Nitrogen (N) is the nutrient most applied in agriculture as fertilizer (as nitrate, Nit; ammonium, A; and/or urea, U, forms) and its availability strongly constrains the crop growth and yield. To investigate the early response (24 hours) of N-deficient tomato plants to these three N forms, a physiological and molecular study was performed.In comparison to N-deficient plants, significant changes in the transcriptional, metabolomic and ionomic profiles were observed. As a probable consequence of N mobility in plants, a wide metabolic modulation occurred in old leaves rather than in young leaves. The metabolic profile of U and A-treated plants was more similar than Nit-treated plant profile, which in turn presented the lowest metabolic modulation with respect to N-deficient condition. Urea and A forms induced some changes at the biosynthesis of secondary metabolites, amino acids and phytohormones. Interestingly, a specific up-regulation by U and down-regulation by A of carbon synthesis occurred in roots. Along with the gene expression, data suggest that the specific N form influences the activation of metabolic pathways for its assimilation (cytosolic GS/AS and/or plastidial GS/GOGAT cycle). Urea induced an up-concentration of Cu and Mn in leaves and Zn in whole plant.This study highlights a metabolic reprogramming depending on the N form applied, and it also provide evidence of a direct relationship between urea nutrition and Zn concentration. The understanding of the metabolic pathways activated by the different N forms represents a milestone in improving the efficiency of urea fertilization in crops.

IntroductionNitrogen (N) and iron (Fe) are involved in several biochemical processes in living organisms, and their limited bioavailability is a strong constraint for plant growth and yield. This work investigated the interplay between Fe and N nutritional pathways in tomato plants kept under N and Fe deficiency and then resupplied with Fe and N (as nitrate, ammonium, or urea) through a physiological, metabolomics and gene expression study. ResultsAfter 24 hours of Fe resupply, the Fe concentration in Fe-deficient roots was dependent on the applied N form (following the pattern: nitrate > urea > ammonium > Fe-deficient control), and whereas in leaves of urea treated plants the Fe concentration was lower in comparison to the other N forms. Untargeted metabolomics pointed out distinctive modulations of plant metabolism in a treatment-dependent manner. Overall, N-containing metabolites were affected by the treatments in both leaves and roots, while N form significantly shaped the phytohormone profile. Moreover, the simultaneous application of Fe with N to Fe-deficient plants elicited secondary metabolites’ accumulation, such as phenylpropanoids, depending on the applied N form (mainly by urea, followed by nitrate and ammonium). After 4 hours of treatment, ammonium- and urea-treated roots showed a reduction of enzymatic activity of Fe(III)-chelate reductase (FCR), compared to nitrate or N-depleted plants (maintained in Fe deficiency, where FCR was maintained at high levels). The response of nitrate-treated plants leads to the improvement of Fe concentration in tomato roots and the increase of Fe(II) transporter (IRT1) gene expression in tomato roots. ConclusionsOur results strengthen and improve the understanding about the interaction between N and Fe nutritional pathways, thinning the current knowledge gap.

A fully mechanistic dynamical model for plant nitrate uptake is presented. Based on physiological and regulatory pathways and based on physical laws, we form a dynamic system mathematically described by seven differential equations. The model evidences the presence of a short-term positive feedback on the high-affinity nitrate uptake, triggered by the presence of nitrate around the roots, which induces its intaking. In the long run, this positive feedback is overridden by two long-term negative feedback loops which drastically reduces the nitrate uptake capacity. These two negative feedbacks are due to the generation of ammonium and amino acids, respectively, and inhibit the synthesis and the activity of high-affinity nitrate transporters. This model faithfully predicts the typical spiking behavior of the nitrate uptake, in which an initial strong increase of nitrate absorption capacity is followed by a drop, which regulates the absorption down to the initial value. The model outcome was compared with experimental data and they fit quite nicely. The model predicts that after the initial exposure of the roots with nitrate, the absorption of the anion strongly increases and that, on the contrary, the intensity of the absorption is limited in presence of ammonium around the roots.

