Functional Analysis Of IQM2 On The Autonomous Pathway Flowering Time Regulation In Arabidopsis

Arabidopsis IQM2 encoded by AT3G13600 is a calmodulin-binding protein?CaMBP?containing one IQ motif.Under conventional experimental condition,mutants of IQM2 flowered later than wild type.Flowering on time is very crucial for most plants to successfully survive and reproduct.Both environmental and genetic factors can regulate flowering time in plants.The flowering regulators constitute at least six closely interlinked but divergent genetic regulation pathways including photoperiod,vernalization,ambient temperature,gibberellin acid,autonomous and aging pathways.Study on the genetic regulation pathway of flowering in which IQM2involves has been reported in this paper and the main results are as follows:1.A transient overexpression vector,P_35s:IQM2-GFP was constructed and transferred into protoplasts of mesophyll cel.The results showed that IQM2 localizes in chloroplast and nucleus.2.Using PCR and RT-PCR,two novel mutant iqm2-3 and iqm2-4 with T-DNA insertion were identified.The results showed that both iqm2-3 and iqm2-4 are loss-of-function mutants.3.The flowering phenotype of iqm2-3 and iqm2-4 was characterized under long–day condition.iqm2-4 had one more rosette leaf than wild type when flowering,while no such addition was observed in iqm2-3.Average flowering time of iqm2-4was 2-3 days later than Ler,while iqm2-3 was 0-1 days later than the Col.These resukts further confirm that IQM2 was related to the flowering regulation.4.Screened by antibiotic,4 complemented iqm2-2 lines were identified and their flowering phenotype was observed.Under long-day condition,iqm2-2 was 3 days later than Ler,and the complemented iqm2-2 lines were similar to Ler.iqm2-2 had 1-2more rosette leaves than wild type,the phenotype of complemented iqm2-2 kines were similar to Ler.These resukts suggest that late-flowering phenotype of iqm2-2resultes from the disruption of IQM2.5.Using the RT-PCR and RT-qPCR,relative expression profiles of IQM2 and FLC at different processes,as well as other autonomous pathway genes at key stage of flowering were analysed.The results showed that IQM2 was expressed stronger at bolting time and significantly down regulated after bolting,suggesting its involvement in flowering regulation.FLC expression profile in iqm2-2 and Ler was similar to that of IQM2,however,the expression level of FLC in iqm2-2 was nuch higher than that in Ler,which is consistent with the late-flowering phenotype of the mutant,and further confirms its involvement of autonomous flowering pathway.In addition,the expression level of other genes in the autonomous flowering pathway do not display significantly differences between iqm2-2 and Ler,indicating that IQM2 is a novel regulator of autonomous pathway and may inderectly promote flowering through suppression of FLC.6.An overexpression vector for plants P_35s:HA-IQM₂ was constructed.In summery,IQM2 is involved in autonomous flowering pathway of Arabidopsis.