Mechanism Of CHR16 Response To Drought Stress In Arabidopsis Thaliana

Chromatin remodeling plays an important role in the regulation of DNA replication,transcription and genome stability in eukaryotes.ATP-dependent chromatin remodeling complexes play an important role in abiotic stresses,such as drought and saline-alkali stress.SNF2 chromatin remodeling complex ATP enzymes play an important role in plant response to stress.CHR16 is an important member of the SNF2 family.In this study,Arabidopsis thaliana was used as material to explore the function of CHR16 Arabidopsis thaliana in drought stress response by means of phenotypic analysis,qRT-PCR,physiology and biochemistry,yeast two-hybrid,BiFC and transcriptome.The main results are as follows:1.Through the experimental analysis of drought stress,we found that compared with WT,chr16 had a higher survival rate under drought stress,and the expression levels of CPK11,Di19 and their downstream drought stress response related genes PR1,PR2,PR3 and PR5 in chr16 mutants were up-regulated.This suggests that CHR16 may negatively regulate the drought tolerance of plants.2.Compared with WT,the activity levels of APX,CAT and POD in chr16 mutants increased significantly after drought stress.This shows that chr16 has a stronger ability to scavenge the production of ROS under stress,thus reducing the damage to plants caused by drought stress.3.Through the experiment that WT and chr16 were treated with mannitol and PEG6000 to simulate the effect of drought stress,it was found that the inhibition of root length of chr16 mutant by mannitol and PEG6000 was weaker than that of WT under different concentrations of osmotic stress,which indicated that chr16 mutant had stronger tolerance to osmotic stress.Compared with WT,the expression level of ABA response gene ABI3 in chr16 mutant increased significantly after mannitol treatment.In addition,the expression level of NCED3 in WT was lower than that in chr16 mutant,and the expression level of CYP707A3 in chr16 mutant was higher than that in chr16 mutant.This indicates that CHR16 can affect the expression levels of ABA pathway response genes,ABA synthesis and catabolism related genes.Therefore,we speculate that the chr16 mutant may improve the tolerance of the mutant plant to osmotic stress through ABA signal pathway.4.Through the analysis of leaf stomata,it was found that the stomatal density and stomatal index of chr16 mutant were significantly lower than those of WT.Compared with WT,the expression level of YDA,MPK3,MPK6 and TMM related to stomatal development in mutant increased,especially after drought stress.Therefore,we speculate that CHR16 may affect stomatal development by negatively regulating the expression of YDA,MPK3,MPK6 and TMM.5.Yeast two-hybrid experiment showed that CHR16 could interact with RD2 and DI19 involved in drought stress response,and BiFC experiment further verified their interaction,which indicated that CHR16 might participate in drought stress response through RD2 and Di19.6.RNA-seq comprehensively analyzed the changes of transcriptional map of chr16 mutants under drought stress.It was found that 1816 genes were associated with CHR16 under drought stress,including members of the signal pathway of ABA.We selected members of the SnRKs family for further analysis.The results of qRT-PCR showed that the expression levels of SnRK2.3 and SnRK2.4 in chr16 mutants were significantly higher than those in WT,which was consistent with the results of RNA-seq data analysis.Moreover,we confirmed the interaction between CHR16 and SnRK2.3 and SnRK2.4 by yeast two-hybrid and BiFC experiments.Therefore,we speculate that CHR16 may be involved in ABA-mediated drought stress response through SnRK2 s.7.A phosphatase PP2C62 from the PP2 Cs family was screened by yeast hybridization,and the interaction between CHR16 and PP2C62 was found by BiFC experiment.qRT-PCR analysis showed that compared with WT,the expression level of PP2C62 in chr16 mutant plants increased significantly after drought stress.This suggests that CHR16 may participate in the response of Arabidopsis thaliana to drought stress by affecting the expression level of PP2C62.We further obtained the homozygous double mutant of chr16 pp2c62 by genetic hybridization.Exogenous ABA was applied to WT,chr16,pp2c62 and chr16 pp2c62 respectively.The results showed that the sensitivity of pp2c62 to ABA was similar to that of WT,and chr16 was hypersensitive to ABA.Compared with chr16,the ABA sensitive phenotype of chr16 pp2c62 double mutant was weaker.These results indicate that the sensitivity of chr16 mutants to ABA is partly dependent on PP2C62.Therefore,we speculate that PP2C62 may regulate the expression of transcription factors in ABA signal pathway by interacting with CHR16.In summary,CHR16 may affect the drought tolerance of Arabidopsis thaliana by regulating stomata.In addition,CHR16 may also participate in drought stress response by interacting with RD2 and DI19 drought stress response genes and with members of ABA signal pathway.This is helpful for us to understand the involvement of chromatin remodeling complex ATP enzyme in plant stress.