Nitrogen (N) is the mineral nutrient required in the greatest amount and its availability is a major factor limiting growth and development of plants. As sessile organisms, plants have evolved different strategies to adapt to changes in the availability and distribution of N in soils. These strategies include mechanisms that act at different levels of biological organization from the molecular to the ecosystem level. At the molecular level, plants can adjust their capacity to acquire different forms of N in a range of concentrations by modulating the expression and function of genes in different N uptake systems. Modulation of plant growth and development, most notably changes in the root system architecture, can also greatly impact plant N acquisition in the soil. At the organism and ecosystem levels, plants establish associations with diverse microorganisms to ensure adequate nutrition and N supply. These different adaptive mechanisms have been traditionally discussed separately in the literature. To understand plant N nutrition in the environment, an integrated view of all pathways contributing to plant N acquisition is required. Towards this goal, in this review the different mechanisms that plants utilize to maintain an adequate N supply are summarized and integrated.

Application of chemical fertilizers, especially nitrogen (N), to crops has increased dramatically in the last half century and therefore developing crop varieties with improved N use efficiency (NUE) is urgent for sustainable agriculture. N utilization procedures generally can be divided into uptake, transport, and assimilation. Transporters for nitrate or ammonium acquisition and enzymes for assimilation are among the essential components determining NUE, and many transcription factors also play a pivotal role in regulating N use-associated genes, thereby contributing to NUE. Although some efforts in improving NUE have been made in various plants, the regulatory mechanisms underlying NUE are still elusive, and NUE improvement in crop breeding is very limited. In this review, the crucial components involved in N utilization and the candidates with the potential for NUE improvement in dicot Arabidopsis and monocot rice are summarized. In addition, strategies based on new techniques which can be used for dissecting regulatory mechanisms of NUE and also the possible ways in which NUE can be improved in crops are discussed.© The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Modern agriculture relies heavily on chemical fertilizers, especially in terms of cereal production. The excess application of fertilizers not only increases production cost, but also causes severe environmental problems. As one of the major cereal crops, rice (Oryza sativa L.) provides the staple food for nearly half of population worldwide, especially in developing countries. Therefore, improving rice yield is always the priority for rice breeding. Macronutrients, especially nitrogen (N) and phosphorus (P), are two most important players for the grain yield of rice. However, with economic development and improved living standard, improving nutritional quality such as micronutrient contents in grains has become a new goal in order to solve the "hidden hunger." Micronutrients, such as iron (Fe), zinc (Zn), and selenium (Se), are critical nutritional elements for human health. Therefore, breeding the rice varieties with improved nutrient use efficiency (NUE) is thought to be one of the most feasible ways to increase both grain yield and nutritional quality with limited fertilizer input. In this review, we summarized the progresses in molecular dissection of genes for NUE by reverse genetics on macronutrients (N and P) and micronutrients (Fe, Zn, and Se), exploring natural variations for improving NUE in rice; and also, the current genetic toolbox and future perspectives for improving rice NUE are discussed.

Crop production is threatened by global climate change, and recent demands for crops to produce bio-fuels have started to affect the worldwide supply of some of the most important foods. How can we support a growing human population in such circumstances? One potential solution is the improvement of crops to increase yield from both irrigated and non-irrigated lands, and to create novel varieties that are more tolerant to environmental stresses. Recent progress has been made in the isolation and functional analyses of genes controlling yield and tolerance to abiotic stresses. In addition, promising new methods are being developed for identifying additional genes and variants of interest and putting these to practical use in crop improvement.

A major challenge in current biology is to understand the genetic basis of variation for quantitative traits. We review the principles of quantitative trait locus mapping and summarize insights about the genetic architecture of quantitative traits that have been obtained over the past decades. We are currently in the midst of a genomic revolution, which enables us to incorporate genetic variation in transcript abundance and other intermediate molecular phenotypes into a quantitative trait locus mapping framework. This systems genetics approach enables us to understand the biology inside the 'black box' that lies between genotype and phenotype in terms of causal networks of interacting genes.

