Study On Seed Dormancy Regulated By Calmodulin-binding Protein IQM4

Ca²⁺is an important second messenger in plant cells,which plays an important role in plant growth,development and stress response.ABA is involved in regulating physiological processes such as seed maturation,dormancy and germination,stomatal movement,seedling growth and development,and mediates rapid response of plants to stress.ABA signal and calmodulin signal play important regulatory functions in plant growth and development and stress response.In our previous study,we found that Arabidopsis calmodulin-binding protein IQM4 contains a highly conserved calmodulin-binding domain--IQ motif,which binds to CaM5 in a Ca²⁺independent manner.IQM4 was involved in the regulation of endogenous ABA content and ABA signal in seeds.The seed dormancy of the mutant iqm4 was decreased,and the seed germination and stomatal closure of the mutant were insensitive to exogenous ABA.Therefore,it is speculated that IQM4 may regulate endogenous ABA synthesis and ABA signal in seeds by mediating CaM signal,and is a positive regulator of ABA signal in plants.In order to further investigate whether IQM4 regulates ABA synthesis and ABA signaling in seeds by mediating CaM signals,three studies were conducted as follows and the following main results were obtained:(1)To construct a functional complementary line of iqm4-2 and a complementary line of IQM4^?143-144with the IQ motif deletion mutation.By analysis of the seed dormancy characteristics and seed germination rate of the IQM4 and IQM4^?143-144gene complementary lines,To explore whether IQM4 is involved in ABA-regulated seed dormancy and germination by mediating CaM signals.The results showed that compared with the wild type,the seed dormancy and seed germination rate of IQM4/iqm4-2 were not different,which proved that IQM4 was complementary to the phenotype of the mutant.However,the growth and development of IQM4^?143-144complementary lines were seriously abnormal,indicating that the binding of IQM4 to CaM is a necessary condition for the function of IQM4.(2)Calmodulin-like At CML37 is the only CaM signaling gene that has been reported to regulate endogenous ABA levels.Do CML37 and IQM4 participate in the regulation of ABA levels and signaling in the same signaling pathway?In this study,the double mutant cml37iqm4-2 was constructed using CRISPR/Cas9 technology,and the seed dormancy,seed germination and seedling growth of the double mutant were analyzed.Genetic studies showed that the dormancy of the double mutant cml37iqm4-2 was lower than that of the wild type,and the dormancy of the single mutant iqm4-2 and cml37 was lower than that of the double mutant.It was speculated that both CML37 and IQM4 were positively regulating the dormancy process of seeds,but they might be antagonistic.Under the treatment of exogenous ABA,Na Cl and mannitol,the seed germination rate of single mutant cml37 and double mutant cml37iqm4-2 was lower than that of wild type,on the contrary,the seed germination rate of iqm4-2 was higher than that of wild type,suggesting that IQM4 and CML37were also involved in the response of seed germination to exogenous ABA,salt and osmotic stress.IQM4 is a positive regulator,while CML37 is a negative regulator.It is speculated that CML37 mediated signal is downstream of IQM4 mediated signal.Single mutants cml37 and iqm4-2 and double mutants cml37iqm4-2 showed weak ABA resistance and similar phenotypes in seedling growth,but no difference in phenotypes between them under Na Cl and mannitol stress,suggesting that IQM4 and CML37 may be involved in the response of seedling growth to exogenous ABA in the same signaling pathway.(3)It has been reported that ABI3 is a major regulator of seed dormancy and germination,and enhances the expression of ABA synthase genes NCED6 and NCED9 in the middle and late stages of seed development,with functional redundancy in NCED6 and NCED9.In this study,a double mutant abi3iqm4-2 and a triple mutant nced6nced9iqm4-2 were constructed using CRISPR/Cas9 technology,and the possible mechanism of IQM4 regulating endogenous ABA synthesis in seeds was investigated by genetic analysis.The results showed that the dormancy degree of the two mutants,abi3iqm4-2,was not different from that of the wild type,while the dormancy degree of the two mutants,ABI3-8 and iqm4-2,was lower than that of the wild type.The dormancy degree of the single mutant nced6 was lower than that of the wild type,but the dormancy degree of the double mutant nced6iqm4-2 was not different from that of the wild type,and the dormancy degree of the double mutant nced9iqm4-2 was lower than that of the wild type.The results showed that IQM4,ABI3,NCED6 and NCED9 positively regulated seed dormancy process.The results showed that single mutant abi3 and iqm4-2 were not sensitive to ABA,and the phenotype of double mutant abi3iqm4-2 was not different from that of wild type.The phenotypes of nced6 and nced6iqm4-2 were not different from those of the wild type.It is speculated that IQM4 and ABI3 also regulate the response of seed germination to exogenous ABA,but the function of IQM4 and ABI3 may be antagonistic,and it is speculated that IQM4 does not participate in the regulation of seed ABA synthesis through ABI3 signaling pathway.