Novel QTL for Lateral Root Density and Length Improve Phosphorus Uptake in Rice (Oryza sativa L.).

The rice root system consists of two types of lateral roots, indeterminate larger L-types capable of further branching, and determinate, short, unbranched S-types. L-type laterals correspond to the typical lateral roots of cereals whereas S-type laterals are unique to rice. Both types contribute to nutrient and water uptake and genotypic variation for density and length of these laterals could be exploited in rice improvement to enhance adaptations to nutrient and water-limited environments. Our objectives were to determine how best to screen for lateral root density and length and to identify markers linked to genotypic variation for these traits. Using different growing media showed that screening in nutrient solution exposed genotypic variation for S-type and L-type density, but only the lateral roots of soil-grown plants varied for their lengths. A QTL mapping population developed from parents contrasting for lateral root traits was grown in a low-P field, roots were sampled, scanned and density and length of lateral roots measured. One QTL each was detected for L-type density (LDC), S-type density on crown root (SDC), S-type density on L-type (SDL), S-type length on L-type (SLL), and crown root number (RNO). The QTL for LDC on chromosome 5 had a major effect, accounting for 46% of the phenotypic variation. This strong positive effect was confirmed in additional field experiments, showing that lines with the donor parent allele at qLDC5 had 50% higher LDC. Investigating the contribution of lateral root traits to P uptake using stepwise regressions indicated LDC and RNO were most influential, followed by SDL. Simulating effects of root trait differences conferred by the main QTL in a P uptake model confirmed that qLDC5 was most effective in improving P uptake followed by qRNO9 for RNO and qSDL9 for S-type lateral density on L-type laterals. Pyramiding qLDC5 with qRNO9 and qSDL9 would be possible given that trade-offs between traits were not detected. Phenotypic selection for the RNO trait during variety development would be feasible, however, the costs of doing so reliably for lateral root density traits is prohibitive and markers identified here therefore provide the first opportunity to incorporate such traits into a breeding program.

Magnesium supply alleviates iron toxicity-induced leaf bronzing in rice through exclusion and tissue-tolerance mechanisms.

Introduction: Iron (Fe) toxicity is a widespread nutritional disorder in lowland rice causing growth retardation and leaf symptoms referred to as leaf bronzing. It is partly caused by an imbalance of nutrients other than Fe and supply of these is known to mitigate the toxicity. But the physiological and molecular mechanisms involved are unknown. Methods: We investigated the effect of magnesium (Mg) on Fe toxicity tolerance in a field study in the Central Highlands of Madagascar and in hydroponic experiments with excess Fe (300 mg Fe L-1). An RNA-seq analysis was conducted in a hydroponic experiment to elucidate possible mechanisms underlying Mg effects. Results and discussion: Addition of Mg consistently decreased leaf bronzing under both field and hydroponic conditions, whereas potassium (K) addition caused minor effects. Plants treated with Mg tended to have smaller shoot Fe concentrations in the field, suggesting enhanced exclusion at the whole-plant level. However, analysis of multiple genotypes showed that Fe toxicity symptoms were also mitigated without a concomitant decrease of Fe concentration, suggesting that increased Mg supply confers tolerance at the tissue level. The hydroponic experiments also suggested that Mg mitigated leaf bronzing without significantly decreasing Fe concentration or oxidative stress as assessed by the content of malondialdehyde, a biomarker for oxidative stress. An RNA-seq analysis revealed that Mg induced more changes in leaves than roots. Subsequent cis-element analysis suggested that NAC transcription factor binding sites were enriched in genes induced by Fe toxicity in leaves. Addition of Mg caused non-significant enrichment of the same binding sites, suggesting that NAC family proteins may mediate the effect of Mg. This study provides clues for mitigating Fe toxicity-induced leaf bronzing in rice.

Transcription factor module NLP-NIGT1 fine-tunes NITRATE TRANSPORTER2.1 expression.

