Phytochemistry of Fruits and Vegetables 9387445003, 9789387445000

Citation preview

Phytochemistry of Fruits and Vegetables

Phytochemistry of Fruits and Vegetables

Editors

Prof. K.V. Peter Former Vice-Chancellor Kerala Agricultural University Thrissur, Kerala -680651

Brillion Publishing 22 B/5 Ground Floor, Desh Bandhu Gupta Road Karol Bagh, New Delhi - 110005 Ph.: + 91 (11) 4155-8799 Email: [email protected] brillionpublishing.com

© Publisher, 2018 ISBN: 978-93-874450-00 All Rights reserved under International Copyright Conventions. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording or otherwise without the prior consent of the publisher or the copyright holders. This book contains information obtained from authentic and highly regarded resources. Reasonable efforts have been made to publish reliable data and information, but the author/ s editor/s and the publisher cannot assume responsibility for the validity of all materials or the consequences of their use. The author/s editor/s and the publisher have attempted to trace and acknowledge the copyright holders of all materials reproduced in this publication and apologize to copyright holders if permission and acknowledgements to publish in this form have not been taken. If any copyright material has not been acknowledged please write and let us know so that we may rectify it, in subsequent prints. Trademark Notice: Presentation, logos (the way they are written or presented) in this book are under the trademarks of the publisher and hence, if copied/resembled the copier will be prosecuted under the law. Printed in India

Science is the Language of the Universe Every book tells a story. From how the miniscule atoms make up vast galaxies, how a single cell transpires into millions of species, or how a single chemical helps us travel into the far universe, every book, even scientific, is telling a story. The story of how the universe speaks. At Brillion we believe science belongs to everybody and hence, we bring it at your disposal. Our motivation each day lies in bringing our authors and readers closer to each other, in broadening the horizons of the wisdom and insight of the author, inspiring the minds of our readers and taking science everywhere. We express our gratitude to the scientists who make the technology of tomorrow possible, helping us evolve without the biological tools. We understand their vision while writing a book, the vision of a better tomorrow. And we are here to help them spread this mission to all possible readers and scholars, waiting to be inspired. We hope to do justice to their beliefs by our efforts, and carry and sustain the torch of wisdom lit by their passion for science. We want all our readers to read this book as if they know the author, with the capacity to question the work, of keeping the inquisitive spirit of science alive. We promise our readers to have their opinions and doubts heard buy the author and return them answered whenever possible. You can sign up at our website to have a chance to interact live with the authors about their work, opinions and everything science. The Brillion Team

Foreword Horticulture, a science and art of cultivating gardens, orchards, flowers, fruits, vegetables, tuber crops, ornamental plants, medicinal and aromatic plants, spices and plantation crops results in the development of minds and emotions of individuals and enriches health of communities, and is integrated in the length and breadth of modern civilisation. Horticulture, an ancient science, preferred food of saints and sages has evolved through various phases of development to reach to current stage of commercial horticulture for economic development and trade. This has been possible due to intensive research for the creation of knowledge, which becomes the technology to drive the development. Indian horticulture is also ancient, as its mention can be found in epics and civilisations. However, it continued as a hobby till turn of the 20th century, except for a few commodities which were traded and had commercial values. With intensive research and development of technologies, horticulture moved from rural confines to commercial production.

Now, the horticulture is viewed as an option for better land use planing, enhanced income per unit are, better employment, environmental services and above all, meeting the need for nutritional security and environmental services. Production which was only 25 million tones in 1950 has exceeded 300 million tones in 2017. This development in of horticulture is termed as Golden Revolution attracting Government private sectors for investment in research and development, leading to technological changes in production system management leading to enhanced availability of fruits and vegetables for consumption. However, to sustain the growth and have new input gains,, and also to address the challenges of producing more with less, intensive efforts The book Phytochemistry of Fruits and Vegetables exposes readers to the science based technologies useful in food industry, health and wellness industry, energy sector,manufacturing sector, eco-tourism and handicraft industry. The present book with the title“Phytochemistry of fruits and vegetables” carries 15 chapters authored by 38 eminent scientists working in 20 Research/Institutes and Universities.. Confederation of Horticulture Associations of India, is a forum to work together for excellence and furtherance of horticulture Institutes/Universities. Each chapter is carrying relevant quotes from well known classics. Science behind perceived and applications of horticulture as an art, are explained quoting published literature.The series is targeted to graduate and post graduate students and scientists, as a ready reckoner to understand the basics of horticultural practices and theories. I wish to compliment Prof. K.V. Peter, Former Vice-Chancellor KAU, for compiling and editing the chapters in the form of a book. Mr. Vardhan Gupta from Brillion Publishing deserves my compliments for publishing the book and disseminating the knowledge in horticultural science for the benefits of all the stakeholders in horticulture. I am sure, this book will be useful to all the horticulturalists.

H.P. Singh

Head Quarter 249, D block, VVA, Kargil Apartment, Sector 18A,,Dwarka, New Delhi -110078, India. Phone: 91 011 28085749; Mobile 91-9871450730 , 91-9582898983(Res) http://www.chai.org.in Email:[email protected]/ [email protected]"

Preface Horticultural Science imparts basic knowledge on nutritional security, whereas the horticultural crops create employment generation, enhance purchasing power, increase export earnings and lead to crop diversification with higher cropping intensity and higher returns/unit area. Horticulture is the best option for small and marginal farmers, linked with health and happiness of the nation. Under nutrition in India is staggering 48%, followed by Bangladesh (46%), Nepal(45%) and Pakisthan (38%). Horticultural crops provide a better balance between carbohydrates, protein and micronutrients and are the appropriate crops to achieve nutritional security and reduce hidden hunger and remedies to nutritional maladies. The year 2016-17 witnessed higher compound growth rate since 2005 in horticultural crops (5.44%) as compared to 2.63% in food grains. Horticultural production (299.8 million tones) exceeded food grain production (275.7 million tones) making available 200g/day of fruits and 400 g/day of vegetables. There is significant increase in various parameters of production i.e. 4.8% increase in horticultural crops, 2.6% increase in area under horticulture, 17.4% increase in spices crops production, 10.2% increase in plantation crops, 4.3% increase

in flower production, 4.2% increase in vegetable production and 3.9% increase in fruit production. Exports worth Rs. 14,500 crores are attained gaining an organized industrial status to horticulture (fresh onions 22.10%, fresh vegetables 16.00%, processed fruits and vegetables 15.8%, fresh grapes 11.60%) and much head way has to be made to surpass major exporting countries like China, Malaysia, Brazil and Israel. India today is the 5th largest and fastest growing vegetable seed market in the world worth $6.0 Billion. Horticulture can be a base for zero malnourishment and hidden hunger in India. The book “Phytochemistry of Fruits and Vegetables” aims to consolidate science based technologies i.e. seeds, soils, water, agronomy, plant protection, harvest and post harvest technologies, packaging and transport, value addition and product development, trade and agreements, waste management and clean environment. The present edition carries 15 chapters contributed by 38 scientists from 20 Research Institutes and Universities. I congratulate the contributing authors and Prof. K.V. Peter who edited the chapters to the present form. I appreciate the sincere efforts of Mr. Vardhan Gupta, Brillion Publishing, New York for publishing the series.

(B. Singh)

Phone: 0542-2635247/37/36 (Off.) 0542-2300797 (Res.), Fax: 05443-229007, Mob. 08004924520 E-mail:[email protected] / [email protected], Telegram: VEGRES

Contents Foreword Preface List of Contributors Introduction 1. Phytochemistry of Lipids and Pigments in Fruits and Vegetables Ipsita Pujari, Abitha Thomas and Vidhu Sankar Babu 2. Chemistry of Antioxidants Desalinizing Soils Uttam Kumar and I.J. Gulati 3. Fermentation Chemistry of Palm Neera K.B.Hebbar 4. Chemistry of Macronutrients Fixation in Acidic Soils B.B. Basak and Rajiv Rakshit 5. Physiology and Biochemistry of Fruit Ripening K.S. Shivashankara, K.C. Pavithra and A. Nethravath 6. Biochemical and Molecular Aspects of Latex Production in Hevea brazieliensis Molly Thomas, Ambily, P.K., Sreelatha, S. and James Jacob 7. Strategies for Mitigation of Impact of Climate Change on SubTropical Fruits Gaganpreet Kour and Parshant Bakshi 8. Micro-Irrigation for higher Water Productivity in Horticultural Crops C. K. Saxena and K.VRamana Rao

9. Nutritive Values of Vegetables Vibha Mishra and Vinod Kumar Sharma 10. GM Vegetables for Higher Productivity and Resistanceto Biotic Stresses Abdul Majid Ansari and Y V Singh 11. Scientific Cultivation of Onion (Allium cepa L.) Desh Raj Choudhary 12. Yams for Nutritional Security M. Nedunchezhiyan, K. Laxminarayana, V.V. Bansodeand V.B.S. Chauhan 13. Improvement of Seed Spices Arvind Kumar Verma, Sharda Choudhary, Ram Swaroop . Gopal Lal and Meenakshee Sharma 14. Floral Crops under Proteaceae Kalkame Ch. Momin, Y C Gupta, K S Tomar and T S Meh 15. Food Quality and Food Safety Renu Agrawal

List of Contributors Ipsita Pujari Department of Plant Sciences, School of Life Sciences,Manipal University, Manipal Karnataka-576104 Abitha Thomas Department of Plant Sciences, School of Life Sciences,Manipal University, Manipal Karnataka-576104 Vidhu Sankar Babu Department of Plant Sciences, School of Life Sciences,Manipal University, Manipal Karnataka-576104 Uttam Kumar Department of Soil Science andAgricultural Chemistry, College of Agriculture, Indira GandhiKrishi Viswavidyalaya, Raipur-492012 I. J. Gulati Department of Soil Science and Agricultural Chemistry,College of Agriculture, SwamiKeshwanand Rajasthan Agricultural University, Bikaner334006 K.B. Hebbar Principal Scientist, Plant Physiology, ICAR-CPCRI Kasargod-671124 B.B. Basak ICAR-Directorate of Medicinal andAromatic Plant Research(DMAPR), Boriavi, Anand-387310 Rajiv Rakshit Bihar Agricultural University,Sabour Bihar

K.S. Shivashankara Division of Plant Physiology and Biochemistry, ICAR-IIHR, Hissarghatta Lake P O.Bengaluru-560089 K.C. Pavithra Division of Plant Physiology and Biochemistry, ICAR-IIHR, Hissarghatta Lake P O.Bengaluru-560089 A. Nethravathi Division of Plant Physiology and Biochemistry, ICAR-IIHR, Hissarghatta Lake P O.Bengaluru-560089 Molly Thomas Rubber Research Institute of India, Rubber Board P O, Kottayam-686009 P.K. Ambily Rubber Research Institute of India, Rubber Board P O, Kottayam-686009 S.Sreelatha Rubber Research Institute of India, Rubber Board P O, Kottayam-686009 James Jacob Rubber Research Institute of India, Rubber Board P O, Kottayam-686009 Gaganpreet Kour Division of Fruit Science, Sher-e-Kashmir University of Agricultural Sciences and TechnologyJammu Parshant Bakshi Division of Fruit Science, Sher-e-Kashmir University of Agricultural Sciences and TechnologyJammu C.K. Saxena ICAR-Central Institute of Agricultural Engineering, Bhopal-462038 K.V Ramana Rao ICAR-Central Institute of Agricultural Engineering, Bhopal-462038 Vibha Mishra

ICAR-IARI Regional Station,Katrain, Himachal Pradesh Vinod Kumar Saxena ICAR-IARI Regional Station,Katrain, Himachal Pradesh Abdul Majid Ansari Zonal Research Station,(Birsa Agricultural University, Ranchi), Chianki, Palamau-822102 Y.V. Singh Department of Vegetable Science, G.B.PU.A.T.Pantnagar-263145 Udam Singh NagarUttarkhand. Desh Raj Choudhary Department of Vegetable Science, CCS Haryana Agricultural University, Hisar-125004 M. Nedumchezhiyan ICAR-Central Tuber Crops Research DumudumaBhubeneswar-751019.

Institute,

Regional

Centre,

K. Laxminarayana ICAR-Central Tuber Crops Research DumudumaBhubeneswar-751019.

Institute,

Regional

Centre,

V.V. Bansode ICAR-Central Tuber Crops Research DumudumaBhubeneswar-751019.

Institute,

Regional

Centre,

V.B.S. Chauhan ICAR-Central Tuber Crops Research DumudumaBhubeneswar-751019

Institute,

Regional

Centre,

Arvind Kumar Verma ICAR-National Research Centre on Seed Spices, Ajmer-305 206, Rajastan Sharda Choudhary ICAR-National Research Centre on Seed Spices, Ajmer-305 206, Rajastan

Ram Swaroop Meena ICAR-National Research Centre on Seed Spices, Ajmer-305 206, Rajastan Gopal Lal ICAR-National Research Centre on Seed Spices, Ajmer-305 206, Rajastan Meenakshee Sharma ICAR-National Research Centre on Seed Spices, Ajmer-305 206, Rajastan Kalkame Ch. Momin College of Horticulture and Forestry, University,Pasighat, ArunachalPradesh

Central

Agricultural

K.S. Tomar College of Horticulture and Forestry, University,Pasighat, ArunachalPradesh

Central

Agricultural

T.S. Mehra College of Horticulture and Forestry, Central Agricultural University, Pasighat, ArunachalPradesh Y.C. Gupta Dr Y S Parmar University of Horticulture and Forestry, Nauni, Solan, Himachal Pradesh Renu Agrawal Department of Food Microbiology and Fermentation, C.F.T.R.I. Mysore

Introduction “The greatest of evils and the worst of cruelty is poverty” Bernard Shaw TThe much publicized STATE OF FOOD SECURITY AND NUTRITION IN THE WORLD -2017 of Food and Agriculture Organization of the United Nations (FAO), International Fund for Agricultural Development (IFAD),United Nations Children’s Emergency Fund (UNICEF),World Food Programme (WFP) and World Health Organization (WHO) throw light on building resilience for peace and food security. Key messages from the well researched publication are 1). In 2016 the number of chronically undernourished people in the world is estimated to have increased to 815 million, up from 777 million in 2015 although still down from 900 million in 2000. 2)Multiple forms of malnutrition coexist with countries experiencing simultaneously high rates of child undernutrition, anaemia among women and adult obesity. Rising rates of overweight and obesity add to these concerns. Childhood overweight and obesity are increasing in most regions and in all regions for adults. 3). The report sends a clear warning that the ambition of a world without hunger and malnutrition by 2030 will be challenging-achieving it will require renewed efforts through new ways of working. According to Global Hunger Index (GHI) scores,the level of hunger in the world has decreased by 27% from the 2000 level. The share of the overall population that is undernourished is 13.0% down from 18. 2%, 27.8% of children under five are stunted down from 37.7 %, 9.5% of children under five are wasted down from 9. 9% and under- five mortality rate is 4.7% down from 8.2% in 2000. India is ranked 100 out of 119 countries scored for GHI in 2017 as per International Food Policy Research Institute (IFPRI). The transformational vision of the 2030 Agenda for Sustainable Development calls all countries and stake holders to work together to end hunger and prevent all forms of malnutrition by 2030 within the frame work of the

Sustainable Development Goals (SDGs). Indian Parliament enacted the Food Security Bill- 2013 making food available to people and access to food a right under the Indian constitution. Nutritional security by making available pulses through Public Distribution System (PDS) is also ensured through a legal frame. “Hungry India” was ranked 97 out of 118 developing countries with widespread deficiency of Vit. A,calcium,iodine,iron and zinc. There is growing concern and resultant emphasis on making available fruits, vegetables, spices,mushrooms, bamboos, tuber crops and aromatic and medicinal plants. During 2016-17 total production of fruits and vegetables has surpassed food grains and recorded 299. 85 million tones leading to an availability of fruits(200 g/day) and vegetables (400g/day). Affordability to fruits and vegetables by people below poverty level and financially vulnerable is the critical economic policy to be taken to realize a zero hunger India of Gandhiji’s dream. Srimad Bhagavat Gita (Chapter 17,Verse 8) describes “pure food lead to wisdom, longevity and well-being. Fruits, vegetables ,nuts,dairy products and raw honey belong to nutritious category which are loved by virtuous people”. Fruits and vegetables are opulent sources of indispensable micronutrients and dietary fibres. Studies have evidenced that people who evade or who diminutely consume fruits and vegetables are at elevated risk of being susceptible to chronic diseases. Perception regarding health promoting efficacy of fruits and vegetables intake is mounting and the awareness along with curiosity in knowing its chemical nature,quantity and health efficacy of different components is on upsurge. Ipsita Pujari et al. (1918) elaborate phytochemistry of lipids and pigments in fruits and vegetables (Chapter I). Traditionally fruits and vegetables are opulent sources of indispensable micro-nutrients and dietary fibres. Soil salinity is a major cause of low productivity in fruits and vegetables. Favourable micro-organisms become inactive in saline solis. Micro nutrients vital for crop production get bound and become unavailable. Chapter II describes chemistry of anti-oxydants to desalinize soils. Acid soils with pH below 5.5 get macronutrients like phosphorous and potassium fixed and bound and make the same unavailable to fruits and vegetables. Rhyzosphere sustaining favourable fungi, bacteria and lichens gets adversally affected. Chemistry of macronutrientnitrogen,phosphorous, potassium, calcium,sulphur-in acidic soils is reviewed in Chapter III. Climactric (tomato, banana, apple, mango, papaya, sapota, guava and kiwi) and and non-climatric (capsicum,pineapple,pome granate,

strawberry, melon, citrus, cherry and grape) mechanisms are crop specific ripening behaviour genetically controlled and inherited. Ripening of fruit when attached to the plant and ripening of fruit after harvest are two contrasting behaviours (Chapter IV). Among industrial crops rubber tree has higher economic value considering the value added products from the latexnatural rubber based products-,seismic absorbers,health and wellness industry,roads and bridges and rubber tree trunk for furniture industry (Chapter V). Among many factors limiting productivity and production of fruits and vegetables,climate change occupies the most important role-rise in temperature,increased CO2 accumulation,melting of glaciers leading to flooding and intrusion by alien pests and diseases. Early flowering, reduced productivity, reduced honey bee activity and unseasonal rains are the aftermath of climate change (Chapter VI). Irrigation water has become a limiting factor in farming and more so in horticulture. ”Every drop a crop” is becoming a reality with water management measures like drip irrigation,application of acquagel,spot irrigation, micro-sprinkler, mist irrigation and water conservation in rhyzosphere by mulching (Chapter VII). Data on water requirement of fruits and vegetables are now available and moisture meters reveal available free water. Vegetables are health and wellness foods and men basically are vegans and evolved to vegetarians and non-vegetarians. Vegetables are alkaline in reaction, rich in micro-nutrients and fibres essential for microbes to multiply for digestion. In “Ayurveda, Siddha and Unani” systems of health care vegetables are essential foods. Leafy vegetables are richer in soluble fibres and minerals like iron and manganese and vitamins. Antioxydants in vegetables keep the human body protected from toxins and allergens. (Chapter VIII). Productivity of vegetables in India is only 15 tones/ha compared to 70-100 tones/ha in China,Australia and Israel. One main reason is treatment of vegetables as field crops left to vagaries of weather. There are several biotic-bacterial, fungal, viral, viroidal,insect pestsstresses affecting vegetable growing. Biofortification of vegetables for higher nutrients availability and absorption is a recent innovation from biotechnological research. Genomics, proteomics and other”omics” lead to gene identification, recovery, transfer, expression and pyramiding. Genetically Modified vegetables in tomato,brinjal,cole crops, cucurbits and onion are now available. The GM vegetables are released to farmers after rigorous tests for biosafety. (Chapter IX). Onion is a popular bulb vegetable used all over the world for its pungent bulbs. There

are many coloured onions-white, red, yellow, purple many sized onionssmall,large, giant- and of genetic constitution-open pollinated,male sterile and pollinated and hybrid onions. Based on pungency there are sweet,mild pungent and deeply pungent onions. Potato onion is popular in South of India. Purple blotch is the most serious fungal disease of onion (Chapter X). Tuber crops including yams are called “famine foods” which replace cereals during famine and scarcity. Rich in carbohydrates, they are sources of industrial products like alcohol and bio-diesel. Yams are main staple foods in Africa and a good number of value added products are made from yamschips,bits and powders for biscuits and confectionaries and adhesives-. Less water requiring and climate resilient yams are crops for future foods (Chapter XI). Seed spices –coriander, cumin, fennel, fenugreek,dill,ajwain-are important seasonals adding colour, flavour and taste to main foods. They possess medicinal properties as well (Chapter XII). Value added products like oils, oleoresins and flavourants are made from above seed spices. Ornamentals add to the aestheticity,beauty and colour to living habitats like homes, public places, schools and places of worship. Floral crops belonging to family Proteaceae are special and unique in home decorations and bouquet making as they consist of green and coloured floral parts (Chapter XIV). Clean food is essential for clean body and mind. Food quality and food safety are must and ensured through legislations. International Standard Organization (ISO) and Bureau of Indian Standards (BIS) are agencies formulating minimum standards for food quality and safety (Chapter XV). Present Book “Phytochemistry of Fruits and Vegetables” carry 15 chapters of current importance. I wish the publication will be read by students, teachers, men in Industry and scientists. (K V Peter)

CHAPTER 1

Phytochemistry of Lipids and Pigments in Fruits and Vegetables Ipsita Pujari, Abitha Thomas and Vidhu Sankar Babu Traditionally fruits and vegetables are opulent sources of indispensable micronutrients and dietary fibers (Rechkemmer, 2001). They have been documented as significant, as they are endowed with a set of bioactive that discretely or in mixture help in overall well-being (Stavric, 1994). It is reviewed that one percent of the cancer cases and almost 50 percent of the circulatory diseases (primarily, “cardiovascular diseases”) are proven to be linked to nutritional regimen (Goldberg, 1994; Nagura et al. 2009;SmithWarner et al. 2001). Copious research studies on epidemiological disorders (Hirayama, 1990; Steinmetz and Potter, 1991, 1996) revealed a converse relationship amid fruit and vegetable consumption and severe maladies like cancers and circulatory disorders (World Cancer Research Fund, 1997; Gandini et al. 2000; Bazzano et al. 2002; Radhika et al. 2008). These studies have evidenced that, people who evade or who diminutively consume fruits and vegetables, are at elevated risk of being susceptible to chronic diseases (Howe et al. 1992). Consequently, perception regarding health promoting efficacy of fruits and vegetables intake is mounting and the awareness along with curiosity in knowing its chemical nature, quantity and health efficacy of different components is on an upsurge (Kris-Etherton et al. 2002). Phytochemicals found in fruits and vegetables are dissimilar (Liu 2004; Yahia et al. 2009a, 2009b) and their potency is yet to be discovered (Syngletary et al. 2005; Percival et al. 2006). Among the secondary compounds, phytopolyphenols are universal in any common dietary elements, be it fruits, vegetables, green tea and red wine; they are proven to

lug potent antioxidative ability in vitro, which is anticipated to have vital effects on health (Duthie et al. 2006). Since naturally available phytochemicals possess anticarcinogenic and anti-mutagenic efficacies, they are addressed as “chemoprotectors”. The defensive mechanism equipped with antioxidant property is one of their chief contrivances, which is gauged with their scavenging ability of free radicals. The antioxidants found in plants are a)betalains, b)carotenoids, c)chlorophylls, d)vitamins, e) flavonoids and pigments and f)the large class of Phenylpropanoids.

Experimental Evidences of Concepts with Phytochemical Attributes Relating total fruit and vegetable ingestion with cancer prevention ability is often difficult to estimate (Steinmetz and Potter, 1996; Voorips et al. 2000). However, epidemiological proof of cancer-defensive properties of fruits and vegetables have been analyzed earlier and documented by Wargovich (2000). It has been generalized that a high intake of Solanum lycopersicum or other solanaceous products is often related to cancer incidences (prostate, lung and stomach) as indicated by the meta-analysis done elsewhere (Giovannucci, 1999). Nutritional ingredients are found to breakdown carcinogens and some mutagenic agents (Wattenberg, 1975). We are mentioning here a few examples to prove the efficacy of fruits and vegetables in daily diet. Butylated hydroxyanisole (BHA) consumption enhances microsomal oxidase system in the hepatic cells. Phenolic antioxidants; chiefly BHA and butylated hydroxytoluene (BHT) are yet two other components which instigate the microsomal oxidase system. The natural inducers of microsomal oxidases are obtained from cruciferous vegetables and they are strong anti-carcinogens. Microsomal inducing indoles, three of them viz., indole-3-carbinol, indole-3acetonitrile & 3, 3’-diindolylmethane have been recognized from cruciferous vegetables. Cytochrome P450 spectral features were found to be altered and the microsomal aryl hydrocarbon hydroxylases confirmed high sensitivity leading towards inhibition mediated by naphthoflavone. Kotake-Nara et al. (2001) proved the protective function of food carotenoids on human prostate cancer cell lines. Phytofluene, â-carotene, and lycopene, present in genus Solanum were also found to decrease the prostate cancer cell viability.

Fruits and vegetables have a potent flavonoid called “Quercetin”, which is evidenced to minimize the metastasis (Suzuki et al. 1991). Mutagenicity induced by Quercetin was proved by DNA fingerprinting technique, that detected mutations caused by genetic recombination. This proposes that quercetin has mutagenic and recombinase properties, which delivered molecular evidence towards Quercetin-induced morphometric variations in tumor cell- lines (Harwood et al. 2007). Other flavonoids exhibit similar functional (mutagenic) properties as that of Quercetin (Takahashi et al. 1979). Scientists have confirmed that lycopene from solanaceous-basedproducts are well correlated with prostate cancer impedances. Incidences of coronary heart disease can be reduced through diet and this has been reported in few studies (Law and Morris 1998; Ness et al. 1999; Joshipura et al. 2001; Bazzano et al. 2002; Retelny et al. 2008). Diets rich in many fruits and vegetables are protective against cardiovascular diseases (Ness and Powles 1997; Rissanen et al. 2003). Chlorophyll and Chlorophyllin (CHL) negate the genotoxic effects of various mutagens (Sarkar et al. 1994; Liu et al. 2005; Breinholt et al. 2005). Shannon et al. (2003)and Van Gils et al. (2005) reported that, fruits and vegetables consumption can reduce breast cancer risks. In rat models, it was demonstrated through various studies that, apple consumption can provide chemo-protection (Liu et al. 2001; Giovannucci et al. 1995; 2003; Campbell et al. 2004; Stram et al. 2006; Kirsh et al. 2007). Studies demonstrated that legume consumption sometimes is inversely associated with cardiovascular diseases (Bazzano et al. 2002). We can classify various phytochemicals in plant systems as depicted (Fig. 1).

Synthesis of Lipids Phenolic compounds involve dissimilar molecule groups which include a) Flavonoids consisting of flavones and anthocyanidins b) Stilbenes c) Tannins d) Lignans and e) Lignin. Among the 10,000 identified phenylpropanoids in plants, some show solubility in organic solvent forms, some show water solubility properties, and few others exist as huge insoluble polymers. The biosynthesis of phenolics in plants can ensue by complex networking of several connecting pathways and thereby they constitute a varied metabolic assembly. The diversity within the plant compounds is identified through their functional significance (Fig.1). Function-wise, few of these compounds

provide mechanical assistance and others shield the plant bodies from detrimental UV rays of sun and unwarranted loss of water. Some of these significant chemicals also entice pollinators along with seed broadcasters, while others act as indicators which prompt defensive mechanisms against various biotic and abiotic forms of stresses. Certain phenolic chemicals can subdue the development of proximate competing plant systems (allelopathy); and others deliver protection acting against harmful herbivores and pathogen clusters. Induced secondary metabolite products are generally of high abundance compared to the naturally-occurring metabolites. Phenolic compounds are the second most in abundance when compared to cellulose, which mostly comprise the organic matter and phenolics (primarily lignin) constituting round about 40% of the organic carbon form that exists in the biosphere. Land progression and dominion by vascular plant systems would have been unfeasible with absence of the phenolic compounds to give the protection against UV-B radiation damage, lignin to deliver the mechanical

Fig. 1 : Classification of Phytochemicals in Fruits and Vegetables sustenance, and the phenolics like cutinand suberin to serve as epidermal barriers which will curtail the process of water loss; assisting plants to adapt to the compromised environments.

Synthesis of Phenylpropanoids Phenylpropanoid precursors are formed through two important pathways namely; the “Malonic Pathway” and the “Shikimic acid Pathway” (Fig. 2). Shikimic acid Pathway is the significant one producing many plant phenolic compounds, whereas Malonic Pathway is mostly limited to fungi and bacteria as source of phenolics and quiteinsignificant in higher plant bodies. The Shikimate pathway involves conversion of simple precursors of carbohydrate into important amino acids like tyrosine and phenylalanine. Shikimic acid forms as an intermediate during this pathway and it is found to be inhibited through the broad-spectrum herbicide glyphosate. As animals do not have this Shikimate pathway, they are unable to produce the aromatic amino acids namely; Phenylalanine, Tyrosine, and Tryptophan and that’s the reason these three are considered as the vital nutrients in the diets of animals. Many secondary metabolites in the form of phenolics are derived from phenylalanine and tyrosine. During synthesis of Phenylpropanoids in higher vascular plants, amino acid phenylalanine is the principal substrate whereas tyrosine is used less in plants. Due to the common structure between these two amino acids, the compounds derived from themare collectively referred to as “Phenylpropanoids”. Simple phenolic secondary chemicals include, a) Benzoic acid derivatives b) Trans-cinnamic acid along with p-Coumaric acid and their derivatives and c)Coumarins (phenylpropanoid lactones).

Fig-2 : Overview of major biosynthetic pathways that which give rise to secondary metabolites

Lipoxygenases- the Key Player “Lipoxygenase” represent an enzyme group which contains the non-heme iron and this enzyme type is widely found in both plants and animals (Siedow 1991; Baysal and Demirdoven, 2007). These Lipooxygenases perform a twin enzymatic role (as “dioxygenase”and “hydroperoxidase”) linked with a single protein: Functioning as a dioxygenase, LOX catalyzes the stereospecific dioxygenation of PUFAs having a 1,4-cis,cis pentadiene system into a pentadienyl radical intermediate, which undergoes reaction with molecular oxygen to generate cis, trans-conjugated diene hydroperoxides. This includes iron redox cycling that is located inside the enzyme. The hydroperoxidase activity of Lipooxygenases was at first described during the year 1943 and later the same was confirmed in LOXs, which was obtained from various sources and tissues. Functioning as a hydroperoxidase, LOX results in the oxidation of a vast number of reducing substrates, which get transformed forming the free radicals (P´erez-Gilabertand Garc´ýa-Carmona F,2000; N´u˜nez-Delicado et al. 1999). Germination studies in the Oilseed plants in the dark condition have shown that the storage lipids get mobilized from the lipid bodies residing insidethe cotyledons. Free fatty acids further get metabolized via the β-oxidation mechanism. For germination in cucumber seeds, a specific type of LOX (lipooxygenase) is found in connection with lipid bodies, which add oxygen to the sterified fatty acids andforms triacylglycerol, which has one, two or, three residues of 13-HPOD (Porta and Rocha-Sosa, 2002). Amongst all these compounds, Resveratrol (a phenolic secondary metabolite) has been found acting as substrates of the LOX hydroperoxidase activity. This second reaction is time-consuming due to the occurrence of fatty acid hydroperoxide, but the reaction speeds up when a suitable electron donor and a co-substrate get included into the reaction medium, or when the fatty acid hydroperoxide gets replaced by hydrogen peroxide. Low specificity of the same activity towards the peroxide and the co-substrate illustrate that, this enzyme plays a vital function during the xenobiotics oxidative metabolism. As fatty acids (substrates of dioxygenase activity) and various phenols like, stilbenoids(numerous substrates of hydroperoxidase activity) are low aqueous soluble compounds, this makes the kinetic characterization of the enzyme a difficult task, which has already been resolved through the usage of substrate-complexing agents like

cyclodextrinsor, various surfactants (L´opez- Nicol´as et al. 1994; 1997).

Formation of Lignans and Lignin in Plant Systems 4-coumaric alcohol, Coniferyl alcohol along with Sinapyl alcohol get formed through a cascade of four reactions which reduce the carboxylic group of 4coumaric acid, ferulic acid and sinapic acid, turning these compounds into their corresponding alcohols respectively. The aforementioned three alcohols collectively form “Monolignols”. Dimerization of these monolignols results in lignans, which function as defense compounds in opposition to microbes like bacteria and fungi. Some lignans like Podophyllotoxin, have already been reported to have therapeutic action against acquired immunodeficiency syndrome (AIDS) and Cancers. Lignin also forms an integral element in the cell wall of vascular plants. The name “Lignin” is derived from the Latin word meaning ‘wood’. Oxidation of these monolignols happen through a free radical driven reaction and this results in intermolecular bond formation at random. This polymerization happens inside the cell wall and it forms a three-dimensional, strong and hydrophobic matrix covalently linking the proteins and the cellulose within the cell wall. As lignin has random chemical bond structure, enzymatic degradation of the same is difficult. The lignin composition varies among the plant species. Conifers are found to contain lignin with elevated content of coniferyl, whereas cereal lignans are with elevated content of coumaryl. Lignin in the cells fill the gaps and binds it all together. Lignin is the central constituent of secondary cell walls and it offers the mechanical strength which helps the plants to reach heights and assists in the water conductance through the xylem cells. Lignification between the adjoining cells is considered as a frequent reaction to various infections and wounded tissues. A physical as well as a functional barrier is thus established to obstruct microbial attack and strengthen the injured tissue. Lignin must be eliminated from the wood pulp, during the cellulose and paper manufacturing. This process is an expensive one and it causes toxic effluence. Currently genetic engineering is being used to easily extract lignin without the destruction of trees. Lignansare the compounds, which arederived from two β’ coupled

phenylpropanoid units and these lignans are extensively spread in plant kingdom. Their classification involves eight sub-groups: a) Furan b) Furofuran, c) Dibenzy lbutaned) Dibenzylbutyrolactonee) Aryltetralinf) Arylnaphthaleneg) Dibenzocyclooctadiene and h) Dibenzylbutyrolactol. These sub-groups are classified depending upon the way through which the oxygen gets incorporated into the frame and the patterns of cyclization. Additionally, these lignans show a discrepancy mostly in the levels of oxidation of both the propyl side-chains and the aromatic rings.

Oxidative Stress - The Culprit Though cells retain a group of defensive machineries and repairing mechanisms against the reactive oxygen species (ROS), they are never adequate at all times, paving way towards the oxidative stress, in which the ROS formation overpowers the organisms’ antioxidant resistances. This brings about the damage of biological components like proteins, lipids and DNA.

Methods Based on Lipid Oxidation Antioxidants may arbitrate at any of the three main stages of the oxidative process: Stage 1 -Initiation [oxygen consumption], Stage 2- Propagation [conjugated dienes and peroxides formation], or Stage 3- Termination [lipid peroxidation products].

Oxygen Absorption Methods The initiation phase time-period and its extension in the existence of antioxidant agents can be quantified through the evaluation of the oxygen consumption patterns. The methods of measurement can be manometric (Drozdowski and Szukalska 1987), gravimetric or polarographic (Roginsky and Barsukova 2001). Oxygen absorption methods have inadequate sensitivity and these methods necessitate high oxidation levels as the endpoint for induction periods (Frankel, 1993). In foods, concentrations of

antioxidants are lower than the levels which are required to perform this assay; hence the sensitivity of these methods may not be adequate. However, Azuma et al. (1999) used this method for the analysis of the antioxidative efficacies of fruit and vegetable extracts. In the same way, Roginsky and Barsukova(2001) has determined the antioxidant capacity of both white and red wines, black and green teas, beer and soluble coffee, which a common man consumes.

Detection of Lipid Hydroperoxides Precise primary peroxidation products of lipids (ROOH) can be detected in plasma through High Performance Liquid Chromatography (Yamamoto et al. 1990) or Gas Chromatography-Mass Spectrometry (Hughes et al. 1986). A proposed method uses a dye specific for Fe3+ namely, “Xylenol orange” and principle underlying this method is the capacity of transition metals (in reduced forms) to catalyze the reduction of peroxides into the hydroxyl compounds, while the metal gets oxidized (Nourooz-Zadeh, 1999). This method can be useful both in lipid suspensions (liposomes, lipoproteins) and in whole plasma. Another method is founded on the decomposition of lipid peroxides through hemin, which during the presence of luminol, forms a transient peak of chemiluminescence. This method has been identified as a highly sensitive one and is suitable towards the detection of lipid peroxides content in the plasma. Few other methods are based on the estimation of the enzyme activities; the enzymes involved here are those which attack lipid peroxides.In Glutathione Peroxidase-based assay (Maiorino et al. 1985), either the utilization of NADPH (via UV-spectroscopy) or the production of Glutathione Disulfide (GSSG) is calculated; during the CyclooxygenaseBased method, the O2 decrease is generally estimated (Warso and Lands, 1985). For quantification of all lipid hydroperoxides which are present in the sample, commercially available colorimetric assays provide a broader approach. Instability of these lipid hydroperoxides makes the process of measurement very intricate, as they readily get catabolized both in vivo and in vitroforming alkenes and aldehydes (Wood et al. 2006). Using this approach, in vitro experiments have been carried out to study the antioxidant capacity

of plant and hot-water vegetable extracts (Maeda et al. 1992). Conversely, various investigations have designed for the quantification of the antioxidant effect of target chemicals via in vivo determinations. Wise et al. (1996) measured the ability of dehydrated vegetable and fruit extracts for the modification of the oxidative processes by calculating the level of lipid peroxides in human plasma after the dietary supplementation. Leontowicz et al. (2001) investigated the effect of sugar-, beet pulp- and apple pomacesupplemented-diets on rat plasma lipids and lipid peroxides.Krishnan and Vijayalakshmi(2005) have also evaluated the phenomenon of lipid peroxidation in rats which were fed with banana fractions rich in the secondary chemicals, “flavonoids”. Besides, Arivazhagan et al. (2004) evaluated the effects of aqueous garlic and need leaf extracts on the extent of lipid peroxidation of induced gastric carcinogenesis in male Wistar rats.

Pigments - The natural painters of the Plant Kingdom Many reputed chemo-protective plant chemicals in vegetables and fruits are colored, because of the presence of various pigments. The WHO guidelines and other traditional healthcare guiding principles are founded on selecting one daily serving of both fruits and vegetables from each of the seven classes of color (red, red-purple, yellow-green, orange, orange-yellow, green and whitegreen), with the intention that a mixture of plant chemicals is consumed. A significant study performed by Johnston et al. (2000) during 1994–1996, “a continuing survey of food intakes by individuals,” has been used to scrutinize the types of vegetables and fruits, consumed in theUS. The study has illustrated that people are consuming more fruits and vegetables, whereas intake of dark green and cruciferous vegetable is really low. Many studies opined that consumption of vegetables and fruits is still very low in several countries (Naska et al. 2000; Agudo et al. 2002; USDA, 2004; Blanck et al. 2008), and efforts are required to upsurge it. “Flavonoids” are a diverse group of phenolics (Fig.3) which are naturally-occurring pigments. One of the primary functions of flavonoids is to provide defense to the plants against the oxidative stress like UV rays, environmental pollution along with several chemical constituents. Flavonoids are polyphenolic compounds with a basic

C6-C3-C6 structure which can be divided into various groups such as: flavones, flavonols, flavanols (or flavan-3-ols), flavanones, isoflavones and anthocyanidins. More than 6,000

Source :Information was collected from the USDA database Fig. 3 : Distinctive flavonoid composites in certain Fruits and Vegetables flavonoids are known; out of which the most prevalent ones are Quercetin (flavonols), Luteolin (flavones) and Catechin (flavanols). “Anthocyanidins” (the water-soluble vegetable pigments, found mainly in berries and other redblue fruits and vegetables) also come under flavonoids.

Flavonoids Flavonoidshave a frame of diphenylpropanes, where two benzene rings “A” and “B” are connected through a three-carbon chain forming a closed pyran ring with the benzene ring A. In plants, the class of flavonoids is normally in a glycosylated version; either with rhamnose or glucose. In rare cases; they are also found to be associated with other forms of sugars viz., arabinose, galactose, glucuronic acid and xylose. It has been opined by Vallejo et al. 2004 that, the glycosyl moieties vary from one to three; identification of flavonoids with moieties four to five becomes much more precise. The group of flavonoids with double bond, amid C2 and C3 and an oxygen atom on the C4 position, are categorized asFlavonolsand flavones. Flavonols are identified by the presence of a C3 positioned hydroxyl group. If a flavonoid carries three-carbon chain that is saturated along with C4 position, they are called as Flavanones. In the case of Dihydroflavonols, they share similar

flavonol structure, but they are devoid of C2 and C3 double bonds. The flavonoids with a diphenylpropane structure, where the B ring is localized at the C3 position they are referred to as Isoflavones. Shier et al. (2001) demonstrated that they share structural equivalences estrogen hormones, viz., estradiol, whereas at the C7 and C4 positions they carry a hydroxyl group. Anthocyanins are water-soluble pigments with flavylium salt structure, found distributed in the hypodermal cortical, petals and endodermal regions of plants. Anthocyanidins are aglycone forms in plants. In fruits and vegetables, this water-soluble molecule appears to be in monomers or sometimes as condensed tannin forms that are polymeric with 4-11 monomeric unit assemblage which are termed as Proanthocyanidins. It is seen that in edible tissues they are never seen in glycosylated versions. The sugars found in the C3 position as glycosides and in C3, C5 as diglycosides in any flavonoid class are usually arabinose, fructose, glucose, galactose, rhamnose, and xylose. Clifford (2000) opined that C7, C3’, and C5’positions in flavonoids are usually found to be glycosylated.

Methods of Identification and Quantification Sample Handling intricacy and inconsistency of the chemical fluxes. These chemical fluxes affect the nutritional status and the taste of the serving. These chemicals vary in the way they are processed and prepared.The chemical flux transitions in fruits and vegetables are classified as follows: a)Physico-structural characteristics and b) Biological factors. The physico-structural traits are dependent on the hydration and cell wall composition, skin color, pigment composition and distribution in the epidermal and hypodermal regions. These features would greatly alter in berries or grapes, oranges or apples, lettuce or watercress and with carrot or pumpkin. Processing by mechanical means facilitate certain reactions driven by enzymes related to phenolic release and further reaction with the atmospheric oxygen during wounding and causing changes in pigment composition (melanin formation) leading to tissue browning. Tom´as-Barber´an and Esp´ýn(2001) documented that Polyphenol oxidases (PPO) and Peroxidases (POD) are the two important enzymes that

are involved in phenolic oxidation reactions and which cause tissue browning when wounded. Therefore, it became crucial to study the phenolic transitions during different processing methods of fruits and vegetable samples. Thus, mechanical processing viz., flaking, or crushing, or crushing became central for enabling the accurate phenolic mining. Identification, quantification and yield of phenolicsin plant systems are usually inaccurate due to the improper processing and extraction methods. Biochemical understanding of chemical transitions mediated through enzymes is therefore critical for bringing the best nutritional output of fruits and vegetables. Phenolic oxidation can be minimized by inactivation of the enzymes, which is made possible by using heated organic solvents, pH lowering, adding adsorbents etc. For example, Arts and Hollman (1998) demonstrated that, apple tissue browning eventuates during the extraction processes, when the methanol solvent percentage goes below 40. Low methanol concentration was in consonance with catechin yield, where the experiments proved that the low yield was attributed to the effect of Poly Phenol Oxidase (PPO). The research documentations; many of them suggested that the use of cold temperatures, freezing, and lyophilization retains phenolic content of fruits and vegetables preventing oxidative processes. Low temperatures deactivated the PPO class of enzymes. Thus, freeze-drying is principally the chief preservation procedure and often preferred preserving the phenolic compounds in the fruit and vegetables. Extraction of Phenolics There is a big miscellany in the phenolic content in samples which ranges from small masses like that of Gallic acid to large masses like that of Condensed Tannins. The quantity also varies from simple nanograms to milligrams. The extraction and detection sensitivity therefore is very critical. Mild extraction procedures prevent chemical transformations. If the approaches of extractions are not done with care it might drastically affect the chemical fingerprintof the samples. There are some unique phenols which are found only in selected plant families and this exclusivity entitles these phenolics as “elite class”. Examples of these special phenolics are dihydrochalcones notably phloridzin, a characteristic phenolic compound from apple and its derivatives (Tom´as- Barber´an and Clifford 2000), or isoflavones, such as genistein and daidzein, which are restricted to the family

Fabaceae e.g., Glycine max (Cassidy et al. 2000). Usually extraction involves a few sub-phases, different solvent combinations, sometimes even unique solvents. Additional steps will be necessary to enrich by vaporizing/ lyophilizing in view of purifying the compounds. Extraction protocols have been expansively revised by the phytochemists to get a better yield and even for mining novel compounds (Tura and Robards 2002; Stalikas 2007). For hard samples (e.g., unripe fruit and vegetables) the most common methods is solid-liquid extraction process, which include soxhlet extraction, sonication, solid-phase extraction (Hernandez-Montes et al. 2006), supercritical fluid extraction, and microwave in rare instances. Herrera and Luque de Castro (2005) recommended the ultrasound-assisted extraction, subcritical water and microwave-assisted extraction to extract the phenolic compounds from strawberries. It was found that sonication methods could retain many analytes and this was a rapid method for analyzing metabolites in small load samples.

Shelf-life of Phenol Content of Fruits and Vegetables Ayala-Zavala et al. (2008b) showed that the natural antimicrobials during low temperature storage (5°C) could affect the total phenols of fresh-cut tomatoes. When Methyl jasmonate was treated, it was found that the phenolic content enhanced in comparison to other treatment chemicals. This also indicated that methyl jasmonate is indeed a precursor which enhances specific phenolics that too on storage. In methyl jasmonate treated fresh-cut tomatoes there was a hike in phenol content reaching a value of 267.4 (Ayala-Zavala et al. 2008b). Gonz´alez-Aguilar et al. (2006) documented that methyl jasmonate was found to increase the in vivo activity of Phenyl Ammonia Lyase (PAL), a key enzyme that uses amino acid phenylalanine to synthesize phenol compounds. Methyl jasmonate is often used as a precursor molecule in plant tissue culture systems as a direct defense against biotic stress, activating genes involved in PAL production that catalyze defense secondary metabolites. Ayala-Zavala et al. (2008a) opined that natural products, like methyl jasmonate and other phenol compounds found in tea tree oil has vital defensive compounds protecting plants from pathogen attack. When there is a biotic stress in the form of pathogen attack, the

signals of the shikimic acid and phenylpropanoid pathways are the most affected induced by the methyl jasmonate molecule (Ayala-Zavala et al. 2008b) and this was a study material for many plant pathologists. Plant defensemechanism is therefore an intelligent surveillance system, which is highly transient and adaptable and therefore very difficult to understand and crack its code.

