OsCBL1 mediates rice response to local nitrate signaling: insights into regulatory networks and gene expression.

Nitrate is a significant source of nitrogen in soils and also serves as a critical signal for root development. Previous studies have demonstrated that the local nitrate supply promotes lateral root elongation primarily through local nitrate signals, rather than nutritional effects. In this study, we report that Calcineurin B-like protein 1 (OsCBL1) positively regulates local nitrate signaling, thereby triggering lateral root colonization, as revealed by a comparative analysis of the phenotype and whole transcriptome of the knockdown mutant (OsCBL1-KD) and the wild-type (WT). In the split-root system, the knockdown of OsCBL1 was found to inhibit local nitrate-induced lateral root growth. Transcriptome analyses identified 398 differentially expressed genes (DEGs) that were under the control of OsCBL1 and associated with the phenotype of nitrate-induced lateral root colonization. Further analysis revealed that the nitrate transporter/sensor gene OsNRT1.1B was up-regulated under Sp-NaNO3 conditions compared to Sp-NaCl in WT but not in OsCBL1-KD plants. Pathway mapping of DEGs (i.e., genes exhibiting a significant change in expression in the Sp-NaNO3 condition compared to the Sp-NaCl condition) revealed a preferential upregulation of genes involved in lignin biosynthesis and a downregulation of genes involved in auxin and salicylic acid signaling. This suggests that OsCBL1 might function as a transmitter within the auxin, salicylic acid signaling, lignin biosynthesis, and nitrate sensor (OsNRT1.1B)-mediated pathways in response to local nitrate signaling. We also identified a transcriptional regulatory network downstream of OsCBL1 in nitrate-rich patches that is centered on several core transcription factors. Our study provides new insights into how plants adapt to an inhomogeneous distribution of nitrogen in the soil.

Nitrate Signaling and Its Role in Regulating Flowering Time in Arabidopsis thaliana.

Plant growth is coordinated with the availability of nutrients that ensure its development. Nitrate is a major source of nitrogen (N), an essential macronutrient for plant growth. It also acts as a signaling molecule to modulate gene expression, metabolism, and a variety of physiological processes. Recently, it has become evident that the calcium signal appears to be part of the nitrate signaling pathway. New key players have been discovered and described in Arabidopsis thaliana (Arabidopsis). In addition, knowledge of the molecular mechanisms of how N signaling affects growth and development, such as the nitrate control of the flowering process, is increasing rapidly. Here, we review recent advances in the identification of new components involved in nitrate signal transduction, summarize newly identified mechanisms of nitrate signaling-modulated flowering time in Arabidopsis, and suggest emerging concepts and existing open questions that will hopefully be informative for further discoveries.

Functional analyses of the NRT2 family of nitrate transporters in Arabidopsis.

Nitrogen is an essential macronutrient for plant growth and development. Nitrate is the major form of nitrogen acquired by most crops and also serves as a vital signaling molecule. Nitrate is absorbed from the soil into root cells usually by the low-affinity NRT1 NO3 - transporters and high-affinity NRT2 NO3 - transporters, with NRT2s serving to absorb NO3 - under NO3 -limiting conditions. Seven NRT2 members have been identified in Arabidopsis, and they have been shown to be involved in various biological processes. In this review, we summarize the spatiotemporal expression patterns, localization, and biotic and abiotic responses of these transporters with a focus on recent advances in the current understanding of the functions of the seven AtNRT2 genes. This review offers beneficial insight into the mechanisms by which plants adapt to changing environmental conditions and provides a theoretical basis for crop research in the near future.

Finding Balance in Adversity: Nitrate Signaling as the Key to Plant Growth, Resilience, and Stress Response.

To effectively adapt to changing environments, plants must maintain a delicate balance between growth and resistance or tolerance to various stresses. Nitrate, a significant inorganic nitrogen source in soils, not only acts as an essential nutrient but also functions as a critical signaling molecule that regulates multiple aspects of plant growth and development. In recent years, substantial advancements have been made in understanding nitrate sensing, calcium-dependent nitrate signal transmission, and nitrate-induced transcriptional cascades. Mounting evidence suggests that the primary response to nitrate is influenced by environmental conditions, while nitrate availability plays a pivotal role in stress tolerance responses. Therefore, this review aims to provide an overview of the transcriptional and post-transcriptional regulation of key components in the nitrate signaling pathway, namely, NRT1.1, NLP7, and CIPK23, under abiotic stresses. Additionally, we discuss the specificity of nitrate sensing and signaling as well as the involvement of epigenetic regulators. A comprehensive understanding of the integration between nitrate signaling transduction and abiotic stress responses is crucial for developing future crops with enhanced nitrogen-use efficiency and heightened resilience.

