Background: Grape ripening represents the third phase of the double sigmoidal curve of berry development and is characterized by deep changes in the organoleptic characteristics. In this process, the skin plays a central role in the synthesis of many compounds of interest (e.g. anthocyanins and aroma volatiles) and represents a fundamental protective barrier against damage by physical injuries and pathogen attacks. In order to improve the knowledge on the role of this tissue during ripening, changes in the protein expression in the skin of the red cultivar Barbera at five different stages fromvéraisonto full maturation were studied by performing a comparative 2-DE analysis.Results: The proteomic analysis revealed that 80 spots were differentially expressed throughout berry ripening. Applying a two-way hierarchical clustering analysis to these variations, a clear difference between the first two samplings (up to 14 days aftervéraison) and the following three (from 28 to 49 days aftervéraison) emerged, thus suggesting that the most relevant changes in protein expression occurred in the first weeks of ripening. By means of LC-ESI-MS/MS analysis, 69 proteins were characterized. Many of these variations were related to proteins involved in responses to stress (38%), glycolysis and gluconeogenesis (13%), C-compounds and carbohydrate metabolism (13%) and amino acid metabolism (10%).Conclusion: These results give new insights to the skin proteome evolution during ripening, thus underlining some interesting traits of this tissue. In this view, we observed the ripening-related induction of many enzymes involved in primary metabolism, including those of the last five steps of the glycolytic pathway, which had been described as down-regulated in previous studies performed on whole fruit. Moreover, these data emphasize the relevance of this tissue as a physical barrier exerting an important part in berry protection. In fact, the level of many proteins involved in (a)biotic stress responses remarkably changed through the five stages taken into consideration, thus suggesting that their expression may be developmentally regulated.

Background: Nitrogen nutrition is one of the major factors that limit growth and production of crop plants. It affects many processes, such as development, architecture, flowering, senescence and photosynthesis. Although the improvement in technologies for protein study and the widening of gene sequences have made possible the study of the plant proteomes, only limited information on proteome changes occurring in response to nitrogen amount are available up to now. In this work, two-dimensional gel electrophoresis (2-DE) has been used to investigate the protein changes induced by NO₃ ^- concentration in both roots and leaves of maize (Zea mays L.) plants. Moreover, in order to better evaluate the proteomic results, some biochemical and physiological parameters were measured. Results: Through 2-DE analysis, 20 and 18 spots that significantly changed their amount at least two folds in response to nitrate addition to the growth medium of starved maize plants were found in roots and leaves, respectively. Most of these spots were identified by Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometry (LC-ESI-MS/MS). In roots, many of these changes were referred to enzymes involved in nitrate assimilation and in metabolic pathways implicated in the balance of the energy and redox status of the cell, among which the pentose phosphate pathway. In leaves, most of the characterized proteins were related to regulation of photosynthesis. Moreover, the up-accumulation of lipoxygenase 10 indicated that the leaf response to a high availability of nitrate may also involve a modification in lipid metabolism. Finally, this proteomic approach suggested that the nutritional status of the plant may affect two different post-translational modifications of phosphoenolpyruvate carboxylase (PEPCase) consisting in monoubiquitination and phosphorylation in roots and leaves, respectively. Conclusion: This work provides a first characterization of the proteome changes that occur in response to nitrate availability in leaves and roots of maize plants. According to previous studies, the work confirms the relationship between nitrogen and carbon metabolisms and it rises some intriguing questions, concerning the possible role of NO and lipoxygenase 10 in roots and leaves, respectively. Although further studies will be necessary, this proteomic analysis underlines the central role of post-translational events in modulating pivotal enzymes, such as PEPCase.

Cadmium (Cd) exposure and sulfate limitation induce root sulfate uptake to meet the metabolic demand for reduced sulfur. Although these responses are well studied, some aspects are still an object of debate, since little is known about the molecular mechanisms by which changes in sulfate availability and sulfur metabolic demand are perceived and transduced into changes in the expression of the high-affinity sulfate transporters of the roots. The analysis of the natural variation occurring in species with complex and highly redundant genome could provide precious information to better understand the topic, because of the possible retention of mutations in the sulfate transporter genes.

The effect of \(\mathrm{NO}_{3}^{{-}}\) uptake on cellular pH was studied in maize roots by an in vivo³¹P-NMR technique. In order to separate the effects on cytoplasmic pH due to \(\mathrm{NO}_{3}^{{-}}\) uptake from those due to \(\mathrm{NO}_{3}^{{-}}\) reduction, tungstate was used to inhibit nitrate reductase (NR). The results confirm that in maize roots tungstate inhibited NR activity. ¹⁵N-NMR in vivo experiments demonstrated the cessation of nitrogen flux from nitrate to organic compounds. Tungstate affected neither \(\mathrm{NO}_{3}^{{-}}\) uptake nor the levels of the main phosphorylated compounds. Slight changes in cytoplasmic pH were observed during \(\mathrm{NO}_{3}^{{-}}\) uptake and reduction (i.e. control). By contrast, in the presence of tungstate, a consistent decrease in cytoplasmic pH occurred. The vacuolar pH did not change in any of the conditions tested. These data show that \(\mathrm{NO}_{3}^{{-}}\) uptake is an acidifying process and suggest a possible involvement of \(\mathrm{NO}_{3}^{{-}}\) reduction in pH homeostasis. In the presence of \(\mathrm{NO}_{3}^{{-}}\) ⁠, a transient depolarization of transmembrane electric potential difference (E_m) was observed in all the conditions analysed. However, in tungstate-treated roots, a lesser depolarization accompanied by a greater ability to recover E_m was found. This was related to a higher activity of the plasma membrane (PM) H⁺-ATPase. When \(\mathrm{NO}_{3}^{{-}}\) was administered as potassium salt, its uptake increased and a greater depolarization of E_m took place, whilst the changes in cytoplasmic pH were remarkably reduced, according to the central role played by K⁺ in the control of plasma membrane activities and cell pH homeostasis. A possible involvement of cytoplasmic pH in the control of PM H⁺-ATPase expression during nitrate exposure is suggested.

The bidirectional fluxes of cadmium and calcium across the plasma membrane were assessed and compared in subapical maize root segments. This homogeneous material provides a simplified system for investigating ion fluxes in whole organs. The kinetic profile of cadmium influx was characterized by a combination of a saturable rectangular hyperbola (K_m = 30.15) and a straight line (k = 0.0013 L h⁻¹ g⁻¹ fresh weight), indicating the presence of multiple transport systems. In contrast, the influx of calcium was described by a simple Michaelis–Menten function (K_m = 26.57 µM). The addition of calcium to the medium reduced cadmium influx into the root segments, suggesting a competition between the two ions for the same transport system(s). The efflux of calcium from the root segments was found to be significantly higher than that of cadmium, which was extremely low under the experimental conditions used. This was further confirmed by comparing cadmium and calcium fluxes across the plasma membrane of inside-out vesicles purified from maize root cortical cells. The inability of the root cortical cells to extrude cadmium may have driven the evolution of metal chelators for detoxifying intracellular cadmium ions.