Li D, Zhang X, Lu W, Yuan Y (2016) Optimization of a cytochrome P450 oxidation system for enhancing protopanaxadiol production in Saccharomyces cerevisiae. Biotechnol Bioeng 113 (8):1787–1795

Genomic Resources for Ginseng Genome Studies

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Murukarthick Jayakodi and Tae-Jin Yang

Abstract

Advances in sequencing technologies have enabled scientists to sequence both DNA and RNA molecules from ginseng (Panax ginseng) and its close relatives. These datasets include transcriptome of various tissues and treatments and reference draft genome assembly. Such a wealth of information has created a necessity to generate databases and tools for retrieving and analysing data to assist researchers and breeders. Ginseng genome database (http://ginsengdb.snu.ac.kr/), an open-access database developed for exploring various genomic features in ginseng. The user-friendly utilities in this database provide free access to genome sequences and various structural and functional annotations. A common BLAST tool included for homology-based comparison of ginseng genes and genome sequences and transcriptome generated by different studies. Visualization

M. Jayakodi
T.-J. Yang (&) Department of Plant Science, Plant Genomics and Breeding Institute, Research Institute of Agriculture and Life Sciences, College of Agriculture and Life Sciences, Seoul National University, Seoul, Korea e-mail: [email protected] M. Jayakodi Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Gatersleben, 06466 Seeland, Germany

of various annotation information in single frame is enabled to track every position of ginseng reference genome. The metabolic pathway annotation and genome-scale metabolic network provided in genome database are invaluable resources for enhanced metabolic production through genetic engineering. In this chapter, we have given a comprehensive overview of ginseng genome database, tools and its broad utilities.

11.1

Introduction

The genomic and evolutionary characterization of the ginseng (Panax ginseng) genome has rapidly been advanced by high-throughput whole genome sequencing (Lee et al. 2017a), transcriptome (Jayakodi et al. 2015, 2014, 2018b) and the availability of reference genome sequences (Kim et al. 2018). The draft genome of ginseng represents the linear form of chromosome fragments, that contain diverse genomic elements including genes, repeats and noncoding RNAs. A user-friendly interface to access these genomic elements leads to better understanding of biological and metabolic process and identification of functional elements. Due to large genome size of ginseng (*3.6 Gb), the resulting genomic data sets are often very large, which cannot be easily handled by non-computational researchers and breeders. Ginseng genome contains more than 50,000 genes classified into

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various gene families. Further, ginseng research communities focus on diverse disciplines ranging from genetic diversity and molecular marker discovery to metabolic profiling and pathway characterization to gene cloning and modification. Information related to gene expression and differential expression between tissues and biotic/abiotic stress condition is also critical. An interactive exploration of genomic features and metabolic pathways allow researchers to easy interpretation and formulate novel hypotheses with relevant evident from multiple genomic data. Therefore, to assist and accelerate diverse domains of ginseng research, a ginseng genome database or resource repository is necessary and implementation of common algorithms for data analysis and visualization including BLAST and genome browser are also essential. Many species having their genome sequenced provide a wealth of information with systematic retrieval systems including families like Poaceae (Beier et al. 2018) and Brassicaceae (Cheng et al. 2011). However, the available genomic resources for the family Araliaceae in which ginseng belonging to are relatively low. This was reflected in slow progress of crop improvement and metabolic enhancement. In the beginning, an adventitious root transcriptome database (http:// im-crop.snu.ac.kr/transdb/index.php) was developed for ginseng cultivars for public usage (Jayakodi et al. 2014). Until 2018, a total of 17,114 expressed sequence tag (EST) have been deposited to NCBI with redundancy. Recently, a comprehensive genome database (http:// ginsengdb.snu.ac.kr) was developed with broad utilities (Jayakodi et al. 2018a). In this chapter, we give you an overview of ginseng genome database and its major options and tools. This will clarify the type and kind of data set can be retrieved and analysed using ginseng genome database.

11.2

Gene Annotations

Ginseng genome database contains various annotation information for genes and genomes. Genes were structurally annotated and

functionally assigned using InterPro (Hunter et al. 2008), Blast2go (Conesa et al. 2005), KEGG (Moriya et al. 2007) and BLASTP search with well-curated protein databases. In addition, genes were classified based on protein domain (Pfam) and InterPro, which encourage users to retrieve and analyse a specific gene family of their interest. Genes encoding enzymes involved in various metabolic pathways were annotated using KAAS server (Moriya et al. 2007). Users can search specific secondary metabolic pathways and retrieve complete set of genes associated with that pathway. Further, the utility of genome-scale metabolic network enabled us to interact with a specific metabolic pathway visually with zoom in and scroll options. The metabolic network includes a total of 4,946 genes covering to 2,194 enzyme-catalyzed and proteinmediated transport reactions involving 2003 unique metabolites across six intracellular compartments. A separate page dedicated for ginsenoside biosynthetic pathway as it has attracted wider attention among ginseng researchers. This page contains list of genes including paralogs associated with each step of ginsenoside pathway (Kang et al. 2018; Lee et al. 2017b). Furthermore, genes were grouped into transcription factors (TF) and transcription regulators (TR), which included a total of 4,439 TF and TR classified into 94 TF families. Moreover, a digital gene expression profile panel was included for showing gene expression in intuitive plot with respect to various tissues and abiotic stress conditions.

11.3

Download and External Links

Ginseng genome database provides a section to bulk download of multiple sets of dataset. This included reference draft genome, CDS and protein sequences in FASTA format, gene structural and repetitive elements annotations in GFF3 format. RNA-Seq raw data generated from various tissues and stress conditions, as part of ginseng genome project can easily be downloaded. Additionally, the genome-scale metabolic

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network can be downloaded, which might be useful to update the metabolic network by different research groups.

ginsenoside biosynthetic pathway. Under this panel, user can find and retrieve the putative biosynthetic pathway for ginsenoside biosynthesis and genes associated with each reaction steps. Lastly, gene expression panel was included to explore the specific expression pattern of user’s gene of interest. This panel included more than 20 available RNA-Seq samples generated from ginseng genome project, and it can be seen according to tissue, developmental stage and abiotic stresses. This resource can be useful for visualization of single genes across tissues or stress conditions.

11.4

Guidelines for Using Ginseng Genome Database

Ginseng genome database contains two major sections, Search and Tools. The search and tools sections included list of panels that facilitate users for their very specific search or browse through the available number of result categories and perform sequences analysis.

11.4.2 Tools Options 11.4.1 Search Options In search panel, user can retrieve number of genes associated with a specific gene family either by entering the common gene ID used in annotation programs such as Pfam, Interpro and KEGG or choosing a browse option and clicking user-desired gene families. Gene annotation panel is specifically designed for researchers who are interested in a specific gene or a small set of genes. This panel provides detailed structural and functional descriptions related to user-defined specific genes. Transcription factors are important regulators, targeted by different functional research groups in ginseng communities. Thus, a specific panel included for retrieving genes grouped into a specific transcription factor or transcription regulator family. Genes encoding enzymes involved in primary and secondary metabolic pathways were annotated in ginseng (Kim et al. 2018). This information can be obtained under metabolic pathway panel. Furthermore, a genome-scale metabolic network constructed in ginseng genome project (Kim et al. 2018) is available under the panel metabolic network visual. This network helps user to explore most of the metabolic pathways visually in different compartments of a cell. Exclusively, a panel is developed for exploring

In tools section, users can download genes/scaffold sequences, perform homology search and visualize the genomic features. Using sequence or subseq retrieve panel, single or multiple set of sequences based unique IDs or genomic physical coordinates can be retrieved for further downstream analysis. For homology search, custom databases were made for ginseng researchers. This included the draft reference genome sequence from cultivar Chunpoong, the annotated genes (CDS and proteins) and the individual transcriptome assemblies used for gene annotations and publicly available transcriptome resources. These can be accessed via http://ginsengdb.snu.ac.kr/blast/blast.php. Further, an interactive graphical interface is for visualizing ginseng genomic features through a fast and scalable instance of Jbrowse (http:// ginsengdb.snu.ac.kr/Browser/). This genome browsers represent scaffolds as linear sequence and allow users to zoom and scroll through each region of contiguous sequence. Functional genomics elements including genes, mRNA/CDS and spliced forms can be seen simultaneously (Fig. 11.2). In addition, this browser integrates resources of gene prediction models, transcriptome assemblies and alignments, repetitive elements and noncoding RNAs (Fig. 11.1).

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Fig. 11.1 Homepage and sections in ginseng genome database. List of navigation menus including Search, Tools, Status, Links and Download at the top of the webpage

11.5

Conclusion and Perspective

The availability of high-quality reference genome provides broad opportunities to facilitate genetic mapping, quantifying variations for diversity, metabolic pathway characterization and gene editing. The continuous accumulation of more data from P. ginseng using different platforms creates necessity to incorporate such information into existing database and make them available to researchers. Ginseng genome

database fulfils and contains datasets for aforementioned goals and research. Currently, ginseng genome database is the only repository for ginseng research community and represents species in the family Araliaceae. The ginseng genome database will help scientists and breeders to fully and efficiently explore the information on genomic, transcriptomic and metabolic datasets. In addition, ginseng genome database serves as a valuable resource for comparative genomics, crop evolution and pharmacogenomics and the breeders of ginseng.

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Fig. 11.2 A genome browser representing ginseng genomic features. The gene features including exon and CDS were shown along with the tracks of predicted gene model and supporting transcriptome evidences

In the future, the continuous developments in the generation of sequencing technology drive further improvements in the reference genome assembly, which lead to the generation of chromosome-scale pseudomolecules for ginseng. The high-quality assemblies will allow cataloguing of genome diversity, high-resolution genetic mapping, structural variations, improved gene models and repetitive elements characterization and gene expression atlas. Further, whole genome methylation and small RNA-Seq data for ginseng might also be generated. Ideally, these datasets can be searchable, interlinked and easily retrieved by users for diverse research purposes. It is anticipated that ginseng genome database should adopt new server technologies and design to incorporate such a wealth of large dataset to become much more user-friendly and quick data retrieval system.

References Beier S, Bolser DM, Scholz U, Spannagl M, Kersey PJ (2018) Databases and tools for the analysis of the barley genome. In: The Barley Genome. Springer, pp 377–394 Cheng F, Liu S, Wu J, Fang L, Sun S, Liu B, Li P, Hua W, Wang X (2011) BRAD, the genetics and genomics database for Brassica plants. BMC Plant Biol 11(1):136 Conesa A, Götz S, García-Gómez JM, Terol J, Talón M, Robles M (2005) Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics 21(18):3674–3676 Hunter S, Apweiler R, Attwood TK, Bairoch A, Bateman A, Binns D, Bork P, Das U, Daugherty L, Duquenne L (2008) InterPro: the integrative protein signature database. Nucleic Acids Res 37(suppl_1):D211–D215 Jayakodi M, Choi B-S, Lee S-C, Kim N-H, Park JY, Jang W, Lakshmanan M, Mohan SV, Lee D-Y, Yang T-J (2018a) Ginseng Genome Database: an openaccess platform for genomics of Panax ginseng. BMC Plant Biol 18(1):62

148 Jayakodi M, Lee S-C, Lee YS, Park H-S, Kim N-H, Jang W, Lee HO, Joh HJ, Yang T-J (2015) Comprehensive analysis of Panax ginseng root transcriptomes. BMC Plant Biol 15(1):138 Jayakodi M, Lee S-C, Park H-S, Jang W, Lee YS, Choi B-S, Nah GJ, Kim D-S, Natesan S, Sun C (2014) Transcriptome profiling and comparative analysis of Panax ginseng adventitious roots. J Ginseng Res 38 (4):278–288 Jayakodi M, Lee S-C, Yang T-J (2018b) Comparative transcriptome analysis of heat stress responsiveness between two contrasting ginseng cultivars. J Ginseng Res Kang KB, Jayakodi M, Lee YS, Nguyen VB, Park H-S, Koo HJ, Choi IY, Kim DH, Chung YJ, Ryu B (2018) Identification of candidate UDP-glycosyltransferases involved in protopanaxadiol-type ginsenoside biosynthesis in Panax ginseng. Sci Rep 8(1):11744 Kim NH, Jayakodi M, Lee SC, Choi BS, Jang W, Lee J, Kim HH, Waminal NE, Lakshmanan M, Van