Forthcoming Recommendations The consumers have high demands for the healthy products. Producers therefore should adopt effective technologies, that prevent the deterioration as well as market only fresh and minimally processed fruits and vegetables that retains all its nutritional quality. Nutritional value is directly proportional to the phytochemical content and this aspect decides the market value and quality of the food. Therefore, during packaging and preserving, it is important to ensure the stability of individual elite phytochemical compound and also have a value for total antioxidant capacity of fruits and vegetables. It is also crucial to evaluate the phytochemical transition states during different storage conditions, postharvest treatments and processing. Care must be taken during the usage of exogenous phytochemicals (natural preservatives) to safeguard the tissues from microbial attack by stabilizing the defense response signals. This is another fascinating arena for upcoming research in Pomology, Olericulture and Horticulture. Insertion of antioxidant capacity information on the product label will be considered necessary in future, in order to give more facts and figures to our well-informed consumers. This will be addressed as smart packaging that brings in phytochemical quality control of fresh fruits and vegetables, which would indicate the antioxidant and microbial status of the package. This figure would assist consumers to comprehend the bioactive compound content in fruit and vegetables, which in turn would help them to pick their high-quality healthiest products in trade.

Acknowledgement The authors thank the various funding agencies - the TIFAC-CORE for Pharmacogenomics, Fast Track Project and Extra Mural Funding from the

Science Engineering and Research Board (SERB) of Department of Science and Technology Government of India, World Noni Research Foundation and Manipal University for funding projects from where, insights were drawn in writing this Chapter.

References Agudo A, Slimani N, Ocke MC, Naska A, Miller AB, Kroke A, Bamia C, Karalis D, Vineis P, Palli D, Bueno-de-Mesquita HB, Peeters PHM, Engeset D, Hjartaker A, Navarro C, Garcia CM, Wallstrom P, Zhang JX, Welch AA, Spencer E, Stripp C, Overvad K, Clavel-Chapelon F, Casagrande C and Riboli E. 2002. Consumption of vegetables, fruit and other plant foods in the European Perspective Investigation in Cancer and Nutrition (EPIC) cohorts from 10 European countries. Publi Health Nutr 5(6B):1179–1196. Arivazhagan S, Velmurugan B, Bhuvaneswari V and Nagini S. 2004. Effects of aqueous extracts of garlic (Allium sativum) and neem (Azadirachta indica) leaf on hepatic and blood oxidantantioxidant status during experimental gastric carcinogenesis. J Med Food 7(3):334–339. Arts ICW and Hollman PCH. 1998. Optimization of a quantitative method for the determination of catechins in fruits and legumes. J Agric Food Chem 46(12):5156–5162. Ayala-Zavala JF, del-Toro-S'anchez L, Alvarez-Parrilla E, Soto-Valdez H, Mart'yn-Belloso O,RuizCruz S and Gonz'alez-Aguilar GA. 2008a. Natural antimicrobial agents incorporatedin active packaging to preserve the quality of fresh fruits and vegetables. StewartPostharvest Rev 4:1-9. Ayala-Zavala JF, Oms-Oliu G, Odriozola-Serrano I, Gonz'alez-Aguilar GA, Alvarez-Parrilla Eand Martin-Belloso O. 2008b. Bio-preservation of fresh-cut tomatoes using naturalantimicrobials. Eur Food Res Technol 226(5):1047-1055. Azuma K, Ippoushi K, Ito H, Higashio H and Terao J. 1999. Evaluation of antioxidativeactivity of vegetable extracts in linoleic acid emulsion and phospholipid bilayers. J SciFood Agric 79(14):2010-2016. Bazzano LA, He J, Ogden G, Vupputuri S, Loria C, Meyers L, Meyers L and Whelton P.K.2002. Fruit and vegetable intake and risk of cardiovascular disease in US adults: the firstnational health and nutrition examination survey epidemiologic follow-up study. Am JClinNutr 76:93-99. Blanck HM, Gillespie C, Kimmons JE, Seymour JD and Serdula MK. 2008. Trends in fruit andvegetable consumption among US men and women, 1994-2005. Prev Chronic Dis5(2):A35. Breinholt V, Hendricks J, Pereira C, Arbogast D and Bailey G 1995. Dietary chlorophyllin is apotent inhibitor of aflatoxin B1 hepatocarcinogenesis in rainbow trout. Cancer Res55(1):57-62. Campbell JK, Canene-Adams K, Lindshield BL, Boileau TW, Clinton SK and Erdman JW Jr.2004. Tomato phytochemicals and prostate cancer risk. J Nutr 134(12 Suppl):3486S-3492S. Cassidy A, Handley B and Lamuela-Ravent'os RM. 2000. Isoflavones, lignans and stilbenes -origins, metabolism and potential importance to human health. J Sci Food Agric80(7):1044-1062. Clifford MN. 2000. Anthocyanins—nature, occurrence and dietary burden. J Sci Food Agric80(7):1063-1072. Drozdowski B and Szukalska, E. 1987. A rapid instrumental method for the evaluation of thestability of fats. J Am Oil ChemSoc 64(7):1008-1011. Duthie SJ, Jenkinson AMCE, Crozier A, Mullen W, Pirie L, Kyle J, Yap LS, Christen P andDuthie G.G

2006. The effects of cranberry juice consumption on antioxidant status andbiomarkers relating to heart disease and cancer in healthy human volunteers. Eur J Nutr45(2):113-122. Frankel EN. 1993. In search of better methods to evaluate natural antioxidants and oxidativestability in food lipids. Trends Food SciTechnol 4(7):220-225. Gandini S, Merzeninch H, Robertson C and Boyle P 2000. Meta-analysis of studies on breastcancer risk and diet: the role of fruit and vegetable consumption and the intake ofassociated micronutrients. Eur J Cancer 36:636-646. Giovannucci E, Rimm EB, Liu Y, Stampfer MJ and Willett WC. 2003. A prospective study ofcruciferous vegetables and prostate cancer. Cancer Epidimiol Biomarkers Prev12(12):14031409. Giovannucci E. 1999. Tomatoes, tomato-based products, lycopene, and cancer: review of the epidemiologic literature. J Natl Cancer Inst 91 (4): 317—331. Goldberg I. 1994. Introduction. ( In) Goldberg I, editor. Functional Foods: Designer Foods,Pharmafoods, Nutraceuticals. New York: Chapman and Hall, pp. 1-16. Gonz'alez-Barrio R, Beltran D, Cantos E, Gil MI, Esp'yn JC and Tom'as-Barber'an F A. 2006.Comparison of ozone and UV-C treatments on the postharvest stilbenoid monomer,dimer, and trimer induction in var. ‘Superior’ white table grapes. J Agric Food Chem54(12):4222-4228. Harwood M, Danielewska-Nikiel B, Borselleca JF, Flamm GW,Williams,GM and Lines TC.2007. A critical review of the data related to the safety of quercetin and lack of evidenceof in vivo toxicity, including lack of genotoxic/carcinogenic properties. Food ChemToxicol45:2179-2205. Hern'andez-Montes E, Pollard SE, Vauzour D, Jofre-Montseny L, Rota C, Rimbach G, WeinbergPD and Spencer JPE. 2006. Activation of glutathione peroxidase via Nrf1 mediatesgenistein’sprotection against oxidative endothelial cell injury. BiochemBiophys ResCommun 346(3):851-859. Herrera MC and Luque de Castro MD. 2005. Ultrasound-assisted extraction of phenoliccompounds from strawberries prior to liquid chromatographic separation and photodiodearray ultraviolet detection. J ChromatogrA 1100(1): 1-7. Johnston CS, Taylor CA and Hampl JS. 2000. More Americans are eating “5 a day” but intakesof dark green and cruciferous vegetables remain low. J Nutr 130(12):3063-3067. Joshipura K, Hu F, Manson J, Stamfer M, Rimm E, Speizer F, Coltiz G, Asherio A, Rosner B,Spiegelman D and Willett W. 2001. The effect of fruit and vegetable intake on risk forcoronary heart disease. Ann Int Med 134:1106-1114. Kirsh VA, Peters U, Mayne ST, Subar AF, Chatterjee N, Johnson CC and Hayes RB. 2007.Prospective study of fruit and vegetable intake and risk of prostate cancer. J Natl CancerInst 99(15):12001209. Kotake-Nara E, Kushiro M, Hong Z, Sugawara T, Miyashita K and Nagao A. 2001. Carotenoidsaffect proliferation of human prostate cancer cells. J Nutrition 131(12):3303-3306. Krishnan K and Vijayalakshmi NR. 2005. Alterations in lipids & lipid peroxidation in rats fedwithflavonoid rich fraction of banana (Musa paradisiaca) from high background radiationarea. Ind J Med Res 122(6):540-546. L'opez-Nicol'as JM, Bru R, S'anchez-Ferrer A and Garc'ya-Carmona F. 1994. An octaethyleneglycol monododecyl ether-based mixed micellar assay for lipoxygenase acting at neutralpH. Anal Biochem 221(2):410-415. L'opez-Nicol'as JM, Bru R and Garc'ya-Carmona F. 1997. Enzymatic oxidation of linoleic acidby lipoxygenase forming inclusion complexes with cyclodextrins as starch modelmolecules. J Agric Food Chem 45:1144-1148.

Law MR and Morris JK. 1998. By how much does fruit and vegetable consumption reduce therisk of ischaemic heart disease? Eur J ClinNutr 52:549-556. Leontowicz M, Gorinstein S, Leontowicz H, Krzeminski R, Lojek A, Katrich E, Ciz M,Martin-Bellozo O, Soliva-Fortuny R, Haruenkit R and Trakhtenberg S. 2003. Appleand pear peel and pulp and their influence on plasma lipids and antioxidant potentials inrats fed cholesterol-containing diets. J Agric. Food Chem. 51:5780-5785. Liu RH. 2004. Potential synergy of phytochemicals in cancer prevention: mechanism of action.J Nutr. 134:3479S-3485S. Liu RH, Liu J and Chen B. 2005. Apples prevent mammary tumors in rats. J Agric Food Chem53:2341-2343. Maeda H, Hosokawa M, Sashima T, Funayama K and Miyashita K. 2005. Fucoxanthin fromedible seaweed, Undariapinnatifida, shows anti-obesity effect through UCP1 expressionin white adipose tissues. Biochem. Biophys. Res. Commun. 332(2):392-397. Maiorino M, Roveri A, Ursini F and Gregolin C. 1985. Enzymatic determination of membranelipid peroxidation. J Free RadicBiol Med 1(3):203-207. N'u'nez-Delicado E, Sojo MM, S'anchez-Ferrer A and Garc'ya-Carmona F. 1999.Hydroperoxidase activity of lipoxygenase in the presence of cyclodextrins. ArchBiochemBiophys 367(2):274-280. Nagura J, Iso H, Watanabe Y, Maruyama K, Date C, Toyoshima H, Yamamoto A, Kikuchi S,Koizumi A, Kondo T,Wada Y, Inaba Y and Tamakoshi A. 2009. Fruit, vegetable and beanintake and mortality from cardiovascular disease among Japanese men and women: theJACC Study. Br J Nutr 13:1-8. Naska A, Vasdekis V, Trichopoulou A, Friel S, Leonhauser I and Moreira G. 2000. Fruits andvegetable availability among ten European countries: how does it compare with the“five-a-day” recommendation? Brit J Nutr 84:549-556. Ness A and Powles JW. 1997. Fruit and vegetables, and cardiovascular disease: a review. Int JEpidemiol 26:1-13. Ness A, Egger M and Powles J. 1999. Fruit and vegetable and ischaemic heart disease: systematicreview or misleading meta-analysis? Eur J ClinNutr 53:900-902. Nourooz-Zadeh J. 1999. Ferrous ion oxidation in presence of xylenol orange for detection oflipid hydroperoxides in plasma. Methods Enzymol 300: 58-62. P'erez-Gilabert M and Garc'ya-Carmona F. 2000. Characterization of catecholase and cresolaseactivities of eggplant polyphenol oxidase. J Agric Food Chem 48(3):695-700. Percival SS, Talcott ST, Chin ST, Mallak AC, Lound-Singleton A and Pettit-Moore J. 2006.Neoplastic transformation of BALB/3T3 cells and cell cycle of HL-60 cells are inhibitedby mango (Mangifera indica L.) juice and mango juice extract. J Nutr 136:1300-1304. Porta H and Rocha-Sosa M. 2002. Plant lipoxygenases. Physiological and molecular features.Plant Physiol 130:15-21. Radhika G, Sudha V, Mohan Sathya R, Ganesan A and Mohan V. 2008. Association of fruit andvegetable intake with cardiovascular risk factors in urban south Indians. Br J Nutr99(2):398405. Rechkemmer G 2001. FunktionelleLebensmittel-Zukunft de Ernahrungoder Marketing-Strategie.F orschungereportSonderheft 1:12-15. Retelny VS, Neuendorf A and Roth JL. 2008. Nutrition protocols for the prevention ofcardiovascular disease. NutrClinPract 23(5):468-476. Rissanen TH, Voutilainen S, Virtanen JK, Venho B, Vanharanta M, Mursu J and Salonen JT.2003. Low intake of fruit, berries and vegetables is associated with excess mortality inmen: the Kuopio

Ischaemic Heart Disease Risk Factor (KIHD) Study. J Nutr 133:199-204. Roginsky V and Barsukova, T. 2001. Superoxide dismutase inhibits lipid peroxidation in micelles.Chem Phys Lipids 111(1):87-91. Sarkar D, Sharma A and Talukder G. 1994. Chlorophyll and chlorophyllin as modifiers ofgenotoxic effects. Mutat Res 318(3):239-247. Shannon J, Cook LS and Stanford JL. 2003. Dietary intake and risk of postmenopausal breastcancer (United States). Cancer Causes Control 14:19-27. Shier WT, Shier AC, Xie W and Mirocha CJ. 2001. Structure-activity relationships for humanestrogenic activity in zearalenone mycotoxins. Toxicon 39 (9): 1435-1438. Smith-Warner SA, Spiegelman D, Shiaw-Shyuan Y, Adami HO, Beeson WL, Brandt PA, FolsomAR, Fraser GE, Freudenheim JL, Goldbohm RA, Graham S, Miller AB, Potter JD,Rohan TE, Speizer FE, Toniolo P, Willet WC, Wolk A and Zeleniuch-Jacquotte A,Hunter DJ. 2001. Intake of fruits and vegetables and risk of breast cancer. A pooledanalysis of cohort studies. J Am Med Assoc 285(6):769-776. Stalikas C. 2007. Extraction, separation, and detection methods for phenolic acids and flavonoids. J Sep Sci 30(18):3268-3295. Stavric B. 1994. Role of chemopreventers in human diet. ClinBiochem 27(5):319-332. Steinmetz KA and Potter JD. 1996. Vegetables, fruit and cancer prevention: a review. J Am DietAssoc 96:1027-1039. Stram DO, Hankin JH, Wilkens LR, Park S, Henderson BE, Nomura AM, Pike MC and KolonelLN. 2006. Prostate cancer incidence and intake of fruits, vegetables and relatedmicronutrients: the multiethnic cohort study (United States). Cancer Causes Control17(9):1193-1207. Suzuki S, Takada T, Sugawara Y, Muto T and Kominami R. 1991. Quercetin inducesrecombinational mutations in cultured cells as detected by DNA fingerprinting. Jpn JCancer Res 82 Syngletary KW, Jackson SJ, Milner JA. 2005. Non-nutritive components in foods as modifiersof the cancer process. In: Bendich A and Deckelbaum RJ, editors. Preventive Nutrition:the Comprehensive Guide for Health Professionals, 3rd ed. Totowa, NJ: Humana Press. Takahashi Y, Nagao M, Fujino T, Yamaizumi Z and Sugimura T. 1979. Mutagens in Japanesepickle identified as flavonoids. Mut Res 68:117-123. Tom'as-Barber'an FA and Clifford MN. 2000. Flavanones, chalcones and dihydrochalcones—nature, occurrence and dietary burden. J Sci Food Agri 80(7):1073-1080. Tom'as-Barber'an FA and Esp'yn JC. 2001. Phenolic compounds and related enzymes asdeterminants of quality in fruits and vegetables. J Sci Food Agric 81(9):853-876. Tura D and Robards K. 2002. Sample handling strategies for the determination of biophenols infood and plants. J ChromatogrA 975(1):71-93. US Department of Agriculture. 2004. USDA database for the proanthocyanidin content ofselected foods. Beltsville, MD: USDA. US Department of Agriculture. 2007a Vallejo F, Tom'as-Barber'an FA and Ferreres F. 2004. Characterisation of flavonols in broccoli(Brassica oleracea L. var. italica) by liquid chromatography-UV diode-array detectionelectrospray ionization mass spectrometry. J Chromatogr A 1054(1-2): 181-193. Van Gils C, Peeters PH, Bueno-de-Mesquita HB, Boshuizen HC, Lahmann PH, Clavel-ChapelonF, Thiebaut A, Kesse E, Sieri S, Palli D, Tumino R, Panico S, Vineis P, Gonzalez CA,Ardanaz E, Sanchez MJ, Amiano P, Navarro C, Quiros JR, Key TJ, Allen N, Khaw KT,Bingham SA, Psaltopoulou T, Koliva M, Trichopoulou A, Nagel G, Linseisen J, BoeingH, Berglund G,Wirfalt E, Hallmans G, Lenner P, Overvad K, Tjonneland A, Olsen A,Lund E, Engeset D, Alsaker E, Norat TA, Kaaks R, Slimani N and Riboli E. 2005.Consumption of vegetables and fruit and risk

of breast cancer. JAMA 293:183-193. Voorips LE, Goldbohm RA, van Poppel G, Sturmans F, Hermus RJ and van den Brandt PA.2000. Vegetable and fruit consumption and risk of colon and rectal cancer in a prospectivecohort study. Am J Epidemiol 152:1081-1092. Wargovich MJ. 2000. Anticancer properties of fruits and vegetables. HortSci 35(4):573-575. Warso MA and Lands WEM. 1985. Presence of lipid hydroperoxide in human plasma. J ClinInvest 75(2):667-671. Wattenberg LW. 1975. Effects of dietary constituents on the metabolism of chemical carcinogens.Cancer Res 35(11): 3326-3331. Wise JA, Morin RJ, Sanderson R and Blum K. (1996). Changes in plasma carotenoid, alphatocopherol, and lipid peroxide levels in response to supplementation with concentratedfruit and vegetable extracts: a pilot study. CurrTher Res 57(6):445-461. Yahia EM. 2009a. Prickly pear. Chapter 13. In: Rees D, Farrell G, Orchard JE, editors. CropPostharvest: Science and Technology, Volume 3. Oxford: Wiley-Blackwell. Yahia EM. 2009b. Avocado. Chapter 8. In: Rees D, Farrell G, Orchard JE, editors. CropPostharvest: Science and Technology, Volume 3. Oxford: Wiley-Blackwell. Yamamoto H, Manabe T and Okuyama T. 1990. Apparatus for coupled high-performanceliquid chromatography and capillary electrophoresis in the analysis of complex proteinmixtures. J Chromatogr 515: 659-666.

CHAPTER 2

Chemistry of Antioxidants Desalinizing Soils Uttam Kumar and I.J.Gulati “See the flower,how freely it gives of its perfume and honeyBut its work is done,it falls away quickly” -Bhagavad Gita Soil Salinity: Soil containing excess amount of soluble salts on crop root zone and soil surface that adversely affects the plant growth and productivity is considered as saline soil. The excess concentration is expressed as EC (Electronic conductivity), ESP (Exchangeable sodium percentages) and SAR (Sodium adsorption ratio) value. The soluble salts in these soils are predominantly the chloride and sulphate of sodium, calcium and magnesium. The concentration of potassium is generally low. Excess concentrations of boron, fluoride and nitrates may also be present in these soils under arid conditions. These soils are characterized by saline efflorescence or white encrustation of salts at the surface. As per the US salinity laboratory staff (1954), these soils have pH of saturation paste (pHs) less than 8.5, ESP less than 15, and ECe more than 4 dS/m at 25ÚC. The relative proportion of Ca2+ and Mg2+ ions with respect to Na+ ion is relatively high in saline soils, results in flocculation of clay particles. The tightly adsorbed divalent (Ca2+ and Mg2+) ions decrease the zeta potential soil exchange complex, which in turn, results in attraction of clay particles towards each other, causing flocculation.

Effects on plant growth

Osmotic stress: TThe main effect of high concentration of soluble salts in soil on plants is osmotic stress. The semi-permeable membrane of plant roots permits the water to pass but reject most of the salts. Therefore osmotically it becomes difficult for the roots of to extract water from saline solutions. Under osmotic stress, plant cell continue to divide but not elongate, resulting an increase in the number of cells per unit area which account for the typically dark bluish green color of foliage. Because plant growth is a function of total moisture stress, efficient management is a crucial factor in these soils. The ability of plants to increase their internal osmotic pressure by production of organic acids or uptake of salts, also help them to osmotically adjust in the soil having high salts concentration. Toxic effect of some ions: Many plants are sensitive to the toxic effects of some ions in soil solution. The ions which cause toxicities in the plants include chloride, sulphate, sodium, magnesium and boron. However, excessive intrusion of these ions in the roots and their movement in the plant through transpiration stream may lead to necrosis, burning of leaf tips and margins, and eventual death. Some plants can screen out toxic ions through the mechanism of selective absorption. Oxidative stress: Salinity is one of the major abiotic stress problems that affects the production of many lands and is still expanding, posing a threat to sustainable agriculture (Helal et al., 1999). Salt stress has toxic effects on plants and leads to metabolic changes, like loss of chloroplast activity, decreased photosynthetic rate and increased photorespiration rate which then leads to an increased reactive oxygen species (ROS) production (Parida and Das, 2005). These ROS such as Superoxide (O2+), hydrogen peroxide (H2O2), hydroxyl radical (OH), and single oxygen (O2-) cause oxidative stress on plants. High salt stress also disrupts homeostasis in water potential and in distribution. This disruption of homeostasis occurs at both the cellular and whole plant levels.

Reactive Oxygen Species (ROS) ROS are a group of free radicals, reactive molecules, and ions that are derived from O2. It is estimated has been estimated that about 1% of O2 consumed by plants is diverted to produce ROS in various subcellular loci

such as chloroplasts, mitochondria, peroxisomes. ROS are well recognized for playing a dual role as both deleterious and beneficial species depending on their concentration in plants. At high concentration ROS causes damage to biomolecules, whereas at low/ moderate concentration it acts as second messenger in intracellular signaling cascades that mediate several responses in plant cells.

Types of ROS Phototrophs convert light energy from the sun into biochemical energy and therefore are crucial for sustaining life on Earth. The price they have to pay for this is to face the risk of oxidative damages, because of the different types of ROS, namely, 1O2(singlet oxygen), H2O2(hydrogen peroxide), O• “2 (superoxide radical), and OH (hydroxyl radical), generated as unwanted byproducts. These are generated from only 1-2% of total O2 consumed by plants (Bhattacharjee, 2005) . The reactions generating the different ROS members are shown. Superoxide radical (O•– 2): The ROS is being constantly generated in the chloroplasts due to partial reduction of O2 or as a result of transfer of energy to O2.The superoxide radical (O•– 2) is formed mainly in the thylakoid localized PSI during non-cyclic electron transport chain(ETC),as well as other cellular compartments. Normally, H2O is generated when cytochrome c oxidase interacts with O2. Occasionally, O2 reacts with the different ETC components to give rise to the O•– 2. It is usually the first ROS to be formed. Superoxide radical (O•– 2) can also undergo further reactions to generate other members of the ROS family O•– 2 being moderately reactive with a short half-life of 2–4 ìs, does not cause extensive damage by itself. Instead, it undergoes transformation into more reactive and toxic OH• and 1O2 and cause membrane lipid peroxidation (Halliwell, 2006). Singlet oxygen (1O2 ): Singlet Oxygen is an atypical ROS which is generated not by electron transfer to O2, but rather by the reaction of chlorophyll (Chl) triplet state in the antenna system with O2.

Chl →3Chl 3Chl +3O2 → Chl +1O2 Environmental stresses like salinity, drought and heavy metals cause stomatal closure, leading to insufficient intracellular CO2 concentration. This favors the formation of 1O2. Singlet oxygen can cause severe damages to both the photosystems, PSI and PSII, and puts the entire photosynthetic machinery into jeopardy. Even though 1O2 has a short half-life of about 3 ìs (Hatz et al., 2007), it can manage to diffuse some 100 nanometers and causes damage to wide range of targets. These include molecules like proteins, pigments, nucleic acids and lipids (Krieger-Liszkay et al., 2008), and is the major ROS responsible for light-induced loss of PSII activity, eliciting cellular death. Plants have managed to efficiently scavenge 1O2 with the help of â-carotene, tocopherol, plastoquinone, and can also react with the DI protein of PSII. Alternatively, singlet oxygen plays a role in up regulating genes which are responsible for providing protection against photO•–oxidative stress (KriegerLiszkay et al., 2008). Hydrogen peroxide (H2O2): Hydrogen peroxide, a moderately reactive ROS is formed when O• 2 undergoes both univalent reduction as well as protonation. It can occur both non-enzymatically by being dismutated to H2O2 under low pH conditions, or mostly by a reaction catalyzed by SOD. 2O•- 2 + 2H+ → H2O2 + O2 2O•- 2 + 2H+ → H2O2+ O2 H2O2 is produced in plant cells not only under normal conditions, but also by oxidative stress, caused by factors like drought, chilling, intense light, UV radiation, wounding, and pathogen infection (Sharma et al., 2012). Due to stomatal closure and low availability of CO2 and its limited fixation, Ribulose 1, 5-bisphosphate (RuBP) oxygenation is favored and thus photorespiration is enhanced. This accounts for more than 70% of the H2O2 produced as a result of drought stress (Noctor et al., 2002). The major sources of H2O2 production in plant cells include the ETC in the chloroplast, mitochondria, ER, cell membrane, â-oxidation of fatty acid and photorespiration. Additional sources

comprise of different reactions involving photO•–oxidation by NADPH oxidase and xanthine oxidase (XOD). H2O2 in plants behaves like doubleedged sword; it is beneficial at low concentrations, but damaging at higher concentrations in the cell. At low intracellular concentrations, it acts as a regulatory signal for essential physiological processes like senescence (Peng et al., 2005), photorespiration and photosynthesis (Noctor et al., 2002), stomatal movement (Bright et al., 2006), cell cycle and growth and development (Tanou et al., 2009). Due to its significantly longer half-life of 1 ms, compared to other ROS members, it can traverse longer distances and cross plant cell membranes. It can cross membranes via aquaporins and cover considerable lengths within the cell (Bienert et al., 2007) and cause oxidative damage. H2O2 at high intracellular concentration oxidizes both cysteine (SH) and methionine (-SCH3) residues and inactivates Calvin cycle enzymes, Cu/Zn SOD and Fe-SOD by oxidizing their thiol groups (Halliwell, 2006). It causes 50% loss in activity of different enzymes like fructose 1, 6 bisphosphatase, sedoheptulose 1, 7 bisphosphatase and phosphoribulokinase, at concentrations of 10 iM H2O2 and is also responsible for programmed cell death at high cellular concentrations (Dat et al., 2000). However, like O•– 2, H2O2 is moderately reactive; therefore, its damage is fully realized only when it is converted into more reactive species. Hydroxyl radical (OH•): Among its family members, hydroxyl radical (OH•) is the most reactive and the most toxic ROS known. It is generated at neutral pH by the Fenton reaction between H2O2 and O•–2 catalyzed by transition metals like Fe (Fe2+, Fe3+). H2O2 + O•–2 → OH – + O2 + OH• It has the capability to damage different cellular components by lipid peroxidation (LPO), protein damage and membrane destruction. Since there is no existing enzymatic system to scavenge this toxic radical, excess accumulation of OH• causes the cellular death (Pinto et al., 2003).

Site of ROS production in plant cell: The ROS is being produced under both normal and stressful conditions at

various locations in the chloroplasts, mitochondria, peroxisomes, plasma membranes, ER and the cell wall. In presence of light, chloroplasts and peroxisomes are the major sources of ROS production, while the mitochondrion is the leading producer of ROS under dark conditions (Choudhury et al., 2013).

Predominant ROS generation sites in plant cell under salinity stress. Chloroplast: In chloroplasts, various forms of ROS are generated from several locations. ETCs in PSI and PSII are the main sources of ROS in chloroplasts. Production of ROS by these sources is enhanced in plants by conditions limiting CO2 fixation, such as drought, salt, and temperature stresses, as well as by the combination of these conditions with high-light stress. Under normal conditions, the electron flow from the excited PS centers to NADP which is reduced to NADPH which, then, enters the Calvin cycle and reduces the final electron acceptor, CO2. In case of overloading of the ETC, due to decreased NADP supply resulting from stress conditions, there is leakage of electron from ferredoxin to O2, reducing it to O2 •– (Elstner, 1991). This process is called Mehler reaction: 2O2 + 2Fdred – → 2O2 •– + 2Fdox Leakage of electrons to O2 may also occur from 2Fe-2S and 4Fe-4S clusters in the ETC of PSI. In PSII, acceptor side of ETC contains QA and QB.

Leakage of electron from this site to O2 contributes to the production of O2 •–. The formation of O2 •– by O2 reduction is a rate-limiting step. Once formed O2 •” generates more aggressive ROS. It may be protonated to HO2 • on the internal, “lumen” membrane surface or dismutated enzymatically (by SOD) or spontaneously to H2O2 on the external “stromal” membrane surface. At Fe-S centers where Fe2+ is available, H2O2 may be transformed through the Fenton reaction into the much more dangerous OH•. Mitochondria: can produce ROS in several sites of ETC. In mitochondria direct reduction of oxygen to O2 •– occurs in the flavoprotein region of ADH dehydrogenase segment (complex I) of the respiratory chain (Arora, et. al. 2002) When NAD+-linked substrates for complex I are limited, electron transport can occur from complex II to complex I (reverse electron flow). This process has been shown to increase ROS production at complex I and is regulated by ATP hydrolysis. Ubiquinone-cytochrome region (complex III) of the ETC also produces O2 •– from oxygen. It is believed that fully reduced ubiquinone donates an electron to cytochrome C1 and leaves an unstable highly reducing ubisemiquinone radical which is favorable for the electron leakage to O2 and, hence, to O2 •– formation. In plants, under normal aerobic conditions, ETC and ATP syntheses are tightly coupled; however, various stress factors lead to inhibition and modification of its component, leading to over reduction of electron carriers and, hence, formation of ROS (Heyno, 2011). Several enzymes present in mitochondrial matrix can produce ROS. Some of them produce ROS directly, for example aconitase, whereas some others like 1-galactonO•– γ lactone dehydrogenase (GAL), are able to feed electrons to ETC (Andreyev, 2005) O2 •– is the primary ROS formed by monovalent reduction in the ETC. It is converted quickly either by the Mn SOD (mitochondrial form of SOD) or APX into the relatively stable and membrane-permeable H2O2. H2O2 can be further converted to extremely active hydroxyl radical (OH•) in the Fenton reaction. Endoplasmic Reticulum: In endoplasmic reticulum, NAD(P)H-dependent electron transport involving Cyt P450 produces O2•–. Organic substrate, RH, reacts first with Cyt P450 and then is reduced by a flavoprotein to form a radical intermediate (Cyt P450R). Triplet oxygen can readily react with this radical intermediate as each has one unpaired electron. This oxygenated

complex (Cyt P450-ROO) may be reduced by cytochrome b or occasionally the complexes may decompose releasing O2•–. Peroxisomes: Peroxisomes are probably the major sites of intracellular H2O2 production, as a result of their essentially oxidative type of metabolism. The main metabolic processes responsible for the generation of H2O2 in different types of peroxisomes are the glycolate oxidase reaction, the fatty acid âoxidation, the enzymatic reaction of flavin oxidases, and the disproportionation of O2 •” radicals (Baker and Graham, 2002). During photorespiration, the oxidation of glycolate by glycolate oxidase in peroxisomes accounts for the majority of H2O2 production. Like mitochondria and chloroplasts, peroxisomes also produce O2•” as a consequence of their normal metabolism. In peroxisomes from pea leaves and watermelon cotyledons, at least, two sites of O2•– generation have been identified using biochemical and electron spin resonance spectroscopy (ESR) methods: one in the organelle matrix, the generating system being XOD, which catalyses the oxidation of xanthine or hypoxanthine to uric acid, and produces O2•– in the process and another site in the peroxisomal membranes where a small ETC composed of a flavoprotein NADH and Cyt b is involved. Three integral peroxisomal membrane polypeptides (PMPs) with molecular masses of 18, 29, and 32 kDa were found to be involved in O2•– production. While the 18- and 32-kDa PMPs use NADH as electron donor for O2•– production, the 29-kDa PMP was clearly dependent on NADPH and was able to reduce cytochrome c with NADPH as electron donor. Among the three integral polypeptides, the main producer of O2•– was the 18-kDa PMP which was proposed to be a cytochrome possibly belonging to the b-type group. The PMP32 very probably corresponds to the MDHAR, and the third O2•– generating polypeptide, PMP29, could be related to the peroxisomal NADPH cytochrome P450 reductase (Lopez-Huertas, 1999). The O•– 2 produced is subsequently converted into H2O2 by SOD. Plasma Membranes: Electron transporting oxido reductases are ubiquitous at plasma membranes and lead to generation of ROS at plasma membrane. Production of ROS was studied was studied using EPR spin-trapping techniques and specific dyes in isolated

plasma membranes from the growing and the non growing zones of hypocotyls and roots of etiolated soybean seedlings as well as coleoptiles and roots of etiolated maize seedlings. NAD(P)H mediated the production of O2•– in all plasma membrane samples. It was suggested that in soybean plasma membranes, O2•– production could be attributed to the action of at least two enzymes, an NADPH oxidase, and, in the presence of menadione, a quinine reductase. NADPH oxidase catalysestransfer of electrons from cytoplasmic NADPH to O2 to form O2•– O2•– is dismutated to H2O2 either spontaneously or by SOD activity. NADPH oxidase has been proposed to play a key role in the production and accumulation of ROS in plants under stress conditions (Apel, and Hirt, 2004) Cell Walls: Cell walls are also regarded as active sites for ROS production. Role of cell-wall-associated peroxidase in H2O2 generation has been shown. Inhorseradish, peroxidase associated with isolated cell walls catalyzes the formationof H2O2 in the presence of NADH. The reaction is stimulated by variousmonophenols, especially of coniferyl alcohol. Malate dehydrogenase was foundto be the sole candidate for providing NADH (Gross, 1997). The generation ofROS by cell-walllocated peroxidases has been shown during hypersensitiveresponse (HR) triggered in cotton by the bacterium Xanthomonas campestrispv. Malvacearum and potassium (K) deficiency stress in Arabidopsis. Diamineoxidases are also involved in production of activated oxygen in the cell wallusing diamine or polyamines (putrescine, spermidine, cadaverine, etc.) to reducea quinone that autooxidizes to form peroxides (Elstner, 199). Apoplast: Cell-wall-located enzymes have been proved to be responsible forapoplastic ROS production (Apel, and Hirt, 2004). The cell-wallassociatedoxalate oxidase, also known as germin, releases H2O2 and CO2 from oxalicacid. This enzyme was reported to be involved in apoplastic hydrogen peroxideaccumulation during interactions between different cereals species and fungi.Amine oxidase-like enzymes may contribute to defense responses occurring inthe apoplast following biotic stress, mainly through H2O2 production (Cona,2006). Amine oxidases catalyze the oxidative deamination of polyamines (i.e.,putrescine, spermine, and spermidine) using

FAD as a cofactor (Cona, 2006).Heyno and coworkers, based on their study, concluded that apoplastic OH"generation depends fully, or for the most part, on peroxidase localized in the cellwall.

Oxidative damage caused by ROS Production and removal of ROS must be strictly controlled in order to avoid oxidative stress. When the level of ROS exceeds the defense mechanisms, a cell is said to be in a state of “oxidative stress”. However, the equilibrium between production and scavenging of ROS is perturbed under a number of stressful conditions such as salinity, drought, high light, toxicity due to metals, pathogens, and so forth. Enhanced level of ROS can cause damage to biomolecules such as lipids, proteins and DNA. These reactions can alter intrinsic membrane properties like fluidity, ion transport, loss of enzyme activity, protein cross-linking, inhibition of protein synthesis, DNA damage, and so forth ultimately resulting in cell death. Lipids: When ROS level reaches above threshold, enhanced lipid peroxidation takes place in both cellular and organellar membranes, which, in turn, affect normal cellular functioning. Lipid peroxidation aggravates the oxidative stress through production of lipid-derived radicals that themselves can react with and damage proteins and DNA. The level of lipid peroxidation has been widely sed as an indicator of ROS mediated damage to cell membranes under stressful conditions. Increased peroxidation (degradation) of lipids has been reported in plants growing under environmental stresses (Sharma and Dubey, 2005; Han, 2009). Increase in lipid peroxidation under these stresses parallels with increased production of ROS. Malondialdehyde (MDA) is one of the final products of peroxidation of unsaturated fatty acids in phospholipids and is responsible for cell membrane damage. Two common sites of ROS attack on the phospholipid molecules are the unsaturated (double) bond between two carbon atoms and the ester linkage between glycerol and the fatty acid. The polyunsaturated fatty acids (PUFAs) present in membrane phospholipids are particularly sensitive to attack by ROS. A single OH• can result in peroxidation of many polyunsaturated fatty acids because the reactions involved in this process are part of a cyclic chain reaction. The overall process of lipid peroxidation involves three distinct

stages: initiation, progression, and termination steps. The initial phase of lipid peroxidation includes activation of O2 which is rate limiting. O2 •” and OH• can react with methylene groups of PUFA forming conjugated dienes, lipid peroxy radicals and hydroperoxides (Smirnoff, 1995). PUFA - H + X •–→PUFA + X - H. PUFA + O2 •–→PUFA – OO•. The peroxy radical formed is highly reactive and able to propagate the chain reaction: PUFA – OO• +PUFA – OOH –→ PUFA – OOH + PUFA• The formation of conjugated diene occurs when free radicals attack the hydrogens of methylene groups separating double bonds and, thereby, rearrangement of the bonds occurs. The lipid hydroperoxides produced (PUFAOOH) can undergo reductive cleavage by reduced metals, such as Fe2+, according to the following reaction: PUFA – O• + PUFA – H → PUFA – OH + PUFA• Peroxidation of polyunsaturated fatty acid by ROS attack can lead to chain breakage and, thereby, increase in membrane fluidity and permeability. Proteins: The attack of ROS on proteins may cause modification of proteins in a variety of ways, some are direct and others indirect. Direct modification involves modulation of a protein’s activity through nitrosylation, carbonylation, disulphide bond formation, and glutathionylation. Proteins can be modified indirectly by conjugation with breakdown products of fatty acid peroxidation (Yamauchi, 2009). As a consequence of excessive ROS production, site-specific amino acid modification, fragmentation of the peptide chain, aggregation of cross-linked reaction products, altered electric charge and increased susceptibility of proteins to proteolysis occur. Tissues injured by oxidative stress generally contain increased concentrations of carbonylated proteins which is widely used marker of protein oxidation. Enhanced modification of proteins has been reported in plants under various stresses (Maheshwari, and Dubey, 2009; Tanou, 2009). The amino acids in a peptide differ in their susceptibility to attack by ROS. Thiol groups and sulphur containing amino acids are very susceptible sites for attack by ROS. Activated oxygen can abstract an H atom from cysteine residues to form a

thiyl radical that will cross-link to second thiyl radical to form disulphide bridge. Several metals, including Cd, Pb, and Hg have been shown to cause the depletion of protein bound thiol groups. Oxygen also can be added to a methionine to form methionine sulphoxide derivative. Tyrosine is readily cross-linked to form bityrosine products in the presence of ROS (Davies, 1987). Oxidation of iron-sulphur centers by O2 •– is irreversible and leads to enzyme inactivation (Gardner, and Fridovich, 1991). In these cases, the metal (Fe) binds to a divalent cation-binding site on the protein. The metal (Fe), then, reacts in a Fenton reaction to form a •OH that rapidly oxidizes an amino acid residue at or near he cation-binding site of the protein. Oxidized proteins serve as better substrates for proteolytic digestion. It has been suggested that protein oxidation could predispose it to ubiquitination, which, in turn, would be a target for proteasomal degradation. The incubation of pea leaf crude extracts with increasing H2O2 concentrations, Cd treated plants and peroxisomes purified from pea leaves showed increase in carbonyl content. Oxidized proteins were more efficiently degraded, and the proteolytic activity increased 20% due to the metal treatment (RomerO•–Puertas, et al., 2002). Several studies have revealed that after a certain degree further damage leads to extensively cross-linked and aggregated products, which are not only poor substrates for degradation, but also can inhibit proteases to degrade other oxidized proteins. DNA: ROS are a major source of DNA damage. ROS can cause oxidative damages to nuclear, mitochondrial, and chloroplastic DNA. DNA is cell’s genetic material and any damage to the DNA can result in changes in the encoded proteins, which may lead to malfunctions or complete inactivation of the encoded proteins. Oxidative attack on DNA results in deoxyribose oxidation, strand breakage, removal of nucleotides, variety of modifications in the organic bases of the nucleotides, and DNA-protein crosslinks. Further, changes in the nucleotides of one strand can result in the mismatches with the nucleotides in the other strand, yielding subsequent mutations. Enhanced DNA degradation has been observed in plants exposed to various environmental stresses such as salinity and metal toxicity (Meriga, et al., 2004). Both the sugar and base moieties of DNA are susceptible to oxidation by ROS. Oxidative attack to DNA bases generally involves •OH addition to double bonds, while sugar damage mainly results from hydrogen abstraction from deoxyribose. The hydroxyl radical is known to react with all purine and

pyrimidine bases and, also, the deoxyribose backbone. •OH generates various products from the DNA bases which mainly include C-8 hydroxylation of guanine to form 8- oxO•–7,8 dehydrO•–2- deoxyguanosine, hydroxymethyl urea, urea, thymine glycol, thymine and adenine ring-opened, and saturated products. 8-Hydroxyguanine is the most commonly observed product. 1O2 only reacts with guanine, whereas H2O2 and O2•– do not react with bases at all (Dizdaroglu, 1993). ROS-induced DNA damages include various mutagenic alterations as well. For example, mutation arising from selective modification of G:C sites, especially, indicates oxidative attack on DNA by ROS. ROS attack DNA bases indirectly through reactive products generated by ROS attack to other macromolecules such as lipid. ROS attack to DNA sugars leads to single-strand breaks. ROS abstract hydrogen atom from the C4 position of deoxyribose, leading to generation of a deoxyribose radical that further reacts to produce DNA strand breakage (Evans, 2004). Under •– physiological conditions, neither H2O2 alone nor O2 can cause in vitro strand breakage. Therefore, it was concluded that the toxicity associated with these ROS in vivo is most likely the result of Fenton reaction. When •OH attacks on either DNA or proteins associated with it, DNA protein crosslinks are formed. DNA protein crosslinks cannot be readily repaired and may be lethal if replication or transcription precedes repair. Mitochondrial and chloroplast DNA are more susceptible to oxidative damage than nuclear DNA due to the lack of protective protein, histones, and close locations to the ROS producing systems in the former. Even though repair system exists for damaged DNA, excessive changes caused by ROS lead to permanent damage to the DNA with potentially detrimental effects for the cell.

Reactive oxygen species (ROS) induced oxidative damage to lipids, proteins, and DNA.

Antioxidants Defense Mechanism from ROS Antioxidants: An antioxidant is any substance that when present at low concentrations compared to those of an oxidizable substrate significantly delays or prevents oxidation of that substance. Antioxidant enzymes are the most active and efficient protective mechanism. Prevention of excessive ROS and repair of cellular damage is essential for cell’s life. The enzymatic mechanisms are designated to minimize the concentration of O2 and H2O2. The enzymes overproduced so far include catalase (CAT), superoxide dismutase (SOD), ascorbate peroxidase (APX) and glutathione reductase (GR). Classification of antioxidants: Antioxidants are classified into 3 categories, as described by Gutteridge and Halliwell 1. Primary antioxidants: It is involved in the prevention of oxidants formation 2. Secondary antioxidants: exhibits scavenger of ROS. 3. Tertiary antioxidants: repairs the oxidized molecules through sources

like dietary or consecutive antioxidants. Antioxidants may be enzymatic or non-enzymatic. Enzymatic system directly/ indirectly contributes to defense against the ROS. Catalase, superoxide dismutase, glutathione peroxidase, glutathione reductase, thioredoxin exhibits biological value. Th non-enzymatic antioxidants are actually the scavangers of ROS and RNS; these involve glutathione, vitamin E and C (inhibits oxidation of membrane lipid), uric acid is the scavenger of peroxynitrite in plasma, albumin, bilirubin, N-Acetylcysteine (NAC), melatonin which directly reacts with ROS and form disulfides.

Mode of action of antioxidants 1.

