Effects Of Nonspecific Phospholipase C On Lipid Metabolism And Growth In Rice

Lipids are not only the main storage form of energy,but also the main component of cell membranes.In addition,lipids,such as diacylglycerol(DAG)and phosphatidic acid(PA)are important molecules for signal transduction in animals and plants.Nonspecific phospholipase C(NPC)hydrolyzes phospholipids to generate DAG that can further phosphorylate to PA.Studies in Arabidopsis thaliana indicate that the NPC family plays important roles in plant response to hormones and stresses.However,nothing was known about the function of NPC family in the important food crop rice.This research was undertaken with the goal to determine the biochemical,molecular,and physiological function of rice NPC.The specific aims of the research were to:1)analyze the rice NPC gene family and their expression patterns;2)genetic manipulation of specific NPC expression by gene knockouts and overexpression;3)characterize the enzymatic function of selected NPCs;4)determine the effect of selected NPCs on rice growth and development;and 5)probe the biochemical and molecular mechanisms by which specific NPCs modulate the growth and development of rice.The main findings are as follows:1.The rice NPC family has five members,designated as NPC 1,2,3,4,and 6 based on the sequence similarity to the 6 members of NPCs in Arabidopsis.All the members of NPC family contain three conserved domains(ENRSFDHxxG,TxPNR and YDExGGxxDHV).2.Based on results from quantitative real-time PCR and microarray data sets,NPC1 is expressed in various tissue of rice,NPC2 is expressed the highest in roots,NPC3 and NPC6 are highest in flowers,whereas NPC4 is expressed only in roots.3.The over-expression of NPC1 leads to brittle stem nodes and panicle nodes and increased seed shattering due to a decrease in mechanical strength in nodes tissues,which results from decreases in contents of cellulose,hemicellulose,and silicon and thinner secondary cell wall in nodes.4.Results from NPC-GFP imaging and two-phase partitioning indicate that NPC1 in rice is associated with the cytoplasm and membranes.5.Enzymatic assaying in vitro indicates that NPC1 uses not only phospholipids but also galactolipids as the substrates to generate DAG.The pH optimum for NPC1 activity on phosphatidylcholine(PC)is at pH 5.0 whereas that on mono galactosyl diglyceride(MGDG)was at pH 6.0.The NPC activity is independent of cations,such as Ca2+,and 2 mM EGTA actually stimulates its activity.6.Quantitative lipid profile shows that the content of galactolipids in the node of NPC1-overexpressing rice is decreased,while the content of some phospholipids,especially the phosphatidic acid(PA),is increased.7.PA binds to the silicon transporter Lsi6,and the lipid-protein interaction promotes Lsi6 binding to silicon and potentially promotes the transport of silicon.The PA-Lsi6 interation was demonstrated by the results of lipid-immunoblotting,liposomal binding,and surface plasma resonance.8.NPC2-knockout rice seedlings are hypersensitive to gibberellin(GA3)with increased length of roots in response to gibberellin treatment.Preliminary data indicate that PA inderacts with the GA receptor(GID1)in rice,raising a promising direction of research on the role of NPC in plant response to GA.