Interplay between NIN-LIKE PROTEINs 6 and 7 in nitrate signaling.

NLP7 (NIN-LIKE-PROTEIN 7) is the major transcriptional factor responsible for the primary nitrate response (PNR), but the role of its homolog, NLP6, in nitrogen signaling and the interplay between NLP6 and NLP7 remain to be elucidated. In this study, we show that, like NLP7, nuclear localization of NLP6 via a nuclear retention mechanism is nitrate dependent, but nucleocytosolic shuttling of both NLP6 and NLP7 is independent of each other. Compared with single mutants, the nlp6nlp7 double mutant displays a synergistic growth retardation phenotype in response to nitrate. The transcriptome analysis of the PNR showed that NLP6 and NLP7 govern ∼50% of nitrate-induced genes, with cluster analysis highlighting 2 distinct patterns. In the A1 cluster, NLP7 plays the major role, whereas in the A2 cluster, NLP6 and NLP7 are partially functionally redundant. Interestingly, comparing the growth phenotype and PNR under high- and low-nitrate conditions demonstrated that NLP6 and NLP7 exert a more dominant role in the response to high nitrate. Apart from nitrate signaling, NLP6 and NLP7 also participated in high ammonium conditions. Growth phenotypes and transcriptome data revealed that NLP6 and NLP7 are completely functionally redundant and may act as repressors in response to ammonium. Other NLP family members also participated in the PNR, with NLP2 and NLP7 acting as broader regulators and NLP4, -5, -6, and -8 regulating PNR in a gene-dependent manner. Thus, our findings indicate that multiple modes of interplay exist between NLP6 and NLP7 that differ depending on nitrogen sources and gene clusters.

Study of vacuole glycerate transporter NPF8.4 reveals a new role of photorespiration in C/N balance.

The photorespiratory intermediate glycerate is known to be shuttled between the peroxisome and chloroplast. Here, localization of NPF8.4 in the tonoplast, together with the reduced vacuolar glycerate content displayed by an npf8.4 mutant and the glycerate efflux activity detected in an oocyte expression system, identifies NPF8.4 as a tonoplast glycerate influx transporter. Our study shows that expression of NPF8.4 and most photorespiration-associated genes, as well as the photorespiration rate, is upregulated in response to short-term nitrogen (N) depletion. We report growth retardation and early senescence phenotypes for npf8.4 mutants specifically upon N depletion, suggesting that the NPF8.4-mediated regulatory pathway for sequestering the photorespiratory carbon intermediate glycerate in vacuoles is important to alleviate the impact of an increased C/N ratio under N deficiency. Thus, our study of NPF8.4 reveals a novel role for photorespiration in N flux to cope with short-term N depletion.

Potential transceptor AtNRT1.13 modulates shoot architecture and flowering time in a nitrate-dependent manner.

Compared with root development regulated by external nutrients, less is known about how internal nutrients are monitored to control plasticity of shoot development. In this study, we characterize an Arabidopsis thaliana transceptor, NRT1.13 (NPF4.4), of the NRT1/PTR/NPF family. Different from most NRT1 transporters, NRT1.13 does not have the conserved proline residue between transmembrane domains 10 and 11; an essential residue for nitrate transport activity in CHL1/NRT1.1/NPF6.3. As expected, when expressed in oocytes, NRT1.13 showed no nitrate transport activity. However, when Ser 487 at the corresponding position was converted back to proline, NRT1.13 S487P regained nitrate uptake activity, suggesting that wild-type NRT1.13 cannot transport nitrate but can bind it. Subcellular localization and ß-glucuronidase reporter analyses indicated that NRT1.13 is a plasma membrane protein expressed at the parenchyma cells next to xylem in the petioles and the stem nodes. When plants were grown with a normal concentration of nitrate, nrt1.13 showed no severe growth phenotype. However, when grown under low-nitrate conditions, nrt1.13 showed delayed flowering, increased node number, retarded branch outgrowth, and reduced lateral nitrate allocation to nodes. Our results suggest that NRT1.13 is required for low-nitrate acclimation and that internal nitrate is monitored near the xylem by NRT1.13 to regulate shoot architecture and flowering time.

Improving nitrogen use efficiency by manipulating nitrate remobilization in plants.