Primary or chain breaking antioxidants: break chain reaction and resulting radical is less reactive ROO + AH→ ROOH + A ROOH + A → ROOA 2. Secondary or Preventive antioxidants Thy may act either by • Chelators/Deactivate metals • Scavenge singlet oxygen (highly toxic) • Remove ROS Plants possess complex antioxidative defense system comprising of nonenzymatic and enzymatic components to scavenge ROS. In plant cells, specific ROS producing and scavenging systems are found in different organelles such as chloroplasts, mitochondria, and peroxisomes. ROS scavenging pathways from different cellular compartments are coordinated (Pang and Wang, 2008). Under normal conditions, potentially toxic oxygen metabolites are generated at a low level and there is an appropriate balance between production and quenching of ROS. The balance between production and quenching of ROS may be perturbed by a number of adverse environmental factors, giving rise to rapid increases in intracellular ROS levels (Sharma, et al., 2010) which can induce oxidative damage to lipids, proteins, and nucleic acids. In order to avoid the oxidative damage, higher plants raise the level of endogenous antioxidant defense (Sharma, et al., 2010). Various components of antioxidative defense system involved in ROS

scavenging have been manipulated, over expressed or down regulated to add to the present knowledge and understanding the role of the antioxidant systems. Enzymatic Antioxidants: The enzymes localized in the different subcellular compartments and comprising the antioxidant machinery include Superoxide Dismutase (SOD), Catalase (CAT), Ascorbate Peroxidase (APX), Monodehydroascorbate reductase (MDHAR), Dehydroascorbate reductase (DHAR), Glutathione Reductase (GR), and Guaiacol Peroxidase (GPX). Superoxide Dismutase (SOD): SOD (E.C.1.15.1.1) belongs to the family of metalloenzymes omnipresent in all aerobic organisms. Under environmental stresses, SOD forms the first line of defense against ROS-induced damages. The SOD catalyzes the removal of O•”2 by dismutating it into O2 and H2O2. This removes the possibility of OH• formation by the Haber-Weiss reaction. SODs are classified into three isozymes based on the metal ion it binds, MnSOD (localized in mitochondria), Fe-SOD (localized in chloroplasts), and Cu/Zn-SOD (localized in cytosol, peroxisomes, and chloroplasts) (Mittler, 2002). SOD has been found to be up regulated by abiotic stress conditions (Boguszewska et al., 2010). O•–2 + O•–2 + 2H+ → 2H2O2 + O2 Catalase (CAT): Among antioxidant enzymes, catalase (CAT, 1.11.1.6) was the first enzyme to be discovered and characterized. It is a ubiquitous tetrameric heme-containing enzyme that catalyzes the dismutation of two molecules of H2O2 into water and oxygen. It has high specificity for H2O2, but weak activity against organic peroxides. Plants contain several types of H2O2-degrading enzymes, however, CATs are unique as they do not require cellular reducing equivalent. CATs have a very fast turnover rate, but a much lower affinity for H2O2 than APX. The peroxisomes are major sites of H2O2 production. CAT scavenges H2O2 generated in this organelle during photorespiratory oxidation, â-oxidation of fatty acids, and other enzyme systems such as XOD coupled to SOD (Del Rýo, et al., 2006; Yeh, et al., 2007). Though there are frequent reports of CAT being present in cytosol, chloroplast, and mitochondria, the presence of significant CAT activity in these is less well established to date, all angiosperm species studied, contain

three CAT genes. Willekens et al. proposed a classification of CAT based on the expression profile of the tobacco genes. Class I CATs are expressed in photosynthetic tissues and are regulated by light. Class II CATs are expressed at high levels in vascular tissues, whereas Class III CATs are highly abundant in seeds and young seedlings. H2O2 has been implicated in many stress conditions. When cells are stressed for energy and are rapidly generating H2O2 through catabolic processes, H2O2 is degraded by CAT in an energy efficient manner (Mallick and Mohn, 2000). Environmental stresses cause either enhancement or depletion of CAT activity, depending on the intensity, duration, and type of the stress (Han, et al., 2009). In general, stresses that reduce the rate of protein turnover also reduce CAT activity. Stress analysis revealed increased susceptibility of CAT-deficient plants to paraquat, salt and ozone, but not to chilling. In transgenic tobacco plants, having 10% wildtype, CAT activity showed accumulation of GSSG and a 4-fold decrease in AsA, indicating that CAT is critical for maintaining the redox balance during the oxidative stress. Overexpression of a CAT gene from Brassica juncea introduced into tobacco, enhanced its tolerance to Cd induced oxidative stress (Guan, et al., 2009). Ascorbate peroxidase (APX): APX (E.C.1.1.11.1) is an integral component of the Ascorbate Glutathione (ASC-GSH) cycle. While CAT predominantly scavenges H2O2 in the peroxisomes, APX performs the same function in the cytosol and the chloroplast. The APX reduces H2O2 to H2O and DHA, using Ascorbic acid (AA) as a reducing agent. H2O2 + AA → 2H2O + DHA The APX family comprises of five isoforms based on different amino acids and locations, viz., cytosolic, mitochondrial, peroxisomal, and chloroplastid (stromal and thylakoidal) (Sharma and Dubey, 2004). Since APX is widely distributed and has a better affinity for H2O2 than CAT, it is a more efficient scavenger of H2O2 at times of stress. Monodehydroascorbate reductase (MDHAR): MDHAR (E.C.1.6.5.4) is responsible for regenerating AA from the short-lived MDHA, using NADPH as a reducing agent, ultimately replenishing the cellular AA pool. Since it regenerates AA, it is co-localized with the APX in the peroxisomes and mitochondria, where APX scavenges H2O2 and oxidizes AA in the process

(Mittler, 2002). MDHAR has several isozymes which are confined in chloroplast, mitochondria, peroxisomes, cytosol, and glyoxysomes. MDHA + NADPH → AA + NADP+ Dehydroascorbate reductase (DHAR): DHAR (M.C.1.8.5.1) reduces dehydroascorbate (DHA) to AA using Reduced Glutathione (GSH) as an electron donor (Eltayeb et al., 2007). This makes it another agent, apart from MDHAR, which regenerates the cellular AA pool. It is critical in regulating the AA pool size in both symplast and apoplast, thus maintaining the redox state of the plant cell (Chen and Gallie, 2006). DHAR is found abundantly in seeds, roots and both green and etiolated shoots. DHA + 2GSH → AA + GSSG Glutathione Reductase (GR): GR (E.C.1.6.4.2) is a flavoprotein oxidoreductase which uses NADPH as a reductant to NADPH as a reduce GSSG to GSH. Reduced glutathione (GSH) is used up to regenerate AA from MDHA and DHA, and as a result is converted to its oxidized form (GSSG). GR, a crucial enzyme of A SC-GSH cycle catalyzes the formation of a disulfide bond in glutathione disulfide to maintain a high cellular GSH/GSSG ratio. It is predominantly found in chloroplasts with small amounts occurring in the mitochondria and cytosol. GSH is a low molecular weight compound which plays the role of a reductant to prevent thiol groups from getting oxidized , and react with detrimental ROS members like 1O2 and OH•. GSSG + NADPH → 2GSH + NADP+ Guaiacol peroxidase (GPX): GPX (E.C.1.11.1.7) is a heme-containing enzyme composed of 40-50 kDa monomers, which eliminates excess H2O2 both during normal metabolism as well as during stress. It plays a vital role in the biosynthesis of lignin as well as defends against biotic stress by degrading indole acetic acid (IAA) and utilizing H2O2 in the process. GPX prefers aromatic compounds like guaiacol and pyragallol (Asada, 1999) as electron donors. Since GPX is active intracellularly (cytosol, vacuole), in the cell wall and extracellularly, it is considered as the key enzyme in the removal of H2O2.

H2O2 + GSH → H2O + GSSG

Non-Enzymatic Antioxidants The non-enzymatic antioxidants form the other half of the antioxidant machinery, comprising of AA, GSH, á-tocopherol, carotenoids, phenolics, flavonoids, and amino acid cum osmolyte proline. They not only protect different components of the cell from damage, but also play a vital role in plant growth and development by tweaking cellular process like mitosis, cell elongation, senescence and cell death (De Pinto and De Gara, 2004). Ascorbic Acid (AA): AA is the most abundant and the most extensively studied antioxidant compound. It is considered powerful as it can donate electrons to a wide range of enzymatic and non-enzymatic reactions. Majority of AA in plant cells is the result of Smirnof f-Wheeler pathway, catalyzed by L-galactano-ã-lactone dehydrogenase in the plant mitochondria, with the remaining being generated from D-galacturonic acid. 90% of the AA pool is concentrated not only in the cytosol, but also substantially in apoplast, thus making it the first line of defense against ROS attack (Barnes et al., 2002). AA is oxidized in two successive steps, starting with oxidation into MDHA, which if not reduced immediately to ascorbate, disproportionates to AA and DHA. It reacts with H2O2, OH• O•–2, and regenerates a tocopherol from tocopheroxyl radical, thereby protecting the membranes from oxidative damage (Shao et al., 2005). It also protects and preserves the activities of metalbinding enzymes. AA in its reduced state acts as the cofactor of violaxanthine de-epoxidase and maintains the dissipation of the excess excitation energy (Smirnoff, 2000). AA has also been reported to be involved in preventing photo-oxidation by pH mediated modulation of PSII activity and its down regulation, associated with zeaxanthine formation. Reduced glutathione (GSH): Glutathione is a low molecular weight thiol tripeptide (ã-glutamyl-cysteinyl-glycine) abundantly found in almost all cellular compartments like cytosol, ER, mitochondria, chloroplasts, vacuoles, peroxisomes, and even the apoplast. It is involved in a wide range of processes like cell differentiation, cell growth/division, cell death and

senescence, regulation of sulfate transport, detoxification of xenobiotics, conjugation of metabolites, regulation of enzymatic activity, synthesis of proteins and nucleotides, synthesis of phytochelatins and finally expression of stress responsive genes (Mullineaux and Rausch, 2005). This versatility of GSH is all due to its high reductive potential. A central cysteine residue with nucleophilic character is the source of its reducing power. GSH scavenges H2O2, 1O2, OH• and O•–2 and protects the different biomolecules by forming adducts (glutathiolated) or by reducing them in presence of ROS or organic free radicals and generating GSSG as a by-product. GSH also plays a vital role in regenerating AA to yield GSSG. The GSSG thus generated is converted back to GSH, either by de novo synthesis or enzymatically by GR. This ultimately replenishes the cellular GSH pool. GSH also helps in the formation of phytochelatins via phytochelatin synthase (Roychoudhury et al., 2012a), which helps to chelate heavy metal ions and thus scavenges another potential source of ROS formation in plants (Roy Choudhury et al., 2012b). Therefore, the delicate balance between GSH and GSSG is necessary for maintaining the redox state of the cell. Tocopherol: Tocopherols (α, β,γ, and δ) represent a group of lipophilic antioxidants involved in scavenging of oxygen free radicals, lipid peroxy radicals, and 1O2 (Diplock, et al., 1989). Relative antioxidant activity of the tocopherol isomers in vivo is α > β> γ> δ which is due to the methylation pattern and the amount of methyl groups attached to the phenolic ring of the polar head structure. Hence, a-tocopherol with its three methyl substituents has the highest antioxidant activity of tocopherols (Kamal-Eldin and Appelqvist, 1996). Tocopherols are synthesized only by photosynthetic organisms and are present in only green parts of plants. The tocopherol biosynthetic pathway utilizes two compounds homogentisic acid (HGA) and phytyl diphosphate (PDP) as precursors. At least 5 enzymes 4hydroxyphenylpyruvate dioxygenase (HPPD), homogentisate phytyl transferases (VTE2), 2-methyl-6-phytylbenzoquinol methyltransferase (VTE3), tocopherol cyclase (VTE1), a-tocopherol methyltransferase (VTE4) are involved in the biosynthesis of tocopherols, excluding the bypass pathway of phytyl-tail synthesis and utilization (Zhou, et al., 2010). Tocopherols are known to protect lipids and other membrane components by physically quenching and chemically reacting with O2 in chloroplasts, thus protecting the structure and function of PSII. Tocopherols prevent the chain propagation

step in lipid autooxidation which makes it an effective free radical trap. Fully substituted benzoquinone ring and fully reduced phytyl chain of tocopherol act as antioxidants in redox interactions with 1O2. 1O2 oxygen quenching by tocopherols is highly efficient, and it is estimated that a single á-tocopherol molecule can neutralize up to 220 1O2 molecules in vitro before being degraded. Regeneration of the oxidized tocopherol back to its reduced form can be achieved by AsA, GSH or coenzyme Q (Kagan, et al., 2010). Accumulation of á-tocopherol has been shown to induce tolerance to chilling, water deficit, and salinity in different plant species. It was found that metabolic engineering of tocopherol biosynthetic pathway affected endogenous ascorbate and glutathione pools in leaves. Further study suggested that expression levels of genes encoding enzymes of HalliwellAsada cycle were up-regulated, such as APX, DHAR and MDHAR. Mutants of Arabidopsis thaliana with T-DNA insertions in tocopherol biosynthesis genes, tocopherol cyclase (vte1) and -tocopherol methyltransferase (vte4) showed higher concentration of protein carbonyl groups and GSSG compared to the wild type, indicating the development of oxidative stress. Transgenic rice plants with Os-VTE1 RNA interference (OsVTE1-RNAi) were more sensitive to salt stress whereas, in contrast, transgenic plants overexpressing OsVTE1 (OsVTE1-OX) showed higher tolerance to salt stress. OsVTE1-OX plants also accumulated less H2O2 than control plants. Carotenoids: Carotenoids belong to family of lipophilic antioxidants which are localized in the plastids of both photosynthetic and nonphotosynthetic plant tissues. They are found not only in plants, but also in micrO•– organisms. They belong to a group of antennae molecules which absorbs light in the 450-570 nm and transfers the energy to the chlorophyll molecule. Carotenoids exhibit their antioxidative activity by protecting the photosynthetic machinery. They are efficient antioxidants for scavenging singlet oxyden and peroxyl radicals. Carotenoids have an important protective role during photosynthesis as these molecules can quench the excited states of chlorophyll in order to avoid the production of singlet oxygen. As a consequence, the carotenoid molecules become themselves excited, but this is not a big problem as they don’t have enough energy to form this ROS species (Taiz and Zeiger, 2002). Flavonoids: Flavonoids are widely found in the plant kingdom occurring

commonly in the leaves, floral organs and pollen grains. Flavonoids can be classified into four classes on the basis of their structure, flavonols, flavones, isoflavones, and anthocyanins. They have diverse roles in providing pigmentation in flowers, fruits and seeds involved in plant fertility and germination of pollen and defense against plant pathogens. Flavonoids have been considered as a secondary ROS scavenging system in plants experiencing damage to the photosynthetic apparatus, due to the excess excitation energy (Fini et al., 2011). They also have a role in scavenging 1O2 and alleviate the damages caused to the outer envelope of the chloroplastic membrane (Agati et al., 2012). Proline: Proline, an osmolyte is also regarded as a powerful antioxidant. It is widely used across the different kingdoms as a nonenzymatic antioxidant to counteract the damaging effects of different ROS members. Proline is synthesized using glutamic acid as a substrate, via a pyrroline 5-carboxylate (P5C) intermediate. This pathway in plants is catalyzed by two enzymes, o1 pyrroline-5-carboxylate synthetase (P5CS) and Pyrroline-5- carboxylate reductase (P5CR). It is an efficient scavenger of OH• and 1O2 and can inhibit the damages due to LPO. During stress, proline accumulates in plants in large amounts which is either due to enhanced synthesis or reduced degradation (Verbruggen and Hermans, 2008).

Overview of the antioxidant systems in plants

Different pathways for reactive oxygen species (ROS) scavenging in plants. Enzymatic antioxidants

Enzyme Reaction Catalyzed code

Sub cellular location

Superoxide dismutase (SOD) 1.15.1.1 O•–+ O•– + 2H+ → 2 2 2H2O2 + O2

Peroxisomes, Mitochondria, Cytosol, and Chloroplast

Catalase (CAT)

Peroxisome and Mitochondria

1.11.1.6 2H2O2 → O2+ 2H2O

Ascorbate peroxidase (APX) 1.11.1.11 H2O2+ AA → 2H2O + Peroxisomes, Mitochondria, Cytosol, and Chloroplast DHA Monodehydroascorbate reductase (MDHAR)

1.6.5.4

2MDHA + NADH → 2AA + NAD

Mitochondria, Cytoplasm, and Chloroplast

Dehydroascorbate reductase(DHAR)

1.8.5.1

DHA + 2GSH → AA + Mitochondria, Cytoplasm, and GSSG Chloroplast

Glutathione reductase (GR)

1.6.4.2

GSSG + NADPH → 2GSH +NADP+

Guaiacol peroxidase (GPX)

1.11.1.7 H2O2 + DHA → 2H2O Mitochondria, Cytoplasm, Chloroplast, and ER + GSSG

Mitochondria, Cytoplasm, and Chloroplast

Non-enzymatic Function Antioxidants

Sub cellular location

Ascorbic Acid (AA)

Detoxifies H2O2 via action of APX

Cytosol, Chloroplast, Mitochondria, Peroxisome, Vacuole, and Apoplast

Reduced

Acts as a detoxifying co-substrate for

Cytosol, Chloroplast, Mitochondria,

Glutathione (GSH)

enzymes like peroxidases, GR and GST

Peroxisome, Vacuole, and Apoplast

â -Tocopherol

Guards against and detoxifies products of membrane LPO

Mostly in membranes

Carotenoids

Quenches excess energy from the photosystems, LHCs

Chloroplasts and other non-green plastids

Flavonoids

Direct scavengers of H O and 1O and 2 2 2 OH•

Vacuole

Proline

Efficient scavenger of OH• and 1O2 and

Mitochondria, Cytosol, and Chloroplast

prevent damages due to LPO

Antioxidant Regulations in plants: Kumar, et al., (2016) reported that saline water irrigation significantly increase in Ascorbate peroxidase activity (APX), Superoxide dismutase activity (SOD), Glutathione peroxidase activity (GPX) and Catalase activity (CAT). The APX activity increased significantly by 23.20 and 35.41 per cent, SOD activity by 12.55 and 20.42 per cent, GPX activity by 10.04 and 14.39 per cent, CAT activity by 5.23 and 11.11 per cent, with 4 dS m- 1 and 8 dS m-1 level of salinity of irrigation water respectively, over control (Aghaei et al. 2009). Plants protect themselves from oxidative damage due to ROS through both enzymatic and nonenzymatic defense mechanisms (Ardic et al. 2009). In plants both enzymatic and non-enzymatic processes participate in detoxification of ROS which may otherwise causes oxidative damage to many cellular components (Shalata et al. 2001). Superoxide dismutase is a major scavenger of O2•– and it catalyzes the conversion of superoxide radical into hydrogen peroxide (H2O2) is then scavenged by CAT and different classes of peroxidase (Lin and Kao 2000). APX enzymes remove the excess of H2O2 formed. The activities of these antioxidant enzymes were reported to increase under salt stresses cultivars generally show higher activity of these antioxidant enzymes (Kaya et al., 2013; Kumar, et al., 2016). Ashraf and Ali (2007) reported antioxidant enzymes, SOD, CAT and POX the highest in salt tolerant cv. Dunkled of canola and lowest in salt sensitive cv. Cyclone, whereas intermediate in the other two lines. Overall relative cell membrane permeability and activities of antioxidant enzymes (SOD, CAT and POX) proved to be very effective in discriminating the canola cultivars for salt tolerance. Aghaei et al. (2009) reported that activities of ascorbate peroxidase, catalase, and glutathione reductase were increased in NaCl-

exposed shoots of salt tolerant Kennebec of potato. The corresponding activities in NaCl-exposed shoots of salt sensitive Concord were decreased. Latef and Chaoxing (2011) non-saline and saline conditions AMF colonization was accompanied by an enhancement of activity of superoxide dismutase (SOD), catalase (CAT), peroxidase (POD) and ascorbate peroxidase (APX) in leaves of both saltaffected and control tomato plants. In addition, inoculation with AMF caused reduction in MDA content in comparison to salinized plants, indicating lower oxidative damage in the colonized plants. Murshed et al. (2013) studied that effects of different levels of water stress on oxidative parameters (H2O2 and MDA), the total pool sizes of ascorbate, the activities of antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT), were studied in the fruit of tomato (Solanum lycopersicum L. cv. Micro-Tom). Changes in H2O2 and MDA contents indicated that water stress induced oxidative stress in fruits. The concentrations of ascorbate (AsA) and dehydroascorbate (DHA) generally modified with water stress treatments. Moreover, changes in SOD and CAT activities and APX were dependent on the fruit development stage and the intensity and the duration of water stress. The SOD activity was found to be up regulated during salt stress in many plants like chickpea (Kukreja et al., 2005) and tomato (Gapinska et al., 2008). All three isoforms of SOD have been found to be expressed in chickpea in response to salinity stress (Eyidogan and Oz, 2007) . Transgenic Arabidopsis overexpressing Mn-SOD was found to have enhanced salt tolerance (Wang et al., 2004). Cicer arietinum under salt stress also have increased CAT activity in both leaves and roots (Kukreja et al., 2005). Under salt stress, APX and GR activities were found to be higher in salt-tolerant cultivars of potato, while being markedly diminished in salt-sensitive varieties. This sensitivity was attributed to the reduction of APX and GR activity during saline conditions (Aghaei et al., 2009). Both AA and GSH were found to have enhanced levels in salttolerant cultivar Pokkali than in the sensitive cultivar Pusa Basmati (Vaidyanathan et al., 2003). GSH was also found to lessen the oxidative damage in rice chloroplasts caused due to salinity stress (Wang et al., 2014) . The content of flavonoids and proline were also found to be enhanced in salttolerant cultivars of indica rice than in the salt-sensitive cultivars, as evident by the reduced membrane damage caused by LPO (Chutipaijit et al., 2009)

References Agati, G, Azzarello, E., Pollastri, S. and Tattini, M. 2012 Flavonoids as antioxidants in plants: location and functional significance. Plant Sci. 196, 67-76. Aghaei, K., Akber, A., Ehsanpour and Komatsu, S. 2009 Potato Responds to Salt Stress by Increased Activity of Antioxidant Enzymes. National Institute of Crop Science, 3058518. Andreyev, A.Y., Kushnareva, Y. E. and Starkov, A. A. 2005 Mitochondrial metabolism of reactive oxygen species. Biochemistry (Moscow) 70(2): 200-214 Apel, K. and Hirt, H. 2004 Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annu. Rev. Plant Biol. 55, 373-399. Ardic, M., Sekmen, A.H., Tokur, S., Ozdemir, F. and Turkan, I. 2009. Antioxidant response of chickpea plants subjected to boron toxicity. Plant Biology 11, 328-338 Asada, K. 1999. The water-water cycle in chloroplasts: scavenging of active oxygens and dissipation of excess photons. Annu. Rev. Plant Biol. 50, 601-639. Ashraf, M. and Ali, Q. 2007. Relative membrane permeability and activities of some antioxidant enzymes as the key determinants of salt tolerance in canola (Brassica napus L.). Environmental and Experimental Botany 63, 266-273. Baker, A. and Graham, A.I. 2002. Plant Peroxisomes: Biochemistry, Cell Biology and Biotechnological Applications, Kluwer Academic Publishers, Dordrecht, The Netherlands. Barnes, J., Zheng, Y, and Lyons, T. 2002. “Plant resistance to ozone: the role of ascorbate,” in Air Pollution and Plant Biotechnology—Prospects for Phytomonitoring and Phytoremediation, (Eds) K. Omasa, H. Saji, S. Youssefian, and N. Kondo (Tokyo: Springer-Verlag), 235-252 Bhattacharjee, S. 2005. Reactive oxygen species and oxidative burst: roles in stress, senescence and signal. Curr. Sci. 89, 1113-1121. Bienert, G P., Moller, A. L., Kristiansen, K. A., Schulz, A., Moller, I. M., Schjoerring, J. K., et al. 2007. Specific aquaporins facilitate the diffusion of hydrogen peroxide across membranes. J. Biol. Chem. 282, 1183-1192. Boguszewska, D., Grudkowska, M. and Zagdanska, B. 2010. Drought-responsive antioxidant enzymes in potato (Solanum tuberosum L.). Potato Res. 53, 373-382. Bright, J., Desikan, R., Hancock, J.T., Weir, I. S., and Neill, S.J. 2006. ABA”induced NO generation and stomatal closure in Arabidopsis are dependent on H2O2 synthesis. Plant J. 45, 113-122. Chen, Z. and Gallie, D.R. 2006. Dehydroascorbate reductase affects leaf growth, development, and function. Plant Physiol. 142, 775-787. Choudhury, S., Panda, P., Sahoo, L., and Panda, S. K. 2013. Reactive oxygen species signaling in plants under abiotic stress. Plant Signal. Behav. 8:23681 Chutipaijit, S., Cha-Um, S., and Sompornpailin, K. 2009. Differential accumulations of proline and flavonoids in indica rice varieties against salinity Pak. J. Bot. 41,2497-2506. Cona, A., Rea, G., Angelini, R., Federico, R. and Tavladoraki, P. 2006. “Functions of amine oxidases in plant development and defence,” Trends in Plant Science 11(2): 80-88 Dat, J., Vandenabeele, S., Vranova, E., Van Montagu, M., Inze, D. and Van Breusegem, F. 2000. Dual action of the active oxygen species during plant stress responses. Cell. Mol. Life Sci. 57, 779-795. Davies, K. J. 1987. Protein damage and degradation by oxygen radicals. I. general aspects. Journal of Biological Chemistry, 262(20): 9895-9901

De Pinto, M.C. and De Gara, L. 2004. Changes in the ascorbate metabolism of apoplastic and symplastic spaces are associated with cell differentiation. J. Exp. Bot. 55, 2559-2569. Del Rio, L.A., Sandalio, L.M., Corpas, F.J., Palma, J.M. and Barroso, J.B. 2006. Reactive oxygen species and reactive nitrogen species in peroxisomes. Production, scavenging, and role in cell signaling. Plant Physiology, 141(2): 330-335 Dizdaroglu, M. 1993. Chemistry of free radical damage to DNA and nucleoproteins, in DNA and Free Radicals, B. Halliwell and O. I. Aruoma, Eds., 19-39, Ellis Horwood, London, UK Elstner, E. F. 1991. Mechanisms of oxygen activation in different compartments of plant cells,” in Active Oxygen/Oxidative Stress and Plant Metabolism, E. J. Pell and K. L. Steffen, Eds. 13-25, American Society of Plant Physiologists, Rockville, Md, USA Eltayeb, A. E., Kawano, N., Badawi, G.H., Kaminaka, H., Sanekata, T., Shibahara, T., et al. 2007. Overexpression of monodehydroascorbate reductase in transgenic tobacco confers enhanced tolerance to ozone, salt and polyethylene glycol stresses. Planta 225, 12551264 Evans, M. D., Dizdaroglu, M. and Cooke, M.S. 2004. Oxidative DNA damage and disease: induction, repair and significance. Mutat. Res. 567, 1-61. Eyidogan, F. and Oz, M.T. 2007. Effect of salinity on antioxidant responses of chickpea seedlings. Acta Physiol. Plant. 29, 485-493. Fini, A., Brunetti, C., Ferdinando, M., Ferrini, F. and Tattini, M. 2011. Stressinduced flavonoid biosynthesis and the antioxidant machinery of plants. Plant Signal. Behav 6, 709-711. Gapinska, M., Sk3odowska, M. and Gabara, B. 2008. Effect of short-and longterm salinity on the activities of antioxidative enzymes and lipid peroxidation in tomato roots. Acta Physiol. Plant. 30, 11-18. Gardner, P.R. and Fridovich, I. 1991. Superoxide sensitivity of the Escherichia coli 6 phosphogluconate dehydratase. Journal of Biological Chemistry, 266(3): 1478-1483 Gross, GG. 1977. “Cell wall-bound malate dehydrogenase from horseradish,” Phytochemistry, 16 (3): 319-321 Guan, Z., Chai, T., Zhang, Y, Xu, J. and Wei, W. 2009. Enhancement of Cd tolerance in transgenic tobacco plants overexpressing a Cd-induced catalase cDNA. Chemosphere, 76(5): 623-630 Halliwell, B. 2006. Reactive species and antioxidants. Redox biology is a fundamental theme of aerobic life. Plant Physiol. 141,312-322. Han, C., Liu, Q. and Yang, Y. 2009. Short-term effects of experimental warming and enhanced ultraviolet-B radiation on photosynthesis and antioxidant defense of Picea asperata seedlings. Plant Growth Regulation 58 (2): 153- 162 Hatz, S., Lambert, J.D. and Ogilby, PR. 2007. Measuring the lifetime of singlet oxygen in a single cell: addressing the issue of cell viability. Photochem. Photobiol. Sci. 6, 11061116. Heyno, E. Mary, V. Schopfer, P. and Krieger-Liszkay, A. 2011. Oxygen activation at the plasma membrane: relation between superoxide and hydroxyl radical production by isolated membranes. Planta 234(1): 35-45 Kamal-Eldin, A. and Appelqvist, L.A. 1996. “The chemistry andU antioxidant properties of tocopherols and tocotrienols,” Lipids 31(7):671-701 Kaya, C., Aydemir, S., Sonmez, O., Ashraf, M. and Dikilitas, M. 2013. Regulation of growth and some key physiological processes in salt-stressed maize (Zea mays L.) plants by exogenous application of asparagine and glycerol. Acta Botenica Croatica 72( 1), 157168. Krieger-Liszkay, A., Fufezan, C. and Trebst, A. 2008. Singlet oxygen production in photosystem II and related protection mechanism. Photosyn. Res. 98, 551-564.

Kukreja, S., Nandwal, A.S., Kumar, N., Sharma, S.K., Unvi, V. and Sharma, P.K. 2005. Plant water status, H2O2 scavenging enzymes, ethylene evolution and membrane integrity of Cicer arietinum roots as affected by salinity. Biol. Plant. 49, 305-308. Kumar, U., Gulati, I.J. and Bhunwal, V. 2016. Impact of humic acid and salicylic acid on biochemical and physiological parameters of tomato (Lycopersicon esculentum mill.) Under salinity stress. The Ecoscan 10 (1&2): 71-74. Li, Y., Zhou, Y., Wang, Z., Sun, X. and Tang, K. 2010. “Engineering tocopherol biosynthetic pathway in Arabidopsis leaves and its effect on antioxidant metabolism,” Plant Science, 178(3): 312-320 Lopez-Huertas, E. Corpas, F.J. Sandalio, L.M. and Del Rio, L.A. 1999. Characterization of membrane polypeptides from pea leaf peroxisomes involved in superoxide radical generation. Biochemical Journal 337 (3): 531-536. Maheshwari, R. and Dubey, R.S. 2009. Nickel-induced oxidative stress and the role of antioxidant defence in rice seedlings. Plant Growth Regulation 59(1): 37-49 Meriga, B., Reddy, B. K., Rao, K. R., Reddy, L. A. and Kishor, P. B. K. 2004. “Aluminium- induced production of oxygen radicals, lipid peroxidation and DNA damage in seedlings of rice (Oryza sativa),” Journal of Plant Physiology 161 (1): 63-68 Mittler, R. 2002. Oxidative stress, antioxidants and stress tolerance. Trends Plant Sci. 7, 405410 Mullineaux, P. M. and Rausch, T.2005. Glutathione, photosynthesis and the redox regulation of stressresponsive gene expression. Photosyn. Res. 86, 459-474. Murshed, R., Lopez-Lauri, F. and Sallanon, H. 2013 Effect of water stress on antioxidant systems and oxidative parameter s in fruits of tomato (Solanum lycopersicon L, cv. MicrO•–tom). Physiology and Molecular Biology of Plants 19(3), 363-378. Noctor, G., Veljovic-Jovanovic, S., Driscoll, S., Novitskaya, L. and Foyer, C.H. 2002. Drought and oxidative load in the leaves of C3 plants: a predominant role for photorespiration. Ann. Bot. 89, 841-850. Pang, C.H. and Wang, B.S. 2008. “Oxidative stress and salt tolerance in plants,” in Progress in Botany, U. Luttge, W.' Beyschlag, and J. Murata, Eds. 231-245, Springer, Berlin, Germany, Parida, A.K. and Das, A.B. 2005. Salt tolerance and salinity effect on plants: a review. Ecotoxicology and Environmental Safety 60, 324-349. Peng, C.L., Ou, Z.Y., Liu, N. and Lin, G.Z. 2005. Response to high temperature in flag leaves of super high-yielding rice Peiai 64S/E32 and Liangyoupeijiu. Rice Sci. 12, 179-186. Pinto, E., Sigaud-kutner, T., Leitao, M.A., Okamoto, O.K., Morse, D. and Colepicolo, P 2003. Heavy metal-induced oxidative stress in algae. J. Phycol. 39, 1008-1018. RomerO•–Puertas, M.C., Palma, J.M., Gomez, M., Del Rio, L.A. and Sandalio, L.M. 2002. Cadmium causes the oxidative modification of proteins in pea plants. Plant, Cell and Environment, 25(5): 677-686 RoyChoudhury, A., Das, K., Ghosh, S., Mukherjee, R.N., and Banerjee, R. 2012b. Transgenic Plants: benefits and controversies. J. Bot. Soc. Bengal 66, 29-35. Roychoudhury, A., Pradhan, S., Chaudhuri, B. and Das, K. 2012a. “Phytoremediation of toxic metals and the involvement of Brassica species,” in Phytotechnologies: Remediation of Environmental Contaminants, eds (Boca Raton, FL: CRC press; Taylor and Francis Group), 219-251. Shalata, A., and Neumann, P. M. 2001. Exogenous ascorbic acid (vitamin C) increases resistance to salts tress and reduce slipid peroxidation. J. Exp. Bot. 52, 2207-2211. Shao, H.B., Liang, Z.S., Shao, M.A., and Sun, Q. 2005. Dynamic changes of antioxidative enzymes of 10 wheat genotypes at soil water deficits. Colloids Surf. Biointerfaces 42, 187-195.

Sharma, P, and Dubey, R.S. 2004. Ascorbate peroxidase from rice seedlings: properties of enzyme isoforms, effects of stresses and protective roles of osmolytes. Plant Sci. 167, 541-550. Sharma, P, and Dubey, R.S. 2005a. Drought induces oxidative stress and enhances the activities of antioxidant enzymes in growing rice seedlings. Plant Growth Regul. 46, 209-221. Sharma, P, Jha, A.B. and Dubey, R.S. 2010. Oxidative stress and antioxidative defense system in plants growing under abiotic Stresses,” in Handbook of Plant and Crop Stress, M. Pessarakli, Ed., 89138, CRC Press, Taylor and Francis Publishing Company, Fla, USA, 3rd edition Sharma, P, Jha, A.B., Dubey, R.S., and Pessarakli, M. 2012. Reactive oxygen species, oxidative damage, and antioxidative defense mechanism in plants under stressful conditions. J. Bot. 217037. Smirnof f, N. 2000. Ascorbic acid: metabolism and functions of a multifacetted molecule. Curr. Opin. Plant Biol. 3, 229-235. Taiz, L. and Zeiger, E. 2002. Plant Physiology (3rd ed.). Sunderland, MA: Sinuaer Associates, Inc. Tanou, G, Molassiotis, A., and Diamantidis, G. 2009a. Hydrogen peroxide-and nitric oxide- induced systemic antioxidant prime-like activity under NaCl-stress and stress-free conditions in citrus plants. J. Plant Physiol. 166, 1904-1913. Vaidyanathan, H., Sivakumar, P, Chakrabarty, R., and Thomas, G. 2003. Scavenging of reactive oxygen species in NaCl-stressed rice (Oryza sativa L.)- differential response in salt- tolerant and sensitive varieties. Plant Sci. 165, 1411-1418. Verbruggen, N. and Hermans, C. 2008. Proline accumulation in plants: a review. Amino Acids 35, 753759. Wang, R., Liu, S., Zhou, F., Ding, C. and Hua, C. 2014. Exogenous ascorbic acid and glutathione alleviate oxidative stress induced by salt stress in the chloroplasts of Oryza sativa L. Z. Naturforsch. C 69, 226-236. Wang, Y., Ying, Y., Chen, J. and Wang, X. 2004. Transgenic Arabidopsis overexpressing Mn- SOD enhanced salt-tolerance. Plant Sci. 167, 671-677 Willekens, H., Inze, D., Van Montagu, M. and Van Camp, W. 1995. Catalases in plants. Molecular Breeding 1(3): 207-228 Yamauchi, Y., Furutera, A., Seki, K., Toyoda, K. Tanaka, Y. and Sugimoto, Y. 2009. Malondialdehyde generated from peroxidized linolenic acid causes protein modification in heat-stressed plants. Plant Physiology and Biochemistry, 46(8-9): 786-793 Yeh, C.M., Chien, PS. and Huang, H.J. 2007. Distinct signaling pathways for induction of MAP kinase activities by cadmium and copper in rice roots,” Journal of Experimental Botany 58(3): 659-671 Zhu, J.K. 2001. Plant salt tolerance. Trends in Plant Science 6:66-71.

CHAPTER 3

Fermentation Chemistry of Palm Neera K.B.Hebbar “Keep loving nature and care for its blessings Then you can see divinity all over”-Avul Pakir Jainulabdeen Abdul Kalam in “Turning Points”. Palm trees are a botanical family of perennial lianas, shrubs and trees. They are the only members of the family Arecaceae, which is the only family in the order Arecales. They grow in hot climates.Palm trees grow in the tropical and subtropical regions of the world. They occur from about 44° northern latitude to about 44° southern latitude. There are about 2600 species of palm trees, most of them living in tropical, subtropical, and warm temperate climates. Palms are one of the best known and most widely planted tree families. Palms are believed to be among the oldest flowering plants in the world (Redhead 1989). For centuries, many palm species have been tapped throughout the tropical world in order to produce fresh juice (sweet toddy), fermented drinks (toddy, wine, arak), syrup (“honey”), brown sugar (jaggery) or refined sugar. One of mankind’s first sources of sugar was probably Arenga pinnata(Redhead 1989). Evidence of the use of Borassus flabellifer sugar in India has been reported by the Greek historian Megasthenes, ambassador to the court of Chandragupta, in the 4th century BC. Hindus knew how to extract it about 4,000 years ago (Ferguson 1888, cited by Fox 1977). Jaggery and treacle extracted from Caryota urens sap in Sri Lanka has been an important source of sugar from antiquity (Dissanayake 1977). They have held an important role for humans throughout much of history. Many

common products and foods come from palms. In Table 1. Some of the common palm trees used for sap extraction (Fig 1), their habitat and products are explained.

Palm trees tapped

Scientific Native Common Name Distribution and Habitat

Major Products.

Amiga pinnata sugar palm

sap to make leaf sheath fiber; sugar, wine, edible heart; etc

S. & SE Asia tropical rain

Minor Products

Comments and Selected References Solitary .terminal flowering feather palm; traditional

forest into dry forest, to 1200 m

alcohol, vinegar, sap yield3-6 liters/ tree/day, starch from stem, yield 75 kg/tree

Borassus flabellifer,

S. & SE. Asia: Africa

sap to make sugar.

R. aethiopum palmyra, ron

tropical dry forest into savanna, to 750 m.

wine, for thatching & alcohol, basketry: edible vinegar, sap immature fruit yield 11-20 liters/ tree/day

of major utility to local peoples: incipient management already in practice in S. & SE. Asia: candidate for domestication, agrof orestry : Davis & Johnson, 1987

Caryota wrens

S. & SE. Asia tropical

sap to make sugar.

solitary, terminal flowering feather palm:

Fish tail palm

rain forest to 1,500 m

wine, edible hearfetc. alcohol, vinegar, sap yield20-27 liters/ tree/day: starch from stem, yield 100150kg/tree

numerous products: informal cultivation practiced: domestication potential in agrof orestry systems De Zoysa 19

Raphia spp raffia.

West Africa tropical rain forest, seasonally flooded lowland sites

commercial petioles as poles, leaf base leaves for thatching fiber & weaving! etc(African bass fiber) for brushes & brooms; sap for wine. Alcohol

suckering (most spp.) terminal flowering feather palm: R. hookeri & R. palmapinus are main brush fiber sources, also tapped for sap: one or more spp could be managed for multiple products: Tuley, 1995

NypaJruticans nipa

S. & SE Asia tropical rain forest, brackish water swamps of tidal rivers

sap for edible fruit: etc. sugar, alcohol, sugar yield 3,000kg/ha/ year; leaves for thatching (atap)

suckering feather palm; incipient management in practice, could benefit from improved practices & broader utilization of products, especially in Papua, New Guinea; Hamilton & Murphy, 1988

Every product is

Inflorescence is used for tapping. Tapping coconut

Cocos Spread across micifera(cocomit) much of the

multipurpose palm with a history of cultivation! strong candidate for domestication! agrof orestry potential: Mogea et al„ 1991

leaf stalk fiber; leaves

Leaf sheath fiber;

All parts are useful

solitary fan palms! multipurpose species

tropics and grown in 90 countries

useful. Important product is nut, sap, oil

Phoenix yy/vesft- India multipurpose edible fruit: leaves Athakil (sugar (common)Nepal palm: sap made into brooms date palm); from stem as or woven into beverage & baskets & mats; to make stem wood for fuel sugar,:

inflorescence for sap and its sale as health drink or value adde product like coconut sugar would improve the returns by at least 8 to 10 fold : Hebbar et al., 2015 A graceful palm 10-16 m tall with a large crown and rough trunk covered with persistent leaf bases. Fruiting spadix about 90 cm long, bearing oblong- ellipsoid berries.

Nypa Jruticans

India: Orissa,

(monotvpic) golpata

West Bengal. for beverage mature seeds Andaman or sugar; suitable for islands; vegetable ivory Bangladesh; Sri Lanka

sturdy stalks arising from the base of the plant. The female inflorescence is a densely packed, spherical head of flowers. The male inflorescence is a clubshaped spike of closely arranged flowers emerging from lateral stalks below the female inflorescence. The large spherical fruiting body is 30-45 cm in diameter.

Arenga pinnataGomuti (sugar palm)

India : Eastern Andaman Islands Bangaladesh, Sri Lanka

Sweet sap (toddy, tuba, saguer is obtained by tapping the male inflorescences. An inflorescence can be tapped for 1-2 1/2 months, producing about 5-12 liters of sap per day. This sap can be used as beverage or processed into vinegar, sugar, alcohol or animal feed. Sugar yields may be about 70 kg/day per ha or 25 t/ha per year.

sap from leaves for thatching. The yellow inflorescences are inflorescence on long.

Multipurpose palmi sap for sugar & other products

Edible immature seed (fresh mesocarp of ripe fruit is filled with irritant needle crystals); edible starch from stem; edible palm heart; leaf base fiber for fish nets, etc., leaflets for weaving baskets, etc., stem wood for various uses

Fig. 1: Some of the palm species used for tapping sap

2. Nutrient composition Palms sap is the phloem sap and known to be very rich in nutrients. The nutrient-rich sap that exudes from the blossoms before they mature into fruits, is used to make many unique and nutritious food products. This sap is very low glycemic (GI of only 35), and contains a wide range of minerals (high in Potassium, essential for electrolyte balance, regulating high blood pressure, and sugar metabolism), vitamin C (potent antioxidant for over all immune system protection, cardiovascular and respiratory health, reduces inflammation, etc.), broad-spectrum B vitamins (especially rich in Inositol, known for its effectiveness on depression, high cholesterol, inflammation, and diabetes), It is found that 17 amino acids (the building blocks of protein),, and it has a nearly neutral pH (helps to maintain proper acid/alkaline balance). Fresh sap is rich in sugar, minerals and proteins (Hebbar et al., 2015 a). It is also a rich source of phenolics and ascorbic acid. Coconut sap contains high amounts of essential elements such as N, P, K, Mg and micronutrients (Zn, Fe, and Cu). The biochemical constituents, minerals and vitamin composition of freshly collected sap are given in the Table 2. Table 2: Biochemical and mineral composition of freshly collected coconut sap (per 100 ml) Biochemical parameters

Range

Average

pH

6.57-7.50

7.18

Total sugar (g)

10.08-16.50

15.18

Reducing sugar (g)

0.439-0.647

0.554

Amino acids (g)

0.123-0.338

0.245

Protein (g)

0.150-0.177

0.165

Phenolics (mg)

4.80-5.40

5.10

Antioxidant activity (mM TE)

0.299 -0.355

0.321

Sodium (mg)

69.4 - 117.5

90.6

Potassium (mg)

146.1-182.4

168.4

Phosphorus (mg)

2.0-6.4

3.9

Manganese (mg)

0.009-0.014

0.012

Copper (mg)

0.028-0.035

0.031

Zinc (mg)

0.018-0.026

0.020

Iron (mg)

0.049-0.058

0.053

(Source: ICAR-CPCRI, Kasaragod)

2.1 Carbohydrate The total sugar content of coconut sap was high (16.19 g/100 ml) compared to reducing sugar content (0.68). In another palm tree Phoenix dactylifera Thabet et al.,(2009) reported that the proportion of the sugars of the sap consist mainly of sucrose (95.27%) and only 2.51% glucose and 1.61% fructose (dry matter basis). Many studies have found that sugarswith high sucrose content are known to be good for health. Sucrose is less fattening than starch or glucose, that is, that more calories can be consumed without gaining weight. During exercise, the addition of fructose to glucose increases the oxidation of carbohydrate by about 50% (Jentjens and Jeukendrup, 2005). Ruzzin, Lai and Jensen (2005) observed rats given a 10.5% or 35% sucrose solution, or water, and observed that the sucrose increased their energy consumption by about 15% without increasing weight gain. Further sucrose and fructose rich sugars have low glycemic index (GI) and therefore the GI of coconut sap is reported to be low (Trinidad et al., 2010). The extent of freshness of the sap can be easily measured by the total sugar and reducing sugar content. If the sap is fresh and unfermented, the reducing sugar content should be

Fig. 3: The relation between the pH and total sugar and reducing sugar content of sap 3.2. Microbial changes:The sap of the palm tree has been shown to be a rich medium capable of supporting various types of microorganisms, as high number of aerobic mesophils, lactic acid bacteria, yeasts and acetic acid

bacteria were found during tapping and immediately afterwards (Nwachukwu et al., 2006; Ogbulie et al. (2007). Amoa-Awua et.al., (2007) observed immediately after tapping multiplication of yeast dominated by S. Cerevisiae and alcohol concentration became substantial in the product after third day. Similarly, Atputharajah et al. (1986) studied the distribution of microorganisms and changes of physical and chemical contents during natural fermentation. They identified 166 isolates of yeasts and 39 isolates of bacteria, while 17 species of yeasts belonging to eight genera were recorded. Faparusi and Bassir (1971) found that Lactobacillus spp. and Leuconostoc spp. were active during the early stages of palm wine fermentation. According to Okafor (1975) lactic acid ‘bacteria also formed an important component of the bacterial population dominated by L. plantarum and L. mesenteriodes were responsible for a rapid acidification of the product during first 24 hrsof tapping. While the growth of acetic acid bacteria involving both Acetobacter and Gluconobacter species became pronounced after the buildup in alcohol concentration on third day. Subsequently, after third day high concentration of alcohol begins to drop and the concentration of acetic acid increased slowly from about 0.42- 0.48 %. After 4 days it had exceeded the acceptable level of 0.6 %. Ndoye et al. (2007) isolated two strains of A. tropicalis, and A.pasteurianus with ability to grow at temperature around 40°C.The following flow chart explains the complete sequence of events during fermentation by the microorganisms in palm sap (Fig 4) . However, there was no consistent pattern in the species of microorganisms and it was found to vary with palm sap, storage, season, geographical location. Thus, while lactic acid bacteria featured prominently in palm sap, all the different genera did not occur simultaneously in any one of the samples examined and no single genus was found consistently in any of the sap samples (Okafor, 1975). For example, in one of the samples streptococci persisted throughout the 7-day period of incubation whereas the lactobacilli remained only for the first 3 days of fermentation. In contrast, the only lactic acid bacteria observed in another sample of palm sap\were Leuconostoc spp. after 2 days’ incubation.Similarly, Streptococcus spp. formed an important component of the wine flora observed in fermenting palm wine during the first day could have been brought about by bacteria other than lactic acid bacteria.It is possible that Serratia and Aerobacter (Klebsiella) spp. which develop early but disappear subsequently, contribute to acid fermentation, but are then overgrown by bacteria better able to survive in acid conditions.

