Phosphatidic acid (PA) has only recently been identified as an important eukaryotic lipid-signalling molecule. In plants, PA formation is triggered by various biotic and abiotic stresses, including wounding, pathogen attack, drought, salinity, cold, and freezing. However, few molecular targets of PA have been identified so far. One of the best characterized is Raf-1, a mammalian MAPKKK. Arabidopsis thaliana CTR1 (constitutive triple response 1) is one of the plant homologues of Raf-1 and functions as a negative regulator of the ethylene signalling pathway. Here, it is shown that PA binds CTR1 and inhibits its kinase activity. Using different PA-binding assays, the kinase domain of CTR1 (CTR1-K) was found to bind PA directly. Addition of PA resulted in almost complete inhibition of CTR1 kinase activity and disrupted the intramolecular interaction between CTR1-K and the CTR1 N-terminal regulatory domain. Additionally, PA blocked the interaction of CTR1 with ETR1, one of the ethylene receptors. The basic amino acid motif shown to be required for PA binding in Raf-1 is conserved in CTR1-K. However, mutations in this motif did not affect either PA-binding or PA-dependent inhibition of CTR1 activity. Subsequent deletion analysis of CTR1's kinase domain revealed a novel PA-binding region at the C-terminus of the kinase.
SummaryPlant cells are continuously exposed to environmental stresses such as hyper-osmolarity, and have to respond in order to survive. When 32 P-labelled Chlamydomonas moewusii cells were challenged with NaCl, the formation of a new radiolabelled phospholipid was stimulated, which was barely detectable before stimulation. The phospholipid was identi®ed as lyso-phosphatidic acid (LPA), and was the only lyso-phospholipid to be accumulated. The increase in LPA was dose-and time-dependent. When other osmotically active compounds were used, the formation of LPA was also induced with similar kinetics, although salts were better inducers than non-salts. At least part of the LPA was generated by phospholipase A 2 (PLA 2 ) hydrolysing phosphatidic acid (PA). This claim is based on PA formation preceding LPA production, and PLA 2 inhibitors decreasing the accumulation of LPA and promoting the conversion of PA to diacylglycerol pyrophosphate. The latter is another metabolic derivative of PA that is implicated in cell signalling. The involvement of multiple lipid-signalling pathways in hyperosmotic stress responses is discussed.
SummaryHyperosmotic stress induces the rapid formation of phosphatidic acid (PA) in Chlamydomonas moewusii via the activation of two signalling pathways: phospholipase D (PLD) and phospholipase C (PLC), the latter in combination with diacylglycerol kinase (DGK) . A concomitant increase in cell Ca 2+ becomes manifest as de¯agellation. When KCl was used as osmoticum we found that two concentration ranges activated de¯agellation: one between 50 and 100 mM and another above 200 mM. De¯agellation in low KCl concentrations was complete within 30 sec whereas in high concentrations it took 5 min. PLC was not activated, as it was by high KCl concentrations that cause hyperosmotic stress. Moreover PLD was activated more strongly by low than by high KCl concentrations. Potassium was the most potent monovalent cation based on the induction of de¯agellation and the formation of PA and PBut. During treatment, the external medium acidi®ed, indicating an increase in H + -ATPase activity in order to re-establish the membrane potential. Activation of PLD and de¯agellation at low KCl concentrations were abrogated by treatment with La 3+ , Gd 3+ and EGTA, indicating the dependency on extracellular Ca 2+ . This suggests that low concentrations of KCl depolarize the plasma membrane, resulting in the activation of H + -ATPases and opening voltage-dependent Ca 2+ T channels, observed as de¯agellation and an increase in PLD activity.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.