Molecular Characterisation of Jatropha curcas; Towards an Understanding of its Potential as a Non-edible Oilseed-Based Source of Biodiesel Siam Popluechai Thesis submitted for the Degree of Doctor of Philosophy Faculty of SAgE Newcastle University Newcastle upon Tyne School of Biology November 2010 Molecular Characterisation of Jatropha curcas; Towards an Understanding of its Potential as a Non-edible Oilseed-Based Source of Biodiesel I certify that no part of the material offered has been previously submitted by me for a degree of other qualification in this or any other University. Siam Popluechai November 2010 ABSTRACT Jatropha curcas, a non-edible oilseed, is becoming popular as a source of biodiesel. However, there are some limitations to using J. curcas such as the presence of carcinogenic substances, unpredictable yield, asynchronous flowering and plant height. Understanding the genetic control of these characteristics will help to find solutions. To accumulate genetic information for J. curcas plant breeding programmes, the genetic and phenotypic characterisation of J. curcas seeds obtained from different geographical locations was undertaken (Chapter 2). The results showed a narrow genetic diversity among accessions but apparent phenetic diversity. The screens employed ranged from phenotypic characterizations of seeds to molecular, biochemical, physiological and genotyping assessments. Seed dimensions and weight measurements provided data on seed density and kernel weight, both important parameters for assessing oilseed quality. Biochemical analysis provided data on oil properties relevant to biodiesel e.g. total oil content, oleic/linoleic acid ratio, saponification number, cetane number and gross energy value. Expression analysis of genes (acc, sad, fad, and dgat) essential for the quantity and quality of seed/oil were targeted as molecular screens. Understanding the molecular mechanism of lipid cycling in J. curcas seeds will guide future improvements in oil yield. However, this is still an area of active research because little information exists for this in J. curcas. With regards to oil storage capacity of seeds, no information previously existed on the biology of oil bodies in Jatropha curcas. In this study, oil bodies from J. curcas were studied at the gene transcript and protein levels (Chapter 3). Three Jatropha oleosin genes were isolated. Sequence analysis showed that all three oleosin genes contained the proline knot conserved domain. The upstream regulatory element of one Jatropha oleosin gene was also isolated. In silico analysis of this upstream sequence indicated the presence of a number of seed specific regulatory motifs. Transcript analysis for the three oleosin genes revealed tissue-specific differences. Immunoassay by western blot analysis using antibodies against Arabidopsis oleosin and the J. curcas specific oleosin revealed a diversity of oleosins. Proteomic analysis of oil bodies revealed additional minor proteins including caleosin, steroleosin, aquaporin and curcin. Information from these studies is essential for varietal improvement of Jatropha and/or developing oleosin gene fusion technology for expression of novel compounds. In this study 623 nucleotides upstream of the JcOleosin3 gene have been isolated and characterized in transgenic Arabidopsis plant using β-glucuronidase (GUS) as the reporter gene (Chapter 4). Result showed that the JcOleosin3 promoter directs ii expression of the β-glucuronidase gene in seed and pollen but not in leaves, root, stem or flower. In addition, in transgenic Arabidopsis, the JcOleosin3 promoter drives GUS activity at the early seedling stage; this was expected based on earlier reports of expression of Jatropha oleosins. To understand the molecular basis of seed germination, proteomic studies were performed on resting and germinating seeds of J. curcas. Such studies using 2-DE and MALDI-TOF MS revealed the presence of patatin-like lipases in the germinating seeds but not in the resting seeds (Chapter 5). These proteins showed sequence similarity and conserved domains as found in the patatin lipases from Solanum tuberosum and Arabidopsis thaliana. Patatin lipases play a significant role in the mobilization and lipolysis of TAGs during the germination of oil seeds. Further, there is little knowledge on the triacylglycerides (TAGs) lipid cycling in J. curcas., Bioinformatic data-mining confirmed the TAGs cycling in J. curcas to include the expected stages of synthesis, storage and lipolysis of fatty acids. Based on the data-mining results for the presence of various genes for TAG storage, synthesis and utilization, a scheme is presented for TAG cycling in J. curcas. The scheme presented in this study provides essential information required for