In plants the communication between organs is mainly carried out via the xylem and phloem. The concentration and the molecular species of some phytohormones, assimilates and inorganic ions that are translocated in the xylem vessel play a key role in the systemic nutritional signaling in plants. In this work the composition of the xylem sap of maize was investigated at the metabolic and ionomic level depending on the N form available in the nutrient solution. Plants were grown up to 7 days in hydroponic system under N-free nutrient solution or nutrient solution containing N in form of nitrate, urea, ammonium or a combination of urea and ammonium. For the first time this work provides evidence that the ureic nutrition reduced the water translocation in maize plants more than mineral N forms. This result correlates with those obtained from the analyses of photosynthetic parameters (stomatal conductance and transpiration rate) suggesting a parsimonious use of water by maize plants under urea nutrition. A peculiar composition in amino acids and phytohormones (i.e. S, Gln, Pro, ABA) of the xylem sap under urea nutrition could explain differences in xylem sap exudation in comparison to plants treated with mineral N forms. The knowledge improvement of urea nutrition will allow to further perform good agronomic strategies to improve the resilience of maize crop to water stress.

The wide use of copper (Cu)-based fungicide has caused a stepwise accumulation of Cu in the environment increasing the occurrence of phytotoxicity in crops. To understand and alleviate this abiotic stress, maize seedlings were grown in hydroponic solution with different combinations of Cu and iron (Fe) forms. Results showed that maize Cu sensitivity is related to the nature of the form supplied and to the chelate-exchange processes that might involve other elements, such as Fe. The use of CuSO4 excess (100 µM) caused severe reduction of plant growth, over accumulation of Cu, high activity of antioxidant enzymes, and impairment of the acquisition of other nutrients. In presence of chelating agents (citrate and ethylenediaminetetraacetic acid, EDTA) the ability of plants to tolerate high Cu-levels depends on the Fe nutritional status. Copper phytotoxicity symptoms do not occur when Cu was supplied chelated by EDTA. The use of synthetic agent EDTA (as Cu-EDTA and Fe-EDTA) prevented the accumulation of toxic Cu-level in plants and allowed a better homeostasis among nutrients. In presence of citrate, high concentration of Cu occurred in plants but its phytotoxicity was limited when even EDTA was available in solution. Results suggest that maize plants can operate a good control of nutritional status when Cu-excess is present concomitantly with a synthetic chelator (as EDTA) even when supplied as a Fe-fertilizer. These results pave the way to provide guidelines for the fertilization managements on Cu-contaminated soils to alleviate phytotoxicity in crops.

The low bioavailability of nutrients, especially nitrogen (N) and phosphorus (P), is one of the most limiting factors for crop production. In this study, under N- and P-free nutrient solution (-N-P), nodulating white lupin plants developed some nodules and analogous cluster root structures characterized by different morphological, physiological, and molecular responses than those observed upon single nutrient deficiency (strong acidification of external media, a better nutritional status than -N+P and +N-P plants). The multi-elemental analysis highlighted that the concentrations of nutrients in white lupin plants were mainly affected by P availability. Gene-expression analyses provided evidence of interconnections between N and P nutritional pathways that are active to promote N and P balance in plants. The root exudome was mainly characterized by N availability in nutrient solution, and, in particular, the absence of N and P in the nutrient solution triggered a high release of phenolic compounds, nucleosides monophosphate and saponines by roots. These morphological, physiological, and molecular responses result from a close interplay between N and P nutritional pathways. They contribute to the good development of nodulating white lupin plants when grown on N- and P-free media. This study provides evidence that limited N and P availability in the nutrient solution can promote white lupin-Bradyrhizobium symbiosis, which is favourable for the sustainability of legume production.

