Physiological And Molecular Regulation Of OsU496A In Rice Seedlings Response To Osmotic Stress

Osmotic stress is a common abiotic stress caused by water shortage.It will lead to changes in the physiological and biochemical status of plants,and then make the growth and development of plants slow down,stagnate,or even die.Plants will accommodate a series of physiological and biochemical mechanisms,such as osmotic adjustment and antioxidant mechanism,to response to osmotic stress.Rice is one of the most important food crops in the world.Its entire growing season requires large amount of water,and osmotic stress has great influence on rice production.Therefore,it is of theoretical and practical significance to study the physiological and molecular mechanisms of rice response to osmotic stress.Previous studies showed that OsU496A,a member of the DUF677 family in rice,was involved in the response of rice to salt stress.In this study,OsU496A RNA interference and overexpression lines and their wild-type rice were used as plant materials,and the physiological and molecular mechanisms of OsU496A in rice response to osmotic stress induced by polyethylene glycol(PEG)and mannitol were researched.The results were as follows:1.The 29 UPF0496 protein 1 found in the NCBI and UniProt databases can be divided into 5 groups according to their evolutionary relationship.The proteins that have a close relationship with OsU496A are all belonged to the Oryza,and the UPF0496 protein 1 of these 9 Oryza species has the same DNDAAAG motif.The physicochemical properties and transmembrane domain analysis of OsU496A protein indicated that OsU496A was a membrane protein,and further,subcellular localization confirmed that OsU496A was mainly localized on the cell membrane.Some stress hormones(abscisic acid,ABA),light induction,and growth and development related promoter elements were obtained by cis-acting elements analysis of the OsU496A promoter.In addition,functional gene interaction analysis also predicted that OsU496A might be closely related to rice growth and stress response.2.The results of real-time fluorescent quantitative PCR showed that OsU496A could be significantly induced by osmotic stress.The OsU496A transgenic seedlings were treated with PEG and mannitol solution,and it was found that the resistance of seedlings to osmotic stress was enhanced after the expression level of OsU496A was down-regulated.Under osmotic stress,the water loss rate of leaves in vitro was the lowest,and the relative water contents of leaves and roots was the highest in OsU496A interference lines.However,the rate of water loss in leaves was the highest and relative water content in leaves and roots was the lowest in the overexpressed lines,and there were significantly differences among all lines.In addition,under osmotic stress,the contents of proline,soluble sugar,hydrogen peroxide,malondialdehyde,relative conductivity and the activities of antioxidant enzyme(APX,CAT,POD and SOD)in the leaves and roots of OsU496A transgenic seedlings all showed an increasing trend.The contents of proline,soluble sugar,activities of antioxidant enzyme(APX,CAT,POD and SOD),expression of genes related to osmotic regulation and antioxidant enzyme synthesis(LEA,APX and CATC)in leaves and roots of OsU496A interference lines were significantly higher than those of wild type and overexpression lines,while the contents of hydrogen peroxide,malondialdehyde and relative conductivity were lower than those of wild-type and Os U496A overexpression lines.3.Transcriptome sequencing results showed that OsU496A could affect the expression of some stress resistance genes,such as hormone level regulation(OsASR5),reactive oxygen species(ROS)scavenging(Pox1),and water transportation(OsPIP2;6)under osmotic stress.GO analysis showed that these genes were mainly enriched in biological processes related to hormone level regulation,ROS metabolism,and stress response.Among the KEGG enrichment pathways,the differential genes were mainly enriched in the phenylpropane biosynthesis,plant MAPK signaling pathway,and plant hormone signaling pathway.Taken together,OsU496A is a cell membrane protein induced by osmotic stress.The resistance of OsU496A RNA interference lines to osmotic stress was enhanced.OsU496A may negatively regulate the response of rice to osmotic stress by regulating the expression of stress resistance genes(OsPIP2;6,LEA3,Pox1,etc.),and thus affect the water retention,osmotic adjustment and ROS scavenging ability of rice seedlings under osmotic stress.These results will provide a new idea for the research of the mechanism of rice response to osmotic stress,and provide plant materials and abundant genetic resources for cultivation of superior rice varieties with stress resistance and stable yield.