In the past 50 years, the application of synthetic nitrogen (N) fertilizer to farmland resulted in a dramatic increase in crop yields but with considerable negative impacts on the environment. New solutions are therefore needed to simultaneously increase yields while maintaining, or preferably decreasing, applied N to maximize the nitrogen use efficiency (NUE) of crops. In this review, we outline the definition of NUE, the selection and development of NUE crops, and the factors that interact with NUE. In particular, we emphasize the challenges of developing crop plants with enhanced NUE, using more classical genetic approaches based on utilizing existing allelic variation for NUE traits. The challenges of phenotyping, mapping quantitative trait loci (QTLs), and selecting candidate genes for NUE improvement are described. In addition, we highlight the importance of different factors that lead to changes in the NUE components of nitrogen uptake efficiency (NUpE) and nitrogen utilization efficiency (NUtE).

Over-application of nitrogen fertilizer in fields has had a negative impact on both environment and human health. Domesticated rice varieties with high nitrogen use efficiency (NUE) reduce fertilizer for sustainable agriculture. Here, we perform genome-wide association analysis of a diverse rice population displaying extreme nitrogen-related phenotypes over three successive years in the field, and identify an elite haplotype of nitrate transporter OsNPF6.1 that enhances nitrate uptake and confers high NUE by increasing yield under low nitrogen supply. OsNPF6.1 differs in both the protein and promoter element with natural variations, which are differentially trans-activated by OsNAC42, a NUE-related transcription factor. The rare natural allele OsNPF6.1, derived from variation in wild rice and selected for enhancing both NUE and yield, has been lost in 90.3% of rice varieties due to the increased application of fertilizer. Our discovery highlights this NAC42-NPF6.1 signaling cascade as a strategy for high NUE and yield breeding in rice.

The indica and japonica rice (Oryza sativa) subspecies differ in nitrate (NO) assimilation capacity and nitrogen (N) use efficiency (NUE). Here, we show that a major component of this difference is conferred by allelic variation at OsNR2, a gene encoding a NADH/NADPH-dependent NO reductase (NR). Selection-driven allelic divergence has resulted in variant indica and japonica OsNR2 alleles encoding structurally distinct OsNR2 proteins, with indica OsNR2 exhibiting greater NR activity. Indica OsNR2 also promotes NO uptake via feed-forward interaction with OsNRT1.1B, a gene encoding a NO uptake transporter. These properties enable indica OsNR2 to confer increased effective tiller number, grain yield and NUE on japonica rice, effects enhanced by interaction with an additionally introgressed indica OsNRT1.1B allele. In consequence, indica OsNR2 provides an important breeding resource for the sustainable increases in japonica rice yields necessary for future global food security.

The drive toward more sustainable agriculture has raised the profile of crop plant nutrient-use efficiency. Here we show that a major rice nitrogen-use efficiency quantitative trait locus (qNGR9) is synonymous with the previously identified gene DEP1 (DENSE AND ERECT PANICLES 1). The different DEP1 alleles confer different nitrogen responses, and genetic diversity analysis suggests that DEP1 has been subjected to artificial selection during Oryza sativa spp. japonica rice domestication. The plants carrying the dominant dep1-1 allele exhibit nitrogen-insensitive vegetative growth coupled with increased nitrogen uptake and assimilation, resulting in improved harvest index and grain yield at moderate levels of nitrogen fertilization. The DEP1 protein interacts in vivo with both the Gα (RGA1) and Gβ (RGB1) subunits, and reduced RGA1 or enhanced RGB1 activity inhibits nitrogen responses. We conclude that the plant G protein complex regulates nitrogen signaling and modulation of heterotrimeric G protein activity provides a strategy for environmentally sustainable increases in rice grain yield.

