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.

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.

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.

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.

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.

A Jasmonate-Activated MYC2-Dof2.1-MYC2 Transcriptional Loop Promotes Leaf Senescence in Arabidopsis.

DNA binding-with-one-finger (Dof) proteins are plant-specific transcription factors closely associated with a variety of physiological processes. Here, we show that the Dof protein family in Arabidopsis (Arabidopsis thaliana) functions in leaf senescence. Disruption of Dof2 1, a jasmonate (JA)-inducible gene, led to a marked reduction in promotion of leaf senescence and inhibition of root development as well as dark-induced and age-dependent leaf senescence, while overexpression of Dof2 1 promoted these processes. Additionally, the dof2 1 knockout mutant showed almost no change in the transcriptome in the absence of JA; in the presence of JA, expression of many senescence-associated genes, including MYC2, which encodes a central regulator of JA responses, was induced to a lesser extent in the dof2 1 mutant than in the wild type. Furthermore, direct activation of the MYC2 promoter by Dof2.1, along with the results of epistasis analysis, indicated that Dof2.1 enhances leaf senescence mainly by promoting MYC2 expression. Interestingly, MYC2 was also identified as a transcriptional activator responsible for JA-inducible expression of Dof2 1 Based on these results, we propose that Dof2.1 acts as an enhancer of JA-induced leaf senescence through the MYC2-Dof2.1-MYC2 feedforward transcriptional loop.

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.

Discovery of nitrate-CPK-NLP signalling in central nutrient-growth networks.

Nutrient signalling integrates and coordinates gene expression, metabolism and growth. However, its primary molecular mechanisms remain incompletely understood in plants and animals. Here we report unique Ca2+ signalling triggered by nitrate with live imaging of an ultrasensitive biosensor in Arabidopsis leaves and roots. A nitrate-sensitized and targeted functional genomic screen identifies subgroup III Ca2+-sensor protein kinases (CPKs) as master regulators that orchestrate primary nitrate responses. A chemical switch with the engineered mutant CPK10(M141G) circumvents embryo lethality and enables conditional analyses of cpk10 cpk30 cpk32 triple mutants to define comprehensive nitrate-associated regulatory and developmental programs. Nitrate-coupled CPK signalling phosphorylates conserved NIN-LIKE PROTEIN (NLP) transcription factors to specify the reprogramming of gene sets for downstream transcription factors, transporters, nitrogen assimilation, carbon/nitrogen metabolism, redox, signalling, hormones and proliferation. Conditional cpk10 cpk30 cpk32 and nlp7 mutants similarly impair nitrate-stimulated system-wide shoot growth and root establishment. The nutrient-coupled Ca2+ signalling network integrates transcriptome and cellular metabolism with shoot-root coordination and developmental plasticity in shaping organ biomass and architecture.

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.

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.

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.

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.

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.

Reduced Expression of APUM24, Encoding a Novel rRNA Processing Factor, Induces Sugar-Dependent Nucleolar Stress and Altered Sugar Responses in Arabidopsis thaliana.

Ribosome biogenesis is one of the most energy-consuming events in the cell and must therefore be coordinated with changes in cellular energy status. Here, we show that the sugar-inducible gene ARABIDOPSIS PUMILIO PROTEIN24 (APUM24) encodes a Pumilio homology domain-containing protein involved in pre-rRNA processing in Arabidopsis thaliana Null mutation of APUM24 resulted in aborted embryos due to abnormal gametogenesis and embryogenesis, whereas reduced expression of APUM24 caused several phenotypes characteristic of ribosome biogenesis or function-related mutants. APUM24 interacted with other pre-rRNA processing factors and a putative endonuclease for the removal of the internal transcribed spacer 2 (ITS2) of pre-rRNA in the nucleolus. The APUM24-containing complex also interacted with ITS2, and reduced APUM24 expression caused the overaccumulation of processing intermediates containing ITS2. Thus, APUM24 likely functions as an ITS2 removal-associated factor. Most importantly, the apum24 knockdown mutant was hypersensitive to highly concentrated sugar, and the mutant showed sugar-dependent overaccumulation of processing intermediates and nucleolar stress (changes in nucleolar size). Furthermore, reduced APUM24 expression diminished sugar-induced promotion of leaf and root growth. Hence, a breakdown in the coordinated expression of ribosome biogenesis-related genes with energy status may induce nucleolar stress and disturb proper sugar responses in Arabidopsis.

Repression of Nitrogen Starvation Responses by Members of the Arabidopsis GARP-Type Transcription Factor NIGT1/HRS1 Subfamily.