Increasing nitrogen use efficiency (NUE) is critical to improve crop yield, reduce N fertilizer demand and alleviate environmental pollution. N remobilization is a key component of NUE. The nitrate transporter NRT1.7 is responsible for loading excess nitrate stored in source leaves into phloem and facilitates nitrate allocation to sink leaves. Under N starvation, the nrt1.7 mutant exhibits growth retardation, indicating that NRT1.7-mediated source-to-sink remobilization of stored nitrate is important for sustaining growth in plants. To energize NRT1.7-mediated nitrate recycling, we introduced a hyperactive chimeric nitrate transporter NC4N driven by the NRT1.7 promoter into the nrt1.7 mutant. NRT1.7p::NC4N::3' transgenic plants accumulated more nitrate in younger leaves, and 15NO3- tracing analysis revealed that more 15N was remobilized into sink tissues. Consistently, transgenic Arabidopsis, tobacco and rice plants showed improved growth or yield. Our study suggests that enhancing source-to-sink nitrate remobilization represents a new strategy for enhancing NUE and crop production.

Nitrate Transport, Signaling, and Use Efficiency.

Nitrogen accounts for approximately 60% of the fertilizer consumed each year; thus, it represents one of the major input costs for most nonlegume crops. Nitrate is one of the two major forms of nitrogen that plants acquire from the soil. Mechanistic insights into nitrate transport and signaling have enabled new strategies for enhancing nitrogen utilization efficiency, for lowering input costs for farming, and, more importantly, for alleviating environmental impacts (e.g., eutrophication and production of the greenhouse gas N2O). Over the past decade, significant progress has been made in understanding how nitrate is acquired from the surroundings, how it is efficiently distributed into different plant tissues in response to environmental changes, how nitrate signaling is perceived and transmitted, and how shoot and root nitrogen status is communicated. Several key components of these processes have proven to be novel tools for enhancing nitrate- and nitrogen-use efficiency. In this review, we focus on the roles of NRT1 and NRT2 in nitrate uptake and nitrate allocation among different tissues; we describe the functions of the transceptor NRT1.1, transcription factors, and small signaling peptides in nitrate signaling and tissue communication; and we compile the new strategies for improving nitrogen-use efficiency.

The Arabidopsis CPSF30-L gene plays an essential role in nitrate signaling and regulates the nitrate transceptor gene NRT1.1.

Plants have evolved sophisticated mechanisms to adapt to fluctuating environmental nitrogen availability. However, more underlying genes regulating the response to nitrate have yet to be characterized. We report here the identification of a nitrate regulatory mutant whose mutation mapped to the Cleavage and Polyadenylation Specificity Factor 30 gene (CPSF30-L). In the mutant, induction of nitrate-responsive genes was inhibited independent of the ammonium conditions and was restored by expression of the wild-type 65 kDa encoded by CPSF30-L. Molecular and genetic evidence suggests that CPSF30-L works upstream of NRT1.1 and independently of NLP7 in response to nitrate. Analysis of the 3'-UTR of NRT1.1 showed that the pattern of polyadenylation sites was altered in the cpsf30 mutant. Transcriptome analysis revealed that four nitrogen-related clusters were enriched in the differentially expressed genes of the cpsf30 mutant. Nitrate uptake was decreased in the mutant along with reduced expression of the nitrate transporter/sensor gene NRT1.1, while nitrate reduction and amino acid content were enhanced in roots along with increased expression of several nitrate assimilatory genes. These findings indicate that the 65 kDa protein encoded by CPSF30-L mediates nitrate signaling in part by regulating NRT1.1 expression, thus adding an important component to the nitrate signaling network.

Influence of differing nitrate and nitrogen availability on flowering control in Arabidopsis.

Nitrogen, an essential macronutrient for plants, regulates many aspects of plant growth and development. Nitrate is one of the major forms of nitrogen taken up by plants from the soil. Nitrate and nitrogen have been reported to regulate flowering; while some studies have shown that lower nitrate/nitrogen promoted flowering, others have reported the opposite trend. To elucidate how nitrate/nitrogen affects flowering, we reviewed the existing literature and conducted experiments to examine flowering time under a wide range of nitrate concentrations using two growth systems. From the literature review and our experiments, we established that differing nitrate availability results in a U-shaped flowering curve, with an optimal concentration of nitrate facilitating flowering and concentrations above or below this optimal concentration delaying flowering. The role of nitrate and nitrogen in regulating flowering has been elucidated by several transcriptomic and mutant studies, which have suggested close interactions between nitrate/nitrogen, phosphate, the circadian clock, photosynthesis, and, potentially, hormones. We discuss several possible molecular mechanisms underlying the U-shaped flowering response.

