Abstract
Understanding how plants integrate environmental and endogenous cues to ensure flowering occurs at a time of highest reproductive success is of significant interest. Flowering time is a key trait in agriculture as flowering determines when crops can be harvested. Medicago truncatula (Medicago) is a legume model plant that is an important food crop for humans and livestock. Therefore, Medicago was used in this study to investigate flowering time, as detailed understanding of flowering time control in this plant family is lacking. Using a forward approach, a late flowering mutant (MtpΦbs) was characterised using RNA sequencing and a single nucleotide change, which produces an early stop codon that disrupts the function of MtPΦBS, was identified as a likely cause for the late flowering phenotype. MtPΦBS functions to synthesize the phytochrome chromophore required for phytochrome activity. Expression analysis identified that the known promotors of flowering FT orthologs MtFTa1 and MtFTb1 were not expressed in the MtpΦbs mutant. This confirms that phytochrome activity is important for regulating expression of the FT genes, and the control of flowering in Medicago. Other potential upstream regulators of the FTs were also investigated. The legume specific protein MtEl was found to be downregulated in the MtpΦbs mutant, which indicates that MtEl promotes FT expression and flowering in Medicago. In contrast E1 represses flowering in the short-day legume soybean, suggesting that the difference in flowering between long day and short day legumes could be based on how E1 functions in each legume species. Finally, attempts were made to identify or produce an MtFTb1 mutant using CRISPR/Cas9 gene editing technology, to further characterise its role in flowering time of Medicago. Although no mutant was identified, this work has helped establish and develop Medicago tissue culture and regeneration protocols in the Macknight laboratory.