Idoia Ariz

Depletion of the heaviest stable N isotope is associated with NH4+/NH3 toxicity in NH4+-fed plants

BackgroundIn plants, nitrate (NO3-) nutrition gives rise to a natural N isotopic signature (δ15N), which correlates with the δ15N of the N source. However, little is known about the relationship between the δ15N of the N source and the 14N/15N fractionation in plants under ammonium (NH4+) nutrition. When NH4+ is the major N source, the two forms, NH4+ and NH3, are present in the nutrient solution. There is a 1.025 thermodynamic isotope effect between NH3 (g) and NH4+ (aq) which drives to a different δ15N. Nine plant species with different NH4+-sensitivities were cultured hydroponically with NO3- or NH4+ as the sole N sources, and plant growth and δ15N were determined. Short-term NH4+/NH3 uptake experiments at pH 6.0 and 9.0 (which favours NH3 form) were carried out in order to support and substantiate our hypothesis. N source fractionation throughout the whole plant was interpreted on the basis of the relative transport of NH4+ and NH3.ResultsSeveral NO3--fed plants were consistently enriched in 15N, whereas plants under NH4+ nutrition were depleted of 15N. It was shown that more sensitive plants to NH4+ toxicity were the most depleted in 15N. In parallel, N-deficient pea and spinach plants fed with 15NH4+ showed an increased level of NH3 uptake at alkaline pH that was related to the 15N depletion of the plant. Tolerant to NH4+ pea plants or sensitive spinach plants showed similar trend on 15N depletion while slight differences in the time kinetics were observed during the initial stages. The use of RbNO3 as control discarded that the differences observed arise from pH detrimental effects.ConclusionsThis article proposes that the negative values of δ15N in NH4+-fed plants are originated from NH3 uptake by plants. Moreover, this depletion of the heavier N isotope is proportional to the NH4+/NH3 toxicity in plants species. Therefore, we hypothesise that the low affinity transport system for NH4+ may have two components: one that transports N in the molecular form and is associated with fractionation and another that transports N in the ionic form and is not associated with fractionation.

Changes in the C/N balance caused by increasing external ammonium concentrations are driven by carbon and energy availabilities during ammonium nutrition in pea plants: the key roles of asparagine synthetase and anaplerotic enzymes

An understanding of the mechanisms underlying ammonium (NH(4)(+)) toxicity in plants requires prior knowledge of the metabolic uses for nitrogen (N) and carbon (C). We have recently shown that pea plants grown at high NH(4)(+) concentrations suffer an energy deficiency associated with a disruption of ionic homeostasis. Furthermore, these plants are unable to adequately regulate internal NH4(+) levels and the cell-charge balance associated with cation uptake. Herein we show a role for an extra-C application in the regulation of C-N metabolism in NH(4)(+) -fed plants. Thus, pea plants (Pisum sativum) were grown at a range of NH(4)(+) concentrations as sole N source, and two light intensities were applied to vary the C supply to the plants. Control plants grown at high NH(4)(+) concentration triggered a toxicity response with the characteristic pattern of C-starvation conditions. This toxicity response resulted in the redistribution of N from amino acids, mostly asparagine, and lower C/N ratios. The C/N imbalance at high NH(4)(+) concentration under control conditions induced a strong activation of root C metabolism and the upregulation of anaplerotic enzymes to provide C intermediates for the tricarboxylic acid cycle. A high light intensity partially reverted these C-starvation symptoms by providing higher C availability to the plants. The extra-C contributed to a lower C4/C5 amino acid ratio while maintaining the relative contents of some minor amino acids involved in key pathways regulating the C/N status of the plants unchanged. C availability can therefore be considered to be a determinant factor in the tolerance/sensitivity mechanisms to NH(4)(+) nutrition in plants.

High irradiance induces photoprotective mechanisms and a positive effect on NH4+ stress in Pisum sativum L.

Photosynthesis provides plant metabolism with reduced carbon (C) but is also the main source of oxidative stress in plants. Likewise, high doses of NH(4)(+) as sole N source have been reported to be toxic for most plants, resulting in reduced plant growth and restricting C availability. The combination of high photosynthetic photon flux densities (PPFD) and NH(4)(+) nutrition may provide higher C availability but could also have a detrimental effect on the plants, therefore the objective of this study is to evaluate whether NH(4)(+) induces photo-oxidative stress that is exacerbated under high light conditions. Pea plants (Pisum sativum cv. sugar-snap) were grown hydroponically with NH(4)(+) (0.5, 2.5, 5 and 10 mM) under high (750 micromol photons m(-2)s(-1)) or low PPFD conditions (350 micromol photons m(-2)s(-1)). High PPFD contributes to a higher tolerance to ammonium by pea plants, as it originated higher biomass content due to higher photosynthetic rates. However, a deficit of N (0.5 and 2.5 mM NH(4)(+)) under high PPFD conditions caused an antioxidant response, as indicated by increased photoprotective pigment and chloroplastic superoxide dismutase contents. Plants grown with higher doses of N and high PPFD showed less need for photoprotection. An increase in the specific leaf weight (SLW) ratio was observed associated not only with high PPFDs but also with the highest NH(4)(+) dose. Overall, these results demonstrate that, despite the activation of some photoprotective responses at high PPFD, there were no photoinhibitory symptoms and a positive effect on NH(4)(+) toxicity, thus suggesting that the harmful effects of NH(4)(+) are not directly related to the generation of photo-oxidative stress.

