Ripening of climacteric fruits is a complex physiological phenomenon, which makes berries attractive/palatable and also determines their nutritional characteristics. We report here a comparative proteomic investigation on tomato fruits from a regional and commercial elite ecotype during maturation. Several hundreds of protein components were resolved on 2-DE gels (pH range 4-7) stained with colloidal Coomassie; almost 57% presented overlapping gel coordinates between the two compared varieties. Specific proteins were recognized in each ecotype as differentially expressed during ripening. Constant and variable components were excised, in-gel digested and analysed by MALDI-TOF-MS and microLC-ESI-IT-MS/MS approaches. Peptide MS and MS/MS data were searched against publicly available protein and EST databases, and 83 protein spots were unambiguously identified by MS. Gel matching procedures allowed further identification of proteins between ecotypes. In general, common variably expressed proteins in both ecotypes during maturation were associated to important physiological processes such as redox status control, defence, stress, carbon metabolism, energy production and cellular signalling. Protein components differentially expressed in each variety were also identified. The role of some of the identified proteins in the berries is discussed in relation to tomato fruit ripening. Accordingly, this investigation provides the first characterization of the tomato fruit proteome and description of its variation during maturation.

A 17-amino acid peptide was selectively cleaved from the highly variant C terminus of the 33-kDa 14-3-3 isoform occurring in fusicoccin receptor preparations from maize and was sequenced. The determined C-terminal sequence was identical to that of the already known maize 14-3-3 homolog GF14-6, thus prompting the use of recombinant GF14-6 in an in vitro protein-protein interaction study. The cDNA of GF14-6 was expressed in Escherichia coli as a 32 P-phosphorylatable glutathione S-transferase fusion protein and was used as a probe in overlay experiments with H ؉ -ATPase partially purified from maize roots. The results demonstrated that the recombinant protein specifically bound to H ؉ -ATPase. The binding was dependent on Mg 2؉ and was strongly increased by fusicoccin. Controlled trypsin digestion of H ؉ -ATPase abolished the association with GF14-6, a finding that was suggestive of an interaction with the C terminus of the enzyme. To confirm this result, the Cterminal domain of H ؉ -ATPase was expressed as a glutathione S-transferase fusion peptide and was used in overlay experiments. GF14-6 was also able to bind to the isolated C terminus, but only in the presence of fusicoccin.

In this review we highlight the advances achieved in the investigation of the role of 14-3-3 proteins in hormone signaling, biosynthesis, and transport. 14-3-3 proteins are a family of conserved molecules that target a number of protein clients through their ability to recognize well-defined phosphorylated motifs. As a result, they regulate several cellular processes, ranging from metabolism to transport, growth, development, and stress response. High-throughput proteomic data and two-hybrid screen demonstrate that 14-3-3 proteins physically interact with many protein clients involved in the biosynthesis or signaling pathways of the main plant hormones, while increasing functional evidence indicates that 14-3-3-target interactions play pivotal regulatory roles. These advances provide a framework of our understanding of plant hormone action, suggesting that 14-3-3 proteins act as hubs of a cellular web encompassing different signaling pathways, transducing and integrating diverse hormone signals in the regulation of physiological processes.

The H؉ -ATPase is a key enzyme for the establishment and maintenance of plasma membrane potential and energization of secondary active transport in the plant cell. The phytotoxin fusicoccin induces H ؉ -ATPase activation by promoting the association of 14-3-3 proteins. It is still unclear whether 14-3-3 proteins can represent natural regulators of the proton pump, and factors regulating 14-3-3 binding to the H ؉ -ATPase under physiological conditions are unknown as well. In the present study in vivo and in vitro evidence is provided that 14-3-3 proteins can associate with the H ؉ -ATPase from maize roots also in a fusicoccin-independent manner and that the interaction depends on the phosphorylation status of the proton pump. Furthermore, results indicate that phosphorylation of H ؉ -ATPase influences also the fusicoccin-dependent interaction of 14-3-3 proteins. Finally, a protein phosphatase 2A able to impair the interaction between H ؉

Ripening of climacteric fruits involves a complex network of biochemical and metabolic changes that make them palatable and rich in nutritional and health-beneficial compounds. Since fruit maturation has a profound impact on human nutrition, it has been recently the object of increasing research activity by holistic approaches, especially on model species. Here we report on the original proteomic characterization of ripening in apricot, a widely cultivated species of temperate zones appreciated for its taste and aromas, whose cultivation is yet hampered by specific limitations. Fruits of Prunus armeniaca cv. Vesuviana were harvested at three ripening stages and proteins extracted and resolved by 1D and 2D electrophoresis. Whole lanes from 1D gels were subjected to shot-gun analysis that identified 245 gene products, showing preliminary qualitative differences between maturation stages. In parallel, differential analysis of 2D proteomic maps highlighted 106 spots as differentially represented among variably ripen fruits. Most of these were further identified by means of MALDI-TOF-PMF and nanoLC-ESI-LIT-MS/MS as enzymes involved in main biochemical processes influencing metabolic/structural changes occurring during maturation, i.e. organic acids, carbohydrates and energy metabolism, ethylene biosynthesis, cell wall restructuring and stress response, or as protein species linkable to peculiar fruit organoleptic characteristics. In addition to originally present preliminary information on the main biochemical changes that characterize apricot ripening, this study also provides indications for future marker-assisted selection breeding programs aimed to ameliorate fruit quality.