Dof1.7 and NIGT1 transcription factors mediate multilayered transcriptional regulation for different expression patterns of NITRATE TRANSPORTER2 genes under nitrogen deficiency stress.

Elucidating the mechanisms regulating nitrogen (N) deficiency responses in plants is of great agricultural importance. Previous studies revealed that decreased expression of NITRATE-INDUCIBLE GARP-TYPE TRANSCRIPTIONAL REPRESSOR1 (NIGT1) transcriptional repressor genes upon N deficiency is involved in N deficiency-inducible gene expression in Arabidopsis thaliana. However, our knowledge of the mechanisms controlling N deficiency-induced changes in gene expression is still limited. Through the identification of Dof1.7 as a direct target of NIGT1 repressors and a novel N deficiency response-related transcriptional activator gene, we here show that NIGT1 and Dof1.7 transcription factors (TFs) differentially regulate N deficiency-inducible expression of three high-affinity nitrate transporter genes, NRT2.1, NRT2.4, and NRT2.5, which are responsible for most of the soil nitrate uptake activity of Arabidopsis plants under N-deficient conditions. Unlike NIGT1 repressors, which directly suppress NRT2.1, NRT2.4, and NRT2.5 under N-sufficient conditions, Dof1.7 directly activated only NRT2.5 but indirectly and moderately activated NRT2.1 and NRT2.4 under N-deficient conditions, probably by indirectly decreasing NIGT1 expression. Thus, Dof1.7 converted passive transcriptional activation into active and potent transcriptional activation, further differentially enhancing the expression of NRT2 genes. These findings clarify the mechanism underlying different expression patterns of NRT2 genes upon N deficiency, suggesting that time-dependent multilayered transcriptional regulation generates complicated expression patterns of N deficiency-inducible genes.

Role of GARP family transcription factors in the regulatory network for nitrogen and phosphorus acquisition.

The GARP (Golden2, ARR-B, Psr1) family proteins with a conserved DNA-binding domain, called the B-motif, are plant-specific transcription factors involved in the regulation of various physiological processes. The GARP family proteins are divided into members that function as monomeric transcription factors, and members that function as transcription factors in the dimeric form, owing to the presence of a coiled-coil dimerization domain. Recent studies revealed that the dimer-forming GARP family members, which are further divided into the PHR1 and NIGT1 subfamilies, play critical roles in the regulation of phosphorus (P) and nitrogen (N) acquisition. In this review, we present a general overview of the GARP family proteins and discuss how several members of the PHR1 and NIGT1 subfamilies are involved in the coordinated acquisition of P and N in response to changes in environmental nutrient conditions, while mainly focusing on the recent findings that enhance our knowledge of the roles of PHR1 and NIGT1 in phosphate starvation signaling and nitrate signaling.

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.

Development of a Manufacturing Process toward the Convergent Synthesis of the COVID-19 Antiviral Ensitrelvir.

We describe the development of the practical manufacturing of Ensitrelvir, which was discovered as a SARS-CoV-2 antiviral candidate. Scalable synthetic methods of indazole, 1,2,4-triazole and 1,3,5-triazinone structures were established, and convergent couplings of these fragments enabled the development of a concise and efficient scale-up process to Ensitrelvir. In this process, introducing a meta-cresolyl moiety successfully enhanced the stability of intermediates. Compared to the initial route at the early research and development stage, the overall yield of the longest linear sequence (6 steps) was improved by approximately 7-fold. Furthermore, 9 out of the 12 isolated intermediates were crystallized directly from each reaction mixture without any extractive workup (direct isolation). This led to an efficient and environmentally friendly manufacturing process that minimizes waste of organic solvents, reagents, and processing time. This practical process for manufacturing Ensitrelvir should contribute to protection against COVID-19.

Chloroplastic Sec14-like proteins modulate growth and phosphate deficiency responses in Arabidopsis and rice.