Nitric Oxide Is Required for Primary Nitrate Response in Arabidopsis: Evidence for S-Nitrosation of NLP7.

Aims: Nitrogen (N) is a necessary nutrient for plant development and seed production, with nitrate (NO3-) serving as the primary source of N in soils. Although several molecular players in plant responses to NO3- signaling were unraveled, it is still a complex process with gaps that require further investigation. The aim of our study is to analyze the role of nitric oxide (NO) in the primary nitrate response (PNR). Results: Using a combination of genetic and pharmacological approaches, we demonstrate that NO is required for the expression of the NO3--regulated genes nitrate reductase 1 (NIA1), nitrite reductase (NIR), and nitrate transporters (nitrate transporter 1.1 [NRT1.1] and nitrate transporter 2.1 [NRT2.1]) in Arabidopsis. The PNR is impaired in the Arabidopsis mutant noa1, defective in NO production. Our results also show that PHYTOGLOBIN 1 (PHYTOGLB1), involved in NO homeostasis, is rapidly induced during PNR in wild type (wt) but not in the mutants of the nitrate transceptor NTR1.1 and the transcription factor nodule inception-like protein 7 (NLP7), suggesting that the NRT1.1-NLP7 cascade modulates PHYTOGLB1 gene expression. Biotin switch experiments demonstrate that NLP7, the PNR-master regulator, is S-nitrosated in vitro. Depletion of NO during PNR intensifies the decrease in reactive oxygen species levels and the rise of catalase (CAT) and ascorbate peroxidase (APX) enzyme activity. Conclusion and Innovation: NO, a by-product of NO3- metabolism and a well-characterized signal molecule in plants, is an important player in the PNR.

The Arabidopsis eIF4E1 regulates NRT1.1-mediated nitrate signaling at both translational and transcriptional levels.

Identifying new nitrate regulatory genes and illustrating their mechanisms in modulating nitrate signaling are of great significance for achieving the high yield and nitrogen use efficiency (NUE) of crops. Here, we screened a mutant with defects in nitrate response and mapped the mutation to the gene eIF4E1 in Arabidopsis. Our results showed that eIF4E1 regulated nitrate signaling and metabolism. Ribo-seq and polysome profiling analysis revealed that eIF4E1 modulated the amount of some nitrogen (N)-related mRNAs being translated, especially the mRNA of NRT1.1 was reduced in the eif4e1 mutant. RNA-Seq results enriched some N-related genes, supporting that eIF4E1 is involved in nitrate regulation. The genetic analysis indicated that eIF4E1 worked upstream of NRT1.1 in nitrate signaling. In addition, an eIF4E1-interacting protein GEMIN2 was identified and found to be involved in nitrate signaling. Further investigation showed that overexpression of eIF4E1 promoted plant growth and enhanced yield and NUE. These results demonstrate that eIF4E1 regulates nitrate signaling by modulating NRT1.1 at both translational and transcriptional levels, laying the foundation for future research on the regulation of mineral nutrition at the translational level.

Nuclear translocation of OsMADS25 facilitated by OsNAR2.1 in reponse to nitrate signals promotes rice root growth by targeting OsMADS27 and OsARF7.

