MAF2 Is Regulated by Temperature-Dependent Splicing and Represses Flowering at Low Temperatures in Parallel with FLM

Abstract

Plants enter their reproductive phase when the environmental conditions are favourable for the successful production of progeny. The transition from vegetative to reproductive phase is influenced by several environmental factors including ambient temperature. In the model plant Arabidopsis thaliana, SHORT VEGETATIVE PHASE (SVP) is critical for this pathway; svp mutants cannot modify their flowering time in response to ambient temperature. SVP encodes a MADS-box transcription factor that directly represses genes that promote flowering. SVP binds DNA in complexes with other MADS-box transcription factors, including FLOWERING LOCUS M (FLM), which acts with SVP to repress the floral transition at low temperatures. Small temperature changes post-transcriptionally regulate FLM through temperature-dependent alternative splicing (TD-AS). As ambient temperature increases, the predominant FLM splice isoform shifts to encode a protein incapable of exerting a repressive effect on flowering. Here we characterize a closely related MADS-box transcription factor, MADS AFFECTING FLOWERING2 (MAF2), which has independently evolved TD-AS. At low temperatures the most abundant MAF2 splice variant encodes a protein that interacts with SVP to repress flowering. At increased temperature the relative abundance of splice isoforms shifts in favour of an intron-retaining variant that introduces a premature termination codon. We show that this isoform encodes a protein that cannot interact with SVP or repress flowering. At lower temperatures MAF2 and SVP repress flowering in parallel with FLM and SVP, providing an additional input to sense ambient temperature for the control of flowering.

Fig 1. Comparison of flowering time in WT and mutant backgrounds.

A,B,C, the columns indicate the number of rosette leaves (in black) plus the number of cauline leaves (in grey). Plants were grown at 21°C. A, Comparison of flowering time between WT and maf2, maf2 svp and svp mutants. (no. of plants analyzed 161) B, Flowering time of WT and plants overexpressing MAF2var1 in WT and svp backgrounds (no. of plants analyzed 100, four independent transformants for each transgenic line). C, Flowering time of flc maf2 double mutants compared to svp mutants (no. of plants analyzed 92). D, Quantitative RT-PCR of FT in WT and maf2 backgrounds. Error bars represent the standard error.

Fig 2. MAF2 splice variants.

MAF2 splice variants observed by RT-PCR are shown with the names used in this report and alternative names reported elsewhere (Rosloski 2013, Ratcliffe 2003 and TAIR website). The names are followed by a schematic representation of the genomic structure of each splice variant. Rectangles represent exons and lines represent introns. The premature termination codon introduced as a result of intron 3 retention in MAF2var2 is indicated (TAA).

Fig 3. MAF2 splice variant analysis.

A, D, Expression of MAF2var1, MAF2var2 and MAF2var5 splice variants at different temperatures analysed by RT-PCR. Elongation Factor (ELF) is used as a control. B, C, Flowering time of plants overexpressing MAF2var2 or MAF2var5 compared to WT at 21°C (no. of plants analyzed 47 and 53, four independent transformants for each transgenic line). The columns represent the number of rosette leaves (in black) plus the number of cauline leaves (in grey). Error bars represent the standard error.

Fig 4. Flowering time of WT and maf2 mutants at different temperatures.

(no. of plants analyzed 257) The columns represent the number of rosette leaves (in black) plus the number of cauline leaves (in grey). Error bars represent the standard error.

Fig 5. Flowering time of WT and maf2, flm, flm maf2 and svp mutants at 16°C.

(no. of plants analyzed 147) The columns represent the number of rosette leaves (in black) plus the number of cauline leaves (in grey). Error bars represent the standard error.