Proteomic changes in the roots of M4 grapevine rootstock in response to nitrate availability

Nitrogen (N) is an essential macronutrient for plants, but very little is known about the biochemical roles played by roots in N acquisition in grapevine (Vitis vinifera L.), an important grafted perennial fruit crop. In recent years, the grapevine rootstock M4 [(V. vinifera × V. berlandieri) × V. berlandieri] was the subject of physiological, transcriptomic and proteomic analysis that highlighted its higher tolerance to drought and salinity stress in comparison with other rootstocks. However, little information is available about its metabolic responses to the availability of nitrate (NO3-), the major form of N nutrient used by plants in agricultural soils. The aim of this study was to determine the metabolic events involved in NO3- acquisition in M4. In details, young M4 plants, grown in a hydroponic system, after a period of N starvation were maintained in the absence of N (control condition) or exposed to 10 mM NO3-. Firstly, the changes of some biochemical parameters (such as NO3-, sugar and amino acid contents) as well as the evaluation by Western blot analyses of the abundances of key enzymes (i.e. Nitrate Reductase and Glutamine Synthetase) were used to define the time course of the metabolic changes occurring in the first 30 h. Taken together, the results showed that root N metabolism significantly increased after 30 h of NO3- availability. To gain a better characterization, a proteomic analysis based on one-dimensional (1D) Gel Liquid Chromatography-Mass Spectrometry (GeLC-MS/MS) was conducted, comparing the root profiles in the control condition and after 30 h of NO3- induction. This approach allowed the identification of some hundreds of proteins, with high reliability and good reproducibility. Many of the proteins found to change in abundance were directly involved in NO3- uptake and assimilation, such as H+-ATPase, Nitrite Reductase and Glutamine Synthetase. According to the induction of N metabolism, some root enzymes involved in protein synthesis, folding and trafficking showed a relevant increment in abundances after 30 h of NO3- availability, while those involved in flavonoid metabolism generally showed a down-accumulation. Moreover, the results underlined the strict relationships between N nutrition, carbon metabolism and the metabolic pathways implicated in cell redox status. Overall, the proteomic analysis reveals that NO3- provision significantly affected the root proteome of M4 grapevine rootstocks, providing novel information about the biochemical pathways involved in N metabolism in this perennial plant. This study lays the bases for a better elucidation of the relations among N nutrition, rootstock/scion interactions and productivity in grapevine.