Nitrogen (N) is often a limiting nutrient whose availability determines plant growth and productivity. Because its availability is often low and/or not uniform over time and space in nature, plants respond to variations in N availability by altering uptake and recycling mechanisms, but the molecular mechanisms underlying how these responses are regulated are poorly understood. Here, we show that a group of GARP G2-like transcription factors, Arabidopsis thaliana NITRATE-INDUCIBLE, GARP-TYPE TRANSCRIPTIONAL REPRESSOR1/HYPERSENSITIVE TO LOW Pi-ELICITED PRIMARY ROOT SHORTENING1 proteins (NIGT1/HRS1s), are factors that bind to the promoter of the N starvation marker NRT2.4 and repress an array of N starvation-responsive genes under conditions of high N availability. Transient assays and expression analysis demonstrated that NIGT1/HRS1s are transcriptional repressors whose expression is regulated by N availability. We identified target genes of the NIGT1/HRS1s by genome-wide transcriptome analyses and found that they are significantly enriched in N starvation response-related genes, including N acquisition, recycling, remobilization, and signaling genes. Loss of NIGT1/HRS1s resulted in deregulation of N acquisition and accumulation. We propose that NIGT1/HRS1s are major regulators of N starvation responses that play an important role in optimizing N acquisition and utilization under fluctuating N conditions.

Light signalling-induced regulation of nutrient acquisition and utilisation in plants.

Light is the foremost regulator of plant growth and development, and the critical role of light signalling in the promotion of nutrient uptake and utilisation was clarified in recent decades. Recent studies with Arabidopsis demonstrated the molecular mechanisms underlying such promotive effects and uncovered the pivotal role of the transcription factor ELONGATED HYPOCOTYL5 (HY5) whose activity is under the control of multiple photoreceptors. Together with a recent finding that phytochrome B, one of photoreceptors, is activated in subterranean plant parts, the discovery that HY5 directly promotes the transcription of genes involved in nutrient uptake and utilisation, including several nitrogen and sulphur assimilation-related genes, expands our understanding of the ways in which light signalling effectively and co-ordinately modulates uptake and utilisation of multiple nutrients in plants. This review presents a summary of the current knowledge regarding light signalling-induced regulation of nutrient uptake and utilisation.

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.

The role of protein-protein interactions mediated by the PB1 domain of NLP transcription factors in nitrate-inducible gene expression.

BACKGROUND: NIN-LIKE PROTEIN (NLP) transcription factors are master regulators of nitrate-inducible gene expression in higher plants. NLP transcription factors contain a nitrate signal-responsive domain in the amino-terminal region, an RWP-RK-type DNA-binding domain in the middle, and a Phox and Bem1 (PB1) domain at the carboxy terminus. Although the PB1 domain of NLP transcription factors appears to mediate protein-protein interactions associated with nitrate-inducible gene expression in higher plants, its precise role in nitrate-inducible gene expression has not previously been characterized. RESULTS: Yeast two-hybrid assays with the PB1 domain of the Arabidopsis transcription factor NLP7 revealed NLP-NLP interactions that required the core amino acid residues (K867, D909, D911, and E913) within the PB1 domain. Consistent with previous speculation on redundant and overlapping functions between different Arabidopsis NLP transcription factors, NLP-NLP interactions were observed between a variety of combinations of different NLP transcription factors. Furthermore, a mutated form of NLP7 that harbored amino acid substitutions at K867, D909, D911, and E913 required a far higher level of expression than wild-type NLP7 to restore nitrate-responsive gene expression and growth of nlp6 nlp7-1 double mutants. Surprisingly, however, the ability to transactivate nitrate-responsive promoters in protoplast transient expression assays was similar between wild-type and mutant forms of NLP7, suggesting that the PB1 domain was not required for transcription from naked DNA. CONCLUSIONS: Protein-protein interactions mediated by the PB1 domain of NLP transcription factors are necessary for full induction of nitrate-dependent expression of target genes in planta. The PB1 domains of NLP transcription factors may act on gene expression from chromosomal DNA via homo- and hetero-oligomerization in the presence of nitrate.

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.

The Transcriptional Cascade in the Heat Stress Response of Arabidopsis Is Strictly Regulated at the Level of Transcription Factor Expression.

Group A1 heat shock transcription factors (HsfA1s) are the master regulators of the heat stress response (HSR) in plants. Upon heat shock, HsfA1s trigger a transcriptional cascade that is composed of many transcription factors. Despite the importance of HsfA1s and their downstream transcriptional cascade in the acquisition of thermotolerance in plants, the molecular basis of their activation remains poorly understood. Here, domain analysis of HsfA1d, one of several HsfA1s in Arabidopsis thaliana, demonstrated that the central region of HsfA1d is a key regulatory domain that represses HsfA1d transactivation activity through interaction with HEAT SHOCK PROTEIN70 (HSP70) and HSP90. We designated this region as the temperature-dependent repression (TDR) domain. We found that HSP70 dissociates from HsfA1d in response to heat shock and that the dissociation is likely regulated by an as yet unknown activation mechanism, such as HsfA1d phosphorylation. Overexpression of constitutively active HsfA1d that lacked the TDR domain induced expression of heat shock proteins in the absence of heat stress, thereby conferring potent thermotolerance on the overexpressors. However, transcriptome analysis of the overexpressors demonstrated that the constitutively active HsfA1d could not trigger the complete transcriptional cascade under normal conditions, thereby indicating that other factors are necessary to fully induce the HSR. These complex regulatory mechanisms related to the transcriptional cascade may enable plants to respond resiliently to various heat stress conditions.