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

• ¹Department of Plant Physiology, Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, Netherlands
• ²Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, IN, USA
• ³Shanghai Center for Plant Stress Biology and Shanghai Institute of Plant Physiology and Ecology, Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences, Shanghai, China

Phosphatidic acid (PtdOH) is emerging as an important signaling lipid in abiotic stress responses in plants. The effect of cold stress was monitored using ³²P-labeled seedlings and leaf discs of Arabidopsis thaliana. Low, non-freezing temperatures were found to trigger a very rapid ³²P-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 ³²P-labeling protocol and a PLD-transphosphatidylation assay, evidence is provided that the rapid ³²P-PtdOH response was primarily generated through DGK. A simultaneous decrease in the levels of ³²P-PtdInsP, correlating in time, temperature dependency, and magnitude with the increase in ³²P-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 ³²P-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.