COMPARATIVE PROTEOMIC ANALYSIS APPLIED FOR STUDY OF MORPHOGENIC COMPETENCE DURING SOMATIC EMBRYOGENESIS IN SUGARCANE ANGELO SCHUABB HERINGER UNIVERSIDADE ESTADUAL DO NORTE FLUMINENSE DARCY RIBEIRO – UENF CAMPOS DOS GOYTACAZES - RJ OUTUBRO – 2016 COMPARATIVE PROTEOMIC ANALYSIS APPLIED FOR STUDY OF MORPHOGENIC COMPETENCE DURING SOMATIC EMBRYOGENESIS IN SUGARCANE ANGELO SCHUABB HERINGER “Tese apresentada ao Centro de Ciências e Tecnologias Agropecuárias da Universidade Estadual do Norte Fluminense Darcy Ribeiro, como parte das exigências para obtenção do título de Doutor em Genética e Melhoramento de Plantas.” Orientador: Prof. Dr. Vanildo Silveira CAMPOS DOS GOYTACAZES - RJ OUTUBRO – 2016 COMPARATIVE PROTEOMIC ANALYSIS APPLIED FOR STUDY OF MORPHOGENIC COMPETENCE DURING SOMATIC EMBRYOGENESIS IN SUGARCANE ANGELO SCHUABB HERINGER “Tese apresentada ao Centro de Ciências e Tecnologias Agropecuárias da Universidade Estadual do Norte Fluminense Darcy Ribeiro, como parte das exigências para obtenção do título de Doutor em Genética e Melhoramento de Plantas.” Aprovada em 25 de outubro de 2016. Comissão Examinadora: _________________________________________________________________ Prof. (D.Sc., Fitotecnia) Virginia Silva Carvalho– UENF _________________________________________________________________ Prof. (D.Sc., Ciências Biológicas) Valdirene Moreira Gomes – UENF _________________________________________________________________ Prof. (D.Sc.,Ciências Biológicas) Miguel Pedro Guerra– UFSC _________________________________________________________________ Prof. Vanildo Silveira (D.Sc., Biotecnologia) – UENF (Orientador) ACKNOWLEDGMENTS I would like to thank the Universidade Estadual do Norte Fluminense Darcy Ribeiro for the quality of education I would like to thank the Genetic and Plant Breeding Graduate Program for the quality of education and for all the support given when I was abroad. I would like to thank my adivisor, Professor Ph.D. Vanildo Silveira, which allowed laboratory structure and education to complete my entire PhD project, and also encouraged me to stay abroad, for one year, as a visiting scholar in University of California Davis. I would like to thank my counselors Professor Ph.D. Gonçalo Apolinário de Souza Filho and Professor Ph.D Virginia Silva Carvalho for education and all suggestions given to my Ph.D project. I would like to thank the Laboratório de Biotecnologia (LBT) and the Unidade de Biologia Integrativa UENF for the laboratory structure that allowed the development of my entire Ph.D. project. I would like to thank the Grupo de Pesquisa em Biotecnologia Vegetal UENF for the scientific collaborations and friendship. I would like to thank the research funding and scholarships were supported by the Carlos Chagas Filho Foundation for Research Support in the State of Rio de Janeiro (FAPERJ), the Coordination for the Improvement of Higher Education ii Personnel (CAPES), and the National Council for Scientific and Technological Development (CNPq). I would like to thank my family for all care and incentives to keep up this research career. I would like to thank all my friends for the familiarity and laughs time. iii SUMMARY RESUMO .............................................................................................................. vii ABSTRACT ............................................................................................................ ix 1. INTRODUCTION .............................................................................................. 1 2. OBJECTIVES ................................................................................................... 4 2.1 General objective ....................................................................................... 4 2.2 Specific objectives...................................................................................... 4 3. CHAPTERS......................................................................................................... 5 3.1 LABEL-FREE QUANTITATIVE PROTEOMICS OF EMBRYOGENIC AND NON-EMBRYOGENIC CALLUS DURING SUGARCANE SOMATIC EMBRYOGENESIS ............................................................................................. 5 3.1.1 INTRODUCTION ..................................................................................... 5 3.1.2 REVIEW .................................................................................................. 9 3.1.2.1 Sugarcane ............................................................................................ 9 3.1.2.2 Somatic embryogenesis in sugarcane ................................................ 10 3.1.3 MATERIALS AND METHODS ............................................................... 11 3.1.3.1 Plant material ...................................................................................... 