Phosphatidic Acid Kinase Research Articles

Differential phosphatidic acid metabolism in barley leaves and roots induced by chilling temperature

Phosphatidic acid (PA) is an important bioactive lipid that mediates chilling responses in barley. Modifications in the lipid composition of cellular membranes during chilling are essential to maintain their integrity and fluidity. First, we investigated the molecular species of PA present in leaves and roots by ESI-MS/MS, to evaluate the modifications that occur in response to chilling. We demonstrated that PA pools in leaves differ from PA fatty acid composition in roots. Compared with plants grown at 25 °C, the short-term and long-term chilling for 3 h and 36 h at 4 °C not produced significant changes in PA molecular species. The endogenous DAG and PA phosphorylation by in vitro DAG and PA kinase activities showed higher activity in leaves compared with that in root, and they showed contrasting responses to chilling. Similarly, PA removal by phosphatidate phosphohydrolase was tested, showing that this activity was specifically increased in response to chilling in roots. The findings presented here may be helpful to understand how the PA signal is modulated between tissues, and may serve to highlight the importance of knowing the basal PA pools in different plant organs.

Phenol modulates lipid kinase activities in Vicia sativa plants

Vicia sativa, is a leguminous species able to germinate, grow in the presence of phenol and remove this contaminant. However, there are not reports concerning the signals triggered by the pollutant and how plants perceive and transduce this signal in order to adapt to adverse conditions. Phosphatidic acid (PA) has been proposed as a key messenger in plants and it can be generated via phospholipase D (PLD) or via phospholipase C (PLC) coupled to diacylglycerol kinase (DGK). Thus, changes in this minor phospholipid and in enzymes involved in its catabolism were analyzed after treatment with phenol (25 and 100mgL−1). The results obtained, seem to suggest that the higher concentration could be sensed as a stressful signal, since a rapid (1.5h) and transient increase in PA, via PLD and a second wave of increase possibly via PLC/DGK was observed after 96h of exposure with 100mgL−1 of phenol. Besides, a markedly increase in enzymes related with PA metabolism, mainly DGK, phosphatidylinositol kinase (PIK) and PA kinase (PAK), was detected after long term treatment. Thus, this study highlighted the key role of minor phospholipids, especially PA, in the transduction pathway induced by phenol.

Differences in phosphatidic acid signalling and metabolism between ABA and GA treatments of barley aleurone cells

Phosphatidic acid (PA) is the common lipid product in abscisic acid (ABA) and gibberellic acid (GA) response. In this work we investigated the lipid metabolism in response to both hormones. We could detect an invivo phospholipase D activity (PLD, EC 3.1.4.4). This PLD produced [(32)P]PA (phosphatidic acid) rapidly (minutes) in the presence of ABA, confirming PA involvement in signal transduction, and transiently, indicating rapid PA removal after generation. The presence of PA removal by phosphatidate phosphatase 1 and 2 isoforms (E.C. 3.1.3.4) was verified in isolated aleurone membranes invitro, the former but not the latter being specifically responsive to the presence of GA or ABA. The invitro DGPP phosphatase activity was not modified by short time incubation with GA or ABA while the invitro PA kinase - that allows the production of 18:2-DGPP from 18:2-PA - is stimulated by ABA. The long term effects (24h) of ABA or GA on lipid and fatty acid composition of aleurone layer cells were then investigated. An increase in PC and, to a lesser extent, in PE levels is the consequence of both hormone treatments. ABA, in aleurone layer cells, specifically activates a PLD whose product, PA, could be the substrate of PAP1 and/or PAK activities. Neither PLD nor PAK activation can be monitored by GA treatment. The increase in PAP1 activity monitored after ABA or GA treatment might participate in the increase in PC level observed after 24h hormone incubation.

