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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.

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.

SUMMARYNitrogen (N) as well as Phosphorus (P) are key nutrients determining crop productivity. Legumes have developed strategies to overcome nutrient limitation by e.g., 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.

Nitrogen deficiency and drought stress are among the major stresses faced by plants with negative consequence on crop production. The use of plant biostimulants is a very promising application in agriculture to improve crop yield, but especially to prevent the effect of abiotic stresses. Algae-derived biostimulants represent an efficient tool to stimulate the root development: while macroalgae have already been widely adopted as a source of biostimulants to improve plants growth and resilience, far less information is available for microalgae. The objective of this work is to investigate the stimulant ability on maize roots of two green algae species, Chlamydomonas reinhardtii and Chlorella sorokiniana, being respectively the model organism for Chlorophyta and one of the most promising species for microalgae cultivation at industrial scale. The results obtained demonstrate that both C. reinhardtii and C. sorokiniana cells promoted the development of maize root system compared to the untreated negative control. C. sorokiniana specifically increased the number of secondary roots, while improved micro-nutrients accumulation on roots and shoots was measured in the case of C. reinhardtii treated plants. When these microalgae-derived biostimulants were applied on plants grown in stress conditions as nitrogen deficiency, improved development of the root system was measured in the case of plants treated with C. sorokiniana biomass. Microalgae cultivation for biostimulant production can thus be considered as a bio-based process providing solutions for improving plant resilience toward stress conditions.

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.

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.

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.

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.

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. Keywords: Fe acquisition, Nutrient transporters, P acquisition, Plant nutrition, RNA-seq, Root transcriptome, Root uptake, Strategy-I

Background 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. Results In this report transcriptomic analysis (NimbleGen microarray) of Fe deficiency response revealed that maize roots modulated the expression levels of 724 genes (508 up- and 216 down-regulated, respectively). As expected, roots of Fe-deficient maize plants overexpressed genes involved in the synthesis and release of 2’-deoxymugineic acid (the main PS released by maize roots). A strong modulation of genes involved in regulatory aspects, Fe translocation, root morphological modification, primary metabolic pathways and hormonal metabolism was induced by the nutritional stress. Genes encoding transporters for Fe2+ (ZmNRAMP1) and P (ZmPHT1;7 and ZmPHO1) were also up-regulated under Fe deficiency. Fe-deficient maize plants accumulated higher amounts of P than the Fe-sufficient ones, both in roots and shoots. The supply of 1 μM 59Fe, as soluble (Fe-Citrate and Fe-PS) or sparingly soluble (Ferrihydrite) sources to deficient plants, caused a rapid down-regulation of genes coding for PS and Fe(III)-PS transport, as well as of ZmNRAMP1 and ZmPHT1;7. Levels of 32P absorption essentially followed the rates of 59Fe uptake in Fe-deficient plants during Fe resupply, suggesting that P accumulation might be regulated by Fe uptake in maize plants. Conclusions The transcriptional response to Fe-deficiency in maize roots confirmed the modulation of known genes involved in the Strategy II and revealed the presence of Strategy I components usually described in dicots. Moreover, data here presented provide evidence of a close relationship between two essential nutrients for plants, Fe and P, and highlight a key role played by Fe and P transporters to preserve the homeostasis of these two nutrients in maize plants. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-3478-4) contains supplementary material, which is available to authorized users.

To limit nitrogen (N) losses from the soil, it has been suggested to provide urea to crops in conjunction with the urease inhibitor N-(n-butyl) thiophosphoric triamide (NBPT). However, recent studies reported that NBPT affects urea uptake and urease activity in plants. To shed light on these latter aspects, the effects of NBPT were studied analysing transcriptomic and metabolic changes occurring in urea-fed maize seedlings after a short-term exposure to the inhibitor. We provide evidence that NBPT treatment led to a wide reprogramming of plant metabolism. NBPT inhibited the activity of endogenous urease limiting the release and assimilation of ureic-ammonium, with a simultaneous accumulation of urea in plant tissues. Furthermore, NBPT determined changes in the glutamine, glutamate, and asparagine contents. Microarray data indicate that NBPT affects ureic-N assimilation and primary metabolism, such as glycolysis, TCA cycle, and electron transport chain, while activates the phenylalanine/tyrosine-derivative pathway. Moreover, the expression of genes relating to the transport and complexation of divalent metals was strongly modulated by NBPT. Data here presented suggest that when NBPT is provided in conjunction with urea an imbalance between C and N compounds might occur in plant cells. Under this condition, root cells also seem to activate a response to maintain the homeostasis of some micronutrients.

