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MESSENGER-RNA DECAY; VERNALIZATION; TRANSCRIPTION; TRANSLATION; POLYMERASE; IMMUNITY; MUTANTS; GENESPlant Sciences; Arabidopsis thaliana; CRISPR/Cas9; FLOWERING LOCUS M (FLM); flowering time; splice isoforms; temperature-dependent alternative splicing;
Abstract:
FLOWERING LOCUS M (FLM), a component of the thermosensory flowering time pathway in Arabidopsis thaliana, is regulated by temperature-dependent alternative splicing (AS). The main splicing variant, FLM-beta, is a well-documented floral repressor that is down-regulated in response to increasing ambient growth temperature. Two hypotheses have been formulated to explain how flowering time is modulated by AS of FLM. In the first model a second splice variant, FLM-delta, acts as a dominant negative isoform that competes with FLM-beta at elevated ambient temperatures, thereby indirectly promoting flowering. Alternatively, it has been suggested that the induction of flowering at elevated temperatures is caused only by reduced FLM-beta expression. To better understand the role of the two FLM splice forms, we employed CRISPR/Cas9 technology to specifically delete the exons that characterize each splice variant. Lines that produced repressive FLM-beta but were incapable of producing FLM-delta were late flowering. In contrast, FLM-beta knockout lines that still produced FLM-delta flowered early, but not earlier than the flm-3 loss of function mutant, as would be expected if FLM-delta had a dominant-negative effect on flowering. Our data support the role of FLM-beta as a flower repressor and provide evidence that a contribution of FLM-delta to the regulation of flowering time in wild-type A. thaliana seems unlikely.
