Diacyglycerol (DAG) is an important class of cellular lipid messengers, but its function in plants remains elusive. Here, we show that knockout of the Arabidopsis thaliana nonspecific phospholipase C (NPC4) results in a decrease in DAG levels and compromises plant response to abscisic acid (ABA) and hyperosmotic stresses. NPC4 hydrolyzes various phospholipids in a calcium-independent manner, producing DAG and a phosphorylated head group. NPC4 knockout (KO) plants display decreased ABA sensitivity in seed germination, root elongation, and stomatal movement and had decreased tolerance to high salinity and water deficiency. Overexpression of NPC4 renders plants more sensitive to ABA and more tolerant to hyperosmotic stress than wild-type plants. Addition of a short-chain DAG or a short-chain phosphatidic acid (PA) restores the ABA response of NPC4-KO to that of the wild type, but the addition of DAG together with a DAG kinase inhibitor does not result in a wild-type phenotype. These data suggest that NPC4-produced DAG is converted to PA and that NPC4 and its derived lipids positively modulate ABA response and promote plant tolerance to drought and salt stresses.

SUMMARYGlycerophosphodiester phosphodiesterase (GDPD), which hydrolyzes glycerophosphodiesters into snglycerol-3-phosphate (G-3-P) and the corresponding alcohols, plays an important role in various physiological processes in both prokaryotes and eukaryotes. However, little is known about the physiological significance of GDPD in plants. Here, we characterized the Arabidopsis GDPD family that can be classified into canonical GDPD (AtGDPD1-6) and GDPD-like (AtGDPDL1-7) subfamilies. In vitro analysis of enzymatic activities showed that AtGDPD1 and AtGDPDL1 hydrolyzed glycerolphosphoglycerol, glycerophosphocholine and glycerophosphoethanolamine, but the maximum activity of AtGDPD1 was much higher than that of AtGDPDL1 under our assay conditions. Analyses of gene expression patterns revealed that all AtGDPD genes except for AtGDPD4 were transcriptionally active in flowers and siliques. In addition, the gene family displayed overlapping and yet distinguishable patterns of expression in roots, leaves and stems, indicating functional redundancy as well as specificity of GDPD genes. AtGDPDs but not AtGDPDLs are up-regulated by inorganic phosphate (P i ) starvation. Loss-of-function of the plastid-localized AtGDPD1 leads to a significant decrease in GDPD activity, G-3-P content, P i content and seedling growth rate only under P i starvation compared with the wild type (WT). However, membrane lipid compositions in the P i -deprived seedlings remain unaltered between the AtGDPD1 knockout mutant and WT. Thus, we suggest that the GDPD-mediated lipid metabolic pathway may be involved in release of P i from phospholipids during P i starvation.

Developing a robust root system is crucial to plant survival and competition for soil resources. Here we report that the non-specific phospholipase C5 (NPC5) and its derived lipid mediator diacylglycerol (DAG) mediate lateral root (LR) development during salt stress in Arabidopsis thaliana. T-DNA knockout mutant npc5-1 produced few to no LR under mild NaCl stress, whereas overexpression of NPC5 increased LR number. Roots of npc5-1 contained a lower level of DAG than wild type, whereas NPC5 overexpressor exhibited an increase in DAG level. Application of DAG, but not phosphatidic acid, fully restored LR growth of npc5-1 to that of wild type under NaCl stress. NPC5 expression was significantly induced in Arabidopsis seedlings treated with NaCl. Npc5-1 was less responsive to auxin-mediated root growth than the wild type. These results indicate that NPC5 mediates LR development in response to salt stress and suggest that DAG functions as a lipid mediator in the stress signalling.