Proteomic changes in the roots of three grapevine rootstocks in response to nitrate availability

Although the biochemical mechanisms involved in nitrate assimilation are overall known, especially in herbaceous species, specific information on perennial plants, such as grapevine, remain yet incomplete. Few studies have investigated the root responses to nitrate availability in this species. Considering that grafting practice is widely used in viticulture, the role of rootstock in nitrogen metabolism as well as its effects on scion must be better characterized. In this view, the responses of roots to nitrogen availability, as well as to its fluctuations in the soil, are among the main factors that influence plant growth and productivity from both quantitative and qualitative point of view. The aim of this study was to analyze the metabolic events occurring in the roots of three grapevine rootstocks, M3, M4 and 1103P, in responses to the addition of 10 mM nitrate, after a period in which the plants were grown in absence of nitrogen. Firstly, the changes of some biochemical parameters (such as nitrate, sugar and amino acid contents) as well as the evaluation of abundances of nitrate reductase and glutamine synthetases by Western blot analyses were used to define the time course of the induction of nitrogen metabolism in roots. Taken together, the results indicated that in all genotypes the nitrogen metabolism significantly increased after 30 h from the addition of nitrate. Moreover, at this time the comparison among the three genotypes revealed a greater assimilative capacity in 1103P. To gain a better characterization of the biochemical responses in roots, a proteomic comparison among the three genotypes, in control condition and after 30 h of induction, was conducted. Proteomic analyses were performed by GeLC-MS/MS, a technique by which proteins are purified by SDS-PAGE, in-gel digested, and then identified and quantified by mass spectrometry. Functional classification was made according to the bin hierarchical tree developed by MapMan ontology. Only proteins showing at least a fold change of 40% in abundance (Student’s t-test, p < 0.05) were considered significantly affected by the treatment. This approach allowed the identification of some hundreds of proteins, with high reliability and good reproducibility, revealing in all genotypes changes of proteins/enzymes involved in nitrogen assimilation as well as in metabolic pathways useful to sustain the requests of carbon skeletons and of energy.