Disruption of the rice nitrate transporter OsNPF2.2 hinders root-to-shoot nitrate transport and vascular development.

Plants have evolved to express some members of the nitrate transporter 1/peptide transporter family (NPF) to uptake and transport nitrate. However, little is known of the physiological and functional roles of this family in rice (Oryza sativa L.). Here, we characterized the vascular specific transporter OsNPF2.2. Functional analysis using cDNA-injected Xenopus laevis oocytes revealed that OsNPF2.2 is a low-affinity, pH-dependent nitrate transporter. Use of a green fluorescent protein tagged OsNPF2.2 showed that the transporter is located in the plasma membrane in the rice protoplast. Expression analysis showed that OsNPF2.2 is nitrate inducible and is mainly expressed in parenchyma cells around the xylem. Disruption of OsNPF2.2 increased nitrate concentration in the shoot xylem exudate when nitrate was supplied after a deprivation period; this result suggests that OsNPF2.2 may participate in unloading nitrate from the xylem. Under steady-state nitrate supply, the osnpf2.2 mutants maintained high levels of nitrate in the roots and low shoot:root nitrate ratios; this observation suggests that OsNPF2.2 is involved in root-to-shoot nitrate transport. Mutation of OsNPF2.2 also caused abnormal vasculature and retarded plant growth and development. Our findings demonstrate that OsNPF2.2 can unload nitrate from the xylem to affect the root-to-shoot nitrate transport and plant development.

A unified nomenclature of NITRATE TRANSPORTER 1/PEPTIDE TRANSPORTER family members in plants.

Members of the plant NITRATE TRANSPORTER 1/PEPTIDE TRANSPORTER (NRT1/PTR) family display protein sequence homology with the SLC15/PepT/PTR/POT family of peptide transporters in animals. In comparison to their animal and bacterial counterparts, these plant proteins transport a wide variety of substrates: nitrate, peptides, amino acids, dicarboxylates, glucosinolates, IAA, and ABA. The phylogenetic relationship of the members of the NRT1/PTR family in 31 fully sequenced plant genomes allowed the identification of unambiguous clades, defining eight subfamilies. The phylogenetic tree was used to determine a unified nomenclature of this family named NPF, for NRT1/PTR FAMILY. We propose that the members should be named accordingly: NPFX.Y, where X denotes the subfamily and Y the individual member within the species.

Two phloem nitrate transporters, NRT1.11 and NRT1.12, are important for redistributing xylem-borne nitrate to enhance plant growth.

This study of the Arabidopsis (Arabidopsis thaliana) nitrate transporters NRT1.11 and NRT1.12 reveals how the interplay between xylem and phloem transport of nitrate ensures optimal nitrate distribution in leaves for plant growth. Functional analysis in Xenopus laevis oocytes showed that both NRT1.11 and NRT1.12 are low-affinity nitrate transporters. Quantitative reverse transcription-polymerase chain reaction and immunoblot analysis showed higher expression of these two genes in larger expanded leaves. Green fluorescent protein and ß-glucuronidase reporter analyses indicated that NRT1.11 and NRT1.12 are plasma membrane transporters expressed in the companion cells of the major vein. In nrt1.11 nrt1.12 double mutants, more root-fed (15)NO3(-) was translocated to mature and larger expanded leaves but less to the youngest tissues, suggesting that NRT1.11 and NRT1.12 are required for transferring root-derived nitrate into phloem in the major veins of mature and larger expanded leaves for redistributing to the youngest tissues. Distinct from the wild type, nrt1.11 nrt1.12 double mutants show no increase of plant growth at high nitrate supply. These data suggested that NRT1.11 and NRT1.12 are involved in xylem-to-phloem transfer for redistributing nitrate into developing leaves, and such nitrate redistribution is a critical step for optimal plant growth enhanced by increasing external nitrate.