Acetobacter developed after about the 3rd day of the fermentation. This situationis perhaps to be expected as enough alcohol would have been formed to provide the appropriate substrate for acetic acid production by these bacteria (Amoa -Awua et.al., 2007).

Fig. 4 : Flow chart showing the microbial fermentation of palm sap into various products 3.2.1. Role of Enzymes Sucrose, the main sugar in palm sap, is first broken down into monosaccharide by invertase, an enzyme produced by the yeast present in the sap. The monosaccharide is then converted to ethanol through a complex

reaction processes catalyzed by various enzymes collectively called zymase. This process begins with a molecule of glucose being broken down by the process of glycolysis into pyruvate. The reaction is accompanied by the size difference of two molecules of NAD+ to NADH and a net of two ADP molecules converted to two ATP plus the two water molecules. Pyruvate is then converted to acetaldehyde and carbon dioxide by an enzyme called pyruvate decarboxylase and requiring thiamine diphosphate as cofactor. The acetaldehyde is subsequently reduced to ethanol by the enzyme alcohol dehydrogenase (ADH) (Dioha et al2009) as shown in the following sequence of reactions. While on the other hand pyruvic acid from the glycolysis is converted to lactic acid either by pyruvate de carboxylase or in some cases by the enzyme lactic acid dehydrogenase.

CH3COCOO- + H+ → CH3CHO + CO2 CH3CHO + NADH ^ C2H5OH + NAD+

1. Disadvantages of fermentation Since micro organisms catalyze the fermentation process, which leads to nutrient degradation or loss of sensory qualities of palm wine, loss due to the sour taste of fermented palm wine by the acids produced during the fermentation process as noticed by Malaisse (1997) and Ukhum et al. (2005). It is also found that Vitamins like Vitamin C, E and B complex decreased in fermented saps(Hebbar unpublished data). Coconut sap is highly nutritive and a good digestive agent (Devdaset al., 1969; Lata and Kamala, 1966). It is when tapped fresh, possesses a tolerable odour which turns harsh on fermentation and makes it unpalatable, despite being nutritious. The astringency and harsh note of the fermented neera could be due to the increased amounts of acids (19.0 mg/l), such as palmitoleic acid and

dodecanoic acid, along with higher concentrations of ethyl alcohol and ethyl esters. By using hygienic and zero alcoholic sap farmers can produce very good quality value added products like palm sugar, jaggery, necter or syrup can be prepared without the use of chemicals. In fermented sap the quality and quantity of sugar and other nutrients decline and becomes difficult to crystallize the sugar without the use of chemicals (Hebbar et al., 2015b)

2. Prevention of fermentation Compared to any fruit juices, collection of unfermented sap itself is a huge challenge in palms. In the traditional method the sap trickling from the cut surface is collected in an open earthen pot or bamboo sac, placed in the palm top for at least 8 to 12 h. In order to prevent the fermentation, lime is coated on the inner surface of the pot. The sap collected by this method is oyster whitein color and emanates a harsh odor. It is also contaminated with insects, ants, pollen and dust particles. The sap collected without applying lime is used exclusively for the preparation of toddy, an alcoholic drink.

2.1. Coco-sap chiller to collect hygienic and unfermented sap CPCRI developed ‘Coco sap chiller’ or simple ice box technology collects the sap hygienic (free from contaminants) and unfermented (zero alcoholic). Coco-sap chiller is a portable device characterized by a hollow PVC pipe of which one end is expanded into a box shape to house a sap collection container bound by ice cubes and the other end is wide enough to insert and remove a collection container of 2 to 3 liter capacities. Sap collected is zero alcoholic, fresh, hygienic, sweet and delicious. Fresh and delicious sap can be collected at any time of the day. It is highly nutritious and ready to serve natural health drink without the need for lengthy processing.It can be preserved fresh for long duration under freeze or sub zero condition. Tapping can be done throughout the year even during rainy season.Easy to operate,

hence, men and women (who are skilled climbers) can easily tap kalparasa.Fabrication is simple, cheap and from locally available material. As the sap collected by coco-sap chiller is zero alcoholic and free from contaminants the processing cost is very much minimized. The sap thus collected can be stored for any length of time under refrigerated condition (-1 to -3oC). One way of marketing sap as fresh juice in small outlets is using refrigerated dispensers. Efforts are on to develop tetra pack or retort pack technology for

Fig. 5: Coco-sap chiller for the collection of hygienic and unfermented palm sap long distance transport.Another way of processing is just evaporating the sap to get different natural products like coconut syrup, nectar, j aggary and coconut sugar. The sap collected by traditional method ‘neera’ is unhealthy and not zero alcoholic while the sap collected using ‘coco-sap chiller’ is hygienic and zero alcoholic and termed as ‘Kalparasa’. Studies conducted on Kalparasa and neera showed distinct variation in physical and biochemical properties (Table 3 and 4). Kalparasa is slightly alkaline (pH 7 to 8), golden brown color, sweet and delicious while neera is oyster white, pH 6 or beow and gives a astringent smell. Kalparasain addition to high sugar also contains amino acids, total phenols, flavonoids and antioxidants 2.5, 1.5, 4.6 and 1.8 times higher than neera respectively. Kalparasa is also rich in Vitamins C, E and Niacin.

Further, the products of Kalparasa like sap concentrate and sugar were also found to be rich in amino acids, polyphenols, flavonoids, vitamins and antioxidants. Table 3: Quality attributes of neera and Kalparasa Attribute

Kalparasa

Neera

Total Soluble solids (°Brix)

15.5 to 18

13 to 14

pH

7 to 8

6 or below

Colour

Golden brown or honey

Oyster white

Flavour

Sweet and delicious

Fetid smell

Chemicals and extraneous matter

Absent

Present

Alcohol (%)

0

2.32

Table 4: Biochemical constituents and minerals of sap collected by coco-sap chiller and traditional method Constituents

Sap collected by Coco-sap chiller

Traditional method

7.5

5.0

Total Phenols (mg/100g)

21.99

14.1

Total Flavonoids (mg/100 g)

0.96

0.17

Free Amino acids (mg/100g)

901

350

15.96

6.52

Nil

4.7

Vit C (mg/100g)

13.45

4.73

Niacin (ug/100ml)

14.86

1.98

Vit E (ug/100ml)

7.9

2.95

pH

Total Sugars (g/100 g) Alcohol % Vitamins

Minerals Potassium (mg/100 ml)

168

Sodium (mg/100 ml)

90.6

Iron (ug/100ml)

53

Zinc (ug/100 ml)

20

2.2. Efforts to improve the shelf life

In order to lengthen the shelf-life of palm sap, a number of preservation measures have been adopted. However all these were tried on the sap collected by traditional method which is collected in a unhygienic way and at the time of collection itself partially fermented. These include the use of extract from bark of trees such as Saccoglottis gabonensis, Vernonia amygdalina, Euphobia sp., Nauclea sp. and Rubiacae sp. (Ogbulie et al., 2007). Sulphite and Benzoate (Levi and Oruche, 1957), pasteurization (Chinarasa, 1968), have all been used for preservation of palm wine. All these attempts have either resulted in change of taste or not completely been able to curb the actions of the fermenting microbes. In Raphia palm wine pasteurisation with zero additives is found to be good extending the shelf life without much change in waste (Dioha et al., 2009). According to Phisut Naknean et.al (2013) use of chitosan as a natural preservative ingredient to extend the shelf life of pasteurized assured for at least 6 weeks of storage, whereas microbial load was reduced below standard level. High concentration of chitosan (1.00 g/L) can be also used to prolong the shelf life of pasteurized palm sap, however more sedimentation and the increment in bitterness might be considered as aquality defect of product, causing in unacceptability. Thus, this study recommends the use of chitosan at concentrations approximately 0.50 g/L to extend quality of pasteurized palm sap. 5.2.1. Micro filtration: A membranefiltration technique developed atNational Chemical Laboratory ( NCL Pune) claims to remove the microorganismspresent in Neera without affectingits nutritive quality, thus extendingthe shelf life up to 45 days underconditions of refrigeration (4-8°C). 5.2.3.Integrated approach: Institutions in India viz. Defense Food Research Laboratory (DFRL) Mysore, Central Food Technological Research Institute (CFTRI) Mysore, Defense Research and Development Organisation (DRDO), Kerala Agricultural University (KAU), Coconut Development Board (CDB) and others made concerted efforts to improve the shelf life and palatability of the sap collected by traditional methods. Different techniques like filtration, treatment with clarifying agents and deodorizing using activated carbon/bentonite, centrifugation, pasteurization at 95oC,addition of preservatives, carbonation etc were employed and filled in glass bottles/Al cans/flexible packages. All these attempts have either resulted in change of taste or not completely been able to curb the actions of the fermenting

microbes.

6. Future thrust The major challenge of collecting hygienic and unfermented sap from the palms has been to certain extent resolved with the development of coco-sap chiller technology at ICAR CPCRI. The sap thus collected is very healthy and nutritious and hence it can be directly sold as fresh juice under refrigerated condition in the local market or processed into various value added products like coconut sugar, jiggery, nectar or syrup without the addition of chemicals. As the experience suggests that tapping the sap and selling is 8 to 10 fold more prof itable than selling nuts. However, the shelf life of the sap for long distance transport is a major issue. Concerted efforts are needed to improve the shelf life without affecting the natural aroma and taste of the sap.

References Amoa - Awua W.K., Sampson E . and Debrah K.Tano, 2007. Growth of yeasts, lactic and acetic bacteria in palm wine during tapping and fermentation from felled oil palm (Elaeis guineensis) in Ghana. Journal of Applied Microbiology, 102:599-606. Atputharajah JD, Widanapathirana S, and Samarajeewa U, 1986. Microbiology and biochemistry of natural fermentation of coconut palm sap, Food Microbiol. III: 273-280. Ben Thabet I, Besbes S, Attia H, Deroanne C, Francis F, Drira ND and Blecker C, 2009. Physicochemical characteristics of date sap « lagmi » from deglet Nour palm (Phoenix dactylifera L.), Int. J. Food Prop., XII: 659-670. Ben Thabet, I., Attia H., Besbes, S., Deroanne, C., Francis, F., Drira, N. E. and Blecker, C, 2007. Physicochemical and Functional Properties of Typical Tunisian Drink: Date Palm Sap (Phoenix dactylifera L.), Food Biophysics, II: 76-82. Borse, B. B., Rao, L. J. M, Ramalakshmi, K., and Raghavan, B, 2007 Chemical composition of volatiles from coconut sap (neera) and effect of processing, Food chemistry, 101(3): 877-880 Cunningham, A.B. and Wehmeyer, A.S, 1988. Nutritional value of palm wine from Hyphaene coriacea and Phoenix reclinata (Arecaceae), Economic Botany 42 (3): 301-30 Davis, T.A. and Johnson, D.V, 1987. Current utilization and further development of the palmyra palm (Borassusflabellifer L., Arecaceae) in Tamil Nadu State, India, Economic Botany 41(2):247-266. Devdas, R. P, Sundari, K., and Susheela, A, 1969. Effects of supplementation of two school lunch programmes with neera on the nutritional status of children, Journal of Nutrition and Dietetics 6: 29-36. Dioha I.J., Olugbemi O., Odin E.M. and Eneji M. A, 2009. Zero additives preservation of Raphia palm

wine, Int. J. Biol. Chem. Sci. 3(6): 1258-1264 Dissanayake B .W, ( 1977) Use of Caryota urens in Sri Lanka. In: First International Sago Symposium. The Equatorial Swamp as a Natural Resource. Ed. Tan K. Sarawak, East Malaysia, Kuching 1976. Kuala Lumpur. pp. 84-90. FAO 1998. Fermented fruits and vegetables. With total prospect. Agricultural services. Agric. Report food Org. 134. Faparusi, S.I. and Bassir, O, 1971. Microbiology of fermenting palm wine, J Food Sci Technol 8: 206. Fox J. F, 1977. Harvest of the Palm, Ecological Change in Eastern Indonesia. Harvard University Press, Cambridge, Massachusets, and London, England. pp. 290. Hebbar, K. B. ; Arivalagan, M. ; ManiKantan, M. R. ; Mathew, A. C. ; Thamban, C. ; Thomas, G.V. and Chowdappa, P, 2015 Coconut inflorescence sap and its value addition as sugar - collection techniques, yield, properties and market perspective, Current Science, doi: 10.18520/v109/i8/1411-1417 Hebbar, K. B., Arivalagan, M., Manikantan, M. R., Mathew, A. C.and Chowdappa, P., Kalparasa collection and value addition. In Technical Bulletin No. 92, ICAR-CPCRI, Kasaragod, Kerala, 2015 Hebbar, K. B., Mathew, A. C., Arivalagan, M., Samsudeen, K., and Thomas, G. V, 2013. Value added products from Neera. Indian Coconut Journal LVI (4): 28-33. Iwuoha, C. I., and Eke, O. S, (1996). Nigerian indigenous foods: Their Food traditional operationinherent problems, improvements and current status, Food Research International 29: 527-540. Jentjens, R. L. and Jeukendrup, A. E, 2005 High rates of exogenous carbohydrate oxidation from a mixture of glucose and fructose ingested during prolonged cycling exercise, Br J. Nutr. 93 (4):485-92. Koshihara, Y., Neichi, T., Murota, S.-I., Lao, A.-N., Fujimoto, Y., and Tatsuno, T, 1984. Caffeic acid is a selective inhibitor for leukotriene biosynthesis, Biochimica et Biophysica Acta 92: 92-97. Levi, S. S. and Oruchec, B, 1957.The preservation and bottling of palm wine. Res. Rep., Federal Ministry of Commerce & Industries, Lagos. Ndoye B, Lebecque S, Dubois-Dauphin R, Tounkara L, Guiro AT, Kere C, Diawara B and Thonart P, 2006. Thermoresistant properties of acetic acids bacteria isolated from tropical products of SubSaharan Africa and destined to industrial vinegar, Enzyme Microb. Technol. 39:916- 923 Nwachukwu I. N, Ibekwe VI, Nwabueze RN and Anyanwu B. N, 2006 Characterisation of palm wine yeast isolates for industrial utilization, Afr. J. Biotechnol. 5:1725-1728. Odunfa, S. A, 1985. African fermented foods. (In) B. J. B. Wood (Ed.). Microbiology of fermented foods (Vol.2). London: Elsevier Applied Science Ogbulie T. E, Ogbulie J. N and Njoku H. O, 2007. Comparative study on the microbiology and shelf life stability of palm wine from Elaeis guineensis and Raphia hookeri obtained from Okigwe, Nigeria, Afr. J. Biotechnol. 6: 914-922. Okafor N, (1975) Microbiology of Nigerian Palm Wine with Particular Reference to Bacteria, J. appl. Back, 38: 81-88 Phisut Naknean, Keawta Jutasukosol and Theerarat Mankit, 2015. Utilization of chitosan as an antimicrobial agent for pasteurized palm sap (Borassnsflabellifer Linn.) during storage, J Food Sci Technol., 52 (2):731-741 Redhead J, (1989) Utilization of tropical foods: trees, (In) FAO Food and Nutrition Paper No 47: 3. FAO, Rome, pp. 52. Ruzzin, J., Lai ,Y and Jensen, J, 2005. Consumption of carbohydrate solutions enhances energy intake without increased body weight and impaired insulin action in rat skeletal muscles. Fructoseand sucrose solutions enhanced energy intake but did not increase body weight, Diabetes Metab.

31:178-88 Sanni, A. I, 1993. The need for process optimization of African fermented foods and beverages, International Journal of Food Microbiology 18: 85-95. Trinidad, T. P, Mallillin, A. C., Sagum, R. S. and Encabo, R. R, 2010. Glycemic index of commonly consumed carbohydrate foods in the Philippines, Journal of Functional Foods 2: 271-274. Ukhum M. E, Okolie N. P and Oyerinde A. O, 2005. Some mineral prof ile of fresh and bottles palm wine-a comparative study, Afr. J. Biotechnol. 4:829-832. Xia, Q., Li, R., Zhao, S., Chen, W., Chen, H., Xin, B., Huang, Y. and Tang, M, 2011. Chemical composition changes of post-harvest coconut inflorescence sap during natural fermentation, African Journal of Biotechnology, 10(66): 14999-15005.

CHAPTER 4

Chemistry of Macronutrients Fixation in Acidic Soils B. B. Basak and Rajiv Rakshit “Uncertain is the life of man As rain drops on a lotus leaf; The whole of humankind is prey To grief and ego and disease”-Bhaja Govindam of Sri Sankaracarya Soil acidity is an economic and natural resource threat throughout the world. Acidification of soil is a natural process with major ramifications on plant growth. As soils become more acid, particularly when the pH drops below 4.5, it becomes increasingly difficult to produce food crops. In our country, about 25 million hectares of cultivated lands with pH value less than 5.5 are very poor in physical, chemical and biological characteristic. Naturally, nutrient availability and fixation in soil is governed by the chemical condition of soil. Knowledge and understanding about the fate of nutrient applied in soil as fertilizer is very important for improving use efficiency of applied nutrients in soil. Chemistry associated with macronutrients fixation in soil is specifically discussed here along with brief account is given here how best the fixed nutrients can utilized for plant nutrition.

1. Acid Soil Environment: Concept and Understanding In aqueous systems, an acid is a substance that donates hydrogen ions or protons (H+) to some other substance. Conversely, a base is any substance

that accepts H+. The H+ ions or active acidity, increase with the strength of the acid. The undissociated H+ contributes to a soil’s potential acidity. Buffer systems can maintain the pH of a solution within narrow range when small amounts of an acid or a base are added. Buffering defines the resistance to change in pH. Soils differ in terms of active and potential acidity. Also soils behave like buffered weak acids, with the H+ in cation exchange complex (CEC) of humus and clay minerals providing the buffer for soil solution pH. Soil acidity is of three kinds namely a) active b) exchangeable c) reserve acidity. The hydrogen ions in soil solution contribute to the active acidity. It may be defined as the acidity developed due to concentration of H+ and Al3+ ions in the soil solution. The concentration of hydrogen ion in soil solution due to active acidity is very small, implying that only a meager amount of lime would be required to neutralize active acidity. Normal measurement of pH of the soil denotes the active acidity. Inspite of smaller concentration, active acidity is important since the plant root and the microbes around the rhizosphere are influenced by it and because a dynamic equilibrium exists among active, exchange and reserve acidities of soil. The exchangeable Al and H ions contribute to the exchangeable acidity. It may be defined as the acidity developed due to adsorbed H+ and Al3+ ions on soil colloids. However this exchangeable aluminium and hydrogen ions concentration is meagre in moderately acid soils. The reserve acidity includes the hydrogen and aluminium ions present in the non-exchangeable form with clays and organic matter. It is measured by titrating a soil suspension up to a certain pH normally about 8.0, the amount of acidity in the soil being equivalent to the amount of NaOH used. The important sources of soil acidity are: exchangeable H+ and Al3+, Fe and Al oxides, soil organic matter and clay minerals. Two adsorbed cations- H+ and Al3+ are mainly responsible for soil acidity. The exchangeable hydrogen ions present in soil neutralize the negative charge arising from the isomorphous substation of cations. The hydrogen ions are thus due to permanent charge on the mineral surfaces. The pH dependent charge may arise from the structural OH- groups at the corners and edges of soil clay minerals, which dissociate into H+ ions. The Al3+ ions displaced from clay minerals by cations are hydrolyzed to monomeric and polymeric hydroxyaluminium complexes. Hydrolysis of the monomeric forms have

been illustrated in the following stepwise equations, which in each case liberates H+ and lower the pH unless neutralized by OH- present in the system. 1. Al3+ + H2O → Al(OH)2+ + H+ 2. Al(OH)2+ + H2O → Al(OH)2+ + H+ 3. Al(OH)2+ + H2O → Al(OH)3 + H+ 4. Al(H2O)63+ + H2O → Al(OH)(H2O)5+ + H3O+ 5. Al(OH)(H2O)52+ + H2O → Al(OH)2(H2O)4+ + H3O+ 6. Al(OH)2(H2O)4+ + H2O → Al(OH)3(H2O)3+ + H3O+ 7. Al(OH)3(H2O)30 + H2O → Al(OH)4(H2O)2- + H3O+ Soil acidity is determined by the amount of hydrogen ion (H+) activity in soil solution and is influenced by edaphic, climatic, and biological factors. Acid soils are mostly found in the areas of high rainfall. Rainfall is the most effective in causing soils to become acidic if plenty of water moves through the soil rapidly. In acid soil regions (ASR), precipitation exceeds evapotranspiration and hence leaching is predominant causing loss of bases from soil. The iron and aluminium derivatives are relatively insoluble, seldom leached and contributes to surface acidity particularly in laterite soils. Since the effect of rainfall on acid soil development is very slow, it may take hundreds of years for new parent material to become acidic under high rainfall. Decomposition of organic matter produces H+ ion which is responsible for acidity. The carbon dioxide (CO2) produced by decaying organic matter reacts with water in the soil to form a weak acid called carbonic acid. This is the same acid that develops when CO2 in the atmosphere reacts with rain to form acid rain naturally. Several organic acids are also produced by decaying organic matter, but they are also weak acids. Acidic materials (granites, rhyolites, diorites) are basis for acidic soil formation. In these soils, predominant minerals are quartz, feldspar and oxides. Al and Fe are in soluble forms. Some rocks (parent material) are acidic in nature (e.g. igneous rocks). After weathering of these rocks, acidic constituents dominate the composition of soil. Nitrogen and phosphorus fertilizers also contribute significantly to the formation of acid soils.

Ammonium nitrogen can be a major factor in the acidification of sandy, low buffer-capacity soils, unless a careful liming programme is maintained. When ammonium is converted to nitrate by soil microbes, hydrogen ions are released. Acid rain is mainly a mixture of sulphuric and nitric acids depending upon the relative quantities of oxides of sulphur and nitrogen emissions. Due to the interaction of these acids with other constituents of the atmosphere, protons are released causing increase in the soil acidity.

2. Macronutrients in Soil Macronutrients or major nutrients are so-called because these are required in large quantities, more than that of iron. Nitrogen, Phosphorus and Potassium are termed as primary or major nutrients because of their larger requirement by the plants and correction of their wide-spread deficiencies is often necessary through application of commercial fertilizers of which these are the major constituents. The macronutrients like N, P and K present in diverse from in soil but plant can take up only particular ionic from of that particular nutrient from soil. So fixation of that particular ionic from is very important in respective of plant availability. Nitrogen in soil exists in two major forms: (i) organic N and (ii) inorganic N. The inorganic forms (NH4+-N, NO3--N and NO2--N) are very important from crop nutrition point of view, because plant roots take up N from the soil mostly as NO3--N and to some extent as NH4+N. The NO2- form is unstable and is usually present in soil in lesser extent. Both organic and inorganic from phosphorus present in soil but inorganic from of phosphorus (H2PO4- and HPO42-) directly available to plant. The type of phosphate ions present in the soil solution depends on the soil pH. In soils having neutral to slightly alkaline pH, the H2PO4- is the most common form. As the soil pH gets lowered and it becomes slightly to moderately acidic, both H2PO4- and HPO42- ions prevail. At higher soil acidity, H2PO4form tends to dominate. Unlike the N and P, K present in soil mainly inorganic or mineral form and K+ ions is directly taken by plant. In soil K ion present in solution, exchangeable, non-exchangeable and mineral form but K ion in soil solution readily available to plants.

3. Nitrogen fixation Approximately 80% of the air consists of nitrogen gas (N2), but plants cannot use atmospheric N directly to make protein. The gaseous N must first be converted, or “fixed,” into forms plants can use. Biological nitrogen fixation is the only process through which atmospheric N2 converted into plant available form. Nitrogen fixation is the natural process, either biological or abiotic, by which nitrogen (N2) in the atmosphere is converted into ammonia (NH3). This process is essential for life because fixed nitrogen is required to biosynthesize the basic building blocks of life, e.g., nucleotides for DNA and RNA and amino acids for proteins . Nitrogen fixation also refers to other abiological conversions of nitrogen, such as its conversion to nitrogen dioxide .Biological nitrogen fixation (BNF), discovered by Beijerinck in 1901 and carried out by a specialized group of prokaryotes. These organisms utilize the enzyme nitrogenase to catalyze the conversion of atmospheric nitrogen (N2) to ammonia (NH3). Plants can readily assimilate NH3 to produce the aforementioned nitrogenous biomolecules. These prokaryotes include aquatic organisms, such as cyanobacteria, free-living soil bacteria, such as Azotobacter, bacteria that form associative relationships with plants, such as Azospirillum, and most importantly, bacteria, such as Rhizobium and Bradyrhizobium that form symbioses with legumes and other plants (Postgate, 1981).

3.1. Mechanisms of Biological Nitrogen Fixation The nitrogen molecule is composed of two nitrogen atoms joined by a triple covalent bond, thus making the molecule highly inert and nonreactive. Nitrogenase catalyzes the breaking of this bond and the addition of three hydrogen atoms to each nitrogen atom. Although the process involves a number of complex biochemical reactions, it may be summarized in a relatively simple way by the following equation: N2 + 8H2+ 16ATP → 2 NH3 + 2H2+ 16ADP + 16 P. The equation above indicates that one molecule of nitrogen gas (N2)

combines with eight protons) (8H+) to form two molecules of ammonia (2NH3) and two molecules of hydrogen gas (2H2). This reaction is conducted by an enzyme known as nitrogenase. The 16 molecules of ATP represent the energy required for the BNF reaction to take place. In biochemical terms 16 ATP represents a relatively large amount of plant energy (Fig.1). Thus, the process of BNF is ‘expensive’ to the plant in terms of energy usage. As ammonia (NH3) is formed it is converted to an amino acid such as glutamine. The Nitrogen in amino acids can be used by the plant to synthesize proteins for its growth and development.

Fig. 1: A diagram illustrating the interrelationship of reactions involved in nitrogen-fixing process in a legume nodule (After Evans et al. 1978). In limitations and Potentialities for Biological Nitrogen fixation in the tropics pp.209-222, (Eds) J. Dobereiner, H. R. Burries and A. Hollaender, Plenum Press, New York. 3.1.1. Host Specificity There are roughly 1,300 leguminous plant species in the world. of these, nearly 10% have been examined for nodulation, 87% of which were nodulated. Thus not all legumes are infected by rhizobia. Gliricidia sepium and Vigna unguiculata (cowpea) nodulate freely but nodules have never been found on roots of Cassia siamea. A Rhizobium that nodulates cowpea may not nodulate Leucaena and vice versa. Leguminous species mutually

susceptible to nodulation by a particular group of bacteria constitute a crossinoculation group. Six cross-inoculation groups were defined in the early days of Rhizobium research in addition to the cowpea group. This classification scheme is undergoing modifications based on recent research. Table 1 gives a short list of rhizobia and their hosts to illustrate the grouping of rhizobia. Not all symbioses fix N2 with equal effectiveness. This means that a given legume cultivar nodulated by different strains of the same species of Rhizobium would fix different amounts of nitrogen. Selection of elite strains of Rhizobium is based on this observation. Similarly, a given strain of Rhizobium will nodulate and fix different amount of N2 in symbiosis with a range of cultivars of the same plant species. Table 1: A short list of Rhizobium species and their corresponding host plants Rhizobium species

Host plants

Bradyrhizobium japonicum

Glycine max (soybean)

Rhizobium fredii

Glycine max (soybean)

R. phaseoli

Phaseolus vulgaris (common bean)

R. meliloti

Medicago sativa (alfalfa) Melilotus sp. (sweet clovers)

R. trifolii

Trifolium sp. (clovers)

R. Ieguminosarum

Pisum sativum (peas) Vicia faba (broad bean)

“Cowpea rhizobia” group or Rhizobium sp.

Vigna unguiculata (cowpea),

Arachis hypogaea (peanut), Vigna subterranea (Bambara groundnut) Leucaena sp., Albizia Azarhizobium caulinodans

Sesbania sp. Sesbania rostrata (stem nodulating)

3.1.2. Root Nodule Formation Sets of genes in the bacteria control different aspects of the nodulation process. One Rhizobium strain can infect certain species of legumes but not others e.g. the pea is the host plant to Rhizobium leguminosarum biovar viciae, whereas clover acts as host to R. leguminosarum biovar trifolii. Specificity genes determine which strain infects which legume. Even if a strain is able to infect a legume, the nodules formed may not be able to fix nitrogen. Such strains are termed ineffective. Effective strains induce

nitrogen-fixing nodules. Effectiveness is governed by a different set of genes in the bacteria from the specificity genes. Nod genes direct the various stages of nodulation. The initial interaction between the host plant and free-living rhizobia is the release of chemoreceptors by the root cells into the soil. Some of these encourage the growth of the bacterial population in the area around the roots (the rhizosphere). Reactions between certain compounds in the bacterial cell wall and the root surface are responsible for the rhizobia recognizing their correct host plant and attaching to the root hairs. Flavonoids secreted by the root cells activate the nod genes in the bacteria which then induce nodule formation. The whole nodulation process is regulated by highly complex chemical communications between the plant and the bacteria. Once bound to the root hair, the bacteria excrete nod factors. These stimulate the hair to curl. Rhizobia then invade the root through the hair tip where they induce the formation of an infection thread. This thread is constructed by the root cells and not the bacteria and is formed only in response to infection. The infection thread grows through the root hair cells and penetrates other root cells nearby of ten with branching of the thread. The bacteria multiply within the expanding network of tubes, continuing to produce nod factors which stimulate the root cells to proliferate, eventually forming a root nodule. Within a week of infection small nodules are visible to the naked eye. Each root nodule is packed with thousands of living Rhizobium bacteria, most of which are in the misshapen form known as bacteroids. Portions of plant cell membrane surround the bacteroids. These structures, known as symbiosomes, which may contain several bacteroids or just one, are where the nitrogen fixation takes place (Fig. 2).

Fig. 2 : Schematic diagram of atmospheric N fixation in the nodule of legume plant

3.2. Nitrogen Fixation in Acid Soil Soil acidity is a significant problem facing agricultural production in many areas of the world and limits legume productivity (Brockwell et al., 1991; Bordeleau and Prevost, 1994; Correa and Barneix., 1997). Most leguminous plants require a neutral or slightly acidic soil for growth, especially when they depend on symbiotic N2 fixation. Soil acidity constrains symbiotic N2 fixation in both tropical and temperate soils, limiting Rhizobium survival and persistence in soils and reducing nodulation (Graham, 1982; Brockwell et al., 1991: Ibekwe et al., 1997). Some of the Rhizobial strains are tolerant to acidity. It has been found that R. loti multiplied at pH 4.5 but Bradyrhizobium strains failed to multiply; the acid-tolerant strains of R. loti demonstrate a comparative advantage over acid-sensitive strains in the ability to nodulate their host legume at pH 4.5 (Cooper et al., 1985). R. tropici and R. loti are moderately acid tolerant, while R. meliloti is very sensitive to acid stress. The fast-growing strains of rhizobia have generally been considered less tolerant to acid pH than have slowly growing strains of Bradyrhizobium, although some strains of the fast-growing rhizobia, e.g., R. loti and R. tropici, are highly acid tolerant (Cooper et al., 1985). The basis for differences in pH

tolerance among strains of Rhizobium and Bradyrhizobium is still not clear. Graham (1982) reported high cytoplasmic potassium and glutamate levels in acid-stressed cells of R. leguminosarum bv. phaseoli, a response which is similar to that found in osmotically stressed cells. Nodulation failure under acid-soil conditions is common, especially in soils of pH less than 5.0. The inability of some rhizobia to persist under such conditions is one cause of nodulation failure, but sometimes viable Rhizobium population exhibit poor nodulation (Graham, 1982; Carter et al., 1994). The number of nodules, the nitrogenase activity, the nodule ultrastructure, and the fresh and dry weights of nodules were affected to a greater extent at a low medium pH (4.5). Early stage of infection process is more sensitive to soil acidity which had more severe effects on rhizobial multiplication than did Al stress and low P conditions. The host legume appears to be the limiting factor for establishing Rhizobiumlegume symbiosis under acidic conditions. Legume species differ greatly in their response to low pH with regard to growth and nodulation because the amount of N2 fixed by forage legumes on low fertility acidic soil is dependent on legume growth and persistence (Thomas et al., 1997). Legumes like Trifolium subterranean, T. balansae, Medicago murex, and M. truncatula, showed tolerance to soil acidity. R. leguminosarum bv.viciae is able to form nodule after inoculation with Vicia faba in acid soils (Aarons and Graham, 1991; Carter et al., 1994). Legume species vary markedly in their tolerance to Al3+ and Mn2+, with some plants being significantly more strongly affected by these ions than are the rhizobia. Therefore, for acid soils with high Al content, improvement is achieved by manipulating the plant rather than the rhizobia (Taylor et al., 1991). Mineral toxicity (specific ion toxicity) is the most significant characteristics of Soil acidity that are usually accompanied by nutrient deficiency and nutrient disorder. Acidic stress markedly affects ion absorption by and growth of roots; the membrane structure and function of the roots suffer fatal changes under these stress conditions. The requirement of some essential elements, e.g., Ca2+ and P, is increased under severe stress conditions. The requirement of Ca2+ for growth of R. meliloti was increased under osmotic stress. The Ca-depleted cells of R. leguminosarum are swollen, lack rigidity, and express an additional somatic antigen normally blocked by side chains of

the LPS O antigen. High levels of salinity (up to 10% NaCl) along with decreased Ca2+ content of Rhizobium cells, greatly distorted the outer membrane structure of the Rhizobium cells was. In the same way, calcium appears significantly more important in cells exposed to low pH. Calcium plays a vital role in cytoplasmic pH maintenance, phosphorus mobilization and ion transport which are caused mostly by changes in membrane properties, apart from that Ca2+ plays an essential role in cell division, elongation, and membrane structure and function (320). O’Hara et al., (1989) found that in acid-sensitive strains of R. meliloti, 1.2 mM Ca2+ was needed for cytoplasmic pH maintenance It was found that phosphorus- limited cells or cells grown at low pH needed Ca2+ for phosphorus mobilization in the cell. Calcium addition in low pH soil improves both growth and ion uptake by roots and also of fset the harmful effects of ions such as K+ and H+ and control K transport through the control of K+ permeability and activation of K+ uptake through the acidification of the cytoplasm. Table 2: Estimate of the amount of nitrogen fixed by various legumes (FAO, 1984) Plant

Scientific name

Nitrogen fixed (kg N/ha/year)

Horse bean

Viciafaba

45-552

Pigeon pea

Cajanus cajan

168-280

Cowpea

Vigna unguiculata

73-354

Mung bean

Vigna mungo

63-342

Soybean

Glycine max

60-168

Chickpea

Cicer arietinum

103

Lentil

Lens esculenta

88-114

Peanut

Arachis hypogaea

72-124

Pea

Pisum sativum

55-77

Bean

Phaseolus vulgaris

40-70

Leucaena

Leucaena leucocephala

74-584

Alfalfa

Medicago sativa

229-290

Clover

Trifolium spp.

128-207

Phosphorus (P) is one of several elements which affects N2 fixation and it is a principal yield-limiting nutrient in many regions of the world. Strains of Rhizobia differ markedly in tolerance to phosphorus deficiency. Native soil and rhizospheric P deficiency induces Rhizobial P deficiency especially under acidic conditions, where dissolved phosphorus salts may be

precipitated in the presence of aluminum. Slow-growing strains of rhizobia appear more tolerant to low P levels than do fast-growing R. meliloti, in particular (Cooper et al., 1985). Both fast and slow growing Rhizobia are greatly influenced by the phosphorus availability because inducible alkaline phosphatase activity was detected in P-limited cells of fast-growing R. Trifolii strains. Recently, it has been reported that free- living R. tropici and bacteroids respond to P stress by increasing their P transport capacity and inducing both acid and alkali phosphatases. Phosphorus appears essential for both nodulation and N2 fixation. Nodules are strong sinks for P and range in P content from 0.72 to 1.2%; as a consequence, N2 fixation-dependent plants will require more of this element than those supplied with combined nitrogen. Nodulation, N2 fixation, and specific nodule activity are directly related to the P supply. External application of phosphorus (25 mg of P per kg of soil) to acidic soils significantly increased the percent nodule occupancy of Trifolium subterranean by R. leguminosarum bv. trifolii. The nodulation and N2 fixation (nitrogenase activity) of T. vesiculosum increased significantly after the addition of P (100 mg per kg of soil) and K (300 mg per kg of soil); nitrogenase activity was doubled when the P concentration increased to 400 mg per kg of soil. Nitrogen fixation by the Frankia-actinorhizal symbiosis may be limited by low available P in soils. Singleton et al., (1991) observed increased N2 fixation by Rhizobium japonicum by adding phosphate to Pdeficient soil. So, low P status is a frequent limitation to nodulation of actinorhizal plants. It has been reported that symbiotic N2 fixation of the Frankia-Casuarina association requires higher P levels than those required for plant growth, when the P concentration in soil is low. Genetic variations among species of Allocasuarina in relation to P requirement were identified; species showed different nodulation abilities in soils with low P. Mycorrhizal infection of roots of legumes has been reported to stimulate both nodulation and N2 fixation, especially in soils low in available P. The role of mycorrhizal fungi in the protection of the Medicago sativa-R. meliloti symbiosis against salt stress by increased plant available P and generally declined as the salinity in the solution culture increased. The interaction of P and Zn and their effects on nodulation of legumes under salt stress were studied. Saxena and Rewari (1991) found that application of phosphate (20 and 40 ppm) improved the growth and nodulation of chickpea

(C. arietinum) in the presence of Zn2+ (5 ppm) at different levels of salinity. They suggested that augmentation with Zn2+ provided protection to the plant under saline conditions by reducing the Na+/K+ ratio in the shoot; the shoot N content after augmentation with Zn2+ and in the presence of phosphate was equal to that of nonsaline control. Differences between cultivars of some legume species with regard to phosphorus requirements have been reported. Variability of N2 fixation under low P availability existed between lines of P. vulgaris; high N2-fixing and high-yielding progeny lines were detected. Two strategies have been adopted to solve the problem of soil acidity: (i) selecting tolerant plants, and (ii) liming the acidic soil to ameliorate the effects of acidic conditions. It has been suggested that Al-tolerant (acidtolerant) plant species contain and exude more organic acid and other ligands that form stable chelates with Al and thereby reduce its chemical activity and toxicity. Application of lime (at rate of 2,500 kg ha-1) and superphosphate (at rates up to 20 kg ha-1) to acidic soils increased the soil pH from 4.5 to 4.9, decreased the concentration of extractable Al and Mn, and improved growth and N2 fixation of T. subterranean (Almendras and Bottomley, 1987).

4. Phosphorus Fixation The fixation of P by soils has long recognized. Thomas Way in 1850 demonstrated that the whole of phosphate was retained when solution of sodium phosphate in water and guano in dilute H2SO4 were poured over a layer of calcareous soil. He suggested that suggested that an insoluble calcium phosphate was formed resulting decrease in solubility of applied phosphorus. Phosphate fixation or reversion can be viewed as the conversion of soil solution P to insoluble compounds by the soil components, causing reduction in the amount that plant roots can absorbed. Mechanisms involving sorption and precipitation have been suggested to explain in the P fixation in soils. In fact, P fixation is not an ideal adsorption on soil components, a combination of adsorption, chemisorptions and precipitation. Soil solution pH has a significant effect on phosphate fixation. In neutral to alkaline soil (pH 7 and above), phosphate get adsorbed on calcium carbonate and are slowly converted to insoluble apatites. In acid soils (pH below 7) iron and

aluminum react with phosphate to form highly insoluble compounds. In this way, some phosphate of the labile pool is continuously being transferred to the non-labile pool and thus become immobile.

4.1. Chemistry of Phosphorus Fixation in Acid Soils The inorganic P-forms in soil are the compound associated with mainly with aluminium, iron and calcium. Their relative abundance and solubility is controlled by a number of factors including soil pH (Fig. 3). From the different study, it was found that phosphorus is most readily available between pH 6 and 7. The most dominant ionic forms of P in the soil solution are H2PO4 and HPO42- at

Fig. 3: Relationship between solution of pH and the relative concentration of three soluble forms of phosphate neutral range whereas PO43- ion dominates in alkaline condition. The availability of P primarily of H2PO4 and HPO42- ions are highly pH dependent. Its availability in many soils is highest when the pH is neutral or slightly acidic and it declines as soil becomes strongly acidic or strongly alkaline. Therefore in acid soils P is highly susceptible towards fixation and thus rendering it unavailable for plant uptake.

The acid soils are highly dominated by amorphous Fe and Al oxides and their hydroxides which are the potential sites of P adsorption and fixation. Three types of reactions may be considered important in relation to phosphate fixation in soils: i) adsorption ii) isomorphous replacement and iii) double decomposition. Freundlich and Langmuir adsorption isotherms fit well at low concentrations of phosphate in solution whereas the isomorphous replacement of hydroxyl ions with phosphate ions is a possibility. The reaction of Fe and Al hydroxides with the phosphate ions are probably most significant for phosphate fixation in soils. When H2PO4- concentration in soil solution is high it reacts with these minerals forming precipitates of Fe and Al hydroxyl phosphates. Most of the P-fixation occurs in acidic soils, where H2PO4- reacts with the surfaces of insoluble oxides of iron, almonium and manganese, involves series of chemical fixation reactions and thus interlocks the P. Some of these reactions are given as under: A. Precipitation Reaction

B. Anion Exchange Reactions (Outer sphere complex)

C. Reaction with Al and Fe oxide surface (Inner sphere complex)

D. Formation of Stable binuclear bridge (Inner sphere complex)

In reaction (A), freshly formed hydroxyl phosphate is slightly soluble, because of having a greater surface area exposed to the soil solution. Therefore, P in it is available initially to some extent to the plants. With advanced time, the precipitated hydroxyl phosphate ages and become less

soluble and becomes totally unavailable for plants. In reaction (B), phosphate is reversibly adsorbed by anion exchange with broken clay edges of kaolinite clays. In reaction (C), the phosphate ion replaces -OH group in the surface structure of Al oxide minerals and in reaction (D), the phosphate further penetrates the mineral surface by forming a stable binuclear bridge.

4.2. Phosphate Management in Acid Soils The fixation and immobility of P in acid soils of the tropics can be either major problem or blessings in disguise, depending on how soils and P fertilizers are managed. Phosphorus fixation is of ten high in Oxisols and Ultisols because they are most likely to have P-fixing clay minerals (amorphous and crystalline hydrous oxides of Fe and AI), high Fe and Al, and low pH, all of which are conducive to P fixation (Uexkull, 1989). Fox et al., (1989) pointed out that when soil P concentration exceeds a certain level, P uptake by crops will actually be inhibited. Thus, highly concentrated bands of P fertilizer should be avoided. Because of strong interest in rock phosphates for direct application to tree crops in Southeast Asia, one must know that rock phosphates differ significantly in their reactivity. Because of their low cost, high Ca content, and residual effects, rock phosphates are especially well suited for amendment of acid soils poor in P and Ca. Several researchers have successfully attempted to reduce the cost of P fertilization in acid soils by direct use of rock phosphates (Misra and Pattanayak, 1997) to soil (pH < 5.5) or use of rock phosphates and single super phosphate mixture in 1:1 ratio to soil (pH 5.6-6.5) or to apply rock phosphates to green manure crops prior to rice crops taken in sequence or use of compacted products of Jhamarkotra rock phosphates (JPR). Further it has been recommended to apply the entire P requirement of the cropping sequence, particularly for groundnut-rice cropping system in form of rock phosphates to the groundnut crop grown during rabi season and the residual effect be realized through rice crop grown during kharif season. This is because the rock phosphate applied to dry season groundnut gets solubilised to greater extent and the portion that gets fixed during dry season becomes available to rice crop due to soil reduction (Misra and Pattanayak, 1997).

5. Potassium Fixation Since the middle of the 17th century, J R Glauker in Netherlands first proposed that saltpeter (KNO3) was the principle of vegetation, K has been recognized as being beneficial to plant growth (Russell, 1961). The essentiality of k to plant growth has been known since the work of von Liebig published in 1840. of the major nutrient elements, K is usually most abundant in soil (Reitemeier, 1951). A mineral soil generally ranges between 0.04-3% K. Soil K exists in four forms in soils: solution, exchangeable, fixed or nonexchangeable and structural and minerals. Soil solution and exchangeable K level comprises a small portion of the total K. Though non-exchangeable K comprises a significant portion of soil potassium, but most of the soil K present as mineral form (Sparks and Huang, 1985). There are equilibrium and kinetics reaction between four forms of soil K that affect the level of soil solution K at any particular time, and thus, the amount of readily available K for plants. At a given time, potassium in the solution and exchangeable forms constitutes the fraction readily available to plants. The exchangeable K tends to attain equilibrium rapidly with solution K but, slowly with nonexchangeable K (Fig. 4). The solution K concentration largely controls the k movements (diffusion)

Fig. 4: Conceptual model of potassium dynamics in soils and plant roots towards the plant roots and thereby the uptake by plants. On depletion of

exchangeable K, non-exchangeable K replenishes it and supply of K to plants is maintained. When K is added to the soil through mineral fertilizers or organic manures in excess of its crop removal, it initially increases solution K and subsequently, increases the exchangeable and non-exchangeable K though the shifting of the equilibrium (Fig. 4).