High irradiance increases NH4 + tolerance in Pisum sativum: Higher carbon and energy availability improve ion balance but not N assimilation

The widespread use of NO(3)(-) fertilization has had a major ecological impact. NH(4)(+) nutrition may help to reduce this impact, although high NH(4)(+) concentrations are toxic for most plants. The underlying tolerance mechanisms are not yet fully understood, although they are thought to include the limitation of C, the disruption of ion homeostasis, and a wasteful NH(4)(+) influx/efflux cycle that carries an extra energetic cost for root cells. In this study, high irradiance (HI) was found to induce a notable tolerance to NH(4)(+) in the range 2.5-10mM in pea plants by inducing higher C availability, as shown by carbohydrate content. This capacity was accompanied by a general lower relative N content, indicating that tolerance is not achieved through higher net N assimilation on C-skeletons, and it was also not attributable to increased GS content or activity in roots or leaves. Moreover, HI plants showed higher ATP content and respiration rates. This extra energy availability is related to the internal NH(4)(+) content regulation (probably NH(4)(+) influx/efflux) and to an improvement of the cell ionic balance. The limited C availability at lower irradiance (LI) and high NH(4)(+) resulted in a series of metabolic imbalances, as reflected in a much higher organic acid content, thereby suggesting that the origin of the toxicity in plants cultured at high NH(4)(+) and LI is related to their inability to avoid large-scale accumulation of the NH(4)(+) ion.

Quantitative proteomics reveals the importance of nitrogen source to control glucosinolate metabolism in Arabidopsis thaliana and Brassica oleracea

Highlight A quantitative proteomic approach demonstrates how ammonium nutrition induces glucosinolate biosynthetic and catabolic pathways in Arabidopsis and broccoli.

Short term physiological implications of NBPT application on the N metabolism of Pisum sativum and Spinacea oleracea.

The application of urease inhibitors in conjunction with urea fertilizers as a means of reducing N loss due to ammonia volatilization requires an in-depth study of the physiological effects of these inhibitors on plants. The aim of this study was to determine how the urease inhibitor N-(n-butyl) thiophosphoric triamide (NBPT) affects N metabolism in pea and spinach. Plants were cultivated in pure hydroponic culture with urea as the sole N source. After 2 weeks of growth for pea, and 3 weeks for spinach, half of the plants received NBPT in their nutrient solution. Urease activity, urea and ammonium content, free amino acid composition and soluble protein were determined in leaves and roots at days 0, 1, 2, 4, 7 and 9, and the NBPT content in these tissues was determined 48h after inhibitor application. The results suggest that the effects of NBPT on spinach and pea urease activity differ, with pea being most affected by this treatment, and that the NBPT absorbed by the plant caused a clear inhibition of the urease activity in pea leaf and roots. The high urea concentration observed in leaves was associated with the development of necrotic leaf margins, and was further evidence of NBPT inhibition in these plants. A decrease in the ammonium content in roots, where N assimilation mainly takes place, was also observed. Consequently, total amino acid contents were drastically reduced upon NBPT treatment, indicating a strong alteration of the N metabolism. Furthermore, the amino acid profile showed that amidic amino acids were major components of the reduced pool of amino acids. In contrast, NBPT was absorbed to a much lesser degree by spinach plants than pea plants (35% less) and did not produce a clear inhibition of urease activity in this species.

Use of Recombinant Iron‐Superoxide Dismutase as A Marker of Nitrative Stress

Superoxide dismutases (SODs; EC 1.15.1.1) are a group of metalloenzymes which are essential to protect cells under aerobic conditions. In biological systems, it has been reported that SODs and other proteins are susceptible to be attacked by peroxynitrite (ONOO(-)) which can be originated from the reaction of nitric oxide with superoxide radical. ONOO(-) is a strong oxidant molecule capable of nitrating peptides and proteins at the phenyl side chain of the tyrosine residues. In the present work, bovine serum albumin (BSA) and recombinant iron-superoxide dismutase from the plant cowpea (Vu_FeSOD) are used as target molecules to estimate ONOO(-) production. The method employs the compound SIN-1, which simultaneously generates *NO and O(2)(-) in aerobic aqueous solutions. First, assay conditions were optimized incubating BSA with different concentrations of SIN-1, and at a later stage, the effect on the tyrosine nitration and catalytic activity of Vu_FeSOD was examined by in-gel activity and spectrophotometric assays. Both BSA and Vu_FeSOD are nitrated in a dose-dependent manner, and, at least in BSA nitration, the reaction seems to be metal catalyzed.