Arabidopsis (Arabidopsis thaliana) high-affinity NITRATE TRANSPORTER2.1 (NRT2.1) plays a dominant role in the uptake of nitrate, the most important nitrogen (N) source for most terrestrial plants. The nitrate-inducible expression of NRT2.1 is regulated by NIN-LIKE PROTEIN (NLP) family transcriptional activators and NITRATE-INDUCIBLE GARP-TYPE TRANSCRIPTIONAL REPRESSOR1 (NIGT1) family transcriptional repressors. Phosphorus (P) availability also affects the expression of NRT2.1 because the PHOSPHATE STARVATION RESPONSE1 transcriptional activator activates NIGT1 genes in P-deficient environments. Here, we show a biology-based mathematical understanding of the complex regulation of NRT2.1 expression by multiple transcription factors using 2 different approaches: a microplate-based assay for the real-time measurement of temporal changes in NRT2.1 promoter activity under different nutritional conditions, and an ordinary differential equation (ODE)-based mathematical modeling of the NLP- and NIGT1-regulated expression patterns of NRT2.1. Both approaches consistently reveal that NIGT1 stabilizes the amplitude of NRT2.1 expression under a wide range of nitrate concentrations. Furthermore, the ODE model suggests that parameters such as the synthesis rate of NIGT1 mRNA and NIGT1 proteins and the affinity of NIGT1 proteins for the NRT2.1 promoter substantially influence the temporal expression patterns of NRT2.1 in response to nitrate. These results suggest that the NLP-NIGT1 feedforward loop allows a precise control of nitrate uptake. Hence, this study paves the way for understanding the complex regulation of nutrient acquisition in plants, thus facilitating engineered nutrient uptake and plant response patterns using synthetic biology approaches.

In-frame mutation in rice TEOSINTE BRANCHED1 (OsTB1) improves productivity under phosphorus deficiency.

Tillering is an important trait in rice productivity. We introduced mutations into the coding region of rice TEOSINTE BRANCHED1 (OsTB1), which is a negative regulator of tillering, using CRISPR/Cas9. The frameshift mutants exhibited substantially enhanced tillering and produced 3.5 times more panicles than the non-mutated plants at maturity. This enhanced tillering resulted in increased spikelet number; however, grain yields did not increase due to substantially reduced filled grain rate and 1,000-grain weight. In contrast, in-frame mutations in OsTB1 had the effect of slightly increasing tiller numbers, and the in-frame mutants had 40% more panicles than non-mutated plants. The grain yield of in-frame mutants also did not increase on nutrient-rich soil; however, under phosphorus-deficient conditions, where tillering is constrained, the in-frame mutants gave a significantly higher grain yield than non-mutated plants due to higher spikelet number and maintained filled grain rate. Rice grassy tiller1 (OsGT1)/OsHox12, which is directly regulated by OsTB1 to suppress tillering, was moderately down-regulated in in-frame mutants, suggesting that OsTB1 with the in-frame mutation shows partial function of intact OsTB1 in regulating OsGT1/OsHox12. We propose that mildly enhanced tillering by in-frame mutation of OsTB1 can improve grain yield under low phosphorus conditions.

QTL mapping for early root and shoot vigor of upland rice (Oryza sativa L.) under P deficient field conditions in Japan and Madagascar.

Upland rice production is limited by the low phosphorus (P) availability of many highly weathered tropical soils and P deficiency is likely to become increasingly limiting in future drier climates because P mobility decreases sharply with soil moisture. Good seedling root development will be crucial to cope with the combined effects of low P and water availability. Upland rice genebank accession DJ123 was used as a donor for P efficiency and root vigor traits in a cross with inefficient local variety Nerica4 and a set of backcross lines were used to characterize the seedling stage response of upland rice to low P availability and to identify associated QTL in field trials in Japan and Madagascar. Ten QTL were detected for crown root number, root, shoot and total dry weight per plant in a highly P deficient field in Japan using the BC1F3 generation. Of these, qPef9 on chromosome 9 affected multiple traits, increasing root number, root weight and total biomass, whereas a neighboring QTL on chromosome 9 (qPef9-2) increased shoot biomass. Field trials with derived BC1F5 lines in a low-P field in Madagascar confirmed a highly influential region on chromosome 9. However, qPef9-2 appeared more influential than qPef9, as the shoot and root biomass contrast between lines carrying DJ123 or Nerica4 alleles at qPef9-2 was +23.8% and +13.5% compared to +19.2% and +14.4% at qPef9. This advantage increased further during the growing season, leading to 46% higher shoot biomass at the late vegetative stage. Results suggest an introgression between 8.0 and 12.9 Mb on chromosome 9 from P efficient donor DJ123 can improve plant performance under P-limited conditions. The QTL identified here have practical relevance because they were confirmed in the target genetic background of the local variety Nerica4 and can therefore be applied directly to improve its performance.

