Nitrate and ammonium are the main forms of inorganic nitrogen available to plants. The present study aimed to investigate the metabolic changes caused by ammonium and nitrate nutrition in maritime pine (Pinus pinaster Ait.). Seedlings were grown with five solutions containing different proportions of nitrate and ammonium. Their nitrogen status was characterized through analyses of their biomass, different biochemical and molecular markers as well as a metabolite profile using 1 H-NMR. Ammonium-fed seedlings exhibited higher biomass than nitrate-fed-seedlings. Nitrate mainly accumulated in the stem and ammonium in the roots. Needles of ammonium-fed seedlings had higher nitrogen and amino acid contents but lower levels of enzyme activities related to nitrogen metabolism. Higher amounts of soluble sugars and L-arginine were found in the roots of ammonium-fed seedlings. In contrast, L-asparagine accumulated in the roots of nitrate-fed seedlings. The differences in the allocation of nitrate and ammonium may function as metabolic buffers to prevent interference with the metabolism of photosynthetic organs. The metabolite profiles observed in the roots suggest problems with carbon and nitrogen assimilation in nitrate-supplied seedlings. Taken together, this new knowledge contributes not only to a better understanding of nitrogen metabolism but also to improving aspects of applied mineral nutrition for conifers.

The amino acids arginine and ornithine are the precursors of a wide range of nitrogenous compounds in all living organisms. The metabolic conversion of ornithine into arginine is catalyzed by the sequential activities of the enzymes ornithine transcarbamylase (OTC), argininosuccinate synthetase (ASSY) and argininosuccinate lyase (ASL). Because of their roles in the urea cycle, these enzymes have been purified and extensively studied in a variety of animal models. However, the available information about their molecular characteristics, kinetic and regulatory properties is relatively limited in plants. In conifers, arginine plays a crucial role as a main constituent of N-rich storage proteins in seeds and serves as the main source of nitrogen for the germinating embryo. In this work, recombinant PpOTC, PpASSY and PpASL enzymes from maritime pine (Pinus pinaster Ait.) were produced in Escherichia coli to enable study of their molecular and kinetics properties. The results reported here provide a molecular basis for the regulation of arginine and ornithine metabolism at the enzymatic level, suggesting that the reaction catalyzed by OTC is a regulatory target in the homeostasis of ornithine pools that can be either used for the biosynthesis of arginine in plastids or other nitrogenous compounds in the cytosol.

Plant growth and development depends on sufficient nutrient availability in soils. Agricultural soils are generally nitrogen (N) deficient and thus, soils need to be supplemented with fertilizers. Ammonium (NH4  +) is a major inorganic N source. However, at high concentrations ammonium nutrition becomes a stress situation that inhibits plant growth. The cause of ammonium stress or toxicity is multi-factorial but the interaction with other nutrients is among the main determinants of plants sensitivity towards high NH4  + supply. In addition, NH4  + uptake and assimilation provokes the acidification of the cell external medium (apoplast/rhizosphere), which has a clear impact on nutrients availability. This review summarizes current knowledge, at both physiological and molecular level, of ammonium nutrition interaction with essential mineral elements absorbed as cations: macronutrients (K +, Ca 2+, Mg 2+) and micronutrients (Fe 2+/3+, Mn 2+, Cu +/2+, Zn 2+, Ni 2+). We hypothesize that considering these nutritional interactions, and soil pH, when formulating fertilizers may be key in order to boost the use of NH4  +-based fertilizers, which have less environmental impact compared to the nitrate-based ones. In addition, we are convinced that better understanding these interactions will help to identify novel targets with potential to improve crop productivity.