The major source of nitrogen for rice (Oryza sativa L.) is ammonium (NH4(+)). The NH4(+) uptake of roots is mainly governed by membrane transporters, with OsAMT1;1 being a prominent member of the OsAMT1 gene family that is known to be involved in NH4(+) transport in rice plants. However, little is known about its involvement in NH4(+) uptake in rice roots and subsequent effects on NH4(+) assimilation. This study shows that OsAMT1;1 is a constitutively expressed, nitrogen-responsive gene, and its protein product is localized in the plasma membrane. Its expression level is under the control of circadian rhythm. Transgenic rice lines (L-2 and L-3) overexpressing the OsAMT1;1 gene had the same root structure as the wild type (WT). However, they had 2-fold greater NH4(+) permeability than the WT, whereas OsAMT1;1 gene expression was 20-fold higher than in the WT. Analogous to the expression, transgenic lines had a higher NH4(+) content in the shoots and roots than the WT. Direct NH4(+) fluxes in the xylem showed that the transgenic lines had significantly greater uptake rates than the WT. Higher NH4(+) contents also promoted higher expression levels of genes in the nitrogen assimilation pathway, resulting in greater nitrogen assimilates, chlorophyll, starch, sugars, and grain yield in transgenic lines than in the WT under suboptimal and optimal nitrogen conditions. OsAMT1;1 also enhanced overall plant growth, especially under suboptimal NH4(+) levels. These results suggest that OsAMT1;1 has the potential for improving nitrogen use efficiency, plant growth, and grain yield under both suboptimal and optimal nitrogen fertilizer conditions.

Rice (Oryza sativa) grown in paddy fields is an ammonium (NH)-preferring crop; however, its AMT-type NH transporters that mediate root N acquisition have not been well characterized yet. In this study, we analyzed the expression pattern and physiological function of the OsAMT1.1 gene of the AMT1 subfamily in rice. OsAMT1.1 is located in the plasma membrane and is mainly expressed in the root epidermis, stele and mesophyll cells. Disruption of the OsAMT1.1 gene decreased the uptake of NH, and the growth of roots and shoots under both low NH and high NH conditions. OsAMT1.1 contributed to the short-term (5 min) NH influx rate by approximately one-quarter, irrespective of the NH concentration. Knockout of OsAMT1.1 significantly decreased the total N transport from roots to shoots under low NH conditions. Moreover, compared with the wild type, the osamt1.1 mutant showed an increase in the potassium (K) absorption rate under high NH conditions and a decrease under low NH conditions. The mutants contained a significantly high concentration of K in both the roots and shoots at a limited K (0.1 mmol/L) supply when NH was replete. Taken together, the results indicated that OsAMT1.1 significantly contributes to the NH uptake under both low and high NH conditions and plays an important role in N-K homeostasis in rice.Copyright © 2016 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Ltd. All rights reserved.

Sheath blight (ShB) significantly threatens rice yield production. However, the underlying mechanism of ShB defence in rice remains largely unknown. Here, we identified a highly ShB-susceptible mutant Ds-m which contained a mutation at the ammonium transporter 1;1 (AMT1;1) D N. AMT1;1 D N interacts with AMT1;1, AMT1;2 and AMT1;3 to inhibit the ammonium transport activity. The AMT1 RNAi was more susceptible and similar to the AMT1;1 D N mutant; however, plants with higher NH uptake activity were less susceptible to ShB. Glutamine synthetase 1;1 (GS1;1) mutant gs1;1 and overexpressors (GS1;1 OXs) were more and less susceptible to ShB respectively. Furthermore, AMT1;1 overexpressor (AMT1;1 OX)/gs1;1 and gs1;1 exhibited a similar response to ShB, suggesting that ammonium assimilation rather than accumulation controls the ShB defence. Genetic and physiological assays further demonstrated that plants with higher amino acid or chlorophyll content promoted rice resistance to ShB. Interestingly, the expression of ethylene-related genes was higher in AMT1;1 OX and lower in RNAi mutants than in wild-type. Also, ethylene signalling positively regulated rice resistance to ShB and NH uptake, suggesting that ethylene signalling acts downstream of AMT and also NH uptake is under feedback control. Taken together, our data demonstrated that the AMT1 promotes rice resistance to ShB via the regulation of diverse metabolic and signalling pathways.© 2022 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Asian cultivated rice (Oryza sativa L.) consists of two main subspecies, indica and japonica. Indica has higher nitrate-absorption activity than japonica, but the molecular mechanisms underlying that activity remain elusive. Here we show that variation in a nitrate-transporter gene, NRT1.1B (OsNPF6.5), may contribute to this divergence in nitrate use. Phylogenetic analysis revealed that NRT1.1B diverges between indica and japonica. NRT1.1B-indica variation was associated with enhanced nitrate uptake and root-to-shoot transport and upregulated expression of nitrate-responsive genes. The selection signature of NRT1.1B-indica suggests that nitrate-use divergence occurred during rice domestication. Notably, field tests with near-isogenic and transgenic lines confirmed that the japonica variety carrying the NRT1.1B-indica allele had significantly improved grain yield and nitrogen-use efficiency (NUE) compared to the variety without that allele. Our results show that variation in NRT1.1B largely explains nitrate-use divergence between indica and japonica and that NRT1.1B-indica can potentially improve the NUE of japonica.