M. Jayakodi and T.-J. Yang Nguyen B (2018) Genome and evolution of the shade-requiring medicinal herb Panax ginseng. Plant Biotechnol J Lee J, Waminal NE, Choi H-I, Perumal S, Lee S-C, Nguyen VB, Jang W, Kim N-H, Gao L-z, Yang T-J (2017a) Rapid amplification of four retrotransposon families promoted speciation and genome size expansion in the genus Panax. Sci Rep 7(1):9045 Lee YS, Park H-S, Lee D-K, Jayakodi M, Kim N-H, Koo HJ, Lee S-C, Kim YJ, Kwon SW, Yang T-J (2017b) Integrated transcriptomic and metabolomic analysis of five Panax ginseng cultivars reveals the dynamics of ginsenoside biosynthesis. Frontiers in plant science 8:1048 Moriya Y, Itoh M, Okuda S, Yoshizawa AC, Kanehisa M (2007) KAAS: an automatic genome annotation and pathway reconstruction server. Nucleic Acids Res 35 (suppl_2):W182–W185

Genomes of Other Species in Panax Linn

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Zhang Guang-hui and Yang Sheng-chao

Abstract

The whole-genome sequencing technology has revolutionized the plant biological studies. In 2000, Arabidopsis thaliana was the first plant to have it genome sequenced (Initiative 2000). Since then, more than 100 plant genomes have been published (Duvick et al. 2007). Genome assembly allows for exploration of functional elements and evolutionary history of plant species. Species in Panax Linn usually have large and highly repetitive genomes. For instance, the genome sizes of P. ginseng and P. quinquefolius are estimated to be 3.12 Gb and 4.91 Gb, respectively (Chen et al. 2017). These features are the main obstacles for obtaining a high-quality reference sequence of Panax species. To date, researchers have successfully done the whole-genome sequencing project on two species in Panax Linn, namely P. ginseng and P. notoginseng by utilizing NGS technologies (Chen et al. 2017; Jayakodi et al. 2018; Jiang et al. 2017). This chapter mainly focuses on the sequencing, assembly, and annotation of the draft genome for P. notogin-

Z. Guang-hui
Y. Sheng-chao (&) National-Local, Joint Engineering Research Center on Germplasm Utilization and Innovation of Chinese Medicinal Materials in Southwest China, Yunnan Agricultural University, Kunming 650201, China e-mail: [email protected]

seng and how this genome sequence will facilitate the associated studies of P. notoginseng and Panax Linn.

12.1

Introduction to Species in Panax Linn

Panax Linn, also known as Panax genus or ginseng genus, belongs to the Araliaceae family. The species in Panax Linn are all perennial herbs and are widely distributed in Eastern Asia and Eastern North America (Lee and Wen 2004; Wen and Zimmer 1996; Zuo et al. 2011). Taxonomic studies revealed more than 18 species in Panax Linn with new species been reported in recent publications (Sharma and Pandit 2009). Currently, 14 species in Panax Linn have been commonly accepted (Table 12.1) (Committee 2013), (Ji et al. 2019). Whole-genome duplications (WGDs) are thought to play a key role in the evolution of plant morphology and physiology diversity (Paterson et al. 2010). Phylogenetic analysis employing multiple chloroplast and nuclear markers suggested that both ancient and recent WGDs have occurred within Panax Linn. The ancient WGDs might have occurred before the establishment of Panax genus as these WGDs happened earlier than the divergence between genera Panax and Aralia. Thereafter, at least two independent recent WGDs events have occurred within Panax Linn, one of which has led to the

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Table 12.1 Information of 14 Panax species (NA: not available) Name

Chromosome numbers

Distribution

Panax japonicus var. bipinnatifidus (Seemann) C. Y. Wu & K. M. Feng

2n = 24/48

Eastern Asian

Panax ginseng C. A. Mey.

2n = 48

Eastern Asian

Panax japonicus (T. Nees) C. A. Mey.

2n = 48

Eastern Asian

Panax japonicus var. angustifolius (Burkill) C. Y. Cheng & C. Y. Chu

NA

Eastern Asian

Panax japonicus var. bipinnatifidus (Seemann) C. Y. Wu & K. M. Feng

NA

Eastern Asian

Panax japonicus var. major (Burkill) C. Y. Wu & K. M. Feng

NA

Eastern Asian

Panax notoginseng (Burkill) F. H. Chen

2n = 24

Eastern Asian

Panax pseudoginseng Wall.

2n = 24

Eastern Asian

Panax quinquefolius L.

2n = 48

Eastern North American

Panax stipuleanatus H. T. Tsai & K. M. Feng

2n = 24

Eastern Asian

Panax trifolius L.

2n = 24

Eastern North American

Panax vietnamensis Ha & Grushv.

2n = 24

Eastern Asian

Panax wangianus S. C. Sun

NA

Eastern Asian

Panax zingiberensis C. Y. Wu & Feng

NA

Eastern Asian

formation of geographically isolated tetraploid (2n = 48) species P. ginseng, P. quinquefolius, and P. japonicus. The other WGDs events had occurred in P. japonicus var. bipinnatifidus, and a species complex possesses both tetraploid and diploid (2n = 24) species/populations (Manzanilla et al. 2018; Shi et al. 2015). These lineage specific duplication events have greatly contributed to the diversification of Panax species.

12.2

Sequencing of P. Notoginseng Genome

Three Panax species, namely P. ginseng, P. quinquefolius, and P. notoginseng, are highly regarded as medicinal plants and are widely cultivated in Eastern Asia (Guo et al. 2010; Jung et al. 2014). The dried root of P. notoginseng (Burk.) F.H. Chen (2n = 2x = 24), also known as sanqi, is used as traditional Chinese medicine and top-class tonic for more than 400 years

(Wang et al. 2016). It is commonly used to treat cardiovascular diseases, pain, inflammation, and trauma as well as internal and external bleeding due to injury (Ng 2010). Aiming to unveil the underlying curative effect of P. notoginseng, substantial efforts have been made in the associated phytochemistry and pharmacology studies. In recent years, the fast development of sequencing technology enabled scientists to study the P. notoginseng in a genomic view. In 2017, the first P. notoginseng whole-genome sequencing (WGS) project has been finished by a group of Chinese researchers (Chen et al. 2017). The P. notoginseng WGS project involves genome sequencing, assembly, and annotation process. Genome sequencing is a technology to determine the order of nucleotides of each chromosome in the genome of an organism. Fresh mature leaves of a single individual from a cultivated line of P. notoginseng were used for genomic DNA extraction. Purified genomic DNA was sheared into smaller fragments with

12

Genomes of Other Species in Panax Linn

random sizes by a focused ultrasonicator (Covaris, MA, USA). DNA fragments were electrophoresed in 0.8% General Purpose Agarose EGel (Invitrogen, CA, USA), by which fragments of desired lengths were obtained. Genomes of species in Panax Linn are featured by large size and high repetitive content. Based on flow cytometry analysis, the genome size of P. notoginseng was estimated to be 2.31 Gb (Chen et al. 2017). The repetitive sequences are the main obstacles in genome assembly project as the short-length reads are unable to span most of the repeat sequences. To overcome this difficulty, 34 libraries with insertion sizes ranging from 200 bp to 20 kb were constructed using the Illumina Paired-End DNA protocol. Short-insert libraries (1 kb) were sequenced on an Illumina Genome HiSeq 2500 machine using either the PE-90 or PE-150 protocol. A total of 1837.6 Gb of raw paired-end sequencing reads were generated from the two Illumina platforms. Inevitably, sequencing errors would be introduced in reads irrespective of different types of sequencing technologies. Related studies have shown that errors of Illumina reads are not random and are likely to be related to sequence context: The Phred-like Q score would drop significantly toward the end of the Illumina reads (Manley et al. 2016). Low-quality reads must be removed before the assembly. The filtering criteria for raw reads are listed as follows: (1) if more than 5% of bases in a read were N or polyA, (2) if more than 30 bases in a read were low quality, (3) if a read was contaminated with adaptor sequence, (4) if the size of a read was too small, and (5) if two copies of the paired-end reads had identical sequence (remove both copies). The resultant reads were then corrected by the SOAPec_v2.0.1 package (http://soap. genomics.org.cn) with default settings. After the strict quality control procedures, 858.6 Gb clean data were obtained for the subsequent genome assembly process.

151

12.3

Assembly of P. Notoginseng Genome

Genome assembly is the process of aligning and merging sequencing reads to reconstruct the genome sequences. De novo assembly pipeline for the P. notoginseng genome comprises four steps to cope with the high repetitive content in the genome, including the initial contig assembly step by Platanus (Kajitani et al. 2014), the first scaffolding step using paired-end reads with insert size below 10 kb by SSPACE (Boetzer et al. 2011), the second scaffolding step using all reads by SOAPdenovo (http://soap.genomics.org.cn/ soapdenovo.html), and the final gap-closing step by GapCloser (Xu et al. 2019). The above steps yielded a reference genome assembly of 2.39 Gb, with a contig N50 size of 16 kb and scaffold N50 size of 96 kb (Table 12.2). The quality of the genome assembly was evaluated by both core eukaryotic genes mapping approach (CEGMA) (Parra et al. 2007) and benchmarking universal single-copy orthologs (BUSCOs) (Simaoet al. 2015). CEGMA revealed that 198 of 248 ultra-conserved genes could be fully annotated in P. notoginseng genome assembly. BUSCO analysis revealed that 1,186 out of 1,440 plant BUSCOs (82.4%) were identified as complete BUSCOs. These results indicated high quality of assembled genome (Fig. 12.1).

12.4

Annotation of P. Notoginseng Genome

Genome annotation is the process of mining biological information in the assembled sequences. With this information, researchers are able to infer the evolutionary history and explore the molecular mechanism beneath the biosynthesis of medicinal compounds in P. notoginseng. Based on the different types of biological information, genome annotation is comprised of three parts: the repeat element annotation, protein-coding gene annotation, and nonprotein-coding gene annotation.

152

Z. Guang-hui and Y. Sheng-chao

Table 12.2 Summary of P. notoginseng genome assembly Benchmarks

Contigs

Scaffolds

Number of sequences

563,700

122,131

Total base pairs (bp)

1,994,716,493

2,394,283,436

Min sequence length (bp)

100

500

Max sequence length (bp)

199,807

834,327

Average sequence length (bp)

3,538.61

19,604.22

Median sequence length (bp)

679

4,169

N25 length (bp)

31,222

178,188

N50 length (bp)

15,987

96,155

N75 length (bp)

6,069

41,148

N90 length (bp)

1,729

11,415

N95 length (bp)

812

5,039

GC content (%)

35.01

28.65

N content (%)

0.00

18.15

Total BUSCOs =1440 71.46% (1029) Complete Single-copy BUSCOs 10.90% (157) Complete Duplicated BUSCOs 3.26% (47) Fragmented BUSCOs 14.38% (207) Missing BUSCOs

Fig. 12.1 BUSCO analysis of P. notoginseng genome assembly

The repeat content of P. notoginseng genome was identified using a hybrid approach combining ab initio and evidence-based methods. In the ab initio method, tandem repeats were identified across the genome with the Tandem Repeats

Finder (Benson 1999) program with default settings. RepeatModeler (Price et al. 2005) and LTR FINDER (Xu and Wang, 2007) were also used to identify novel repetitive elements in the P. notoginseng genome. In terms of evidencebased prediction, RepeatMasker (TaralioGraovac and Chen 2004) and RepeatProteinMask were used to identify known repeat sequences in the genome using Repbase as reference (Bao et al. 2015) (Release 16.10; http:// www.girinst.org/repbase/index.html). The final result reveals 1.71 Gb transposable element, accounting for about 75.94% of the genome. About 127.6 Mb of the genome assembly (5.32%) was identified as tandem repeats. Specifically, long terminal repeats (LTRs) made up about 66.72% of the genome assembly (Table 12.3). Hence, characteristics (large in size and high repetitive ratio) of P. notoginseng genome might be explained by extremely high LTR content as LTR is considered to be the key determinants of angiosperm genome size and genome content variation (Bennetzen and Wang 2014). The protein-coding gene annotation of P. notoginseng was performed with a combination of de novo, homology-based and transcriptsbased prediction methods. Protein sequences of all the protein-coding genes for Arabidopsis