The study aims to investigate at a physiological and molecular level the root absorption of nitrogen (N) during the annual cycle of grapevine. The study was performed on potted Pinot Gris plants grafted on Kober 5BB and grown for two consecutive years in a semi-controlled environment (Northeast, Italy). The study compared the response of plants N-fertilized in spring (T1), or in spring and in post-harvest (T2) or no-N fertilized (C). Results showed that under our climate conditions nitrate was taken up by the grapevines when applied both in spring or in spring and post-harvest. The nitrate acquisition in T1 roots is mediated at molecular and physiological level by a higher activation of high-affinity nitrate-transport system to take up nitrate in comparison to no-fertilized plants. Comparing the two N fertilization managements, the dynamic of nitrate uptake rates showed different patterns with an overall late response of High-Affinity Transport System when the application of part of N-fertilization was delayed to post-harvest (T2) in comparison to T1. Nevertheless, during the 2 years of the trial the fractionation of N fertilization applied in spring and post-harvest did not negatively affect the yield and quality parameters of vines. An increase of N concentration in T2 roots after two consecutive growing seasons may suggest that the fractionation can lead to beneficial effects on long period. Results of this work contribute to improve the comprehension of N acquisition in grapevine in order to optimize the use of N inputs in vineyard.

To date urea and ammonium are two nitrogen (N) forms widely used in agriculture. Due to a low production cost, urea is the N form most applied in agriculture. However, its stability in the soil depends on the activity of microbial ureases, that operate the hydrolysis of urea into ammonium. In the soil ammonium is subjected to fast volatilization in form of ammonia, an environmental N loss that contributes to the atmospheric pollution and impacts on farm economies. Based on these considerations, the optimization of N fertilization is useful in order to maximize N acquired by crops and at the same time limit N losses in the environment. The use of mixed nitrogen forms in cultivated soils allows to have urea and ammonium simultaneously available for the root acquisition after a fertilization event. A combination of different N-sources is known to lead to positive effects on the nutritional status of crops. It is plausible suppose that N acquisition mechanisms in plants might be responsive to N forms available in the root external solution, and therefore indicate a cross connection among different N forms, such as urea and ammonium.This DIB article provides details about the elemental composition and transcriptional changes occurring in maize seedlings when ammonium and urea mixture is applied to nutrient solution. An extensive and complete characterization of seedling response to urea and ammonium treatments is shown in the research article “Characterization of physiological and molecular responses of Zea mays seedlings to different urea-ammonium ratios” Buoso et al. [1].Maize seedlings were grown under hydroponic system with N applied to nutrient solution in form of urea and or ammonium, hence five different urea (U) to ammonium (A) ratios were tested (100U, 75U:25A, 50U:50A, 25U:75A, 100A). As control maize were fed with nitrate as sole N source, or were maintained in N deficiency (-N). After 1 or 7 days of N-treatment, maize seedlings were collected, and physiological and transcriptional analyses were performed on maize roots.Depending on nutritional treatment, no significant changes in seedling biomass were observed comparing N treatments. At both sampling times, an overall higher N accumulation in shoots and roots were detected when the inorganic N sources were applied to nutrient solutions (as ammonium or nitrate). 15N experiments indicated that in comparison to -N seedlings, urea fed seedlings showed an increase of N accumulation and data showed that ureic-N was taken up by seedlings in lower amounts than inorganic N-forms. Through EA-IRMS, ICP-OES and ICP-MS a multielemental composition of maize tissues was performed as well as gene expression analyses by Real-time RT-PCR that allowed to monitor the expression profile of genes most involved in urea and ammonium nutritional pathways.