A large proportion of the nitrate (NO(3)(2-)) acquired by plants from soil is actively transported via members of the NRT families of NO(3)(2-) transporters. In Arabidopsis, the NRT1 family has eight functionally characterised members and predominantly comprises low-affinity transporters; the NRT2 family contains seven members which appear to be high-affinity transporters; and there are two NRT3 (NAR2) family members which are known to participate in high-affinity transport. A modified reciprocal best hit (RBH) approach was used to identify putative orthologues of the Arabidopsis NRT genes in the four fully sequenced grass genomes (maize, rice, sorghum, Brachypodium). We also included the poplar genome in our analysis to establish whether differences between Arabidopsis and the grasses may be generally applicable to monocots and dicots. Our analysis reveals fundamental differences between Arabidopsis and the grass species in the gene number and family structure of all three families of NRT transporters. All grass species possessed additional NRT1.1 orthologues and appear to lack NRT1.6/NRT1.7 orthologues. There is significant separation in the NRT2 phylogenetic tree between NRT2 genes from dicots and grass species. This indicates that determination of function of NRT2 genes in grass species will not be possible in cereals based simply on sequence homology to functionally characterised Arabidopsis NRT2 genes and that proper functional analysis will be required. Arabidopsis has a unique NRT3.2 gene which may be a fusion of the NRT3.1 and NRT3.2 genes present in all other species examined here. This work provides a framework for future analysis of NO(3)(2-) transporters and NO(3)(2-) transport in grass crop species.

The nitrate transport accessory protein OsNAR2 plays a critical role in root-growth responses to nitrate and nitrate acquisition in rice (). In this study, a pull-down assay combined with yeast two-hybrid and coimmunoprecipitation analyses revealed that OsNAR2.1 interacts with OsNIT1 and OsNIT2. Moreover, an in vitro nitrilase activity assay indicated that indole-3-acetonitrile (IAN) is hydrolyzed to indole-3-acetic acid (IAA) by OsNIT1, the activity of which was enhanced 3- to 4-fold by OsNIT2 and in excess of 5- to 8-fold by OsNAR2.1. Knockout (KO) of was accompanied by repressed expression of both and, whereas KO of and in the and mutant lines did not affect expression of or the root nitrate acquisition rate. and displayed decreased primary root length and lateral root density. Double KO of and caused further decreases in lateral root density under nitrate supply. Ammonium supply repressed expression whereas it upregulated and expression. Both and showed root growth hypersensitivity to external ammonium; however, less root growth sensitivity to external IAN, higher expression of three IAA-amido synthetase genes, and a lower rate of H-IAA movement toward the roots were observed. Taken together, we conclude that the interaction of OsNIT1 and OsNIT2 activated by OsNAR2.1 and nitrogen supply is essential for maintaining root growth possibly via altering the IAA ratio of free to conjugate forms and facilitating its transportation.© 2020 American Society of Plant Biologists. All Rights Reserved.

