Interaction of ammonium nutrition with essential mineral cations.

Plant growth and development depend 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, NH4+ becomes a stressor that inhibits plant growth. The cause of NH4+ stress or toxicity is multifactorial, but the interaction of NH4+ with other nutrients is among the main determinants of plants' sensitivity towards high NH4+ supply. In addition, NH4+ uptake and assimilation provoke the acidification of the cell external medium (apoplast/rhizosphere), which has a clear impact on nutrient availability. This review summarizes current knowledge, at both the physiological and the molecular level, of the interaction of NH4+ nutrition with essential mineral elements that are absorbed as cations, both macronutrients (K+, Ca2+, Mg2+) and micronutrients (Fe2+/3+, Mn2+, Cu+/2+, Zn2+, Ni2+). 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 with nitrate-based ones. In addition, we are convinced that better understanding of these interactions will help to identify novel targets with the potential to improve crop productivity.

Knock-down of phosphoenolpyruvate carboxylase 3 negatively impacts growth, productivity, and responses to salt stress in sorghum (Sorghum bicolor L.).

Phosphoenolpyruvate carboxylase (PEPC) is a carboxylating enzyme with important roles in plant metabolism. Most studies in C4 plants have focused on photosynthetic PEPC, but less is known about non-photosynthetic PEPC isozymes, especially with respect to their physiological functions. In this work, we analyzed the precise roles of the sorghum (Sorghum bicolor) PPC3 isozyme by the use of knock-down lines with the SbPPC3 gene silenced (Ppc3 lines). Ppc3 plants showed reduced stomatal conductance and plant size, a delay in flowering time, and reduced seed production. In addition, silenced plants accumulated stress indicators such as Asn, citrate, malate, and sucrose in roots and showed higher citrate synthase activity, even in control conditions. Salinity further affected stomatal conductance and yield and had a deeper impact on central metabolism in silenced plants compared to wild type, more notably in roots, with Ppc3 plants showing higher nitrate reductase and NADH-glutamate synthase activity in roots and the accumulation of molecules with a higher N/C ratio. Taken together, our results show that although SbPPC3 is predominantly a root protein, its absence causes deep changes in plant physiology and metabolism in roots and leaves, negatively affecting maximal stomatal opening, growth, productivity, and stress responses in sorghum plants. The consequences of SbPPC3 silencing suggest that this protein, and maybe orthologs in other plants, could be an important target to improve plant growth, productivity, and resistance to salt stress and other stresses where non-photosynthetic PEPCs may be implicated.

Ammonium nutrition interacts with iron homeostasis in Brachypodium distachyon.

Most plant species develop stress symptoms when exposed to high ammonium (NH4+) concentrations. The root is the first organ in contact with high NH4+ and therefore the first barrier to cope with ammonium stress. In this work, we focused on root adaptation to ammonium nutrition in the model plant Brachypodium distachyon. Proteome analysis revealed changes associated with primary metabolism, cell wall remodelling, and redox homeostasis. In addition, it showed a strong induction of proteins related to methionine (Met) metabolism and phytosiderophore (PS) synthesis in ammonium-fed plants. In agreement with this, we show how ammonium nutrition impacts Met/S-adenosyl-Met and PS metabolic pathways together with increasing root iron content. Nevertheless, ammonium-fed plants displayed higher sensitivity to iron deficiency, suggesting that ammonium nutrition triggers impaired iron utilization and root to shoot transport, which entailed an induction in iron-related responses. Overall, this work demonstrates the importance of iron homeostasis during ammonium nutrition and paves a new way to better understand and improve ammonium use efficiency and tolerance.

Arabidopsis MYB28 and MYB29 transcription factors are involved in ammonium-mediated alterations of root-system architecture.

Ammonium (NH4+) is known to produce alterations in root-system architecture, notably, by inhibiting primary root elongation and stimulating lateral root branching. This stimulation is associated with higher auxin transport promoted by apoplast acidification. Recently, we showed that MYB28 and MYB29 transcription factors play a role in ammonium tolerance, since its double mutant (myb28myb29) is highly hypersensitive toward ammonium nutrition in relation to altered Fe homeostasis. In the present work, we observed that primary root elongation was lower in the mutant with respect to wild-type plants under ammonium nutrition. Moreover, ammonium-induced lateral root branching was impaired in myb28myb29 in a Fe-supply dependent manner. Further research is required to decipher the link between MYB28 and MYB29 functions and the signaling pathway leading to root-system architecture modification by NH4+ supply.