0.00

0.00

34,126

252,431,968

328

–

264,117,857

SINE

LTR

Other

Unknown

Total

11.05

10.54

0.00

0.04

1,020,231

LINE

% in genome

443,659,497

– 18.58

0.00

17.63 0.00

421,992,896 –

0.14 0.00

3,454,752

0.81

–

19,366,906

Length (Bp)

% in genome

0.47

Length (Bp)

11,193,242

DNA

TE proteins

Repbase TEs

Table 12.3 Summary of transposable elements in P. notoginseng genome assembly

1,709,677,280

27,932,985

–

1,589,886,305

1,149,317

31,764,260

148,198,669

Length (Bp)

De novo % in genome

75.13

1.17

0.00

66.40

0.05

1.33

6.19

1,715,125,042

27,932,985

328

1,597,380,754

1,181,907

33,971,836

157,780,522

Length (Bp)

Combined TEs

75.94

1.17

0.00

66.72

0.05

1.42

6.59

% in genome

12 Genomes of Other Species in Panax Linn 153

154

Z. Guang-hui and Y. Sheng-chao

thaliana, Fragaria vesca, Malus domestica, Oryza sativa, Prunus Mume, Prunus persica, Pyrus communis, and Vitis vinifera were used in the homology-based gene annotation. In brief, protein sequences of the aforementioned species were searched against the P. notoginseng genome using TBLASTN (Camacho et al. 2009) with a cutoff E-value of 1e-5. The genomic regions of identified hits from TBLASTN were further evaluated by GeneWise (Birney et al. 2004) to predict gene models. In de novo method, AUGUSTUS (Stanke et al. 2006) and GlimmerHMM (Majoros et al. 2004) were used to predict the structures of protein-coding genes using parameters trained from A. thaliana. Transcripts of P. notoginseng were assembled using Trinity (Grabherr et al. 2011) and fed to PASA (Haas et al. 2008) to generate transcriptome-based gene models. Finally, EVidenceModeler (Haas et al. 2008) was used to combine the above gene models into weighted consensus gene structures. A total of 36,790 protein-coding genes were identified in P. notoginseng genome (Table 12.4).

Nonprotein-coding genes include tRNA, rRNA, snRNA, and miRNA. The miRNAs and snRNAs were predicted using INFERNAL v0.81 (Nawrocki et al. 2009) (http://infernal.janelia.org) and covariance models from the Rfam database (Release 11.0) (Griffithsjones et al. 2004). Annotation of tRNAs was performed using tRNAscanSE (Lowe and Eddy 1997). The rRNAs were predicted by conducting a homology search against rRNA sequences using BLAST (v2.2.29 +) with a cutoff E-value of 1e-10. A total of 8,446 nonprotein-coding genes were identified in P. notoginseng genome, as listed in Table 12.5.

12.5

Phylogenetic Analysis

To study the evolution of Panax species, root transcriptomes of three Panax species, P. ginseng, P. notoginseng, and P. quinquefolius were used to construct the phylogenetic tree. The result suggested that the diploid P. notoginseng diverged from the other two tetraploid Panax plants before a putative lineage-specific WGD

Table 12.4 Summary of protein-coding gene annotation of P. notoginseng genome assembly Methods De novo Homolog

RNA-Seq

AUGUSTUS

Gene number

Average mRNA lengths (bp)

Average CDS lengths (bp)

32,420

2,662.85

996.61

GlimmerHMM

36,927

2,017.43

754.86

A. thaliana

32,487

3,475.85

981.78

F. vesca

32,318

3,623.00

1,002.24

M. domestica

38,614

3,087.62

884.73

O. sativa

36,599

2,633.95

773.90

P. mume

34,899

3,489.65

984.79

P. persica

33,964

3,619.11

1,014.39

P. communis

35,187

3,603.42

972.83

V. vinifera

36,331

3,418.14

958.32

Flower

38,564

4,105.35

1,971.78

Fruit

36,605

4,194.35

1,960.81

Leaf

33,719

4,231.62

1,939.68

Primary root

30,596

4,260.11

1,889.56

Secondary root

36,316

4,042.09

1,920.81

Stem

34,860

4,188.53

1,908.34

36,790

3,307.48

942.43

Evidence Modeler

12

Genomes of Other Species in Panax Linn

155

Table 12.5 Summary of nonprotein-coding gene annotation of P. notoginseng genome Type

Copy number

Average lengths (bp)

Total length (bp)

Percentage in genome (%)

miRNA

557

119.38

66,493

0.00278

tRNA

1,665

75.28

125,345

0.00524

rRNA

1,762

201.45

354,962

0.01483

18S

234

732.75

171,463

0.00716

28S

466

132.13

61,572

0.00257

5.8S

96

144.57

13,879

0.00058

5S

966

111.85

108,048

0.00451

snRNA

4,462

113.50

506,433

0.02115

CD-box

3,888

107.27

417,052

0.01742

HACA-box

50

147.58

7,379

0.00031

Splicing

524

156.49

82,002

0.00343

Total

8,446

124.70

1,053,233

0.04399

P. notoginseng

P. ginseng

WGD

P. quinquefolius Divergence, substitutions/site 0

0.006

0.012

0.018

0.024

0.03

Fig. 12.2 Phylogenetic analysis of three Panax species

event (Fig. 12.2). This finding is consistent with the previous evolutionary studies employing expressed sequence tags (Choi et al. 2013), (Kim et al. 2014).

12.6

Conclusions and Perspectives

After the first release of P. notoginseng genome assembly, another two versions of P. notoginseng draft genome assemblies were reported (Zhang et al. 2017; Fan et al. 2018). However, no significant improvements were made regarding the completeness of the genome as both studies mainly use short reads from Illumina sequencing platform for genome assembly. The advancement in sequencing and assembly techniques (long

read sequencing, Hi-C, optical mapping, etc.) has made it possible for generating chromosomescale genome assembly of plant species with highly complex genome. Such genomic data would better assist researchers in unveiling the evolutionary history of Panax species and in conservation and utilization of these valuable plants.

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Z. Guang-hui and Y. Sheng-chao DNA barcodes in Panax (Araliaceae). Mol Ecol Res 19(5):1333–1345 Jiang X, Yang C, Baosheng L, Shuiming X, Qinggang Y, Rui B, Jun Q (2017) Panax ginsenggenome examination for ginsenoside biosynthesis. GigaScience 6 (11):1–15 Jung J, Kim KH, Yang K, Bang KH, Yang TJ (2014) Practical application of DNA markers for highthroughput authentication of Panax ginseng and Panax quinquefolius from commercial ginseng products. J Ginseng Res 38(2):123–129 Kajitani R, Toshimoto K, Noguchi H, Toyoda A, Ogura Y, Okuno M, Yabana M, Harada M, Nagayasu E, Maruyama H, Kohara Y, Fujiyama A, Hayashi T, Itoh T (2014) Efficient de novo assembly of highly heterozygous genomes from whole-genome shotgun short reads. Genome Res Aug 24(8):1384–95. https://doi.org/10.1101/gr.170720.113 Kim N-H, Choi H-I, Kim KH, Jang W, Yang T-J (2014) Evidence of genome duplication revealed by sequence analysis of multi-loci expressed sequence tag–simple sequence repeat bands in Panax ginseng Meyer. J Ginseng Res 38(2):130–135 Lee C, Wen J (2004) Phylogeny of Panax using chloroplast trnC–trnD intergenic region and the utility of trnC–trnD in interspecific studies of plants. Mol Phylogenet Evol 31(3):894–903 Lowe TM, Eddy SR (1997) tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res 25(5):955–964 Majoros WH, Pertea M, Salzberg SL (2004) TigrScan and GlimmerHMM: two open source ab initio eukaryotic gene-finders. Bioinformatics 20(16):2878–2879 Manley LJ, Ma D, Levine SS (2016) Monitoring error rates in Illumina sequencing[J]. J Biomol Tech: JBT 27(4):125 Manzanilla V, Kool A, Nhat LN, Van HN, Thu HLT, De Boer H (2018) Phylogenomics and barcoding of Panax: toward the identification of ginseng species. BMC Evol Biol 18(1):44 Nawrocki EP, Kolbe DL, Eddy SR (2009) Infernal 1.0: inference of RNA alignments. Bioinformatics 25 (13):1335–1337 Ng TB (2010) Pharmacological activity of sanchi ginseng (Panax notoginseng). J Pharm Pharmacol 58(8):1007– 1019 Parra G, Bradnam K, Korf IF (2007) CEGMA: a pipeline to accurately annotate core genes in eukaryotic genomes. Bioinformatics 23(9):1061–1067 Paterson AH, Freeling M, Tang H et al (2010) Insights from the comparison of plant genome sequences. Ann Rev Plant Biol 61:349–372 Price AL, Jones NC, Pevzner PA (2005) De novo identification of repeat families in large genomes. Bioinformatics 21:i351–i358 Sharma SK, Pandit MK (2009) A new species of Panax L. (Araliaceae) from Sikkim Himalaya, India. India. Syst Bot 34(2):434–438 Shi F-X, Li M-R, Li Y-L, Jiang P, Zhang C, Pan Y-Z, Li L-F (2015) The impacts of polyploidy, geographic and

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Synthetic Biology of Ginsenosides Pingping Wang, Lu Yu, Chaojing Li, Chengshuai Yang, Zhihua Zhou, and Xing Yan

Abstract

Ginsenosides, a group of triterpenoid saponins, are the signature compounds from plants in the genus Panax. Ginsenosides display a variety of pharmaceutical activities and have potential applications in various fields such as medicine, food, and cosmetics. However, the contents of these compounds, especially rare ginsenosides, are extremely low in Panax plants. Current approaches for the manufacture of ginsenosides mainly rely on the extraction of total saponins from Panax plants, followed by biological or chemical deglycosylation; such methods are high cost with low efficiency and are difficult to scale up. In recent decades, major developments have been achieved in synthetic biology, and many successful cases of the production of

Pingping Wang and Lu Yu contributed equally to this work P. Wang
L. Yu
C. Li
C. Yang
Z. Zhou (&)
X. Yan CAS-Key Laboratory of Synthetic Biology, CAS Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Fenglin Rd., Shanghai 200032, China e-mail: [email protected] C. Li University of Chinese Academy of Sciences, Beijing 100049, China

13

natural products via synthetic biology approaches have been reported. These studies demonstrate that synthetic biology provides potential alternative methods for the manufacture of ginsenosides. In this chapter, we briefly review ginsenoside bioactivity, chemical structure, and traditional methods of their manufacture, with a focus on tracing the recent progress of synthetic biology for ginsenoside production, including the elucidation of biosynthetic pathways and construction of cell factories for both natural and non-natural ginsenosides.

13.1

Background

13.1.1 Structural Diversity of Ginsenosides Ginsenosides are the main pharmacological active compounds distributed within plants in the genus Panax, including Panax ginseng Meyer, Panax quinquefolius L, and Panax notoginseng (Burk.) F. H. Chen (Nicol et al. 2002; Cho et al. 2006; Wang 2016). These natural products display a wide spectrum of pharmacological activities, including antitumor, antioxidation, antiaging, anti-inflammatory, and immune system enhancing activities (Leung and Wong 2010). Ginsenosides are the oligosaccharide glycosides of dammarane or oleanane-type triterpenoids. Based on the structural differences of saponins,

© Springer Nature Switzerland AG 2021 J. Xu et al. (eds.), The Ginseng Genome, Compendium of Plant Genomes, https://doi.org/10.1007/978-3-030-30347-1_13

159

160

P. Wang et al.

Fig. 13.1 Chemical a structures of ginsenosides. a PPD-type; b PPT-type; c Oleanane-type ginsenosides; and d OT-type (Nicol et al. 2002)

b

R1

R2

PPD

OH

OH

PPT

OH

OH

CK

OH

OGlc

F1

OH

OGlc

Rh2

OGlc

OH

Rh1

OGlc

OH

F2

OGlc

OGlc

Rg1

OGlc

OGlc

Rg3

OGlc(2-1)Glc

OH

Rg2

OGlc(2-1)Rha

OH

Rd

OGlc(2-1)Glc

OGlc

Re

OGlc(2-1)Rha

OGlc

Rb1

OGlc(2-1)Glc

OGlc(6-1)Glc

Rf

OGlc(2-1)Glc

OH

Rb2

OGlc(2-1)Glc

OGlc(6-1)Ara(pry)

F3

OH

OGlc(6-1)Ara(pyr)

Rb3

OGlc(2-1)Glc

OGlc(6-1)Xyl

Ra1

OGlc(2-1)Glc

OGlc(6-1)Ara(pry)(4-1)Xyl

Ra2

OGlc(2-1)Glc

OGlc(6-1)Ara(fur)(2-1)Xyl

Ra3

OGlc(2-1)Glc

OGlc(6-1)Glc(3-1)Xyl

Rc

OGlc(2-1)Glc

OGlc(6-1)Ara(fur)

Rs1

OGlc(2-1)Glc-Ac

OGlc(6-1)Ara(pyr)

Rs2

OGlc(2-1)Glc-Ac

OGlc(6-1)Ara(fur)

Rs3

OGlc(2-1)Glc-Ac

OH

R1

R2

c

R1

R2

Oleanolic acid

OH

OH

Ro

OGlc(2-1)Glc

OGlc

d

R Octillol

OH

Pseudoginsenoside F11

OGlc(2-1)Rha

ginsenosides can be reasonably classified into six different subtypes: protopanaxadiol (PPD) type, protopanaxatriol (PPT) type, octillol (OT) type, oleanolic acid (OA) type, C17 side-chain varied type, and a series of miscellaneous triterpenes (Yang 2014). Of these, the PPD, PPT, OA, and OT types are the four most common subtypes of ginsenosides (Fig. 13.1). Currently, more than 100 ginsenosides have been identified from Panax species (Yang 2014). Despite their significant medicinal and commercial value, it is difficult to obtain large quantities of ginsenosides due to their extremely low contents in nature. According to their contents in Panax plants, ginsenosides can be divided into the rich and rare ginsenosides.

