Mutant and transcriptomic analysis of candidate photoperiodic flowering pathway genes in Medicago truncatula

Abstract

Legumes are an important staple food group with numerous environmental and health benefits associated with their cultivation and consumption. However there is significant potential for genetic improvement across the family with flowering time likely to play a substantial role, given its notable influence on plant productivity. Work in the long-day and vernalisation responsive reference species Medicago truncatula has identified a FLOWERING LOCUS T (FT) homologue MtFTa1 as a key component of the transition to flowering in cool season legumes. Whether additional FT-like genes present in M. truncatula also participate in the regulation of flowering time remains unclear, as is how MtFTa1 and the other FT-like genes are regulated since homologues of the core regulators of FT in Arabidopsis thaliana are absent. This thesis investigates the role the additional FT-like genes play in the regulation of flowering in M. truncatula and evaluates potential regulators, predominantly via screening of mutants originating from the Tnt1 retrotransposon mutant population maintained by the Noble Research Institute (Ardmore, OK, USA). In the course of this study I identified a novel Mtfta1 mutant which, consistent with previously described mutants, caused severe late-flowering and a Mtftb2 mutant with only a very slight late flowering phenotype in long-day photoperiod conditions. Efforts to identify mutant lines which knocked-down MtFTa2 and MtFTb1 were unsuccessful. I also screened Tnt1 lines for insertions in potential FT-like gene regulators MtE1 and MtFE. Disruption of MtE1 caused a modest delay in flowering in one out of two previously reported mutants indicating that MtE1 plays a less prominent role in M. truncatula flowering time regulation than the homologue E1 does in tropical legume Glycine max. Additionally, a Mtfe Tnt1 line was found to have a late flowering phenotype in long-day photperiod conditions. Functional assays found that MtFE can complement the A. thaliana fe mutant and that the MtFE protein can interact with NUCLEAR FACTOR Y proteins in yeast-2-hybrid assays. This is similar to FE/ALTERED PHLOEM DEVELOPMENT (APL) in A. thaliana suggesting a conservation of function in M. truncatula. Lastly I analysed existing RNA-Seq datasets to identify novel flowering time candidate genes. This analysis characterised the transcriptomic response in M. truncatula leaves to a short-day to long-day photperiod shift. The results presented in this thesis contribute to the growing understanding of how flowering time is regulated in legumes, which differs significantly from previously studied species.