Molecular Genetic Study On The Involvement Of IQM3 In The Regulation Of Flower Formation In The Photoperiodic Pathway

In Arabidopsis thaliana,each member of IQM family contains an IQ motif through which IQM can bind with calmodulin,IQM3 is one of them,which is encoded by At3g52870.IQM3 participates suppression of flowering in short day;CO（CONSTANS）is a key gene involved in the integrated regulation of photoperiodic and circadian rhythm signals;Phy B is a light receptor that senses red light and regulates the stability of CO protein.Hence,they are involved in the regulation of flowering through the photoperiodic pathway in Arabidopsis.In this study,genetics,molecular biology,bioinformatics,plant physiology and other research methods were employed to research on the molecular mechanism of IQM3 mediating calmodulin signalling in floral regulation,the photoperiodic expression pattern of IQM3,and the genetic relationship between IQM3 and Phy B and CO,And the following results were obtained.1)Determination of IQM3 mediating calmodulin signaling to regulate flowering.The point mutation complementary expression vector of IQ motif,the key motif of calmodulin binding with IQM3,was successfully constructed,and introduced to iqm3-2 successfully.The transgenic plants failed to complement the mutation of IQM3 due to their dwarf phenotype.Therefore,it has not been determined that calmodulin signalling mediated by IQM3 was involved in flowering regulation.It is speculated that IQM3 is toxic protein.When possessing a normal IQ motif,Ca M can bind to it and inhibite its toxicity.When possessing a abnormal IQ motif,Ca M cannot bind to it and release its toxicity,severely inhibiting plant growth and development.2)IQM3 may not be involved in flowering regulation by temperature pathway in Arabidopsis.Neither high temperature（27°C）nor low temperature（16°C）affected the main flowering phenotypes of iqm3-1 and iqm3-2,such as rosette leaf number,flowering time and flowering time course,indicating that IQM3 is not involved in the temperature-pathway flowering regulating in Arabidopsis.3)IQM3 has a photoperiodic expression pattern and its mutations affect the photoperiodic expression patterns of key genes and circadian rhythm genes.The results from RT-q PCR analysis showed that the longer the illumination time,the higher the expression level of IQM3,while the longer the darkness time,the lower its expression level.It is indicated that the expression of IQM3 is regulated by the photoperiod.Moreover,mutation of IQM3 altered the expression patterns of photoperiod key genes CO,FT,and important circadian rhythm genes LHY,GI,TOC1,CCA1,FKF1,and ZTL.4)The IQM3 mutation did not affect hypocotyl elongation under red light.Hypocotyls of iqm3-1 and iqm3-2 were not significantly different from the wild type when grown under continuous red light conditions at 1.41μmol/m²·s^-1and 0.17μmol/m²·s^-1for 5,10 or 15 days.5)A new CO（At3g 52870）mutant,co-12,has been discovered..The sequencing results showed that 9 bases of"ACTTGCTAG"were missing in the first exon of the gene,containing the restriction endonuclease Bfa I cleavage site"CTAG",based on which a molecular marker for the identification of the mutant was established.The phenotype analysis results indicate that co-12 has important phenotypes of other mutants of the gene,namely late flowering under long day,early flowering under short day,and short root in seedling.This mutant can be subsequently used to construct the double mutants of CO and IQM3.6)IQM3 functions downstream of Phy B to regulate CO expression is involved in short-day inhibition of flowering.RT-q PCR analysis showed that the transcript level of Phy B are unaffected and that of CO are upregulated in iqm3-2;that of IQM3 were down-regulated and that of CO were up-regulated in phyb-9;the transcript levels of both Phy B and IQM3were unaffected in co-12.It is inferred that IQM3 acts downstream of Phy B and upstream of CO under short day.When grown in short day condition,the double mutant phyb-9 iqm3-2flowered earlier than iqm3-2,and its flowering phenotype was biased towards phyb-9,suggesting that the iqm3 did not affect the early flowering phenotype of phyb,and indicating that IQM3 acts downstream of Phy B to inhibite flowering.Under the same conditon,the double mutant co-12 iqm3-2 flowered earlier than iqm3-2 and later than co-12,and its flowering phenotype was biased towards co-12,suggesting that the early flowering phenotype of the iqm3 required CO,and indicating that IQM3 inhibits flowering through CO namely IQM3 acts upstream of CO.These confirm the results of the above gene expression analysis.In summary,IQM3 exhibits a photoperiodic expression pattern,functions downstream of Phy B to inhibite CO expression,and ultimately inhibits flowering in short day.This study provides the molecular genetics evidence for the involvement of IQM3 in the regulation of flowering in the photoperiodic pathway.