Role Of Rice Phospholipase C In Salt Tolerence And Phospholipase D In Seeds Accelerated Aging

Phospholipases are grouped in to three classes, phospholipase A (PLA), phospholipase C (PLC) and phospholipase D (PLD) according to their differerent hydrolysis site. Phospholipases have been implicated in different cellular processes in plant growth, development, and stress responses. But phospholipase are consisting of many genes, the physiological functions of them were not characterized very well. In this study, we found that OsPLC3is a PI-PLC, and postive regulated salt stress in rice.We also characterized the relationship between the changes of phospholipids and PLD during rice seed aging.PLC consists of two types, non-specific phospholipase C (NPC) and phosphoinositide-specific phospholipase C (PI-PLC) according to their substrates. PI-PLC is an effector molecule in the signal transduction process. It generates two second messengers, inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) from phosphatidylinositol4,5-bisphosphate (PI(4,5)P2). Through genome-wide analysis, four PLC members found in chromosome3,5,7and12have been identified in rice. Expression pattern analysis indicates that most PLC-encoding genes are differentially expressed in various tissues. Expression levels of OsPLC1are lower in all tissues; OsPLC2are highly expression in roots and young leaves; the expression levels of OsPLC3are higher in old leaves than other tissues; OsPLC4levels are abundant in roots. They are induced by low tempature, ABA, salt and drought conditions, especially OsPLC2and OsPLC3. Under salt treatment, OsPLC1levels aren’t induced; OsPLC2, OsPLC3and OsPLC4are all induced. Among them, OsPLC3levels are induced dramatically during salt treatment. We characterized the mutant of OsPLC2and OsPLC3, and the functions of them are discussed in this work. First we determined whether both genes indeed encode catalytically active PI-PLCs. For this we expressed the genes in E. coli (strain BL21) as glutathione S-transferase (GST)-fusion constructs. Both OsPLC2and OsPLC3displayed phosphoinositide-specific lipase acitvity as they are both able to hydrolyse PI(4)P and PI(4,5)P2. The PC could not be hydrolysed by OsPLC2and OsPLC3. There is no enzymatic activity present in the GST-only control too. These results indicated that OsPLC2and OsPLC3belong to the PI-PLC. According to the transient expression methods in Rice protoplasts, we found that OsPLC2-YFP and OsPLC3-YFP are located in the plasma membrane. We also transformed the fusion sonstruct into onion epidermis cells. The location of PLCs in onion epidermis cells are further confirmed this result.We further analyzed the function of OsPLC2and OsPLC3using the Tosl7insertion lines. The content of DAG and PA in osplc3is lower than in WT, and the agronomic traits of osplc3and WT are widely diverse, such as height and setting rate. During the drought and salt stress, we found only osplc3showed susceptible to salt treatment. The Na+rised extremely after the salt treatment in osplc3. These results indicated that OsPLC3must play a key role in salt tolerance in Rice. Based on this theory, we dected the expression levels of some genes which response to salt tolerance in WT and osplc3seedlings. The transcription levels of many genes were disturbed in plc3seedlings, such as OsbZIP23, OsP5CS,OsHKT2;1,OsMSR2, OsLEA3and OsCDPK7. Interestingly, OsMSR2, OsLEA3and OsCDPK7are all regulated by Ca2+signal, and the Ca2+should be released by IP3form endoplasmic reticulum. This implies that OsPLC3may, or partially, regulate salt tolerance through the IP3-Ca2+pathway.Aging during storage negatively affects rice seed viability and nutrition quality. Lipid degradation caused by phospholipase D (PLD) activity is known to be responsible for seed aging in Arabidopsis, but the mechanisms of this process in monocotyledonous plant rice remain unclear. In this study, we carried out lipid profiling analysis for rice seeds, and revealed that the main membrane lipids phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) declined during accelerated aging. In contrast, phosphatidic acid (PA), a hydrolysis product produced by PLD, increased. Moreover, the decreased molecular species in PC and PE were well fit with those that were increased in PA, suggesting that PA may be derived from PLD-mediated lipid hydrolysis. OsPLDal and OsPLD a3were the major PLDas in rice seeds, and their transcription increased significantly during the aging process. The aging treatment induced OsPLDa protein expression and activation. Three lipoxygenases (LOXs) were analyzed, and OsLOX2transcription was induced significantly during seed aging. Our results reveal detailed information regarding lipid degradation during rice seed aging, and we go on to discuss the relationship between lipid degradation and peroxidation during seed aging.In conclusion, we characterized the OsPLC3is a PI-PLC, and positive regulated salt tolerance in rice using genetical, biological and physiological methods. We also found that OsPLDas are involved in rice seed aging. These results further confirm the roles of phospholipase and the changes of phospholipids in abiotic stress and aging.