However, even the contents of rich saponins are usually below 1% of the dry weight of P. ginseng, while the contents of rare saponins are even lower, usually only * 1‱ of the dry weight, and sometimes their levels are undetectable (Shibata 2001). The pharmacological activities of ginsenosides have been confirmed to be related to their sugar moieties. When total ginsenosides are taken orally, rich ginsenosides with a higher number of sugar moieties are hydrolyzed to rare ginsenosides with fewer sugar moieties by intestinal microorganisms or the liver (Hasegawa 2004). This indicates that the aglycone and rare ginsenosides are more easily absorbed by the body. For example, ginsenoside compound K (CK) has been proven

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to have multifarious medicinal properties including anticancer, anti-inflammation, and anti-diabetes activities (Yang et al. 2015).

13.1.2 Traditional Methods of Ginsenoside Production Extraction of total saponins rich in triterpenoids from Panax plants is the traditional method of ginsenoside production. The total saponins are further separated by several methods such as macro-porous resin column chromatography, gel column chromatography, thin layer chromatography, and high-performance liquid chromatography so as to obtain the saponin monomer (Ruan et al. 2010; Wang et al. 2009). However, it is not feasible to extract rare ginsenosides directly from plants due to their extremely low contents. At present, rare ginsenosides, such as CK, Rg3, Rh2, Rh1, and F1, are mainly obtained by chemical or biological hydrolysis of rich PPD/PPT-type ginsenosides. Chemical hydrolysis selectively hydrolyzes the glycosyl at different sites of ginsenosides on the basis of their variable stability in different chemical conditions. For example, the glycosyl group at C-3 is more stable than that at C-20 in weak acid conditions; therefore, Rh2 and Rg3 with C-3 glycosylation can be prepared from total ginsenosides by hydrolyzing the glycosyl at C-20 (Bae et al. 2004). Similarly, Rh1 can be obtained by chemically hydrolyzing the C-20 glycosyl of Rg1 (Han et al. 1982). However, acid hydrolysis often produces several by-products and causes configuration changes of ginsenosides. By contrast, biological hydrolysis, including enzymatic hydrolysis and microbial transformation, has become popular in recent years because of its high specificity, mild conditions, and greater stability. Enzymatic hydrolysis uses various glycosidases to selectively hydrolyze the glycosyl groups on saponins. In the microbial transformation method, ginsenosides are hydrolyzed by microbes isolated from the rhizosphere soil of ginseng plants or by the intestinal bacteria of animals. Park et al. reviewed the preparation

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of rare ginsenosides by biotransformation and analyzed the possible biotransformation routes of PPD and PPT-type ginsenosides (Park et al. 2010). However, the above methods rely completely on the supply of ginsenoside resources. The limited natural resources cannot meet the growing demand for ginsenosides in medicine and research. Long-term dependence on the collection of wild Panax plants as drug sources will inevitably lead to the exhaustion of natural resources and seriously damage the ecological balance. In addition, artificial cultivation of Panax plants requires a growth and cultivation cycle of 4–15 years and faces problems such as pesticide residues, heavy metal pollution, and variety degradation, all of which constrain largescale cultivation. Meanwhile, the hydrolysis of total saponins to produce rare ginsenosides generates by-products that cannot be used, resulting in wasted resources.

13.1.3 Advantages of Synthetic Biology as an Alternative Approach to Ginsenoside Production Synthetic biology is the emerging science of reengineering, constructing, and applying biological systems and processes. It is predicted to become one of the top ten world-changing technologies (Cameron et al. 2014; Paddon and Keasling 2014). Synthetic biology excavates and characterizes bioparts related to natural drug biosynthesis, then designs and standardizes these components using engineering principles. Through the assembly and integration of bioparts into chassis cells, the biosynthetic pathways and metabolic networks of natural medicine can be reconstructed. Thus, the highly efficient one-pot heterologous synthesis of the active components from simple sugars can be realized (Fig. 13.2). In the field of natural drug design and synthesis, the application of synthetic biology has enabled scientists to more precisely control metabolic pathways. It is also possible to design

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Fig. 13.2 Comparison of synthetic biology approach and traditional methods with plant extraction and transformation for ginsenoside manufacture

and construct synthetic super-producing microbial cell factories capable of producing important natural drugs. The desired target compounds can be obtained directly by fermentation of these cell factories. This is expected to become one of the most promising green technologies for drug production. At present, synthetic biology approaches have been successfully used in the sustainable production of artemisinic acid (Paddon et al. 2013). Other natural products involving the production of terpenes (Yan et al. 2014), flavonoids (Rodriguez et al. 2015), polyphenols (Li et al. 2015), and alkaloids (Galanie et al. 2015) have also been realized. In addition, several advances have been made in the study of the biosynthetic pathways and reaction mechanisms of ginsenosides in recent years (Wang et al. 2015; Wei et al. 2015; Wang et al. 2019), laying a solid foundation for ginsenoside synthetic biology.

13.2

Biosynthetic Pathways of Ginsenosides

13.2.1 Biosynthesis of Common Precursors of Triterpenoids Isopentenyl pyrophosphate (IPP) and dimethylallyl diphosphate (DMAPP) are common

precursors for the biosynthesis of isoprenoids, which are sequentially converted into geranyl diphosphate and farnesyl diphosphate (FPP) by prenyltransferases. Two molecules of FPP are catalyzed into squalene (a linear C30 molecule) by squalene synthase (SQS). Squalene is converted by squalene epoxidase (SQE) into 2,3oxidosqualene (2,3-epoxysqualene), which is the last common intermediate for triterpenes. IPP and DMAPP can be produced in plants through the 2methylerythritol phosphate (MEP) and mevalonate (MVA) pathways. Generally, the MEP pathway occurs in plastids and primarily provides IPP and DMAPP for the biosynthesis of monoterpenes and diterpenes, while the MVA pathway occurs in the cytosol and mainly provides IPP and DMAPP for the biosynthesis of sesquiterpenes and triterpenoids. Ginsenosides belong to the triterpenoids; however, recent investigations have suggested that both the MEP and MVA pathways contribute to ginsenoside biosynthesis by providing the precursors of ginsenosides such as IPP. For example, a series of specific suppression experiments on either or both pathways indicated that both the MVA and MEP pathways might affect ginsenoside biosynthesis in P. ginseng, though the ginsenoside was preferably synthesized via the MVA pathway (Zhao et al. 2014). A more recent study based on transcriptomic profiling data indicated that the transcriptional levels of MEP

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pathway genes were much higher than those of MVA pathway genes in the leaves of P. ginseng and were comparable to those of MVA pathway genes in the root of the plant (Xue et al. 2019). Further efforts to confirm whether and how the MVA and MEP pathways contribute to ginsenoside biosynthesis in different tissues and different stages are required.

13.2.2 Biosynthesis of Ginsenoside Aglycones 13.2.2.1 Oxidized Squalene Cyclases (OSCs) 2,3-epoxysqualene, the last common intermediate for triterpenes, can be converted into various scaffolds of triterpenoids by different OSCs. Modifications are then made by tailor enzymes including cytochromes P450 (CYPs), UDPglycosyltransferases (UGTs), acetyltransferases, and so on, to produce triterpenes with more structural diversity. Dammarenediol-II (DM) is a type of tetracyclic triterpenoid scaffold, mainly found in Panax plants, but is also present in other plants including Gynostemma pentaphyllum and Centella asiatica. Triterpene aglycones PPD and PPT and their ginsenosides are modified from this triterpene scaffold. In 2006, Tansakul et al. designed a degenerate primer targeting the conserved domains of OSCs and cloned the first OSC gene (PNA or PgDDS) encoding a DM synthase from ginseng hairy root (Tansakul et al. 2006). When PgDDS was expressed in the lanosterol synthase deficient (erg7) Saccharomyces cerevisiae strain GIL77, DM could be detected in the fermentation liquid. In addition, b-amyrin synthases are widely distributed in plants, and two b-amyrin synthase genes, PNY and PNY2, which catalyze 2,3-epoxysqualene to produce b-amyrin, have been identified from P. ginseng (Han et al. 2006). 13.2.2.2 Cytochromes P450 CYPs are a class of monooxygenases involved in many metabolism and biosynthesis processes in living organisms. For the biosynthesis of

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triterpenoids, CYPs are often involved in the oxidative modification of the triterpene skeleton by introducing a hydroxyl, ketone, aldehyde, carboxyl, or epoxy group to form a new compound. Since each plant genome encodes hundreds of different CYP genes with high sequence diversity, it is difficult to predict their functions and it is especially challenging to identify the specific CYP genes involved in the modification of triterpenoids by sequence. According to the draft genome of P. ginseng, more than 488 CYPs have been annotated (Li et al. 2013). Methyl jasmonate (MeJA) treatment can stimulate ginsenoside production in P. ginseng; therefore, the transcriptome variation following MeJA treatment may provide important clues about which genes, including CYP genes, take part in the biosynthesis of ginsenosides. In 2011, a Korean group cloned nine CYP candidate genes from MeJA-treated adventitious ginseng roots. One of these genes, CYP716A47, was the most likely to be an essential gene responsible for ginsenoside biosynthesis, because its transcription was not only induced by MeJA, but was also activated in a transgenic ginseng that overexpresses SQS and overproduces ginsenosides. The CYP gene was heterologously expressed in S. cerevisiae WAT21 bearing the Arabidopsis thaliana NADPH-CYP reductase gene (ATR2), and in vitro enzymatic experiments confirmed that CYP716A47 participated in the hydroxylation of DM at the C-12 position to produce PPD (Han et al. 2011). The results also showed that the CYP716A subfamily may play an important role in the modification of ginsenosides. Therefore, the same group tested the functions of two CYPs belonging to this subfamily (CYP716A52v2 and CYP716A53v2) that were also cloned from adventitious ginseng roots in a method similar to that used for CYP716A47. They found that CYP716A53v2 catalyzes the hydroxylation of PPD at the C-6 position to yield PPT (Han et al. 2012), and CYP716A52v2 catalyzes the introduction of a carboxyl group at the C-28 position of b-amyrin to produce OA, a precursor of oleanane-type ginsenosides (Han et al. 2013).

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13.2.2.3 NADPH-Cytochrome P450 Reductase (CPR) Plant-derived CYPs generally belong to the Class II CYPs, which localize to the intracellular endoplasmic reticulum and require CPR to transfer electrons from NADPH/NADH. Based on the conserved sequences of plant CPRs, we acquired two CPR candidates, PgCPR1 and PgCPR2, from the Panax cDNA database. The two genes were cloned, and subsequent functional analysis demonstrated that both genes can be coupled with CYP716A47 for the conversion of DM into PPD and are more efficient than ATR2.1, a previously commonly used CPR from A. thaliana (Yan et al. 2014).

