High-affinity Nitrate Transporter Research Articles

Transcriptome analysis of potato shoots, roots and stolons under nitrogen stress

Potato crop requires high dose of nitrogen (N) to produce high tuber yield. Excessive application of N causes environmental pollution and increases cost of production. Hence, knowledge about genes and regulatory elements is essential to strengthen research on N metabolism in this crop. In this study, we analysed transcriptomes (RNA-seq) in potato tissues (shoot, root and stolon) collected from plants grown in aeroponic culture under controlled conditions with varied N supplies i.e. low N (0.2 milli molar N) and high N (4 milli molar N). High quality data ranging between 3.25 to 4.93 Gb per sample were generated using Illumina NextSeq500 that resulted in 83.60–86.50% mapping of the reads to the reference potato genome. Differentially expressed genes (DEGs) were observed in the tissues based on statistically significance (p ≤ 0.05) and up-regulation with ≥ 2 log2 fold change (FC) and down-regulation with ≤ −2 log2 FC values. In shoots, of total 19730 DEGs, 761 up-regulated and 280 down-regulated significant DEGs were identified. Of total 20736 DEGs in roots, 572 (up-regulated) and 292 (down-regulated) were significant DEGs. In stolons, of total 21494 DEG, 688 and 230 DEGs were significantly up-regulated and down-regulated, respectively. Venn diagram analysis showed tissue specific and common genes. The DEGs were functionally assigned with the GO terms, in which molecular function domain was predominant in all the tissues. Further, DEGs were classified into 24 KEGG pathways, in which 5385, 5572 and 5594 DEGs were annotated in shoots, roots and stolons, respectively. The RT-qPCR analysis validated gene expression of RNA-seq data for selected genes. We identified a few potential DEGs responsive to N deficiency in potato such as glutaredoxin, Myb-like DNA-binding protein, WRKY transcription factor 16 and FLOWERING LOCUS T in shoots; high-affinity nitrate transporter, protein phosphatase-2c, glutaredoxin family protein, malate synthase, CLE7, 2-oxoglutarate-dependent dioxygenase and transcription factor in roots; and glucose-6-phosphate/phosphate translocator 2, BTB/POZ domain-containing protein, F-box family protein and aquaporin TIP1;3 in stolons, and many genes of unknown function. Our study highlights that these potential genes play very crucial roles in N stress tolerance, which could be useful in augmenting research on N metabolism in potato.

Biostimulant Action of Dissolved Humic Substances From a Conventionally and an Organically Managed Soil on Nitrate Acquisition in Maize Plants.

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.

Bacillus subtilis strain L1 promotes nitrate reductase activity in Arabidopsis and elicits enhanced growth performance in Arabidopsis, lettuce, and wheat.

Plant growth promoting rhizobacteria (PGPR) are a group of bacteria that promote plants growth in the rhizosphere. PGPRs are involved in various mechanisms that reinforce plant development. In this study, we screened for PGPRs that were effective in early growth of Arabidopsis thaliana when added to the media and one Bacillus subtilis strain L1 (Bs L1) was selected for further study. When Bs L1 was placed near the roots, seedlings showed notably stronger growth than that in the control, particularly in biomass and root hair. Quantitative reverse transcription polymerase chain reaction analysis revealed a high level of expression of the high affinity nitrate transporter gene, NRT2.1 in A. thaliana treated with Bs L1. After considering how Bs L1 could promote plant growth, we focused on nitrate, which is essential to plant growth. The nitrate content was lower in A. thaliana treated with Bs L1. However, examination of the activity of nitrate reductase revealed higher activity in plants treated with PGPR than in the control. Bs L1 had pronounced effects in representative crops (wheat and lettuce). These results suggest that Bs L1 promotes the assimilation and use of nitrate and plant growth.

