Arabidopsis Phospholipase C3 is Involved in Lateral Root Initiation and ABA Responses in Seed Germination and Stomatal Closure

: Phospholipase C (PLC) is well known for its role in animal signaling, where it generates the second messengers, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) by hydrolyzing the minor phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP2) upon receptor stimulation. In plants, PLC’s role is still unclear, especially because the primary targets of these second messengers are lacking, i.e. the ligand-gated Ca2+ channel and protein kinase C, but also because PIP2 levels are extremely low. Nonetheless, the Arabidopsis genome encodes 9 PLCs. We used a reversed-genetic approach to explore PLC’s function in Arabidopsis and report here that PLC3 is required for proper root development, seed germination and stomatal opening. Two independent knock-down mutants, plc3-2 and plc3-3, were found to exhibit reduced lateral root densities by 10-20%. Mutant seeds germinated slower but were less sensitive to ABA to prevent germination. Guard cells of plc3 were also compromised in ABA-dependent stomatal closure. Promoter-GUS analyses confirmed PLC3 expression in guard cells and germinating seeds, and revealed that the majority is expressed in vascular tissue, most likely phloem companion cells, i.e. in roots, leaves and flowers. In vivo 32Pi-labeling revealed that ABA stimulated the formation of PIP2 in germinating seeds and guard cell-enriched leaf peels, which was significantly reduced in plc3 mutants. Overexpression of PLC3 had no effect on root system architecture or seed germination, but increased the plant’s tolerance to drought. Our results provide genetic evidence for PLC’s involvement in plant development and ABA signaling, and confirm earlier observations that overexpression increases drought tolerance. Potential molecular mechanisms for the above observations are discussed. Phospholipase C3 is Involved in Lateral Root Initiation and ABA Responses in Seed Germination and Stomatal Closure. important intermediate in lipid and key roles in cell signaling, either as a second messenger itself or as a precursor of phosphatidic acid. Methods to identify distinct DAG pools have proven difﬁcult because biochemical fractionation the pools, and concentrations are limiting. Here, we validate the use of a genetically encoded DAG biosensor in plant cells. did not locate to membranes but was present in the cytosol and nucleus. Treatment with short-chain DAG or PMA (phorbol-12-myristate-13-acetate), a phorbol ester that binds the C1a domain of PKC, caused the recruitment of the biosensor to the plasma membrane. These results indicate that the biosensor works and that the basal DAG concentration in the cytoplasmic leaﬂet of membranes (i.e. accessible to the biosensor) is in general too low, and conﬁrms that the known pools in plastids, the endoplasmic reticulum and mitochondria are located at the luminal face of these compartments (i.e. inaccessible to the biosensor). Nevertheless, detailed further analysis of different cells and tissues discovered four novel DAG pools, namely at: (i) the trans -Golgi network; (ii) the cell plate during cytokinesis; (iii) the plasma membrane of root epidermal cells in the transition zone, and (iv) the apex of growing root hairs. The results provide new insights into the spatiotemporal dynamics of DAG in plants and offer a new tool to monitor this in vivo. , phosphatidylinositol 4,5-bisphosphate; PKC, protein kinase C; PLC, phospholipase C; PMA, phorbol-12-myristate-13-acetate; PPI, polyphosphoinositide; PS, phosphatidylserine; TAG, triacylglycerol; TGN, trans -Golgi network; YFP, yellow ﬂuorescent protein.