Phosphoinositide Specific Phospholipase C4 Modulates Rice Response To Osmotic Stress And Growth And Development

High salinity and drought adversely affect rice seedling growth,development and crop yield.Many studies have indicated that the transcript of plant phosphatidylinositol-specific phospholipase C（PI-PLC）gene is up-regulated by multiple abiotic stress treatment and its function is involved in plant response to abiotic stress.However,the mechanism by which PI-PLC mediated abiotic stress response remains unclear.Therefore,this study was undertaken to determine the function and mechanism of action of OsPLC4 in rice response to osmotic stress.The main results are as follows:1.Four OsPLCs were identified in the rice genome.All rice PLCs contains three conserved domains,including PI-PLC-X,PI-PLC-Y and C2 domains.The sequences of rice PLCs were not in the same clade as those of PLCs in Arabidopsis.OsPLC4 has a low sequence homology with any other plant PI-PLCs.The biochemical assasy indicated that OsPLC4 was a catalytically active PI-PLC.OsPLC4 hydrolyzes phosphatidylinositol（PI）,phosphatidylinositol 4-phosphate（PI4P）,and phosphatidylinositol-4,5-bisphosphate（PIP₂）with the activity PIP₂﹥PI4P﹥PI.OsPLC4 showed a Ca²⁺concentration-dependent hydrolytic activity.Its maximum activity toward PI was at 50 mmol/L Ca²⁺concentration with the pH optimum between 6.5 to 7.5.OsPLC4 was mainly expressed in root and leaf at young seedling stage and the tillering stage.OsPLC4 was noticeably up-regulated by high salinity,dehydration,abscisic acid（ABA）and indole-3-acetic acid（IAA）treatment in rice leaf or root.OsPLC4 was also significantly up-regulated by high salinity and dehydration treatment in leaf at translation level,and OsPLC4 was mainly localized on the endoplasmic reticulum membrane and plasma membrane.2.OsPLC4 positively modulates rice response to osmotic stress via lipid-mediated and Ca²⁺signaling.Both two independent knockout mutants,plc4-1,and plc4-2,were more sensitive to NaCl or drought stress treatment than wild type（WT）,while genetic complementation could restore the sensitivity of plc4-1 to that of WT under osmotic stress conditions.By constrast,OsPLC4-overexpresssion（OE）plants were more resistant to osmotic stress,as compared to WT.It is found that phosphatidic acid（PA）,inositol triphosphate（IP₃）and free cytoplasmic Ca²⁺([Ca²⁺]_cyt)were closely related to OsPLC4 in response to osmotic stress.Under salt stress,the increase of PA was less in plc4-1 but more in OsPLC4-OE plants than that in WT.In addition,application of DAG or PA both restored the growth of plc4-1 to that of WT under salt stress.However,in the presence of a DAG kinase inhibitor,DAG failed to rescue the growth defect of plc4-1 under salt stress.Moreover,loss of OsPLC4 compromised the increase of IP₃ and[Ca²⁺]_cytyt and inhibited the induction of genes involved in Ca²⁺sensor and osmotic stress responses to salt stress.3.OsPLC4 was also involved in rice growth and development or other physiological progress except the osmotic stress response.Abolished OsPLC4 in rice compromised seed dormancy,reduced inhibitory effect of ABA on seed germination and increased water loss in detached leaves.Loss of OsPLC4 reduced crown root number,whereas OsPLC4-OE exhibited constitutive response to IAA.In addition,loss of OsPLC4resulted in reduction in pollen viability and seed setting rate.These findings indicate that OsPLC4 exhibits multiple effects including plant growth,development and osmotic stress and OsPLC4 modulates rice response to osmotic stress via lipid-mediated and Ca²⁺signaling.