Screening And Validation Of IQM3-Interacting Proteins In Arabidopsis Thaliana

Arabidopsis IQM3(IQ motif-containing protein 3)is the third member of the IQM family,and contains one IQ motif.Previous studies have shown that iqm3-1 and iqm3-2,the loss-of-function mutants of IQM3,flower early under short day,indicating that IQM3 may be involved in the inhibition of flowering in short day,but the mechanism involved is not yet clear.In this paper,the yeast two-hybrid technique(Y2H),bimolecular luciferase complementation(Bi LC),and immunoprecipitation-MS(IP-MS)were used to search for the interacting proteins of IQM3,to provide molecular evidences for further understanding of the involvement of IQM3 in the regulation of flowering through photoperiodic pathway,and to provide materials and references for construction the signaling network mediated by IQM3.The main results obtained are as follows.1)The calmodulin binding activity of IQM3 was confirmed by using yeast twohybrid technique.Firstly,bioinformatics analysis revealed that its spatial structure was favorable for IQ motif to bind calmodulin(Ca M).Secondly,by yeast two-hybrid analysis,IQM3 was found to bind to Ca M1.1,Ca M2.1,Ca M3.1,Ca M4.1,Ca M5.1,Ca M6.1,Ca M7.1.Finally,the ability of IQM3 to bind to Ca M1.1 in plants was verified by using Bi LC assay.The above results demonstrate that IQM3 is a calmodulin-binding protein and that the binding of IQM3 to Ca M is dependent on the spatial structure with Ca M in additional to the IQ motif.2)IQM3 can bind to BBX30 and BBX31 in yeast.Using yeast two-hybrid assay,it was verified that IQM3 bind to BBX30 and BBX31,the repressors of CO,a key regulator of the photoperiod flowering pathway,in yeast,providing a molecular basis for the involvement of IQM3 in the regulation of photoperiod pathway flowering.3)Y2H and Bi LC assays revealed that IQM3 can bind to HCF244,HSP17.6A,HIR2 and CAT2 in yeast and in planta,indicating that IQM3 may be involved in calcium signaling,peroxide scavenging,photosynthetic,combination of male and female gametophyte,and stress response.4)To identify the factors that interact with IQM3 and participate in the regulation of flowering in short day in planta during the critical phase of flowering by IP-MS,vectors harbouring IQM3-GFP or GFP driven by the IQM3 promoter and Ca MV35 S promoter were introduced into iqm3-2 respectively.Preliminary phenotypic observations showed that the primary root lengths of the seedlings of the plants transformed with IQM3-GFP were similar to the wild type,and significantly shorter than that of iqm3-2.It suggested that the transfer of IQM3-GFP into iqm3-2 had complemented the IQM3 mutation,further indicating that the transferred IQM3-GFP was performing normal functions.These transgenic plants can be used in the subsequent IP-MS screening for IQM3-interacting proteins in planta.5)Preliminary microscopic observation on the above PIQM3:IQM3-GFP transformd plants showed that IQM3 is mainly present in the epidermal cells of leaf.This study demonstrated that IQM3 has calmodulin-binding activity,and it can bind to Ca M family members in yeast and plants;IQM3 binds to BBX30 and BBX31,repressors of the key regulator of the photoperiodic flowering pathway CO,in yeast.This provides a reference for further analysis of the involvement of IQM3 in photoperiodic regulation of flowering.IQM3 may be mainly present in epidermal cells.IQM3 also binds to HSP17.6A and HIR2 in yeast;binding to HCF244,and CAT2 both in yeast and in plants.suggesting that IQM3 may also be involved in biological processes such as calcium signaling regulation,peroxide scavenging,photosynthesis,combination of male and female gametophyte and response to environmental stresses.It also provides a reference for a comprehensive understanding for the biological functions of IQM3.