13.2.3 Conversion of Aglycones into Diverse Ginsenosides by UDPGlycosyltransferases

P. Wang et al.

respectively, which is the first UGT catalyzing tetracyclic triterpene to be reported (Fig. 13.3) (Yan et al. 2014; Wei et al. 2015). To date, we have cloned and functionally characterized more than 100 UGTs from ginseng, and UGTs that transfer a glucose moiety to free hydroxyls at the C-3, C-6, or C-20 positions of PPD or PPT have all been identified (Yan et al. 2014; Wang et al. 2015; Wei et al. 2015). In addition, UGTs transferring another glucose moiety to extend the first glucose moiety attached at various sites (C-3, C-6, or C-20) of PPD or PPT have also been identified (Patent PCT/CN2013/088819). Moreover, several UGTs transferring one xylose moiety to the first glucose moiety attached to PPD or PPT were also identified. These advances will pave the way toward production of these triterpene saponins using a synthetic biology approach.

13.3 Although more than 100 ginsenosides have been isolated from ginseng, the aglycones are mainly limited to PPD, PPT, and OA. Glycosylation modification not only greatly increases their structural diversity, but also plays key roles in the chemical properties and bioactivity of ginsenosides. At least 200 UGT genes are estimated to exist in the genome of P. ginseng according to transcriptome data, so the identification of UGTs involved in ginsenoside biosynthesis remains a challenge (Li et al. 2013). To systematically examine the UGTs of Panax species, we collected the transcriptome data of plants affiliated with the Panax genus from the National Center for Biotechnology Information and re-assembled them. All UGTs were annotated according to the conserved plant secondary product glycosyltransferase (PSPG) box, and thus, a database of UGT bioparts from Panax plants was established. At the same time, a standard method for the characterization of UGT bioparts was also developed. Using this database and characterization method, we quickly identified UGTPg1, capable of catalyzing C-20 glycosylation of PPD and PPT to produce the rare ginsenosides CK and F1,

Cell Factories Built for Ginsenosides

13.3.1 Cell Factories for Natural Ginsenosides By artificially introducing biosynthetic pathways and precisely regulating these exogenous pathways in chassis cells, many high value-added natural products can be produced safely, efficiently, and inexpensively via synthetic biology approaches. Since the first elucidation of the ginsenoside pathway, many microbial and plant cell factories have been developed for the biosynthesis of ginsenosides and their precursors (Table 13.1).

13.3.1.1 Saccharomyces Cerevisiae S. cerevisiae is the most commonly used chassis for the construction of ginsenoside cell factories due to its ease of manipulation and integrated membrane system for the expression of key genes from plants. PPD is the common precursor of all dammarane-type ginsenosides. In recent years, there have been several reports on high-efficiency production of PPD in S. cerevisiae. For example,

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Fig. 13.3 Pathway for the biosynthesis of natural and non-nature ginsenosides

by integrating the PPD biosynthesis pathway into a yeast chromosome and overexpressing the ratelimiting gene tHMG1 and three key precursor genes, the resulting strain could produce PPD at 148.1 mg L−1 in shake flasks, and the titer reached more than 1 g L−1 in fed-batch fermentation (Dai et al. 2013). Recently, to overcome the poor coupling between P. ginseng CYP-type PPD synthase (PPDS) and the A. thaliana CYP reductase ATR1 in PPD-producing yeast recombinants, the fusion of PPDS and ATR1 encoding genes was introduced and PPD production increased significantly, reaching 265.7 mg L−1 in shake flasks and 4.25 g L−1 in 5-L fed-batch fermentation, which is the highest PPD yield reported to date (Zhao et al. 2016; Zhao et al. 2017). Recently, we redesigned and constructed a PPD-producing chassis via modular engineering of the mevalonic acid pathway and optimization of CYP expression levels, enabling PPD production at 529.0 mg L−1 in shake flasks and 11.02 g L−1 in 10-L fed-batch fermentation (Wang et al. 2019).

Since 2014, we have resolved the biosynthetic pathways of more than 15 ginsenosides, including rare ginsenosides CK, Rh2, Rg3, Rh1, and F1. Based on these novel UGTs and a PPDproducing chassis, cell factories for one-pot production of CK, Rh2, Rg3, F1, and Rh1 from simple sugars have been created, with production yields of 0.8, 16.9, 48.9, 42.1, and 92.8 mg L−1 in shake flasks (Yan et al. 2014; Wang et al. 2015; Wei et al. 2015), respectively. At the same time, a Korean research group also reported the isolation of two glycosyltransferase genes involved in the biosynthesis of ginsenosides Rh2 and Rg3 and reported de novo production of Rg3 at 1.3 mg L−1 in S. cerevisiae. In 2017, Zhuang et al. reported the construction of an Rh2 cell factory by repurposing the inherently promiscuous UGT51 from S. cerevisiae. Based on a structure-guided semi-rational engineering approach, the catalytic efficiency of UGT51 in converting PPD into ginsenoside Rh2 increased by more than 1800-fold. The yeast cell factory with this engineered glycosyltransferase could

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Table 13.1 Cell factories for the production of ginsenoside and its precursors Product

Platform organism

Production

Refs.

DM

N. tobacum

20-30 lg/g DCW

(Lee et al. 2012)

DM

N. tobacum

0.573 mg/g FW (5.2 mg/L)

(Han et al. 2014)

PPD

N. tobacum

0.167 mg/g DCW in shake flask and 0.981 mg/g DCW in airlift bioreactor culture

(Chun et al. 2015)

PPD and PPT

O. sativa

16.4 µg/g DCW of PPD 4.5 µg/g DCW of DM

(Han 2019)

DM

P. pastoris

0.736 mg/g DCW without the addition of squalene, with the addition of 0.5 g/L squalene in shake flask

(Liu et al. 2015)

DM

P. pastoris

0.1 mg/g DCW in shake flask

(Zhao et al. 2016)

CK

Y. lipolytica

161.8 mg/L fed-batch fermentation in a 5-L fermenter

(Li et al. 2019)

DM

E.coli

8.63 mg/L in shake flask

(Li et al. 2016)

DM

S. cerevisiae

0.25 mg/L in shake flask

(Liang et al. 2012)

PPD

S. cerevisiae

17.32 lg/g FW in shake flask

(Han et al. 2011)

PPT

S. cerevisiae

Not given

(Han et al. 2012)

PPD

S. cerevisiae

18.09 lg/g DCW in shake flask

(Wang et al. 2014)

DM and PPD

S. cerevisiae

8.2 mg/g DCW of DM and 13.11 mg/g DCW of PPD in shake flask; 1548 mg/L of DM and 1189 mg/L of PPD in 7.5L fed-batch fermentation

(Dai et al. 2013)

PPD

S. cerevisiae

265.7 mg/L in shake flask 1436.6 mg/L in 5L fed-batch fermentation

(Zhao et al. 2016)

PPD, PPT and Oleanolic acid

S. cerevisiae

17.2 mg/L of PPD, 15.9 mg/L of PPT and 21.4 mg/L of oleanolic acid

(Dai 2014)

PPD

S. cerevisiae

4.25 g/L in 5L fed-batch fermentation

(Zhao et al. 2017)

PPD

S. cerevisiae

529.0 mg/Lin shake flasks

(Dai et al. 2018)

PPD

S. cerevisiae

11.02 g/L in 10-L fed-batch fermentation

(Dai et al. 2018)

CK

S. cerevisiae

0.8 mg/L in shake flask

(Yan et al. 2014)

Rh2

S. cerevisiae

16.9 mg/L in shake flask,

(Wang et al. 2015)

Rh2

S. cerevisiae

300 mg/L in a 5-L fed-batch fermentation system

(Zhuang et al. 2017)

Rh2

S. cerevisiae

179.3 mg/L in shake flasks and 2.25 g/L in 10-L fed-batch fermentation

(Wang et al. 2019)

Rg3

S. cerevisiae

48.9 mg/L in shake flask

(Wang et al. 2015)

Rg3

S. cerevisiae

1.3 mg/L in shake flask

(Jung et al. 2014)

Rh1, F1

S. cerevisiae

92.9 mg/L of Rh1 and 42.0 mg/L of F1

(Wei et al. 2015)

3b-O-Glc-DM, 20S-O-Glc-DM

S. cerevisiae

2.4 g/L of 3b-O-Glc-DM and 5.6 g/L of 20S-O-Glc-DM in a 3 L bioreactor

(Hu et al. 2019)

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produce Rh2 at approximately 300 mg L−1 in a 5-L fed-batch fermentation system (Zhuang et al. 2017). However, the production yield of Rh2 by these cell factories is still very low, far from commercialization. In our group, we used the highest PPD-producing yeast chassis and focused on improving the C3-OH glycosylation efficiency of the cell factory. Efforts included tuning the copy numbers and promoter strengths of UGT bioparts and optimizing the UGT efficiency by screening UGT bioparts from other plant species and mutants originating from the direct evolution of UGTPg45. Finally, we obtained a yeast cell factory with Rh2 production of 179.3 mg L−1 in shake flasks and 2.25 g L−1 in 10-L fed-batch fermentation (Wang et al. 2019).

13.3.1.2 Nicotiana Tabacum In 2011, transgenic tobacco was constructed by expressing the P. ginseng DM synthase gene (PgDDS) under the control of the Cauliflower mosaic virus 35S promoter. The accumulation of DM in leaves was approximately 20–30 lg g−1 dry weight (DW) in the transgenic lines (Lee et al. 2012). The production of DM was further established via cell suspension culture of transgenic tobacco in 2014. The amount of DM produced in the cell suspension reached 573 lg g−1 DW after 3 weeks of culture (Han et al. 2014). In 2015, a PPD production system was established via cell suspension culture of transgenic tobacco cooverexpressing the genes for PgDDS and CYP716A47. PPD production reached 166.9 lg g−1 DW in a 5-L airlift bioreactor culture (Chun et al. 2015). The biosynthesis of CK in transgenic tobacco was subsequently realized in 2017. By co-overexpressing a glycosyltransferase UGT71A28 gene (a highly homologous gene of UGTPg1) together with PgDDS and CYP716A47, the concentration of CK in the leaves ranged from 1.55 to 2.64 lg g−1 DW, depending on the transgenic line (Gwak et al. 2017). Recently, the genes CYP716A47, PgDDS, and CYP716A53v2 were co-expressed in tobacco via Agrobacterium-mediated transformation. Production of PPT was achieved via cell suspension culture, and the PPT content was 104.3 lg g−1 DW (Gwak et al. 2018).

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13.3.1.3 Oryza Sativa Transgenic rice plants overexpressing the P. ginseng PgDDS and CYP716A47 genes driven by a rice endosperm-specific a-globulin promoter were constructed by a Korea group recently. The transgenic grains produced not only PPD but also DM, and the mean concentrations of PPD and DM in rice grains were 16.4 and 4.5 µg g−1 dry weight, respectively (Han 2019). 13.3.1.4 Escherichia Coli E. coli is one of the most commonly used chassis in synthetic biology and has been engineered for the synthesis of many natural products. By reconstituting the DM biosynthetic pathway in E. coli and co-expressing SQS, SQE, and CPR from S. cerevisiae, SQE from Methylococcus capsulatus, and CPR from A. thaliana, DM was detected and the production yield reached 8.63 mg L−1 under shake flask conditions (Li et al. 2016). 13.3.1.5 Pichia Pastoris Based on the native triterpene synthetic pathway, a DM synthetic pathway was established in P. pastoris by introducing a PgDDS gene. To enhance productivity, a strategy of increasing supply and reducing competitive consumption of 2,3-epoxysqualene was used. By overexpressing ERG1, a key precursor gene for 2,3epoxysqualene synthesis, and replacing the ERG7 promoter with the repressor promoter, the consumption of 2,3-epoxysqualene was reduced and the yield of DM was increased to 0.736 mg g−1 DW. In addition, the DM yield was increased to 1.073 mg g−1 DW by feeding squalene at 0.5 g L−1 in the medium (Liu et al. 2015). 13.3.1.6 Yarrowia Lipolytica An efficient biosynthetic pathway of CK was recently constructed in metabolically engineered Y. lipolytica, which produced CK at 5.1 mg L−1. The production of CK was further increased by 5.96-fold to 30.4 mg L−1 with overexpression of key genes in the MVA pathway and fusion of CYP monooxygenase (PPDS) and NADPH-CYP reductase. Finally, a CK production yield of

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161.8 mg L−1 was achieved by fed-batch fermentation in a 5-L fermenter using the strain YLMVA-CK.