Comparative transcriptome analysis reveals a rapid response to phosphorus deficiency in a phosphorus-efficient rice genotype.

Phosphorus (P) is an essential plant nutrient. Most rice growing lands lack adequate P, requiring multiple P fertiliser applications to obtain expected yields. However, P fertiliser is environmentally damaging, and already unaffordable to the marginal farmers. This warrants developing P-efficient rice varieties that require less P to produce the expected yield. However, genetic factors underlying P-use efficiency (PUE) in rice remain elusive. Here, we conducted comparative transcriptome analysis using two rice varieties with contrasting PUE; a P-efficient landrace DJ123 and a P-inefficient modern cultivar IR64. We aimed to understand the transcriptomic responses in DJ123 that allow it to achieve a high PUE under low P conditions. Our results showed that both DJ123 and IR64 had replete tissue P concentrations after 48 h of P deprivation. Yet, DJ123 strongly responded to the external low P availability by inducing P starvation-inducible genes that included SPX2, PHO1, PAPs and SQDs, while these genes were not significantly induced in IR64. We envisage that the ability of DJ123 to rapidly respond to low P conditions might be the key to its high PUE. Our findings lay a valuable foundation in elucidating PUE mechanism in rice, thus will potentially contribute to developing P-efficient modern rice variety.

Below-ground plant-soil interactions affecting adaptations of rice to iron toxicity.

Iron toxicity is a major constraint to rice production, particularly in highly weathered soils of inland valleys in sub-Saharan Africa where the rice growing area is rapidly expanding. There is a wide variation in tolerance of iron toxicity in the rice germplasm. However, the introgression of tolerance traits into high-yielding germplasm has been slow owing to the complexity of the tolerance mechanisms and large genotype-by-environment effects. We review current understanding of tolerance mechanisms, particularly those involving below-ground plant-soil interactions. Until now these have been less studied than above-ground mechanisms. We cover processes in the rhizosphere linked to exclusion of toxic ferrous iron by oxidation, and resulting effects on the mobility of nutrient ions. We also cover the molecular physiology of below-ground processes controlling iron retention in roots and root-shoot transport, and also plant iron sensing. We conclude that future breeding programmes should be based on well-characterized molecular markers for iron toxicity tolerance traits. To successfully identify such markers, the complex tolerance response should be broken down into its components based on understanding of tolerance mechanisms, and tailored screening methods should be developed for individual mechanisms.

Corrigendum: QTL mapping for early root and shoot vigor of upland rice (Oryza sativa L.) under P deficient field conditions in Japan and Madagascar.

[This corrects the article DOI: 10.3389/fpls.2022.1017419.].

Environmental Control of Phosphorus Acquisition: A Piece of the Molecular Framework Underlying Nutritional Homeostasis.

Homeostasis of phosphorus (P), an essential macronutrient, is vital for plant growth under diverse environmental conditions. Although plants acquire P from the soil as inorganic phosphate (Pi), its availability is generally limited. Therefore, plants employ mechanisms involving various Pi transporters that facilitate efficient Pi uptake against a steep concentration gradient across the plant-soil interface. Among the different types of Pi transporters in plants, some members of the PHOSPHATE TRANSPORTER 1 (PHT1) family, present in the plasma membrane of root epidermal cells and root hairs, are chiefly responsible for Pi uptake from the rhizosphere. Therefore, accurate regulation of PHT1 expression is crucial for the maintenance of P homeostasis. Previous investigations positioned the Pi-dependent posttranslational regulation of PHOSPHATE STARVATION RESPONSE 1 (PHR1) transcription factor activity at the center of the regulatory mechanism controlling PHT1 expression and P homeostasis; however, recent studies indicate that several other factors also regulate the expression of PHT1 to modulate P acquisition and sustain P homeostasis against environmental fluctuations. Together with PHR1, several transcription factors that mediate the availability of other nutrients (such as nitrogen and zinc), light, and stress signals form an intricate transcriptional network to maintain P homeostasis under highly diverse environments. In this review, we summarize this intricate transcriptional network for the maintenance of P homeostasis under different environmental conditions, with a main focus on the mechanisms identified in Arabidopsis.

NIGT1 family proteins exhibit dual mode DNA recognition to regulate nutrient response-associated genes in Arabidopsis.

