Analysis Of The Mechanism For Calcium-Dependent Protein Kinase32 In Flowering Time Regulation In Arabidopsis

In all eukaryotes,Ca2+ plays critical roles in maintaining the charge balance.Calcium is not only an important structural component but also serves as one of the most second ubiquitous secondary messengers.As one kind of the calcium sensors,CDPKs play important roles in responses to various internal and environmental signals,including development,hormones,nutrition metabolism,pathogen elicitors and abiotic stresses.Flowering is the crucial transition from the adult vegetative state to the reproductive state.Floral initiation in appropriate conditions is required for seeds production and the survival of the species.Floral initiation is regulated by a very fine and complicated network.In agriculture,crop flowering on schedule is a decisive factor for breeding and the production improvement.Based on the late-flowering phenotype of cpk32 mutant,we explored the role of the calcium dependent protein kinase mediated phosphorylation in Arabidopsis flowering time regulation.We found cpk32 mutant flowering was later in long/short photoperiod conditions when compared to wild type plants.cpk32 mutant positively respond to vernalization and GA treatment.It is speculated that CPK32 may regulate flowering time through autonomous pathway.Northern blot results showed that the expression of FLC,a key regulatory factor in the autonomous pathway,was significantly increased in cpk32 mutant.Meanwhile,the expression of FT and SOC1,which are down-regulated by FLC,were decreased in cpk32 mutant.Repressing the expression of FLC in cpk32 background(cpk32 flc)reversed the late-flowering phenotype.Our data suggest that CPK32 regulates the flowering time through autonomous pathway via the repression of FLC.Y2H,Pull-down and BiFC analyses further confirmed that CPK32 interacts with FCA.In vitro phosphorylation assay demonstrated that CPK32 phosphorylates the C terminal of FCA.Northern blot indicated the expression of FLC in cpk32fca double mutant was more close to that in fca single mutant.As known that FCA regulates the splicing processing of its own pre-mRNA.RT-qPCR analyses showed that the ratio of FCA-y?/FCA-? was decreased in cpk32 mutant.These data suggest that CPK32 affects the functional regulation of FCA.In addition,FCA regulates microRNA172 processing in thermosensory flowering.Northern blot analyses showed that the expression of microRNA172 decreased in cpk32.This implied that CPK32 participated in FCA mediated microRNA172 processing.In summary,CPK32 promotes floral transition through the autonomous pathway via the repression of FLC.