Preliminary Study On Arabidopsis Thaliana IQM3 Involved In Photoperiod Flowering Regulation

Flowering is the core of individual development of higher plants.Flowering process marks the transformation of plant from vegetative growth stage to reproductive development stage.Flowering is precisely regulated by the network of flowering-regulating genes.There are six genetic regulation pathways in the whole network of flowering-regulating genes: temperature,age,gibberellin,vernalization,photoperiod and autonomous pathway.These pathways not only interact with each other,but also independently regulate the flowering of plants.Like other IQM family members,AtIQM3 encoded by AT3G52870 contains a calmodulin binding protein with an IQ motif.Its loss-of-function mutants iqm3-1 and iqm3-2 show early flowering under short day condition,while their flowering period is similar to that of wild type under long day condition,suggesting that IQM3 may regulate flowering through photoperiod pathway.In this study,physiological experiments,gene expression analysis and yeast two-hybrid were used to study the role of IQM3 in the regulation of photoperiod flowering.The following results were obtained:1)Identification of mutants of IQM3.Homozygotes of iqm3-1 and iqm3-2 were obtained by resistance screening and molecular identification.Backcross experiments confirmed that iqm3-1 and iqm3-2 were single gene mutation.2)Preliminary determination of genetic pathways involved in flowering regulation in IQM3.iqm3-1 and iqm3-2 showed an early-flowering phenotype under short-day conditions,but no phenotype under long-day conditions,suggesting that IQM3 might be involved in the regulation of photoperiod flowering.The cotyledon openness of iqm3-1 and iqm3-2 was significantly different form that of wild type Col-0 under red light,although there was no phenotypes in hypocotyl length and seed germination rate under blue light and far red light.It is further suggested that IQM3 is involved in the regulation of photoperiod flowering.In addition,the flowering process of iqm3-1,iqm3-2 and Col-0 was basically the same in vernalization treatment,indicating that IQM3 was not involved in the regulation of vernalization pathway.The flowering time of iqm3-1,iqm3-2 and Col-0 was basically the same when treated withPaclobutrazol(PAC),an inhibitor of gibberellin biosynthesis,which indicated that IQM3 was not involved in the regulation of gibberellin pathway flowering.It was also found that the sensitivity of iqm3-1,iqm3-2 and wild-type seeds to PAC was similar,further indicating that IQM3 was not involved in the synthesis of gibberellin.The structure and the number of epidermal hair of iqm3-1 and iqm3-2 was not different from that of wild-type seeds,indicating that IQM3 was not involved in the regulation of age-related flowering.3)The effect of IQM3 mutation on the expression of key genes in flowering.Compared with wild-type Col-0,iqm3-1 and iqm3-2 showed a high transcriptional levels of CO,FT,SOC1 and LFY,key factors of photoperiod flowering pathway,but the same levels of key genes in other pathways.Meanwhile,FT protein levels of iqm3-1 and iqm3-2 were up-regulated in both leaf and stem apical tissues.These results suggest that IMQ3 participates in the photoperiodic flowering of Arabidopsis,which further confirms the results of phenotypic observation.4)Analysis on the binding activity of IQM3 calmodulin.The yeast two-hybrid experiment showed that IQM3 can to CaM5.When the first amino acid residue V in IQ motif of IQM3 mutated into E,however,two proteins cannot bind with each other,indicating that IQ motif and its first amino acid V were the key to the binding of IMQ3 and CaM.5)In addition,the complementary expression vectors of iqm3 were constructed and transferred into iqm3-1 and iqm3-2 plants,and the homozygous complementary lines were screened out.The overexpression vectors of IQM3 were also constructed and transferred into Col-0 plants,and the homozygous overexpression lines were screened out,too.Under long-day conditions,the flowering time and number of rosette leaves of the complementary lines were basically the same as Col-0,while the overexpression lines flowered later and had more rosette leaves than wild type.