Nitrate is an important nitrogen source and signaling molecule that regulates plant growth and development. Although several components of the nitrate signaling pathway have been identified, the detailed mechanisms are still unclear. Our previous results showed that OsMADS25 can regulate root development in response to nitrate signals, but the mechanism is still unknown. Here, we try to answer two key questions: how does OsMADS25 move from the cytoplasm to the nucleus, and what are the direct target genes activated by OsMADS25 to regulate root growth after it moves to the nucleus in response to nitrate? Our results demonstrated that OsMADS25 moves from the cytoplasm to the nucleus in the presence of nitrate in an OsNAR2.1-dependent manner. Chromatin immunoprecipitation sequencing, chromatin immunoprecipitation qPCR, yeast one-hybrid, and luciferase experiments showed that OsMADS25 directly activates the expression of OsMADS27 and OsARF7, which are reported to be associated with root growth. Finally, OsMADS25-RNAi lines, the Osnar2.1 mutant, and OsMADS25-RNAi Osnar2.1 lines exhibited significantly reduced root growth compared with the wild type in response to nitrate supply, and expression of OsMADS27 and OsARF7 was significantly suppressed in these lines. Collectively, these results reveal a new mechanism by which OsMADS25 interacts with OsNAR2.1. This interaction is required for nuclear accumulation of OsMADS25, which promotes OsMADS27 and OsARF7 expression and root growth in a nitrate-dependent manner.

Genome-wide identification of BcGRF genes in flowering Chinese cabbage and preliminary functional analysis of BcGRF8 in nitrogen metabolism.

Growth-regulating factors (GRFs) are a unique family of transcription factors with well-characterized functions in plant growth and development. However, few studies have evaluated their roles in the absorption and assimilation of nitrate. In this study, we characterized the GRF family genes of flowering Chinese cabbage (Brassica campestris), an important vegetable crop in South China. Using bioinformatics methods, we identified BcGRF genes and analyzed their evolutionary relationships, conserved motifs, and sequence characteristics. Through genome-wide analysis, we identified 17 BcGRF genes distributed on seven chromosomes. A phylogenetic analysis revealed that the BcGRF genes could be categorized into five subfamilies. RT-qPCR analysis showed that BcGRF1, 8, 10, and 17 expression clearly increased in response to nitrogen (N) deficiency, particularly at 8 h after treatment. BcGRF8 expression was the most sensitive to N deficiency and was significantly correlated with the expression patterns of most key genes related to N metabolism. Using yeast one-hybrid and dual-luciferase assays, we discovered that BcGRF8 strongly enhances the driving activity of the BcNRT1.1 gene promoter. Next, we investigated the molecular mechanism by which BcGRF8 participates in nitrate assimilation and N signaling pathways by expressing it in Arabidopsis. BcGRF8 was localized in the cell nucleus and BcGRF8 overexpression significantly increased the shoot and root fresh weights, seedling root length, and lateral root number in Arabidopsis. In addition, BcGRF8 overexpression considerably reduced the nitrate contents under both nitrate-poor and -rich conditions in Arabidopsis. Finally, we found that BcGRF8 broadly regulates genes related to N uptake, utilization, and signaling. Our results demonstrate that BcGRF8 substantially accelerates plant growth and nitrate assimilation under both nitrate-poor and -rich conditions by increasing the number of lateral roots and the expression of genes involved in N uptake and assimilation, providing a basis for crop improvement.

A molecular framework underlying low-nitrogen-induced early leaf senescence in Arabidopsis thaliana.

Nitrogen (N) deficiency causes early leaf senescence, resulting in accelerated whole-plant maturation and severely reduced crop yield. However, the molecular mechanisms underlying N-deficiency-induced early leaf senescence remain unclear, even in the model species Arabidopsis thaliana. In this study, we identified Growth, Development and Splicing 1 (GDS1), a previously reported transcription factor, as a new regulator of nitrate (NO3-) signaling by a yeast-one-hybrid screen using a NO3- enhancer fragment from the promoter of NRT2.1. We showed that GDS1 promotes NO3- signaling, absorption and assimilation by affecting the expression of multiple NO3- regulatory genes, including Nitrate Regulatory Gene2 (NRG2). Interestingly, we observed that gds1 mutants show early leaf senescence as well as reduced NO3- content and N uptake under N-deficient conditions. Further analyses indicated that GDS1 binds to the promoters of several senescence-related genes, including Phytochrome-Interacting Transcription Factors 4 and 5 (PIF4 and PIF5) and represses their expression. Interestingly, we found that N deficiency decreases GDS1 protein accumulation, and GDS1 could interact with Anaphase Promoting Complex Subunit 10 (APC10). Genetic and biochemical experiments demonstrated that Anaphase Promoting Complex or Cyclosome (APC/C) promotes the ubiquitination and degradation of GDS1 under N deficiency, resulting in loss of PIF4 and PIF5 repression and consequent early leaf senescence. Furthermore, we discovered that overexpression of GDS1 could delay leaf senescence and improve seed yield and N-use efficiency (NUE) in Arabidopsis. In summary, our study uncovers a molecular framework illustrating a new mechanism underlying low-N-induced early leaf senescence and provides potential targets for genetic improvement of crop varieties with increased yield and NUE.