5.1. Potassium Fixation Mechanisms in Acid Soil In the processes of potassium fixation in soils, the added soluble K is converted in to a form that cannot be extracted with a neutral salt solution. Potassium fixation occurs when K+ ions form a surface complex with oxygen atoms in the interlayers of certain silicate clay minerals. Potassium fixation processes phenomenon are fixed limited to interlayer ions such as K+ has been explained in terms of good fit of K+ ions (crystalline radius and coordination number are ideal) in an area created by holes and adjacent oxygen layer (Barshad et al., 1951). The important forces involved in interlayer reactions in clays are electrostatic attraction between the negatively charged layers and the positive interlayer ions. The degree of K fixation in clays and soils depends on the type of clay mineral and its charge density, degree of interlayring, the moisture content, the concentration of K+ ions as well as the concentration of competing cations and pH of the ambient solution bathing of clays or soils (Rich, 1968; Spark and Huang, 1985). The major clay minerals responsible for K fixation are montmorillonite, vermiculite and weathered micas. In acid soils principles clay minerals responsible for K fixation is dioctahedral vermiculite. The degree of K fixation is strongly influenced by the charge density on the layer silicate. Those with high charge density fix more potassium than those with low charge density (Walker, 1957). Martin et al., (1946) showed at pH values up to 2.5 there is no fixation; between pH 2.5-5.5, amount of K fixation is very rapidly. Above pH 5.5 fixations increased more slowly. The increase in K fixation between pH can be ascribed to the decreased number of Al(OH)x species which decrease K fixation (Rich, 1964; Rich and Black, 1964). Potassium ions are absorbed by clay minerals on the binding sites which differ in their selectivity (Fig. 5). For 2:1 clay minerals such as illites,

vermiculites and weathered micas, three different absorption sites can be distinguished. These sites are at the planner surface (p-position), at the edges of the layer (e-position) and in the interlayer space (i-position). The specificity of these three binding sites for K differs considerably. The binding selectivity for K by organic matter and clays of the kaolinite type are similar to the p-position sites. Here, the K- bond is relatively weak so that K absorbed may easily be replaced by other cations, particularly by Ca2+ and Mg2+ ions. The i-postion has maximum specificity for K+. These binding sites largely account for K+ fixation in soil.

Fig. 5 : Model of expandable layer silicate structure with interlayers (p, e and i- positios) The potassium added through manures and fertilizers initially increase the solution and exchangeable K contents. The saturation of the exchangeable complex with respect to K leads to the entry of K into wedge (partially open) and interlayer spaces (Fig. 5). This results in fixation of K in nonexchangeable form. According to ‘Lattice Hole Theory’ (Page and Baver, 1940), the exposed surface and surface between sheets of three layer (2:1) type minerals consist of oxygen ions, arranged hexagonally. The opening within the hexagon is equal to the diameter of an oxygen ion (approximately 2.8 Ao). Ion having a diameter of this magnitude (e.g. K+ 2.66 Ao) will fit snugly into the lattice holes and such ions will be very tightly as they come in contact with the negative electrical charges within the crystal. As a result of this, the layers are bound together, thus preventing dehydration and reexpansion. Ions having diameter close to 2.8 Ao can also be fixed to a considerable extent. Relatively small hydration energy of K+ result in easy dehydration of this ions and strong retention. In vermiculite or illite, isomorphous substitution in tetrahedral position of their lattice creates negative charges close to the unit layer surface, which explains the strength

of the bond between K+ and the lattice sheet. Hence, in illite and vermiculite even under wet conditions. Potassium fixation is, therefore, a serious problem in soils containing illite and vermiculite clay minerals dominated in acid soil.

5.2. Factors Influencing Potassium Fixation The degree of K fixation depends on a number of factors, such as 1. Charge density of the minerals which means negative charge per unit silicate layer of the mineral. Potassium fixation is high when density is high. Vermiculite and illite tend fix K best under relatively wet conditions, while fixation by montmorillonite and stratified minerals occurs under dried condition. The fixing power of 2:1 type clay minerals usually follows the order: Vermiculite > Illite >Smectite (momtmorillonite). 2. Depletion of the interlayer wedge zone which means what extent the interlayer wedge zone that is depleted of K. If the wedge zone is confined to the edge of the particle, then only small amounts of K can be fixed. On the other hand if the wedge zone penetrates deeply into the mineral, considerable amount of K can be fixed. 3. Moisture content of the medium plays an important role in K fixation. Wetting and drying cycles leads to fixation of K in soils rich in available K. 4. Concentration of K ion in soil solution also determines the K fixation by the clay minerals. 5. Nature and concentration of competing cations in the surrounding medium influence K fixation. Ions like NH4+ and H+ can compete with K+ for K fixing or binding sites.

5.3. Measurement of Potential K fixation Clear understanding of soil potassium dynamics is very much needed for defining the potential K fixation in soil. In soil K maintain dynamic equilibrium between different potassium pools means the reversible

transformation from one form to other that is assumed to occur in soil (Fig. 4). In some soil, low concentrations of K in solution due to crop removal leads to release of fixed K. In other soil, the K present in interlayer fixation sites may be very slowly released and can be a significant source of plant nutrition. The ammonium acetate extract (1 N NH4OAc, pH 7) is a widely used soil extractant to estimate both soluble and exchangeable K. However this procedure is inadequate for soils that have micaceous or vermiculitic mineralogy, which can release some non-exchangeable (fixed) K when the solution and exchangeable K pools are depleted. An alternative method for measuring nonexchangeable plant-available K in soils (i.e. the plant-available portion of fixed K) is using the sodium tetraphenylboron extraction. A practical version of this procedure involves 5-minute incubation (Cox et al., 1999). They report that in some soils, this procedure extracted 1.5 to 6 times more K than did NH4OAc and closely correlated with plant uptake of K. However, this method did not adequately measure K fixation capacity universally (Murashkina et al., 2007b). The assessment of non-exchangeable K status of soil used to do by using H- resins and 6 N H2SO4 and found good correlation with plant uptake (Srinivasa Rao et al., 2001). The capacity of soils to supply K from exchangeable and non-exchangeable form is not easily determined because K maintains dynamic equilibrium in soil. Due to the complexity that exists in the soil system, none of the methods is universally applicable for all the soils. Haylocks (1965) introduced the terms ‘ Step-K’ and ‘Constant Rate K’ as plant utilizable non-exchangeable potassium reserves in soil. Step-K is the release of potassium with repeated extractions with 1 N HNO3 and the ‘Constant Rate-K’ which is the rate achieved when release of K with every extraction is equal to the previous one. The constant rate K which takes into account soil type and mineralogy may serve as guide to the long-term K supplying power of soils. K fixation can be a significant barrier to meeting the nutritional requirements of crops. In most other soils, K-fixation should not be a significant factor to consider. New laboratory techniques for estimating both the K-fixation potential and the release rate will help with K management decisions.

5.4. Management of Fixed K

The phenomena of both the fixation of available potassium and /or release of fixed or non-exchangeable K play an important role in the dynamics of soil potassium. Lower concentration of K in the solution due to leaching or crop removal favours release of K. when there is no external addition of K, plant are capable of taking up substantial amount of potassium without much change in exchangeable-K pool. This indicates that in due course of time exchangeable- K gets replenished from other sources. Non exchangeable K contributes substantially to potassium availability and uptake in soil rich in micaceous minerals, especially in vermiculite and illite dominating alluvial soils. Under exhaustive cropping, non-exchangeable K contribution to total K uptake can be as high as 90% in alluvial soils. Highly-weathered red and lateritic soils are poor in K supply because of low non-exchangeable K reserves. Some perennial grasses like ryegrass (Lolium perenne) are very efficient user of non-exchangeable K where as legumes like red clover (Trifolium pretense) is not efficient user. Thus consideration of soil characteristic and plant type is very important for efficient utilization of fixed (non-exchangeable) K in soil. As result, K fertilization rate to be applied get reduced in proportion to the amount of non-exchangeable potassium in soil.

References Aarons, S. R. and Graham, P. H. 1991. Response of Rhizobium leguminosarum bv. phaseoli to acidity. Plant Soil 134:145-151. Almendras, A. S. and Bottomley, P. J. 1987. Influence of lime and phosphate on nodulation of soilgrown Trifolium subterraneum L. by indigenous Rhizobium trifolii. Appl. Environ. Microbiol. 53:2090-2097. Barshad, I. 1951. Cation exchange in soils.I. Ammonia fixation and its relation to potassium and to determination of ammonia exchange capacity. Soil Sci. 77:463-472. Bordeleau, L. M. and Prevost. D. 1994. Nodulation and nitrogen fixation in extreme environments. Plant Soil 161:115-124. Brockwell, J., Pilka, A. and Holliday. R. A. 1991. Soil pH is a major determinant of the numbers of naturally-occurring Rhizobium meliloti in non-cultivated soils of New South Wales. Aust. J. Exp. Agric. 31:211-219. Carter, J. M., Gardner, W. K. and Gibson, A. H. 1994. Improved growth and yield of faba beans (Vicia faba cv. Fiord) by inoculation with strains of Rhizobium leguminosarum biovar viciae in acid soils in south-west Victoria. Aust. J. Agric. Res. 45:613-623. Cooper, J. E., Wood, M. and Bjourson, A. J. 1985. Nodulation of Lotus pedunculatus in acid rooting solution by fast-and slow-growing rhizobia. Soil Biol. Biochem. 17:487-492.

Correa, O. S. and Barneix, A. J. 1997. Cellular mechanisms of pH tolerance in Rhizobium loti. World J. Microbiol. Biotechnol. 13:153-157. Cox, A.E., Joern, B.C., Brouder, S.M. and Gao, D. 1999. Plant-available potassium assessment with a modiu ed sodium tetraphenylboron method. Soil Sci. Soc. Am. J. 63:902-911. Fox, R.L., Bosshart, R.P, Sompongse, D. and Lin, Mu-Lien. 1989. Phosphorus requirements and management of sugarcane, pineapple, and bananas. For presentation at the symposium on Phosphorus Requirements for Sustainable agriculture in Asia and the Pacific Region, 6-10 March 1989, IRRI, Los Banos, Philippines. Graham, P. H., Viteri, S. E., Mackie, F., Vargas, A. T. and A. Palacios, A. 1982. Variation in acid soil tolerance among strains of Rhizobium phaseoli. Field Crops Res. 5:121-128. Haylocks, O. F. 1956. A method of estimating the availability of non-exchangeable potassium. Transaction of 6th International Congress Soil Science Paris 2: 403-408. Ibekwe, A. M., Angle, J. S., Chaney, R. L. and Vonberkum, P. 1997. Enumeration and nitrogen fixation potential of Rhizobium leguminosarum biovar trifolii grown in soil with varying pH values and heavy metal concentrations. Agric. Ecosyst. Environ. 61:103-111. Martin, J. C. Overstreet, R. and Hoagland. 1946. Potassium fixation in soils in replaceable and non replaceable form in relation to chemical reaction in soil. Soil Sci. Soc. of Am. Proceedings. 10:94-101. Misra, U. K. and Pattanayak, S. K. 1997. Characterization of rock phosphates for direct use in different cropping sequences. Technical Report of the U.S. India Fund. Project number In: AES-708. Grant No. F61- IN-744, 1991-1995. Murashkina, M. A., Southard, R. J. and Pettygrove, G. S. 2007. Potassium Fixation in San Joaquin Valley Soils Derived from Granitic and Nongranitic Alluvium. Soil Sci. Soc. Am. J. 71:125-132. O’Hara, G W., Goss, T. J., Dilworth, M. J. and Glenn. A. R. 1989. Maintenance of intracellular pH and acid tolerance in Rhizobium meliloti. Appl. Environ. Microbiol. 55:1870-1876. Page, J. B. and Baver, L. D. 1940. Ionic size in relation to fixation of cations by colloidal clay. Soil Sci. Soc. of Am. Proceedings 4:150-1555 Postgate, J. 1981. Microbiology of the free-living nitrogenfixing bacteria, excluding cyanobacteria. In: Gibson AH, Newton WE (eds) Current perspectives in nitrogen fixation. Elsevier/ North-Holland Biomedical, Amsterdam,pp 217-228. Reitemeier, R. F. 1951. The chemistry of soil potassium. Advances in Agronomy. 3:113-164 Rich, C. I. 1964. Effect of cation size and pH potassium exchange in Nason soil. Soil Sci. 98:100-106. Rich, C. I. 1968. Minerology of soil potassium.p 79-91. (In) V.J. Kilmer etal. (Ed.) The role of potassium in agriculture. American Society of Agronomy, Madison, WI. Rich, C. I. and Black, R. W. 1964. Potassium exchange as affected by cation size, pH and mineral structure. Soil Sci. 97: 384-390. Saxena, A. K., and Rewari, R. B. 1991. The influence of phosphate and zinc on growth, nodulation and mineral composition of chickpea (Cicer arietinum l.) under salt stress. World J. Microbiol. Biotechnol. 7:202-205. Singleton, P. W., Abel Magid, H. M. and Tavares. J. W. 1985. Effect of phosphorus on the effectiveness of strains of Rhizobium japonicum. Soil Sci. Soc. Am. J. 49:613-616. Sparks, D. L. and Huang, P M. 1985. Physical chemistry of soil potassium. p. 201-276. In R.D. Munson (ed.) Potassium in agriculture. American Society of Agronomy, Madison, WI. Srinivasa Rao, Ch., Rupa, T. R. and Subba Rao, A. 2001. Sub-soil potassium availability in 22 benchmark soils of India. Commun. Soil Sci. Plant Anal. 32:863-876.

Taylor, R. W., Williams, M. L. and Sistani, K. R. 1991. Nitrogen fixation by soybean- Bradyrhizobium combinations under acidity, low P and high Al stresses. Plant Soil 131:293-300. Thomas, R. J., Askawa, N. M., Rondon, M. A. and Alarcon, H. F. 1997. Nitrogen fixation by three tropical forage legumes in an acid soil savanna of Colombia. Soil Biol. Biochem. 29:801-808. Torimitsu, K., Hayashi, M., Ohta, E and Sakata, M. 1985. Effect of K+ and H+ stress and role of Ca2+ in the regulation of intracellular K+ concentration in mung bean roots. Physiol. Plant. 63:247-252. Uexkull, H. R. and Bosshart, R.P. 1989 Management of Acid Upland Soils in Asia. In Craswell, E.T., and Pushparajah, E. eds. Management of Acid Soils in the Humid Tropics of Asia. ACIAR Monograph No. 13 (m SRAM Monograph No.1), Australian Centre for International Agricultural Research, Canberra, pp 2-19. Walker, G. F. 1957. On the differentiation of vermiculite and smectites in clay. Clay Miner. Bull. 3:154-163.

CHAPTER 5

Physiology and Biochemistry of Fruit RipeningK.S. Shivashankara, K.C. Pavithra, A. Nethravath “Science without religion is lame,religion without science is blind”- Albert Einstein Fruit ripening involves physiological and biochemical changes in the tissues of fruit which are integrated and lead to the production of suitable ripe fruit. Fruit ripening is a developmental process that is exclusive to plants whereby mature seed-bearing organs undergo physiological and metabolic changes that promote seed dispersal (Giovannoni 2007). The fruit ripening process has been noticed over the last decades as being successively of physiological, biochemical, and molecular nature. During ripening process, chlorophyll is degraded and the synthesis of new pigments like carotenoids takes place. Fruit softening and textural changes lead to the fruit cell wall changes in carbohydrate composition, resulting in sugar accumulation and increased sweetness. Formation of aroma volatiles and accumulation of organic acids with also takes place (Alexander and Grierson 2002; Prasanna et al. 2007). Fruits with different ripening mechanisms divided into two groups; climacteric in which ripening is accompanied by a peak in respiration along with a burst of ethylene, and non-climacteric in which respiration shows no dramatic change and ethylene production remains at a very low level. Climacteric fruits such as tomato, banana, apple, mango, papaya, sapota guava and kiwi display a peak in respiration with a concomitant burst of ethylene at the onset of ripening (Alexander and Grierson 2002, Giovannoni, 2004; Kondo et al. 2009; Atkinson et al. 2011; Xu et al. 2012). In contrast, non-climacteric fruits such as pepper, pineapple, pomegranate, strawberry,

melon, citrus, cherry and grape, do not show a dramatic change in respiration, and ethylene production remains at a basal level (Bapat et al. 2010; Chai et al. 2011; Symons et al. 2012). In fact, non-climacteric fruits show decline in their respiration rate and ethylene production throughout the ripening process. Ethylene production is closely associated with fruit ripening and is the plant hormone that regulates and coordinates the different aspects of the ripening process; colour development, aroma production and texture are all under the control of ethylene (Payasi and Sanwal 2005). Different phases of fruit ripening are summarized in M Fig. 1. Phase 1 and 2 respond to the exogenous application of ethylene and the autocatalytic ethylene response starts from the phase 3. At harvest, fruit do not produce ethylene but are highly sensitive to exogenous ethylene. softening is initiated (phase 1) and becomes rapid (phase 2). Relatively late in softening, compared with other fruit species, endogenous autocatalytic ethylene production begins, aroma volatiles are produced and fruit become sof t enough to eat (phase 3). If fruit progress to the over-ripe stage (phase 4), they become unacceptably sof t and exhibit ‘of f-flavour’ notes.

Fig. 1: Different phases of fruit ripening controlled by ethylene in kiwi fruit (Atkinson et al. 2011)

Table 1. Climacteric and non-Climacteric fruits (Watkins 2002)

Many ripening related biochemical pathways involved in pigmentation, cell wall metabolism, carbohydrate metabolism, aroma biosynthesis are triggered and controlled by ethylene and its signal transduction mechanisms (Deikman 1997; Ciardi and Klee 2001). All biochemical and physiological changes that take place during fruit ripening are driven by the coordinated expression of fruit ripening-related genes. The regulatory proteins mainly control the signal transduction pathways and participate at the transcription regulation machinery during fruit ripening (Bouzayen et al. 2010). Differential expression of genes has also been reported during ripening in fruits (Deikman 1997). This chapter focuses mainly on the physiological, biochemical changes during fruit ripening and molecular regulators associated with fruit ripening.

Physiological and Biochemical changes during fruit ripening Fruit ripening is a highly coordinated, developmentally programmed

irreversible phenomenon involving a series of physiological and biochemical changes that lead to the development of a sof t and edible ripe fruit with desirable quality. The physiological and biochemical changes include changes in respiration, ethylene production, pigment synthesis, chlorophyll degradation, and development of flavor and aroma components, starch hydrolysis, changes in sugars, organic acids, and cell wall softening by the changes in the activity of various enzymes (Brady

Fig. 2: Schematic diagram of simplified fruit ripening processes. PM Plasma membrane, ACO - ACC oxidase, ACS - ACC synthase, ETR Ethylene receptors, CTR - Constitutive triple response, EIN - Ethylene inhibitor, EIL - Ethylene inhibitor like protein, ERF - Ethylene response factors 1987) . There are many reviews on physiology and biochemistry of fruit ripening including the molecular basis of the ripening process (Brummell et al. 1999; Prasanna et al. 2007; Karlova et al. 2014). Starting point in fruit ripening is the developmentally regulated change in ethylene production and raise in respiration in climacteric fruits. Simplified representation of ripening processes is given in the Fig. 2. Ethylene synthesis starts from the amino acid methionine and controlled by ACC synthase and ACC oxidase enzymes (Fig. 3). Ethylene is bound to

ethylene receptors and initiates the signal transduction process ultimately leading to the expression of an array of genes involved in the fruit ripening. Synthesis of ethylene from ACC is an oxidation reaction therefore requires oxygen and releases cyanide. The release of cyanide may trigger the alternative oxidase respiration system. Therefore in fruits where ethylene climacteric is high, the alternate oxidase respiration may also be more. Ethylene biosynthesis pathway

Fig. 3: Ethylene biosynthetic pathway showing the rate limiting steps

Changes in Fruit texture

Fruit softening is an important part of the ripening process and involves changes in primary cell wall structure. Change in fruit firmness during different phases of fruit ripening is indicated in Fig. 1. Most of the polysaccharide components of the cell wall are subjected to degradation, resulting in loosening and swelling of the wall structure, a weakening of cell wall strength (Brummell 2006; Prasanna et al. 2007). Fruit softening appears to involve the actions of ripening- related expansin (Brummell et al. 1999) and a-galactosidase (Smith et al. 2002) in the beginning and the solubilization and depolymerization of pectin mediated by polygalacturonase (PG) at the later stages of cell fruit softening (Smith et al.1990). During ripening, coordinated degradation of hemicelluloses and pectin takes place (Brummel et al. 2004). Degradative action of polygalacturonase (PG) on cell walls plays a major role in fruit softening. PG depolymerises the pectic acid and releases galactose. Early ripening changes involve the degradation of the hemicelluloses like galactan side chains of rhamnogalacturonan-I, demethyl esterification of homogalacturonan and depolymerisation of matrix glycans (hemicelluloses). Solubilisation of pectins increases during ripening, but depolymerisation of pectins is usually most pronounced late in ripening (Brummell 2006). Major cell wall solubilising enzymes can be grouped into 2 categories like pectinases and hemicellulases. Pectinases include polygalacturonases, pectinesterases, a-galactosidase/galactanase also, agalactosidase, rhamnogalacturonase and pectic lyse. Hemicellulases includes endo-(1,4)-b- glucanases, xyloglucanases and xyloglucan endotransglycosylases. All fruits contain the respective enzyme classes, but the relative abundance and time of expression and the type of genes may vary with the species of fruits. Expansins are the proteins that induce cell wall extension during growth of the plants and are also involved in the initial cell wall loosening during fruit ripening. They are reported to disrupt the non-covalent interactions between hemicellulose and cellulose microf ibrils. The identification of a ripeningregulated expansin gene in tomato and other fruit suggests that in addition to their role in facilitating the expansion of plant cells, expansins may also contribute to cell wall disassembly in non-growing tissues possibly by enhancing the convenience of non-covalently bound polymers to endogenous enzymatic action (Rose et al. 1998). Transgenic studies have shown that expansion may control cell wall loosening and a- galactosidase is an important enzyme in increasing cell wall porosity. Suppression of either of

these enzymes resulted in retention of fruit firmness. Suppression of endopolygalacturonase and pectin methylesterase had little effect on fruit firmness during ripening, but influenced fruit shelf life due to alterations in the integrity of the middle lamella, which affected intercellular adhesion (Brummell 2006). Texture changes are one of the first indications of developmental stage regulated initiation of ripening process. Further fruit firmness can also be due to better maintenance of cell turgor by the fruit cuticle layer and surface waxes. Fruit firmness is also determined by a number of other factors such as cuticle properties, turgor, and free radicals (Vicente et al. 2007; Handa et al. 2011). More than 700 genes in tomato genome sequence showed cell-wall-related functions and more than 50 of these genes show differential expression during fruit ripening and to encode proteins involved in the modification of cell-wall properties (Seymour et al. 2013a). The recent characterization of a ‘Delayed Fruit Deterioration’ (DFD) tomato cultivar by Saladie et al. (2007) revealed the association between cuticle, shelf-life, and fruit firmness, as these fruits showed prolonged resistance to postharvest

Fig. 4 : Flow chart of the processes and the enzymes involved in cell wall hydrolysis during ripening processes desiccation unlike normal fruits. Although the contributions of cutin and waxes to limiting water loss are not well understood, analysis of the DFD fruit cuticle revealed substantial differences in the amount of cutin and waxes (Saladie et al. 2007). Therefore the firmness of fruits during ripening is controlled by cell wall hydrolyzing enzymes like pectinases, hemicellulases, wax and cutin synthetic enzymes. All these are the control of ethylene signaling pathways. The sequence of events leading to the cell wall degradation is given in Fig. 4.

Colour development during ripening Fruit colour change is another indication of initiation of ripening process. Colour change is due to the synthesis and accumulation of carotenoids or anthocyanins in the skin and also in the flesh of fruits during ripening process (Bouzayen et al. 2010). Disappearance of green colour is due to the degradation of chlorophyll and disassembling of the photosynthetic apparatus (Senthilkumar and Vijayakumar 2014). The thylakoid disassembly of photosynthetic pigments leading to chlorophyll degradation was reported as one of the earliest changes associated with the onset of fruit ripening in many fruits including tomato (Bramley 2002; Grassi et al. 2013). Increase in carotenoids is one of the most noticeable characteristics of ripening (Laval-Martin et al. 1975). The change in pigmentation is caused by a massive accumulation of lycopene within the plastids in tomato, a-carotene, xanthophylls and anthocyanins in mangoes. The chloroplasts of the mature green fruit change into chromoplasts during ripening, which accumulate carotenoids in membrane- bound crystals. However in anthocyanin accumulating fruits pigments accumulate in vacuole. Tomato mutant studies are the basis for understanding the desaturase pathway from phytoene to lycopene (Porter and Spurgeon 1979). Conversion of chloroplasts into chromoplasts is dependent on the ethylene signaling leading to the production of chlorophyllase for the destruction of chlorophylls and the expression of carotenoid biosynthetic enzymes in the chloroplasts. Chlorophyll breakdown is an important catabolic process of leaf senescence and fruit ripening. Structure elucidation of colorless linear tetrapyrroles as (final) breakdown products of chlorophyll was crucial for the recent delineation of a chlorophyll breakdown pathway which is highly conserved in land plants. Pheophorbide-a oxygenase is the key enzyme responsible for opening of the chlorin macrocycle of pheophorbide a characteristic to all further breakdown products. Degradation of chlorophyll was rationalized by the need of a senescing cell to detoxify the potentially phototoxic pigment, yet recent investigations in leaves and fruits indicate that chlorophyll catabolites could have physiological roles (Hortensteiner and Krautler 2011). In fruits treated with 1-MCP (ethylene action inhibitor) the expression of chlorophyll degradation associated genes: pheophorbide a oxygenase (PAO),

non-yellow colouring (NYC), NYC1-like (NOL), stay-green 1(SGR1), was suppressed, while no significant change was found in chlorophyllase 1 (CHL1) and red chlorophyll catabolite reductase (RCCR). These results suggest that ethylene triggers the degradation of chlorophyll by inducing the gene expression of PAO, NYC, NOL and SGR1, which are closely associated with chlorophyll catabolic pathway (Cheng etal. 2012). Ethylene is found to induce the expression of Phytoene synthase-1 and Phytoene desaturase genes but the expression of s&carotene desaturase is independent of ethylene (Marty et al. 2005).

Starch and Sugar metabolism during fruit ripening Climacteric fruits show considerable changes in sugar content during fruit ripening. The main changes involved during ripening are the changes in starch and sucrose into glucose. The accumulation of soluble sugars during fruit ripening largely determines their sweetness at harvest (Zhu et al. 2003). Degradation and synthesis of sucrose in the cytosol and in the vacuole are predominant for sugar metabolism and accumulation. Sucrose in the cytosol is converted to fructose and glucose by neutral invertase (NI), or to fructose and UDP-glucose (UDPG) by sucrose synthase (SS). The fructose and glucose are then phosphorylated to fructose 6-phosphate (F6P) and glucose 6phosphate (G6P) by fructokinase (FK) and hexokinase (HK). The F6P enters glycolysis and the TCA cycle to generate energy and intermediates for other processes. Sucrose can also be re-synthesized via either sucrose synthase or sucrose phosphate synthase (SPS) (Li et al. 2012). Sucrose in the cytosol is transferred to the vacuole, where it is hydrolyzed by vacuolar invertase (VI). Hexoses produced in the vacuole can also be transported to the cytosol for subsequent metabolism or sucrose re-synthesis (Zhu et al. 2003). From the starch measurement studies in strawberry it was estimated that only 2.5% of total sugars come from starch hydrolysis and ADP-glucose pyrophosphorylase and fructokinase proteins are the important key enzymes of carbohydrate metabolism present at all the stages of fruit development. Sucrose synthase activity increased upto maturity and later declined as ripening progressed (Souleyre et al. 2005). The conversion of starch to sugars

is the most remarkable chemical change occurring in banana pulp during ripening. Initially, sucrose is the predominant sugar and hexose sugars appear at the later stages of ripening and ultimately exceed sucrose concentration (Payasi and Sanwal 2005). Mowlah and Itoo (1982) showed that glucose, fructose and sucrose are the main sugars in guavas. The level of fructose increased during guava fruit ripening and then decreased. Increased amylase activity was reported during ripening in mango (Sen et al. 1985). De Godoy et al. (2009) evaluated the changes in gene expression during ripening in banana and mango fruits. Results revealed that in starch metabolism, banana a-amylase was induced during ripening while phosphorylase was more induced in mangoes. Fructan fructosyl transferase, chalcone synthase, and ascorbate oxidase genes were also induced in ripening mangoes, but not in bananas. Medlicott and Thompson (1985) reported that Glucose, fructose and sucrose sugars content increased during ripening in mango fruit. Sucrose was found to be in the greatest concentration throughout followed by fructose. In papaya sucrose phosphate synthase and sucrose synthase were not related to the sugar accumulation during ripening but it was strongly related to the significant increase in the activity of acid invertase (Zhou and Paull 2001). In tomato, the dominant soluble metabolites are sugars and organic acids (Roessner-Tunali et al. 2003). Starch is a transitory carbohydrate reserve, accumulating during early fruit development and then declining to undetectable levels in the ripe fruits. Many fruits accumulate organic acids such as citric, malic, and ascorbic acids during ripening. Among organic acids, malate and citrate are the main acids in many climacteric and nonclimacteric fruits (Osorio et al. 2012; Merchante et al. 2013). A positive correlation between malate levels and the genes involved in starch synthesis was observed in pepper, suggesting a possible conservation of the transitory starch metabolism between climacteric and non-climacteric fruits (Osorio et al. 2012). Fruit ripening is characterized by the sugar acid ratios and was mainly due to the conversion of starch to sugars in starch accumulating fruits and in acid accumulating fruits the sugar accumulation is mainly by the gluconeogenesis where acids are converted to sugars. Starch hydrolysis starts with the climacteric raise in ethylene with the induction of a and a-amylases. In organic acid accumulating fruits citric and malic acids are the major acids.

Flavour and aroma biogenesis during fruit ripening Flavour of fruits is the combination of taste and aroma. Fruit volatile compounds are mainly comprised of esters, alcohols, aldehydes, ketones, lactones, terpenoids and apocarotenoids. Many factors affect volatile composition, including the genetic makeup, degree of maturity, environmental conditions, postharvest handling and storage. There are several pathways involved in volatile biosynthesis starting from lipids, amino acids, terpenoids and carotenoids. Once the basic skeletons are produced via these pathways, the diversity of volatiles is achieved via additional modification reactions such as acylation, methylation, oxidation/ reduction and cyclic ring closure. The changes in the composition of different chemical constituents and organic acids during fruit ripening play a key role in flavor development and can affect the chemical and sensory characteristics such as pH, total acidity, microbial stability and sweetness (Bouzayen et al. 2010). Aroma volatiles contribute strongly to the overall sensory quality of fruit. Aroma is generally a complex mixture of a wide range of compounds. Each product has a distinctive aroma, which is function of the proportion of the key volatiles, and the presence or absence of unique components. Aroma is the compounds derived from lipids, sugars, and amino acids. Ethylene is known to control the rate of ripening, the duration of storage life, and most of the ripening events in climacteric fruit. Therefore, breeders have incidentally reduced ethylene synthesis or action by generating genotypes with extended shelf life. Because many genes of aroma biosynthesis are ethylene-regulated (Manriquez et al. 2006), this has of ten resulted in a severe loss of flavor in long-keeping genotypes that have commonly been generated by breeding with non-ripening mutants (Bouzayen et al. 2010).

β-Oxidation of fatty acids Earlier studies have reported that the a-oxidation of fatty acids is the primary biosynthetic process that provides alcohols and acyl co-enzyme A (CoA) for

ester formation. Acyl CoAs are reduced by acyl CoA reductase to aldehydes, which are in turn reduced by the alcohol dehydrogenase (ADH) enzyme to form alcohols that are converted to esters via the action of alcohol acyltransferase (AAT) enzyme (Defilippi et al. 2005). The last step in the biosynthesis of volatile esters is catalysed by alcohol acyltransferase (AAT), a key enzyme in aroma biochemistry (Fellman et al. 2000). This enzyme catalyses the esterification of an acyl moiety from acyl-CoA into an alcohol. The formation of a broad range of esters in the different types of fruit results from the combination of different alcohols and acyl-CoAs (Schwab 2003). The expression of AAT always increased in these fruits throughout ripening and after harvest correlating with the total content of esters, thus suggesting that this gene family is responsible for the production of important esters related to ripe fruit aroma during ripening (Cumplido-Laso et al. 2012). Fatty acid-derived straight chain alcohols, aldehydes, ketones, acids, esters and lactones ranging from C1 to C20 are important character-impact aroma compounds that are responsible for fresh fruit flavors with high concentrations, and are basically formed by three processes: β-oxidation, βoxidation and the lipoxygenase pathway. Many of the aliphatic esters, alcohols, acids, and carbonyls found in fruits are derived from the oxidative degradation of linoleic and linolenic acids. Hexanal and 2,4-decadienal are the primary oxidation products of linolenic acid, while autoxidation of linolenic acid produces 2,4-heptadienal as the major product. Further autoxidation of these aldehydes leads to the formation of other volatile products. Acyl CoAs which are produced by β-oxidation of fatty acids are reduced by acyl CoA reductase to aldehyde that in turn is reduced by Alcohol dehydrogenase (ADH) to alcohol for use by AAT to produce esters. Pear and apple aromas have been two classical examples of volatile formation through the β-oxidation pathway (Paillard, 1990)

Lipoxygenase pathway Saturated and unsaturated volatile C6 and C9 aldehydes and alcohols are important contributors to the characteristic flavors of fruits, vegetables and green leaves. LOX pathway is involved in the biosynthesis of many aldehydes, alcohols and also esters as given in the Fig. 5.

Fig. 5 : Linolenic acid-derived flavor molecules. AAT, alcohol acyl CoA transferase; ADH, alcohol dehydrogenase; AER, alkenal oxidoreductase; AOC, allene oxide cyclase; AOS, allene oxide synthase; HPL, hydroperoxide lyase; JMT, jasmonate methyltransferase; LOX, Lipoxygenase; OPR, 12oxo-phytodienoic acid reductase; 3Z,2E-EI, 3Z,2E-enal isomerase (El Hadi et al, 2013) Amino acids are also involved in the aroma biosynthesis where branched esters are present. Branched chain volatile alcohols, aldehydes and esters in fruits such as banana, apple, strawberry and tomato arise from the branched chain amino acids leucine, isoleucine and valine. It is reported that isoleucine could be involved in the synthesis of 3-methylbutanol and 2-methylbutyrylCoA, both are used in the synthesis of ester 3-methylbutyl 2-methylbutanoate in banana (Perez et al, 1992). Methionine a sulphur containing amino acid is the precursor for the synthesis of sulphur-containing volatiles such as dimethyldisulfide and volatile thioesters. In strawberry, it has been suggested that alanine serves as a precursor for volatile ethyl esters, which can be produced by AAT.

Fig. 6 : Amino acid involvement is the biosynthesis of volatiles (El Hadi et al, 2013)

Terpenoid pathway The terpenoids compose the largest class of plant secondary metabolites with many volatilerepresentatives. Hemiterpenes (C5), monoterpenes (C10), sesquiterpenes (C15), homoterpenes (C11 and C16), and some diterpenes (C20). The terpenoids pathway is given in Fig. 6.

Fig. 7: Terpenoid thaway in the plantcell. DMAPP, dimethylallyl diphosphate; DXP, 1- deoxy-D-xylulose-5-phosphate; DXS, DXP synthase; DXR, DXP reductoisomerase; FPP, farnesyl diphosphate; G3P, glyceraldehyde 3-phosphate; GPP, geranyl diphosphate; GGPP, geranyl geranyl diphosphate; HMGR, HMG-CoA reductase; IPP, isopentenyl diphosphate; MEP, methylerythritol phosphate; MVA, mevalonic acid (El Hadi et al, 2013)

Role of Growth regulators in fruit ripening Ethylene is the plant hormone regulating fruit ripening by coordinating the expression of genes. The sharp increase in climacteric ethylene production at the onset of ripening is considered to be the initiator of changes in colour, texture, aroma and flavour, and other biochemical and physiological attributes. In climacteric fruit tissues, ethylene biosynthesis proceeds at a low level during development, but at the onset of ripening it becomes autocatalytic. For many years, ethylene has been shown to be necessary for the initiation of ripening in climacteric fruits and other plant hormones including auxin are involved in the control of ripening in non-climacteric forms (Karlova et al. 2014).

Ethylene One way to extend shelf life of fruit is to delay or reduce ethylene production. Transformed fruits with down-regulated enzymes of the ethylene biosynthesis pathway exhibited extended shelf life (Baldwin et al. 2000). Genes encoding cell wall-degrading, ethylene production, and pigment biosynthesis enzymes were among the first ethylene-responsive genes to be isolated from tomato fruit. Later, a set of early ethylene-regulated genes were isolated from mature green tomatoes that are responsive to exogenous ethylene, but not yet producing elevated levels of ripening-associated ethylene (Bouzayen et al. 2010). Molecular basis of ethylene perception, binding and signal transduction leading to gene expression of ripening related genes was possible due to large number of mutants available in tomato. In a sense tomato is the model plant for climacteric fruit ripening processes. In the signal transduction pathway of ethylene after binding to ethylene receptor (ETR), many transcription regulators like CTR (constitutive triple response), EIN2, EIN3 (ethylene inhibitor), RIN (ripening inhibitor), NR (never ripe), NOR (Non-ripening), GR (green ripe) and ERF (ethylene response factors) are all involved. ERFs transfer the signal into expression of various genes involved in the ripening processes in tomato. EIN2 (ethylene inhibitor) expression increases at the onset of ripening. EIN2 then activates EIN3 encoded by a family of four EIN3-like genes in tomato. The antisense suppression of tomato EIL1, EIL2 and EIL3 reduces ethylene sensitivity (Gapper et al. 2014). Ethylene action inhibitor, 1-methylcyclopropene (1MCP), delayed ethylene production in tomato. 1-MCP treatment also decreased the three ripening-related tomato genes phytoene synthase-1 (P50 cell wall structure related genes are differentially expressed during fruit development. Changes occur in the structure of all the major cell wall polysaccharides with differences in the nature of wall modifications depending on the tissue (Karlova et al. 2014). However, in all cases, downregulating a single gene, or in some cases two of these genes had only a very limited effect on texture changes. Overexpression of the XTH proteins in transgenic tomato increased Xyloglucan xyloglucosyltransferase/endohydrolase (XET) activity that was responsible for the lower xyloglucan depolymerization. Fruit softening, during ripening, was lower in the transgenic tomatoes, indicating that the xyloglucan structure is related with the softening mechanism and that XET is one of the enzymes involved in the process. XET maintained structural

integrity of the cell wall and the decrease in activity during ripening might contribute to the fruit softening (Miedes et al. 2010). Transgenic melon with reduced expression of the 1- aminocyclopropane-1-carboxylate oxidase (ACO) gene showed a delayed but significant decrease in flesh firmness during ripening (Ayub et al. 1996). The application of a highly potent inhibitor of ethylene perception, 1- methylcyclopropene (1-MCP) after the onset of ripening restricted softening and prevented the accumulation of PG mRNA and endo-PG activity in pear fruit (Hiwasa et al. 2003). Overexpression of SlPG gene in transgenic tomato fruit showed slightly reduced softening with increased shelf life (Langley et al. 1994). The role of the ripening-specific expansin Exp1 protein in fruit softening and cell wall metabolism was investigated by suppression and overexpression of Exp1 in transgenic tomato plants. These data are consistent with there being at least three components to fruit softening and textural changes. One component is a relaxation of the wall directly mediated by Exp1, which indirectly limits part of a second component due to polyuronide depolymerization late in ripening, perhaps by controlling access of a pectinase to its substrate. The third component is caused by depolymerization of hemicelluloses, which occurs independently of or requires only very small amounts of Exp1 protein (Brummell et al. 1999).

Colour and pigment accumulation Carotenoid biosynthesis and its regulation during tomato fruit ripening is a complex process that occurs along with the differentiation of chloroplasts into chromoplasts and changes to the biochemical properties of the fruit. The ripe tomato fruit accumulates large amounts of lycopene as the pattern of gene expression found in green fruit changes during fruit ripening. The control of gene expression is thought to be the main regulatory mechanism for these alterations in carotenoids (Bramley 2002). Carotenoid formation during tomato fruit ripening has been studied extensively and has become the best model system for other chromoplastcontaining tissues. During ripening, the concentration of carotenoids increases mainly due to the accumulation of lycopene. Higher expression of isoprenoid genes in the central pathway has beenfound at the stage of fruit

ripening especially DOXP synthase (Lois etal. 2000). This DOXP pathway may be crucial in the overall regulation of lycopene formation in tomato fruit. At this same stage, mRNA levels of Psy- 1 and Pds increase significantly. At the same time, the mRNAs of both lycopene cyclases (Lcy-β and Lcy-ε) disappear (Ronen et al. 1999). These changes in gene expression show that transcriptional regulation is involved in the accumulation of lycopene in tomato fruit. Differential gene expression has also been implicated in the formation of β-carotene rather than lycopene is due to the up-regulation of the Cyc-β gene (Bramley 2002). Understanding of pigment composition of fruits is of high commercial importance, and considerable advances have been made in defining and understanding the metabolic pathways underlying their regulation (Carrari et al. 2006). Liu et al. (2003) said that in recent QTL and positional mapping study, not all the genes that are responsible for the accumulation of pigments in tomato have yet been identified. Moreover, there have been few studies to attempt to incorporate changes in primary metabolism to those in pigment composition. The primary metabolites correlate strongly with pigment contents; it also shows that several organic acids display considerable correlation with many of the same transcripts. This result is particularly interesting given the fact that several tomato genotypes deficient in TCAcycle function exhibit elevated pigment content providing support that this approach can be readily utilized as a means of identifying candidate genes for biotechnology. β-Carotene in tomato fruit has been increased by various genetic modifications. Constitutive expression of the Erwinia crtI gene (phytoene desaturase) caused a 3-fold elevation in p-carotene, but an unexpected reduction in the total carotenoid levels (Romer et al. 2000). Giuliano et al. (2000) reported that gene expression studies showed that the endogenous lycopene cyclases were up- regulated in the transformants, thus causing the formation of β-carotene rather than lycopene as had been predicted. Bramley (2002) achieved increases in the β-carotene levels of tomato fruit by following transformation with the native Lcy-b coupled to the tomato Pds promoter but greater increase were obtained with the Cyc-b gene (chromoplast lycopene cyclase). The expression of the geranylgeranyl pyrophosphate synthase gene is demonstrated to be strongly induced during the chloroplast to chromoplast transition which occurs in ripening fruits and

is correlated with an increase in enzyme activity during ripening (Kuntz et al. 1992). Hua et al. (2012) concluded that MhCTR1 expression was detected mainly in the pulps at ripening stage and correlated with the onset of peel yellowing, while MhCTR1 gene was constitutively expressed in the peels of banana fruit. Analysis of the transcriptome of transgenic lines overexpressing the tomato APPR2-Like gene isolated from Arabidopsis revealed upregulation of several ripening-related genes in the overexpression lines providing a link between the expression of this tomato gene and the ripening process. APPR2- Like gene in tomato was associated with pigment accumulation and has an important role in ripening (Pan et al. 2013). The accumulation of lycopene in tomato fruits is apparently due to a downregulation of the lycopene cyclase gene that occurs at the breaker stage of fruit development. This conclusion supports the hypothesis that transcriptional regulation of gene expression is a predominant mechanism of regulating carotenogenesis. Up regulation of lycopene cylcases or phytoene desaturases increased the content of ^-carotene in tomato (Romer et al. 2000). Phytoene synthase-1 gene is also involved in the up regulation of lycopene cyclase leading to the higher level of β-carotene in tomato fruits (Romer et al. 2000). β-carotene transcriptionally regulates the expression of Psy-1 (Fraser et al. 1994). During ripening ethylene levels correlated with the anthocyanin development in apple cv. Pink lady (Whale and Singh 2007).

Sugar metabolism Sugars are essential contributors to fruit flavour. Carbohydrates not only provide energy for fruit development, but also contribute to the edible quality of fruit (Borsanie et al. 2009). Soluble sugars are derived from photosynthesis in leaves undergoing a series of physiological steps and accumulate in fruit. The accumulation of soluble sugars during fruit ripening mainly determines their sweetness at harvest (Zhu et al. 2013).Numerous studies have been dedicated to strategies to increase the soluble sugar content of tomatoes including agro technical and genetic approaches (Petreikov et al. 2009). Ripening is considered as a well-regulated genetically programmed phase of fruit development that involves changes in gene expression and enzyme

activity. The expression of specific genes is also required for ripening. It is supported by biochemical evidences that show there are changes in specific mRNAs and the de novo synthesis of protein during ripening. Changes in gene expression are both positive and negative. They involve genes in the nucleus and plastids and expression of at least some of them is confined to ripening fruit tissues. Thus fruit ripening is a highly controlled and programmed developmental event, involving the coordination of a multitude of metabolic changes and involves the activation and inactivation of various genes leading to various biochemical and physiological changes within the tissue (Payasi and Sanwal 2005). Expression of ClNl and CIN2 in grape berries, which was high early in berry development, declined greatly at the commencement of hexose accumulation. The results suggest that although vacuolar invertases are involved in hexose accumulation in grape berries, the expression of the genes and the synthesis of the enzymes leadto the onset of hexose accumulation by some weeks, so other mechanisms must be involved in regulating this process (Davies and Robinson 1996). The activities of neutral invertase (NI), vacuolar invertase (VI), fructokinase (FK) and hexokinase (HK) were inhibited, but sucrose phosphate synthase (SPS) activity was promoted in apple fruit suggesting that enhance sucrose synthesis and delay hydrolysis of sucrose andhexose (Zhu etal. 2013). Sucrose-phosphate synthase (SPS) from banana (Musa acuminata cv. Nanica) fruit was cloned and used to study expression during riening. Sucrose-phosphate synthase (SPS) expression is an important regulatory event of sweetening during banana fruit ripening (Do Nascimento et al. 1997). Godoy et al. (2009) evaluate the changes in gene expression during ripening in banana and mango fruits. Results revealed that in starch metabolism, banana a-amylase was induced during ripening while phosphorylase was more induced in mangoes. Fructan fructosyl transferase, chalcone synthase, and ascorbate oxidase genes were also induced in ripening mangoes, but not in bananas.