Cloning and Molecular Characterization of CcNRT2.1/CcNAR2, a Putative Inducible High Affinity Nitrate Transport System in Capsicum chinense Jacq. Roots

NRT2 family of nitrate transporters normally require a partner protein, NAR2 (nitrate assimilation related protein), to transport nitrate to a high-affinity (high affinity nitrate transport system, HATS), although its role is still not well understood. In this study, the CcNRT2.1 and CcNAR2 cDNAs from of Capsicum chinense were cloned and characterized in terms of their structure, phylogeny, and their organ-specific transcriptional regulation, and by ligh/dark cycle, N and sucrose. CcNRT2.1 (putative molecular mass 57.5 kDa) and CcNAR2 (22.66 kDa) retained the characteristic domains of NRT2 and NAR2 family, respectively. CcNRT2.1 is expressed exclusively in the root, and both western blot and protein immunolocalization, demonstrated its exclusive location in root epidermal and cortical cells. CcNAR2 is expressed mainly in the root, but also in fruits of 25 days post-anthesis (DPA). The presence of two protein bands (~60 and ~80 kDa) detected in the western blot, suggested that CcNRT2.1 monomer and CcNRT2.1/CcNAR2 heterodimer work together for nitrate transport function in roots. Both genes were induced by NO3−, NH4+ and glutamate, and CcNRT2.1 was induced by sucrose. The co-expression of both genes in the specific zone of the root (2–4 cm from the apex) in which the high-affinity nitrate transport occurs (evaluated by root NO3− endogenous content, extracellular pH alkalinization, and H+ net flux by MIFE technique), strongly supports that CcNRT2.1/CcNAR2 belong to a HATS, with a functional similarity to AtNRT2.1/AtNAR2.1 of Arabidopsis. These are the first components of a HATS characterized in the Capsicum genus.

Phosphorylation at Ser28 stabilizes the Arabidopsis nitrate transporter NRT2.1 in response to nitrate limitation.

Nitrate is one of the main inorganic nitrogen sources for plants. Nitrate absorption from soils is achieved through the combined activities of specific nitrate transporters. Nitrate transporter 2.1 (NRT2.1) is the major component of the root high-affinity nitrate transport system in Arabidopsis thaliana. Studies to date have mainly focused on transcriptional control of NRT2.1. Here, we show that NRT2.1 protein stability is also regulated in response to nitrogen nutrition availability. When seedlings were transferred to nitrate-limited conditions, the apparent half-life of NRT2.1 in roots increased from 3 to 9 h. This stabilization of NRT2.1 protein occurred rapidly, even prior to the transcriptional stimulation of NRT2.1. Furthermore, we revealed that phosphorylation at serine 28 (Ser28) of NRT2.1 is involved in regulating the stability of this protein. Substitution of Ser28 by alanine resulted in unstable NRT2.1, and this loss-of-phosphorylation mutant (NRT2.1S28A ) failed to complement the growth-restricted phenotype of the nrt2.1 mutant when a low concentration of nitrate was the sole nitrogen source. These results demonstrate that phosphorylation at Ser28 is crucial for NRT2.1 protein stabilization and accumulation in response to nitrate limitation.

Methods for Measuring Nitrate Reductase, Nitrite Levels, and Nitric Oxide from Plant Tissues.

Nitrogen (N) is one of the most important nutrients which exist in both inorganic and organic forms. Plants assimilate inorganic form of N [nitrate (NO3-), nitrite (NO2-) or ammonium (NH4+)] and incorporate into amino acids. The metabolism of N involves a series of events such as sensing, uptake, and assimilation. The initial stage is sensing, triggered by nitrate or ammonium signals initiating signal transduction processes in N metabolism. The assimilation pathway initiates with NO3-/NH4+ transport to roots via specific high and low affinity (HATs and LATs) nitrate transporters or directly via ammonium transporters (AMTs). In cytosol the NO3- is reduced to NO2- by cytosolic nitrate reductase (NR) and the produced NO2- is further reduced to NH4+ by nitrite reductase (NiR) in plastids. NR has capability to reduce NO2- to nitric oxide (NO) under specific conditions such as hypoxia, low pH, and pathogen infection. The produced NO acts as a signal for wide range of processes such as plant growth development and stress. Here, we provide methods to measure NR activity, NO2- levels, and NO production in plant tissues.

