The Histone Deacetylase 6 Mediates Epigenetic Changes To Negatively Regulate Drought Tolerance In Apple

With its important economic value and extensive cultivation area,apple is one of the four major fruits in the world.However,at present,many advantageous apple-producing areas,such as the Loess Plateau region in northwest China,have suffered a large-scale reduction in apple production due to the long-term drought stress,which restricts the innovative development of the apple industry.Therefore,identification and characterization of the resistance related genes in apple will have a meaningful significance for the improvement of stress-resistant germplasm resources.Histone deacetylation modifiers are widely involved in various stages of plant growth and play an important role in plant response to drought stress.In this research,we identified the biological function of the apple histone deacetylase gene MdHDA6 under drought stress and to further explode the molecular mechanism of this gene in response to drought stress.The main results were as follows:1.Identification and analysis of MdHDACs in appleA total of 24 HDAC members were identified in the apple genome,among which 15members belonged to the HDA subfamily.All members of this subfamily have a conserved Hist_-deacetyl domain and they are expressed in various tissues of apple.In this study,we found these HDAs can respond to abiotic stress such as drought,cold,salt and ABA treatment.We screened a candidate gene MdHDA6 that can be significantly induced by drought stress from these genes.We analyzed its genetic characteristic and found that the CDS of MdHDA6 is 1407bp,encoding 468 amino acids in total.In addition,we found MdHDA6 locates in the nucleus,and the purified MdHDA6 had a catalytic activity of histone deacetylation in vitro.2.MdHDA6 acts as a negative regulator for drought tolerance in apple treesWe obtained three MdHDA6 OE and three MdHDA6 RNAi transgenic lines by agrobacterium-mediated genetic transformation.After long-term drought treatment,the relative growth of plant height,root-shoot ratio and photosynthetic rate of MdHDA6 plants were lower than those of GL-3,suggesting that MdHDA6 could negatively regulate the adaptability of apple to drought stress.After short-term drought treatment,MdHDA6 OE plants were found to have higher ion leakege,more H2O2content and lower survival rate than GL-3,indicating that MdHDA6 can negatively regulate the response of apple to drought stress.3.The molecular regulation mechanism of MdHDA6 in apple response to drought stress(1)MdHDA6 regulates the expression of drought-responsive genes.Transcriptome analysis showed that the number of differentially expressed genes in MdHDA6 OE plants was significantly higher than GL-3 after drought treatment,indicating that MdHDA6 OE plants were more sensitive to drought stress.After drought treatment,the expression levels of Md WRKY57,Md MYB4 and other drought positive regulation genes in MdHDA6 OE plants were significantly lower than GL-3.While the expression levels of Md CYP707A1/A4,Md UGT71B6 and other drought negative regulation genes in MdHDA6 OE were significantly higher than GL-3.These results suggest that MdHDA6 can respond to drought stress by regulating the expression of drought-responsive genes.(2)MdHDA6 inhibits the expression of drought-responsive genes through histone H3deacetylation.The results of western blot assays showed that the genome acetylation level of histone H3 in MdHDA6 OE plants was significantly lower than that of GL-3.Ch IP-seq and Ch IP-q PCR assays further confirmed that the histone H3 acetylation levels of Md WRKY57,Md MYB4,Md GRX480 and Md MYB4 decreased significantly in MdHDA6 OE plants,and their expression levels also decreased significantly.These results indicated that MdHDA6 can inhibit the expression of positive drought regulation genes through histone H3 deacetylation and then negatively regulate drought stress.(3)MdHDA6 interacts with Md JAZ3 and Md ABI5 respectively.To further elucidated the regulatory mechanism of MdHDA6-targeted drought-responsive genes,we screened Md JAZ3 and Md ABI5 can interact with MdHDA6 respectively through Y2H system.Co-IP,Bi FC,Split-Luc and Y2H experiments confirmed the interaction between them in vivo and in vitro.In addition,the truncation interaction analysis of MdHDA6 showed that Md JAZ3interactes with the C-terminus of MdHDA6,while Md ABI5 interactes with the N-terminus(containing the Hist-deacetyl domain)of MdHDA6,speculating that MdHDA6 may act with these two factors to regulate drought response in apple.