A GEF-to-phospholipase molecular switch caused by PA, RAC and jak tyrosine kinase, that explains leukocyte cell migration

Phospholipase D2 (PLD2) is a cell-signaling molecule that bears two activities: a guanine-nucleotide exchange factor (GEF) and a lipase that reside in the PX/PH domains and in two HKD domains, respectively. Upon cell stimulation, the GEF activity yields Rac2-GTP and the lipase activity yields phosphatidic acid (PA). In the present study, we show for the first time that these activities regulate one another. Upon cell stimulation, both GEF and lipase activities are quickly (within ∼3 min) elevated. As soon as it is produced, PA positively feeds back on the GEF and further activates it. Rac2-GTP, on the other hand, is inhibitory to the lipase activity. PLD2 would remain downregulated if it were not for the contribution of the tyrosine kinase Janus kinase 3 (JAK3), which restores lipase action (by phosphorylation at Y415). Conversely, the GEF is inhibited upon phosphorylation by JAK3 and is effectively terminated by this action and by the increasing accumulation of PA at >15 min of cell stimulation. This PA interferes with the ability of the GEF to bind to its substrate (Rac2-GTP). Thus, both temporal inter-regulation and phosphorylation-dependent mechanisms are involved in determining a GEF-lipase switch within the same molecule. Human neutrophils stimulated by interleukin-8 follow a biphasic pattern of GEF and lipase activation that can be explained by such an intramolecular switch. This is the first report of a temporal inter-regulation of two enzymatic activities that reside in the same molecule with profound biological consequences in leukocyte cell migration.

Arabidopsis thaliana lipid phosphate phosphatase 2 is involved in abscisic acid signalling in leaves

Lipid phosphate phosphatases (LPPs, E.C. 3.1.3.4) catalyse the dephosphorylation of diacylglycerol pyrophosphate (DGPP) and phosphatidic acid (PA), which are secondary messengers in abscisic acid (ABA) signalling. In this study, we investigated the effect of ABA on the expression of AtLPP genes as they encode putative ABA-signalling partners. We observed that AtLPP2 expression was down-regulated by ABA and we performed experiments on Atlpp2-2, an AtLPP2 knockout mutant, to determine whether AtLPP2 was involved in ABA signalling. We observed that Atlpp2-2 plantlets contained about twice as much PA as the wild-type Col-0 and exhibited higher PA kinase (PAK) activity than Col-0 plants. In addition, we showed that ABA stimulated diacylglycerol kinase (DGK) activity independently of AtLPP2 activity but that the ABA-stimulation of PAK activity recorded in Col-0 was dependent on AtLPP2. In order to evaluate the involvement of AtLPP2 activity in guard cell function, we measured the ABA sensitivity of Atlpp2-2 stomata. The inhibition of stomatal opening was less sensitive to ABA in Atlpp2-2 than in Col-0. Watered and water-stressed plants of the two genotypes accumulated ABA to the same extent, thus leading us to consider Atlpp2-2 an ABA-signalling mutant. Taken together our observations show that AtLPP2 is a part of ABA signalling and participate to the regulation of stomatal movements.

La signalisation lipidique chez les plantes et son rôle dans la transduction des signaux en réponse aux contraintes hydriques

Plants are sessile organisms that have developed the capacity to detect slight variations of their environment. They are able to perceive these environmental signals and to transduce them by signaling pathways in order to trigger adaptative responses. Lipid signaling elements play a central role in these pathways in plants. A key element is phosphatidic acid (PA), which can be produced by two pathways. In the first one, phospholipids are hydrolysed by phospholipase D (PLD) to release PA. In the second one, PA is produced through the activity of phospholipase C (PLC) to produce diacylglycerol (DAG) which is then phosphorylated by DAG kinase (DAGK). The amount of PA in the cell is regulated by PA kinase, which phosphorylates PA to produce diacylglycerolpyrophosphate (DGPP), considered as a second messenger as well. PLCs play a dual role in cell signaling by regulating the amount of intracellular Ca(2+), another essential second messenger. Phosphoinositides, such as PI3P, PI4P and PI(4,5)P(2), are substrates of PLCs and PLDs and are considered as second messengers also. In this review, we present recent data regarding the specific features of these lipid signaling pathways in plant compared with other eukaryotes.

Nod factor-induced phosphatidic acid and diacylglycerol pyrophosphate formation: a role for phospholipase C and D in root hair deformation.

