This study investigates the role of phospholipase Dα (PLDα) in freezing-induced lipid changes in Arabidopsis. Using electrospray ionization tandem mass spectrometry, researchers profiled membrane lipid molecular species in Arabidopsis plants under cold and freezing stress. Freezing at sublethal temperatures reduced phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylglycerol (PG) levels but increased phosphatidic acid (PA) and lysophospholipids. Analysis of PLDα-deficient plants showed that PC levels dropped only half as much, and PA levels rose only half as high as in wild-type plants, suggesting that PC is the major in vivo substrate of PLDα. PLDα-deficient plants exhibited improved freezing tolerance, likely due to reduced PC loss and increased PA, which destabilizes membrane bilayer structure and promotes membrane fusion and cell death in wild-type plants. Eukaryotic membranes contain diverse lipid molecular species that change in response to internal and external cues. Understanding these changes is crucial for understanding membrane and cell functions. The study used electrospray ionization tandem mass spectrometry to analyze lipid composition in Arabidopsis, which requires simple sample preparation and small samples to identify and quantify lipid molecular species. This approach can help understand lipid functions in plant growth, development, and stress responses. Freezing injury in plants is primarily due to membrane damage, with lipid hexagonal II phase formation being a major form of damage. Lipid hydrolysis is proposed to be responsible for these changes, but the role of lipid hydrolysis in freezing injury and tolerance is not clear. Several lipolytic enzymatic activities have been described in plants, including phospholipase D (PLD), phospholipase C, phospholipase A, nonspecific acyl hydrolase, and galactolipases. PLD, which hydrolyzes phospholipids to phosphatidic acid (PA) and free head groups, is particularly abundant in plants. Arabidopsis has multiple PLD genes, and PLDα is the most common plant PLD. PLDα-deficient plants show enhanced freezing tolerance, suggesting that PLDα plays a key role in freezing injury and tolerance. The study used an automated ESI-MS/MS strategy to profile plant lipids and determine lipid changes in Arabidopsis after cold acclimation and freezing. Comparative lipid profiling of wild-type and PLDα-deficient plants revealed that PLDα is the major in vivo substrate for PC hydrolysis. The results suggest that PLDα and PC hydrolysis play key roles in freezing injury and point to a new avenue for improving freezing tolerance. The study also highlights the importance of lipid composition in freezing tolerance, with the findings indicating that membrane lipid composition can have a profound impact on the tendency of aThis study investigates the role of phospholipase Dα (PLDα) in freezing-induced lipid changes in Arabidopsis. Using electrospray ionization tandem mass spectrometry, researchers profiled membrane lipid molecular species in Arabidopsis plants under cold and freezing stress. Freezing at sublethal temperatures reduced phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylglycerol (PG) levels but increased phosphatidic acid (PA) and lysophospholipids. Analysis of PLDα-deficient plants showed that PC levels dropped only half as much, and PA levels rose only half as high as in wild-type plants, suggesting that PC is the major in vivo substrate of PLDα. PLDα-deficient plants exhibited improved freezing tolerance, likely due to reduced PC loss and increased PA, which destabilizes membrane bilayer structure and promotes membrane fusion and cell death in wild-type plants. Eukaryotic membranes contain diverse lipid molecular species that change in response to internal and external cues. Understanding these changes is crucial for understanding membrane and cell functions. The study used electrospray ionization tandem mass spectrometry to analyze lipid composition in Arabidopsis, which requires simple sample preparation and small samples to identify and quantify lipid molecular species. This approach can help understand lipid functions in plant growth, development, and stress responses. Freezing injury in plants is primarily due to membrane damage, with lipid hexagonal II phase formation being a major form of damage. Lipid hydrolysis is proposed to be responsible for these changes, but the role of lipid hydrolysis in freezing injury and tolerance is not clear. Several lipolytic enzymatic activities have been described in plants, including phospholipase D (PLD), phospholipase C, phospholipase A, nonspecific acyl hydrolase, and galactolipases. PLD, which hydrolyzes phospholipids to phosphatidic acid (PA) and free head groups, is particularly abundant in plants. Arabidopsis has multiple PLD genes, and PLDα is the most common plant PLD. PLDα-deficient plants show enhanced freezing tolerance, suggesting that PLDα plays a key role in freezing injury and tolerance. The study used an automated ESI-MS/MS strategy to profile plant lipids and determine lipid changes in Arabidopsis after cold acclimation and freezing. Comparative lipid profiling of wild-type and PLDα-deficient plants revealed that PLDα is the major in vivo substrate for PC hydrolysis. The results suggest that PLDα and PC hydrolysis play key roles in freezing injury and point to a new avenue for improving freezing tolerance. The study also highlights the importance of lipid composition in freezing tolerance, with the findings indicating that membrane lipid composition can have a profound impact on the tendency of a