13.3.2 Cell Factories for Non-natural Ginsenosides Taking advantage of the regiospecificity or substrate promiscuity of UGT bioparts from plants or microbes, glycosylation modification of ginsenoside skeletons that are not naturally occurring in Panax plants can be realized, and the resulting ginsenoside-like compounds with novel structures can be referred to as non-natural ginsenosides. Although glycosylation modified products of DM have never been isolated from P. ginseng or other Panax plants, two native UGTs identified from P. ginseng, PgUGT74AE2 and UGTPg1, have been shown to catalyze C-3 and C-20 glycosylation of DM, respectively. Based on these two UGT bioparts, Hu et al. reconstructed the complete biosynthetic pathways of 3-O-b-Dglucopyranosyl-dammar-24-ene-3b, 20S-diol (3b-O-Glc-DM) and 20-O-b-D-glucopyranosyldammar-24-ene-3b,20S-diol (20S-O-Glc-DM) in S. cerevisiae. Multistep metabolic engineering strategies were applied, including optimization of the chassis cell, multi-copy integration of heterologous genes via the CRISPR/Cas9 system, increasing precursor supply by overexpressing rate-limiting enzymes, and downregulation of the competitive pathway to redirect the metabolic flux towards the target products. Finally, titers of 2.4 g L−1 for 3b-O-Glc-DM and 5.6 g L−1 for 20S-O-Glc-DM were achieved through fed-batch fermentation in a 3-L bioreactor (Hu et al. 2019). In another case, the promiscuous Bs-YjiC from Bacillus subtilis was used to synthesize ginsenoside Rh2 and non-natural ginsenoside F12 (3-O-b-d-glucopyranosyl-12-O-b-d-glucopy ranosyl-20(S)-protopanaxadiol). Bs-YjiC could catalyze the C3-OH and C12-OH glycosylation

P. Wang et al.

of PPD to yield ginsenoside Rh2 and non-natural ginsenoside F12. Coupling this enzyme with a UDP-glucose regeneration system powered by sucrose synthase, an in vitro one-pot ginsenoside synthesis platform was established to produce 0.2 g L−1 of Rh2 and 3.98 g L−1 of F12 using PPD as the substrate (Dai et al. 2018).

13.4

Conclusion and Future Prospects

With the development of omics data of Panax plants and the functional characterization of key genes, including OSCs, CYPs, CPRs, and UGTs, involved in the ginsenoside biosynthetic pathway, more and more ginsenoside pathways have been completely elucidated. Meanwhile, the introduction of synthetic biology techniques offers a novel and attractive approach for one-pot ginsenoside production. We have now reached a new stage of ginsenoside synthetic biology. In the future, with the complete genome sequences of Panax plants and the improvement of our ability to functionally screen and characterize key genes, the biosynthetic pathways of ginsenosides with increasingly complex structures will be elucidated, especially ginsenosides with longer sugar chains and diverse types of sugar modifications. The systematic engineering and optimization of ginsenoside cell factories will boost ginsenoside production, and the downstream fermentation, product extraction, and purification systems will promote the industrialization of ginsenosides via a synthetic biology approach. Acknowledgements This work was financially supported by the Natural Science Foundation of China (No. 21672228), the National Basic Research Program of China (2015CB755703), the Key Deployment Projects of the Chinese Academy of Sciences (No. KFZD-SW-215), the Strategic Biological Resources Service Network Plan of the Chinese Academy of Sciences (ZSYS-016), and the International Great Science Program of the Chinese Academy of Sciences (No. 153D31KYSB20170121).

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Synthetic Biology of Ginsenosides

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169 ginsenoside biosynthesis in Panax ginseng. Plant Cell Physiol 54(12):2034–2046 Han JY, Wang HY, Choi YE (2014) Production of dammarenediol-II triterpene in a cell suspension culture of transgenic tobacco. Plant Cell Rep 33 (2):225–233 Hasegawa H (2004) Proof of the mysterious efficacy of ginseng: basic and clinical trials: metabolic activation of ginsenoside: deglycosylation by intestinal bacteria and esterification with fatty acid. J Pharmacol Sci 95:153–157 Hu Z-F et al (2019) Construction and optimization of microbial cell factories for sustainable production of bioactive dammarenediol-II glucosides. Green Chem 21(12):3286–3299 Jung SC et al (2014) Two ginseng UDPglycosyltransferases synthesize ginsenoside Rg3 and Rd. Plant Cell Physiol 55(12):2177–2188 Lee MH et al (2012) Dammarenediol-II production confers TMV tolerance in transgenic tobacco expressing Panax ginseng dammarenediol-II synthase. Plant Cell Physiol 53(1):173–182 Leung KW, Wong AS-T (2010) Pharmacology of ginsenosides: a literature review. Chin Med 5:1–7 Li C et al (2013) Transcriptome analysis reveals ginsenosides biosynthetic genes, microRNAs and simple sequence repeats in Panax ginseng C. A. Meyer. BMC Genomics 14:245 Li M et al (2015) De novo production of resveratrol from glucose or ethanol by engineered Saccharomyces cerevisiae. Metab Eng 32:1–11 Li DS et al (2016) Heterologous biosynthesis of triterpenoid dammarenediol-II in engineered Escherichia coli. Biotechnol Lett 38(4):603–609 Li D et al (2019) Production of triterpene ginsenoside compound K in the non-conventional yeast yarrowia lipolytica. J Agric Food Chem 67(9):2581–2588 Liang YL et al (2012) Heterologous expression of dammarenediol synthase gene in an engineered Saccharomyces cerevisiae. Letters in Appl Microb 55 (5):323–329 Liu XB et al (2015) Metabolic engineering of Pichia pastoris for the production of dammarenediol-II. J Biotechnol 216:47–55 Nicol RW, Traquair JA, Bernards MA (2002) Ginsenosides as host resistance factors in American ginseng (Panax quinquefolius). Can J Bot 80(5):557–562 Paddon CJ, Keasling JD (2014) Semi-synthetic artemisinin: a model for the use of synthetic biology in pharmaceutical development. Nat Rev Microbiol 12 (5):355–367 Paddon CJ et al (2013) High-level semi-synthetic production of the potent antimalarial artemisinin. Nature 496(7446):528–532 Park CS et al (2010) Biotransformation of ginsenosides by hydrolyzing the sugar moieties of ginsenosides using microbial glycosidases. Appl Microbiol Biotechnol 87(1):9–19

170 Rodriguez A et al (2015) Establishment of a yeast platform strain for production of p-coumaric acid through metabolic engineering of aromatic amino acid biosynthesis. Metab Eng 31:181–188 Ruan CC et al (2010) Isolation and characterization of a new ginsenoside from the fresh root of Panax Ginseng. Molecules 15(4):2319–2325 Shibata S (2001) Chemistry and cancer preventing activities of ginseng saponins and some related triterpenoid compounds. J Korean Med Sci 16 (Suppl):S28–S37 Tansakul P et al (2006) Dammarenediol-II synthase, the first dedicated enzyme for ginsenoside biosynthesis. Panax ginseng. FEBS Lett 580(22):5143–5149 Wang J et al (2009) Isolation and purification of ginsenosides from plant extract of Panax quinquefolium L. by high performance centrifugal partition chromatography coupled with ELSD. Chromatographia 71(3–4):267–271 Wang L et al (2014) The isolation and characterization of dammarenediol synthase gene from Panax quinquefolius and its heterologous co-expression with cytochrome P450 gene PqD12H in yeast. Functi Inte Gen 14(3):545–557 Wang P et al (2015) Production of bioactive ginsenosides Rh2 and Rg3 by metabolically engineered yeasts. Metab Eng 29:97–105 Wang T et al (2016) Traditional uses, botany, phytochemistry, pharmacology and toxicology of Panax notoginseng (Burk.) F.H. Chen: a review. J Ethnopharmacol 188:234–58 Wang P et al (2019) Synthesizing ginsenoside Rh2 in Saccharomyces cerevisiae cell factory at highefficiency. Cell Discov 5:5 Wei W et al (2015) Characterization of Panax ginseng UDP-Glycosyltransferases catalyzing protopanaxatriol and biosyntheses of bioactive ginsenosides F1 and Rh1 in metabolically engineered yeasts. Mol Plant 8 (9):1412–1424

P. Wang et al. Xue L et al (2019) Transcriptomic profiling reveals MEP pathway contributing to ginsenoside biosynthesis in Panax ginseng. BMC Genom 20(1):383 Yan X et al (2014) Production of bioactive ginsenoside compound K in metabolically engineered yeast. Cell Res 24(6):770–773 Yang WZ et al (2014) Saponins in the genus Panax L. (Araliaceae): a systematic review of their chemical diversity. Phytochemistry 106:7–24 Yang XD et al (2015) A review of biotransformation and pharmacology of ginsenoside compound K. Fitoterapia 100:208–220 Zhao S et al (2014) Both the mevalonate and the nonmevalonate pathways are involved in ginsenoside biosynthesis. Plant Cell Rep 33(3):393–400 Zhao F et al (2016a) Optimization of a cytochrome P450 oxidation system for enhancing protopanaxadiol production in Saccharomyces cerevisiae. Biotechnol Bioeng 113(8):1787–1795 Zhao CC et al (2016b) Enhancing biosynthesis of a ginsenoside precursor by self-assembly of two key enzymes in pichia pastoris. J Agri Food Chem 64 (17):3380–3385 Zhao FL et al (2016c) Optimization of a cytochrome P450 oxidation system for enhancing protopanaxadiol production in saccharomyces cerevisiae. Biotech Bioengin 113(8):1787–1795 Zhao FL et al (2017) Enhancing Saccharomyces cerevisiae reactive oxygen species and ethanol stress tolerance for high-level production of protopanoxadiol. Biores Technol 227:308–316 Zhuang Y et al (2017a) Biosynthesis of plant-derived ginsenoside Rh2 in yeast via repurposing a key promiscuous microbial enzyme. Metabol Eng 42:25– 32 Zhuang Y et al (2017b) Biosynthesis of plant-derived ginsenoside Rh2 in yeast via repurposing a key promiscuous microbial enzyme. Metab Eng 42:25–32

Gut Microbiome for Ginseng Medicine

14

Xiao Shuiming and Zhang Xiaoyan

Abstract

In the past few decades, using traditional Chinese medicines (TCMs), including Panax ginseng as pharmaceutical, nutraceutical, and healthcare products has expanded globally. TCMs can exert its pharmacological effects as a single herb, as well as in a variety of herbal compatibility forms. And in general, ginseng is orally administered, which make their constituents inevitably brought into contact with gastrointestinal microbiota in the intestine. The gut microbiota possesses the corresponding enzyme system that can metabolize some of the ginsenoside prototypes, which found almost exclusively in ginseng and regarding as the major pharmacological ingredients with therapeutic activities, into metabolites with higher bioavailability and efficacy. Therefore, understanding the role of the gut microbiota in ginsenosides metabolism in vivo and therefore targeting the specific bacterial strains/metabolic function by probiotics, prebiotics, or other micro-ecological modulators potentially to become an attractive approach to enhance the ginseng efficiency.