Physiological and Molecular Osmotic Stress Responses in Three Durum Wheat (Triticum Turgidum ssp Durum) Genotypes

This study aims to investigate the activities and expression of enzymes of primary metabolism and relate these data with the growth performance of three different durum wheat genotypes (Maali; YT13; and ON66) under osmotic stress. Growth traits—including plant height, dry weight (DW) and relative water content (RWC)—were measured to classify genotypes depending on their tolerance to stress. Several enzymes were investigated: Ascorbate peroxidase (APX), Glutamine Synthetase (GS), Glutamine dehydrogenase (GDH), Glutamate synthase (GOGAT), Glucose 6-phosphate dehydrogenase (G6PDH), and Phosphoenolpyruvate Carboxylase (PEPC). The expression of the cytosolic and plastidic glutamine synthetase (TaGS1 and TaGS2), high affinity nitrate transporters (TaNRT2.3) and Glutamate dehydrogenase (TaGDH) were also detected by qRT-PCR. The results indicated different growth performances among genotypes, indicating Maali and YT13 as tolerant genotypes and ON66 as a drought-susceptible variety. Data showed a decrease in PEPC and increase in APX activities under osmotic stress; a slight decrease in GS activity was observed, together with an increase in G6PDH in all genotypes; GS and NRT2 expressions changed in a similar pattern in the different genotypes. Interestingly, Maali and YT13 showed higher transcript abundance for GDH under stress compared to ON66, suggesting the implication of GDH in protective phenomena upon osmotic stress.

Identification and Expression Analyses of the Nitrate Transporter Gene (NRT2) Family Among Skeletonema species (Bacillariophyceae).

High-affinity nitrate transporters are considered to be the major transporter system for nitrate uptake in diatoms. In the diatom genus Skeletonema, three forms of genes encoding high-affinity nitrate transporters (NRT2) were newly identified from transcriptomes generated as part of the marine microbial eukaryote transcriptome sequencing project. To examine the expression of each form of NRT2 under different nitrogen environments, laboratory experiments were conducted under nitrate-sufficient, ammonium-sufficient, and nitrate-limited conditions using three ecologically important Skeletonema species: S.dohrnii, S.menzelii, and S.marinoi. Primers were developed for each NRT2 form and species and Q-RT-PCR was performed. For each NRT2 form, the three Skeletonema species had similar transcriptional patterns. The transcript levels of NRT2:1 were significantly elevated under nitrogen-limited conditions, but strongly repressed in the presence of ammonium. The transcript levels of NRT2:2 were also repressed by ammonium, but increased 5- to 10-fold under nitrate-sufficient and nitrogen-limited conditions. Finally, the transcript levels of NRT2:3 did not vary significantly under various nitrogen conditions, and behaved more like a constitutively expressed gene. Based on the observed transcript variation among NRT2 forms, we propose a revised model describing nitrate uptake kinetics regulated by multiple forms of nitrate transporter genes in response to various nitrogen conditions in Skeletonema. The differential NRT2 transcriptional responses among species suggest that species-specific adaptive strategies exist within this genus to cope with environmental changes.

Expression of the cassava nitrate transporter NRT2.1 enables Arabidopsis low nitrate tolerance

The cassava grows well on low-nutrient soils because of its high-affinity to absorb nitrate. However, the molecular mechanisms by which cassava adapts itself to this environment remain elusive, although we have cloned a putative gene named MeNRT2.1 which has a crucial role in high-affinity nitrate transporter from cassava seeding. Here, the expression pattern of MeNRT2.1 was further assessed using the GUS activity driven by MeNRT2.1 promoter in Arabidopsis transformation plants. The GUS activity was monitored over time following the reduction of nitrate supply. The GUS gene expression not only peaked in roots after 12 h in 0.2mM nitrate media, but also stained stems and leaves. Arabidopsis plants with overexpression of MeNRT2.1 increased the biomass compared to the wild type on rich nitrogen (N-full) media. However, chlorate sensitivity analysis showed that Arabidopsis plants expressing MeNRT2.1 were more susceptable to chlorate than wild type. Significantly, after growing for 15 days on media containing 0.2mM nitrate concentration, wild-type plants became yellowor died, while the transgenic MeNRT2.1 Arabidopsis plants maintained normal growth. With significant increases in the amount of 15NO- 3 uptake in roots, the MeNRT2.1 plants also increased the contents of chlorophyll and nitrate reductase. Taken together, these results demonstrate that MeNRT2.1 has an important role in adaptation to low nitrate concentration as a nitrate transporter.