Despite the wide use of urea and ammonium as N-fertilizers, no information is available about the proper ratio useful to maximize the efficiency of their acquisition by crops. Ionomic analyses of maize seedlings fed with five different mixes of urea and ammonium indicated that after 7 days of treatment, the elemental composition of plant tissues was more influenced by ammonium in the nutrient solution than by urea. Within 24 h, similar high affinity influx rates of ammonium were measured in ammonium-treated seedlings, independently from the amount of the cation present in the nutrient solution (from 0.5 to 2.0 mM N), and it was confirmed by the similar accumulation of 15N derived from ammonium source. After 7 days, some changes in ammonium acquisition occurred among treatments, with the highest ammonium uptake efficiency when the urea-to-ammonium ratio was 3:1. Gene expression analyses of enzymes and transporters involved in N nutrition highlight a preferential induction of the cytosolic N-assimilatory pathway (via GS, ASNS) when both urea and ammonium were supplied in conjunction, this response might explain the higher N-acquisition efficiency when both sources are applied. In conclusion, this study provides new insights on plant responses to mixes of N sources that maximize the N-uptake efficiency by crops and thus could allow to adapt agronomic practices in order to limit the economic and environmental impact of N-fertilization.

The simultaneous presence of different N-forms in the rhizosphere leads to beneficial effects on nitrogen (N) nutrition in plants. Although widely used as fertilizers, the occurrence of cross connection between urea and ammonium nutrition has been scarcely studied in plants. Maize fed with a mixture of urea and ammonium displayed a better N-uptake efficiency than ammonium- or urea-fed plants (Buoso et al., Plant Physiol Biochem, 2021a; 162: 613-623). Through multiomic approaches, we provide the molecular characterization of maize response to urea and ammonium nutrition. Several transporters and enzymes involved in N-nutrition were upregulated by all three N-treatments (urea, ammonium, or urea and ammonium). Already after 1 day of treatment, the availability of different N-forms induced specific transcriptomic and metabolomic responses. The combination of urea and ammonium induced a prompt assimilation of N, characterized by high levels of some amino acids in shoots. Moreover, ZmAMT1.1a, ZmGLN1;2, ZmGLN1;5, ZmGOT1, and ZmGOT3, as well transcripts involved in glycolysis-TCA cycle were induced in roots by urea and ammonium mixture. Depending on N-form, even changes in the composition of phytohormones were observed in maize. This study paves the way to formulate guidelines for the optimization of N fertilization to improve N-use efficiency in maize and therefore limit N-losses in the environment.

Nitrogen (N) as well as Phosphorus (P) are key nutrients determining crop productivity. Legumes have developed strategies to overcome nutrient limitation by, for example, forming a symbiotic relationship with N-fixing rhizobia and the release of P-mobilizing exudates and are thus able to grow without supply of N or P fertilizers. The legume-rhizobial symbiosis starts with root release of isoflavonoids that act as signaling molecules perceived by compatible bacteria. Subsequently, bacteria release nod factors, which induce signaling cascades allowing the formation of functional N-fixing nodules. We report here the identification and functional characterization of a plasma membrane-localized MATE-type transporter (LaMATE2) involved in the release of genistein from white lupin roots. The LaMATE2 expression in the root is upregulated under N deficiency as well as low phosphate availability, two nutritional deficiencies that induce the release of this isoflavonoid. LaMATE2 silencing reduced genistein efflux and even more the formation of symbiotic nodules, supporting the crucial role of LaMATE2 in isoflavonoid release and nodulation. Furthermore, silencing of LaMATE2 limited the P-solubilization activity of lupin root exudates. Transport assays in yeast vesicles demonstrated that LaMATE2 acts as a proton-driven isoflavonoid transporter.