Leaf δ15N as a physiological indicator of the responsiveness of N2-fixing alfalfa plants to elevated [CO2], temperature and low water availability

The natural 15N/14N isotope composition (δ15N) of a tissue is a consequence of its N source and N physiological mechanisms in response to the environment. It could potentially be used as a tracer of N metabolism in plants under changing environmental conditions, where primary N metabolism may be complex, and losses and gains of N fluctuate over time. In order to test the utility of δ15N as an indicator of plant N status in N2-fixing plants grown under various environmental conditions, alfalfa (Medicago sativa L.) plants were subjected to distinct conditions of [CO2] (400 vs. 700 μmol mol−1), temperature (ambient vs. ambient +4°C) and water availability (fully watered vs. water deficiency—WD). As expected, increased [CO2] and temperature stimulated photosynthetic rates and plant growth, whereas these parameters were negatively affected by WD. The determination of δ15N in leaves, stems, roots, and nodules showed that leaves were the most representative organs of the plant response to increased [CO2] and WD. Depletion of heavier N isotopes in plants grown under higher [CO2] and WD conditions reflected decreased transpiration rates, but could also be related to a higher N demand in leaves, as suggested by the decreased leaf N and total soluble protein (TSP) contents detected at 700 μmol mol−1 [CO2] and WD conditions. In summary, leaf δ15N provides relevant information integrating parameters which condition plant responsiveness (e.g., photosynthesis, TSP, N demand, and water transpiration) to environmental conditions.

Expression and Localization of a Rhizobium-Derived Cambialistic Superoxide Dismutase in Pea (Pisum sativum) Nodules Subjected to Oxidative Stress

Two phylogenetically unrelated superoxide dismutase (SOD) families, i.e., CuZnSOD (copper and zinc SOD) and FeMn-CamSOD (iron, manganese, or cambialistic SOD), eliminate superoxide radicals in different locations within the plant cell. CuZnSOD are located within the cytosol and plastids, while the second family of SOD, which are considered to be of bacterial origin, are usually located within organelles, such as mitochondria. We have used the reactive oxygen species-producer methylviologen (MV) to study SOD isozymes in the indeterminate nodules on pea (Pisum sativum). MV caused severe effects on nodule physiology and structure and also resulted in an increase in SOD activity. Purification and N-terminal analysis identified CamSOD from the Rhizobium leguminosarum endosymbiont as one of the most active SOD in response to the oxidative stress. Fractionation of cell extracts and immunogold labeling confirmed that the CamSOD was present in both the bacteroids and the cytosol (including the nuclei, plastids, and mitochondria) of the N-fixing cells, and also within the uninfected cortical and interstitial cells. These findings, together with previous reports of the occurrence of FeSOD in determinate nodules, indicate that FeMnCamSOD have specific functions in legumes, some of which may be related to signaling between plant and bacterial symbionts, but the occurrence of one or more particular isozymes depends upon the nodule type.

Inhibition of endogenous urease activity by NBPT application reveals differential N metabolism responses to ammonium or nitrate nutrition in pea plants : a physiological study

Background and aimsUrea is the predominant form of N applied as fertilizer to crops, but it is also a significant N metabolite of plants themselves. As such, an understanding of urea metabolism in plants may contribute significantly to subsequent N fertilizer management. It currently appears that arginase is the only plant enzyme that can generate urea in vivo. The aim of this work was, therefore, to gain a more in-depth understanding of the significance of the inhibition of endogenous urease activity and its role in N metabolism depending on the N source supplied.MethodsPea (Pisum sativum cv. Snap-pea) plants were grown with either ammonium or nitrate as the sole N source in the presence or absence of the urease inhibitor NBPT.ResultsWhen supplied, NBPT is absorbed by plants and translocated from the roots to the leaves, where it reduces endogenous urease activity. Different N metabolic responses in terms of N-assimilatory enzymes and N-containing compounds indicate a different degree of arginine catabolism activation in ammonium- and nitrate-fed plants.ConclusionsThe arginine catabolism is more highly activated in ammonium-fed plants than in nitrate-fed plants, probably due to the higher turnover of substrates by enzymes playing a key role in N recycling and remobilization during catabolism and in early flowering and senescence processes, usually observed under ammonium nutrition.