LncRNA DANA1 Together With DIP1 Cooperatively Regulate Histone Acetylation To Affect Drought Tolerance In Arabidopsis Thaliana

In recent years,due to the intensification of global temperature changes,precipitation has decreased,depletion of water resources,and drought has occurred frequently.With the intensification of drought,global crop yields have been greatly affected.China is also one of the countries most affected by drought stress.Frequent drought disasters have led to a significant decline in crop yields,seriously endangering China’s food security.Understanding how plants respond to drought stress can help increase crop yields and ensure food supply.When suffering from drought,a variety of mechanisms are initiated to response to drought stress,and it is particularly important to clarify these mechanisms to solve the drought problem.Previous study shows that a lncRNA DANA1 involved in plant responses to drought in Arabidopsis thaliana.And our group have identified a novel RNA binding protein DIP1 by yeast three-hybrid screening.Then,when the drought stress assay was carried out,danal and dip1 mutant plants exhibited more drought sensitivity than Col-0 plants.Moreover,the results show that DANA1 and DIP1 can positively regulate the expression level of ABA2,which is drought response gene.Based on our previous studies,the aim of this research is to analyze the molecular mechanism of the co-regulation of ABA2 by DANA1 and DIP1.The main results are as follows:1.DIP1 interacts with PWWP1/2.PWWP1 and PWWP2,the member of the PEAT complex,interact with DIP1 by yeast two-hybrid in vitro.In addition,protein pull-down experiments were used to further prove that PWWP1 and PWWP2 were interact with DIP1 in vitro.Further applying bimolecular fluorescence complementation(BiFC)assay,we showed that PWWP2 can interact with DIP1 in Arabidopsis protoplast.2.PWWP2 positively regulates drought response in plant.Homozygous T-DNA insertion allele of PWWP2 exhibited PEG sensitive phenotype.In agreement with the drought sensitive phenotype,detached leaves from pwwp2 mutant exhibited increased water loss.Also,pwwp2 mutants had a greater stomatal aperture index when compared to WT.The qRT-PCR results show that PWWP2 is positively regulate the expression level of ABA2.Therefore,it is speculated that PWWP2 can positively regulate plant responses to drought stress.3.HAM1/2 positively regulates drought response in plant.PWWP2 is known to belong to the PEAT complex that associates with HAM1/2 histone acetyltransferases,and HAM1/2 can regulate gene expression level through histone acetylation.Because PWWP2 is positively regulate the expression level of ABA2,we focus on the function of HAM1/2 in this pathway.Because ham1/ham2 is lethal,we generated heterozygous HAM1/ham1 HAM2/ham2 mutant plants by crossing ham 1-1 with ham2-1.We find heterozygous HAM1/ham1 HAM2/ham2 mutant plants with more than 40%reduction of both HAM1 and HAM2 transcripts.Phenotyping of heterozygous HAM1/ham1 HAM2/ham2 mutant revealed that the mutants were substantially more sensitive to PEG and drought stress when compared to WT plants.In agreement with the drought sensitive phenotype,detached leaves from heterozygous HAM1/ham1 HAM2/ham2 mutants exhibited increased water loss.Also,the mutants had a greater stomatal aperture index and enhanced ABA-induced stomatal closure when compared to WT.And Stomata of HAM1 over-expressing plants had smaller aperture than did Col-0 plants.Phenotypes of heterozygous HAM1/ham1 HAM2/ham2 mutant plants are consistent with the trend presented by danal and dip1 mutants.4.Analysis of histone acetylation level at ABA2 locus.Laboratory classmates found that DANA1 was associated to ABA2 locus in vivo by ChIRP assay.Then,we also demonstrated DIP1 protein could bind to the ABA2 promoter.In agreement,we found that H4K5ac active marks were decreased at the ABA2 locus in dana1-2,dip1-1 and heterozygous HAM1/ham1 HAM2/ham2 mutants.Collectively,these findings show that DANA1 and DIP1 can regulate ABA2 expression during the plant response to drought by regulating histone acetylation.In summary,these results together demonstrate that DANA1 interacts with DIP1 to regulate ABA2 expression during the plant response to drought by regulating histone acetylation.The results of this study not only enrich the molecular mechanism of DANA1 regulating the expression of ABA2,but also improve the regulatory model of histone acetylate modification involved in plant response to drought stress.Our studies provide new ideas for improving crop drought tolerance.