Fine-tuning of nutrient uptake and response is indispensable for maintenance of nutrient homeostasis in plants, but the details of underlying mechanisms remain to be elucidated. NITRATE-INDUCIBLE GARP-TYPE TRANSCRIPTIONAL REPRESSOR 1 (NIGT1) family proteins are plant-specific transcriptional repressors that function as an important hub in the nutrient signaling network associated with the acquisition and use of nitrogen and phosphorus. Here, by yeast two-hybrid assays, bimolecular fluorescence complementation assays, and biochemical analysis with recombinant proteins, we show that Arabidopsis NIGT1 family proteins form a dimer via the interaction mediated by a coiled-coil domain (CCD) in their N-terminal regions. Electrophoretic mobility shift assays defined that the NIGT1 dimer binds to two different motifs, 5'-GAATATTC-3' and 5'-GATTC-N38-GAATC-3', in target gene promoters. Unlike the dimer of wild-type NIGT1 family proteins, a mutant variant that could not dimerize due to amino acid substitutions within the CCD had lower specificity and affinity to DNA, thereby losing the ability to precisely regulate the expression of target genes. Thus, expressing the wild-type and mutant NIGT1 proteins in the nigt1 quadruple mutant differently modified NIGT1-regulated gene expression and responses towards nitrate and phosphate. These results suggest that the CCD-mediated dimerization confers dual mode DNA recognition to NIGT1 family proteins, which is necessary to make proper controls of their target genes and nutrient responses. Intriguingly, two 5'-GATTC-3' sequences are present in face-to-face orientation within the 5'-GATTC-N38-GAATC-3' sequence or its complementary one, while two 5'-ATTC-3' sequences are present in back-to-back orientation within the 5'-GAATATTC-3' or its complementary one. This finding suggests a unique mode of DNA binding by NIGT1 family proteins and may provide a hint as to why target sequences for some transcription factors cannot be clearly determined.

Genetic and physiological traits for internal phosphorus utilization efficiency in rice.

Phosphorus (P) is an essential macronutrient for plant growth and development. Phosphorus is usually applied as fertilizer obtained from rock phosphate which is a non-renewable resource. Therefore, developing rice varieties that can use P more efficiently is crucial. Here, we investigated genotypic differences in traits related to internal Phosphorus Utilization Efficiency (PUE) in five rice genotypes grown under P-deficient conditions. P-efficient rice genotypes showed higher total biomass. This was partly due to higher root biomass, which in turn relied on preferential allocation of P to roots in these genotypes. Changes in P content and tissue P concentrations were analyzed in individual leaves at different time points. Genotypes belonging to the high-PUE group responded more quickly to P starvation in terms of reducing leaf P concentrations and they were able to reduce these concentrations to a lower level compared to the low-PUE group. Changes in P concentrations were reflected in gene expression levels for genes involved in lipid remodeling. Sulfolipid (OsSQD2) and galactolipid (OsMGD and OsDGD) synthesis-related genes were generally induced due to P starvation with most pronounced up-regulation in OsDGD1 and OsMGD3, but patterns differed between genotypes. A significantly higher expression of OsDGD5 and OsMGD1 & 2 was detected in the youngest fully expanded leaf of the high-PUE genotype group, whereas expression levels were reversed in older leaves. This pattern would confirm that P efficient genotypes react faster to P starvation in terms of freeing P for redistribution to growing tissues and replacing phospholipids with galactolipids in younger leaves may contribute to this aspect.

Metabolomic markers and physiological adaptations for high phosphate utilization efficiency in rice.

Utilizing phosphate more efficiently is crucial for sustainable crop production. Highly efficient rice (Oryza sativa) cultivars have been identified and this study aims to identify metabolic markers associated with P utilization efficiency (PUE). P deficiency generally reduced leaf P concentrations and CO2 assimilation rates but efficient cultivars were reducing leaf P concentrations further than inefficient ones while maintaining similar CO2 assimilation rates. Adaptive changes in carbon metabolism were detected but equally in efficient and inefficient cultivar groups. Groups furthermore did not differ with respect to partial substitutions of phospholipids by sulfo- and galactolipids. Metabolites significantly more abundant in the efficient group, such as sinapate, benzoate and glucoronate, were related to antioxidant defence and may help alleviating oxidative stress caused by P deficiency. Sugar alcohols ribitol and threitol were another marker metabolite for higher phosphate efficiency as were several amino acids, especially threonine. Since these metabolites are not known to be associated with P deficiency, they may provide novel clues for the selection of more P efficient genotypes. In conclusion, metabolite signatures detected here were not related to phosphate metabolism but rather helped P efficient lines to keep vital processes functional under the adverse conditions of P starvation.