Root nitrate uptake is well known to adjust to the plant's nitrogen demand for growth. Long-distance transport and/or root storage pools are thought to provide negative feedback signals regulating root uptake. We have characterized a vascular specific nitrate transporter belonging to the high-affinity Nitrate Transporter2 (NRT2) family, OsNRT2.3a, in rice (Oryza sativa ssp. japonica 'Nipponbare'). Localization analyses using protoplast expression, in planta promoter-β-glucuronidase assay, and in situ hybridization showed that OsNRT2.3a was located in the plasma membrane and mainly expressed in xylem parenchyma cells of the stele of nitrate-supplied roots. Knockdown expression of OsNRT2.3a by RNA interference (RNAi) had impaired xylem loading of nitrate and decreased plant growth at low (0.5 mm) nitrate supply. In comparison with the wild type, the RNAi lines contained both nitrate and total nitrogen significantly higher in the roots and lower in the shoots. The short-term [(15)N]NO(3)(-) influx (5 min) in entire roots and NO(3)(-) fluxes in root surfaces showed that the knockdown of OsNRT2.3a in comparison with the wild type did not affect nitrate uptake by roots. The RNAi plants showed no significant changes in the expression of some root nitrate transporters (OsNRT2.3b, OsNRT2.4, and OsNAR2.1), but transcripts for nia1 (nitrate reductase) had increased and OsNRT2.1 and OsNRT2.2 had decreased when the plants were supplied with nitrate. Taken together, the data demonstrate that OsNRT2.3a plays a key role in long-distance nitrate transport from root to shoot at low nitrate supply level in rice.

Plant proteins belonging to the NPF (formerly NRT1/PTR) family are well represented in every genome and function in transporting a wide variety of substrates. In this study, we showed that rice OsNPF2.4 is located in the plasma membrane and is expressed mainly in the epidermis, xylem parenchyma, and phloem companion cells. Functional analysis in oocytes showed that OsNPF2.4 is a pH-dependent, low-affinity NO₃⁻ transporter. Short-term (¹⁵NO₃⁻) influx rate, long-term NO₃⁻ acquisition by root, and upward transfer from root to shoot were decreased by disruption of OsNPF2.4 and increased by OsNPF2.4 overexpression under high NO₃⁻ supply. Moreover, the redistribution of NO₃⁻ in the mutants in comparison with the wild type from the oldest leaf to other organs, particularly to N-starved roots, was dramatically changed. Knockout of OsNPF2.4 decreased rice growth and potassium (K) concentration in xylem sap, root, culm, and sheath, but increased the shoot:root ratio of tissue K under higher NO₃⁻. We conclude that OsNPF2.4 functions in acquisition and long-distance transport of NO₃⁻, and that altering its expression has an indirect effect on K recycling between the root and shoot.© The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Low availability of nitrogen (N) is often a major limiting factor to crop yield in most nutrient-poor soils. Arbuscular mycorrhizal (AM) fungi are beneficial symbionts of most land plants that enhance plant nutrient uptake, particularly of phosphate. A growing number of reports point to the substantially increased N accumulation in many mycorrhizal plants; however, the contribution of AM symbiosis to plant N nutrition and the mechanisms underlying the AM-mediated N acquisition are still in the early stages of being understood. Here, we report that inoculation with AM fungus remarkably promoted rice () growth and N acquisition, and about 42% of the overall N acquired by rice roots could be delivered via the symbiotic route under N-NO supply condition. Mycorrhizal colonization strongly induced expression of the putative nitrate transporter gene in rice roots, and its orthologs in and in OsNPF4.5 is exclusively expressed in the cells containing arbuscules and displayed a low-affinity NO transport activity when expressed in oocytes. Moreover, knockout of resulted in a 45% decrease in symbiotic N uptake and a significant reduction in arbuscule incidence when NO was supplied as an N source. Based on our results, we propose that the NPF4.5 plays a key role in mycorrhizal NO acquisition, a symbiotic N uptake route that might be highly conserved in gramineous species.Copyright © 2020 the Author(s). Published by PNAS.