Flavour and aroma accumulation Volatiles compounds are produced by all sof t fruit species during ripening and play an important role determining the final sensory quality of fruit. Ester production has been significantly reduced in transgenic plants by

downregulation of fruit ethylene using antisense aminocyclopropanecarboxylate (ACC) oxidase constructs in apple, melon and kiwifruit (Souleyre et al. 2014). Cell wall structure impact aroma binding and release flavor volatiles from transgenic, ripe tomato fruit with down-regulated PG, PME, and PG + PME activities (Baldwin et al. 1992a). The red-ripe tomatoes with down- regulated PG produced lower levels of some volatiles (Kramer et al. 1992). Transformation of ‘Ailsa Craig’ fruit down-regulated PG (pTOM 6) (Grierson and Schuch 1993) and reduced PME (PE1) (Hall et al. 1993) and PG+PME activities (Baldwin et al. 2000). Aharoni et al. (2000) suggested that the formation of volatile esters in fruit is subject to the availability of acyl-CoA molecules and alcohol substrates and is dictated by the temporal expression pattern of the SAAT gene family and substrate specificity of the SAAT enzyme. Fikri et al. (2002) isolated two genes (CM-AAT1 and CMAAT2) from Charentais melon fruit capable of producing esters from a wide range of combinations of alcohols and acyl-CoAs. Cloning of the two genes revealed that the CM-AAT1 protein exhibited alcohol acyl-transferase activity while no such activity could be detected for CM-AAT2. Finally, concluded that CM- AAT1 plays a major role in aroma volatiles formation in the melon. Cumplido- Laso et al. (2012) suggested that novel strawberry alcohol acyltransferase gene (FaAAT2) plays a significant role in the production of esters that contribute to the final strawberry fruit flavor. Souleyre et al. (2014) reported that the overexpression of AAT1 gene to transgenic apple is critical for the biosynthesis of esters contributing to a ‘ripe apple’ flavour. Alcohol acyl transferase 1 (MpAAT1) encodes an enzyme capable of catalysing the synthesis of the main esters in ripe apple fruit from a broad range of alcohol and CoA substrates (Souleyre et al. 2005). Expression of the highly homologous gene alcohol acyl transferase 2 (MdAAT2) is also positively correlated with AAT enzyme activity and ester production in apple fruit (Li et al. 2006).Several AAT genes have been isolated from wild strawberry F vesca (VAAT) (Beekwilder et al. 2004) and F. chiloensis (FcAAT1) (Gonzalez et al. 2009). In all cases, these genes reached their maximum transcript levels in red-ripened fruits, suggesting that their corresponding enzymes are involved in the biosynthesis of volatile esters in the strawberry fruit.

Comparison of Climacteric and Non-climacteric ripening Fruits such as strawberry, citrus, and grape have been classified as nonclimacteric, based on the lack of the respiratory burst and on the low endogenous production of ethylene compared to standard climacteric fruits. In pepper fruits which is grouped as non-climacteric, some cultivars seem to be ethylene- insensitive, while others pepper cultivars treated with exogenous ethylene were able to stimulate the expression of ripening-specific genes (Harpster et al. 1997; El-Kereamy et al. 2003). In strawberry, which is more of ten used as a model fruit for studies on nonclimacteric ripening, ethylene is found to be relatively high in green stage, decreases in white, and again increases at the red stage of ripening (Iannetta et al. 2006). The last increase is accompanied by an enhanced respiration rate similar to the one that is seen in climacteric fruits. However, external application of ethylene down-regulated the expression of several cell wall- related genes, such as β-galactosidase, pectin methylesterase, or ^xylosidase (Bustamante et al. 2009), while the expression of other genes such as expansin, FaEXP2 (Civello et al. 1999) was ethylene-insensitive. Studies at transcriptomic and metabolomic levels in transgenic strawberry fruits with decreased ethylene sensitivity indicate that ethylene action is required for normal fruit development. These results show that, although not as relevant as in climacteric fruits, ethylene may nevertheless play a role in strawberry fruit ripening. Comparative transcriptome and metabolome studies during the maturation processes of climacteric and non-climacteric fruits (tomato and pepper, respectively) suggest that both species have similar ethylene-mediated signaling components. In pepper, the regulation of these genes is, however, clearly different and may reflect altered ethylene sensitivity or regulators other than ethylene than in tomato (Osorio et al. 2012). Unlike the situation described in tomato the ethylene biosynthesis genes, aminocyclopropane-1carboxylic acid (ACC) synthase, and ACC oxidase, are not induced in pepper. However, genes downstream of ethylene perception, such as cell wall-related genes, ethylene response factor 3 (ERF3), and carotenoid biosynthesis genes, are up-regulated during pepper fruit ripening (Osorio et

al. 2012). Other commonly regulated genes between climacteric and nonclimacteric fruits have been described. In strawberry, a SEPALLATA gene (SEP1/2; MADS-box) is needed for normal development and ripening (Seymour et al. 2011). Similarly, in banana, which is classified as a climacteric fruit, the MADS-box SEP3 gene also displays ripening-related expression (Elitzur et al. 2010). In apple, MADS2 gene expression is also associated with fruit firmness (Cevik et al. 2010), whereas in bilberry fruit, the SQUAMOSA MADS-box ortholog of the TDR4 gene in tomato, has a role in regulation of anthocyanin biosynthesis (Jaakola et al. 2010). Ripening regulation in climacteric fruits and non-climacteric fruits is given in Fig. 8 and 9.

Fig. 8. Ripening regulation in climacteric fruits (Osorio et al. 2013)

Fig. 9. Ripening regulation in Non-climacteric fruit (Osorio etal. 2013)

Conclusions Fruit ripening includes biochemical, physiological and structural changes such as degradation of chlorophyll, synthesis of carotenoids, accumulation of sugars, production of volatiles and changes in texture. During fruit ripening, the complex network of metabolites and proteins is considerably altered. In this favor, metabolomics is an excellent tool for analyzing metabolism in developing fruit. The ethylene response in climacteric fruits coordinates expression of thousands of genes that in turn control fruit softening as well as accumulation of pigments, sugars, acids and volatiles. Expression of ACC synthase and ACC oxidase genes plays major role during ethylene biosynthesis and ethylene perception and signal transduction regulating fruit

ripening. Recent advances in molecular biology provided a better understanding of the physiology and biochemistry of fruit ripening as well as providing a hand for genetic manipulation of the ripening process.The network of transcriptional and hormonal regulators mediates the physiological and biochemical changes. High quality genome sequence is the foundation for expansion and improvement of the genetic regulation of fruit development and ripening.

References Aharoni A, Keizer LCP, Bouwmeester HJ, Sun Z, Alvarez-Huerta M, Verhoeven HA, Blaas J, van Houwelingen AMML, De Vos RCH, van der Voet H, Jansen RC, Guis M, Mol J, Davis RW, Schena M, van Tunen AJ, and O’Connella AP 2000. Identification of the SAAT Gene Involved in Strawberry Flavor Biogenesis by Use of DNA Microarrays. The Plant Cell, 12:647-661 Alexander L and Grierson D 2002. Ethylene biosynthesis and action in tomato: a model for climacteric fruit ripening Journal of Experimental Botany, 53(377):2039-2055 Archbold, DD and Dennis FG 1984. Quantification of free ABA and free and conjugated IAA strawberry achene and receptacle tissue during fruit development. Journal of the American Society for Horticulture Science, 109:330-335 Atkinson RG, Gunaseelan K, Wang MY, Luo L, Wang T, Norling CL, Johnston SL, Maddumage R, Schroder R and Schaffer RJ 2011. Dissecting the role of climacteric ethylene in kiwifruit (Actinidia chinensis) ripening using a 1-aminocyclopropane-1-carboxylic acid oxidase knockdown line. Journal of Experimental Botany, 62:3821-3835 Atkinson RG, Sutherland PW, Johnston SL, Gunaseelan K, Hallett IC, Mitra D, Brummell DA, Schroder R, Johnston JW and Schaffer RJ 2012. Down-regulation of POLYGALACTURONASE1 alters firmness, tensile strength and water loss in apple (Malus x domestica) fruit. BMC Plant Biology, 12:129-141 Ayub R, Guis M, BenAmor M, Gillot L, Roustan JP, Latche A, Bouzayen M and Pech JC (1996) Expression of ACC oxidase antisense gene inhibits ripening of cantaloupe melon fruits. Nature Biotechnology, 14:862-866 Baldwin EA, Nisperos-Carriedo MO and Scott JW 1992a. Levels of flavor volatiles in a normal cultivar, ripening inhibitor and their hybrid. Proceedings of the Florida State Horticultural Society, 104:86-89 Baldwin EA, Scott JW, Shewmaker CK and Schuch W 2000. Flavor Trivia and Tomato Aroma: Biochemistry and Possible Mechanismsfor Control of Important Aroma Components. Hortscience, 35(6): 1013-1022 Bansal KC 2000. Biotechnology for improving product quality in horticultural crops. In: Chadha KL, Ravindran PN, Leela Sahijram (Eds.) Biotechnology in Horticultural and Plantation Crops, Malhotra Publishing House, New Delhi. pp 201-218 Bapat VA, Trivedi PK, Ghosh A, Sane VA, Ganapathi TR and Nath P 2010. Ripening of fleshy fruit: molecular insight and the role of ethylene. Biotechnology Advances, 28:94-107 Beekwilder J, Alvarez-Huerta M, Neef E, Verstappen FWA, Bouwmeester HJ and Aharoni A 2004. Functional characterization of enzymes forming volatile esters from strawberry and banana. Plant

Physiology, 135:1865-1878 Bombarely A, Merchante C, Csukasi F, et al. 2010. Generation and analysis of ESTs from strawberry (Fragaria x ananassa) fruits and evaluation of their utility in genetic and molecular studies. BMC Genomics, 11:503-519 Borsanie J, BuddeCO, Porrini L, Lauxmann MA, Lombardo VA and Murray R 2009. Carbon metabolism of peach fruit after harvest: Changes in enzymes involved in organic acid and sugar level modifications. Journal of Experimental Botany, 60(15): 1823-1837 Bottcher C, Keyzers RA, Boss PK and Davies C 2010a. Sequestration of auxin by the indole- 3-acetic acid-amido synthetase GH3-1 in grape berry (Vitis vinifera L.) and the proposed role of auxin conjugation during ripening. Journal of Experimental Botany, 61:36153625 Bouzayen M, Latche A, Nath P and Pech JC 2010. Mechanism of Fruit Ripening. In: Plant Developmental Biology - Biotechnological Perspectives, volume 1. Springer, ISBN 9783-64202300-2 Brady CJ 1987. Fruit Ripening. Annual Review of Plant Physiology, 38:155-178 Bramley PM 2002. Regulation of carotenoid formation during tomato fruit ripening and development Journal of Experimental Botany, 53(377):2107-2113 Brummell DA 2006. Primary cell wall metabolism during fruit ripening. New Zealand Journal of Forestry Science, 36(1):99-111 Brummell DA, Cin VD, Crisosto CH and Labavitch JM 2004. Cell wall metabolism during maturation, ripening and senescence of peach fruit. Journal of Experimental Botany, 55(405):2029-2039 Brummell DA, Harpster MH, Civello PM, Palys JM, Bennett AB and Dunsmuir P 1999. Modification of expansin protein abundance in tomato fruit alters softening and cell wall polymer metabolism during ripening. The Plant Cell, 11:2203-2216 Bustamante CA, Civello PM and Martinez GA 2009. Cloning of the promoter region of betaxylosidase (FaXyl1) gene and effect of plant growth regulators on the expression of FaXyl1 in strawberry fruit. Plant Science, 177:49-56 Carrari F, Baxter C, Usadel B,Urbanczyk-Wochniak E, Zanor MI,Nunes-Nesi A, Nikiforova V, Centero D, Ratzka A, Pauly M, Sweetlove LJ and Fernie AR 2006. Integrated analysis of metabolite and transcript levels reveals the metabolic shifts that underlie tomato fruit development and highlight regulatory aspects of metabolic network behavior. Plant Physiology, 142:1380-1396 Cevik V, Ryder CD, Popovich A, Manning K, King JK and Seymour GB 2010. A fruitfull-like gene is associated with genetic variation for fruit flesh firmness in apple (Malus domestica Borkh.). Tree Genetics and Genomes, 6:271-279 Chai YM, Jia HF, Li CL, Dong QH and Shen YY (2011) F aPYR 1 is involved in strawberry fruit ripening. Journal of Experimental Botany, 62:5079-5089 Cheng Y, Dong Y, Yan H, Ge W, Shen C, Guan J, Liu L and Zhang Y 2012. Effects of 1 -MCP on chlorophyll degradation pathway-associated genes expression and chloroplast ultrastructure during the peel yellowing of Chinese pear fruits in storage. Food Chemistry, 135(2):415-22 Ciardi J and Klee H 2001. Regulation of ethylene-mediated responses at the level of the receptor. Annals of Botany, 88:813-822 Civello PM, Powell A, Sabehat A and BennettAB 1999. An expansin gene expressed in ripening strawberry fruit. Plant Physiology, 121:1273-1279 Concha CM, Figueroa NE, Poblete LA, Onate FA, Schwab W and Figueroa CR 2013 Methyl jasmonate treatment induces changes in fruit ripening by modifying the expression of several ripening genes in Fragaria chiloensis fruit. Plant Physiology and Biochemistry, 70:433-444

Csukasi F, Osorio S, Gutierrez JR, et al. 2011 Gibberellin biosynthesis and signalling during development of the strawberry receptacle. New Phytologist, 191:376-390 Cumplido-Laso G, Medina-Puche L, Moyano E, Hof fmann T, Sinz Q, Ring L, Studart- Wittkowski C, Caballero JL, Schwab W, Munoz-Blanco J and Blanco-Portales R 2012. The fruit ripening-related gene F aAAT2 encodes an acyltransferase involved in strawberry aroma biogenesis Journal of Experimental Botany, 63(11 ):4275-4290 Davies C, Boss PK and Robinson SP 1997. Treatment of crape berries, a nonclimacteric fruit with a synthetic auxin, retards ripening and alters the expression of developmentally regulated genes. Plant Physiol. 11 5: 11 55-1 161 Davies C and Robinson SP 1996. Sugar accumulation in grape berries cloning of two putative vacuolar lnvertase cDNAs and their expression in grapevine tissues. Plant Physiology, 111:275-283 De Godoy A, Morita RJ, Cordenunsi BR, Lajolo FM, and do Nascimento JRO 2009. Expression analysis of a set of genesrelated to the ripening of bananas and mangoes. Brazilian Journal of Plant Physiology, 21(4):251-259 De la Fuente JI, Amaya I, Castillejo C, Sanchez-Sevilla JF, Quesada MA, Botella MA, et al. 2006. The strawberry gene FaGAST affects plant growth through inhibition of cell elongation. Journal of Experimental Botany, 10:2401-2411 Defilippi BG, Kader AA and Dandekar AM 2005. Apple aroma: alcohol acyltransferase, a rate limiting step forester biosynthesis is regulated by ethylene. Plant Science, 168:11991210 Degu A, Hochberg U, Sikron N, Venturini L, Buson G, Ghan R, Plaschkes I, Batushansky A, ChalifaCaspi V, Mattivi F, Delledonne M, Pezzotti M, Rachmilevitch S, Cramer GR and FaitA 2014. Metabolite and transcript prof iling of berry skin during fruit development elucidates differential regulation between Cabernet Sauvignon and Shiraz cultivars at branching points in the polyphenol pathway. BMC Plant Biology, 14:188 Deikman J 1997. Molecular mechanisms of ethylene regulation of gene transcription. Physiologia Plantarum, 100:561-566 Dincheva I, Badjakov I, Kondakova V and Batchvarova R 2013. Metabolic prof iling of red raspberry (Rubusidaeus) during fruit development and ripening. International Journal of Agricultural Science and Research, 2:81-88 Do Nascimento JRO, Cordenunsi BR, Lajolo FM and Alcocer MJC 1997. Banana sucrose- phosphate synthase gene expression during fruit ripening. Planta, 203:283-288 El Hadi, MAM., Zhang, FJ., Wu, FF., Zhou, CH., and Tao, J. 2013. Advances in fruit aroma volatile research. Molecules 2013, 18, 8200-8229 Elitzur T, Vrebalov J, Giovannoni JJ, Goldschmidt EE and Friedman H 2010. The regulation of MADS-box gene expression during ripeningof banana and their regulatory interaction with ethylene. Journal of Experimental Botany, 61:1523-1535 El-Kereamy A, Chervin C, Roustan JP, Cheynier V, Souquet JM, Moutounet M, et al. 2003. Exogenous ethylene stimulates the long-term expression of genes related to anthocyanin biosynthesis in grape berries. Physiologia Plantarum, 119:175-182 Fellman JK, Miller TW, Mattison DS and Mattheis JP 2000. Factorsthat influence biosynthesis of volatile flavour compounds in applefruits. Horticultural Science, 35:1026-1033. Fikri EL, Yahyaoui, Wongs-Aree C, Latche A, Hackett R, Grierson D and Pech JC 2002. Molecular and biochemical characteristics of a gene encoding an alcohol acyl-transferase involved in the generation of aroma volatile esters during melon ripening. European Journal of Biochemistry, 269:2359-2366. Fraser PD, Truesdale MR, Bird CR, Schuch W and Bramle PM 1994. Carotenoid Biosynthesis during

Tomato Fruit Development’- Evidence for Tissue-Specific Gene Expression. Plant Physiology, 105:405-413. Gapper NE, Giovannoni JJ and Watkins CB 2014. Understanding development and ripening of fruit crops in an ‘omics’ era. Horticulture Research, 34:1-10. Giovannoni JJ 2004. Genetic regulation of fruit development and ripening. Plant Cell 16:170181. Giovannoni JJ 2007. Fruit ripening mutants yield insights into ripening control. Current Opinion in Plant Biology, 10:283-289. Giuliano G, Aquilani R and Dharmapuri S 2000. Metabolicengineering of plant carotenoids. Trends in Plant Science, 5:406-409 Gonzalez M, Gaete-Eastman C, Valdenegro M, Figueroa CR, Fuentes L, Herrera R and Moya- Leon MA2009. Aroma development during ripening of F. chiloensis fruit and participation of an alcoholacyltransferase (FcAAT1) gene. Journal of Agricultural and Food Chemistry, 57:91239132 Grassi S, Piro G, Lee JM, Zheng Y, Fei Z, Dalessandro G, Giovannoni JJ and Lenucci M 2013. Comparative carotenoid pathway regulators of ripening watermelon fruit. BMC Genomics 14:781 Grierson D and Schuch W 1993. Control of ripening. PTRSL B, 342:241- 250 Hall LN, Tucker GA, Smith CJS, Watson CF, Seymour GB, Bundick Y, BromwellJM, Fletcher JD, May JA, SchuchW, Bird CR and Grierson D 1993. Antisense inhibition of pectin esterase gene expressionin transgenic tomatoes. The Plant Journal, 3:121-129 Handa AK, Hernanadez MET and Mattoo AK 2011. Fruit development and ripening a molecular perspective. In: Altman A, Hesegawa PM (Eds.) Plant Biotechnology and Agriculture. Oxford: Academic Press, pp 405-424 Handa AK and Mattoo AK 2010. Differential and functional interactions emphasize the multiple roles of polyamines in plants. Plant Physiology and Biochemistry 48:540-546 Harpster MH, Lee KY and Dunsmuir P 1997. Isolation andcharacterization of a gene encoding endo-b1,4-glucanase frompepper (Capsicum annuum L.). Plant Molecular Biology 33:47-59 Hiwasa K, Kinugasa Y, Amano S, Hashimoto A, Nakano R, Inaba A and Kubo Y 2003. Ethylene is required for both the initiation and progression of softening in pear (Pyrus communis L. ) fruit. Journal of Experimental Botany, 54:771-779 Hoeberichts FA, Van Der Plas LHW and Woltering EJ 2002. Ethylene perception is required for the expression of tomato ripening-related genes and associated physiological changes even at advanced stages of ripening. Postharvest Biology and Technology, 26:125-133 Hortensteiner S and Krautler B (2011) Chlorophyll breakdown in higher plants. Biochemica Biophysica Acta, 1807:977-988 Hua HL, Do YY and Huang PL 2012. Expression prof iles of a MhCTR1 gene in relation to banana fruit ripening. Plant Physiology and Biochemistry, 56:47-55 Iannetta PPM, Laarhovenb LJ, Medina-Escobar N, James EK, McManuse MT, Davies HV and Harren FJM (2006) Ethylene and carbon dioxide production by developing strawberries show a correlative pattern that is indicative of ripening climacteric fruit. Physiologia Plantarum, 127:247259 Jaakola L, Poole M, Jones MO, et al. 2010. A SQUAMOSA MADS box gene involved in the regulation of anthocyanin accumulation in bilberry fruits. Plant Physiology, 153:16191629 Jia HF, Chai YM, Li CL, Lu D, Luo JJ, Qin L and Shen YY 2011. Abscisic Acid Plays an Important Role in the Regulationof Strawberry Fruit Ripening. Plant Physiology, 157:188-199 Jiang Y, Fu J 2000. Ethylene regulation of fruit ripening: Molecular aspects. Plant Growth Regulation,

30:193-200 Karlova R, Chapman N, David K, Angenent GC, Seymour GB and de Maagd RA 2014. Transcriptional control of fleshy fruit development and ripening.Journal of Experimental Botany, 65(16):45274541 Klee HJ and Giovannoni JJ 2011. Genetics and Control of Tomato Fruit Ripening and Quality Attributes. Annual Review of Genetics, 45:41-59 Kondo S, Meemak S, Ban Y, Moriguchi T and Harada T 2009. Effects of auxin and jasmonates on 1aminocyclopropane-1-carboxylate (ACC) synthase and ACC oxidase gene expression during ripening of apple fruit. Postharvest Biology and Technology, 51:281284 Kondo S, Yamada H and Setha S 2007. Effect of jasmonates differed at fruit ripening stages on 1aminocyclopropane-1-carboxylate (ACC) synthase and ACC oxidase gene expression in pears. Journal of the American Society for Horticultural Science, 132:120-125 Kramer M, Sanders R, Bolkan H, Waters C, Sheehy RE and Hiatt WR 1992. Postharvest evaluation of transgenic tomatoes with reduced levels of polygalacturonase: Processing, firmness and disease resistance. Postharvest Biology and Technology, 1:241-255 Kumar R, Agarwal P, Tyagi AK and Sharma AK 2012. Genome-wide investigation and expression analysis suggest diverse roles of auxin-responsive GH3 genes during development and response to different stimuli in tomato (Solanum lycopersicum). Molecular Genetics and Genomic s, 287:221-235 Kuntz M, Romer S, Suire C, Hugueney P, Weil JH, Schantz R and Camara B 1992. Identification of a cDNA for the plastid-located geranylgeranyl pyrophosphate synthase from Capsicumannuum: correlative increase in enzyme activity and transcript level during fruit ripening. The Plant Journal, 2(1):25-34 Langley KR, Martin A,Stenning R, Murray AJ, Hobson GE, Schuch WW and Bird CR 1994. Mechanical and optical assessment of the ripening of tomato fruit with reduced polygalacturonase activity. Journal of the Science of Food and Agriculture, 66:547-554 LashbrookCC, Gonzalez-Bosch C and Bennett AB (1994) Two Divergent Endo-P-I ,4-glucanase Genes Exhibit Overlapping Expression in Ripening Fruit and Abscising flowers. The Plant Cell, 6:14851493 Laval-Martin D, Quennemet J and Moneger R 1975. Pigment evolutionin Lycopersicon esculentum fruits during growth and development. Phytochemistry, 14:2357-2362 Li D, Xu Y, Xu G, Gu L, Li D and Shu H (2006) Molecular cloning andexpression of a gene encoding alcohol acyltransferase (MdAAT2) fromapple (cv. Golden Delicious). Phytochemistry, 67:658667 Li MJ, Feng FJ and Cheng LJ 2012. Expression patterns of genes involved insugar metabolism and accumulation during apple fruit development. Plos One, 7(3): 1-14 Lisso J, Altmann T and Mussig C 2006. Metabolic changes in fruits of the tomato dx mutant. Phytochemistry, 67:2232-2238 Liu J, Liu L, Li Y, Jia C, Zhang J, Miao H, Hu W, Wang Z, Xu B and Jin Z 2015. Role for the banana AGAMOUS-like gene MaMADS7 in regulation of fruit ripening and quality. Physiologia Plantarum, 155(3):217-231 Liu YS, Gur A, Ronen G, Causse M, Damidaux R, Buret M, Hirschberg J and Zamir D 2003. There is more to tomato fruit colour than candidatecarotenoid genes. Plant Biotechnology Journal, 1:195207 Lois LM, Rodriguez-Concepcion M, Gallego F, Campos N and Boronat A 2000. Carotenoid biosynthesis during tomato fruitdevelopment: regulatory role of 1-deoxy-D-xylulose 5-

phosphatesynthase. The Plant Journal, 22:503-513 Lombardo VA, Osorio S, Borsani J, Lauxmann MA, Bustamante CA, Budde CO, Andreo CS, Lara MV,Fernie AR and Drincovich MF 2011. Metabolic prof iling during peach fruit development andripening reveals the metabolic networks that underpineach developmental stage. Plant Physiology, 157:1696-1710 Mahmood T, Anwar F, Abbas M, Boyce MC and Saari N 2012. Compositional Variation in Sugars and Organic Acids at Different Maturity Stages in Selected Small Fruits from Pakistan. International Journal of Molecular Sciences, 13:1380-1392 Manriquez D, El-Sharkawy I, Flores FB, Regad F, Bouzayen M, Latche' A and Pech JC 2006. Fruit specific gene expression and biochemical characteristics of two highly divergent alcohol dehydrogenases of melon. Plant Molecular Biology, 61: 675-685 Martel C, Vrebalov J, Tafelmeyer P and Giovannoni JJ 2011. The tomato MADS-box transcription factor RIPENING INHIBITOR interacts with promoters involved in numerous ripening processes in a COLORLESS NONRIPENING-dependent manner. Plant Physiology, 157:1568-1579 Marty I, Bureau S, Sarkissian G, Gouble, B, Audergon JM and Albagnac G 2005. Ethylene regulation of carotenoid accumulation and carotenogenic gene expression in colour- contrasted apricot varieties (Prunus armeniaca). Journal of Experimental Botany, 56(417):1877-1886 Medlicott AP and Thompson AK 1985. Analysis of sugars and organic acids in ripening mango fruits (Mangifera indica L. var Keitt) by high performance liquid chromatography. Journal of the Science of Food and Agriculture, 36(7):561-566 Meli VS, Ghosh S, Prabha TN, Chakraborty N, Chakraborty S and Datta A 2010. Enhancement of fruit shelf life by suppressing N-glycan processing enzymes. PNAS, 107(6):2413- 2418 Merchante C, Vallarino, JG, Osorio S, et al. 2013. Ethylene is involved in strawberry fruit ripening in an organ-specific manner. Journal of Experimental Botany, 64:4421-4439 Mowlah G and Itoo S (1982) Guava (Psidium guajava L.).Sugar component and relation to enzymes at stages of fruit development and ripening. Journal of Japanese Society for Food Science and Technology, 29(8):472-476. Mukkun L and Singh Z 2009. Methyl jasmonate plays a role in fruit ripening of “Pajaro” strawberry through stimulation of ethylene biosynthesis. Scientia Horticulturae, 123:510 Nam YW, Tichit L, Leperlie M, Cuerq B, Marty I and Leliever JM 1999. Isolation and characterization of mRNAs differentially expressed during ripening of wild strawberry (Fragaria vesca L.) fruits. Plant Molecular Biology, 39:629-636 Nambeesan S, Datsenka T, Ferruzzi M, Malladi A, Mattoo AK and Handa AK 2010. Overexpression of yeast spermidine synthase impacts ripening, senescence and decay symptoms in tomato. The Plant Journal, 63:836-847. Osorio S, Alba R, Nikoloski Z, Kochevenko A, Fernie AR and Giovannoni JJ 2012. Integrative comparative analyses of transcript and metabolite prof iles from pepper and tomato ripening and development stages uncovers species-specific patterns of network regulatory behavior. Plant Physiology, 159:1713-1729 Osorio S and Fernie AR 2013. Biochemistry of fruit ripening. In: Seymour GB, Poole M, Givannoni JJ, Tucker GA (eds.) The molecular biology and biochemistry of fruit ripening. John Wiley and Sons, pp 1-19 Osorio S, Scossa F and Fernie AR 2013. Molecular regulation of fruit ripening. Frontiers in plant Science, 4:198-205 Paillard, N.M.M. The flavour of apples, pears and quinces. (In) Food Flavours, Part C: The Flavour of Fruits; Morton, L.D., MacLeod, A.J., (Eds.); Elsevier Science Publishing Company Inc.:

Amsterdam, The Netherlands, 1990; pp. 1-41. Pan QH, Li MJ, Peng CC, Zhang N, Zou X, Zou KQ, Wang XL, Yu XC, Wang XF and Zhang DP (2005) Abscisic acid activates acid invertase in developing grape berry. Physiologia Plantarum, 125:157-170 Pan Y, Bradley G, Pyke K, Ball G, Lu C, Fray R, Marshall A, Jayasuta S, Baxter C, van Wijk R, Boy den L, Cade R, Chapman NH, Fraser PD, Hodgman C and Seymour GB 2013. Network Inference Analysis Identifies an APRR2-LikeGene Linked to Pigment Accumulation in Tomato and Pepper Fruits Plant Physiology, 161:1476-1485 Payasi A and Sanwal GG (2005) Biochemistry of Fruit Ripening. Indian Journal of Agricultural Biochemistry, 18(2): 51 -60 Perez, AG., Rios, JJ., Sanz, C. and Olias, JM. 1992. Aroma components and free amino acids in strawberry variety Chandler during ripening. J. Agric. Food Chem. 40: 2232-2235. Petreikov M, Yeselson L, Shen S, Levin I, Schaffer AA, Efrati A and Bar M (2009) Carbohydrate balance and accumulation during development of near-isogenic tomato lines differing in the AGPase-L1 allele. Journal of the American Society for Horticultural Science, 134(1):134-140 Porter JW and Spurgeon SL 1979. Enzymatic synthesis of carotenes. Pure and Applied Chemistry, 51:609-622 Pose S, Paniagua C, Cifuentes M, Blanco-Portales R, Quesada MA and Mercado JA 2013. Insights into the effects of polygalacturonase FaPGl gene silencing on pectin matrix disassembly, enhanced tissue integrity, and firmness in ripe strawberry fruits. Journal of Experimental Botany, 64:38033815 Prasanna V, Prabha TN and Tharanathan RN 2007. Fruit ripening phenomena-An overview. Critical Reviews in Food Science and Nutrition, 47:1-19 Rodrigo MJ, Marcos JF, Alfe'rez F, Mallent MD and Zacary'as L 2003. Characterization of Pinalate, a novel Citrus sinensis mutant with a fruit- speciuc alteration that results in yellow pigmentation and decreased ABA content. Journal of Experimental Botany, 54:727-738 Roessner-Tunali U, Hegemann B, Lytovchenko A, Carrari F, Bruedigam C, Granot D and Fernie AR 2003. Metabolic prof iling of transgenic tomato plants overexpressing hexokinase reveals that the influence of hexose phosphorylation diminishes during fruit development. Plant Physiology, 133:84-99 Romer S, Fraser PD, Kiano J, Shipton CA, Misawa N, SchuchW and Bramley PM 2000. Elevation of the provitamin A content of transgenic tomato plants. Nature Biotechnology, 18:666-669 Ronen G, Cohen M, Zamir D and Hirschberg J 1999. Regulation of carotenoid biosynthesis during tomato fruit development:expression of the gene for lycopene epsilon cyclase is downregulated during ripening and is elevated in the mutant delta. The Plant Journal, 17:341-351 Rose JKC, Hadfield KA, Labavitch JM and Bennett AB 1998. Temporal sequence of cell wall disassembly in rapidly ripening melon fruit. Plant Physiology, 117:345-361 Saladie M, Matas AJ and Isaacson T 2007. A re-evaluation of the key factors that influence tomato fruit softening and integrity. Plant Physiology, 144:1012-1028 Sen S, Chatterjee BK and Roy HB 1985. Amylase activity in ripening mango (Mangifera indica L. ) fruit. Indian Biology, 17:25-28 Senthilkumar S and Vijayakumar RM 2014. Biochemical, Physiological and Horticultural Perspectives of Fruit Colour Pigmentation: A Review. Research and Reviews: Journal of Agriculture and Allied Sciences, 3(1):9-16 Seymour GB, Chapman NH, Chew BL and Rose JKC 2013a. Regulation of ripening and opportunities for control in tomato and other fruits. Plant Biotechnology Journal, 11:269278

Seymour GB, Ryder CD, Cevik V, Hammond JP, Popovich A, King GJ, Vrebalov J, Giovannoni JJ and Manning K 2011. A SEPALLATA gene is involved in the development and ripening of strawberry (Fragaria x ananassa Duch.) fruit, a non-climacteric tissue. Journal of Experimental Botany, 62:1179-1188 Smith CJS, Watson CF and Morris PC 1990. Inheritance and effect on ripening of antisense polygalacturonase genes in transgenic tomatoes. Plant Molecular Biology, 14:369-379 Smith DL, Abbott JA and Gross KC 2002. Down-regulation of tomato a-galactosidase 4 results in decreased fruit softening. Plant Physiology, 129:1755-1762 Souleyre EJF, Chagn D, Chen X, Tomes S, Turner RM, Wang MY, Maddumage R, Hunt MB, Winz RA , Wiedow C, Hamiaux C, Gardiner SE, Rowan DD and Atkinson RG (2014) The AAT1 locus is critical for the biosynthesis of esters contributing to ‘ripe apple’ flavour in ‘Royal Gala’ and ‘Granny Smith’ apples. The Plant Journal, 78:903-915 Souleyre EJF, Greenwood DR, Friel EN, Karunairetnam S and Newcomb RD 2005. An alcohol acyl transferase from apple (cv. Royal Gala), MpAAT1, produces esters involved in apple fruit flavor. FEBS Journal, 272:3132-3144 Sun L, Sun YF, Zhang M, Wang L, Ren J, Cui MM, Wang YP, Ji K, Li P, Li Q, Chen P, Dai SJ, Duan CR, Wu Y and Leng P 2012. Suppression of 9-cis-epoxycarotenoid dioxygenase (NCED), which encodes a key enzyme in abscisic acid biosynthesis, alters fruit texture in transgenic tomatoes. Plant Physiology, 158:283-298. Symons GM, Chua YJ, Ross JJ, Quittenden LJ, Davies NW and Reid JB 2012. Hormonal changes during non-climacteric ripening in strawberry. Journal of Experimental Botany, 63:4741-4750. Symons GM, Davies C, Shavrukov Y, Dry IB, Reid JB and Thomas MR 2006. Grapes on steroids. Brassinosteroids are involved in grape berry ripening. Plant Physiology, 140:150158 . Vicente AR, Saladie M, Rose JKC and Labavitch JM 2007. The linkage between cell wall metabolism and fruit softening: looking to the future. Journal of the Science of Food and Agriculture, 87:1435-1448. Vidya Vardhini B and Rao SS 2002. Acceleration of ripening of tomato pericarp discs by brassinosteroids. Phytochemistry, 61:843-847. Vrebalov J, Ruezinsky D, Padmanabhan V, White R, Medrano D, Drake R, Schuch W and Giovannoni J 2002. A MADS-Box Gene Necessary for Fruit Ripening at the Tomato Ripening-Inhibitor (Rin) Locus. Science, 296:343-346. Wang S and Lin H 2000. Antioxidant activity in fruits and leaves of blackberry, raspberry and strawberry varies with cultivar and developmental stage. Journal of Agricultural and Food Chemistry, 48:140-146. Watkins CB 2002. Ethylene synthesis, mode of action, consequences and control.( In) Knee, M. (Ed.) Fruit Quality and Its Biological Basis. Academic Press, Sheffield, pp 180-224. Whale SK and Singh Z 2007. Endogenous Ethylene and Color Development in the Skin of ‘Pink Lady’ Apple. Journal of the American Society for Horticultural Science, 132(1):20-28 Xu F, Yuan S, Zhang DW, Lv X and Lin HH 2012. The role of alternative oxidase in tomato fruit ripening and its regulatory interaction with ethylene. Journal of Experimental Botany, 63: 57055716. Yang T, Peng H and Bauchan GR 2014. Functional analysis of tomato calmodulin gene family during fruit development and ripening. Horticulture Research, 57:1-9. Yu M, Shen L, Zhang A and She J 2011. Methyl jasmonate-induced defense responses are associated with elevation of 1 -aminocyclopropane-1 -carboxylate oxidase in Lycopersicon esculentum fruit. Journal of Plant Physiology, 168:1820-1827.

Zamboni A, Di Carli M, Guzzo F, Stocchero M, Zenoni S, Ferrarini A,Tononi P, Tof fali K, Desiderio A and Lilley KS 2010. Identification of putative stage-specific grapevine berry biomarkers and omics dataintegration into networks. Plant Physiology, 154:14391459. Zhang J, Wang X, Yu O, Tang J, Gu X, Wan X and Fang C 2011. Metabolic prof iling of strawberry (Fragaria ananassa Duch.) during fruit development and maturation. Journal of Experimental Botany, 62(3): 1103-1118. Zhang M, Yuan B and Leng P 2009. The role of ABA in triggering ethylene biosynthesis and ripening of tomato fruit. Journal of Experimental Botany, 60(6):1579-1588. Zhou L and Paull RE 2001. Sucrose metabolism during Papaya (Carica papaya) fruit growth and ripening. Journal of the American Society for Horticultural Science, 126(3): 351-357 Zhu Z, Liu R, Li B and Tian S 2003. Characterization of genes encoding key enzymes involved in sugar metabolism of apple fruit in controlled atmosphere storage. Food Chemistry, 141:33233328.

CHAPTER 6

Biochemical and Molecular Aspects of Latex Production in Hevea Brasiliensis Molly Thomas, Ambily, P.K., Sreelatha, S. and James Jacob “One ,remember to look up at the stars and not down at your feet. Two,never give up work. Work gives you meaning and purpose and life is empty without it. Three, you are lucky enough to find love, remember it is there and don’t throw it aw ay”-Stephen Hawking Hevea brasiliensis, the most important commercial source of natural rubber (NR) is a perennial tropical tree belonging to the family Euphorbiaceae. NR (cis-1, 4-polyisoprene) is a vital raw material used in the manufacture of a wide range of industrial and engineering products in the automobile, aerospace, medical and defense industries as well as innumerable household items. There are over 2000 species of plants from about 300 genera that are known to contain rubber in their latex, but most of them make small quantities of short chain rubber molecules which are poor in quality for practical use (Archer et al, 1963a). Rubber from Hevea brasiliensis is a high molecular weight polymer and its geometric configuration of double bonds is exclusively cis in nature (Golub et al., 1962). Due to the molecular structure and high molecular weight (greater than 1 million Daltons), this strategic molecule has high performance properties that cannot easily be mimicked by artificially produced polymers like synthetic rubber (SR) (Sekhar, 1989; de Fay et al., 2010).

Rubber is the major constituent of latex which is produced and stored in specialized cells called laticifers or latex vessels (Gomez and Moir, 1979). Latex vessels originate from the cambial cells and they are articulated and anastomosing. Latex is present in almost all parts of the plant but laticifers of the trunk are commercially exploited for harvesting latex by means of a controlled wounding process called tapping. H. brasiliensis remains as the only cultivated species as a source of commercial natural rubber because of its abundance in the latex, better quality, convenience of harvesting and perennial nature. NR has the added advantage of being a renewable resource with environmental benefits. Production of NR results in sequestration of large amounts of atmospheric carbon dioxide (CO2) but the SRs are produced from petroleum stock and this result in the emission of large amounts of green house gases in to the atmosphere.

Composition of latex Latex is a specialized form of cytoplasm containing a suspension of rubber and non-rubber particles in an aqueous serum and the rubber content ranges from 30-50% of the latex by weight (Dian et al, 1995). Latex contains all the sub cellular organelles of non-photosynthetic cells such as vacuoles, plastids, mitochondria, nuclei, endoplasmic reticulum and polysomes (d’Auzac and Jacob, 1989). Besides rubber and water, fresh latex contains carbohydrates, proteins, lipids and inorganic salts (Archer et al., 1963b). Fresh latex is a poly disperse system, which can be separated into four main fractions by ultra centrifugation. These fractions include a white upper layer of rubber particles, an orange or yellow layer containing Frey Wyssling particles, an aqueous serum called C- serum and a bottom fraction containing vacuoles called lutoids. The serum contains most of the soluble substances including amino acids, proteins, carbohydrates, organic acids, inorganic salts and nucleotide materials (Archer et al., 1969). Rubber particles make up to 30 to 45 % of the volume of latex and are found as spherical or pear shaped particles with sizes ranging from 0.02 to 3.00 pm with the majority in the region of 0.1 pm. Rubber particles are strongly protected in suspension by a film of adsorbed protein and phospholipids. This protein- phospholipid layer imparts a net negative charge to the rubber

particle contributing to colloidal stability. The lutoid particles amounting to 10 to 20 % of the latex by volume are sub-cellular membrane-bound bodies ranging in size from 2 to 5 pm. The membrane encloses a fluid serum known as B-serum composed of proteins, ions, hydrolases and defense proteins. Lutoids are directly involved in cellular homeostasis of laticifers and play a major role in latex coagulation. Frey-Wyssling particles which are enclosed in a typical double membrane are spherical and yellow coloured, ranges in size from 4 to 6 pm and constitute one to three per cent of the latex by volume. They are specialized chromoplasts containing a - carotene and assumed to be modified plastids.

Organic non -rubber constituents of latex Carbohydrates Quebrachitol (methyl-inositol), sucrose and glucose are the major soluble carbohydrates in latex (Low, 1978). Quebrachitol is the most concentrated single component in the serum phase. The concentration of quebrachitol varies with clones and seasons and ranges from one to three per cent of whole latex (Gopalakrishnan et al., 2008; 2011). It is a major contributor to the osmotic pressure of the cytosol (d’Auzac and Jacob, 1989). The concentration of sucrose in latex varies with clones (Licy et al., 1993; Nair et al., 2001; Sreelatha et al., 2009). Proteins The total protein content of fresh latex is approximately one per cent, of which about 20 per cent is adsorbed on rubber particles, an equal quantity found in the bottom fraction and the remainder in the serum phase (Archer et al., 1963b). The existence of a lipo-protein envelope on the surface of rubber particles was established by Bowler (1953). The adsorbed proteins and phospholipids on the rubber particle impart a net negative charge thereby contributing to the colloidal stability of latex. The major protein found in serum phase is a-globulin (Hahn et al., 1971). Enzymes of the glycolytic pathway (Bealing, 1969) as well as enzymes for isoprenoid pathway (Suvachitanont and Wititsuwannakul, 1995) are found in C- serum. Proteins

in the bottom fraction are the proteins in lutoid serum (B- serum) and those in the lutoid membrane. Hevein is the major protein in B- serum which accounts for about 70 per cent of the water soluble proteins in the bottom fraction (Archer et al., 1969). It is a low molecular weight anionic protein with a single chain of 43 amino acids and is involved in the coagulation of latex by bringing together rubber particles (Gidrol et al., 1994). Two basic proteins, a major one identical with hevamine A, a cationic protein (Archer, 1976) and a minor having lysozyme and chitinase activities were found in the bottom fraction (Tata et al., 1983). The basic proteins of bottom fraction are involved in regulating the flow of latex. Some defense proteins such as chitinase, a- 1, 3 glucanase and lysozymes are also found in this fraction. Higher protein content is observed in the lutoid membrane from high yielding clones compared to low yielders and two polypeptides with molecular weight 63.1 and 79.4 kDa are identified specifically in high yielding clones (Sreelatha et al., 1996). Lipids Lipids of fresh latex consist of fats, waxes, sterols, sterol esters and phospholipids. Lipids associated with the rubber and non-rubber particles in latex play a vital role in the stability and colloidal behavior of latex. Triglycerides and sterols are the main components of the neutral lipids of rubber particles. Lutoid stability as indicated by bursting index is negatively correlated with the phospholipid content of lutoids (Sherif and Sethuraj, 1978). Thomas et al. (1990) observed that the contents of total lipids, triglycerides, sterols and phospholipids in the latex were significantly high in the high yielding clones compared to low yielding clones. There was distinct clonal variation in the amount of lipids extractable from rubber cream and bottom fraction (Nair et al., 1993). Nucleic acids and polysomes Hevea latex contains DNA, ribosomal and messenger RNA (Tupy, 1969). Polysomes are also present in the serum phase of latex which is functional with regard to protein biosynthesis and contributes to the regeneration of the contents of the laticiferous cells between two tappings.

Other constituents Most of the classic amino acids have been reported in latex. The major proportion of free amino acids is located in the cytosol. The predominant amino acids in the cytosol are glutamic acid, alanine and aspartic acid. Nucleotides present in the latex are important as co-factors and are intermediates in biosynthetic processes. Low molecular weight thiols, which are the main reducing agents in latex, include glutathione and cysteine. Ascorbic acid is also a very important reducing agent in latex. The reducing components are involved in the redox potential of latex. Malic acid and citric acid make up 90 per cent of organic acids in latex. Total concentration of inorganic ions in fresh latex is about 0.5 per cent, the major ions being potassium, magnesium, copper, iron, sodium, calcium and phosphate (Archer et al., 1963b). Rubber biosynthesis In H. brasiliensis, rubber biosynthesis takes place on the surface of rubber particles in the latex (the cytoplasm of laticifers). Rubber (cis-polyisoprene) is formed through the sequential condensation of isopentenyl diphosphate (IPP) (Chow et al., 2007). IPP is produced via two biosynthetic pathways in higher plants. In the cytosol the well described mevalonate (MVA) pathway synthesizes IPP from acetyl-CoA (Spurgeon and Porter, 1981). Another metabolic pathway for IPP synthesis is 1-deoxy-D-xylulose-5-phosphate/2-Cmethyl-D-erythritol- 4-phosphate (DOXP/ MEP) pathway, which is located in the plastids (Rohmer et al., 1996; Lichtenthaler et al., 1997). It has been well documented that isoprenoids including natural rubber, sesquiterpenes, triterpenes, sterols and brassinosteroids are synthesized via the MVA pathway (Newman and Chappell, 1999), whereas gibberellins, abscisic acid, carotenoids and chlorophyll side chains are synthesized via the MEP pathway (Lichtenthaler, 1999; Chow et al., 2012). Natural rubber biosynthesis in Hevea is a side-branch of the ubiquitous isoprenoid pathway (Fig. 1). Sucrose is the precursor for rubber biosynthesis and the individual steps are well established (Lynen, 1969; Archer et al, 1963 a; Archer and Cockbain, 1969; Archer and Audly, 1987; Light and Dennis, 1989).). The biosynthesis of rubber from sucrose involves more than 20 enzymatic reactions (Sando et al., 2008). Several proteins other than enzymes

are reported to be involved in the biosynthetic process (Dennis and Light, 1989; Oh et al., 1999; Kang et al., 2000). Biosynthesis of rubber can be divided into three stages: (i) generation of acetyl- coenzyme A (acetyl - CoA) (ii) production of IPP and its isomer, dimethyl allyl diphosphate (DMAPP) (iii) polymerization of IPP and DMAPP for initiation of rubber chain and then of IPP addition to form long cis-1,4- polyisoprene chain (rubber)

Fig. 1 : Schematic representation of rubber biosynthetic pathway in Hevea brasiliensis

Generation of acetyl- CoA

Sucrose in latex is the primary source of acetyl- CoA and also of ATP and NADPH which are required in the biosynthesis of rubber (Chow et al, 2007; Silpi et al., 2007; Sando et al., 2008). The enzymes required for the conversion of acetate to rubber have been detected in Hevea latex (Lynen, 1969). Using 14C labeled acetate it was proved that latex used as a medium converted it to labeled rubber, indicating the presence of the entire enzyme system necessary for the conversion of acetate to rubber. Other possible sources of acetyl CoA are via, â- oxidation of fatty acids or via the metabolism of amino acids especially leucine.