Gene regulatory network and its constituent transcription factors that control nitrogen-deficiency responses in rice.

Increase in the nitrogen (N)-use efficiency and optimization of N response in crop species are urgently needed. Although transcription factor-based genetic engineering is a promising approach for achieving these goals, transcription factors that play key roles in the response to N deficiency have not been studied extensively. Here, we performed RNA-seq analysis of root samples of 20 Asian rice (Oryza sativa) accessions with differential nutrient uptake. Data obtained from plants exposed to N-replete and N-deficient conditions were subjected to coexpression analysis and machine learning-based pathway inference to dissect the gene regulatory network required for the response to N deficiency. Four transcription factors, including members of the G2-like and bZIP families, were predicted to function as key regulators of gene transcription within the network in response to N deficiency. Cotransfection assays validated inferred novel regulatory pathways, and further analyses using genome-edited knockout lines suggested that these transcription factors are important for N-deficiency responses in planta. Many of the N deficiency-responsive genes, including those encoding key regulators within the network, were coordinately regulated by transcription factors belonging to different families. Transcription factors identified in this study could be valuable for the modification of N response and metabolism.

Nitrate-inducible NIGT1 proteins modulate phosphate uptake and starvation signalling via transcriptional regulation of SPX genes.

Nitrogen and phosphorus are two major soil nutrients required for plant growth. Because requirements of both these elements are interdependent, acquisition of one must be balanced with that of the other. However, the mechanism underlying this balanced acquisition remains unclear. Here, we show by in vivo luciferase imaging that the presence of nitrogen sources is a pre-requisite for strong activation of phosphate starvation responses. In addition, we also show that nitrate rather than ammonium is a potent modulator of phosphate starvation-induced gene expression. Furthermore, protoplast-based transient expression assay and chromatin immunoprecipitation assay demonstrate that NIGT1 GARP-type transcriptional repressors, which are encoded by nitrate-inducible genes, directly bind to and repress the promoters of genes encoding SPX proteins. Consistent with the role of SPX proteins in the suppression of the PHR1 transcriptional activator, the master regulator for phosphate starvation responses, nitrate-dependent enhancement of phosphate starvation responses, such as accumulation of anthocyanin and promotion of root hair growth and phosphate uptake, was less evident in the nigt1.1-nigt1.4 quadruple mutant. Consistently, NIGT1 overexpression alleviated the reduction in phosphate uptake under phosphate-replete conditions. We further reveal the intricate feedback regulations involving PHR1, NIGT1, and SPX family proteins in the phosphate starvation signalling network. Importantly, results of mutant protoplast-based assays and in planta analysis using NIGT1 overexpression in the spx1 spx2 double mutant indicated that the NIGT1-SPX-PHR cascade mediates nitrogen status-responsive regulation of phosphate uptake and starvation signalling. These findings uncover the mechanism underlying the balanced acquisition of nitrogen and phosphorus.

Enhanced ascorbate level improves multi-stress tolerance in a widely grown indica rice variety without compromising its agronomic characteristics.

A biotechnological approach was adopted for increasing foliar ascorbate levels as a strategy to adapt a widely grown high yielding rice variety to multiple abiotic stresses. The variety IR64 (Oryza sativa L. ssp. indica) was engineered to express the ascorbate biosynthesis gene GDP-L-galactose phosphorylase (AcGGP) from kiwifruit (Actinidia chinensis Planch.) under the control of a leaf-specific promoter of the Leaf Panicle 2 (LP2) gene. Transgene expression increased foliar ascorbate levels up to >2.5 fold but did not lead to any changes in morphological traits (seed yield, sterility rate, grain weight, and biomass) in non-stress conditions. We then hypothesized that enhanced foliar ascorbate would confer multi-stress tolerance. Indeed transgenic lines were more tolerant to salinity in terms of lipid peroxidation and foliar symptoms, and to drought in terms of lipid peroxidation and post-drought recovery (number of dead leaves). A significantly better performance in ozone stress was seen only when ozone coincided with salinity. However, no differences between transgenic lines and wild types occurred when plants were subjected to toxicities in redox-active transition metals, i.e. iron and manganese, although plants showed clear symptoms of oxidative stress. Moreover, no differential response to zinc deficiency was observed, because the background genotype IR64 was not sensitive to this stress. Taken together, our study helps to identify stress conditions that can be mitigated by enhancing foliar ascorbate levels, and therefore facilitates an adaptive breeding approach for multiple stresses that would not imply any yield penalty.