Arabidopsis thaliana transcription factors MYB28 and MYB29 shape ammonium stress responses by regulating Fe homeostasis.

Although ammonium (NH4+ ) is a key intermediate of plant nitrogen metabolism, high concentrations of NH4+ in the soil provoke physiological disorders that lead to the development of stress symptoms. Ammonium nutrition was shown to induce the accumulation of glucosinolates (GSLs) in leaves of different Brassicaceae species. To further understand the link between ammonium nutrition and GSLs, we analysed the ammonium stress response of Arabidopsis mutants impaired in GSL metabolic pathway. We showed that the MYB28 and MYB29 double mutant (myb28myb29), which is almost deprived of aliphatic GSLs, is highly hypersensitive to ammonium nutrition. Moreover, we evidenced that the stress symptoms developed were not a consequence of the lack of aliphatic GSLs. Transcriptomic analysis highlighted the induction of an iron (Fe) deficiency response in myb28myb29 under ammonium nutrition. Consistently, ammonium-grown myb28myb29 plants showed altered Fe accumulation and homeostasis. Interestingly, we showed overall that growing Arabidopsis with increased Fe availability relieved ammonium stress symptoms and that this was associated with MYB28 and MYB29 expression. Taken together, our data indicated that the control of Fe homeostasis was crucial for the Arabidopsis response to ammonium nutrition and evidenced that MYB28 and MYB29 play a role in this control.

Molecular and biochemical analysis of XDH from Phaseolus vulgaris suggest that uric acid protects the enzyme against the inhibitory effects of nitric oxide in nodules.

Xanthine dehydrogenase (XDH) is essential for the assimilation of symbiotically fixed nitrogen in ureidic legumes. Uric acid, produced in the reaction catalyzed by XDH, is the precursor of the ureides, allantoin and allantoate, which are the main N-transporting molecules in these plants. XDH and uric acid have been reported to be involved in the response to stress, both in plants and animals. However, the physiological role of XDH under stressful conditions in ureidic legumes remains largely unexplored. In vitro assays showed that Phaseolus vulgaris XDH (PvXDH) can behave as a dehydrogenase or as an oxidase. Therefore, it could potentially protect against oxidative radicals or, in contrast, it could increase their production. In silico analysis of the upstream genomic region of XDH coding gene from P. vulgaris revealed the presence of several stress-related cis-regulatory elements. PvXDH mRNA and enzymatic activity in plants treated with stress-related phytohormones or subjected to dehydration and stressful temperatures showed several fold induction. However, PvXDH activity was in vivo and in vitro inhibited by nitric oxide in leaves but not in nodules. In extracts from RNAi PvXDH silenced nodules, with lower levels of uric acid, XDH activity was inhibited by SNP which indicates that uric acid produced by XDH in the nodules of this ureidic legume could help to protect XDH against the inhibitory effects of nitric oxide.

Isotopic labelling reveals the efficient adaptation of wheat root TCA cycle flux modes to match carbon demand under ammonium nutrition.

Proper carbon (C) supply is essential for nitrogen (N) assimilation especially when plants are grown under ammonium (NH4+) nutrition. However, how C and N metabolic fluxes adapt to achieve so remains uncertain. In this work, roots of wheat (Triticum aestivum L.) plants grown under exclusive NH4+ or nitrate (NO3-) supply were incubated with isotope-labelled substrates (15NH4+, 15NO3-, or [13C]Pyruvate) to follow the incorporation of 15N or 13C into amino acids and organic acids. Roots of plants adapted to ammonium nutrition presented higher capacity to incorporate both 15NH4+ and 15NO3- into amino acids, thanks to the previous induction of the NH4+ assimilative machinery. The 15N label was firstly incorporated into [15N]Gln vía glutamine synthetase; ultimately leading to [15N]Asn accumulation as an optimal NH4+ storage. The provision of [13C]Pyruvate led to [13C]Citrate and [13C]Malate accumulation and to rapid [13C]2-OG consumption for amino acid synthesis and highlighted the importance of the anaplerotic routes associated to tricarboxylic acid (TCA) cycle. Taken together, our results indicate that root adaptation to ammonium nutrition allowed efficient assimilation of N thanks to the promotion of TCA cycle open flux modes in order to sustain C skeleton availability for effective NH4+ detoxification into amino acids.