X. Shuiming (&)
Z. Xiaoyan Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China e-mail: [email protected]

14.1

Introduction

Up to 80% of the global population depend on plant-derived medicines for their principal health care (Romanelli et al. 2015). The Chinese government will integrate traditional Chinese medicines (TCMs) into the healthcare system by 2020 (Xinhuanet. 2016). TCMs are deemed to have profound impacts on health, survival, and reproduction of the Chinese people by their diseases prevention and treatment (Tu 2011; Liu et al. 2013a). In addition to the direct medical value of the active ingredients, TCMs also contain protein, polysaccharides, and other nutrients, which potentially make them possess a variety of pharmacological effects (Kano et al. 2012; Wu 2012). However, most of the chemicals from TCMs are proven to be with little bioavailability, which seriously obstruct their clinical applications (Gao et al. 2013). Panax ginseng C. A. Mey, as a precious herbal medicine, has been used in the clinical practice and healthcare nourishing for several millennia in China, Japan, South Korea, and other East Asian countries (Hemmerly 1977). Modern pharmacological research has confirmed that ginsenosides, the major bioactive compounds of P. ginseng, exhibit multiple therapeutic activities, including antitumor, antihypertensive, antivirus, and immune modulatory activities. (Leung and Wong 2010). However, the bioavailability of ginsenosides prototype after oral administration is poor thus difficult to achieve full pharmacological

© Springer Nature Switzerland AG 2021 J. Xu et al. (eds.), The Ginseng Genome, Compendium of Plant Genomes, https://doi.org/10.1007/978-3-030-30347-1_14

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activity. For example, the absorption rate of ginsenoside Rb1 is only about 1.0%, Rb2 is 3.4%, and Rg1 is 1.9%, respectively (Li et al. 2009). The limitation is also widespread in other active ingredients of TCMs, including other saponins, flavonoids (such as daidzein), isoflavones (such as puerarin), alkaloids (such as berberine), and monoterpenoids (such as paeoniflorin) (Gao et al. 2013) and become the bottleneck to hinder their clinical application. To exert these pharmacological actions in vivo, the major ginsenosides (ginsenosides Rb1, Rb2, Rc, etc.) are deglycosylated by glycoside hydrolases (such as b-glucosidase, a-rhamnosidase, xylosidase enzymes) residing in gut microbiota to 20-O-b-Dglucopyranosyl-20(S)-protopanaxadiol [ginsenoside compound K (CK)] (Akao et al. 1998a; b). The ginsenoside CK is considered as the major bioactive metabolite since possessing the pharmacological activities more potently than its precursors (Hasegawa 2004; Wang et al. 2012). Furthermore, the extremely low oral bioavailability (0.28–1.18%) of prototypical Rb1 will increase to 1.8–35.0% after being transformed to ginsenoside CK (Kim 2012). However, due to the 5–7 years of cultivation periods and  5 years of crop rotation cycles for Panax plants; the industrial production of CK by chemical total synthesis is still unpractical, the availability of ginsenosides for CK manufacturing is limited (Dürr et al. 2004; Sivakumar et al. 2010). Nowadays, a variety of formulation methods have been developed to improve the solubility and bioavailability of TCMs, such as “herbpairs,” absorption enhancers (Aungst 1993), chemical modification (Wang et al. 2003), different dosage forms (Araya et al. 2005; Yin et al. 2009), drug delivery systems (Cui et al. 2009; He and He 2010; Yang et al. 2013; Norvaisas and Ziemys 2014). Though variety dosage forms have been developed, because that oral decoctions of TCMs have been used for centuries, oral administration still is the most common way due to its convenience and better compliance of patients (Chen et al. 2010; Lin et al. 2010). Oral administration leads to the constituents of TCMs inevitably contact with gut microbiota in the human intestine (Crow 2011). In humans,

X. Shuiming and Z. Xiaoyan

the gastrointestinal tract harbors > 1,000 microbial species, which is about 10 times the number of body cells (Crow 2011; Lozupone et al. 2012). The active ingredients of TCMs are transformed into metabolites with more potent pharmacological activity by the intestinal bacteria before being absorbed from the gastrointestinal tract (Park et al. 2007; 郭亚雄 et al. 2010). Besides that, gut microbiota regulates the synergism and antagonism between multiple components in TCMs (Li and Jiang 2006; Shu et al. 2009). Thus, the gut microbiota is viewed as a “hidden organ” (Thomson 2016) of the body and has coevolved long term with host to participate in the metabolism (O’Hara and Shanahan 2006). Recently, “pharmacomicrobiomics” field reemergences and seeks to understand how interindividual gut microbiome compositional and functional variations shape drug efficacy, fate, and toxicity (Saad et al. 2012; Elrakaiby et al. 2014; Doestzada et al. 2018). Furthermore, Turnbaugh et al. (2019) proposed that precision medicine should goes microscopic by engineering the gut microbiome to improve drug outcomes. To be specific, considering the crucial role of gut microbiota in ginsenoside CK formation, modulating the gut microbiota metabolic function probably would be an effective strategy to perform the pharmacological effects of ginseng’s oral administration. In current chapter, we summarized the role of gut microbiota in mediating the metabolism and enhanced bioavailability of ginseng. Firstly, the role of gut microbiota involved in the metabolism of ginsenosides, the most important active component of ginseng, is briefly introduced; Secondly, we discuss the potential for gut microbiota-targeted intervention to promote the bioavailability of ginsenosides.

14.2

Ginsenosides Metabolism Involves Gut Microbiota

Polar compounds which extracted from TCMs by water or ethanol for oral administration often possess low bioavailability due to their poor lipophilicity (Liu et al. 2013b). For example,

14

Gut Microbiome for Ginseng Medicine

hydrolysis by gastric, passive diffusion absorption mechanism, especially the poor intestinal permeability caused by increased hydrogen bond count, polar surface area, and molecular flexibility of sugar moieties, result the glycosides with a limited intestinal absorption (Liu et al. 2009). Therefore, gut microbiota-mediated biotransformation, mainly deglycosylation, hydrolyzes gradually the glycosyl moieties from the backbone to converts the molecules into secondary glycoside and/or aglycones which with better intestinal absorption and thereby better bioavailability (Laparra and Sanz 2010). The genera Bacteroides, Bifidobacterium, Eubacterium, Prevotella, Lactobacillus, and Fusobacterium, belonging to the most abundant phyla Bacteroidetes and Firmicutes (Table 14.1) in human gut, are encoded with abundant glycoside hydrolase genes to perform glycoside hydrolysis (Abdessamad et al. 2013). In addition to hydrolase, gut microbiota also secrete the oxidase, reductase, esterase, lyase, transferase, etc. to perform wide range of other catalytic reactions on TCMs varied components (Abdessamad et al. 2013). From the above, the gut microbiota colonized in gastrointestinal tract plays an important role in determining the metabolic fate of TCMs. According to the structure of aglycones skeleton, ginsenosides are classified into two groups, namely dammarane-type and oleananetype. Ginsenosides within the dammarane-type consist mainly of three types: protopanaxadiol (PPD) (20(S)-PPDs Rb1, Rb2, Rc, Rd, Rg3, and Rh2), protopanaxatriol (PPT) (20(S)-PPTs Re, Rf, Rg1, Rg2, and Rh1), and ocotillol, whereas oleanane-type ginsenosides are classified according to their aglycone oleanolic acid (ginsenoside Ro, Rh3, and Ri). The major ginsenosides Rb1, Rb2, Rc, Rd, Rg1, and Re, which belonging to PPD and PPT, normally account for over 90% of the total ginsenoside content in ginseng roots (Christensen et al. 2006; Wang et al. 2006). As mentioned above, ginsenosides are the main active ingredients of the ginseng, which are not easily absorbed by the body through the intestines due to their hydrophilicity. Ever-increasing evidence suggests that gut microbiota-mediated in vivo metabolism and

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pharmacokinetics of ginsenosides produce the secondary glycosides and/or aglycones, which normally harbor greater absorption and bioactivity than their precursors. Below we will summarize the metabolic pathway in vivo by gut microbiota of representative ginsenosides from PPD and PPT, respectively.

14.2.1 20(S)-Protopanaxadiol-Type Ginsenosides (PPD) The biotransformation of ginsenosides in the gastrointestinal tract has been largely studied using in vitro and in vivo. Currently, the metabolic pathway of ginsenoside Rb1 is relatively clear (Fig. 14.1). Orally taken prototypical ginsenoside Rb1 is hardly absorbed by intestinal tract with extremely low oral bioavailability (0.28  1.18%) (Kim 2012). Meanwhile, comparing to its secondary glycosides and/or aglycones (compound K and/or 20(S)protopanaxadiol), ginsenoside Rb1 exhibits almost no significant pharmacological activities in in vitro/in vivo (intraperitoneal injection) models which without gut microbiota conversion. The metabolic pathway of ginsenoside Rb1 is degraded by b-D-glucosidases residing in mammalian gut microbiota via stepwise cleavage of the sugar moieties in C-20, C-3, and C-3, that is, followed by the formation of ginsenoside Rd, ginsenoside F2, and ultimately generate ginsenoside compound K and 20(S)-protopanaxadiol (molar ratio: 1:1). The detail process is that: ginsenoside Rd is formed after removing one molecule of glucose from C-20 by broken of the terminal glycosidic bond; then cleavage of second glucose molecule from C-3 position to produce ginsenoside F2; and furthermore, after hydrolyzing the another glucose molecule at position C-3 to forming ginsenoside Compound K; Finally, ginsenoside Compound K takes off a molecule of glucose at the C-20 position to produces aglycone PPD (Akao et al. 1998a; Bae et al. 2000; Chi and Ji 2005). Under such circumstances, ginsenoside Compound K and 20 (S)-protopanaxadiol are absorbed well and possess better oral bioavailability (1.8  35.0% and

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Table 14.1 Ginsenosides precursors, main active metabolites, involved main enzymes, and gut microbiota (宋玮 et al. 2018) Ginsenoside

Strains

Source

Metabolites

Enzymes activities

Refs

G-Rb1

Eubacterium sp. A-44, Bifidobacterium sp., Streptococcus sp., Prevotella oris

Human feces isolates

G-Rd ! GCK

b-D-glucosidase a-glucosidase b-Nacetylglucosaminidase a-fucosidase

(Hasegawa et al. 1997; Akao et al. 1998b; Bae et al. 2000)

Fusobacterium K-60

Human feces isolates

Gypenoside XVII ! GCK

b-glucosidase

(Bae et al. 2000)

Bifidobacterium sp. Int57, Bifidobacterium sp. SJ32

Human feces isolates

G-Rd ! GF2 ! G-CK

b-glucosidase

(Chi and Ji 2005)

Aspergillus usamii, Lactobacillus delbrueckii

KCTO purchase

G-Rd ! GF2 ! G-Rh2

b-glucosidase

(Chi and Ji 2005)

Bifidobacterium sp. SH5

Human feces isolates

G-Rd ! GF2

b-glucosidase

(Chi and Ji 2005)

G-Rb2

Bifidobacterium cholerium, Fusobacterium K-60, Bacteroides sp.

Human feces isolates

G-Rd/GCO ! G-CK

b-glucosidase

(Bae et al. 2000)

G-Rc

Bifidobacterium K-103, Eubacterium A-44

Human feces isolates

G-Rd ! GCK

/

(Bae et al. 2002)

Bacteroides HJ-15, Bifidobacterium K-506

Human feces isolates

G-Mb ! GCK

/

(Bae et al. 2002)

Bacteroides sp., Bifidobacterium sp., Eubacterium sp

Human feces isolates

GRh2 ! PPD

/

(Bae et al. 2004)

Fusobacterium sp

Human feces isolates

G-Rh2

/

(Bae et al. 2004)

Bifidobacterium K-103

Human feces isolates

G-Rg1

/

(Bae et al. 2005)

Bifidobacterium K-525, Bacteroides HJ15, Aspergillus niger, Leuconostoc paramesenteroides

Human feces isolates

G-Rg1 ! GRh1

b-glucosidase

(Bae et al. 2005; Chi and Ji 2005)

Bacteroides JY6

Human feces isolates

G-Rg1 ! GRh1 ! GF1 ! 20 (S)PPT

a-rhamnosidase b-glucosidase

(Bae et al. 2005)

Fusobacterium K-60

Human feces isolates

G-Rg1 ! GRh1 ! G-F1

/

(Bae et al. 2005)

Bifidobacterium sp. Int57, Bifidobacterium sp. SJ32, Aspergillus niger

G-Rg2 ! GRh1

b-glucosidase

(Chi and Ji 2005)

Aspergillus usamii, Bifidobacterium sp. SH5

G-Rg2

b-glucosidase

(Chi and Ji 2005)

G-Rg3

G-Re

G-: ginsenoside CK: compound K CO: compound O KCTO: Korean Collection for Type Cultures PPD: protopanaxadiol PPT: protopanaxatriol

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Gut Microbiome for Ginseng Medicine

