Comparative proteomics of organelles in maize (Zea mays L.) roots in response to different availabilities of nitrate and ammonium

Nitrogen (N) is one of the main factors limiting agricultural productivity, especially in cereals such as maize (Zea mays L.), and nitrate (NO3-) and ammonium (NH4+) represent the predominant forms of N in agricultural soils. Plants are differently affected by the availability of these two N forms at morphological, physiological and metabolic levels, especially during the early stages of development. In particular, roots responses to different N availabilities involve the modulation of distinct metabolic pathways, the regulation of ion compartmentation and the balancing of cellular homeostasis. In this context, the strict coordination between the biochemical activities of cell organelles, among which mitochondria, plastids and vacuole, is crucial. Hence, subcellular proteomics represents a powerful method to study these metabolic interactions. The aim of this research was to study the roles of organelles in the adaptation to different N availabilities in maize roots, through the combination of physiological, metabolic and proteomic analyses. The research was conducted in roots of three-day-old maize plants, grown by hydroponic system, and exposed to different N availabilities: absence of N, 2.5 mM NO3-, 2.5 mM NH4+, 1.25 mM NO3- + 1.25 mM NH4+. This experimental design allowed to appreciate differences in plant growth and metabolic status, with variations in plant biomass accumulation and in the shoot/root ratios, in the contents of NO3- and NH4+ and metabolites, such as sugars and amino acids, and in the abundance of key enzymes in N assimilation (i.e. Nitrate Reductase and Glutamine Synthetase). Root organelles were enriched by differential centrifugation techniques and the sub-proteomic profiles were analysed by means of one- dimensional (1D) Gel Liquid Chromatography-Mass Spectrometry (1D GeLC-MS/MS). This approach allowed to identify and quantify a total of 365 proteins, showing, by means of bioinformatic analysis, a good degree of enrichment of the target proteomes (77.7%), and revealing that the 33% of the proteins were differently accumulated in the four conditions (one-way ANOVA, p ≤ 0.01). The results showed that the N forms induced different changes in abundance of proteins involved in the assimilation of N at the plastid level, in protein synthesis, in respiratory metabolism, and in the exchange of metabolites between organelles. Interestingly, some classes of mitochondrial carrier proteins, such as Mitochondrial dicarboxylate/tricarboxylate transporter (DTC), Mitochondrial phosphate carrier protein 3 and ADP/ATP carrier protein showed the highest abundance in NH4+-fed plants, highlighting a specific involvement of mitochondrion metabolism in root adaptations to this nutrient. Differently, the availability of NO3- affected the accumulation of some tonoplast proteins, such as aquaporins (TIP) and V-type proton ATPase subunits, and of some proteins involved in cell calcium network, providing new hints about the role of this nutrient both as osmolyte and metabolic signal. Overall, this research confirms the fundamental roles of roots in N acquisition and provides novel information about the responses induced by the different N nutrients in multiple sub-cellular compartments in young maize plants.