Membrane Rigidity and Phosphatidic Acid (PtdOH) Signal: Two Important Events in Acinetobacter guillouiae SFC 500-1A Exposed to Chromium(VI) and Phenol.

The remodeling of membrane lipids is a mechanism that allows microorganisms to survive in unfavorable environments such as industrial effluents, which often contain inorganic and organic pollutants, like chromium and phenol. In the present work, we evaluated the effect of Cr(VI) and phenol on the membrane of Acinetobacter guillouiae SFC 500-1A, a bacterial strain isolated from tannery sediments where such pollutants can be found. The presence of lipid kinases and phospholipases and the changes in their activities under exposure to these pollutants were determined. Cr(VI) and Cr(VI) + phenol caused the membrane to become more rigid for up to 16 h after exposure. This could be due to an increase in cardiolipin (Ptd2 Gro) and a decrease in phosphatidylethanolamine (PtdEtn), which are indicative of more order and rigidity in the membrane. Increased phospholipase A activity (PLA, EC 3.1.1.4) could be responsible for the decrease in PtdEtn levels. Moreover, our results indicate that Cr(VI) and Cr(VI) + phenol trigger the phosphatidic acid (PtdOH) signal. The finding of significantly increased phosphatidylinositol-4-phosphate (PtdIns-4-P) levels means this is likely achieved via PtdIns-PLC/DGK. This report provides the first evidence that A. guillouiae SFC 500-1A is able to sense Cr(VI) and phenol, transduce this signal through changes in the physical state of the membrane, and trigger lipid-signaling events.

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.

Lipid signalling mediated by PLD/PA modulates proline and H2O2 levels in barley seedlings exposed to short- and long-term chilling stress.

Phospholipase D (PLD) hydrolyses phospholipids to yield phosphatidic acid (PA) and a head group, and is involved in responses to a variety of environmental stresses, including chilling and freezing stress. Barley responses to chilling stress (induced by incubating seedlings at 4 °C) are dynamic and the duration of stress, either short (0-180 min) or long-term (24-36 h) had a significant impact on the response. We investigated the roles of PLD/PA in responses of barley (Hordeum vulgare) seedlings to short and long-term chilling stress, based on regulation of proline and reactive oxygen species (ROS) levels. Short-term chilling stress caused rapid and transient increases in PLD activity, proline level, and ROS levels in young leaves. PLD has the ability to catalyse the transphosphatidylation reaction leading to formation of phosphatidylalcohol (preferentially, to PA). Pre-treatment of seedlings with 1-butanol significantly increased proline synthesis but decreased ROS (H2O2) formation. These observations suggest that PLD is a negative regulator of proline synthesis, whereas PA/PLD promote ROS signals. Exogenous PA pre-treatment reduced the proline synthesis but enhanced H2O2 formation. Effects of long-term chilling stress on barley seedlings differed from those of short-term chilling stress. E.g., PLD activity was significantly reduced in young leaves and roots, whereas proline synthesis and ROS signals were increased in roots. Exogenous ROS application enhanced proline level while exogenous proline application reduced ROS level and modulated some effects of long-term chilling stress. Our findings suggest that PLD contributes to signalling pathways in responses to short-term chilling stress in barley seedling, through regulation of the balance between proline and ROS levels. In contrast, reduced PLD activity in the response to long-term chilling stress did not affect proline level. Increased ROS levels may reflect an antioxidant system that is affected by chilling stress and positively compensated by changes in proline level. Implications of our findings are discussed in regard to adaptation strategies of barley seedlings to low temperatures.

Arsenic stress induces changes in lipid signalling and evokes the stomata closure in soybean.

Soybean (Glycine max) is often exposed to high arsenic (As) level in soils or through irrigation with groundwater. In previous studies on As-treated soybean seedlings we showed deleterious effect on growth, structural alterations mainly in root vascular system and induction of antioxidant enzymes. However, there are not reports concerning signal transduction pathways triggered by the metalloid in order to develop adaptive mechanisms. Phosphatidic acid (PA), a key messenger in plants, can be generated via phospholipase D (PLD) or via phospholipase C (PLC) coupled to diacylglycerol kinase (DGK). Thus, changes in PA and in an enzyme involved in its metabolism (PLD) were analysed in soybean seedlings treated with 25 µM AsV or AsIII. The present study demonstrated that As triggers the PA signal by PLD and also via PLC/DGK mainly after 48 h of As treatment. DGPP, other lipid messenger produced by phosphorylation of PA by PAK increased in As treated roots. Arsenic also induced rapid and significant stomatal closure after 1.5 h of treatment, mainly with AsIII, probably as an adaptive response to the metalloid to reduce water loss by transpiration. This report constitute the first evidence that shows the effects of As on lipid signalling events in soybean seedlings which would be crucial in adaptation and survival of soybean seedlings under As stress.