175

36.8 ± 12.4%, respectively) over their precursor ginsenoside Rb1. In addition to the improved bioavailability, various kinds of pharmacological activities comparisons by in vivo and in vitro models actually support that compound K and/or 20(S)-protopanaxadiol should be the effective substance(s) of orally taken ginsenoside Rb1. For example, only compound K protected chemically injured HepG2 cells in vitro comparing to ginsenoside Rb1; however, ginsenoside Rb1 also presented hepato-protective effect as well as compound K when orally administration in mice, whereas without such effect by intraperitoneal injection (Fig. 14.1) (Xu et al. 2017). Besides above major metabolic pathway, ginsenoside Rb1 possesses other minor pathway in human gut: ginsenoside Rb1 ! gypenoside XVII ! gypenoside LXXV ! compound K PPD (Bae et al. 2000). In a similar vein,

ginsenoside Rb2 can be metabolized via stepwise deglycosylation by gut microbiota to ginsenoside Rd and compound K in rats; meanwhile, the terminal metabolite of ginsenoside Rc is compound K and 20(S)-protopanaxadiol by human gut microbiota (Bae et al. 2000, 2002). Based on illumination of the metabolic pathway, the gut bacterial strain members who participate exclusively in the process have been screened and identified by comparative pharmacokinetic study of ginsenoside Rb1 in such normal and germ-free model mice. Akao et al. (1998b) compared the metabolites of ginsenoside Rb1 after orally consuming in germ-free rats and gnotobiote rats mono-associated with 31 defined intestinal strains from man, respectively, and found that only Eubacterium sp. A-44 transformed ginsenoside Rb1 into compound K (70.3%) via ginsenoside Rd (16.8%). Prevotella

Fig. 14.1 Crucial role of gut microbiota in intestinal biotransformation of ginsenoside Rb1 in mammalian host. For arrows in the “bioactive comparison” box: solid

arrows denote positive/strong effect; dotted arrows denote weak effect; solid arrows with an “X” mark denote negative effect (Xu et al. 2017)

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oris strains were also isolated as a major bacterial species possessing the potential of ginsenoside Rb1 hydrolyzing activity from 58 human subjects fecal specimens (Hasegawa et al. 1997). Eubacterium sp. A-44 and Prevotella oris these two strains were demonstrated as the competent ones to transform ginsenoside Rb1 into compound K. Some other bacterial species were identified to cooperate on the gradual metabolism of ginsenosides. Bae et al. (2004) found that among the bacteria isolated from human fecal microflora, Bacteroides sp., Bifidobacterium sp., and Eubacterium sp. potently transformed ginsenoside Rg3 to ginsenoside Rh2, ultimately compound K in an “efficient division cooperation system” (Fig. 14.2).

14.2.2 20(S)-Protopanaxatriol-Type Ginsenosides (PPT) Comparing to PPD, the C-6 of 20(S)protopanaxatriol-type ginsenosides (PPT) attaches to a hydroxyl group, resulting a similar metabolic pathway in the intestine between them. That is namely under the action of the gut bacterium, the cleavage of the glycosidic bond occurs stepwise from the outer side of sugar chain on the C-3 or C-20. As the representative of PPT, ginsenoside Re is catalyzed to

X. Shuiming and Z. Xiaoyan

ginsenoside Rg1, Rg2, Rh1, F1, and 20(S)-protopanaxatriol successively by the intestinal flora. The entire metabolic process is performed by series of intestines bacteria together, including Prevotella oris, Eubacterium sp. A-44, Bifidobacterium sp. K506, Bacteroides sp. JY6, and Fusobacterium sp. K-60 (Hasegawa et al. 1997; Bae et al. 2005; Chi and Ji 2005; Yang et al. 2009).

14.3

Individual Varied in Ginsenosides Biotransformation

Many drugs exhibit greatly varied efficacy and toxicity in individuals, and thus the resulted adverse drug reactions cause considerable patient harm and financial burden (Classen et al. 1997). Besides genetic polymorphisms, environmental factors, such as lifestyle, nutritional status, diseases and age, also affect individual response to drugs (Schnackenberg 2007). Among these environmental factors, a neglected but critical component, namely gut microbiota has evoked people’s attention to its influence on drug action, fate, and even toxicity (Nicholson et al. 2012; Zhao et al. 2012). It is speculated that more than 60% of drug responses are related to the individual-specific gut microbiota, through direct

Fig. 14.2 Gut microbial metabolisms of ginsenosides Rb1 (Niu et al. 2013)

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Gut Microbiome for Ginseng Medicine

microbial biotransformation or modulating host enzymes of drug metabolism indirectly (Haiser and Turnbaugh 2012). Therefore, Nicholson et al. (2003, 2005) proposed the concepts of “global systems biology” and “pachinko model,” which integrated gut microbiota into human global metabolism, to explain the variation in drug metabolic responses. In recent years, pharmacomicrobiomics field has focused on the interplay of gut microbiota variation and drugs response and disposition to approach personalized medicine (Doestzada et al. 2018). Therefore, gut microbiota becomes the attractive target for enhancing drug, especially the TCMs efficacy since the plasticity of its structure composition and metabolic function. These individual-specific gut microbiota composition and/or metabolic function potential, for instance, varied a/b glucosidase activities, make them become a source of individual physiological variability. Choi et al. (2011) measured the metabolic activities of ginseng, ginsenoside Rb1 and Rg1 by 100 fecal specimens to evaluate the difference in expressing pharmacological effects of ginseng and came to a conclusion that the intestinal bacterial metabolic activities of ginseng components are variable in individuals. On this basis, Kim et al. (2013) reported that the individuals which with fecal activity potently metabolizing ginsenoside Rb1 to CK (FPG) processed higher population levels of phylum Bacteroidetes and Tenericutes, genus Clostridiales_uc_g, Oscillibacter, Ruminococcus, Holdemania, and Sutterella. Especially, the levels of Bacteroides and Bifidobacterium were dramatically increased in FPG since they were considered to metabolize ginsenoside Rb1 to CK. This phenomenon that fecal bacterial enzymatic activities related to the pharmacological actions of herbal medicines including ginseng were variable among individuals also confirmed by other reports (Lee et al. 2002; Bae et al. 2004; Yim et al. 2004; Kim et al. 2008). Interestingly, sometimes this individual variation is not caused directly by the shift of the intestinal bacteria structure/composition, but the enzyme activities which affected by dietary change and physiological factors (Reddy et al. 1980; Mallett et al. 1988; H et al. 2010).

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14.4

Improving the Bioavailability of Ginsenosides by Gut Microbiota-Targeted Intervention

The absorption of parental ginsenoside is low in the intestine, such as the intestinal absorption rate of ginsenoside Rb1, Rb2, and Rg1 is 1.0%, 3.4%, 1.9%, respectively (Li et al. 2009; 王菡 et al. 2012). As a “natural active precursor” of ginsenosides was removed the glycosyl by various glycosidases (such as b-glucosidase and aarabinosidase), which was secreted by the intestinal microflora and convert to more potent pharmacological effect and less sugar-based saponins or aglycones (Hasegawa 2004). Therefore, targeting gut microbiota to improve the specific species abundance or glucosidase activities by probiotics (Marteau et al. 1990; Goossens et al. 2003) and prebiotics (Khadka et al. 2014) would probably increase the formation and subsequent absorption of CK. In our unpublished work, Sprague Dawley (SD) rats were oral administered of Rb1 after 2 weeks intervention with three commercial prebiotic fibers, including fructooligosaccharide (FOS), galactooligosaccharide (GOS), and fibersol-2, respectively. Pharmacokinetics analysis of ginsenoside Rb1 and its metabolites were performed, while gut microbiota was taxonomically and functionally analyzed using total 16S rDNA microbiota and metagenome shotgun sequencing. Our results showed that the pharmacokinetic parameters, such as the peak plasma concentration (Cmax) and the area under concentration–time curve (AUC0-t) of ginsenoside Rb1 and its intermediate metabolite ginsenoside Rd, F2, and CK from the three prebiotics intervention groups, were increased in varying degrees than the control group. The overall gut microbiota responded to the prebiotics intervention showed significant differences both at the taxonomical and functional level. Most notably, the genera Prevotella, which possesses the potential function to hydrolyze ginsenoside Rb1 to CK, were significantly elevated in prebiotics groups (P < 0.05). The gut metagenome results

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indicated that the functional enrichment of genes occurs for functions such as terpenoids and polyketides metabolism, glycolysis/ gluconeogenesis, propanoate metabolism and the citrate cycle in prebiotics intervention groups. These results indicated that the prebiotics treatment may promote selectively the proliferation of certain stains with glycoside hydrolases, namely strengthen the hydrolytic activity of gut microbiota to convert ginsenosides Rb1 into its metabolites, including ginsenosides CK. Another a 3-week intervention by fermented dairy product that contains 2 probiotics: Lactobacillus acidophilus and Bifidobacterium bifidum on healthy volunteers increased the fecal bglucosidase activity (Marteau et al. 1990). Furthermore, Kim et al. (2014) investigated the effect of a soluble prebiotic fiber, NUTRIOSE, on the absorption of ginsenoside Rd after administered ginseng extract. When the fecal microbiota of rat were anaerobically cultured with or without NUTRIOSE in vitro and measured the ginsenoside Rd-forming activity. The results showed that the metabolism of ginsenoside Rb1 to ginsenoside Rd was induced 3.4 fold compared with model group. When ginseng extract (2000 mg/kg) was orally administered to rats fed NUTRIOSE-containing diets (2.5%, 5%, and 10%). The AUC of ginsenoside Rd increased by 1.13, 1.08, and 1.34 fold, respectively, and the Cmax of ginsenoside Rd is also significantly increased 1.08, 1.19, 1.35 fold, respectively, compared with the model group. These findings reveal that intestinal microflora promote metabolic conversion of ginseng extract to ginsenoside Rd and promote its absorption into the blood in rats. Kim et al. (2015) also investigated the effect of NUTRIOSE on the pharmacokinetics of ginsenoside CK. Firstly, the effect of NUTRIOSE on CK pharmacokinetics were determined by HPLC-MS/MS when orally administered of ginseng extract (2000 mg/kg) after receiving control or NUTRIOSE-containing diet (2.5%, 5%, or 10%) in rats. The AUC values

X. Shuiming and Z. Xiaoyan

of compound K increased by 1.22, 1.43, and 2.82 fold, and the Cmax of compound K is also significantly increased 1.00, 1.61, and 2.26 fold, respectively, when compared with the model group. The biotransformation of ginsenosides to compound K involves glycosidic bond cleavage by gut microbial hydrolysis. Further, NUTRIOSE can increase the number of intestinal Lactobacillus spp. and Bacteroides (Lefranc‐ Millot 2010; Lefranc-Millot et al. 2012). In the study, the effects of NUTRIOSE on glycosidase activities of intestinal microbiota were investigated, the results showed that the activities of aD-glucosidase, b-D-glucosidase, b-D-xylosidase, and a-L-rhamnosidase were increased by NUTRIOSE treatment. The resulting data showed the metabolic rates of ginsenoside Rb1 to CK by fecal bacteria in the 5% and 10% NUTRIOSE-treated groups were > 10 times higher than that in the control group. Those findings display that prebiotic diets, such as NUTRIOSE, may alter the composition of intestinal microbes and thereby promote the metabolic conversion of ginsenosides to CK and the subsequent absorption of CK in the gastrointestinal tract. Zhou et al. (2016) explored the effect of ginseng polysaccharides on the intestinal metabolism and absorption of ginsenosides by suffering successive over-fatigue and acute cold stress model. The high-performance gel permeation chromatography (HPGPC) chromatogram showed that ginseng polysaccharides had a wide molecular weight distribution from 1.00 kDa to 1308.98 kDa with the weight-average molecular weight and number-average molecular weight of 39.56 kDa and 3.57 kDa, respectively. Eleven main chemicals were analyzed by ultraperformance liquid chromatography quadrupole time of flight mass spectrometry (UPLC-QTOFMS) containing ginsenosides Re, Rg1, Rf, Rb1, 20(S)-Rg2, Rc, Rb2, Rd, F2, 20(S)-Rg3, CK. The results indicated that ginseng polysaccharides can effectively regulate the endogenous metabolites

14

Gut Microbiome for Ginseng Medicine

of tryptophan, phenylalanine, lysophosphatidylcholine, cholic acid, cresol sulfate, trimethylamine-N-oxide, isocitrate and 4-methyl phenol and restored OACS-induced disorder of endogenous metabolism. Influence of ginseng polysaccharides on the gut microbiota of OACS model was investigated. Ginseng polysaccharides reversed the OACS-induced gut microbial dysbiosis at phyla level to approach the homeostasis by increasing the relative abundance of Firmicutes and decreasing that of Bacteroidetes. The PCoA results of OTUs further confirmed a tendency: the clusters of the ginseng polysaccharides and blank groups intertwined mutually, but detached with that of the model group. The relative abundances of Bacteroides spp. and Lactobacillus spp. were increased by ginseng polysaccharides treatments with significant (P < 0.05) or near-significant (0.05 < P