Molecular Mechanism Of OsDRT1 In Regulating Drought Resistance In Rice

As a semi-aquatic crop,rice requires a large amount of water resources during its growth process,and also emitting a significant amount of methane.In recent years,extreme climate events have become more frequent,and drought stress has significantly constrained rice production,threatening human food security.Therefore,analyzing the mechanism of drought resistance in rice,mining drought-resistant genes,and cultivating drought-resistant rice varieties are effective ways to cope with environmental stress.We identified a gene associated with drought-resistant by genome-wide association analysis(GWAS)in our previous study,which was named OsDRT1.Here,we explored the function of OsDRT1.Main results were listed below:1.OsDRT1 encodes a membrane protein containing three transmembrane domains.The subcellular localization results showed that OsDRT1 was mainly localized on the endoplasmic reticulum membrane and the cell membrane,and the deletion of either transmembrane domain alone could still localize to the cell membrane.When two transmembrane domains were deleted at the same time,they mainly localized in the cytoplasm,but could not enter to the nucleus.In the absence of three transmembrane domains,OsDRT1 was localized in both the nucleus and cytoplasm.2.OsDRT1 showed significant differentiation between indica and japonica.Sanger sequencing of 270 rice core germplasm showed that OsDRT1 was significantly differentiated in CDS region of indica and japonica rice.The subcellular localization results showed that the indica-haplotype of OsDRT1 was more concentrated in the cytoplasm,while the japonica-haplotype of OsDRT1 was mainly distributed in the endoplasmic reticulum membrane.3.Bimolecular fluorescence complementation assay showed that OsDRT1 interacted with two aquaporins Os PIP1;1 and Os PIP2;6.Yeast double hybrid experiment showed there is also an interaction between Os PIP1;1 and Os PIP2;6.4.Using CRISPR-Cas9 technology,osdrt1,ospip1;1,ospip2;6 mutants and OsDRT1 overexpressing plants were generated.The results showed that the survival rate of osdrt1 plants was significantly higher than that of wild-type(Nipponbare)under drought stress,while the survival rate of the overexpressing plants was significantly lower than that of the wild-type,indicating that OsDRT1 negatively regulates rice drought resistance.The survival rate of ospip1;1 and ospip2;6 mutant plants were significantly lower than that of the wild-type,indicating that Os PIP1;1 and Os PIP2;6 positively regulate rice drought resistance.Measurement of water loss rate of detached leaves from osdrt1 mutants and wild-type plants showed that the water loss rate of osdrt1 mutant leaves was lower than that of the wild-type.5.The tissue expression pattern of OsDRT1 was analyzed,and the results showed that OsDRT1 was expressed in all tissues of rice,with the highest expression in leaves.6.Investigation of phenotype showed that under normal water management,there was no significant difference in tiller number and effective panicle number between the osdrt1 mutants and wild-type(Nipponbare),while plant height,biomass,grain filling rate,and yield per plant were significantly higher than the wild-type.Under drought stress conditions,the effective panicle number,biomass,and yield per plant of the osdrt1 mutants were signficantly higher than the wild-type,and there was no significant difference in plant height,tiller number,and grain filling rate.The above results indicate that osdrt1 mutants may improve rice drought resistance.In summary,OsDRT1 protein may affect the functions of Os PIP1;1 and Os PIP2;6 to regulate the absorption or loss of water and negatively regulate rice drought resistance.However,the molecular mechanism underlying it still requires further study.