Changes in membrane lipid composition play important roles in plant adaptation to and survival after freezing. Plant response to cold and freezing involves three distinct phases: cold acclimation, freezing, and post-freezing recovery. Considerable progress has been made toward understanding lipid changes during cold acclimation and freezing, but little is known about lipid alteration during post-freezing recovery. We previously showed that phospholipase D (PLD) is involved in lipid hydrolysis and Arabidopsis thaliana freezing tolerance. This study was undertaken to determine how lipid species change during post-freezing recovery and to determine the effect of two PLDs, PLD
1 and PLD, on lipid changes during post-freezing recovery. During post-freezing recovery, hydrolysis of plastidic lipids, monogalactosyldiacylglycerol and plastidic phosphatidylglycerol, is the most prominent change. In contrast, during freezing, hydrolysis of extraplastidic phospholipids, phosphatidylcholine and phosphatidylethanolamine, occurs. Suppression of PLD
1 decreased phospholipid hydrolysis and phosphatidic acid production in both the freezing and post-freezing phases, whereas ablation of PLD increased lipid hydrolysis and phosphatidic acid production during post-freezing recovery. Thus, distinctly different changes in lipid hydrolysis occur in freezing and postfreezing recovery. The presence of PLD
1 correlates with phospholipid hydrolysis in both freezing and post-freezing phases, whereas the presence of PLD correlates with reduced lipid hydrolysis during post-freezing recovery. These data suggest a negative role for PLD
1 and a positive role for PLD in freezing tolerance.