Diacylglycerol pyrophosphate binds and inhibits the glyceraldehyde-3-phosphate dehydrogenase in barley aleurone.

The aleurona cell is a model that allows the study of the antagonistic effect of gibberellic acid (GA) and abscisic acid (ABA). Previous results of our laboratory demonstrated the involvement of phospholipids during the response to ABA and GA. ABA modulates the levels of diacylglycerol, phosphatidic acid and diacylglycerol pyrophosphate (DAG, PA, DGPP) through the activities of phosphatidate phosphatases, phospholipase D, diacylglycerol kinase and phosphatidate kinase (PAP, PLD, DGK and PAK). PA and DGPP are key phospholipids in the response to ABA, since both are capable of modifying the hydrolitic activity of the aleurona. Nevertheless, little is known about the mechanism of action of these phospholipids during the ABA signal. DGPP is an anionic phospholipid with a pyrophosphate group attached to diacylglycerol. The ionization of the pyrophosphate group may be important to allow electrostatic interactions between DGPP and proteins. To understand how DGPP mediates cell functions in barley aleurone, we used a DGPP affinity membrane assay to isolate DGPP-binding proteins from Hordeum vulgare, followed by mass spectrometric sequencing. A cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) was identified for being bound to DGPP. To validate our method, the relatively abundant GAPDH was characterized with respect to its lipid-binding properties, by fat western blot. GAPDH antibody interacts with proteins that only bind to DGPP and PA. We also observed that ABA treatment increased GAPDH abundance and enzyme activity. The presence of phospholipids during GAPDH reaction modulated the GAPDH activity in ABA treated aleurone. These data suggest that DGPP binds to GAPDH and this DGPP and GAPDH interaction provides new evidences in the study of DGPP-mediated ABA responses in barley aleurone.

Phosphatidylinositol kinase activities in Trypanosoma cruzi epimastigotes.

Phosphatidylinositol (PtdIns) metabolism through phosphatidylinositol kinase (PIKs) activities plays a central role in different signaling pathways. In Trypanosoma cruzi, causative agent of Chagas disease, PIKs have been proposed as target for drug design in order to combat this pathogen. In this work, we studied the classes of PI4K, PIPK and PI3K that could participate in signaling pathways in T. cruzi epimastigote forms. For this reason, we analyzed their enzymatic parameters and detailed responses to avowed kinase inhibitors (adenosine, sodium deoxycholate, wortmannin and LY294002) and activators (Ca(2+), phosphatidic acid, spermine and heparin). Our results suggest the presence and activity of a class III PI4K, a class I PIPK, a class III PI3K previously described (TcVps34) and a class I PI3K. Class I PI3K enzyme, here named TcPI3K, was cloned and expressed in a bacterial system, and their product was tested for kinase activity. The possible participation of TcPI3K in central cellular events of the parasite is also discussed.

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 in vivo 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 in vitro, the former but not the latter being specifically responsive to the presence of GA or ABA. The in vitro DGPP phosphatase activity was not modified by short time incubation with GA or ABA while the in vitro PA kinase - that allows the production of 18:2-DGPP from 18:2-PA - is stimulated by ABA. The long term effects (24 h) 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 24 h hormone incubation.

Comparative phytohormone profiles, lipid kinase and lipid phosphatase activities in barley aleurone, coleoptile, and root tissues.

We analyzed lipid kinase and lipid phosphatase activities and determined endogenous phytohormone levels by liquid chromatography-tandem mass spectrometry in root and coleoptile tissues following germination of barley (Hordeum vulgare) seeds. The enzymes showing highest activity in aleurone cells were diacylglycerol kinase (DAG-k, EC 2.7.1.107) and phosphatidate kinase (PA-k). The ratio of gibberellins (GAs) to abscisic acid (ABA) was 2-fold higher in aleurone than in coleoptile or root tissues. In coleoptiles, phosphatidylinositol 4-kinase (PI4-k, EC 2.7.1.67) showed the highest enzyme activity, and jasmonic acid (JA) level was higher than in aleurone. In roots, activities of PI4-k, DAG-k, and PA-k were similar, and salicylic acid (SA) showed the highest concentration. In the assays to evaluate the hydrolysis of DGPP (diacylglycerol pyrophosphate) and PA (phosphatidic acid) we observed that PA hydrolysis by LPPs (lipid phosphate phosphatases) was not modified; however, the diacylglycerol pyrophosphate phosphatase (DGPPase) was strikingly higher in coleoptile and root tissues than to aleurone. Relevance of these findings in terms of signaling responses and seedling growth is discussed.