oil manipulation in J. curcas. iii Acknowledgements I wish to express my sincere gratitude and appreciation to Dr Ajay Kohli for his supervision, encouragement, guidance, friendship throughout my study. I also would like to express my sincere gratitude to Professor Angharad M. Gatehouse for her supervision, support and encouragement throughout my PhD study. I would like to express my gratitude to Dr Diego Breviario (IBBA-CNR, Italy) for his valuable guidance, support and collaboration. I am also grateful to Dr. Thierry Chardot (INRA, France) and Dr. Maria Manuela R. Costa (Portugal) for their collaboration on oleosin work and their permission for using our publication as a part of my thesis I would like to express my sincere thanks to Professor Anthony G. O‘Donnell for his initial support and guidance for my PhD study. I would like to thank the following authors who allowed me to use our publications as a part of my thesis: Professor John K. Syers, Dr Sujatha Mulpuri, Dr Harinder P.S. Makkar, Dr Attipalli R Reddy, Dr Enrico Palchetti, Dr Marine Froissard, Dr Pascale Jolivet, Mr Manish Raorane, Miss Udhaya Kannan, Mr. Ibrahim Khalil Adam, Mr João Alexandre Pereira Raimundo. I would like to thank Dr Silvia Gianni for her help and valuable guidance throughout my work at IBBA-CNR. I would like to thank Dr Prapassorn D. Eungwanichayapant (Mae Fah Luang University, Thailand) for financially supporting part of the work on oleosin genes expression carried out in Thailand. I also appreciate the help and pleasant working atmosphere from members of the Plant-Insect Interaction Laboratory, Newcastle University. I would like to thank members of Ecotub Laboratory, Milan for their help during my stay at IBBA-CNR. I also wish to thank Mr Nikolaos Tsakirpaloglou, Mr Manish Raorane, and members of the Thai society in Newcastle for their sincere friendship. I am greatly indebted to the Royal Thai Government who provided me with a PhD scholarship. Finally, I wish to express my special thanks to my family and friends for their love, understanding, support and encouragement. I dedicate this thesis to my mother whom I love the most. iv PUBLICATIONS RELEVANT TO THIS THESIS Popluechai S, Raorane M, Syers KJ, O‘Donnell AG. Kohli A. 2008. Research needs to make Jatropha a viable alternate oilseed crop. Proceedings of the International Workshop on Feasibility of Non-Edible Oil Seed Crops for Biofuel Production, May 25 – 27, 2007, Mae Fah Luang University, Chiang Rai, Thailand. Eds. Syers KJ and Wood D. MFLU publication, p 56 - 60 Popluechai S., Chardot T., Gatehouse AMR., Kohli A. Characterization of Jatropha curcas oil bodies using a systems biology approach. Jatropha International Congress 2008, 17-18 December 2008, Grand Copthorne Waterfront Hotel, Singapore Popluechai, Siam, Breviario, Diego, Mulpuri, Sujatha, Makkar, Harinder, Raorane, Manish, Reddy, Attipalli, Palchetti, Enrico, Gatehouse, Angharad, Syers, Keith, O‘Donnell, Anthony, and Kohli, Ajay. Narrow genetic and apparent phenetic diversity in Jatropha curcas: initial success with generating low phorbol ester interspecific hybrids. Available from Nature Precedings <http://hdl.handle.net/10101 /npre.2009.2782.1> (2009) Kohli A, Raorane M, Popluechai S, Kannan U, Syers KJ, O‘Donnell AG. 2009. Jatropha curcas as a novel, non-edible oilseed plant for biodiesel. In: Ferry N, Gatehouse AMR, eds. Environmental impact of genetically modified novel crops. Chapter 14 CAB International, London, UK. p 294 -322 Siam Popluechai, Marine Froissard, Pascale Jolivet, Diego Breviario, Angharad MR Gatehouse, Anthony G O'Donnell, Thierry Chardot, Ajay Kohli. (2010) Understanding Jatropha curcas seed oil storage components: characterization of seed oilbody oleosins. Plant Physiology and Biochemistry (accepted with minor modification, 05/10/10) Siam Popluechai, João Alexandre Pereira Raimundo, Angharad M.R. Gatehouse, Maria Manuela R. Costa, Ajay Kohli (2010) Jatropha seed-specific, JcOleosin3 gene, promoter in transgenic Arabidopsis. Manuscript under preparation v TABLE OF CONTENT STATEMENT OF ORIGINALITY…………………………………..…………….…i ABSTRACT………………………………………………………….……………….ii ACKNOWLEDGEMENTS………………………………………………………......iv PUBLICATIONS RELEVANT TO THIS THESIS…………………………….….....v TABLE OF CONTENTS……………………………………………………………..vi LIST OF FIGURES………………………………………………………………...….x LIST OF TABLES…………………………………………………………………...xiv Chapter 1: Literature review: Jatropha curcas as a novel, non-edible oilseed plant for biodiesel 1.1 Summary…………………………………………………………………..…1 1.2 The need for renewable sources of energy………………………………..….2 1.2.1 Depletion of non-renewable resources………………………………....2 1.2.2 Environmental damage……………………………………………..…..3 1.2.3 Rising prices……………………………………………………….…...4 1.3 The case for renewables……………………………………………………...5