A sensitive approach based on electrospray ionization tandem mass spectrometry has been employed to profile membrane lipid molecular species in Arabidopsis undergoing cold and freezing stresses. Freezing at a sublethal temperature induced a decline in many molecular species of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylglycerol (PG) but induced an increase in phosphatidic acid (PA) and lysophospholipids. To probe the metabolic steps generating these changes, lipids of Arabidopsis deficient in the most abundant phospholipase D, PLD␣, were analyzed. The PC content dropped only half as much, and PA levels rose only half as high in the PLD␣-deficient plants as in wild-type plants. In contrast, neither PE nor PG levels decreased significantly more in wild-type plants than in PLD␣-deficient plants. These data suggest that PC, rather than PE and PG, is the major in vivo substrate of PLD␣. The action of PLD␣ during freezing is of special interest because Arabidopsis plants that are deficient in PLD␣ have improved tolerance to freezing. The greater loss of PC and increase in PA in wild-type plants as compared with PLD␣-deficient plants may be responsible for destabilizing membrane bilayer structure, resulting in a greater propensity toward membrane fusion and cell death in wild-type plants.Eukaryotic membranes contain diverse lipid molecular species, and the lipid composition changes in response to both internal and external cues. Knowing how lipid molecular species change and how the changes are generated is important to the understanding of membrane and cell functions. Detailed study of membrane lipid changes, however, has been technically challenging because of the complexity of lipid molecular species and analytical procedures. Recently, an approach based on electrospray ionization tandem mass spectrometry (ESI-MS/ MS) 1 has been developed to comprehensively analyze lipid composition in animal and yeast cells (1-9). It requires only simple sample preparation and small samples to identify and quantify lipid molecular species. Expansion of this approach to plants, which harbor unique lipids, such as galactosylglycerolipids, should greatly facilitate the understanding of lipid functions in plant growth, development, and stress responses.Plant stress caused by freezing has been an area of intensive research for many years, but the molecular and cellular mechanisms of freezing injury and tolerance are not well understood (10 -12). The best documented freezing injury occurs at the membrane level. One major form of freezing damage is due to the formation of lipid hexagonal II phase in regions where the plasma membrane and the chloroplast envelope are closely apposed (13,14). Changes in membrane lipid composition occur when plants are exposed to freezing temperatures (15). Lipid hydrolysis has been proposed to be mainly responsible for the change, but the role of lipid hydrolysis in freezing injury and tolerance is not clear.In plants, several lipolytic enzymatic activities have been described, ...

Freezing injury is a major environmental limitation on the productivity and geographical distribution of plants. Here we show that freezing tolerance can be manipulated in Arabidopsis thaliana by genetic alteration of the gene encoding phospholipase Ddelta (PLDdelta), which is involved in membrane lipid hydrolysis and cell signaling. Genetic knockout of the plasma membrane-associated PLDdelta rendered A. thaliana plants more sensitive to freezing, whereas overexpression of PLDdelta increased freezing tolerance. Lipid profiling revealed that PLDdelta contributed approximately 20% of the phosphatidic acid produced in wild-type plants during freezing, and overexpression of PLDdelta increased the production of phosphatidic acid species. The PLDdelta alterations did not affect the expression of the cold-regulated genes COR47 or COR78 or alter cold-induced increases in proline or soluble sugars, suggesting that the PLD pathway is a unique determinant of the response to freezing and may present opportunities for improving plant freezing tolerance.

Physical and optical properties of binary amorphous selenium-antimony thin films

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.