Perception, transduction, and integration of nitrogen and phosphorus nutritional signals in the transcriptional regulatory network in plants.

Nitrate and phosphate ions are major sources of nitrogen and phosphorus for plants. In addition to their vital roles as indispensable macronutrients, these ions function as signalling molecules and induce a variety of responses. Plants adapt to different levels of nutrients by altering their gene expression profile and subsequent physiological and morphological responses. Advances made in recent years have provided novel insights into plant nutrient sensing and modulation of gene expression. Key breakthroughs include elucidation of the mechanisms underlying post-translational regulation of NIN-LIKE PROTEIN (NLP) and PHOSPHATE STARVATION RESPONSE (PHR) family transcription factors, which function as master regulators of responses to nitrate and phosphate starvation, respectively. Determination of the mechanisms whereby these nutrient signals are integrated through NIGT1/HHO family proteins has likewise represented important progress. Further studies have revealed novel roles in nutrient signalling of transcription factors that have previously been shown to be associated with other signals, such as light and phytohormones. Nitrate and phosphate signals are thus transmitted through an intricate gene regulatory network with the help of various positive and negative transcriptional regulators. These complex regulatory patterns enable plants to integrate input signals from various environmental factors and trigger appropriate responses, as exemplified by the regulatory module involving NIGT1/HHO family proteins. These mechanisms collectively support nutrient homeostasis in plants.

Overexpression of a Brix Domain-Containing Ribosome Biogenesis Factor ARPF2 and its Interactor ARRS1 Causes Morphological Changes and Lifespan Extension in Arabidopsis thaliana.

The Brix domain is a conserved domain in several proteins involved in ribosome biogenesis in yeast and animals. In the Arabidopsis genome, six Brix domain-containing proteins are encoded; however, their molecular functions have not been fully characterized, as yet. Here we report the functional analysis of a Brix domain-containing protein, ARPF2, which is homologous to yeast Rpf2 that plays an essential role in ribosome biogenesis as a component of the 5S ribonucleoprotein particle. By phenotypic characterization of arpf2 mutants, histochemical GUS staining, and analysis using green fluorescence protein, we show that ARPF2 is an essential and ubiquitously expressed gene encoding a nucleolar protein. Co-immunoprecipitation and split-GFP-based bimolecular fluorescence complementation assays revealed that ARPF2 interacts with a protein named ARRS1, which is homologous to yeast Rrs1 that forms a complex with Rpf2 in yeast. Furthermore, the result of RNA immunoprecipitation assay indicated that ARPF2 interacts with 5S ribosomal RNA (rRNA) or the precursor of 5S rRNA, as well as with the internal transcribed spacer 2 in the precursors of 25S rRNA. Most intriguingly, we found that the overexpression of ARPF2 and ARRS1 leads to characteristic phenotypes, including short stem, abnormal leaf morphology, and long lifespan, in Arabidopsis. These results suggest that the function of Brix domain-containing ARPF2 protein in ribosome biogenesis is intimately associated with the growth and development in plants.

A NIGT1-centred transcriptional cascade regulates nitrate signalling and incorporates phosphorus starvation signals in Arabidopsis.

Nitrate is a nutrient signal that triggers complex regulation of transcriptional networks to modulate nutrient-dependent growth and development in plants. This includes time- and nitrate concentration-dependent regulation of nitrate-related gene expression. However, the underlying mechanisms remain poorly understood. Here we identify NIGT1 transcriptional repressors as negative regulators of the Arabidopsis NRT2.1 nitrate transporter gene, and show antagonistic regulation by NLP primary transcription factors for nitrate signalling and the NLP-NIGT1 transcriptional cascade-mediated repression. This antagonistic regulation provides a resolution to the complexity of nitrate-induced transcriptional regulations. Genome-wide analysis reveals that this mechanism is applicable to NRT2.1 and other genes involved in nitrate assimilation, hormone biosynthesis and transcription. Furthermore, the PHR1 master regulator of the phosphorus-starvation response also directly promotes expression of NIGT1 family genes, leading to reductions in nitrate uptake. NIGT1 repressors thus act in two transcriptional cascades, forming a direct link between phosphorus and nitrogen nutritional regulation.