Phosphorus is an essential nutrient acquired from soil as phosphate (Pi), and its deficiency severely reduces plant growth and crop yield. Here, we show that single nucleotide polymorphisms (SNPs) at the PHOSPHATIDYLINOSITOL TRANSFER PROTEIN7 (AtPITP7) locus, which encodes a chloroplastic Sec14-like protein, are associated with genetic diversity regarding Pi uptake activity in Arabidopsis (Arabidopsis thaliana). Inactivation of AtPITP7 and its rice (Oryza sativa) homolog (OsPITP6) through T-DNA insertion and CRISPR/Cas9-mediated gene editing, respectively, decreased Pi uptake and plant growth, regardless of Pi availability. By contrast, overexpression of AtPITP7 and OsPITP6 enhanced Pi uptake and plant growth, especially under limited Pi supply. Importantly, overexpression of OsPITP6 increased the tiller number and grain yield in rice. Targeted metabolome analysis of glycerolipids in leaves and chloroplasts revealed that inactivation of OsPITP6 alters phospholipid contents, independent of Pi availability, diminishing the reduction in phospholipid content and increase in glycolipid content induced by Pi deficiency; meanwhile, overexpression of OsPITP6 enhanced Pi deficiency-induced metabolic alterations. Together with transcriptome analysis of ospitp6 rice plants and phenotypic analysis of grafted Arabidopsis chimeras, these results suggest that chloroplastic Sec14-like proteins play an essential role in growth modulations in response to changes in Pi availability, although their function is critical for plant growth under any Pi condition. The superior traits of OsPITP6-overexpressing rice plants also highlight the potential of OsPITP6 and its homologs in other crops as additional tools for improving Pi uptake and plant growth in low Pi environments.

Histone chaperone NUCLEOSOME ASSEMBLY PROTEIN 1 proteins affect plant growth under nitrogen deficient conditions in Arabidopsis thaliana.

Nitrogen (N) availability is one of the most important factors regulating plant metabolism and growth as it affects global gene expression profiles. Dynamic changes in chromatin structure, including histone modifications and nucleosome assembly/disassembly, have been extensively shown to regulate gene expression under various environmental stresses in plants. However, the involvement of chromatin related changes in plant nutrient responses has been demonstrated only in a few studies to date. In this study, we investigated the function of histone chaperone NUCLEOSOME ASSEMBLY PROTEIN1 (NAP1) proteins under N deficient conditions in Arabidopsis. In the nap1;1 nap1;2 nap1;3 triple mutant (m123-1), the expression of N-responsive marker genes and growth of lateral roots were decreased under N deficient conditions. In addition, the m123-1 plants showed a delay in N deficiency-induced leaf senescence. Taken together, these results suggest that NAP1s affect plant growth under N deficient conditions in Arabidopsis.

The Arabidopsis NLP7-HB52/54-VAR2 pathway modulates energy utilization in diverse light and nitrogen conditions.

In plants, nitrate is the dominant nitrogen (N) source and a critical nutrient signal regulating various physiological and developmental processes.1,2,3,4 Nitrate-responsive gene regulatory networks are widely believed to control growth, development, and life cycle in addition to N acquisition and utilization,1,2,3,4 and NIN-LIKE PROTEIN (NLP) transcriptional activators have been identified as the master regulators governing the networks.5,6,7 However, it remains to be elucidated how nitrate signaling regulates respective physiological and developmental processes. Here, we have identified a new nitrate-activated transcriptional cascade involved in chloroplast development and the maintenance of chloroplast function in Arabidopsis. This cascade consisting of NLP7 and two homeodomain-leucine zipper (HD-Zip) class I transcription factors, HOMEOBOX PROTEIN52 (HB52) and HB54,8,9 was responsible for nitrate- and light-dependent expression of VAR2 encoding the FtsH2 subunit of the chloroplast FtsH protease involved in the quality control of photodamaged thylakoid membrane proteins.10,11 Consistently, the nitrate-activated NLP7-HB52/54-VAR2 pathway underpinned photosynthetic light energy utilization, especially in high light environments. Furthermore, genetically enhancing the NLP7-HB52/54-VAR2 pathway resulted in improved light energy utilization under high light and low N conditions, a superior agronomic trait. These findings shed light on a new role of nitrate signaling and a novel mechanism for integrating information on N nutrient and light environments, providing a hint for enhancing the light energy utilization of plants in low N environments.

NIN-like protein 7 transcription factor is a plant nitrate sensor.

Nitrate is an essential nutrient and signaling molecule for plant growth. Plants sense intracellular nitrate to adjust their metabolic and growth responses. Here we identify the primary nitrate sensor in plants. We found that mutation of all seven Arabidopsis NIN-like protein (NLP) transcription factors abolished plants' primary nitrate responses and developmental programs. Analyses of NIN-NLP7 chimeras and nitrate binding revealed that NLP7 is derepressed upon nitrate perception via its amino terminus. A genetically encoded fluorescent split biosensor, mCitrine-NLP7, enabled visualization of single-cell nitrate dynamics in planta. The nitrate sensor domain of NLP7 resembles the bacterial nitrate sensor NreA. Substitutions of conserved residues in the ligand-binding pocket impaired the ability of nitrate-triggered NLP7 to control transcription, transport, metabolism, development, and biomass. We propose that NLP7 represents a nitrate sensor in land plants.

Arabidopsis nitrate-induced aspartate oxidase gene expression is necessary to maintain metabolic balance under nitrogen nutrient fluctuation.

