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, ...

We have investigated the ability of methionine sulfoxide reductase (MsrA) to maintain optimal calmodulin (CaM) function through the repair of oxidized methionines, which have been shown to accumulate within CaM in senescent brain [Gao, J., Yin, D. H., Yao, Y., Williams, T. D., and Squier, T. C. (1998) Biochemistry 37, 9536-9548]. Oxidatively modified calmodulin (CaMox) isolated from senescent brain or obtained by in vitro oxidation was incubated with MsrA. This treatment restores the functional ability of CaMox to activate the plasma membrane (PM) Ca-ATPase, confirming that (i) the decreased ability of CaM isolated from senescent animals to activate the PM Ca-ATPase results solely from methionine sulfoxide formation and (ii) MsrA can repair methionine sulfoxides within cytosolic proteins. We have used electrospray ionization mass spectrometry to investigate the extent and rates of methionine sulfoxide repair within CaMox. Upon exhaustive repair by MsrA, there remains a distribution of methionine sulfoxides within functionally reactivated CaMox, which varies from three to eight methionine sulfoxides. The rates of repair of methionine sulfoxides within individual tryptic fragments of CaMox vary by a factor of 2, where methionine sulfoxides located within hydrophobic sequences are repaired in preference to methionines that are more solvent accessible within the native structure. However, no single methionine sulfoxide is completely repaired in all CaM oxiforms. Decreases in the alpha-helical content and a disruption of the tertiary structure of CaM have previously been shown to result from methionine oxidation. Repair of selected methionine sulfoxides in CaMox by MsrA results in a partial refolding of the secondary structure, suggesting that MsrA repairs methionine sulfoxides within unfolded sequences until native-like structure and function are re-attained. The ability of CaMox isolated from senescent brain to fully activate the PM Ca-ATPase following repair by MsrA suggests the specific activity of MsrA is insufficient to maintain CaM function in aging brain. These results are discussed in terms of the possible regulatory role MsrA may play in the modulation of CaM function and calcium homeostasis under conditions of oxidative stress.

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At a Zn2+/INS molar ratio comparable to that in regular INS preparations, Zn2+-INS was susceptible to MCO. Both His residues of INS were converted partially to 2-oxo-His, with 10His(B) possessing ca. three times greater susceptibility to MCO than 5His(B).

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