Molecular Characterization of ZosmaNRT2, the Putative Sodium Dependent High-Affinity Nitrate Transporter of Zostera marina L.

One of the most important adaptations of seagrasses during sea colonization was the capacity to grow at the low micromolar nitrate concentrations present in the sea. In contrast to terrestrial plants that use H+ symporters for high-affinity NO3− uptake, seagrasses such as Zostera marina L. use a Na+-dependent high-affinity nitrate transporter. Interestingly, in the Z. marina genome, only one gene (Zosma70g00300.1; NRT2.1) is annotated to this function. Analysis of this sequence predicts the presence of 12 transmembrane domains, including the MFS domains of the NNP transporter family and the “nitrate signature” that appears in all members of the NNP family. Phylogenetic analysis shows that this sequence is more related to NRT2.5 than to NRT2.1, sharing a common ancestor with both monocot and dicot plants. Heterologous expression of ZosmaNRT2-GFP together with the high-affinity nitrate transporter accessory protein ZosmaNAR2 (Zosma63g00220.1) in Nicotiana benthamiana leaves displayed four-fold higher fluorescence intensity than single expression of ZosmaNRT2-GFP suggesting the stabilization of NRT2 by NAR2. ZosmaNRT2-GFP signal was present on the Hechtian-strands in the plasmolyzed cells, pointing that ZosmaNRT2 is localized on the plasma membrane and that would be stabilized by ZosmaNAR2. Taken together, these results suggest that Zosma70g00300.1 would encode a high-affinity nitrate transporter located at the plasma membrane, equivalent to NRT2.5 transporters. These molecular data, together with our previous electrophysiological results support that ZosmaNRT2 would have evolved to use Na+ as a driving ion, which might be an essential adaptation of seagrasses to colonize marine environments.

Genetic analyses of nitrogen assimilation enzymes in Brassica juncea (L.) Czern & Coss.

Nitrogen (N) is a critical input for plant growth and development. A better understanding of N uptake and utilization is important to develop plant breeding strategies for improving nitrogen use efficiency (NUE). With that objective in mind, we assayed a SNP-genotyped association panel comprising 92 inbred lines for the activities of nitrate reductase (NR), nitrite reductase (NIR), glutamine synthetase (GS) and glutamate synthase (GOGAT). All these enzymes are associated with N assimilation. The experiments were carried out at two levels of N application: no added N (N0) and agrnomically recommened dose (100kg/ha) of N application (N100). Genome wide association studies (GWAS) helped to identify several marker-trait associations (MTAs), involving chromosomes A01, A06, A08, B02, B04, B05 and B08. These explained high phenotypic variation (up to 32%). Annotation of the genomic region(s) in or around significant SNPs allowed prediction of genes encoding high affinity nitrate transporters, glutamine synthetase 1.3, myb-like transcription factor family protein, bidirectional amino acid transporter 1, auxin signaling F-box 3 and oxidoreductases. This is the first attempt to use GWAS for identification of enzyme QTLs to explain variation for nitrogen assimilation enzymes in Brassica juncea.

A Transcription Factor, OsMADS57, Regulates Long-Distance Nitrate Transport and Root Elongation.