Lipid profiling by electrospray ionization tandem mass spectrometry and the identification of lipid phosphorylation by kinases in potato stolons.

There is limited information about the involvement of lipids and esterified fatty acids in signaling pathways during plant development. The purpose of this study was to evaluate the lipid composition and molecular species of potato (Solanum tuberosum L., cv. Spunta) stolons and to identify phosphorylated lipids in the first two developmental stages of tuber formation. Lipid profiling was determined using ESI-MS/MS, a useful method for the determination of the biosynthesis and catabolism of lipids based on their fatty acid composition. The most prevalent compound identified in this study was phosphatidic acid (PA); digalactosyldiacylglycerol (DGDG) was the second most abundant compound. A 34:2 species was identified in PA, phosphatidylcholine (PC), phosphatidylinositol (PI), and phosphatidylethanolamine (PE). The identification of lipid phosphorylation by kinases was revealed by the presence of the phosphorylated lipids. PA was metabolized to diacylglycerol pyrophosphate (DGPP) by phosphatidic acid kinase (PAK). This work establishes a correlation between lipid fatty acid composition and lipid metabolism enzymes at the beginning of tuber formation and is the first report of PAK activity in the early events of potato tuber formation.

Diacylglycerol pyrophosphate inhibits the alpha-amylase secretion stimulated by gibberellic acid in barley aleurone.

ABA plays an important regulatory role in seed germination because it inhibits the response to GA in aleurone, a secretory tissue surrounding the endosperm. Phosphatidic acid (PA) is a well-known intermediary in ABA signaling, but the role of diacylglycerol pyrophosphate (DGPP) in germination processes is not clearly established. In this study, we show that PA produced by phospholipase D (E.C. 3.1.4.4) during the antagonist effect of ABA in GA signaling is rapidly phosphorylated by phosphatidate kinase (PAK) to DGPP. This is a crucial fact for aleurone function because exogenously added dioleoyl-DGPP inhibits secretion of alpha-amylase (E.C. 3.2.1.1). Aleurone treatment with ABA and 1-butanol results in normal secretory activity, and this effect is reversed by addition of dioleoyl-DGPP. We also found that ABA decreased the activity of an Mg2+-independent, N-ethylmaleimide-insensitive form of phosphatidate phosphohydrolase (PAP2) (E.C. 3.1.3.4), leading to reduction of PA dephosphorylation and increased PAK activity. Sequence analysis using Arabidopsis thaliana lipid phosphate phosphatase (LPP) sequences as queries identified two putative molecular homologues, termed HvLPP1 and HvLPP2, encoding putative Lpps with the presence of well-conserved structural Lpp domains. Our results are consistent with a role of DGPP as a regulator of ABA antagonist effect in GA signaling and provide evidence about regulation of PA level by a PAP2 during ABA response in aleurone.

Phosphatidylinositol kinases as regulators of GA-stimulated alpha-amylase secretion in barley (Hordeum vulgare).

Phosphorylated derivatives of phosphatidylinositol, in association with phosphatidylinositol 3-kinase (PI3 kinase, EC 2.7.1.137) and phosphatidylinositol 4-kinase (PI4 kinase, EC 2.7.1.67), play a key role in regulation of fundamental cell processes. We present evidence for a relationship between alpha-amylase (EC 3.2.1.1) secretion regulated by GA and levels of phosphatidylinositol 3-phosphate and phosphatidylinositol 4-phosphate (PtdIns(4)P) in barley (Hordeum vulgare). Microsomal membranes were incubated in the presence of [gamma-(32)P]ATP, and radiolabeled membrane lipids were extracted and separated by TLC using a boric acid system. Treatment of aleurone layers with GA for short or long periods of time increased PI4 kinase activity. To evaluate the effect of PtdIns(4)P levels on GA signaling, we used phenylarsine oxide (PAO), an inhibitor of PI4 kinase activity. PAO reversibly reduced the alpha-amylase secretion and protoplast cell vacuolation in a dose-dependent manner. Wortmannin showed a similar inhibitory effect on alpha-amylase secretion and PI4 kinase activity. GA evoked only a long-term increase in PI3 kinase activity, which was also affected by PAO. The effect of PAO was suppressed by the reducing agent 2,3-dimercapto-1-propanol (BAL), leading to restoration of secretion, vacuolation and PI4 kinase activity. In contrast, the effect of PAO on PI3 kinase activity was not abolished by BAL, suggesting that PI3 kinase is not involved in the secretion process. Likewise, the compound LY294002 inhibited PI3 kinase but had no effect on the secretion process. These findings indicate that PI4 kinase acts as a positive regulator of early GA signaling in aleurone.