Roles Of Cytidinediphosphate Diacylglycerol Synthase5 In Phospholipid Homeostasis And Osmotic Stress Response

Cytidinediphosphate diacylglycerol synthase(CDS)catalyzes phosphatidic acid(PA)and cytidinetriphosphate(CTP)to produce cytidinediphosphate-diacylglycerol(CDP-DAG),an active intermediate for phosphatidylglycerol(PG)and phosphatidylinositol(PI)synthesis.PA is an intermediate for glycerolipid metabolism and is emerging as an important signal molecule involved in various biological processes including ABA signaling,osmotic stress response,root hair formation,and nutrition starvation.Therefore,CDS alteration may result in significant impact on lipid metabolism and physiological effects.However,the knowledge of CDS remains limited in plants,especially in crop plants.In this study,rice CDS5 was identified and was found to be important for lipid homeostasis,thylakoid membrane modeling and plant growth.In addition,CDS5 is negatively involved in osmotic stress functioning as a suppressor in PLD-derived PA signaling.The main results are as following:The rice genome contains five CDS genes,namely CDS1-5,and the knockout mutants for CDS2,CDS3 and CDS5 were isolated.Loss of either CDS2 or CDS3 did not exhibit visible phenotype under normal growth conditions.However,loss of CDS5resulted in pale color leaf,defective thylakoid grana architecture,and reduced solube sugar accumulation at early seedling stage.Loss of CDS5 also led to a growth arrest,accompanied by multiple defects including reduced tiller number,shorter panicles,less pollen viability,reduced seed setting,and decreased seed yield relative to wild type(WT)plants.These defects in the cds5 mutant were rescued by CDS5 complementation(COM),suggesting that CDS5 is responsible for thylakoid grana modeling and plant growth.The CDS5-GFP fused protein was transiently expressed in cells of tobacco leaves.The results showed that CDS5-GFP was overlaid with endoplasmic reticulum(ER)marker protein and also overlaid with red fluorescent chloroplasts,suggesting that CDS5is localized to both ER and chloroplasts.The CDS5 transcript was detected in roots,stems and leaves with relatively stable level.In vitro activity assay showed that CDS5 is able to catalyze PA to produce CDP-DAG.These results suggest that CDS5 is involved in lipid metabolism through both prokaryotic pathway in chloroplasts and eukaryotic pathway in ER.Lipid profiling revealed that loss of CDS5 resulted in reduced PG and PI level,and a corresponding elevated PA.The reduced PG resulted from decreased 34:4-,34:3-,and32:1-PG.Reduced PI come from less 34:3-and 34:2-PI,which are similar to those elevated PA fatty acid species.The opposite changes between PA and PI/PG were corrected by CDS5 complementation,suggesting that CDS5 uses PA as substrate for PG and PI synthesis in rice plants.To probe the source of the elevated PA in the cds5 mutant,1-butanol,which suppresses PLD-produced PA,was applied to rice plants,and 2-butanol,which is not a substrate of PLD,was used as a control.The 1-butanol supplement greatly suppressed PA production.Moreover,the 1-butanol treatment abolished the elevated PA in cds5,and the levels of PA,PG,and PI level in cds5 were not significantly different from that of WT and COM plants.By comparison,in 2-butanol-treated plants,the PA level in cds5 was higher whereas the PG and PI levels were lower than that of WT and COM plants.The results suggest that the PA substrate utilized by CDS5 is derived from PLD activity.Exogenous supplementations of CDP-DAG and PG in the cds5 mutant partially relieved the pale leaf and growth arrest phenotype,whereas supplemented PI rescued the cds5 mutant growth arrest phenotype to WT.The results suggest that PG is responsible for normal green leaves,while PI is required for growth.The enhanced growth in the cds5 mutant by PI supplement was partially suppressed by wortmannin,an inhibitor of PI3K and PI4K,suggesting PI derivatives,such as PI3P,PI4P and PI(4,5)P₂,are responsible for growth promotion in the mutant in some extent.Enhanced growth by PI requires PA cooperation as demonstrated by PI/PA supplement and PA inhibitor.Phospholipase D(PLD)produced PA plays an important role in mediating ABA signaling to regulate stomatal closure.PA signal sequestering remains less understood.This study also showed that loss of CDS5 resulted in enhanced ABA response,decreased stomatal aperture and less water transpiration,thus enhanced the cds5 mutant tolerance to salt and drought due to elevated PA,as compared to WT and COM plants.Additional inhibitor of PLD-derived PA formation suppressed the enhanced tolerance in the cds5mutant.The results suggest that CDS5 catalyzes the PA that is derived from PLD activity,thus negatively involved in PLD-derived PA mediated ABA signaling in stomatal closure in response to osmotic stress.