Root nitrate uptake adjusts to the plant's nitrogen demand for growth. Here, we report that OsMADS57, a MADS-box transcription factor, modulates nitrate translocation from rice (Oryza sativa) roots to shoots under low-nitrate conditions. OsMADS57 is abundantly expressed in xylem parenchyma cells of root stele and is induced by nitrate. Compared with wild-type rice plants supplied with 0.2 mM nitrate, osmads57 mutants had 31% less xylem loading of nitrate, while overexpression lines had 2-fold higher levels. Shoot-root 15N content ratios were 40% lower in the mutants and 76% higher in the overexpression lines. Rapid NO3 - root influx experiments showed that mutation of OsMADS57 did not affect root nitrate uptake. Reverse transcription quantitative PCR analysis of OsNRT2 nitrate transporter genes showed that after 5 min in 0.2 mM nitrate, only OsNRT2.3a (a vascular-specific high-affinity nitrate transporter) had reduced (by two-thirds) expression levels. At 60 min of nitrate treatment, lower expression levels were also observed for three additional NRT2 genes (OsNRT2.1/2.2/2.4). Conversely, in the overexpression lines, four NRT2 genes had much higher expression profiles at all time points tested. As previously reported, OsNRT2.3a functions in nitrate translocation, indicating the possible interaction between OsMADS57 and OsNRT2.3a Yeast one-hybrid and transient expression assays demonstrated that OsMADS57 binds to the CArG motif (CATTTTATAG) within the OsNRT2.3a promoter. Moreover, seminal root elongation was inhibited in osmads57 mutants, which may be associated with higher auxin levels in and auxin polar transport to root tips of mutant plants. Taken together, these results suggest that OsMADS57 has a role in regulating nitrate translocation from root to shoot via OsNRT2.3a.

A novel function of N-signaling in plants with special reference to Trichoderma interaction influencing plant growth, nitrogen use efficiency, and cross talk with plant hormones.

Trichoderma spp. is considered as a plant growth promoter and biocontrol fungal agents. They colonize on the surface of root in most of the agriculture crops. They secrete different secondary metabolites and enzymes which promote different physiological processes as well as protect plants from various environmental stresses. This is part of their vital functions. They are widely exploited as a biocontrol agent and plant growth promoter in agricultural fields. Colonization of Trichoderma with roots can enhance nutrient acquisition from surrounding soil to root and can substantially increase nitrogen use efficiency (NUE) in crops and linked with activation of plant signaling cascade. Among Trichoderma species, only some Trichoderma species were well characterized which help in the uptake of nitrogen-containing compound (especially nitrate form) and induced nitric oxide (NO) in plants. Both nitrate and NO are known as a signaling agent, involved in plant growth and development and disease resistance. Activation of these signaling molecules may crosstalk with other signaling molecule (Ca2+) and phytohormone (auxin, gibberellins, cytokinin and ethylene). This ability of Trichoderma is important to agriculture not only for increased plant growth but also to control plant diseases. Recently, Trichoderma strains have been shown to encompass the ability to regulate transcripts level of high-affinity nitrate transporters and probably it was positively regulated by NO. This review aims to focus the usage of Trichoderma strains on crops by their abilities to regulate transcript levels, probably through activation of plant N signaling transduction that improve plant health.

OsNAR2.1 Positively Regulates Drought Tolerance and Grain Yield Under Drought Stress Conditions in Rice

Drought is an important environmental factor that severely restricts crop production. The high-affinity nitrate transporter partner protein OsNAR2.1 plays an essential role in nitrate absorption and translocation in rice. Our results suggest that OsNAR2.1 expression is markedly induced by water deficit. After drought stress conditions and irrigation, compared with wild-type (WT), the survival rate was significantly improved in OsNAR2.1 over-expression lines and decreased in OsNAR2.1 RNAi lines. The survival rate of Wuyunjing7 (WYJ), OsNRT2.1 over-expression lines and OsNRT2.3a over-expression lines was not significantly different. Compared with WT, overexpression of OsNAR2.1 could significantly increase nitrogen uptake in rice, and OsNAR2.1 RNAi could significantly reduce nitrogen uptake. Under drought conditions, the expression of OsNAC10, OsSNAC1, OsDREB2a, and OsAP37 was significantly reduced in OsNAR2.1 RNAi lines and increased substantially in OsNAR2.1 over-expression lines. Also, the chlorophyll content, relative water content, photosynthetic rate and water use efficiency were decreased considerably in OsNAR2.1 RNAi lines and increased significantly in OsNAR2.1 over-expression lines under drought conditions. Finally, compared to WT, grain yield increased by about 9.1 and 26.6%, in OsNAR2.1 over-expression lines under full and limited irrigation conditions, respectively. These results indicate that OsNAR2.1 regulates the response to drought stress in rice and increases drought tolerance.

A combined association mapping and t-test analysis of SNP loci and candidate genes involving in resistance to low nitrogen traits by a wheat mutant population.

