Study On Arabidopsis IQM1 And IQM4 Involved In The Regulation Of Stomatal Movement

Stomata are the gateways for the exchange of gas and moisture between plants and the external environment,and they play an important role in plant life activities.Previous studies have shown that there are 6 members of the IQM gene family of calmodulin-binding proteins in Arabidopsis,among which IQM1 is involved in stomatal movement regulated by light and ABA,and IQM4 is involved in ABA-induced seed dormancy and ABA-inhibited seed germination,which positively regulates seeds ABA signal transduction.In the IQM family of Arabidopsis,the amino acid sequence similarity of IQM1 and IQM4 is 86%.It is speculated that IQM1 and IQM4 may have similar functions of stomatal movement regulation.This paper plans to conduct in-depth research on the molecular mechanism of Arabidopsis IQM1 and IQM4 involved in the regulation of stomatal movement on the basis of previous research,including five aspects of research:?1?By analyzing the stomatal phenotypes of iqm1,iqm4 and iqm1iqm4 mutants and iqm4 complementary strains and IQM4 overexpressing strains,it is clear that IQM1 and IQM4 jointly participate in stomatal movement regulated by light and ABA;?2?By carrying out the pharmacological experiment of the rosette leaf skin strips of iqm1,iqm4 and iqm1iqm4 mutants and the experiment of determining the active oxygen content of guard cells,we analyze the signal pathways of IQM1 and IQM4 regulating stomatal movement.?3?By studying the difference in the transcriptome and ABA signal gene expression of rosette leaves of iqm1 and iqm4 mutants,we analyze the molecular mechanism of IQM1 and IQM4 regulating stomatal movement;?4?By knocking out ABA and ROS signal gene GHR1 of iqm1 and iqm4 mutants using CRISPR/Cas9technology,we determine the genetic pathways of IQM1 and IQM4 regulating stomatal movement;?5?By constructing IQM4-GFP and IQM1-GFP transgenic plants,we analyze the expression of IQM1 and IQM4 protein.The results of the paper are as follows:?1?Under light conditions,the stomatal opening of iqm4 deletion mutants was significantly smaller than that of wild-type,and ABA cannot induce the stomatal closure.The stomatal phenotypes of IQM4 overexpression lines and iqm4complemented lines were the same as wild-type.iqm1iqm4 double mutant and single mutant had the same phenotype,which proves that IQM4 and IQM1 have the same stomatal movement regulation function,and there may be the same regulation pathway.?2?Epidermal strip pharmacological experiments and ROS content analysis found that the loss of IQM1 and IQM4 functions increased the level of ROS in guard cells.IQM1 depended on Ca²⁺and IQM4 does not depend on Ca²⁺to participate in regulating stomatal movement,and IQM1 is located upstream of IQM4.?3?Transcriptome analysis found that there were significant differences in the expression of environmental stress response genes in the rosette leaves of the iqm1mutant under light conditions,while there were significant differences in the expression of the cells/cell components and environmental stress response genes of the iqm4 mutant.The differentially expressed genes of leaves participate in the biosynthesis of secondary metabolites of the iqm1 and iqm4 mutant.RT-qPCR analysis showed that light enhanced the expression of IQM1 and IQM4 in rosette leaves,and 100?M ABA treatment for 6 h inhibited the expression of IQM1 and IQM4.The expression of KAT1 in rosette leaves of iqm1 and iqm1iqm4 mutants increased,while the expression of KAT1 in iqm4 mutants did not change;after ABA treatment,the expression of Rboh F and PP2A genes in rosette leaves of iqm1 and iqm4 mutants was down-regulated,proving that IQM1 and IQM4 may be in different positions Points participate in stomatal regulation signals.?4?Genetic experiments of iqm1ghr1 and iqm4ghr1 double mutants had proved that IQM1,IQM4 and GHR1 had the same genetic pathway to regulate stomatal movement,and IQM1/IQM4 play a role upstream of GHR1.?5?Transgenic lines containing IQM4-GFP and IQM1-GFP fusion genes were constructed.IQM1 was initially expressed in root vascular bundles and leaves,and IQM4 was expressed in root tips and leaves.In summary,the Arabidopsis calmodulin-binding protein IQM1 and IQM4 genes affect the regulation of light and ABA on stomatal movement by regulating the level of ROS in guard cells.IQM1 is located upstream of IQM4,IQM1 depends on Ca²⁺,and IQM4 does not depend on Ca²⁺ to regulate stomatal movement;IQM1/IQM4 has the same signaling pathway as the guard cell ABA/ROS signal gene GHR1,and IQM1/IQM4 is upstream of GHR1.