Genetic and molecular analysis of two new loci controlling flowering in garden pea By A S M Mainul Hasan School of Natural Sciences Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy University of Tasmania, July 2018 Declaration of originality This thesis contains no material which has been accepted for a degree or diploma by the University or any other institution, except by way of background information and duly acknowledge in the thesis, and to the best of my knowledge and belief no material previously published or written by another person except where due acknowledgement is made in the text of the thesis, nor does the thesis contain any material that infringes copyright. Authority of access This thesis may be made available for loan. Copying and communication of any part of this thesis is prohibited for two years from the date this statement was signed; after that time limited copying and communication is permitted in accordance with the Copyright Act 1968. Date: 6-07-2018 A S M Mainul Hasan i Abstract Flowering is one of the key developmental process associated with the life cycle of plant and it is regulated by different environmental factors and endogenous cues. In the model species Arabidopsis thaliana a mobile protein, FLOWERING LOCUS T (FT) plays central role to mediate flowering time and expression of FT is regulated by photoperiod. While flowering mechanisms are well-understood in A. thaliana, knowledge about this process is limited in legume (family Fabaceae) which are the second major group of crops after cereals in satisfying the global demand for food and fodder. Due to its short generation time, ability to reproduce via self or cross-pollination and availability of diverse lines, garden pea (Pisum sativum) serves as a model legume species for flowering time studies. Isolation and characterization of mutants have been a key research strategy in order to identify genes responsible for flowering in pea. The current study involved investigation of two novel EMS mutants namely late3 and late4 in the background of a cultivated pea line NGB5839 which are extremely late flowering indicating that LATE3 and LATE4 are essential for normal promotion of flowering in pea. Detailed phenotypic characterization carried out in the present study showed that both the loci positively regulate various vegetative, reproductive and yield related traits across different growth stages strongly suggesting potential global regulatory role for the underlying genes. Prior to the present study, synteny between pea and a closely related species, M. truncatula was exploited in order to roughly map LATE3 and LATE4 at the middle of pea LGIII and bottom of LGV respectively. These positions were significantly refined in the present study as LATE3 and LATE4 were mapped within a narrow genetic intervals in the syntenic regions of M. truncatula consisted of 62 (chromosome 3) and 54 (chromosome 7) genes respectively. Usage of the high-throughput RNA sequencing technology along with phylogenetic, co- segregation analysis and direct sequencing assisted in determining LATE3 and LATE4 genes as the pea homologue of Cyclin Dependent Kinase 8 (PsCDK8) and Cyclin C (PsCYCC1). CDK8 and CYCC1 are components of highly conserved CDK8 module (along with MED12 and MED13) of eukaryotic mediator complex that negatively regulate transcription of various genes involved in different biological processes. Analysis of the entire mediator complex revealed that this ii complex is highly conserved in A. thaliana, M. truncatula and pea. Phenotypic characterization of loss-of-function mutant of novel flowering gene CYCC1 in A. thaliana exhibited delayed flowering similar to already known similar mutants of AtCDK8, AtMED12 and AtMED13 which was consistent with the notion that these four genes act together to carry out the same regulatory process. Genetic interaction and yeast two hybrid assay unveiled complementarity and strong physical interaction between LATE3 and LATE4 which strongly suggested that these two genes act in the same regulatory process in a likely inter-dependent manner. Further genetic and regulatory interaction studies involving already known key pea flowering loci showed that LATE3 and LATE4 mediate flowering by regulating expression of known genes such as FTa1, FTc, PIM (PsAP1), VEG1(PsFULc), UNI (PsLFY)and VEG2 (PsFD) and LF (PsTF1c). The present study also undertook a systems biology approach for generating relevant hypothesis about the function and regulation of LATE3 and LATE4 genes. To this end, STRING v10.5 predicted the potential interactome network of AtCDK8 and AtCYCC1 consisted of members from cell cycle and mediator complex indicating a conserved role for them. In addition, predicted functions of MtCDK8 and MtCYCC1 genes obtained from AraNET v2.0 database was similar to that of PsCDK8, PsCYCC1, AtCYCC1 genes, thus giving hints of potential conservation of the function of both the genes in plant system. Moreover, PlantTFDB v4.0 database was used to forecast the transcription factors that may regulate the function of CDK8 and