Nitrate is a nutrient signal that regulates growth and development through NLP transcription factors in plants. Here we identify the L-aspartate oxidase gene (AO) necessary for de novo NAD+ biosynthesis as an NLP target in Arabidopsis. We investigated the physiological significance of nitrate-induced AO expression by expressing AO under the control of the mutant AO promoter lacking the NLP-binding site in the ao mutant. Despite morphological changes and severe reductions in fresh weight, the loss of nitrate-induced AO expression resulted in minimum effects on NAD(H) and NADP(H) contents, suggesting compensation of decreased de novo NAD+ biosynthesis by reducing the growth rate. Furthermore, metabolite profiling and transcriptome analysis revealed that the loss of nitrate-induced AO expression causes pronounced impacts on contents of TCA cycle- and urea cycle-related metabolites, gene expression profile, and their modifications in response to changes in the nitrogen nutrient condition. These results suggest that proper maintenance of metabolic balance requires the coordinated regulation of multiple metabolic pathways by NLP-mediated nitrate signaling in plants.

RWP-RK domain-containing transcription factors in the Viridiplantae: biology and phylogenetic relationships.

The RWP-RK protein family is a group of transcription factors containing the RWP-RK DNA-binding domain. This domain is an ancient motif that emerged before the establishment of the Viridiplantae-the green plants, consisting of green algae and land plants. The domain is mostly absent in other kingdoms but widely distributed in Viridiplantae. In green algae, a liverwort, and several angiosperms, RWP-RK proteins play essential roles in nitrogen responses and sexual reproduction-associated processes, which are seemingly unrelated phenomena but possibly interdependent in autotrophs. Consistent with related but diversified roles of the RWP-RK proteins in these organisms, the RWP-RK protein family appears to have expanded intensively, but independently, in the algal and land plant lineages. Thus, bryophyte RWP-RK proteins occupy a unique position in the evolutionary process of establishing the RWP-RK protein family. In this review, we summarize current knowledge of the RWP-RK protein family in the Viridiplantae, and discuss the significance of bryophyte RWP-RK proteins in clarifying the relationship between diversification in the RWP-RK protein family and procurement of sophisticated mechanisms for adaptation to the terrestrial environment.

Two independent cis-acting elements are required for the guard cell-specific expression of SCAP1, which is essential for late stomatal development.

Regulating the stomatal aperture to adapt to environmental changes is critical for plants as stomatal guard cells are responsible for gas exchange between plants and the atmosphere. We previously showed that a plant-specific DNA-binding with one finger (Dof)-type transcription factor, SCAP1, functions as a key regulator in the final stages of guard cell differentiation. In the present study, we performed deletion and gain-of-function analyses with the 5' flanking region of SCAP1 to identify the regulatory region controlling the guard cell-specific expression of SCAP1. The results revealed that two cis-acting elements, 5'-CACGAGA-3' and 5'-CACATGTTTCCC-3', are crucial for the guard cell-specific expression of SCAP1. Consistently, when an 80-bp promoter region including these two cis-elements was fused to a gene promoter that is not active in guard cells, it functioned as a promoter that directed gene expression in guard cells. Furthermore, the promoter region of HT1 encoding the central regulator of stomatal CO2 signaling was also found to contain a 5'-CACGAGA-3' sequence, which was confirmed to function as a cis-element necessary for guard cell-specific expression of HT1. These findings suggest the existence of a novel transcriptional regulatory mechanism that synchronously promotes the expression of multiple genes required for the stomatal maturation and function.

Ribosome biogenesis factor OLI2 and its interactor BRX1-2 are associated with morphogenesis and lifespan extension in Arabidopsis thaliana.

Mutations that reduce the expression of ribosomal proteins (RPs) or limit the activity of ribosome biogenesis-related factors frequently cause physiological and morphological changes in Arabidopsis. Arabidopsis OLI2/NOP2A, a homolog of yeast Nop2, encodes a nucleolar methyltransferase that is required for the maturation of the 25S ribosomal RNA of the 60S large ribosomal subunit. Mutant oli2 plants exhibit pointed leaves and shortened primary roots. In this study, detailed phenotypic analysis of oli2 mutant and OLI2 overexpressor lines revealed a range of phenotypes. Seeds produced by oli2 mutant and OLI2 overexpressor plants were lighter and heavier than wild-type seeds, respectively. Seeds of the oli2 mutant also showed delayed germination, whereas seeds from the OLI2 overexpressor lines germinated earlier than the wild type. The oli2 mutant also had fewer and shorter lateral roots than the wild type. The lateral root development phenotype in the oli2 mutant was similar to that of auxin-related mutants, but was not enhanced by exogenously supplied auxin. Furthermore, the oli2 mutant and OLI2 overexpressor lines were hypersensitive and less sensitive to high concentrations of sugar, respectively. Split-GFP-based bimolecular fluorescence complementation analysis revealed that OLI2 interacted with a nucleolar protein, BRX1-2, which is involved in rRNA processing for the large ribosomal subunit. Moreover, overexpression of OLI2 and BRX1-2 caused similar morphological changes, including extension of plant lifespans. These results suggest that the functions of OLI2 and its interactor BRX1-2 are intimately associated with a range of developmental events in Arabidopsis.

