Functional Analysis Of A DGK Gene Encoding A Diacylglycerol Kinase In Arabidopsis

DGKs,known as diacylglycerol kinase,are members of a unique and conserved family of lipid kinase that catalyze the phosphorylation of diacylglycerol?DAG?,thus generating phosphatidic acid?PA?.The majority of diacylglycerol?DAG?is generated by hydrolysis of phosphatidylinositol-4,5-bisphosphate?PIP2?by the enzyme phospholipase C?PLC?,but DAG can also be generated when phosphatidic acid phosphatases remove the phosphate headgroup from phosphatidic acid?PA?.Both PA and DAG are play an important role in acting as signaling lipids and also intermediating in the lipid biosynthetic pathways,so it is crucial that the intracellular levels DAG and PA be tightly regulated,which is accomplished by the diacylglycerol kinases?DGKs?and phosphatidic acid phosphatases?PAP?.As such,to understand the biological role of DGKs is also important.The model plant Arabidopsis thaliana has 7 DGK members.Previous data show that AtDGK2 is expressed in a variety of tissues,include roots,leaves and flowers.Furthermore,its expression is induced by cold and injury and it may play a role in resistance of cold in plant.The functional role of DGKs are widely studied in animals,but the plant counterparts of DGKs need further investigation.In this work,molecular cell biological and reverse genetic strategies were used to study the functions of AtDGKs in Arabidopsis.The major results are presented as follows:1.N-terminal region deleted DGK^a+c and DGK^a are localized in the cytoplasmBy using stable Arabidopsis transgenic plants,YFP-tagged DGK^a+c and DGK^a were found to be localized in the cytoplasm of the root and leaf pavement cell.2.Overexpression of YFP-DGK^a+c influences plant developmentPhenotype analysis showed that overexpression of YFP-DGK^a+c,DGK^a+cinfluence both embryonic and postembryonic development.Over-expressing YFP-DGK^a+c caused embryo defects in various developmental stages,such as abnormal cell divisions at early embryogenesis and lead to defects in the establishment of the root pole.At the postembryonic stage,over-expression of YFP-DGK^a+c resulted in disturbed floral phytaxis,and fasciated stem as well as defective flower organs.These data indicate that perturbation of YFP-DGK^a+c affects plant development.3.Overexpression of DGK^a+c influences embryo development Under the control of embryonic expressed RPS5A promoter,our data show that,overexpressing DGK^a+c also have abnormal cell divisions which lead to defects in early embryogenesis and the establishment of the root pole.4.T-DNA insertion mutations in AtDGK influence lateral root developmentPhenotype analysis of AtDGKs T-DNA insertion mutant,showed that dgks had shorter primary roots and increased lateral roots compared to the wild type.These results suggest that AtDGKs regulate root development,especially in lateral root development.Taken together,AtDGKs is involved in embryogenesis and the postembryonic development,and might play an important role in both embryo and lateral root development.