Arabidopsis thaliana cell suspension cultures have been used to investigate the effects of salinity and hyperosmotic stress on plant cellular proteins. We show that 200 mM NaCl and 400 mM sorbitol treatments induce extracellular medium acidification in Arabidopsis cell cultures, a typical response of plant cells to salt and hyperosmotic stress. Using (35)S-labelled amino acids, we demonstrated that NaCl causes a transient suppression of de novo protein synthesis, from which the cells recover within 4 h. Changes in the abundance of cellular proteins 6 h post NaCl and sorbitol treatments were analysed by 2-DE. Of a total of 2,949 protein spots detected on the gels, 266 showed significant changes in abundance across five independent experiments. Using MALDI-TOF MS, we identified 75 salt and sorbitol responsive spots. These fall into 10 functional categories that include H(+) transporting ATPases, signal transduction related proteins, transcription/translation related proteins, detoxifying enzymes, amino acid and purine biosynthesis related proteins, proteolytic enzymes, heat-shock proteins, carbohydrate metabolism-associated proteins and proteins with no known biological functions.

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With the completion of the Arabidopsis genome, many hypothetical proteins have been predicted without any information on their expression, subcellular localisation and function. We have performed proteomic analysis of proteins sequentially extracted from enriched Arabidopsis cell wall fractions and separated by two-dimensional gel electrophoresis (2-DE). The proteins were identified by peptide mass fingerprinting using matrix-assisted laser desorption/ionisation-time of flight (MALDI-TOF) mass spectrometry and genomic database searches. This is part of a targeted exercise to establish the entire Arabidopsis secretome database. We report evidence for new proteins of unknown function whose existence had been predicted from genomic sequences and, furthermore, localise them to the cell wall. In addition, we observed an unexpected presence in the cell wall preparations of proteins whose known biochemical activity has never been associated with this compartment hitherto. We discuss the implications of these findings and present results suggesting a possible involvement of cell wall kinases in plant responses to pathogen attack.

ATP is a vital molecule used by living organisms as a universal source of energy required to drive the cogwheels of intracellular biochemical reactions necessary for growth and development. Animal cells release ATP to the extracellular milieu, where it functions as the primary signaling cue at the epicenter of a diverse range of physiological processes. Although recent findings revealed that intact plant tissues release ATP as well, there is no clearly defined physiological function of extracellular ATP in plants. Here, we show that extracellular ATP is essential for maintaining plant cell viability. Its removal by the cell-impermeant traps glucose-hexokinase and apyrase triggered death in both cell cultures and whole plants. Competitive exclusion of extracellular ATP from its binding sites by treatment with b,g-methyleneadenosine 59-triphosphate, a nonhydrolyzable analog of ATP, also resulted in death. The death response was observed in Arabidopsis thaliana, maize (Zea mays), bean (Phaseolus vulgaris), and tobacco (Nicotiana tabacum). Significantly, we discovered that fumonisin B1 (FB1) treatment of Arabidopsis triggered the depletion of extracellular ATP that preceded cell death and that exogenous ATP rescues Arabidopsis from FB1-induced death. These observations suggest that extracellular ATP suppresses a default death pathway in plants and that some forms of pathogen-induced cell death are mediated by the depletion of extracellular ATP.

Acyl carrier protein (ACP) is an essential cofactor in biosynthesis of fatty acids and many other reactions that require acyl transfer steps. We have determined the first crystal structures of an acylated form of ACP from E. coli, that of butyryl-ACP. Our analysis of the molecular surface of ACP reveals a plastic hydrophobic cavity in the vicinity of the phosphopantethylated Ser36 residue that is expanded and occupied by the butyryl and beta-mercaptoethylamine moieties of the acylated 4'-phosphopantetheine group in one of our crystal forms. In the other form, the cavity is contracted, and we propose that the protein has adopted the conformation after delivery of substrate into the active site of a partner enzyme.