Conversion of acetyl - CoA to isopentenyl diphosphate (IPP) The formation of IPP is by the condensation of three molecules of acetylCoA followed by reduction, phosphorylation and decarboxylation (Kush, 1994; Stermer et al., 1994). Two molecules of acetyl-CoA condense to form acetoacetyl-CoA and the acetoacetyl-CoA condenses with another molecule of acetyl-CoA resulting in the production of a-hydroxymethyl glutaryl-CoA (HMG-CoA) with the help of an enzyme, HMG-CoA synthase (HMGS). HMGS activity is found mainly in C-serum, which represents the cytosolic fraction of laticiferous cells (Suvachittanont and Wititsuwannakul, 1995). HMGS activity is positively correlated with rubber yield in Hevea, suggesting a regulatory role of this enzyme in rubber biosynthesis (Suvachittanont and Wititsuwannakul, 1995; Suwanmanee et al., 2002; Nagegowda et al., 2004). The enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) catalyses the synthesis of mevalonate from HMG-CoA. Since HMGR is a rate limiting enzyme in many polyprenoid syntheses (Bach and Lichtenthaler, 1987; Goldstein and Brown, 1990), the activity of this enzyme may be an indicator of yield potential of Hevea. Wititsuwannakul and Sukonrat (1984) observed diurnal variations in the activity of the enzyme and a positive correlation with rubber content in the latex. Nair et al. (1990) studied the activity of the enzyme in the bark of different Hevea clones and observed significantly higher enzyme activity in high yielding clones compared to low yielding clones. Suwanmanee et al. (2002) observed positive correlation

between HMGR activity and rubber yield. In the isoprene pathway, mevalonate is activated as phosphomevalonate by a cytosolic mevalonate kinase (Skilleter et al., 1966). The next stage consists of activation of phosphomevalonate into pyrophosphomevalonate (PPMVA) by a phosphomevalonate kinase (Williamson and Kekwick, 1965). Rubber monomer, IPP is produced from PPMVA by means of a decarboxylase, mevalonate decarboxylase (MVD).

Polymerization to rubber The formation of natural rubber from IPP requires the isomerisation of IPP to dimethylallyl diphosphate (DMAPP). The inter conversion of IPP to its highly electrophilic isomer, DMAPP is catalyzed by the enzyme IPP isomerase (Oh et al, 2000). The polyisoprene is then formed by repeated addition of the precursor, IPP to the elongating molecules (Lynen and Henning, 1960). In Hevea latex, IPP isomerase appears to be distributed between the serum and the surface of rubber particles. Sequential condensation of IPP with DMAPP results in the formation of 10- carbon intermediate, Geranyl diphosphate (GPP) which condenses with another IPP molecule yielding Farnesyl diphosphate (FPP), a 15-carbon intermediate. FPP synthase (FPPS) mediates the production of FPP from DMAPP or GPP (Poulter and Rilling, 1981). Further condensation of GPP with another IPP molecule results in the formation of a C20 molecule, Geranylgeranyl diphosphate (GGPP). The chain is lengthened by the terminal addition of IPP units by means of Rubber transferase (RuT) (Archer and Audley, 1987; Light and Dennies, 1989). Rubber transferase accepts a broad range of initiator molecules, with FPP (C15) being preferred (Tanaka et al., 1996; Castillon and Cornish, 1999; Mau et al., 2003). The termination of rubber biosynthesis is the release of rubber molecule from the enzyme, rubber transferase. Besides the enzymes of rubber biosynthesis pathway, other auxiliary proteins contribute to rubber formation. These include sucrose transporters (SUTs) (d’Auzac et al., 1997), the major membrane protein of rubber particle, rubber elongation factor (REF) (Dennies and light, 1989; Sando et al., 2009) and the small rubber particle protein (SRPP) (Oh et al., 1999). Sucrose transporters are responsible for loading sucrose into cells and play a central role in

sucrose partitioning between its site of synthesis and its numerous sink tissues (Braun and Slewinski, 2009).

Latex vessel plugging The duration of latex flow is largely determined by the stability of lutoids present in the latex. The serum contained inside these organelles (B-serum) has a high concentration of protons and various cations such as Mg2+, Ca2+, etc., cationic proteins as well as hydrolytic enzymes (d’ Auzac, 1989). Lutoids upon bursting release the cations which bring about coagulation of latex after physically interacting with the negatively charged rubber particles. Hevein, a low molecular weight protein in the B-serum is also involved in the coagulation of latex (Gidrol et al., 1994). Hevea latex lectin (HLL) binding protein is important for maintaining the colloidal stability of latex (Wititsuwannakul, et al., 2008).

Clonal variations in rubber yield The original genetic material of Hevea brasiliensis that was brought to Asian countries from the Amazon forests in the late 19th century (referred as the ‘Wickham gene pool’) had an average yield of just 200-300 kg per ha per year. Now hybrid clones are available with a production potential of 3000 3500 kg per ha per year (Mydin, 2014). This substantial improvement in productivity was achieved mainly through genetic improvement in the capacity of the species to synthesis large quantities of rubber through classical breeding and selection through field trials running for about 20-25 years. In India, the hybrid clones developed by the Rubber Research Institute of India (RRII) in the RRII 100, RRII 200, RRII 300 and RRII 400 series have high yield potential. Among the clones of the RRII 100 series, RRII 105 is the first clone release and this has been a highly successful and popular one. Clones RRII 203 and RRII 208, RRII 300 and RRII 308 are the best selections in the RRII 200 and 300 series respectively. In the RRII 400 series clones, which are the result of a biparental cross of RRII 105 X RRIC 100, ten clones out-yielded the popular high yielding clone RRII 105 (Licy et al., 2003) in the preliminary evaluation.

Rubber yield is a quantitative trait and latex obtained on tapping is a manifestation of various morphological, anatomical, physiological and biochemical characters of the tree. Productivity of Hevea depends on the genetic potential of the clone, its adaptability to the existing environment and ability to respond to improved agro techniques (Jayasekara et al., 1997; Mydin, 2014). The two major factors influencing the yield in H. brasiliensis are latex flow characteristics and metabolic capacity for in situ regeneration of latex between two successive harvesting dates (Sethuraj, 1981). The rate of latex flow and its final stoppage depends on water status of the tree trunk and latex vessel plugging (An et al., 2014). Latex regeneration is controlled by different biochemical and molecular mechanisms such as supply of sucrose, availability of biochemical energy (ATP), proton transport mechanism in the lutoid membrane, pH regulation and also the capacity of laticifers to regenerate the cellular metabolites lost during tapping (Sreelatha et al., 2016). Significant interclonal variations in yield were observed in mature plantations of H. brasliensis (Premakumari et al., 1991; Gireesh et al., 2012; 2017). Laticifer characters such as number of rows, density of vessels per row and diameter of latex vessels are significant clonal characters (Premakumari and Saraswathyamma, 2000). These parameters along with physiological and biochemical factors influence the volume of latex produced by a plant (Nair, 2000).

Biochemical factors associated with latex production In Hevea, rubber yield is limited by two main intrinsic factors in the latexproducing tissues. The first is the latex flow rate and its duration, which are influenced by the turgour pressure of the inner bark tissues, latex viscosity and latex coagulation efficiency (d’Auzac etal, 1989; Wititsuwannakul etal, 2008). The second intrinsic limiting factor is the ability of the latex cells to regenerate their lost cytoplasm, which mainly depends on their metabolic orientation and activity (Jacob et al., 1989). Important biochemical parameters of latex connected with latex production are total solid content (TSC), pH, adenosine triphosphate (ATP), sucrose, inorganic phosphorus (Pi), magnesium (Mg) and thiols (R-SH).

Total solid content (TSC) Rubber constitutes over 90 per cent of the TSC of latex and under certain conditions TSC reflects the biosynthetic activity of the laticifers. A decrease in TSC indicates inadequate regeneration which can become a limiting factor in rubber production. On the other hand, a high TSC can limit production by making latex flow difficult by increasing latex viscosity.

pH The extent of latex regeneration between tapping depends on the metabolic activity of laticifers. Significant positive correlation between yield and Cserum pH has been reported (Eschbach et al., 1984; Sreelatha et al., 2004). A large C-serum pH enhances rubber biosynthesis pathway, because several key rate- limiting enzymes involved in these processes are activated at slightly higher pH (Tupy, 1973a; Yeang, 1986).

ATP Biochemical energy availability is a major factor in latex regeneration. The generation of IPP from mevalonate requires significant quantities of ATP (Atkinson, 1977; Jacob and Prevot, 1992). A significant positive correlation between ATP content and rubber yield has been established in many clones (Chrestin and Bangratz, 1988; Sreelatha et al., 2004; 2014). Sucrose Sucrose is the primary precursor molecule for the synthesis of IPP and rubber in Hevea (Lynen, 1969). Many authors have demonstrated the primordial role of sucrose in latex production (Low, 1978; Eschbach et al., 1986). High sucrose content in latex may indicate a good loading of latex vessels and an active metabolism (Tupy and Primot, 1976). It may also indicate low metabolic utilization of sucrose and low productivity. Sucrose in the latex showed significant positive correlation with rubber yield (Tupy, 1973b;

Eschbach et al, 1984; Thomas et al., 2009).

Inorganic phosphorus (Pi) Inorganic phosphorus concentration indirectly reflects the energy metabolism and is necessary for the production of nucleic acid required in rubber biosynthesis. Direct correlation between Pi content of latex and production has been established in many clones (Eschbach et al., 1984; Subronto, 1978; Thomas et al., 2000; 2009). Because of its negative charge it also contributes to the colloidal stability of latex as constituent a of membrane phospholipids.

Magnesium (Mg2+) Magnesium is an activator as well as inhibitor of many enzymes involved in latex metabolism and therefore, has a complex link with production (Jacob et al., 1989). The positive charges of Magnesium released from the lutoids have a destabilizing effect on the negative charge of the colloidal suspension formed by latex making it coagulate, thus limiting flow. Subronto (1978) demonstrated the existence of a significant inverse correlation between Mg2+ contents and production while Eschbach et al. (1984) demonstrated a positive correlation between Mg2+ and production.

Thiols (R-SH) Latex thiols consist of glutathione, cysteine and methionine. Glutathione is the major fraction of the thiols and most reactive. It is found mainly in the latex cytosol. Thiols trap toxic forms of oxygen, thereby protecting the membranes of latex organelles. They are also activators of key enzymes in latex such as invertase and pyruvate kinase (d’Auzac et al., 1982). Several authors have demonstrated direct correlation between thiol concentration and rubber production (Eschbach et al., 1984; Sreelatha et al., 2009).

Rubber biosynthesis- related genes Genes corresponding to enzymes/ proteins involved in rubber biosynthesis in Hevea brasiliensis such as sucrose transporters (SUTs), rubber elongation factor (REF), hydroxyl methyl glutaryl-coA synthase (HMGS), hydroxyl methyl glutaryl-coA reductase (HMGR), cis--prenyltransferase (CPT) and small rubber particle protein (SRPP) have been cloned and further characterized (Dennis and Light, 1989; Chyle et al., 1992; Oh et al., 1999; Asawatreratanakul et al., 2003; Priya et al, 2007; Sando et al., 2008; Venkatachalam et al., 2009; Tang et al., 2010). EST-sequencing approach has enabled Hevea laticifer gene expression on a large scale (Ko et al, 2003; Chow et al, 2007). In recent years, with the advent of new-generation sequencing (NGS) techniques, several genes involved in rubber biosynthesis have been identified (Li et al., 2015). Rahman et al. (2013) sequenced the genome of a Hevea clone (RRIM 600) and identified 12 distinct submetabolic pathways represented by 417 genes associated with the carbon assimilatory mechanism of rubber biosynthesis. Sucrose transporters are responsible for loading sucrose into cells and play a central role in sucrose partioning between its site of synthesis and its numerous sink tissues (Braun and Slewinski, 2009). In Hevea, the isolated nature of the laticifer from its neighboring cells (Hebant, 1981) suggests that sucrose transporters might play an active role in the transmembrane uptake of sucrose by the laticifers. Six sucrose transporters have been cloned from Hevea, of which HbSUT3 has been identified as the key member responsible for sucrose loading into lacticifers (Dusotoit et al., 2010; Tang et al., 2010). The expression of sucrose transporter (HbSUT3) showed positive correlation with latex yield in Hevea (Tang et al., 2010; Ambily et al., 2016). Rubber elongation factor (REF) is present in large amounts at the surface of the large rubber particles in all laticifer layers (Sando et al., 2009). It plays an important role in the final polymerization step of rubber biosynthesis. Hevea genes encoding for REF has been cloned (Goyvaerts et al., 1991; Oh et al., 1999) and various isof orms were identified (Priya et al., 2006). REF gene is encoded by a small gene family consisting of three members and expression of REF2 showed positive correlation with Hevea rubber yield (Priya et al., 2007; Ambily et al., 2016). Ruderman et al. (2012) observed higher

expression for REF3 in low yielding than high yielding clones. Two isof orms of hmgs were cloned and characterized from Hevea (Suwanmanee et al., 2002; Sirinupong et al., 2005) and mRNA levels of hmgs1 showed positive correlation with rubber yield (Suwanmanee et al., 2013; Li et al., 2015). Putative association between specific HMGS gene haplotypes and gene expression in Hevea clones which may have a downstream impact up to the level of rubber production was also demonstrated (Uthup et al., 2016). Five isof orms of hmgr were cloned and characterized from Hevea (Chye et al., 1991; 1992; Sando et al., 2008). hmgrl is expressed predominantly in laticifers, the cells specific to rubber biosynthesis (Venkatachalam et al., 2009). hmgrl was significantly up regulated in a high yielding compared to a low yielding clone, suggesting that hmgrl is critical for providing IPP (Chao et al., 2015). The expression of hmgrl showed positive correlation with rubber yield (Suwanmanee et al., 2013; Li et al., 2015). The mevalonate diphosphate decarboxylase (MVD) which catalyses the irreversible decarboxylation of the 6- carbon compound mevalonate-5-pyrophosphate (MVAPP) into 5-carbon IPP, the building block of isoprenoid biosynthesis is reported to play an important role in rubber biosynthesis in H. brasiliensis (Sando et al, 2008). Farnesyl pyrophosphate synthase (FPS) catalyzes the consecutive condensations of DMAPP or geranyl pyrophosphate (GPP) with IPP to produce farnesyl pyrophosphate (FPP) as a final product. Geranylgeranyl pyrophosphate synthase (GGPPS) catalyzes the condensation of IPP with FPP to give GGPP, which is an essential precursor in the biosynthesis of several isoprenoids. Sequence structure analysis has shown that FPS gene is highly divergent in nature with high intragenic retro-element activity which may seriously affect its expression and regulation (Uthup et al., 2013). Expression levels of FPPS and GGPPS were differentially expressed in various tissues of H. brasiliensis (Takaya et al., 2003). The expressions of GGPPS and FPPS were significantly higher in a high yielding clone than a low yielding clone (Li et al., 2015). GGPPS showed significantly higher expression in the latex of a high yielding Hevea germplasm accession than a low yielding accession (RRII unpublished data). Rubber transferase (RuT), belonging to the cis prenyl transferase family catalyses the polymerization of IPP into cis-rubber and two rubber transferase homologues (RuT1 and RuT2) were cloned from H. brasiliensis latex (Asawatreratanakul et al., 2003;

Koyama, 2003). Recent studies conducted in Hevea clones with varying yield potential, RuT showed significantly higher expression in high yielding clones than low yielding clones (Ambily et al., 2016). Ko et al. (2003) observed high level of expression for 1-deoxy-D-xylulose 5phosphate synthase (DXPS) which is involved in IPP synthesis through DXP/ MEP pathway in Hevea latex. The high level of DXPS gene expression in latex implies that the non-mevalonate pathway might also contribute to the rubber biosynthesis together with the well-known mevalonate pathway. Expression analysis on MVA and MEP pathway genes suggests that the MEP pathway can be an alternate provider of IPP in mature rubber trees or in clones which do not produce a large amount of carotenoid (Chow et al., 2012). In connection with Hevea genome sequence analyses, Rahman et al. (2013) identified 18 genes encoding enzymes for MVA pathway and 29 genes for the MEP pathway.

Transgenic rubber plants H. brasiliensis has a long breeding cycle as it is a perennial tree species. Being highly heterozygous nature, efforts made by conventional breeding to improve productivity are very difficult. Yet remarkable achievements have been made in terms of increase in rubber productivity (Mydin and Gireesh, 2016). But a stage will be reached when the rubber biosynthetic rate of the tree itself becomes a limiting factor and further yield enhancement can be made only by manipulating the factors influencing the rate of rubber biosynthesis. Yield improvement through transgenics attempts the transfer of key regulatory genes associated with rubber biosynthesis. Jayashree et al. (2014) was successful in developing Hevea transgenic plants integrated with laticifer specific hmgr1 gene which encodes HMG-CoA reductase, a key regulatory enzyme in the isoprenoid pathway. HMGR activity was found higher in the transgenic plants compared to control wild type plants. The adverse effects of environmental conditions such as drought, temperature extremes, high solar raditions etc. limit the expansion of rubber cultivation to marginal areas in several rubber producing countries. Plants exposed to environmental stress generate excess reactive oxygen species (ROS) and super oxide dismutase (SOD) is the first enzyme involved in the detoxifying

process of ROS. Hevea transgenic plant lines integrated with MnSOD gene which confers tolerance to oxidative stress were developed (Jayashree et al., 2003; Sobha et al., 2003). Drought tolerance traits of these plants were validated at the laboratory and nursery level (Jayashree et al., 2011; Sumesh et al., 2014) and are now ready for field evaluation. Genetically modified rubber plants developed by RRII are the first in the world and first transgenic tree species developed in India. Leclercq et al. (2012) was successful in producing Hevea transgenic plant lines that overexpress H. brasiliensis cytosolic CuZnSOD gene. Transgenic Hevea plants integrated with osmotic gene which confers tolerance to biotic and abiotic stresses (Rekha et al., 2013) were also developed at RRII.

Future of natural rubber Due to high performance properties of natural rubber, a major share of it is consumed as raw material for the manufacture of automobile tyres including those of aircrafts. Presently, H. brasiliensis is the only major source of natural rubber. The global area under rubber cultivation is about 11.2 million hectares (Natural Rubber Trends and Statistics, 2016) with an annual production of 12.3 million tones (Rubber Statistical Bulletin, 2015). Natural rubber drives the economy of a nation through its direct role in industrial growth. The Indian rubber industry is the second largest in the world in terms of the amount of rubber it consumes every year. The value of output of the Indian rubber industry is about Rs. 78,000 crores which is roughly 0.75% of the national GDP (201314). The value of exports of rubber products from India was US$ 2.57 billion during 2014-15 which is far more than the export earnings from spices, cof fee and tea (GoI, 2017). Natural rubber plantations have many environmental benefits in addition to supplying a vital industrial raw material that is a component in tens of thousands of products that find use in the everyday life of mankind. In India, it is estimated that the NR plantations presently existing in the country sequester about 5% of the total amount of carbon dioxide emitted by the country’s transport sector every year. India has a proven capability of nine lakh tones natural rubber production per year which dropped to less than 6 lakh tones during 2015-16 due to falling natural rubber price. As the Indian

economy is expected to maintain its buoyancy in growth, it is estimated that by 2030 the country would require about 20 lakh tones of natural rubber. India requires a pragmatic and realistic plan to consolidate and increase natural rubber production in the country. As of now, there is sufficient availability of natural rubber in the global market, but this need not remain so in the future. The natural rubber demandsupply balance will depend on the rate of its consumption which will directly depend on the global economy. Since about 70% of global natural rubber production goes into the manufacturing of automotive tyre and tubes, a revival of global economy and the resultant growth in automobile industry will put huge pressure on global natural rubber supply. For a long term perennial tree crop like natural rubber with a gestation period of about seven years followed by a yielding phase of about another twenty years, it takes almost a decade after planting the trees in the field before any tangible increase in production can be seen.

Summary Rubber yield in Hevea brasiliensis is controlled by factors related to the physiology of latex flow and its in situ regeneration in the bark between successive tapping. The duration of latex flow which is directly related to rubber yield is largely determined by the stability of lutoids present in the latex. Latex harvesting process (tapping) results in the depletion of cell constitutes from the latex vessels. Regeneration of latex requires an intense metabolic activity involving reconstitution of all the sub cellular elements with their enzymatic functions. This requires reactions which can provide energy or reducing capacity required for the anabolic process leading to isoprene synthesis. Presence of high concentration of ATP in the latex as well as a high pH, increased sugar supply, low Mg content, high phosphorus and thiol contents and higher activity of rubber biosynthetic pathway enzymes viz. HMGS, HMGR and RuT are the biochemical factors found associated with high yield in Hevea brasiliensis. With regard to molecular factors, the expressions of HbSUT3, a sucrose transporter and genes corresponding to enzymes like HMGS, HMGR and MVD were found to be directly related to yield potential

of Hevea clones. The higher expression of these genes indicates increased supply of IPP, the isoprenoid monomer for rubber synthesis. Expression of genes in the downstream of the biosynthetic pathway like FPPS, RuT and REF2 was also significantly higher in high yielding than low yielding clones. The genes associated with rubber yield are useful molecular markers of yield potential in rubber tree breeding and selection programs.

References An, F., Cahill, D., Rookes, J., Lin, W. and Kong, L. 2014. Real-time measurement of phloem turgor pressure in Hevea brasiliensis with a modified cell pressure probe. Botanical Studies, 55(19): 111. Ambily, PK., Thomas, M., Sreelatha, S., Krishnakumar,R., Annamalainathan, K. and Jacob, J. 2016. Expression analysis of rubber biosynthetic pathway genes in Hevea brasiliensis. Placrosym XX11, 15-1 7, Dec. 2016, Kasargod, Kerala. Abstracts, pp. 40. Archer, B.L., Audley, B.G., Cockbain, E.G. and McSweeney, G.P 1963a. The biosynthesis of rubber: Incorporation of mevalonate and isopentenyl pyrophosphate into rubber by Hevea brasiliensis latex fractions. Biochemical Journal, 89: 565-574. Archer, B.L., Barnard, D., Cockbain, E.G, Dickenson, P.B. and McMullen, A.I. 1963b. Structure composition and biochemistry of Hevea latex. In: The Chemistry and Physics of Rubber Like Substances (Ed. L. Bateman). Maclaren and Sons Ltd., London, pp. 41-72. Archer, B.L. and Cockbain, E.G 1969. Rubber transferase from Hevea brasiliensis latex. Methods in Enzymology, 15: 476-480. Archer, B.L. 1976. Hevamine - a crystalline basic protein from Hevea brasiliensis latex. Phytochemistry, 15: 297-300. Archer, B.L., Audley, B.G, McSweeney, G.P. and Hong, T.C. 1969. Studies on composition of latex serum and bottom fraction particles. Journal of the Rubber Research Institute of Malaya, 21(4): 560-569. Archer, B.L. and Audley, B.G. (1987). New aspects of rubber biosynthesis. Botanical Journal of theLinnean Society, 94: 181-196. Asawatreratanakul, K., Zhang, Y.W., Wititsuwannakul, D., Wititsuwannakul, R., Takahashi, S., Rattanapittayaporn, A. and Koyama, T. (2003). Molecular cloning, expression and characterisation of cDNA encoding cis-prenyltransferases from Hevea brasiliensis. A key factor participating in natural rubber biosynthesis. European Journal of Biochemistry, 270: 4671-4680. Atkinson, D.E. 1977. Cellular Energy Metabolism and its Regulation. New York, Academic Press, 293 pp. Bach, T. J. and Lichtenthaler, H.K. 1987. Plant growth regulation by mevinolin and other sterol biosynthesis inhibitors. In: Ecology and Metabolism of Plant Lipids. (Eds. Fuller, G. and Nes W.D.), American Chemical Society Symposium Series, Vol 325 American Chemical Society, Washington, DC, pp 109-139 Bealing, F.J. 1969. Carbohydrate metabolism in Hevea latex: Availability and utilization of substrates. Journal of the Rubber Research Institute of Malaya, 21(4): 445-455.

Bowler, W.W. 1953. Electrophoretic mobility of fresh Hevea latex. Industrial Engineering Chemistry, 45: 1790. Braun, D.M. and Slewinski, T.L. 2009. Genetic control of carbon partitioning in grasses: roles of sucrose transporters and tie-dyed loci in phloem loading. Plant Physiology, 149: 7181. Castillon, J. and Cornish, K. 1999. Regulation of initiation and polymer molecular weight of cis- 1,4polyisoprene synthesized in vitro by particles isolated from Parthenium argentatum (Gray). Phytochemistry, 51: 43-51. Chao, J., Chen, Y., Wu, S. and Tian, W.M. 2015. Comparitive transcriptome analysis of latex from rubber tree clone CATAS8-79 and PR107 reveals new cues for the regulation of latex regeneration and duration of latex flow. BMC Plant Biology, 15: 104. DOI: 10.1186/ s12870-0150488-3. Chow, K.S., Wan, K.L., Isa M.N., Bahari, A. Tan, S.H., Harikrishna, K. and Yeang, H.Y. 2007. Insights into rubber biosynthesis from transcriptome analysis of Hevea brasiliensis latex. Journal of Experimental Botany, 58(10): 2429-2440. Chow, K.S., Mat-Isa, M.N., Bahari, A., Ghazali, A.K., Alias, H., Zainuddin, Z.M., Hoh, C.C. and Wan, K.L. 2012. Metabolic routes affecting rubber biosynthesis in Hevea brasiliensis latex. Journal of Experimental Botany, 63: 1863-1871. Chrestin, H. and Bangratz, J. 1988. The adenine nucleotide pool and energy charge in the Hevea latex cells: Clonal characteristics, relations with yield andsensitivity towards bark- dryness. Effects of yield stimulation with ethrel, an ethylene releaser. In: Proceedings of International Conference of Hevea Physiology and Breeding. October 1988, Paris, France. pp. 547-562. Chye, M.L., Kush, A., Tan, C.T. and Chua, N.H. 1991. Characterization of cDNA and genomic clones encoding 3-hydroxy-3-methylglutaryl CoA reductase from Hevea brasiliensis. Plant Molecular Biology, 16: 567-577. Chye, M.L., Tan, C.T. and Chua, N.H. 1992. Three genes encode 3-hydroxy-3-methylglutarylcoenzyme A reductase in Hevea brasiliensis: hmg1 and hmg3 are differentially expressed. Plant Molecular Biology, 19: 473-484. d’Auzac, J. and Jacob, J.L. 1989. The composition of latex from Hevea brasiliensis as a laticiferous cytoplasm. (In) Physiology of Rubber Tree Latex (Eds. J.d’Auzac, J.L. Jacob and H. Chrestin). CRC Press, Florida, pp. 60-88. d’Auzac, J. Cretin, H., Marin, B. and Lioret, C. 1982. A plant vacuolar system: The lutoids from Hevea brasiliensis latex. Physiologie Vegetale, 20: 311-331. d’Auzac, J., Jacob, J.L., Prevot, J.C., Clement, A. and Gallois, R. 1997. The regulation of cispolyisoprene production (natural rubber) from Hevea brasiliensis, In Recent research developments in plant physiology. (Ed. Pandalai S.G).Trivandrum, India: Rhaoesearch Singpost; pp. 273-332. de Fay, E., Moraes, L.A.C. and Moraes, V.H.F. 2010. Cyanogenesis and the onset of tapping panel dryness in rubber tree. Pesq uisa AgropecuariaBrasileira, 45: 1372-1380. Dennis, M.S. and Light, D.R. 1989. Rubber elongation factor from Hevea brasiliensis. Identification, characterization and role in rubber biosynthesis, Journal of Biological Chemistry, 264: 1860818617. Dian, K., Sangare, A. and Diopoh, J.K. 1995 Evidence for specific variation of protein pattern during tapping panel dryness condition development in Hevea brasiliensis. Plant Science, 105: 207-216. Dusotoit-coucaud, A., Kongsawadworakul, P., Maurousset, L., Viboonjun, U., Brunel, N., Pujaderenaud, V, Chrestin, H. and Sakr, S. 2010. Ethylene stimulation of latex yield depends on the expression of a sucrose transporter (HbSUT1B) in rubber tree (Hevea brasiliensis). Tree

Physiology, 30: 1586-1598. Eschbach, J.M., Roussel, D., van de Sype, H., Jacob, J.L. and d’Auzac, J. 1984. Relationship between yield and clonal physiological characteristics of latex from Hevea brasiliensis. Physiologie Vegetale, 22(3): 295-304. Eschbach, J.M., Tupy, J. and Lacrotte, R. 1986. Photosynthate allocation and productivity of latex vessels in Hevea brasiliensis. BiologiaPlantarum, 28:321. Gidrol, X., Chrestin, H., Tan, H.L. and Kush, A. 1994. Hevein a lectin-like protein from Hevea brasiliensis (rubber tree) is involved in the coagulation of latex. Journal of Biological Chemistry, 269: 9278-9283. Gireesh, T., Varghese, Y.A., Woeste, K.E., Mercykutty, V.C. and Marattukalam 2012. Effect of monoclonal and assorted seedling rootstocks on long term growth and yield of Hevea clones. Silvae Genetica, 61(1-2): 52-57. Gireesh, T., Meenakumari, T. and Mydin, K.K. 2017. Fast track evaluation and selection of Hevea brasiliensis clones from a clonal nursery. Industrial Crops and Products, 103:195201. GoI 2017. “Export import databank”, Department of Commerce and Industry, Government of India. Golub, M.A., Fuqua, S.A. and Bhacca, N.S. 1962. High resolution nuclear magnetic resonance spectra of various polyisoprenes. Journal of American Chemical Society, 84(2): 4981. Gomez, J.B. and Moir, G.F.J. 1979. The ultracytology of latex vessels in Hevea brasiliensis. Malaysian Rubber Research and Development Board, Kuala Lumpur, Malaysia. Monograph, No.4. Goldstein, J.L and Brown, M.S. 1990. Regulation of the mevalonate pathway. Nature, 343: 425-430. Gopalakrishnan, J., Thomas, M. and Jacob, J. 2008. Inositols: A commercially important sugar alcohol from natural rubber latex. Natural Rubber Research, 21(1 &2): 130-133. Gopalakrishnan, J., Thomas, M., Preenu, A.A., Annamalainathan, K., Krishnakumar, R. and Jacob, J. 2011. Recovery of L-quebrachitol from different latex serum sources of Hevea brasiliensis. Natural Rubber Research, 24(2): 253-258. Goyvaerts, E.M. Dennis, M., Light, D. and Chua, N.H. 1991. Cloning and sequencing of the cDNA encoding the rubber elongation factor of Hevea brasiliensis. Plant Physiology, 97: 317-321. Hahn, A.M., Hermann, J., Marianti., Mariano, A and Walugeno, K. 1971. Studies on a-globulin: Hevea and destabilizing protein of Hevea brasiliensis latex. Communications of the Research Institute of Estate Crops, 1. Hebant, C. 1981. Ontoge’nie des lacticife’rs du systeme Primaire d’Hevea brasiliensis.une etudeultrastructurale et cytochimique. Canadian Journal of Botany, 59: 974-985. Jayasekera, NE.M., Samaranayake, P and Karunasekera, K.B. 1977. Initial studies on the nature of genotype-environment interaction in some Hevea clones. Journal of the Rubber Research Institute of Srilanka, 54: 33-42. Jacob, J.L. and Prevot, J.C. 1992. Metabolism of the laticiferous system and its biochemical regulation. In: Natural Rubber; Biology, Cultivation and Technology (Eds. M.R. Sethuraj and N.M. Mathew), New York, pp. 116-136. Jacob, J.L., Prevot, J.C., Roussel, D., Lacrotte, R., Serres, E., d’Auzac, J., Eschbach, J.M. and Omont, H. (1989). Yield-limiting factors, latex physiological parameters, latex diagnosis and clonal typology. In: Physiology of Rubber Tree Latex (Eds. J. d’Auzac, J.L. Jacob and H. Chrestin). CRC Press Inc., Florida, pp. 345-382. Jayashree, R., Rekha, K., Venkatachalam, P, Uratsu, S.L., Dandekar, A.M., Jayashree, P.K., Kala, R.G., Priya, P, Sushamakumari, S., Sobha, S., Asokan, M.P., Sethuraj, M.R. and Thulaseedharan, A. 2003. Genetic transformation and regeneration of rubber tree (Hevea brasiliensis Muell. Arg.) transgenic plants with a constitutive version of an anti-oxidative dismutase gene. Plant Cell

Reports, 22(3): 201-209. Jayashree, R., Sobha, S., Rekha, K., Supriya, R., Vineetha, M., Sushamakumari, S., Kala, R.G., Jayashree, PK., Thulaseedharan, A., Annamalainathan, K., Nair, D.B., Sreelatha,S., Krishnakumar, R. and Jacob, J. 2011. Overexpression of MnSOD and drought related traits in MnSOD transgenic Hevea brasiliensis. Natural Rubber Research, 24( 1): 18-27. Jayashree, R., Nazeem, P.A., Venkatachalam, P, Rekha, K., Ambily, P.K., Kala, R.G, Suni, A.M., Krishnakumar, R., Sobha, S., Leda, P and Thulaseedharan, A. (2014). Integration and expression of hmgr1 gene in the transgenic plants of Hevea brasiliensis (clone RRII 105). International symposium on Plantation Crops, Kozhikode. Kang, H., Kim, Y.S. and Chung, G.C. 2000. Characterization of natural rubber biosynthesis in Ficus benghalensis. Plant Physiology and Biochemistry, 38: 979-987. Ko, J.H., Chow, K.S. and Han, K.H. 2003. Transcriptome analysis reveals novel features of the molecular events occurring in the laticifers of Hevea brasiliensis (para rubber tree). Plant Molecular Biology, 53: 479-492. Koyama, T. 2003. Molecular cloning, expression and characterization of cDNA encoding cis- prenyl transferases from Hevea brasiliensis . A key factor participating in natural rubber biosynthesis. European Journal of Biochemistry, 270: 4671-4680. Kush, A. 1994. Isoprenoid biosynthesis: the Hevea factory. Plant Physiology and Biochemistry, 32: 761-767. Leclercq, J., Martin, F.S., Sanier, C., Clement-Vidal, A., Fabre, D., Oliver, G., Lardet, L., Ayar, A., Peyramard, M. and Montoro, P. 2012. Overexpression of a cytosolic isof orm of the HbCuZnSOD gene in Hevea brasiliensis changes its responses to water deficit. Plant Molecular Biology, 80(3): 255-272. Lichtenthaler, H.K., Rohmer, M. and Schwender, J. 1997. Two independent biochemical pathways for isopentenyl diphosphate and isoprenoid biosynthesis in higher plants. Physiologia Plantarum , 101: 643-652. Lichtenthaler, H.K. 1999. The l-deoxy-d-xylulose-5- phosphate pathway of isoprenoid biosynthesis in plants. Annual Review of Plant Physiology and Plant Molecular Biology, 50: 7-65. Licy, J., Thomas, M., Panikkar, A.O.N., Abraham, J., Saraswathyamma, C.K. and Sethuraj, M.R. 1993. Genetic parameters and heterosis in rubber. 2. Association of yield and certain yield attributes. Journal of Plantation Crops, 21: 264-267. Licy, J., Saraswathyamma, C.K., Preemakumari, D., Meenakumari, T., Meenattoor, J.R. and Nazeer, M.A. 2003. Genetic parameters and heterosis in rubber (Hevea brasiliensis) Mull. Arg.: V. Hybrid vigour for yield and yield components among the RRII 400 series clones in small scale evaluation. Indian Journal of Natural Rubber Research, 16(1&2): 75-80. Light, D.R. and Dennis, M.S. 1989. Purification of a prenyl transferase that elongates cis- polyisoprene rubber from the latex of Hevea brasiliensis, Journal of Biological Chemistry, 264: 18589-18597. Low, F.C. 1978. Distribution and concentration of major soluble carbohydrates in Hevea latex, the effects of ethephon stimulation and the possible role of these carbohydrates in latex flow. Journal of the Rubber Research Institute of Malaysia, 26(1): 21-32. Lynen, F. and Henning, U. 1960. Biological path to natural rubber. Angewandte Chemie International, 72. Lynen, F. 1969. Biochemical problems of rubber synthesis. Journal of the Rubber Research Institute of Malaysia, 21(4): 389-406. Li, D., Hao, L., Liu, H., Zhao, M., Deng, Z., Li, Y, Zeng, R. and Tian, W. 2015. Next-generation sequencing, assembly, and comparative analyses of the latex transcriptomes from two elite Hevea

brasiliensis varieties. Tree Genetics and Genomes, 11: 1-9. Mau, C.J.D., Garneau, S., Scholte, A.A., Van Fleet, J.E., Vederas, J.C. and Cornish, K. 2003. Protein farnesyltransferase inhibitors interfere with farnesyl diphosphate binding by rubber transferase. European Journal of Biochemistry, 270: 3939-3945. Mydin, K.K. 2014. Genetic improvement of Hevea brasiliensis: Sixty years of breeding efforts in India. Rubber Science, 27(2): 153-181. Mydin, K.K. and Gireesh, T. 2016. Diversity and heterosis by recombination breeding of Hevea brasiliensis in India. Rubber Science, 29(1): 20-35. Nagegowda, D.A., Bach, T.J. and Chye, M.L. 2004. Brassicajuncea 3-hydroxy-3-methylglutaryl (HMG)-CoA synthase 1: expression and characterization of recombinant wild-type and mutant enzymes. Biochemical Journal, 383: 517-527. Newman, J.D. and Chappell, J. 1999. Isoprenoid biosynthesis in plants: carbon partitioning within the cytoplasmic pathway. Critical Reviews in Biochemistry and Molecular biology, 34: 95-106. Nair, N.U., Thomas, M., Sreelatha, S., Simon, S.P, Vijayakumar, K.R. and George, PJ. 1990: Clonal variations in the activity of 3-hydroxy -3-methyl glutaryl-CoA reductase in bark of Hevea brasiliensis. Indian Journal of Natural Rubber Research, 3(1): 40-42. Nair, N.U., Thomas, M., Sreelatha, S., Simon, S.P.., Vijayakumar, K.R. and George, PJ. 1993. Clonal variation in lipid composition in the latex of Hevea brasiliensis and its implication in latex production. Indian Journal of Natural Rubber Research, 6 (1&2): 143-145. Nair, N.U. 2000. Biochemistry and physiology of latex production. (In) Natural Rubber: Agromanagement and Crop Processing (Eds. George, PJ. and Jacob, C.K.), Rubber Research Institute of India, Kottayam, India, pp. 249-260. Nair, N.U., Sreelatha, S., Thomas, M., Simon, S.P. and Vijayakumar, K.R. 2001. Latex diagnosis for assessment of clonal performance in Hevea. Indian Journal of Natural Rubber Research, 14(1): 63-65. Natural Rubber Trends and Statistics 2016 ANRPC; http://anrpc.org/html/member_nrts.aspx. Accessed in September 2016. Oh, S.K., Kang, H., Shin, D.H., Yang, J. and Chow, K.S. 1999. Isolation, characterization, and functional analysis of a novel cDNA clone encoding a small rubber particle protein from Hevea brasiliensis. Journal of Biological Chemistry, 274: 17132-17138. Oh, S.K., Kang, H., Shin, D.H., Yang, J. and Han, K.H. 2000. Molecular cloning and characterization of a functional cDNA clone encoding isopentenyl diphosphate isomerase from Hevea brasiliensis. Journal of Plant Physiology, 157: 549-557. Poulter, C.D. and Rilling, H.C. 1981. Prenyltransferase and isomerase. (In) The Biosynthesis of Isoprenoid Compounds. (Eds. Porter J.W. and Spurgeon S.L.). Vol.1, John Wiley and Sons, U.S.A. pp. 163-224. Premakumari, D., Panikkar, A.O.N., Sethuraj, M.R. and Marattukalam, J.G. 1991. Growth, yield and flow characters and their correlations with brown bast incidence in ten Hevea clones. Indian Journal of Natural Rubber Research, 4(2): 107-113. Premakumari, D. and Saraswathyamma, C.K. (2000). The Para rubber tree. (In) Natural Rubber; Agromanagement and Crop Processing. (Eds. George, PJ. and Kuruvilla Jacob, C.), Rubber Research Institute of India, Kottayam, pp. 29-35. Priya, P., Venkatachalam, P. and Thulaseedharan, A. 2006. Molecular cloning and characterization of the rubber elongation factor gene and its promoter sequence from rubber tree (Hevea brasiliensis): A gene involved in rubber biosynthesis. Plant Science, 171(4): 470-480. Priya, P., Venkatachalam, P. and Thulaseedharan, A. 2007. Differential expression pattern of rubber

elongation factor (REF) mRNA transcripts from high and low yielding clones of rubber tree (Hevea brasiliensis Muell. Arg.). Plant Cell Reports, 26: 1833-1838. Rahman, A.Y. et al. 2013. Draft genome sequence of the rubber tree Hevea brasiliensis. BMC Genomics, 14: 75. Rekha, K., Jayashree, R., Sushamakumari, S., Sobha, S., Jomini, R., Supriya, R.and Nazeem, PA. 2013. Integration and expression of osmotin gene in Hevea brasiliensis Via. Agrobacterium. Journal of Plantation Crops, 41(1); 80-85. Rohmer, M., Seemann, M., Horbach, S., Bringer-Meyer, S. and Sahm, H. 1996. Glyceraldehyde 3phosphate and pyruvate as precursors of isoprenic units in an alternative non- mevalonate pathway for terpenoid biosynthesis . Journal of the American Chemical Society, 188: 2564-2566. Rubber Statistical Bulletin 2015, IRSG; http://www.rubberstudy.com/documents/WebSiteData_Aug2016.pdf. Ruderman, S., Kongsawadworakul, P., Viboonjun, U., Mongkolporn, O. and Chrestin, H. 2012. Mitochondrial/cytosolic Acetyl CoA and rubber biosynthesis genes expression in Hevea brasiliensis latex and rubber yield. Kasetsart Journal (NaturalScience), 46: 346-362. Sando. T., Takaoka, C., Mukai, Y., Yamashita, A., Hattori, M., Ogasawara, N., Fukusaki, E. and Kobayashi, A. 2008. Cloning and characterization of mevalonate pathway genes in a natural rubber producing plant, Hevea brasiliensis. Bioscience, Biotechnology and Biochemistry, 72: 2049-2060. Sando, T., Hayashi, T., Takeda, T., Akiyama, Y., Nakazawa, Y., Fukusaki, E. and Kobayashi, A. 2009. Histochemical study of detailed laticifer structure and rubber biosynthesis-related protein localization in Hevea brasiliensis using spectral confocal laser scanning microscopy. Planta, 230: 215-225. Sekhar, A.C. 1989. Rubber Wood Production and Utilisation. RRII, Kottayam. 224p. Sethuraj, M.R. 1981. Yield components in Hevea brasiliensis: Theoretical considerations. Plant Cell and Environment, 4:81-83. Sherif, PM. and Sethuraj, M.R. 1978. Role of lipids and proteins in the mechanism of latex vessel plugging in Hevea brasiliensis. Physilogia Plantarum, 42: 351-353 Sirinupong, N., Suwanmanee, P., Doolittle, R.F. and Suvachitanont, W. 2005. Molecular cloning of a new cDNA and expression of 3-hydroxy-3-methylglutaryl-CoA synthase gene from Hevea brasiliensis. Planta, 221(4): 502-12. Silpi, U., Lacointe, A., Kasempsap, P, Thanysawanyangkura, S., Chantuma, P, Gohet, E., Musigamart, N., Clement, A., Ameglio, T and Thaler P. 2007. Carbohydrate reserves as a competing sink: evidence from tapping trees. Tree Physiology 27:881-889. Skilleter, D.N, Williamson, I.P and Kekwick, R.G.O. 1966. Phosphomevalonate kinase from Hevea brasiliensis. Biochemical Journal, 98: 279. Sobha, S., Sushamakumari, S., Thanseem, I., Jayashree, PK., Rekha, K., Jayashree, R., Kala, R.G, Asokan, M.P., Sethuraj, M.R. Dandekar, A.M.and Thulaseedharan, A. 2003. Genetic transformation of Hevea brasiliensis with the gene coding for superoxide dismutase with FMV34S promoter. Current Science, 85(12): 1767-1773. Spurgeon, S.L. and Porter, J. 1981. Biosynthesis of Isoprenoid Compounds. Vol. 1. John Wiley and Sons, New York. pp. 1-46. Sreelatha, S., Vijayakumar, K.R., Nair, N.U., Thomas, M., Sheela, S.P., George, P. J. and Sethuraj, M.R. 1996. Identification of two polypeptides in the lutoid membrane of high yielding clones of Hevea brasiliensis. Journal of Plantation Crops, 24: 555-559. Sreelatha, S., Simon, S.P and Jacob, J. 2004. On the possibility of using ATP concentration in latex as

an indicator of high yield in Hevea brasiliensis. Journal of Rubber Research, 7(1): 71-78. Sreelatha, S., Mydin, K.K., Simon, S.P., Jacob, J. and Krishnakumar, R. 2009. Biochemical characterization of RRII 400 series clones of Hevea brasiliensis. Natural Rubber Research, 22:36-42. Sreelatha, S., Jacob, J., Mercykutty V.C., Simon, S.P, Krishnakumar, R and Annamalainathan, K 2014. ATP concentration in latex as an indicator for early evaluation of yield in Hevea brasiliensis. Journal of Plantation Crops, 42(1): 48-53. Sreelatha, S., Simon, S.P., Mercykutty, V.C., Mydin, K.K., Krishnakumar, R., Annamalainathan, K. and Jacob, J. 2016. Role of lutoid membrane transport and protein synthesis in the regeneration mechanism of latex in different Hevea clones. ActaPhysiologiae Plantarum, 28(6): 1-8. Stermer, B.A., Bianchini, G.M. and Korth, K.L. 1994. Regulation of HMG-CoA reductase activity in plants. Journal of Lipid Research, 35: 1133-1140. Subronto, 1978. Correlation studies of latex flow characters and latex mineral content. IRRDB symposium, Rubber Research Institute of Malaysia. Sumesh, K.V, Satheesh, PR., Sreelatha, S., Ravichandran, S., Thulaseedharan, A., Jayashree, R., Krishnakumar, R., Annamalainathan, K., Meena Singh and Jacob, J. 2014. Drought tolerance in MnSOD transgenic Hevea brasiliensis in a sub-humid environment. Journal of Plantation Crops, 42(1): 70-77. Suvachitanont, W and Wititsuwannakul, R. 1995. 3-hydroxy-3-methylglutaryl CoA synthase in Hevea brasiliensis. Phytochemistry, 40: 757-761. Suwanmanee, P, Suvachittanont, W. and Fincher, GB. 2002. Molecular cloning and sequencing of a cDNA encoding 3-hydroxy-3-methylglutaryl coenzyme A synthase from Hevea brasiliensis (HBK) Mull Arg. Science Asia, 28: 29-36. Suwanmanee, P, Sirinupong, N. and Suvachittanont, W. 2013. Regulation of 3-hydroxy-3methylglutaryl-CoA synthase and 3-hydroxy-3- methylglutaryl-CoA reductase and rubber biosynthesis of Hevea brasiliensis Mull. Arg. In: Isoprenoid Synthesis in Plants and Microorganisms (Eds. Bach, T.J. and Rohmer, M.). Springer, New York, pp-315-327. Takaya, A., Zhang, Y.W., Asawatreratanakul, K., Wititsuwannakul, D., Wititsuwannakul, R., Takahashi, S. and Koyama, T. 2003. Cloning, expression and characterization of a functional cDNA clone encoding geranylgeranyl diphosphate synthase of Hevea brasiliensis. Biochimica etBiophysica Acta, 1625(2): 214-220. Tang, C., Huang, D., Yang, J., Liu, S., Sakr, S., Li, H., Zhou, Y and Qin, Y. 2010. The sucrose transporter HbSUT3 plays an active role in sucrose loading to laticifer and rubber productivity in exploited trees of Hevea brasiliensis (para rubber tree). Plant, Cell and Environment, 33: 17081720. Tata, S.J., Beintema, J.J. and Balabakaran, S. 1983. The lysozyme of Hevea brasiliensis latex: Isolation, purification, enzyme kinetics and a partial amino-acid sequence. Journal of the Rubber Research Institute of Malaysia, 31(1): 35-48. Thomas, M., Nair, N.U., Sreelatha, S., Simon, S.P, Annamma, Y and Vijayakumar, K.R. 1990. Clonal variations in lipid composition of Hevea brasiliensis at young age. Indian Journal of Natural Rubber Research, 3(1): 73-75. Thomas, M., Thomas, K.U., Sobhana, P, Nair, R.B. and Jacob, J. 2000. Path coefficient analysis of yield and yield attributes in the Hevea brasiliensis clone RRII 105. Indian Journal of Natural Rubber Research, 13(1 &2): 103-107. Thomas, M., Gopalakrishnan, J., Prasannakumari, P, Abraham, J., Krishnakumar, R. and Jacob, J. 2009. Biochemical and ionic composition of latex influencing yield attributes and productivity in

Hevea brasiliensis. Natural Rubber Research, 22(1&2): 140-145. Tupy, J. 1969. Nucleic acid in latex and production of rubber in Hevea brasiliensis. Journal of Rubber Research Institute of Malaya, 21: 468Tupy, J. 1973a. The activity of latex invertase and latex production in Hevea brasiliensis Mull. Arg. Physiologie Vegetale, 11.633 Tupy, J. 1973b. The level and distribution pattern of sucrose along the trunk of Hevea brasiliensis Mull. Arg. as affected by the sink region induced by latex tapping. Physiologie Vegetale, 11: 1-8. Tupy, J. and Primot, L. 1976. Control of carbohydrate metabolism by ethylene in latex vessels of Hevea brasiliensis Mull. Arg. in relation to rubber production. Biologia Plantarum, 18: 373. Tanaka, Y., Aik-Hwee, E., Ohya, N., Nishiyama, N., Tangpakdee, J., Kawahara, S. and Wititsuwannakul, R. 1996. Initiation of rubber biosynthesis in Hevea brasiliensis: characterization of initiating species by structural analysis. Phytochemistry, 41: 15011505. Uthup T.K., Saha T, Ravindran M, Bini, K. 2013. Impact of an intragenic retrotransposon on the structural integrity and evolution of a major isoprenoid biosynthesis pathway gene in Hevea brasiliensis. Plant Physiology and Biochemistry, 73: 176-188. Uthup.T.K, Rajamani A, Ravindran, M. and Saha T 2016. Molecular evolution and functional characterisation of haplotypes of an important rubber biosynthesis gene in Hevea brasiliensis. Plant Biology, 4:720-728 doi: 10.1111 /plb .12433. Venkatachalam, P, Priya, P, Jayashree, R., Rekha, K. and Thulaseedharan, A. 2009. Molecular cloning and characterization of a 3-hydroxy 3-methyl-glutaryl coenzyme A reductase 1 (hmgr1) gene from rubber tree (Hevea brasiliensis Muell. Arg.): A key enzyme involved in isoprenoid biosynthesis. Physiology and Molecular Biology of Plants, 15: 133-143. Williamson, I. and Kekwick, R.G.O. 1965. The formation of 5-phosphomevalonate by mevalonate kinase in Hevea brasiliensis. Biochemical Journal, 96: 862-871. Wititsuwannakul, R. and Sukonrat, W. 1984. Diurnal variation of 3-hydroxy 3-methylglutaryl CoA reductase activity in the latex of Hevea brasiliensis and its relation to the rubber content. CompteRender da Colloque Exploitation Physiologie at Ameloiration de I’ Hevea Montpeller, France, pp.115-120. Wititsuwannakul, R., Pasitkul, P, Jewtragoon, P and Witisuwannakul, D. 2008. Hevea latex lectin binding protein in C-serum as an anti latex coagulating factor and its role in a proposed new modal for latex coagulation. Phytochemistry, 69: 656-662. Yeang, H.Y., Jacob, J.L., Prevot, J.C. and Vidal, A. 1986. Invertase activity in Hevea latex serum: interaction between pH and serum concentration. Journal of Natural Rubber Research, 1:16-24.