Rhizobium-secreted nodulation factors are lipochitooligosaccharides that trigger the initiation of nodule formation on host legume roots. The first visible effect is root hair deformation, but the perception and signalling mechanisms that lead to this response are still unclear. When we treated Vicia sativa seedlings with mastoparan root hairs deformed, suggesting that G proteins are involved. To investigate whether mastoparan and Nod factor activate lipid signalling pathways initiated by phospholipase C (PLC) and D (PLD), seedlings were radiolabelled with [(32)P]orthophosphate prior to treatment. Mastoparan stimulated increases in phosphatidic acid (PA) and diacylglycerol pyrophosphate, indicative of PLD or PLC activity in combination with diacylglycerol kinase (DGK) and PA kinase. Treatment with Nod factor had similar effects, although less pronounced. The inactive mastoparan analogue Mas17 had no effect. The increase in PA was partially caused by the activation of PLD that was monitored by its in vivo transphosphatidylation activity. The application of primary butyl alcohols, inhibitors of PLD activity, blocked root hair deformation. Using different labelling strategies, evidence was provided for the activation of DGK. Since the PLC antagonist neomycin inhibited root hair deformation and the formation of PA, we propose that PLC activation produced diacylglycerol (DAG), which was subsequently converted to PA by DGK. The roles of PLC and PLD in Nod factor signalling are discussed.

Nod factor‐induced phosphatidic acid and diacylglycerol pyrophosphate formation: a role for phospholipase C and D in root hair deformation

SummaryRhizobium‐secreted nodulation factors are lipochitooligosaccharides that trigger the initiation of nodule formation on host legume roots. The first visible effect is root hair deformation, but the perception and signalling mechanisms that lead to this response are still unclear. When we treated Vicia sativa seedlings with mastoparan root hairs deformed, suggesting that G proteins are involved. To investigate whether mastoparan and Nod factor activate lipid signalling pathways initiated by phospholipase C (PLC) and D (PLD), seedlings were radiolabelled with [32P]orthophosphate prior to treatment. Mastoparan stimulated increases in phosphatidic acid (PA) and diacylglycerol pyrophosphate, indicative of PLD or PLC activity in combination with diacylglycerol kinase (DGK) and PA kinase. Treatment with Nod factor had similar effects, although less pronounced. The inactive mastoparan analogue Mas17 had no effect. The increase in PA was partially caused by the activation of PLD that was monitored by its in vivo transphosphatidylation activity. The application of primary butyl alcohols, inhibitors of PLD activity, blocked root hair deformation. Using different labelling strategies, evidence was provided for the activation of DGK. Since the PLC antagonist neomycin inhibited root hair deformation and the formation of PA, we propose that PLC activation produced diacylglycerol (DAG), which was subsequently converted to PA by DGK. The roles of PLC and PLD in Nod factor signalling are discussed.

Diacylglycerol- and phosphatidic acid-kinase studies in plant cell suspension cultures

Diacylglycerol- and phosphatidic acid-kinase studies in plant cell suspension cultures

Phosphatidate Kinase, a Novel Enzyme in Phospholipid Metabolism (Purification, Subcellular Localization, and Occurrence in the Plant Kingdom).

Microsomal membranes from suspension-cultured Catharanthus roseus cells possess an enzymic activity that catalyzes the ATP-dependent phosphorylation of phosphatidic acid (PA) to form diacylglycerol pyrophosphate (H. Behrbohm, J.B. Wissing [1993] FEBS Lett 315: 95-99). This enzyme activity, PA kinase, was purified and characterized. Plasma membranes, obtained from C. roseus microsomes by aqueous two-phase partitioning, were extracted, and PA kinase was purified 3200-fold by applying different chromatographic steps that resulted in a specific activity of about 10 [mu]mol min-1 mg-1. Sodium dodecyl sulfate-gel electrophoresis of the fractions obtained from the final chromatographic step revealed a 39-kD protein that correlated with the enzyme activity; PA kinase activity could be eluted from this protein band. Subcellular localization, investigated with C. roseus cells, showed that the activity was confined to membrane fractions, and at least 80% was associated with plasma membranes. The data revealed the same distribution within the cellular membranes of PA kinase as reported for diacylglycerol kinase, which is a typical plasma membrane-located enzyme. Furthermore, PA kinase activity was detected in the calli of 16 different plant species and in the different organs of C. roseus plants and obviously occurs ubiquitously in the plant kingdom.