Background It is well known that in the rhizosphere soluble Fe sources available for plants are mainly represented by a mixture of complexes between the micronutrient and organic ligands such as carboxylates and phytosiderophores (PS) released by roots, as well as fractions of humified organic matter. The use by roots of these three natural Fe sources (Fe-citrate, Fe-PS and Fe complexed to water-extractable humic substances, Fe-WEHS) have been already studied at physiological level but the knowledge about the transcriptomic aspects is still lacking. Results The 59Fe concentration recorded after 24 h in tissues of tomato Fe-deficient plants supplied with 59Fe complexed to WEHS reached values about 2 times higher than those measured in response to the supply with Fe-citrate and Fe-PS. However, after 1 h no differences among the three Fe-chelates were observed considering the 59Fe concentration and the root Fe(III) reduction activity. A large-scale transcriptional analysis of root tissue after 1 h of Fe supply showed that Fe-WEHS modulated only two transcripts leaving the transcriptome substantially identical to Fe-deficient plants. On the other hand, Fe-citrate and Fe-PS affected 728 and 408 transcripts, respectively, having 289 a similar transcriptional behaviour in response to both Fe sources. Conclusions The root transcriptional response to the Fe supply depends on the nature of chelating agents (WEHS, citrate and PS). The supply of Fe-citrate and Fe-PS showed not only a fast back regulation of molecular mechanisms modulated by Fe deficiency but also specific responses due to the uptake of the chelating molecule. Plants fed with Fe-WEHS did not show relevant changes in the root transcriptome with respect to the Fe-deficient plants, indicating that roots did not sense the restored cellular Fe accumulation. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-2331-5) contains supplementary material, which is available to authorized users.

Despite the widespread use of urease inhibitors in agriculture, little information is available on their effect on nitrogen (N) uptake and assimilation. Aim of this work was to study, at physiological and transcriptional level, the effects of N-(n-butyl) thiophosphoric triamide (NBPT) on urea nutrition in hydroponically grown maize plants. Presence of NBPT in the nutrient solution limited the capacity of plants to utilize urea as a N-source; this was shown by a decrease in urea uptake rate and (15)N accumulation. Noteworthy, these negative effects were evident only when plants were fed with urea, as NBPT did not alter (15)N accumulation in nitrate-fed plants. NBPT also impaired the growth of Arabidopsis plants when urea was used as N-source, while having no effect on plants grown with nitrate or ammonium. This response was related, at least in part, to a direct effect of NBPT on the high affinity urea transport system. Impact of NBPT on urea uptake was further evaluated using lines of Arabidopsis overexpressing ZmDUR3 and dur3-knockout; results suggest that not only transport but also urea assimilation could be compromised by the inhibitor. This hypothesis was reinforced by an over-accumulation of urea and a decrease in ammonium concentration in NBPT-treated plants. Furthermore, transcriptional analyses showed that in maize roots NBPT treatment severely impaired the expression of genes involved in the cytosolic pathway of ureic-N assimilation and ammonium transport. NBPT also limited the expression of a gene coding for a transcription factor highly induced by urea and possibly playing a crucial role in the regulation of its acquisition. This work provides evidence that NBPT can heavily interfere with urea nutrition in maize plants, limiting influx as well as the following assimilation pathway.

While nitrate acquisition has been extensively studied, less information is available on transport systems of urea. Furthermore, the reciprocal influence of the two sources has not been clarified, so far. In this review, we will discuss recent developments on plant response to urea and nitrate nutrition. Experimental evidence suggests that, when urea and nitrate are available in the external solution, the induction of the uptake systems of each nitrogen (N) source is limited, while plant growth and N utilization is promoted. This physiological behavior might reflect cooperation among acquisition processes, where the activation of different N assimilatory pathways (cytosolic and plastidic pathways), allow a better control on the nutrient uptake. Based on physiological and molecular evidence, plants might increase (N) metabolism promoting a more efficient assimilation of taken-up nitrogen. The beneficial effect of urea and nitrate nutrition might contribute to develop new agronomical approaches to increase the (N) use efficiency in crops.

Even though urea and nitrate are the two major nitrogen (N) forms applied as fertilizers in agriculture and occur concomitantly in soils, the reciprocal influence of these two N sources on the mechanisms of their acquisition are poorly understood. Therefore, molecular and physiological aspects of urea and nitrate uptake were investigated in maize (Zea mays), a crop plant consuming high amounts of N. In roots, urea uptake was stimulated by the presence of urea in the external solution, indicating the presence of an inducible transport system. On the other hand, the presence of nitrate depressed the induction of urea uptake and, at the same time, the induction of nitrate uptake was depressed by the presence of urea. The expression of about 60,000 transcripts of maize in roots was monitored by microarray analyses and the transcriptional patterns of those genes involved in nitrogen acquisition were analyzed by real-time reverse transcription-PCR (RT-PCR). In comparison with the treatment without added N, the exposure of maize roots to urea modulated the expression of only very few genes, such as asparagine synthase. On the other hand, the concomitant presence of urea and nitrate enhanced the overexpression of genes involved in nitrate transport (NRT2) and assimilation (nitrate and nitrite reductase, glutamine synthetase 2), and a specific response of 41 transcripts was determined, including glutamine synthetase 1-5, glutamine oxoglutarate aminotransferase, shikimate kinase and arogenate dehydrogenase. Also based on the real-time RT-PCR analysis, the transcriptional modulation induced by both sources might determine an increase in N metabolism promoting a more efficient assimilation of the N that is taken up.