Crop productivity is highly dependent on the application of N fertilizers, but ever-increasing N application is causing serious environmental impacts. To facilitate the development of new wheat cultivars that can thrive in low N growth conditions, key loci and genes associated with wheat responses to low N must be identified. In this GWAS and t-test study of 190 M6 mutant wheat lines (Jing 411-derived) based on genotype data from the wheat 660k SNP array, we identified a total of 221 significant SNPs associated four seedling phenotypic traits that have been implicated in resistance to low N: relative root length, relative shoot length, relative root weight, and relative shoot weight. Notably, we detected large numbers of significantly associated SNP in what appear to be genomic ‘hotspots’ for resistance to low N on chromosomes 2A and 6B, strongly suggesting that these regions are functionally related to the resistance phenotypes that we observed in some of the mutant lines. Moreover, the candidate genes, including genes encoding high-affinity nitrate transporter 2.1, gibberellin responsive protein, were identified for resistance to low N. This study raises plausible mechanistic hypotheses that can be evaluated in future applied or basic efforts by breeders or plant biologists seeking to develop new high-NUE wheat cultivars.

Shoot nitrate underlies a perception of nitrogen satiety to trigger local and systemic signaling cascades in Arabidopsis thaliana

ABSTRACT Nitrate is an important ion for plant growth and development. It serves not only as a building block for amino acid synthesis but also as a signaling molecule. Changes in the exogenous nitrate concentrations affect the expression of nitrate-responsive genes within minutes. Following these rapid transcriptional events, nitrate and its downstream organic nitrogen (N) compounds accumulate in the plant body, inducing secondary responses to the internal N level. Nevertheless, the respective roles of nitrate and organic N in triggering plant responses to internal N remain to be clarified. Several studies have implied that internal nitrate levels regulate root N uptake independent of the levels of N assimilation products. However, little is known about the specific effects of internal nitrate levels on plant growth and gene expression. To manipulate the internal nitrate levels independently of internal organic N, we grew wild-type Arabidopsis and a nitrate reductase (NR)-null mutant under a series of modified N conditions. Using their shoots and roots, we performed analyses of plant growth and RNA sequencing. The results showed that elevated shoot nitrate accumulation in the NR-null mutant was accompanied by increased expression of nitrate assimilatory genes in the shoots, decreased gene expression of high-affinity nitrate and ammonium uptake transporters in the roots, and decreased lateral root growth. Furthermore, the genes normally induced by N deficiency were significantly downregulated both in the shoots and roots of the NR-null mutant, compared with the wild-type. Our transcriptional profiling suggests that the transcription factors NLP7 and NIGT mediate a wide range of these transcriptional responses. Taken together, we conclude that shoot nitrate acts as a N satiety signal to trigger local and systemic signaling cascades in A. thaliana. The present study illustrates an adaptive strategy of plants to survive in N-limited environments, depending on the residual nitrate storage.

Overexpression of a High-Affinity Nitrate Transporter OsNRT2.1 Increases Yield and Manganese Accumulation in Rice Under Alternating Wet and Dry Condition.

Nitrate and manganese (Mn) are necessary elements for the growth and development of rice in paddy soil. Under physiological conditions, we previously reported that the uptake of Mn in roots can be improved by the addition of external nitrate but not ammonium. To investigate the mechanism(s) of this phenotype, we produced plant lines overexpressing OsNRT2.1 and assessed Mn uptake under alternating wet and dry (AWD) and waterlogged (WL) conditions. Under AWD condition, we observed a 31% reduction in grain yields of wild type (WT) plants compared to WL condition. Interestingly, the overexpression of OsNRT2.1 could recover this loss, as OsNRT2.1 transgenic lines displayed higher grain yields than WT plants. We also observed 60% higher grain Mn in the transgenic lines in AWD condition and approximately 30% higher Mn in the grain of transgenic lines in WL condition. We further found that the overexpression of OsNRT2.1 did not alter Mg and Fe in the seeds in either growth condition. The reasons for the increased Mn content in OsNRT2.1 transgenic seeds in AWD condition could be explained by the elevated expression of OsNRAMP family genes including OsNRAMP3, OsNRAMP5, and OsNRAMP6 in node I, the panicle-neck, and the flag leaves. The mechanism(s) underpinning the upregulation of these genes requires further investigation. Taken together, our results provide a new function of OsNRT2.1 in improving rice yields and grain Mn accumulation during water-saving cultivation patterns. This represents a new strategy for maintaining yield and improving food quality in a sustainable agricultural system.

