Functional analysis of candidate flowering time genes from the model legume Medicago truncatula
Flowering at optimal times promotes the success of plant sexual reproduction and agronomic productivity and yield. Legumes are the second most important group of crop plants, but their flowering gene networks are less well understood than in the Brassicaceae and Poaceae. Medicago truncatula (Medicago) is a legume model plant with powerful genetic resources and which flowers in response to long day length and vernalisation (winter cold) conditions. A putative Medicago Polycomb Repressive Complex 2 (PRC2) subunit VERNALISATION2 (MtVRN2) was characterised previously in the Putterill laboratory, whose mutation led to early flowering. This indicated that MtVRN2 represses the transition to flowering in Medicago, opposite to VRN2 function in Arabidopsis thaliana (Arabidopsis). In addition, candidate floral integrator and homeotic genes had elevated expression in the Mtvrn2 mutant. An attempt was made to further characterise MtVRN2 function by analysing a candidate protein interactor CURLY LEAF (CLF, a candidate PRC2 member) using yeast two hybrid and pull down assays and selected candidate target genes (FLOWERING LOCUS Ta1, APETALA1, AGAMOUS-LIKE11 and SEPALLATA3b) using Chromatin Immunoprecipitation (ChIP)-PCR. In addition, a second candidate PRC2 member Medicago EMBRYONIC FLOWER2 (EMF2) was aimed to be functionally characterised. Third, the function of six candidate flowering genes related to SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1) and FRUITFULL (FUL), some with elevated expression in the Mtvrn2 mutant, was examined by ectopic expression in Arabidopsis. The results of ChIP-PCR using transgenic Medicago plants overexpressing epitope HA-tagged MtVRN2 showed that MtVRN2 did not appear to bind directly to the selected candidate target genes. The protein interaction studies suggested that MtVRN2 might not physically interact with MtCLF via their VEFS and C5 domains respectively. The investigation of the cDNA clones of three MtEMF2-like genes revealed several different potential coding sequences from the annotated genomic sequence. Functional analysis revealed that MtEMF2 did not appear to play the same role as AtEMF2 in flowering time control. Three MtFUL genes and three MtSOC1 genes were identified in Medicago, as opposed to one of each in Arabidopsis. The overexpression of these six genes in Arabidopsis indicated the potential role of three of them, MtFULa, MtFULb and MtSOC1a in flowering time control because they accelerated Arabidopsis flowering.
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