Conversion of conventional farming (CF) to organic farming (OF) is claimed to allow a sustainable management of soil resources, but information on changes induced on dissolved organic matter (DOM) are scarce. Among DOM components, dissolved humic substances (DHS) were shown to possess stimulatory effects on plant growth. DHS were isolated from CF and OF soil leacheates collected from soil monolith columns: first in November (bare soils) and then in April and June (bare and planted soils). DHS caused an enhancement of nitrate uptake rates in maize roots and modulated several genes involved in nitrogen acquisition. The DHS from OF soil exerted a stronger biostimulant action on the nitrate uptake system, but the first assimilatory step of nitrate was mainly activated by DHS derived from CF soil. To validate the physiological response of plants to DHS exposure, real-time RT-PCR analyses were performed on those genes most involved in nitrate acquisition, such as ZmNRT2.1, ZmNRT2.2, ZmMHA2 (coding for two high-affinity nitrate transporters and a PM H+-proton pump), ZmNADH:NR, ZmNADPH:NR, and ZmNiR (coding for nitrate reductases and nitrite reductase). All tested DHS fractions induced the upregulation of nitrate reductase (NR), and in particular the OF2 DHS stimulated the expression of both tested transcripts encoding for two NR isoforms. Characteristics of DHS varied during the experiment in both OF and CF soils: a decrease of high molecular weight fractions in the OF soil, a general increase in the carboxylic groups content, as well as diverse structural modifications in OF vs. CF soils were observed. These changes were accelerated in planted soils. Similarity of chemical properties of DHS with the more easily obtainable water-soluble humic substance extracted from peat (WEHS) and the correspondence of their biostimulant actions confirm the validity of studies which employ WEHS as an easily available source of DHS to investigate biostimulant actions on agricultural crops.

Iron and phosphorus are abundant elements in soils but poorly available for plant nutrition. The availability of these two nutrients represents a major constraint for fruit tree cultivation such as apple ( Malus × domestica ) leading very often to a decrease of fruit productivity and quality worsening. Aim of this study was to characterize common and specific features of plant response to Fe and P deficiencies by ionomic, transcriptomic and exudation profiling of apple roots. Under P deficiency, the root release of oxalate and flavonoids increased. Genes encoding for transcription factors and transporters involved in the synthesis and release of root exudates were upregulated by P-deficient roots, as well as those directly related to P acquisition. In Fe-deficiency, plants showed an over-accumulation of P, Zn, Cu and Mn and induced the transcription of those genes involved in the mechanisms for the release of Fe-chelating compounds and Fe mobilization inside the plants. The intriguing modulation in roots of some transcription factors, might indicate that, in this condition, Fe homeostasis is regulated by a FIT-independent pathway. In the present work common and specific features of apple response to Fe and P deficiency has been reported. In particular, data indicate similar modulation of a. 230 genes, suggesting the occurrence of a crosstalk between the two nutritional responses involving the transcriptional regulation, shikimate pathway, and the root release of exudates.

Nitrogen (N) deficiency is one of the major stresses that crops are exposed to. It is plausible to suppose that a stress condition can induce a memory in plants that might prime the following generations. Here, an experimental setup that considered four successive generations of N-sufficient and N-limited Arabidopsis was used to evaluate the existence of a transgenerational memory. The results demonstrated that the ability to take up high amounts of nitrate is induced more quickly as a result of multigenerational stress exposure. This behavior was paralleled by changes in the expression of nitrate responsive genes. RNAseq analyses revealed the enduring modulation of genes in downstream generations, despite the lack of stress stimulus in these plants. The modulation of signaling and transcription factors, such as NIGTs, NFYA and CIPK23 might indicate that there is a complex network operating to maintain the expression of N-responsive genes, such as NRT2.1, NIA1 and NIR. This behavior indicates a rapid acclimation of plants to changes in N availability. Indeed, when fourth generation plants were exposed to N limitation, they showed a rapid induction of N-deficiency responses. This suggests the possible involvement of a transgenerational memory in Arabidopsis that allows plants to adapt efficiently to the environment and this gives an edge to the next generation that presumably will grow in similar stressful conditions.

