Functional Characterization Of Rice Histone Deacetylase Gene HDA710 Under Salt Stress

Rice is one of the most important staple crops that provide food to 50 percent of the world population.To this day,abiotic stress remained a significant problem to be addressed for sustainable crop production.Histone deacetylase?HDAC?plays an important role in the regulation of cellular processes,normal growth and development,and stress response in plants.Although the function of HDACs in Arabidopsis thaliana has been extensively studied,their function in rice is largely unexplored.A deeper understanding of the HDAC molecular mechanism will help us improve crops through genetic engineering.The purpose of this study was to investigate the regulation mechanism of rice HDA710/Os HDAC2 gene under salt stress.In this study,the function of HDA710 was explored by analyzing its phenotypic characteristics,subcellular localization,histone acetylation regulation,expression analysis and response to ABA,drought and salt stress.The main results are as follows:1.Expression profile analysis of HDA710 showed that under normal conditions,except for callus and root,HDA710 has low expression levels in most parts of the plant such as stem,internode,panicle and pistil.However,the expression can be induced under conditions of salt stress,drought,darkness,and flooding,among which salt stress had the most obvious response.In addition,plant hormones such as abscisic acid?ABA?,jasmonic acid?JA?,GA₃,2,4-D and NAA also induced HDA710 expression.2.HDA710 knockout mutants?hda710-1 and hda710-2? were constructed using CRISPR/Cas9 system,and overexpression plants were also created using p U1301 vector?Ubi+HDA710 CDS?.Phenotype observation showed that the HDA710 overexpression material grew slower than the wild-type in the seedling stage,and the plant height became shorter,while the plant height of hda710 was not significantly different from that of the wild-type.The seed setting rate of the hda710 mutant at maturity was significantly lower than that of the wild-type.3.The subcellular localization analysis of HDA710 using rice protoplasts and tobacco leaf showed that HDA710 is distributed in the nucleus and cytoplasm.4.Immunoblot analysis showed that HDA710 deacetylate histone H3 and H4 in rice.HDA710 mutation significantly increased the level of acetylation modification of histone H3 and H4,while the level of acetylation modification of histone H3 and H4 in plants overexpressing HDA710 was significantly reduced.Further research found that HDA710can specifically remove acetylation of H4K5 and H4K16.5.Under drought and salt stress treatment,the hda710 mutant plants showed increased resistance,while overexpressing plants were more sensitive.6.To study the role of HDA710 in the ABA signaling,hda710 mutants and overexpressing plants were treated with ABA.The results showed that the hda710mutants showed lower sensitivity compared to the wild-type plants,while the overexpressing plants were highly sensitive to ABA.Further research showed that the seed germination rate of mutants and overexpressed materials was also affected by ABA,of which hda710 seeds showed insensitivity to ABA and normal germination rate,while overexpression seeds showed higher sensitivity and lower germination rate.7.The expression analysis of ABA treatment and salt stress response genes showed that the expression levels of OsLEA3,OsABI5,OsbZIP72 and OsNHX1 in the mutant plants of HDA710 were significantly up-regulated compared with the wild-type.These expression differences corresponded with higher levels of histone H4 acetylation in the gene promoter regions in hda710 compared with the wild type under ABA and salt-stress treatment.In summary,this study shows that the reduced potassium dependency 3/histone deacetylase 1?RPD3/HDA1?family protein,histone deacetylase HDA710/Os HDAC2plays a key role in response to salt stress in rice by regulating ABA-and salt-stress-responsive genes by altering H4 acetylation levels in their promoters.Besides,HDA710 plays an important regulatory role in rice growth and development.This study further deepens the understanding of the apparent regulation of diverse biological processes in rice,and provides a theoretical basis for the regulation mechanism and function of histone deacetylases in rice.