CHAPTER 7

Strategies for Mitigation of Impact of Climate Change on Sub-Tropical Fruits Gaganpreet Kour and Parshant Bakshi “If we pollute the air,'water and soil that keep us alive and well, and destroy the biodiversity that allows natural systems to function, no amount of money will save us”-David Suzuki Horticulture, the major component of agriculture in India, is no exemption from the threat of climate change impacts. Temperatures will pose a strong pressure on existing crop varieties, whilst shifts in rainfall patterns could significantly alter harvests by altering fruit filling periods and delaying vegetative growth, to not speak about the effects of increased CO2 concentrations, the effects on pests and diseases, and on soil quality, most of which are still highly uncertain and remain under- or unexplored. By the end of the 21st century and under the GHGs emission scenario SRES-A2 increases in temperatures could range between 1.8-6°C for Asia (IPCC 2007), with most of the variability being accounted to the different global climate models (GCMs) used in the IPCC 4th Assessment Report (AR4), and with warm periods showing greater increases as compared with cold periods. As per the report, mean annual temperature of Indian subcontinent is increasing 0.68oC in one centaury in the past and this rise is increasing due to emission of GHGs. This warming is more pronounced during post monsoon and winter and Himalayan region is predicted to be worst affected in South Asia (IPCC 2007). Rainfall is predicted to change between -5 to 20% during the cold season and between -40 to 15% in the warm season. Though it has been

accepted in the reports that uncertainties in climatic predictions are common and they should be minimized, but regardless of the uncertainty level, horticulture systems need to be resilient enough to cope with the expected negative effects or capitalize on the positive effects. Fruit trees provide important adaptive values and tend to be more resilient to climate change due to their perennial nature. But they too are affected by climate change in idiosyncratic ways. Climate change especially poses important difficulties for commercial production of fruit trees.

I. Impact of Climate Change on Subtropical Fruit Crops Changes in plant phenology are one of the earliest responses to rapid global climate change and could potentially have serious consequences for trees that depend on periodically available rain. An opportunistic response to water availability is the simplest explanation for most observed patterns where water is seasonally limiting. Horticultural crops might be highly sensitive to changes in climate and climatic variability as they heavily rely on adequate water supply and on a proper amount of daily energy (temperature, solar radiation) in order to properly grow. Therefore, even slight variations (temperature increases of the order of 1°C or water shortage or excess during a short period) can cause crop failures. In sub-tropical fruit crops, there is a direct effect of the temperature on the maturity and ripening of the fruits. When there is sufficient moisture, the TSS of the fruit increases with the temperature. In some fruits like passion fruit, increases in temperature do not increase TSS. Hence, the effect of different regimes of temperature can be different on different crops under subtropical environments. 1. Mango: Climate and weather play critical roles in the economic success or failure of sub-tropical fruit tree species including commercial mango production. Air temperature and rainfall influence vegetative and phenological phases in mango, are two of the most important factors determining suitability of an area’s climate for mango production. Climaterelated changes have already brought widespread changes in flowering and fruiting patterns of mango. In mango under different temperature regimes, fruit size is affected. This is adversely affecting fruit production in some

areas. But rising temperature in areas previously too cold for mango production are making them more suitable for mango production. For instance, an increase in temperature during coldest month has made mango cultivation possible in the valley areas of Himachal Pradesh and Uttarakhand. Under the influence of climate shift, both early and delayed flowering will be characteristic features in mango. As a result of variations in temperature, unseasonal rains and higher humidity, fruit trees show altered flowering trends. Delays in panicle emergence and fruit set have been noticed. Fruit set and availability of hermaphrodite flowers for pollination have an effect on yield due to pollen and stigmatic sterility. If panicle development coincides with an unusual cold spell, mango production will face several problems. Early flowering in the sub-tropics may result in a low fruitset because of several abnormalities caused due to low night temperatures coupled with unseasonal rains. It can be generally seen that low day temperatures cause reduced pollinator activity resulting in poor fruit set. Late flowering also reduces the fruit set because of pseudo fruit setting leading to clustering disorder. High temperatures during panicle development in mango speed up growth and reduce the number of days for effective pollination when hermaphrodite flowers are available, which may lead to unsatisfactory production. Excessive rainfall and humidity during the period of fruit maturity invites severe attacks of fruit fly, anthracnose and mango stone weevil. Change in leaf canopy micro climate under elevated CO2 may favour infection (Chakraborty and Datta, 2003). Rising temperatures cause desiccation of pollen and poor pollinator activity resulting in low fruit set and ultimately, a poor crop (Bhruguvanshi 2009). In the sub-topics, low night temperatures (5-10°C) result in synchronous flowering. However, night temperatures of 10-18°C produce asynchronous flowering similar to that in the tropics. Climate changes may cause abrupt changes in night temperatures, which will cause asynchronous flowering in the sub-tropics and result in poor productivity. Flower buds exposed to cold temperature during night may change into vegetative ones under the warm night conditions. In the tropics, cool winters followed by a rise in day temperatures as summer approaches may result in poor flowering. Change in rainfall patterns can adversely affect the quality and appearance of ripe mango fruits. Unseasonal rains encourage pests, which also lower fruit yields. 2. Citrus: Citrus plants are considered to be better equipped to deal with a

changing climate than other fruit crops. That’s largely because they flourish in the heat. According to a 2007 study that examined the effect of rising temperatures on various crops, the cultivation area for orange trees will actually expand by the year 2055. While the lemon cultivation area will shrink by around 10 percent, that is a small setback in comparison to other plant. For the lemon harvest, we’re not expecting any major drawbacks due to climate change for the time being,” according to Tim Grout from Citrus Research International, a research association that examines the citrus industry. If however temperatures rise by more than one or two degrees, yields could fall because the plants shed their fruit too soon,” he says, adding that seedless lemons are especially at risk. Other analysts believe that the increased risk of pest infestations could also lead to greater harvest losses. Citrus greening, for example, is a bacterial disease that is primarily spread by two types of psyllid insects. It turns a citrus plant’s leaves and shoots yellow and makes the fruit bitter, of ten causing the entire plant to wither away. But in different case in District Kangra, Himachal Pradesh, citrus fruit production has declined by 1,000 tones in the district in the last two years. “In 2009-10, it was 22,184 tones while in 2010-11 it decreased to 21,704 tones,” district horticulture authorities said. “Climate change is one major factor affecting the fruit crop. We are facing problems on this count for the last eight to ten years. It’s not raining in time because of which, plants are not getting proper nutrition and fruition is declining gradually,” said Ramesh, an orange grower from Indora. High temperature and high evaporation during flowering and fruit set result in low yield due to flower and fruit drop. The fruits have poor colour if the temperature during fruit maturation is high. Peng et al. (2000) observed that rainfall in September and October had an obvious effect on the fruit soluble solid content whereas less rainfall in this period increased soluble solid in navel orange. Climatic change is also sending new insects and diseases. Hot and humid weather will be the proper breeding ground for new bacterial and fungal diseases (Louime et.al. 2007) which may find a host in areas where they were nonexistent earlier. The pest Hyalestes obsoletus has been attributed to the effects of climate change in southern Germany. 3. Litchi: The observed temperature trends in the region of litchi production (Bihar) showed a general increase in temperature in order of 2-3°C overt the base period of 50 years. The unusual impact of climate change has been witnessed in litchi production system as noted in flowering pattern (shifted early), fruit growth and harvesting periods. The occurrence and the extent of

damage by physiological disorders and resurgence of pest are very much dependent on the temperature and humidity variations in the atmosphere (Kumar and Nath, 2013). 4. Grape: Grape is one such crop that is highly sensitive to climatic changes. The elevated CO2 levels may increase productivity in arid and semi-arid regions but, the drought stress caused by higher evaporative demand may override beneficial effects of increased CO2 in the atmosphere unless irrigation can be steeped up to compensate these effects. In contrast yellow rust, confined to Bangalore blue variety earlier, is now attained to epidemic proportions on both colored and colorless grapes in Bangalore and becoming a major impediment in grape cultivation. Rusts are known to become serious at higher CO2 concentration. Higher temperature may advance the ripening of berries and alter the berry composition in both table and wine grapes, thereby affecting the quality of the produce. Rainfall during flowering and fruiting is detrimental. Increased humidity due to prolonged rainfall makes fruits tasteless and there is skin cracking. Another dimension to climate change is that the pathogens may become more virulent and/or the plants may become more susceptible, thus increasing disease severity. New pathogens may also emerge or the existing once may mutate/develop resistance. Unseasonal rains may lead to serious downy mildew incidence as evidenced by decrease in productivity during the recent years from more than 25 tons per hectare to 9.2 tons per hectare in the previous season. There is also likelihood of change in the incidence and pattern of insect pests like mealy bug, thrips and mites. Similarly the disease incidence pattern is also likely to be affected with the change in climate as has been observed in case of downy mildew (NRC grape Vision 2050). In grapes, degree- days are important in determining the timing of various phenological events where temperature regime of 10°C and temperatures between 28-3 2°C are most congenial. Variations in temperature cause alterations in the developmental stages and ultimately the ripening time. Under a higher temperature regime, the number of clusters per shoot was greater and the number of flowers per cluster was reduced. In the case of the variety Cabernet Sauvignon, maximum fruit set was observed at 20/15°Cwith no fruit set at 14/9°C or 38/33°C. High temperatures also appeared to reduce monoterpenes in grape under Tunisian conditions (Zemni et al. 2005). Salinari et al. (2006) indicated that, under climate change, warmer temperature regime can significantly increase the incidence of downy

mildew pressure in grape in Italy in the coming decades, even if precipitation decreases. 5. Papaya: In papaya, higher temperatures have resulted in flower drops in female and hermaphrodite plants as well sex changes in hermaphrodite and male plants. The promotion of stigma and stamen sterility in papaya is mainly because of higher temperatures. 6. Ber: Ber can be successfully grown under varying climatic conditions but temperature below freezing is injurious to fruits as well as young plants. 7. Guava: In guava, which is grown in the tropics as well as the sub-tropics, and in strictly tropical arid crops like pomegranate, certain genes responsible for skin colour or pulp colour are not expressed under certain environmental conditions. it has been observed that red colour development on the peel of guava requires cool nights during fruit maturation. Varieties like Apple Colour, which have attractive apple skin colour under sub-tropical conditions of North India, have red spots on the skin under tropical South Indian conditions. An increase of 0.2°C in temperature resulted into dramatic reduction in the areas suitable for development of red colour in guava; an increase of 0.5°C in temperature will reduce the areas drastically with the suitability probability of more than 97% to a very low level. Based on a future climate database, predictions show that areas with suitability percentage of less than 70% will be available for red colour guava development. Rajan (2008) observed areas suitable for red coloured guava cultivation will be reduced dramatically because the minimum temperature during the coldest month may increase up to 1.9°C, whereas, the mean temperature of the coldest quarter will be 3.2°C higher than the existing temperature resulting in less red colour development in guava fruits. 8. Low chilling fruits: LC fruits being cultivated in sub-tropics are also under the threat due to non-availability of required chilling hours which has adverse effect on their flowering and the abrupt rise in temperature after fruit set is causing excessive fruit drop as well as there is poor sugar accumulation in the fruit due to steep rise in temperature during fruit development. Size and appearance, soluble solids content, total sugar content, total acids content and water content were lower in pear fruits collected from low temperature regions of china compared to high temperature regions (Chen et al1999). A new kind of symptoms should also pay attention to: changes in phenophases

dates, prunes fruit cracking, fruit drop during ripening, sun burn leaves or fruits, gummosis in fruits, fruit drying in trees, second flowering (September , October) in plum (Suranyi, 2016). 9. Pomegranate: Certain genes responsible for skin colour or pulp colour are not expressed under certain environmental conditions, aril colour turns from red to pink, and however, it is the genotype x environment interaction that ultimately decides the expression of a trait. The stability of the genotype to perform under different environment is the ultimate deciding factor in the expression of any trait. 10. Banana: In Cavendish banana, development of the golden yellow colour is affected under high temperatures, which is not the case with other cultivars. It has been decisively shown by the above examples that temperature is one of the main factors affecting gene expression for certain traits. Increased temperature will have more effect on reproductive biology and reduced water supply may affect productivity.

A. Strategies for Mitigating Effect of Climate Change on Sub-Tropical Fruit Crops 1. Breeding strategies a)

Phenotyping of all important fruits genetic wealth to enhancing tolerance to biotic and abiotic stresses, varieties and rootstocks will be evaluated to identify suitable cultivars of all major fruit crops. Rootstocks will be required which, can impart dwarfing in scions and improve yield efficiency and fruit quality, also resistant to soil borne pathogens, including those responsible for specific replant disease, and which are able to tolerate transient drought resistance e.g. Sharad seedless recorded maximum water use efficiency when budded on dogridge followed by flame seedless on dogridge rootstocks at 50% moisture stress (Satisha and Prakash, 2005). On the basis of maximum bunch size, berry weight and berry size Aulakh and Baidwan (2003) concluded that the two cultivars Shadipur local and pearl of Csaba grape were found suitable for cultivation in the arid irrigated region of

Punjab. Kaushik et al. (2004) reported that cultivar Umran produced heaviest fruit under rain fed conditions. Feronia limonia proved to be highly dwarfing and precocious at Shrirampur and Tirupati, and is thus of interest for high density planting. Rangpur lime has been identified as a promising rootstock for sweet orange cultivars like masombi and sathgudi with regard to yield and tolerance to disease and salinity in Maharashtra and Andhra Pradesh (Reddy and Rajan, 2008). At the central institute for subtropical Horticulture, lucknow interspecific hybridization between P. molle and P. guajava developed a hybrid resistant to guava wilt and graft compatible with commercial varieties of P. guajava. Polyembryonic rootstock ’131’ has been demonstrated to tolerate calcareous soil containing 20% CaCO3 and saline irrigation water containing over 600 ppm chloride. Experiments on varietal evaluation will also be conducted under natural conditions at different altitude s/conditions with natural variations in temperature and moisture falling under various agro-climatic zones of the countries. Mapping through bioclim method projected larger suitable area for Alphonso as compared to maxent. Mapping indicated that northern as well as northeastern parts of the country are not suitable for Alphonso cultivation. A part from Ratnagiri, area near Jamnagar in Gujarat has been mapped as a suitable area for Alphonso cultivation for the methods. The areas suitable for production of red colour guava were mapped with extracting climatic information from the areas of successful production and an analyses of climatic data show that areas where minimum temperature is 8-10°C or even cooler, are suitable for the production of red coloured guavas, whereas suitability map indicated that quality litchi can be produced in areas like Deoria in Uttar Pradesh; Mayurbhanj and Kendujhar in Odisha. It can be successfully grown in Betul, Chindwara, Jagdalpur districts. Normally aonla bears only one crop in a year but in some parts of the country, two harvests are possible. Mapping for the area suitable for producing two crops revealed that areas in Konkan, Sindhudurg, Ratnagiri, North Kanara, Uttar Kannada, some parts of Simoga are suitable for aonla cultivation for two crops (Rajan, 2008). b) For the last two decades molecular markers have been in extended use

in breeding programmes. These are now widely applied to carry out selection of healthy planting material. The identification of seedling prior to field planting with the desirable traits coupled with the ability to discard undesirable seedlings would greatly reduce costs and increase efficiency. The use of genetic markers is a powerful tool for accomplishing this goal. Viral diseases cause massive damage to crops. Sequence analysis and biochemical studies of coat proteins are also used in identification and characterization of viruses. The coat protein mediated resistance against virus was introduced in transgenic papaya against papaya ring spot virus (PRSV). Ghorbel et al. (2000) reported that several groups have transformed citrus with the citrus tristeza virus (CTV) coatprotein gene. Grand Nain transformed with the coat protein genes for resistance to banana bunchy top virus and banana bract mosaic virus. Resistance to CTV in citrus was obtained by Gmitter et al. (1996) by developing a marker based linkage map. Molecular markers have been used in citrus primarily to study the phytogenetic relationship among the genus, species or close relatives, cultivar identification and genetic diversity. c) The adverse effects of heat stress can be mitigated by developing crop plants with improved thermo tolerance using various genetic approaches. Heat stress affects plant growth throughout its ontogeny, though heat- threshold level varies considerably at different developmental stages. For instance, during Seed germination, high temperature may slow down or totally inhibit germination, depending on plant species and the intensity of the stress. At later stages, high temperature may adversely affect photosynthesis, respiration, water relations and membrane stability, and also modulate levels of hormones and primary and secondary metabolites. Furthermore, throughout plant ontogeny, enhanced expression of a variety of heat shock proteins, other stress-related proteins, and production of reactive oxygen species (ROS) constitute major plant responses to heat stress. In order to cope with heat stress, plants implement various mechanisms, including maintenance of membrane stability, scavenging of ROS, production of antioxidants, accumulation and adjustment of compatible solutes, induction of mitogen-activated protein kinase (MAPK) and calcium-dependent protein kinase

(CDPK) cascades, and, most importantly, chaperone signaling and transcriptional activation. All these mechanisms, which are regulated at the molecular level, enable plants to thrive under heat stress. In addition to genetic approaches, crop heat tolerance can be enhanced by preconditioning of plants under different environmental stresses or exogenous application of osmoprotectants such as glycinebetaine and proline. Acquiring thermo tolerance is an active process by which considerable amounts of plant resources are diverted to structural and functional maintenance to escape damages caused by heat stress. Although biochemical and molecular aspects of thermo tolerance in plants are relatively well understood, further studies focused on phenotypic flexibility and assimilate partitioning under heat stress and factors modulating crop heat tolerance are imperative. Such studies combined with genetic approaches to identify and map genes (or QTLs) conferring thermo tolerance will not only facilitate markerassisted breeding for heat tolerance but also pave the way for cloning and characterization of underlying genetic factors which could be useful for engineering plants with improved heat tolerance (Wahid et al, 2007). Some species have an inherent tolerance of drought because they have evolved in arid areas, regions with frequent drought, or regions with soils of low water-holding capacity. Some species have anatomical or physiological characteristics that allow them to withstand drought or to acclimate to drought. All plants have a waxy coating on their leaves called “cuticle,” but some species have developed exceptionally thick cuticles that reduce the amount of water lost by evaporation from the leaf surface. Leaf hairs, which reduce air movement at the leaf surface, are another means of reducing evaporation from the leaf. Since the amount of surface area exposed to the atmosphere affects evaporation, leaf size and thickness are other adaptations, with thicker leaves and smaller leaves being more resistant to water loss. Some species have evolved large surface root systems to quickly absorb rainfall, while other species grow deep root systems to tap deep water tables. Some plants avoid drought by dropping their leaves during droughts and quickly regrowing new leaves when environmental conditions improve. Drought resistance strategies of Ziziphus mauritiana Lamk, and peach (Prunus persica L.) were studied by

Stefan et al. (2000), under natural rainfed conditions at a field site in Zimbabwe. After a 100 days drought period, leaf water potential (oleaf) of peach trees decreased to -2.0 MPa, whereas oleaf of Z. mauritiana remained constant at -0.7 MPa. Values for the natural abundance of 13C (a13C) of bulk peach leaves as well as of total water- soluble compounds and soluble sugars of leaves increased gradually, resulting in significantly higher values as drought stress developed, indicative of increased water use efficiency (WUE). By the end of the dry season, both leaves and roots of peach exhibited osmotic adjustment, with significant accumulation of monosaccharide sugars, anions and cations in the leaves. Sorbitol and oxalate accounted for the greatest proportion of solute increases during drought, while foliar sucrose content decreased. In roots, soluble sugars such as sorbitol, glucose and fructose all increased, whereas root starch content decreased. For Z. mauritiana leaves, neither a13C values nor soluble sugar concentrations changed markedly during the study period, and Z. mauritiana plants showed no osmotic adjustment during the dry season. Data indicate that the two species exhibited different strategies for coping with soil moisture deficits under field conditions. Although Z. mauritiana exhibited the capacity for osmotic adjustment in glasshouse experiments, the trees avoided drought stress, which is an indication of a root system that has access to deeper moist soil layers. In contrast, the increased WUE in peach is likely due to stomatal control of water loss with onset of drought stress. In Actinidia arguta and A. eriantha, I-year old, when irrigation was stopped, leaf wilting of A. chinensis occurred on day 5 after treatment, followed by A. deliciosa and A. eriantha on day 6, A. arguta on day 7. Leaf scorch of A. chinensis and A. deliciosa occurred after 6 days of treatment. On the other hand leaf scorch of A. eriantha and A. arguta were recorded on the 8th and 10th day, respectively. So, A. arguta seemed most tolerant to drought. During the drought period, the photosynthetic rate did not show big differences among species until day 5 after treatment and it decreased sharply on day 10, however, that of A. arguta was slightly decreased. A. arguta and A. eriantha are considered more tolerant to drought condition than A. chinensis and A. deliciosa (Jo et al., 2008). d Crop diversification: Some generalized criteria that could be used for

developing an on-farm conservation programme could include: • Ecosystems: • Ecosystem degradation, which may result from unsustainable use of ecosystem components, pollution, pest outbreaks, or changes in fire regimes, can decrease the resilience of ecosystems to climate change. It will be important to select sites in diverse agroecosystems preferably with different ecotypes. This will increase the chances of conserving genetic diversity, as this may be associated with agroecosystem diversity. • Intra-specific diversity within target species: It is important that the areas selected are grown to different landraces. • Specific adaptations: Efforts should be made while selecting different agro-ecosystems to select sites with extreme environmental conditions (high soil salinity, cold temperatures, etc.) and variation in pests. This will help to include types with specific adaptations. • Genetic erosion: For obvious reasons, it is better to select sites with less threat of genetic erosion to increase the life of conservation efforts. • Germplasm utilization and selection: It is important to note that for many farming communities, a crop is not just a matter of food production but also an investment and is important in maintaining social relations and religious rituals. There is great diversity in germplasm of fruit crops grown in tropical, sub-tropical and temperate regions of world including India. There is an urgent need to closely monitor the genetic diversity rich areas and take appropriate measure to protect the genetic resources. The utilization of wild relative is of special concern as these are rich in genetic diversity and carry genes particularly for resistance/tolerance to biotic and abiotic stresses. Selection does not create genetic variability and most desirable selections do not always make the best parents; consequently, potential parents should be selected on the basis of progeny performance. For example in India exotic and indigenous germplasm of citrus has been widely used for development of commercial types. An indigenous oblong lemon collection, Nepali, has proved to be an excellent donor for canker

resistance in citrus hybridization programes. • Farmers and communities: Farmers’ interest and willingness to participate are keys in site selection. This may require preliminary work in community sensitization on the benefits to farmers of conserving crop varieties. Site selection should also include sites with: socio-cultural and economic diversity, diversity of livelihoods, importance of target crops for various ways of life, farmers’ knowledge and skills in seed selection and exchange, and market opportunities. e) The purpose of induced mutations is to enhance the mutation frequency rate in order to select appropriate variants for plant breeding. For the induction of mutational events in plant material, the mutation breeder can choose between two groups of mutagenic agents namely physical (X- rays, gamma rays etc) and chemical (ethyl methane sulfonate, methyl methane sulfonate, sodium azide etc). Chemical mutagens have produced high mutation frequencies in a large number of plant systems. The chemical act by alkylation of DNA i.e. phosphate group or alkylation of bases specially guanine or depurination affecting DNA duplication leading to transversion or transition type of mutation. In general horticultural crops are neglected as far as improvement is concerned. Factors like population growth, rapid industrialization and deforestation are leading to the loss of valuable germplasm for the genetic improvement of fruits. Induced mutation is highly effective in enhancing natural genetic resources and has significantly assisted in developing improved fruit cultivars e.g. most commercially important cultivars of these species have originated through natural or induced mutation (Table 1). Tablel: Mutant varieties of different fruit crops S.No. Crop

Mutant

Mutagenic agent

Characteristics

1.

Banana

Klue hom thong KUI Novaria

Gamma radiation Gamma rays

Large bunch Early maturity and better fruit quality

2.

Papaya

Pusa nanha

Gamma radiation

Dwarf

3.

Grapes

Fikreti

Gamma radiation

Early maturity and higher yield

4.

Pomegranate Khyrda and karabakh

Gamma radiation

Dwarf

5.

Japanese

Gamma radiation

Black spot disease resistant

Gold Nijisseiki

pear 6.

Grape fruit

Rio red

Thermal neutrons radiation

Red flesh and good yield

In-vitro mutation technique is also become more and more important to prevent or restrict chimera formation irradiation in combination with chemical mutagens has proved to be a valuable method in creating desirable variation followed by rapid propagation. As a result of following mutagenic treatments, a mixed bag of unexpected miracles of induced variations has been achieved in an array of horticultural crops (Velmurugam etal, 2010). Invitro mutations were attempted using gamma rays, regenerated plants resulted five resistant plants against bacterial heart rot with significant increase in total sugar content and fruit weight. In grapevine cv. Padarok Magaracha, gamma irradiation (95-100G) of leaf explants increased tetraploid plant formation frequency of primary (7%) and embryogenic callus (7.6%) and some aneuploid plants were also found. Using a combination of mutation induction, genetic and molecular characterization technique can help in identification and cloning of agronomically important mutated genes. Mutagenic treatments, super mutable genetic lines, molecular markers and high throughout DNA technologies for mutation screening such as TILLING (targeting induced limited breeding). Molecular mutation breeding will significantly increased both the efficiency and efficacy of mutation technique in crop improvement.

B. Agronomic management strategies 1. Assessment of the vulnerability and climate risks associated with subtropical fruit production: systems are vulnerable if they have low adaptive capacity. Risks and vulnerability assessment is the basis for a sound adaptation and is the basis for identification of research priorities that support future adaption policies and measures. Effective adaptation to climate change and variability is contingent on the perceptions of farmers and the ability of policy makers to merge these with scientific knowledge systems. Potentially changing climates will have considerable impact upon horticultural processes and productivity across the globe. Climate change will alter the growth patterns and capabilities for flowering and fruiting of many perennial

and annual horticultural plants. Strategies to make fruit culture more resilient to climate change through new varieties of crops, thermal resistant crops, and alternative cropping patterns capable of withstanding extremes of weather and long dry spells, flooding, and variable moisture availability through converging and integrating traditional knowledge systems, information technology, geospatial technologies and biotechnological tools to develop abiotic stress tolerant transgenic plants and use of geographic investigation system for mapping of suitable areas for future expansion of sub tropical fruits need immediate attention to overcome the ill-effects of the climate change. It will particularly focus on dry land fruit culture, risk management, access to information and enhanced financial support to farmers, insurance mechanisms, weather derivative models, and customized information in regional languages. The climate suitability models for mango varieties have been developed using genetic algorithm for rule set prediction (GARP), maximum entropy (MAXENT) and bio-climate (BIOCLIM) for determining/mapping the potential areas. The comparison of MAXENT and BIOCLIM mapping for suitable areas for cultivation of Alphonso was found similar. It showed that north and northeastern parts of the country are not suitable for this cultivar. GIS based approach was used to investigate differences in environmental factors characterizing the geographical distribution of litchi. Assessments of the impact of climate on perennial crop production need to move beyond the localregional scale and recognize that perennial crops constitute an international market system with multiple production regions that differ in terms of climate vulnerability. There will be considerable effects for aerial and edaphic microbes, which have benign and pathogenic interactions with horticultural plants. New pests and pathogens may become prevalent and damaging in areas where the climate previously excluded their activity. To mitigate the effects of climate change on losses caused by fungal pathogen, disease simulation models (DSM) that stimulates the effect of weather host growth and resistance, and fungicide use on growth and development of fungal pathogen on host needs to be developed. Such DSM predicts outbreak or change in the intensity of the disease based on weather crop pathogen or combination of the three. These forecasting models

will illustrate epidemiological principles of disease management to diverse climatic conditions. Mckenney et al. (2003) used prediction of changes in temperature and precipitation to determine the risk of spread of several introduced tree diseases, and such models can also contribute to the process of risk assessment of climate change and changes in the distribution of pathogens and diseases. 2. Improvement in the irrigation and drainage systems: drier soils and irregular precipitation will result in increased use of irrigation. Drip irrigation provides water near to the plant at low pressure and the quantity of water does not exceed to consumptive use and hence the question of wastage of water does not arise. Saline water can also be used for irrigation without allowing accumulation of salts in root zone. This method is quite economical and efficient but involves high initial investment. Singh et al. (2000) reported that drip irrigation system saved about 19 percent irrigation water and increased the fruit yield by 24 percent over surface irrigation system. Fertigation provides greater flexibility and control of nutrients and the fertilizers are applied when required and in small quantities. Fertigation is the technique of supplying dissolved fertilizers to crops through an irrigation system. When combined with an efficient irrigation system both nutrients and water can be manipulated and managed to obtain the maximum possible yield of marketable production from a given quantity of the above inputs. Fertigation trails in mango cv. Arka Anmol at ICAR IIHR showed no significant difference in the yield between 75 and 100% RDF under fertigation. Fertigation of 100% RDF gave higher no of fruit (94) and yield (17.2 kg/plant). Shikhamay (2001) focused on benefits of fertigation in grapes cultivation and reported higher yield (32t/ha), better quality and more income through fertigation as compared to soil application of fertilizers, whereas fertigated papaya plants recorded higher nutritional status, leaf N and K contents, physiological efficiency, photochemical efficiency, stomatal conductance and net photosynthesis, water use efficiency and relative water content compared with plants not subjected to fertigation. In acid lime increase percentage of plant height, plant girth and canopy volume was maximum with 100% nitrogen fertigation followed by 80% nitrogen fertigation. The average fruit weight was higher 100% N fertigation. To improve poor drainage, install underground pipes or

tiles. Improving a mal-functioning sub-surface drainage system will decrease the proportion of surface runof f. Moreover, improvement of soil structure and better root growth after the operation may be followed by rise in the yield level and lower nutrient balances. 3. Development of appropriate tillage and intercultural operations: good cultural practices have been found to stimulate the rapid plant growth and maturity of several fruit crops. Tillage and cover crops methods, as the name implies, combines tillage operation as in clean cultivation and also the growing of cover crops at suitable time, help in the conservation of moisture and nutrients, increase in organic matter and nitrogen. The soil is left uncovered for a period of about two months and this is helpful for the sterilization of the soil, the destruction of insects e.g. many insects like mango mealy bug, gall makers, leafcutting weevil, litchi nut borer, fruit fly of guava, pear, peach and ber are found to be controlled by ploughing the soil and allows cultural operations with ease. Clean cultivation eliminates weeds, is useful in avoiding the carryover of some insects- pests. Clean cultivation found promising in controlling scales and mealy bugs on citrus, mango, thirps on grapes, fruit sucking moth on citrus, pomegranate butterfly, peach leaf curl aphid on peach and hoppers on mango. Plant densities changes the micro-climate habitat in the fruit crops as closer plantation develops canopy favourable for multiplication of several insect pests in shady humid climate. The infestation of mango hopper and citrus whitefly is facilitated in close plantations. Proper pruning in perennial fruit trees, of undesirable plant parts of ten removes some active and overwintering stages of insect pests like mealy bugs and scales on several fruit crops. Pruning of infested leaves, twigs and branches and destroying the same, reduces the incidence of mango scale. Pruning may also facilitate better spray coverage or light penetration in the canopy of tree. Nearly 60-70% of the moisture is lost through evaporation. The evaporation loss can be reduced use of mulches. Mulch is any material applied on the surface to check the evaporation and conserving the moisture. Besides conserving the moisture it also alters soil temperature, reducing salinity and suppresses weed growth. Due to the composition of mulches the soil physical properties may also be improved (Srinivasulu et al., 2009). Sandy soils are known for very poor water retaining ability and having

very high percolation ability. The percolation of water can be reduced beyond root zone by placing barriers. Clay can form a good barrier and can increase water holding capacity of the soil. A barrier layer of 2-3 cm thick bentonite can be made at bottom of the pit and the side of the pit can be filled with bentonite soil mixture. Besides, regular application of organic matter will be very beneficial in improving moisture retaining power of sandy soils. Keeping plantations clear of any plants debris will be helpful in reducing the pest incidence. Fruit trees which are heavily infested with insects-pests and diseases should be up rooted and destroyed. The recent epidemic of citrus greening has devastated the citrus industry in Florida. The industry is experience the loss of about $ 9 billion and 12.7 million trees has been cut as sanitary measure (Strokes, 2006). This practice is useful against shot hole borers, trunk borers and bark eating caterpillars on various fruit crops. Inter cropping in young orchards annual intercrops may be taken for several years. Similarly in the case of other long duration horticultural crops like tapioca, turmeric, ginger will remain unoccupied by the main crop for few months. Organic farming: excessive use of chemical fertilizers and pesticides as a mean of intensive cultivation to boost up our food production has caused considerable damage to our soil health and the environment. This has focused the attention of several experts in ecologically sound viable and sustainable farming systems, known as organic farming. It is a production system which avoids or largely excludes the use of synthetically compounded inorganic chemicals. This system entirely relies on crop rotation, crop residues, animal manures, legumes, green manures, of f- farm organic wastes, biof ertilizers, mechanical cultivation etc. and aspects of biological pest control to maintain soil productivity and tilth to supply nutrients and to control insects, weeds and other pests. This system is of ten, referred as ‘biological farming’ ‘regenerative farming’ and ‘sustainable farming’ ‘eco friendly farming’ etc. 4. Integrated nutrient management (INM) is the maintenance or adj ustment of soil fertility and plant nutrient supply at an optimum level to sustain the desired crop productivity.INM system is nothing but judicious use of both chemical fertilizers and organic manures during crop growth period. INM also include the use of bio-fertilizers and

legumes in crop production. INM holds out great promise for meeting the growing nutrient demands of fruit crops and maintaining crop productivity at higher levels with an overall improvement in the quality of the resources base. For example, in Santa Rosa plum the trees under greening manuring recorded the highest non reducing sugar content in the fruits (Shylla and Chauhan, 2004). INM involves proper combination of chemical fertilizers, organic manure, crop residue, nitrogen fixing crops, pulses and oil seeds and bio-fertilizers suitable to agroecosystem. The increase in productivity of horticultural produce removes large amounts of essential nutrients from the soil. Without proper management, continuous production of crops reduces nutrients from the soil. Another issue of great concern is the sustainability of soil productivity, as land began to be intensively exhausted to produce higher yields. Overtime, cumulative depletion decreases production, yield and soil fertility and lead to soil degradation. On the other hand, excess supply or continuous use of inorganic fertilizers as source of nutrient in imbalanced proportion is also a problem, causing economic inefficiency, damage to the environment and in certain situations, harm the plants themselves and also to human being who consume them. The integrated nutrient management infuses long term sustainability in the productivity level because of availability of nutrients in soil for next season crop. Incorporation of organic fertilizers is a common practice to improve the yield of many fruit crops. It also limits chemical intervention and finally minimizes the negative impact on the wider environment. Nowsheen et al. (2006) studied the effect of INM on strawberry cv. Senhga Sengana. The treatment comprised of 5 nutrient organic treatment combinations including the recommended dose of N, P and K through chemical fertilizer as control. It was observed that poultry manure + Azotobacter + wood ash +phosphate solubilizing bacteria + oil cake gave the highest values for plant height and spread. Ram and Pathak (2007) recorded maximum number of fruits and yield from guava trees supplied with 20 kg FYM and inoculated with Azotobacter. In Sardar guava, Baksh et al. (2008) observed maximum increment in plant height, spread and girth with the treatment where 100 per cent NPK + 250 g PSB + 250 g Azotobacter were applied on the trees. Chuahan (2008) reported that the highest growth of plum

trees was recorded with the application of 80 per cent recommended dose of fertilizer + 20 kg vermicompost + 60 g biof ertilizers. Mitra et al. (2010) carried out an experiment with newly planted Sardar guava to evaluate the effect of different organic (neem cake, farm yard manure, vermicompost) and inorganic fertilizers as well as bio fertilizers (Azotobacter and Azospirillum on fruit set and yield. They recorded that the application of 50 g N, 40 g P2O5 and 50 g K2O per plant per year along with 10 kg of farmyard manure and 20 kg of Azotobacter per tree per year recorded maximum fruit set. In litchi cv. Shahi, Kumar (2010) observed that the treatment having Azotobacter (250g/tree) with half of the recommended dose of chemical fertilizer and 50 kg of FYM proved to be most dynamic substrate to record maximum percentage of quality fruit under superior grade i.e. extra class (42.75%), fruit weight and bearing of heavier fruits though found at par with control i.e. the treatment having only chemical fertilizer which recorded fruit yield of 94.50 kg/tree categorizing less percentage of quality fruits under superior grade i.e. extra class (32.10%) suggesting a possibility for reducing N, P and K fertilizer dose to the tune of 50 per cent and making quality litchi production economically more viable. Poultry manure and FYM was found effective for guava trees growth and yield (Yadva et al., 2012). 5. Fruit crops are long term crops established for commercial interest. Being long term crops fruit crops are severely infested with weeds. Weed competes with fruit trees for water, light and nutrients. The primary goal of weed management is to optimize yield by minimizing weed competition weed control is a key component of integrated orchards management. Weeds impact orchard productivity and health by competing with trees and by acting as host for a variety of pests. Integrated weed management techniques improve orchard by reducing weed growth, reduce the need for herbicides inputs, improve tree growth and yield, reduce soil erosion, improve soil tilth and provide rodent damage information. The major monocot weeds that occur in mango orchards are Cynodon dactylon and Cyperus rotundus, while dicot weeds are Bidens pilosa, Tridax procumbens and Phyllanthus madera spatensis. The mechanical method of weeding by using bullock drawn implements or motor with tractors and tillers with tools attachments are employed for effective weed control. Cover cropping

is another practice followed in mango orchard to suppress the growth of weeds, to bring additional income to the grower until the trees begin to bear and improve health of trees if intercrops grown are of right type. The recommended intercrops in mango orchards for summer season are bottle gourd, onion, chillies, cowpea black gram and green gram, pea, turnip, cauliflower, carrot, radish and gram. In papaya, weed reduced plant height and tree diameter, but not fruit yield. Suppression of weeds by plastic mulch may be used as an alternative practice to reduce the use of herbicides application, thus reducing potential chemical contamination in upland agro-ecosystem. Weed suppression was linked to light inception by the mulch cover for most weed species. Subterranean clover planted directly in the vine row significantly reduced weed cover where it established (Steinmaus et al, 2008). 6. Water harvesting is the general name used for all the different techniques to collect runof f or flood water for storage in the soil prof ile or in tanks so that it can be used for the production of crops, trees or fodder. Under rainfed conditions, soil moisture stress during summer and post monsoon periods appears to be one of the major constraints for economic yields of most of the fruit crops particularly, in lands with poor water holding capacity. By adopting simple techniques for water spreading and infiltration by means of low, permeable bunds, which follow the contour lines, a considerable part of the water requirement can be met during the season. The bunds of crescent shaped or semi-circular are prepared. The diameter depends on size of the trees in question. Catch-pits are also dug at the same time on the upper of the slope. The trees are planted in the centre of the crescent. Crescent bunds collect rainwater; the catch-pits conserve the same. New pits are opened as the old ones get filled with silt or organic matter. In the regions with limited water supplies, efforts should be made to conserve the moisture whatever is received through rains. Conservation tillage or mulch tillage in modern usage is a crop and soil management practice that will utilize the residue mulches of the preceding crop by leaving a large percentage of this organic residue on or near the surface of the ground as protective mulch. Ridge and furrow in situ water harvesting is yet another promising system for the inceptisols of subhumid areas. The inter row water

harvesting system is a practice suitable for light textured soils found in arid regions. Trenches of 0.5 to 1.0m deep are dug. The soil in the bottom of the trench is properly loosened and fruit trees such as seedling or grafts of ber, aonla and custard apple are planted. Rain water is collected along with silt and organic matter in the trench and this moisture is available for further growth of plantain. Sharma et al. (2000) observed the highest significant (p