In agricultural soil, the bioavailability of iron (Fe) and phosphorus (P) is often below the plant's requirement causing nutritional deficiency in crops. Under P-limiting conditions, white lupin (Lupinus albus L.) activates mechanisms that promote P solubility in the soil through morphological, physiological and molecular adaptations. Similar changes occur also in Fe-deficient white lupin roots; however, no information is available on the molecular bases of the response. In the present work, responses to Fe and P deficiency and their reciprocal interactions were studied. Transcriptomic analyses indicated that white lupin roots upregulated Fe-responsive genes ascribable to Strategy-I response, this behaviour was mainly evident in cluster roots. The upregulation of some components of Fe-acquisition mechanism occurred also in P-deficient cluster roots. Concerning P acquisition, some P-responsive genes (as phosphate transporters and transcription factors) were upregulated by P deficiency as well by Fe deficiency. These data indicate a strong cross-connection between the responses activated under Fe or P deficiency in white lupin. The activation of Fe- and P-acquisition mechanisms might play a crucial role to enhance the plant's capability to mobilize both nutrients in the rhizosphere, especially P from its associated metal cations.

Improvement of plant iron nutrition as a consequence of metal complexation by humic substances (HS) extracted from different sources has been widely reported. The presence of humified fractions of the organic matter in soil sediments and solutions would contribute, depending on the solubility and the molecular size of HS, to build up a reservoir of Fe available for plants which exude metal ligands and to provide Fe-HS complexes directly usable by plant Fe uptake mechanisms. It has also been shown that HS can promote the physiological mechanisms involved in Fe acquisition acting at the transcriptional and post-transcriptional level. Furthermore, the distribution and allocation of Fe within the plant could be modified when plants were supplied with water soluble Fe-HS complexes as compared with other natural or synthetic chelates. These effects are in line with previous observations showing that treatments with HS were able to induce changes in root morphology and modulate plant membrane activities related to nutrient acquisition, pathways of primary and secondary metabolism, hormonal and reactive oxygen balance. The multifaceted action of HS indicates that soluble Fe-HS complexes, either naturally present in the soil or exogenously supplied to the plants, can promote Fe acquisition in a complex way by providing a readily available iron form in the rhizosphere and by directly affecting plant physiology. Furthermore, the possibility to use Fe-HS of different sources, size and solubility may be considered as an environmental-friendly tool for Fe fertilization of crops.

This DIB article provides details about transcriptional and physiological response of Fe- and P-deficient white lupin roots, an extensive and complete description of plant response is shown in the research article "Physiological and transcriptomic data highlight common features between iron and phosphorus acquisition mechanisms in white lupin roots" Venuti et al. [1]. White lupin plants were grown under hydroponic system and three different nutritional regimes: Fe deficiency (-Fe), P deficiency (-P), or Fe and P sufficiency (+P + Fe). Depending on nutritional treatment, white lupin plants showed changes in the fresh weights, in root external acidification and FeIII-reductase activity. Moreover, the transcriptomic changes occurring in apices and clusters of Fe-deficient lupin roots were investigated and compared with differences of gene expression occurring in P-deficient plants (-P) and in Fe- and P-sufficient plants (+P + Fe). Transcriptomic data are available in the public repository Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo) under the series entry (GSE112220). The annotation, mapping and enrichment analyses of differentially modulated transcripts were assessed. (C) 2019 The Authors. Published by Elsevier Inc.

•WEHS promoted the induction of high affinity transport system of nitrate in maize.•The WEHS presence enhanced the transcriptional response of maize roots to nitrate.•Genes coding for nitrate transport and assimilation were highly responsive to WEHS.•WEHS modulated genes related to transcriptional regulation and hormone metabolism.•These genes might be involved in the fast induction of nitrate uptake by WEHS.

The rate of nitrate (NO3 −) uptake and changes in rhizosphere properties were studied growing seedlings of two maize inbred lines differing in nitrogen use efficiency (NUE) in rhizoboxes.

Under limited iron (Fe) availability maize, a Strategy II plant, improves Fe acquisition through the release of phytosiderophores (PS) into the rhizosphere and the subsequent uptake of Fe-PS complexes into root cells. Occurrence of Strategy-I-like components and interactions with phosphorous (P) nutrition has been hypothesized based on molecular and physiological studies in grasses.