Nitrate-responsive NIN-like protein transcription factors perform unique and redundant roles in Arabidopsis.

Upon sensing nitrate, NODULE INCEPTION (NIN)-like protein (NLP) transcription factors alter gene expression to promote nitrate uptake and utilization. Of the nine NLPs in Arabidopsis, the physiological roles of only three NLPs (NLP6-NLP8) have been characterized to date. To evaluate the unique and redundant roles of Arabidopsis NLPs, we assessed the phenotypes of single and higher order nlp mutants. Unlike other nlp single mutants, nlp2 and nlp7 single mutants showed a reduction in shoot fresh weight when grown in the presence of nitrate as the sole nitrogen source, indicating that NLP2, like NLP7, plays a major role in vegetative growth. Interestingly, the growth defect of nlp7 recovered upon the supply of ammonium or glutamine, whereas that of nlp2 did not. Furthermore, complementation assays using chimeric constructs revealed that the coding sequence, but not the promoter region, of NLP genes was responsible for the differences between nlp2 and nlp7 single mutant phenotypes, suggesting differences in protein function. Importantly, nitrate utilization was almost completely abolished in the nlp septuple mutant (nlp2 nlp4 nlp5 nlp6 nlp7 nlp8 nlp9), suggesting that NLPs other than NLP2 and NLP7 also assist in the regulation of nitrate-inducible gene expression and nitrate-dependent promotion of vegetative growth in Arabidopsis.

Enhanced NRT1.1/NPF6.3 expression in shoots improves growth under nitrogen deficiency stress in Arabidopsis.

Identification of genes and their alleles capable of improving plant growth under low nitrogen (N) conditions is key for developing sustainable agriculture. Here, we show that a genome-wide association study using Arabidopsis thaliana accessions suggested an association between different magnitudes of N deficiency responses and diversity in NRT1.1/NPF6.3 that encodes a dual-affinity nitrate transporter involved in nitrate uptake by roots. Various analyses using accessions exhibiting reduced N deficiency responses revealed that enhanced NRT1.1 expression in shoots rather than in roots is responsible for better growth of Arabidopsis seedlings under N deficient conditions. Furthermore, polymorphisms that increased NRT1.1 promoter activity were identified in the NRT1.1 promoter sequences of the accessions analyzed. Hence, our data indicated that polymorphism-dependent activation of the NRT1.1 promoter in shoots could serve as a tool in molecular breeding programs for improving plant growth in low N environments.

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.

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.

Multilayered Regulation of Membrane-Bound ONAC054 Is Essential for Abscisic Acid-Induced Leaf Senescence in Rice.

In most plants, abscisic acid (ABA) induces premature leaf senescence; however, the mechanisms of ABA signaling during leaf senescence remain largely unknown. Here, we show that the rice (Oryza sativa) NAM/ATAF1/2/CUC2 (NAC) transcription factor ONAC054 plays an important role in ABA-induced leaf senescence. The onac054 knockout mutants maintained green leaves, while ONAC054-overexpressing lines showed early leaf yellowing under dark- and ABA-induced senescence conditions. Genome-wide microarray analysis showed that ABA signaling-associated genes, including ABA INSENSITIVE5 (OsABI5) and senescence-associated genes, including STAY-GREEN and NON-YELLOW COLORING1 (NYC1), were significantly down-regulated in onac054 mutants. Chromatin immunoprecipitation and protoplast transient assays showed that ONAC054 directly activates OsABI5 and NYC1 by binding to the mitochondrial dysfunction motif in their promoters. ONAC054 activity is regulated by proteolytic processing of the C-terminal transmembrane domain (TMD). We found that nuclear import of ONAC054 requires cleavage of the putative C-terminal TMD. Furthermore, the ONAC054 transcript (termed ONAC054α) has an alternatively spliced form (ONAC054ß), with seven nucleotides inserted between intron 5 and exon 6, truncating ONAC054α protein at a premature stop codon. ONAC054ß lacks the TMD and thus localizes to the nucleus. These findings demonstrate that the activity of ONAC054, which is important for ABA-induced leaf senescence in rice, is precisely controlled by multilayered regulatory processes.

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.