UNIVERSIDADE FEDERAL DO CEARÁ CENTRO DE CIÊNCIAS DEPARTAMENTO DE BIOQUÍMICA E BIOLOGIA MOLECULAR PROGRAMA DE PÓS-GRADUAÇÃO EM BIOQUÍMICA MOHIBULLAH SHAH PROTEOME ANALYSIS OF DEVELOPING SEEDS OF Jatropha curcas L. FORTALEZA 2014 MOHIBULLAH SHAH PROTEOME ANALYSIS OF DEVELOPING SEEDS OF Jatropha curcas L. Tese apresentada ao Curso de Doutorado em Bioquímica do Departamento de Bioquímica e Biologia Molecular da Universidade Federal do Ceará, como parte dos requisitos para obtenção do título de Doutor em Bioquímica. Área de concentração: Bioquímica vegetal. Orientador: Prof. Francisco A. P. Campos. Co-orientador: Prof. Fabio C. S. Nogueira. FORTALEZA 2014 Dados Internacionais de Catalogação na Publicação Universidade Federal do Ceará Biblioteca de Ciências e Tecnologia S537p Shah, Mohibullah. Proteome analysis of developing seeds of Jatropha curcas L. / Mohibullah Shah. – 2014. 168 f. : il., color., enc. ; 30 cm. Tese (doutorado) – Universidade Federal do Ceará, Centro de Ciência, Departamento de Bioquímica e Biologia Molecular, Programa de Pós-Graduação em Bioquímica, Fortaleza, 2014. Área de Concentração: Bioquímica vegetal. Orientação: Prof. Dr. Francisco de Assis de Paiva Campos. Coorientação: Prof. Dr. Fabio César Sousa Nogueira. 1. Plantas oleaginosas. 2. Biodiesel. 3. Pinhão-manso. 4. Espectrometria de massa. I. Título. CDD 574-192 ACKNOWLEDGEMENTS First of all I would like to thank to ALLAH (SWT) , for blessing me good health, courage, strength during the difficult moments and making me enable to reach here. I would also like to thank and appreciate some people who directly or indirectly support me in successful completion of this work. First and foremost, I express my wholehearted gratitude to my respected supervisor, Prof. Francisco A. P. Campos (Prof. Chico), for his trust, unfading interest, consistent encouragement, stimulating suggestions, expert advices, guidance and above all for his friendly relationship throughout this period. I am also thankful to his family, especially his wife Dr. Ursula Wille , for her respect and loving attitudes. Thanks to Prof. Gilberto B. Domont (UFRJ), for keeping the doors of his lab (Proteomic Unit) open for me whenever I visited, also for his friendship, guidance, encouragement and being a source of inspiration. My gratitude to his wife Prof. Solange Guimaraes (UFRJ), for her loving and friendly attitude, and for inviting me a number of times in her home during my stay in Rio de Janeiro. Special respect for Prof. Fabio C.S. Nogueira (UFRJ), my friend and co- supervisor, among the few competent young scientists I met so far, for his guidance and sharing of knowledge with me. In each of our discussions I always learned new things from him. I am also very thankful for his continuous guidance and help in the development of this project. Warm thanks to Dr. Paulo C. Carvalho (FIOCRUZ-PR), a competent young scientist and a great example of simplicity, for his help and collaboration in the analysis of data. I highly acknowledge his prompt reply whenever I contacted him for assistance in data analysis during the entire period of my PhD course. I am thankful to Prof. Arlete A. Soares (Department of Biology, UFC), for her help in histological analysis of the seeds. My sincere compliments to Dr. Jonas E. A. Perales together with Prof. Gilberto, Prof. Fabio and Dr. Paulo , for accepting the invitation to be a member of the evaluation committee of my PhD thesis. I appreciate Emanoella Lima Soares (Manu) and Camila B. Pinheiro (minha amigas de mais tempo), for their great company, friendship, fruitful discussions, and continuous support since my first day in the Lab. I want to specially thank Manu , for her help in the analysis of data and preparation of this thesis. My appreciation for Dr. Fabiano M. Texeira (meu amigo de mais tempo), and his wife Alice and mother Antonia, for the friendship , accepting me as a member of their family, inviting me in all the moments of happiness and for the help I needed during difficult moments. I am also thankful to Fabiano, for sharing his apartment with me after my arrival in Brazil, and for his great company. Thanks to all the former and new members of the Bioplant lab , including Veronica, Magda, Carlos, Isabelle, Washington, Antonio, Tiago and Muciana , for their pleasant company in the lab. I would like to extend my special thanks to all the members of the Proteomic unit (UFRJ), especially Gabriel and Prof. Magno , for their help in conducting mass spectrometry experiments. I am also thankful to my friends Anwar and Rizwan (UFRJ) for giving me space in their apartment, for their moral support during my stay in Rio de Janeiro and for conducting the mass spectrometry experiments. Special regards to my close friends, Mr. Javeed (South Korea), Dr. Umar (Unicamp), Dr. Asif (USP) and Dr. Asifullah (China) for their great moral support during completion of this work. Very special respect to my loving parents , who have always been so close to me and I found them whenever I needed, for their unconditional love, dedication and support throughout my carrier; a continuous source of motivation to set higher goals in life. Sincere regards to my late grandfather Dr. Mohammad Shah , for his love and efforts, in providing me with quality education that enabled me to reach here. Without his support, life would have been very difficult. I am thankful to my brothers and sisters for their love and support. I would like to pay my very special thanks to my life partner , for her loving company, that greatly helped me in understanding the true meaning of life and without her moral help the completion of my PhD would not have been possible. Thank you for every thing. My Parents in law, for their affection, dedication and love. I am also thankful to my Brother in law Mr. Noman , for solving all my problems, especially bureaucratic things that I needed from Pakistan. Special thanks to Third World Academy of Sciences (TWAS) and Brazilian National Council for Scientific and Technological Development (CNPq), for their financial support. Kind regards to the Department of Biochemistry and Molecular Biology of Plants , Federal University of Ceará and to the Department of Biochemistry , Institute of Chemistry, Federal University of Rio de Janeiro, for providing the infrastructure. ABSTRACT Physic nut ( Jatropha curcas L.) is an important crop due to its ability of storing high content of oil in the seeds, which can serve as raw material for biodiesel production. Because of the presence of toxic constituents like phorbol esters (PEs) and curcins, the seed cake produced as a result of oil extraction cannot be utilize for animal feed. Development of the genotypes better suited for the industrial applications and biodiesel production as well as with lower level of toxic constituents is being hampered by a lack of understanding about the a) proteins related to the biosynthesis and degradation of fatty acids (FAs) and triacylglycerides (TAGs), b) role of proteins deposited during seed development and c) proteins related to the synthesis and storage of toxic compounds during seed development. Agreeing with this, we have performed the anatomical analysis of the developing seeds of J. curcas , followed by the proteome analysis of the endosperm isolated from the seeds of J. curcas at five different developmental stages, which resulted into the identification of the 1517, 1256, 1033, 752 and 307 proteins, from Stage 6, 7, 8, 9 and 10, respectively, summing up to a total of 1760 proteins. Proteins with similar expression pattern were grouped into five different clusters and protein quantification based on spectral counts was determined. Besides identification of the proteins involved in the biosynthesis and degradation of the FAs and TAGs, we also identified a large number of proteins involved in the metabolism of the carbohydrates, which are important for supplying energy and carbon source for the synthesis of TAGs in heterotrophic seeds. Among the members of different classes of seed storage proteins (SSPs), we have identified four SSPs named as nutrients reservoir, which in contrast to the other SSPs showed decreasing deposition pattern during seeds development and revealed to have special role during seed development. In addition, peptidases belong to different mechanistic classes were identified, which have a range of functions, highlighting the role in reserve mobilization during germination. Isoforms of curcin were also identified in this proteome analysis which were absent in our previous proteome analysis of the other tissues from these seeds, suggesting that the deposition of these toxic proteins only occur in the endosperm. Similarly, several enzymes involved in the biosynthesis of diterpenoid precursors were identified in this proteome analysis but, like in our previous proteome analysis of the other tissues from J. curcas seeds,we were unable to identify any terpene synthase/cyclase, enzymes responsible for the synthesis of PEs, which collectively suggesting that the synthesis of PEs may not occur in seeds of this plant. In conclusion, the strategy used here enabled us to provide a first in depth proteome analysis of the endosperm from J. curcas developing seeds, which along with providing information regarding important aspects of the seed development, also set the foundation of a proteomic approach to study biotechnologically important plant species. Key words: Endosperm, proteome, oilseeds, biodiesel, mass spectrometry, seed development, seed proteins RESUMO Pinhão manso ( Jatropha