Elevated CO2-induced production of nitric oxide differentially modulates nitrate assimilation and root growth of wheat seedlings in a nitrate dose-dependent manner.

Wheat is a major staple food crop worldwide contributing approximately 20% of total protein consumed by mankind. The nitrogen and protein concentration of wheat crop and grain often decline as a result of exposure of the crop to elevated CO2 (EC). The changes in nitrogen (N) assimilation, root system architecture, and nitric oxide (NO)-mediated N signaling and expression of genes involved in N assimilation and high affinity nitrate uptake were examined in response to different nitrate levels and EC in wheat. Activity of enzyme nitrate reductase (NRA) was downregulated under EC both in leaf and root tissues. Plants grown under EC displayed enhanced production of NO and more so when nitrate supply was high. Based on exogenous supply of NO, inhibitors of NO production, and NO scavenger, regulatory role of NO on EC mediated changes in root morphology and NRA was revealed. The enhanced NO production under EC and high N levels negatively regulated the transcript abundance of NR and high affinity nitrate transporters (HATS).

Unraveling the Functional Role of NPF6 Transporters.

The nitrate transporter 1/peptide transporter (NPF) family represents a growing list of putative nitrate permeable transport proteins expressed within multiple cell types and tissues across a diverse range of plant species. Their designation as nitrate permeable and/or selective transporters is slowly being defined as more genes are characterized and their functional activities tested both in planta and in vitro. The most notable of the NPF family has been the Arabidopsis thaliana homolog, AtNPF6.3, previously known as AtNRT1.1 or CHL1. AtNPF6.3 has traditionally been characterized as a dual-affinity nitrate transporter contributing to root nitrate uptake in Arabidopsis. It has also been identified as a nitrate sensor which regulates the expression of high-affinity nitrate transport proteins NRT2s and lateral root development as a part of the primary nitrate response in plants. The sensor function of AtNPF6.3 has also been attributed to its auxin transport activity. Other homologs of AtNPF6.3 are now being described highlighting the variability in their functional capabilities (alternative substrates and kinetics) linking to structural aspects of the proteins. This review focusses on NPF6.3-like transport proteins and the knowledge that has been gained since their initial discovery over two decades ago. The review will investigate from a structural point of view how NPF6.3-like proteins may transport nitrate as well as other ions and what can be learned from structural uniqueness about predicted activities in plants.

Copper excess reduces nitrate uptake by Arabidopsis roots with specific effects on gene expression

Nitrate uptake by plants is mediated by specific transport proteins in roots (NRTs), which are also dependent on the activity of proton pumps that energize the reaction. Nitrogen (N) metabolism in plants is sensitive to copper (Cu) toxicity conditions. To understand how Cu affects the uptake and assimilation processes, this study assesses the inhibitory effects of elevated Cu levels on the expression of genes related to N absorption, transport and assimilation in roots of Arabidopsis. Plants were grown hydroponically for 45 days, being exposed to a range of Cu concentrations in the last 72 h or alternatively exposed to 5.0 μM Cu for the last 15 days. High Cu levels decreased the uptake and accumulation of N in plants. It down-regulated the expression of genes encoding nitrate reductase (NR1), low-affinity nitrate transporters (NRT1 family) and bZIP transcription factors (TGA1 and TGA4) that regulate the expression of nitrate transporters. Cu toxicity also specifically down-regulated the plasma membrane proton pump, AHA2, whilst having little effect on AHA1 and AHA5. In contrast, there was an up-regulation of high-affinity nitrate transporters from the NRT2 family when exposed to medium level of Cu excess, but this was insufficient for restoring N absorption by roots to control levels. These results demonstrate that plants display specific responses to Cu toxicity, modulating the expression of particular genes related to nitrate uptake, such as low-affinity nitrate transporters and proton pumps.