CYCC1 genes in M. truncatula and pea. As various mediator complex genes in A. thaliana are known to be involved in the regulatory processes driving response to different environmental factors, therefore relevant experimentation have shown that both LATE3 and LATE4 genes are important for controlling response to general light/darkness, ambient temperature, UVB, heat, mechanical wound and salt stress in pea. Overall, the present study provided significant understanding about the role of LATE3/PsCDK8 and LATE4/PsCYCC1 in mediating diverse range of reproductive, developmental and adaptive characteristics in pea where flowering time was the most important trait. iii Acknowledgements During my journey as a PhD student at the School of Natural Sciences, University of Tasmania I have received assistance from many individuals and I would like to humbly convey my gratitude to all of them. First of all, I would like to thank my primary supervisor and mentor Dr. Jim Weller who always managed time for me whenever I sought guidance for any aspects of the project be it laboratory/glasshouse work, writing the thesis or attending conferences. I indeed believe that I have become a better scientist over the past three and half years where his leadership played a significant role. Likewise, I am thankful to my second supervisor Dr. Valerie Hecht whose technical expertise and in-depth knowledge about various aspects of flowering time regulation have contributed immensely towards my development as a PhD student. Special thanks goes to my colleague Jackie Vander Schoor who has played significant role in the development of this project at different stages. Another person who has offered guidance and inspiration regarding my academic responsibilities and ownership of my PhD project is my graduate research coordinator Dr. Erik Wapstra and therefore I am beholden to him. Next, I would like to acknowledge helps that I have received from my colleague Raul Ortega for useful discussion about various issues related to my PhD project. I am also conveying similar humility to Dr. Lu Wang specifically for the A. thaliana part of the project. In addition, I am grateful for various suggestions and scientific brainstorming by my former and current group mates Dr. Frances Sussmilch, Dr. Steven Ridge, Dr. Vinodan Rajendran, Owen Williams, Beatriz Contreras, Andy Rubenach and Kelsey Picard. Colleagues in the glass house Michelle Lang and Tracy Winterbottom were fantastic and cooperative and their help from time to time is highly appreciated. The same humbleness goes to recognize the assistance of colleagues from CSL molecular biology laboratory Adam Smolenski and Sharee McCammon. I would also like to thank the bigger family of the School of Natural Sciences specially those with whom I worked as part of the school’s student organization in organizing various social events in the first year of the PhD. In addition, I would like to thank University of Tasmania and Australian Research council for providing financial iv support for this project. Moreover, I am thankful to Australian Society of Plant Scientists (ASPS) for providing grant in order to be able to attend the Combio 2017 conference in Adelaide. I would like to specifically mention the contributions being made by my previous professional mentors Prof. Dr. Dr. Wolfgang Friedt, late Dr. Patrick Schweizer and Dr. Thorsten Schnurbusch towards my development which enabled me to undertake intensive plant molecular genetics based research in the form of a PhD. I am highly indebted to my lovely wife who constantly provided support and took care of various aspects of my life during this period. At this specific junction of my life, I would also like to convey utmost respect and gratitude to my mother, late father and elder brothers without whom I would not have been where I am today. Besides, I would like to thank my fellow compatriots living in Hobart for their consistent assistance and care about different matters during my stay as a PhD student in this city. v Conference proceedings from this project Invited talk: A.S.M Mainul Hasan, Valerie Hecht, Jacqueline K. Vander Schoor, Jim Weller, October 30-November 1, 2017. Genetic and molecular analysis of two new loci controlling flowering in garden pea, Pisum sativum. In: Australasian Genomics Technologies Association Conference, Hobart, Australia. Poster presentation: A.S.M Mainul Hasan, Valerie Hecht, Jacqueline K. Vander Schoor, Jim Weller, October 4-6, 2017. Genetic and molecular analysis of two new loci controlling flowering in garden pea, Pisum sativum. In: Combio 2017, Adelaide Convention Centre, Adelaide, Australia. vi Abbreviation 3AT 3-amino-1,2,4-triazole ABA Abscisic acid AFR SAP30 FUNCTION-RELATED AGL18 AGAMOUS LIKE 18 ANOVA Analysis of variance AP1 APETALA1 bp Base pair bZIP Basic leucine zipper CAPS Cleaved amplified polymorphic sequence CDF CYCLING DOF FACTOR CDK8 Cyclin Dependent Kinase 8 cDNA Complemetary Deoxyribonucleic Acid cM Centi Morgan CO CONSTANS Col-0 Columbia 0 CRY2 CRYPTOCHROME 2 CSM Cryptic splice motif CSS Cryptic splice site cv.