New Insights on Arabidopsis thaliana Root Adaption to Ammonium Nutrition by the Use of a Quantitative Proteomic Approach.

Nitrogen is an essential element for plant nutrition. Nitrate and ammonium are the two major inorganic nitrogen forms available for plant growth. Plant preference for one or the other form depends on the interplay between plant genetic background and environmental variables. Ammonium-based fertilization has been shown less environmentally harmful compared to nitrate fertilization, because of reducing, among others, nitrate leaching and nitrous oxide emissions. However, ammonium nutrition may become a stressful situation for a wide range of plant species when the ion is present at high concentrations. Although studied for long time, there is still an important lack of knowledge to explain plant tolerance or sensitivity towards ammonium nutrition. In this context, we performed a comparative proteomic study in roots of Arabidopsis thaliana plants grown under exclusive ammonium or nitrate supply. We identified and quantified 68 proteins with differential abundance between both conditions. These proteins revealed new potential important players on root response to ammonium nutrition, such as H⁺-consuming metabolic pathways to regulate pH homeostasis and specific secondary metabolic pathways like brassinosteroid and glucosinolate biosynthetic pathways.

Leaves play a central role in the adaptation of nitrogen and sulfur metabolism to ammonium nutrition in oilseed rape (Brassica napus).

BACKGROUND: The coordination between nitrogen (N) and sulfur (S) assimilation is required to suitably provide plants with organic compounds essential for their development and growth. The N source induces the adaptation of many metabolic processes in plants; however, there is scarce information about the influence that it may exert on the functioning of S metabolism. The aim of this work was to provide an overview of N and S metabolism in oilseed rape (Brassica napus) when exposed to different N sources. To do so, plants were grown in hydroponic conditions with nitrate or ammonium as N source at two concentrations (0.5 and 1 mM). RESULTS: Metabolic changes mainly occurred in leaves, where ammonium caused the up-regulation of enzymes involved in the primary assimilation of N and a general increase in the concentration of N-compounds (NH4+, amino acids and proteins). Similarly, the activity of key enzymes of primary S assimilation and the content of S-compounds (glutathione and glucosinolates) were also higher in leaves of ammonium-fed plants. Interestingly, sulfate level was lower in leaves of ammonium-fed plants, which was accompanied by the down-regulation of SULTR1 transporters gene expression. CONCLUSIONS: The results highlight the impact of the N source on different steps of N and S metabolism in oilseed rape, notably inducing N and S assimilation in leaves, and put forward the potential of N source management to modulate the synthesis of compounds with biotechnological interest, such as glucosinolates.

Functional specialization of one copy of glutamine phosphoribosyl pyrophosphate amidotransferase in ureide production from symbiotically fixed nitrogen in Phaseolus vulgaris.

Purines are essential molecules formed in a highly regulated pathway in all organisms. In tropical legumes, the nitrogen fixed in the nodules is used to generate ureides through the oxidation of de novo synthesized purines. Glutamine phosphoribosyl pyrophosphate amidotransferase (PRAT) catalyses the first committed step of de novo purine synthesis. In Phaseolus vulgaris there are three genes coding for PRAT. The three full-length sequences, which are intron-less genes, were cloned, and their expression levels were determined under conditions that affect the synthesis of purines. One of the three genes, PvPRAT3, is highly expressed in nodules and protein amount and enzymatic activity in these tissues correlate with nitrogen fixation activity. Inhibition of PvPRAT3 gene expression by RNAi-silencing and subsequent metabolomic analysis of the transformed roots shows that PvPRAT3 is essential for the synthesis of ureides in P. vulgaris nodules.