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Nickel and cobalt are obligate nutrients for the gammaproteobacteria but when present at high concentrations they display toxic effects. These two metals are present in the environment, their origin being either from natural sources or from industrial use. In this study, the effect of inhibitory concentrations of Ni or Co was assessed on the soil bacterium Pseudomonas putida KT2440 using a proteomic approach. The identification of more than 400 spots resulted in the quantification of 160 proteins that underwent significant variations in cells exposed to Co and Ni. This analysis allowed us to depict the cellular response of P. putida cells toward metallic stress. More precisely, the parallel comparison of the two proteomes showed distinct responses of P. putida to Ni or Co toxicity. The most striking effect of Co was revealed by the accumulation of several proteins involved in the defense against oxidative damage, which include proteins involved in the detoxification of the reactive oxygen species, superoxides and peroxides. The up-regulation of the genes encoding these enzymes was confirmed using qRT-PCR. Interestingly, in the Ni-treated samples, sodB, encoding superoxide dismutase, was up-regulated, indicating the apparition of superoxide radicals due to the presence of Ni. However, the most striking effect of Ni was the accumulation of several proteins involved in the synthesis of amino acids. The measurement of the amount of amino acids in Ni-treated cells revealed a strong accumulation of glutamate.

Although essential in many cellular processes, metals become toxic when they are present in excess and constitute a global environmental hazard. To overcome this stress, fungi have evolved several mechanisms at both intracellular and extracellular levels. In particular, fungi are well known for their ability to secrete a large panel of proteins. However, their role in the adaptation of fungi to metal toxicity has not yet been investigated. To address this question, here, the fungus Botrytis cinerea was challenged to copper, zinc, nickel or cadmium stress and secreted proteins were collected and separated by 2D-PAGE. One hundred and sixteen spots whose volume varied under at least one tested condition were observed on 2D gels. Densitometric analyses revealed that the secretome signature in response to cadmium was significantly different from those obtained with the other metals. Fifty-five of these 116 spots were associated with unique proteins and functional classification revealed that the production of oxidoreductases and cell-wall degrading enzymes was modified in response to metals. Promoter analysis disclosed that PacC/Rim101 sites were statistically over-represented in the upstream sequences of the 31 genes corresponding to the varying unique spots suggesting a possible link between pH regulation and metal response in B. cinerea.

The characterization of proteins isolated from skin tissue is apparently an essential parameter for understanding grape ripening as this tissue contains the key compounds for wine quality. It has been particularly difficult to extract proteins from skins for analysis by two-dimensional electrophoresis gels and, therefore, a protocol for this purpose has been adapted. The focus was on the evolution of the proteome profile of grape skin during maturation. Proteome maps obtained at three stages of ripening were compared to assess the extent to which protein distribution differs in grape skin during ripening. The comparative analysis shows that numerous soluble skin proteins evolve during ripening and reveal specific distributions at different stages. Proteins involved in photosynthesis, carbohydrate metabolisms, and stress response are identified as being over-expressed at the beginning of colour-change. The end of colour-change is characterized by the over-expression of proteins involved in anthocyanin synthesis and, at harvest, the dominant proteins are involved in defence mechanisms. In particular, increases in the abundance of different chitinase and beta-1,3-glucanase isoforms were found as the berry ripens. This observation can be correlated with the increase of the activities of both of these enzymes during skin ripening. The differences observed in proteome maps clearly show that significant metabolic changes occur in grape skin during this crucial phase of ripening. This comparative analysis provides more detailed characterization of the fruit ripening process.

In Podospora anserina previous investigations showed that mutations in genes involved in the control of protoplasmic incompatibility cause defects at various stages of differentiation during the life cycle and also modify properties of the plasma membrane. To establish these relationships in another way, a new method for screening mutations has been developed as a first step. Eighty-five new mutants were selected for resistance to toxic products (sorbose or thiourea); in a second step these mutants were tested for modifications of protoplasmic incompatibility and cellular differentiation. Seven of the sorbose or thiourea resistant-mutants (i.e., 8%) exhibited new patterns of protoplasmic incompatibility. Genetic analyses were carried out with three mutants. Mutation X25 suppresses protoplasmic incompatibility resulting from all allelic interactions and restores the fertility of the crosses female symbol Vx male symbol V1 and female symbol Z1 x male symbol Z2. Mutation V41 is an allele of the v locus with new properties. Mutation X61 totally suppresses the V/V'1 interaction and weakens all of the other allelic incompatibility systems; X61 strains are defective in protoperithecia differentiation. Electrophoresis of plasma membrane proteins showed that X61 strains lack two polypeptides whose apparent molecular weights are 41,000 and 44,000. This new screening method is especially efficient for obtaining new mutants and identifying additional genes involved in incompatibility. These results provide further support demonstrating the relationships between protoplasmic incompatibility, cellular differentiation and plasma membrane.