Rapid phosphatidic acid accumulation in response to low temperature stress in Arabidopsis is generated through diacylglycerol kinase.

Phosphatidic acid (PtdOH) is emerging as an important signaling lipid in abiotic stress responses in plants. The effect of cold stress was monitored using (32)P-labeled seedlings and leaf discs of Arabidopsis thaliana. Low, non-freezing temperatures were found to trigger a very rapid (32)P-PtdOH increase, peaking within 2 and 5 min, respectively. In principle, PtdOH can be generated through three different pathways, i.e., (1) via de novo phospholipid biosynthesis (through acylation of lyso-PtdOH), (2) via phospholipase D hydrolysis of structural phospholipids, or (3) via phosphorylation of diacylglycerol (DAG) by DAG kinase (DGK). Using a differential (32)P-labeling protocol and a PLD-transphosphatidylation assay, evidence is provided that the rapid (32)P-PtdOH response was primarily generated through DGK. A simultaneous decrease in the levels of (32)P-PtdInsP, correlating in time, temperature dependency, and magnitude with the increase in (32)P-PtdOH, suggested that a PtdInsP-hydrolyzing PLC generated the DAG in this reaction. Testing T-DNA insertion lines available for the seven DGK genes, revealed no clear changes in (32)P-PtdOH responses, suggesting functional redundancy. Similarly, known cold-stress mutants were analyzed to investigate whether the PtdOH response acted downstream of the respective gene products. The hos1, los1, and fry1 mutants were found to exhibit normal PtdOH responses. Slight changes were found for ice1, snow1, and the overexpression line Super-ICE1, however, this was not cold-specific and likely due to pleiotropic effects. A tentative model illustrating direct cold effects on phospholipid metabolism is presented

Plant phosphatidic acid metabolism in response to environmental stress

Planten staan bloot aan snelle veranderingen in omgevingsfactoren zoals temperatuur, de beschikbaarheid van water en het zoutgehalte in de bodem. In de loop van honderden miljoenen jaren evolutie hebben ze het vermogen ontwikkeld om daarop adequaat te reageren, zodat de negatieve impact op het functioneren beperkt blijft. Op het cellulaire niveau worden zulke ‘stressresponsen' gereguleerd door signaalmoleculen en een daarvan is het fosfolipide fosfatidylzuur. Steven Arisz onderzocht de stress-geïnduceerde vorming - binnen minuten - van deze stof in twee plantensoorten: de groenalg Chlamydomonas en de zandraket. Het metabolisme van fosfatidylzuur is ingewikkeld, omdat het een centrale positie heeft in de synthese en afbraak van lipiden. Gebruikmakend van verschillende experimentele benaderingen toonde Arisz aan welke biochemische wegen betrokken zijn bij de accumulatie van fosfatidylzuur tijdens blootstelling aan koude en hoge zoutconcentraties. Het enzym diacylglycerol kinase lijkt hierbij een cruciale rol te spelen. Fundamenteel begrip van de regulatie van stressresponsen kan handvatten bieden voor het ontwikkelen van meer bestendige gewassen

The salt stress-induced LPA response in Chlamydomonas is produced via PLA(2) hydrolysis of DGK-generated phosphatidic acid

The unicellular green alga Chlamydomonas has frequently been used as a eukaryotic model system to study intracellular phospholipid signaling pathways in response to environmental stresses. Earlier, we found that hypersalinity induced a rapid increase in the putative lipid second messenger, phosphatidic acid (PA), which was suggested to be generated via activation of a phospholipase D (PLD) pathway and the combined action of a phospholipase C/diacylglycerol kinase (PLC/DGK) pathway. Lysophosphatidic acid (LPA) was also increased and was suggested to reflect a phospholipase A(2) (PLA(2)) activity based on pharmacological evidence. The question of PA's and LPA's origin is, however, more complicated, especially as both function as precursors in the biosynthesis of phospho- and galactolipids. To address this complexity, a combination of fatty acid-molecular species analysis and in vivo (32)P-radiolabeling was performed. Evidence is provided that LPA is formed from a distinct pool of PA characterized by a high α-linolenic acid (18:3n-3) content. This molecular species was highly enriched in the polyphosphoinositide fraction, which is the substrate for PLC to form diacylglycerol. Together with differential (32)P-radiolabeling studies and earlier PLD-transphosphatidylation and PLA(2)-inhibitor assays, the data were consistent with the hypothesis that the salt-induced LPA response is primarily generated through PLA(2)-mediated hydrolysis of DGK-generated PA and that PLD or de novo synthesis [via endoplasmic reticulum - or plastid-localized routes] is not a major contributor