Iron (Fe) sources available for plants in the rhizospheric solution are mainly a mixture of complexes between Fe and organic ligands, including phytosiderophores (PS) and water-extractable humic substances (WEHS). In comparison with the other Fe sources, Fe-WEHS are more efficiently used by plants, and experimental evidences show that Fe translocation contributes to this better response. On the other hand, very little is known on the mechanisms involved in Fe allocation in leaves. In this work, physiological and molecular processes involved in Fe distribution in leaves of Fe-deficient Cucumis sativus supplied with Fe-PS or Fe-WEHS up to 5days were studied combining different techniques, such as radiochemical experiments, synchrotron micro X-ray fluorescence, real-time reverse transcription polymerase chain reaction and in situ hybridization. In Fe-WEHS-fed plants, Fe was rapidly (1day) allocated into the leaf veins, and after 5days, Fe was completely transferred into interveinal cells; moreover, the amount of accumulated Fe was much higher than with Fe-PS. This redistribution in Fe-WEHS plants was associated with an upregulation of genes encoding a ferric(III)-chelate reductase (FRO), a Fe2+ transporter (IRT1) and a natural resistance-associated macrophage protein (NRAMP). The localization of FRO and IRT1 transcripts next to the midveins, beside that of NRAMP in the interveinal area, may suggest a rapid and efficient response induced by the presence of Fe-WEHS in the extra-radical solution for the allocation in leaves of high amounts of Fe. In conclusion, Fe is more efficiently used when chelated to WEHS than PS and seems to involve Fe distribution and gene regulation of Fe acquisition mechanisms operating in leaves.

Plants mainly rely on a mixture of Fe complexes with different organic ligands, like carboxylates and soluble fractions of water-extractable humic substances (WEHSs), to sustain the supply of this micronutrient. It has been demonstrated that the Fe-WEHS complex is more efficiently acquired by plant roots as it enhances functionality of the mechanisms involved in Fe acquisition at the root and leaf levels, allowing a faster recovery of the Fe-deficiency symptoms. The aim of this work is to verify whether this recovery involves also the allocation and accumulation of nutrients other than Fe to and within the leaf tissues. Iron-deficient plants treated with Fe-WEHS recovered more quickly the functionality both to uptake nitrate at the root level and to fixate CO2 in the leaves than those supplied with Fe-citrate. Concomitantly, Fe-WEHS-treated plants also accumulated other cationic nutrients faster and at a higher extent. Synchrotron 2D-scanning μ-X-ray fluorescence analyses of the leaves revealed that the recovery promotes a change in the allocation of these nutrients from the vascular system (K, Cu, and Zn) or trichomes (Ca and Mn) to the entire leaf blade. Fe-WEHS treatment efficiently promotes the recovery from Fe-deficiency-induced chlorosis with an enhanced allocation of other nutrients into the leaves and promoting their distribution into the entire leaf blade.

Background Despite its extensive use as a nitrogen fertilizer, the role of urea as a directly accessible nitrogen source for crop plants is still poorly understood. So far, the physiological and molecular aspects of urea acquisition have been investigated only in few plant species highlighting the importance of a high-affinity transport system. With respect to maize, a worldwide-cultivated crop requiring high amounts of nitrogen fertilizer, the mechanisms involved in the transport of urea have not yet been identified. The aim of the present work was to characterize the high-affinity urea transport system in maize roots and to identify the high affinity urea transporter. Results Kinetic characterization of urea uptake (

Background Plants react to iron deficiency stress adopting different kind of adaptive responses. Tomato, a Strategy I plant, improves iron uptake through acidification of rhizosphere, reduction of Fe3+ to Fe2+ and transport of Fe2+ into the cells. Large-scale transcriptional analyses of roots under iron deficiency are only available for a very limited number of plant species with particular emphasis for Arabidopsis thaliana. Regarding tomato, an interesting model species for Strategy I plants and an economically important crop, physiological responses to Fe-deficiency have been thoroughly described and molecular analyses have provided evidence for genes involved in iron uptake mechanisms and their regulation. However, no detailed transcriptome analysis has been described so far. Results A genome-wide transcriptional analysis, performed with a chip that allows to monitor the expression of more than 25,000 tomato transcripts, identified 97 differentially expressed transcripts by comparing roots of Fe-deficient and Fe-sufficient tomato plants. These transcripts are related to the physiological responses of tomato roots to the nutrient stress resulting in an improved iron uptake, including regulatory aspects, translocation, root morphological modification and adaptation in primary metabolic pathways, such as glycolysis and TCA cycle. Other genes play a role in flavonoid biosynthesis and hormonal metabolism. Conclusions The transcriptional characterization confirmed the presence of the previously described mechanisms to adapt to iron starvation in tomato, but also allowed to identify other genes potentially playing a role in this process, thus opening new research perspectives to improve the knowledge on the tomato root response to the nutrient deficiency.