Using membrane transporters to improve crops for sustainable food production.

With the global population predicted to grow by at least 25 per cent by 2050, the need for sustainable production of nutritious foods is critical for human and environmental health. Recent advances show that specialized plant membrane transporters can be used to enhance yields of staple crops, increase nutrient content and increase resistance to key stresses, including salinity, pathogens and aluminium toxicity, which in turn could expand available arable land.

Uptake, allocation and signaling of nitrate.

Plants need to acquire nitrogen (N) efficiently from the soil for growth. Nitrate is one of the major N sources for higher plants. Therefore, nitrate uptake and allocation are key factors in efficient N utilization. Membrane-bound transporters are required for nitrate uptake from the soil and for the inter- and intracellular movement of nitrate inside the plants. Four gene families, nitrate transporter 1/peptide transporter (NRT1/PTR), NRT2, chloride channel (CLC), and slow anion channel-associated 1 homolog 3 (SLAC1/SLAH), are involved in nitrate uptake, allocation, and storage in higher plants. Recent studies of these transporters or channels have provided new insights into the molecular mechanisms of nitrate uptake and allocation. Interestingly, several of these transporters also play versatile roles in nitrate sensing, plant development, pathogen defense, and/or stress response.

Arabidopsis nitrate transporter NRT1.9 is important in phloem nitrate transport.

This study of the Arabidopsis thaliana nitrate transporter NRT1.9 reveals an important function for a NRT1 family member in phloem nitrate transport. Functional analysis in Xenopus laevis oocytes showed that NRT1.9 is a low-affinity nitrate transporter. Green fluorescent protein and ß-glucuronidase reporter analyses indicated that NRT1.9 is a plasma membrane transporter expressed in the companion cells of root phloem. In nrt1.9 mutants, nitrate content in root phloem exudates was decreased, and downward nitrate transport was reduced, suggesting that NRT1.9 may facilitate loading of nitrate into the root phloem and enhance downward nitrate transport in roots. Under high nitrate conditions, the nrt1.9 mutant showed enhanced root-to-shoot nitrate transport and plant growth. We conclude that phloem nitrate transport is facilitated by expression of NRT1.9 in root companion cells. In addition, enhanced root-to-shoot xylem transport of nitrate in nrt1.9 mutants points to a negative correlation between xylem and phloem nitrate transport.

Integration of nitrogen and potassium signaling.

Sensing and responding to soil nutrient fluctuations are vital for the survival of higher plants. Over the past few years, great progress has been made in our understanding of nitrogen and potassium signaling. Key components of the signaling pathways including sensors, kinases, miRNA, ubiquitin ligases, and transcriptional factors. These components mediate the transcriptional responses, root-architecture changes, and uptake-activity modulation induced by nitrate, ammonium, and potassium in the soil solution. Integration of these responses allows plants to compete for limited nutrients and to survive under nutrient deficiency or toxic nutrient excess. A future challenge is to extend the present fragmented sets of data to a comprehensive signaling network. Then, such knowledge and the accompanying molecular tools can be applied to improve the efficiency of nutrient utilization in crops.

Nitrate, ammonium, and potassium sensing and signaling.

Plants acquire numerous nutrients from the soil. In addition, nutrients elicit many physiological and morphological responses especially in roots. Recently, there has been significant progress in identifying the sensing and regulatory mechanisms of several essential nutrients. In this review, we describe the newly identified signaling components of nitrate, ammonium, and potassium, focusing specifically on the initial sensing steps.

Nitrate signaling: adaptation to fluctuating environments.

Nitrate (NO(3)(-)) is a key nutrient as well as a signaling molecule that impacts both metabolism and development of plants. Understanding the complexity of the regulatory networks that control nitrate uptake, metabolism, and associated responses has the potential to provide solutions that address the major issues of nitrate pollution and toxicity that threaten agricultural and ecological sustainability and human health. Recently, major advances have been made in cataloguing the nitrate transcriptome and in identifying key components that mediate nitrate signaling. In this perspective, we describe the genes involved in nitrate regulation and how they influence nitrate transport and assimilation, and we discuss the role of systems biology approaches in elucidating the gene networks involved in NO(3)(-) signaling adaptation to fluctuating environments.