Nitrogen-use efficiency of indica varieties of rice is superior to that of japonica varieties. We apply 16S ribosomal RNA gene profiling to characterize root microbiota of 68 indica and 27 japonica varieties grown in the field. We find that indica and japonica recruit distinct root microbiota. Notably, indica-enriched bacterial taxa are more diverse, and contain more genera with nitrogen metabolism functions, than japonica-enriched taxa. Using genetic approaches, we provide evidence that NRT1.1B, a rice nitrate transporter and sensor, is associated with the recruitment of a large proportion of indica-enriched bacteria. Metagenomic sequencing reveals that the ammonification process is less abundant in the root microbiome of the nrt1.1b mutant. We isolated 1,079 pure bacterial isolates from indica and japonica roots and derived synthetic communities (SynComs). Inoculation of IR24, an indica variety, with an indica-enriched SynCom improved rice growth in organic nitrogen conditions compared with a japonica-enriched SynCom. The links between plant genotype and root microbiota membership established in this study will inform breeding strategies to improve nitrogen use in crops.

Eight mutants of barley (Hordeum vulgare cv Maris Mink) lacking the chloroplast isozyme of glutamine synthetase (EC 6.3.1.2.) were isolated by their inability to grow under photorespiratory conditions. The cytoplasmic isozyme of glutamine synthetase was present in the leaves of all the mutants, with activities comparable to the wild-type (10-12 nanokatals per gram fresh weight). The mutant plants developed normally and were fully fertile under conditions that minimize photorespiration. In 1% O(2) the rate of CO(2) fixation in leaves of one of the mutants, RPr 83/32, was the same as the wild-type, but in air this rate declined to 60% of the wild-type after 30 minutes. During this time the ammonia concentration in leaves of the mutant rose from 1 to 50 micromoles per gram fresh weight. Such ammonia accumulation in air was found in all the mutant lines. In back-crosses with the parent line, F(1) plants were viable in air. In the F(2) generation, nonviability in air and the lack of chloroplast glutamine synthetase co-segregated, in both the lines tested. These two lines and four others proved to be allelic; we designate them gln 2a-f. The characteristics of these mutants conclusively demonstrate the major role of chloroplast glutamine synthetase in photorespiration and its associated nitrogen recycling.

The high affinity nitrate transport system (HATS) plays an important role in rice nitrogen acquisition because, even under flooded anaerobic cultivation when NH(4)(+) dominates, significant nitrification occurs on the root surface. In the rice genome, four NRT2 and two NAR2 genes encoding HATS components have been identified. One gene OsNRT2.3 was mRNA spliced into OsNRT2.3a and OsNRT2.3b and OsNAR2.1 interacts with OsNRT2.1/2.2 and OsNRT2.3a to provide nitrate uptake. Using promoter-GUS reporter plants and semi-quantitative RT-PCR analyses, it was observed that OsNAR2.1 was expressed mainly in the root epidermal cells, differently from the five OsNRT2 genes. OsNAR2.1, OsNRT2.1, OsNRT2.2, and OsNRT2.3a were up-regulated by nitrate and suppressed by NH(4)(+) and high root temperature (37 °C). Expression of all these genes was increased by light or external sugar supply. Root transcripts of OsNRT2.3b and OsNRT2.4 were much less abundant and not affected by temperature. Expression of OsNRT2.3b was insensitive to the form of N supply. Expression of OsNRT2.4 responded to changes in auxin supply unlike all the other NRT2 genes. A region from position -311 to -1, relative to the translation start site in the promoter region of OsNAR2.1, was found to contain the cis-element(s) necessary for the nitrate-, but not light- and sugar-dependent activation. However, it was difficult to define a conserved cis-element in the promoters of the nitrate-regulated OsNRT2/OsNAR2 genes. The results imply distinct physiological functions for each OsNRT2 transporter, and differential regulation pathways by N and C status.