11 3.1.3.2 Maturation treatment........................................................................... 12 3.1.3.3 Total protein extraction ....................................................................... 13 3.1.3.4 Protein digestion ................................................................................. 13 3.1.3.5 Mass spectrometry analysis ............................................................... 14 3.1.3.6. Histological analysis .......................................................................... 15 iv 3.1.4 RESULTS .............................................................................................. 16 3.1.4.1 Maturation and morphogenetic changes............................................. 16 3.1.4.2 Protein identification during maturation treatment .............................. 20 3.1.4.3 Protein functional classification during maturation treatment .............. 27 3.1.5 DISCUSSION ........................................................................................ 29 3.1.5.1 Maturation and morphogenetic changes............................................. 29 3.1.5.2 Unique proteins that are abundant in embryogenic (E-0) callus ......... 31 3.1.5.3 Unique proteins that are abundant in maturated embryogenic (E-21) callus .............................................................................................................. 35 3.1.5.4 Unique proteins abundant in non-embryogenic (NE-0) and maturated non-embryogenic (NE-21) callus .................................................................... 36 3.1.5.5 Common proteins in embryogenic and non-embryogenic callus ........ 37 3.1.6 CONCLUSION ....................................................................................... 38 3.2 COMPARATIVE PROTEOMICS ANALYSIS OF THE EFFECT OF COMBINED RED AND BLUE LIGHTS ON SUGARCANE SOMATIC EMBRYOGENESIS ........................................................................................... 40 3.2.1 INTRODUCTION ................................................................................... 40 3.2.2.1 Somatic embryos maturation .............................................................. 42 3.2.2.2 Temporary immersion system (TIS).................................................... 43 3.2.2.3 Light emitting diode (LED) .................................................................. 46 3.2.2.4 Proteomics in sugarcane somatic embryogenesis .............................. 47 3.2.3 MATERIAL AND METHODS ................................................................. 48 3.2.3.1 Plant Material ...................................................................................... 48 3.2.3.2 Maturation treatment with combinations of red and blue wavelengths 49 3.2.3.3 Somatic embryo conversion with a RITA temporary immersion system combining red and blue wavelengths ............................................................. 50 3.2.3.4 Plantlet elongation and acclimatization ............................................... 50 3.2.3.5 Protein extraction ................................................................................ 51 3.2.3.6 Protein digestion ................................................................................. 51 3.2.3.7 Mass spectrometry analysis ............................................................... 52 3.2.4 RESULTS AND DISCUSSION .............................................................. 54 3.2.4.1 Maturation and conversion of somatic embryos with different combinations of red and blue wavelengths ..................................................... 54 v 3.2.4.2 Proteomic analysis of maturation callus subjected to different combinations of red and blue wavelengths ..................................................... 60 3.2.5 CONCLUSION ....................................................................................... 72 REFERENCES...................................................................................................... 74 APPENDICES ....................................................................................................... 93 APPENDICE A ................................................................................................... 93 APPENDICE B ................................................................................................. 130 vi RESUMO HERINGER, Angelo Schuabb; D.Sc.; Universidade Estadual do Norte Fluminense Darcy Ribeiro; Setembro, 2016; Comparative proteomic analysis applied for study of morphogenic competence during somatic embryogenesis in sugarcane Orientador: Prof. Dr. Vanildo Silveira; Conselheiros: Prof. Dr. Gonçalo Apolinário de Souza Filho e Profa. Dra. Virginia Silva Carvalho. O desenvolvimento