This study investigates the role of phospholipase Dα (PLDα) in freezing-induced lipid changes in Arabidopsis. Using electrospray ionization tandem mass spectrometry (ESI-MS/MS), the authors profiled membrane lipid molecular species in Arabidopsis subjected to cold and freezing stresses. Freezing at a sublethal temperature led to a decline in phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylglycerol (PG) but increased phosphatidic acid (PA) and lysophospholipids. PLDα-deficient plants showed reduced changes in PC and PA levels compared to wild-type plants, suggesting that PC is the major in vivo substrate of PLDα. The greater loss of PC and increase in PA in wild-type plants may contribute to destabilizing membrane bilayer structure, leading to membrane fusion and cell death. The study also found that PLDα-deficient plants have improved freezing tolerance, which may be due to the reduced hydrolysis of PC and increased PA levels. These findings highlight the importance of PLDα in freezing injury and provide insights into the mechanisms of plant stress responses.This study investigates the role of phospholipase Dα (PLDα) in freezing-induced lipid changes in Arabidopsis. Using electrospray ionization tandem mass spectrometry (ESI-MS/MS), the authors profiled membrane lipid molecular species in Arabidopsis subjected to cold and freezing stresses. Freezing at a sublethal temperature led to a decline in phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylglycerol (PG) but increased phosphatidic acid (PA) and lysophospholipids. PLDα-deficient plants showed reduced changes in PC and PA levels compared to wild-type plants, suggesting that PC is the major in vivo substrate of PLDα. The greater loss of PC and increase in PA in wild-type plants may contribute to destabilizing membrane bilayer structure, leading to membrane fusion and cell death. The study also found that PLDα-deficient plants have improved freezing tolerance, which may be due to the reduced hydrolysis of PC and increased PA levels. These findings highlight the importance of PLDα in freezing injury and provide insights into the mechanisms of plant stress responses.