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.

Nitrate signaling and use efficiency in crops.

Nitrate (NO3-) is not only an essential nutrient but also an important signaling molecule for plant growth. Low nitrogen use efficiency (NUE) of crops is causing increasingly serious environmental and ecological problems. Understanding the molecular mechanisms of NO3- regulation in crops is crucial for NUE improvement in agriculture. During the last several years, significant progress has been made in understanding the regulation of NO3- signaling in crops, and some key NO3- signaling factors have been shown to play important roles in NO3- utilization. However, no detailed reviews have yet summarized these advances. Here, we focus mainly on recent advances in crop NO3- signaling, including short-term signaling, long-term signaling, and the impact of environmental factors. We also review the regulation of crop NUE by crucial genes involved in NO3- signaling. This review provides useful information for further research on NO3- signaling in crops and a theoretical basis for breeding new crop varieties with high NUE, which has great significance for sustainable agriculture.

The Nitrate Transporter MtNPF6.8 Is a Master Sensor of Nitrate Signal in the Primary Root Tip of Medicago truncatula.

Nitrate is not only an essential nutrient for plants, but also a signal involved in plant development. We have previously shown in the model legume Medicago truncatula, that the nitrate signal, which restricts primary root growth, is mediated by MtNPF6.8, a nitrate transporter. Nitrate signal also induces changes in reactive oxygen species accumulation in the root tip due to changes in cell wall peroxidase (PODs) activity. Thus, it was interesting to determine the importance of the role of MtNPF6.8 in the regulation of the root growth by nitrate and identify the POD isoforms responsible for the changes in POD activity. For this purpose, we compared in M. truncatula a npf6.8 mutant and nitrate insensitive line deficient in MtNPF6.8 and the corresponding wild and sensitive genotype for their transcriptomic and proteomic responses to nitrate. Interestingly, only 13 transcripts and no protein were differently accumulated in the primary root tip of the npf6.8-3 mutant line in response to nitrate. The sensitivity of the primary root tip to nitrate appeared therefore to be strongly linked to the integrity of MtNPF6.8 which acts as a master mediator of the nitrate signal involved in the control of the root system architecture. In parallel, 7,259 and 493 genes responded, respectively, at the level of transcripts or proteins in the wild type, 196 genes being identified by both their transcript and protein. By focusing on these 196 genes, a concordance of expression was observed for most of them with 143 genes being up-regulated and 51 being down-regulated at the two gene expression levels. Their ontology analysis uncovered a high enrichment in POD genes, allowing the identification of POD candidates involved in the changes in POD activity previously observed in response to nitrate.

Spatiotemporal analysis identifies ABF2 and ABF3 as key hubs of endodermal response to nitrate.

Nitrate is a nutrient and a potent signal that impacts global gene expression in plants. However, the regulatory factors controlling temporal and cell type-specific nitrate responses remain largely unknown. We assayed nitrate-responsive transcriptome changes in five major root cell types of the Arabidopsis thaliana root as a function of time. We found that gene-expression response to nitrate is dynamic and highly localized and predicted cell type-specific transcription factor (TF)-target interactions. Among cell types, the endodermis stands out as having the largest and most connected nitrate-regulatory gene network. ABF2 and ABF3 are major hubs for transcriptional responses in the endodermis cell layer. We experimentally validated TF-target interactions for ABF2 and ABF3 by chromatin immunoprecipitation followed by sequencing and a cell-based system to detect TF regulation genome-wide. Validated targets of ABF2 and ABF3 account for more than 50% of the nitrate-responsive transcriptome in the endodermis. Moreover, ABF2 and ABF3 are involved in nitrate-induced lateral root growth. Our approach offers an unprecedented spatiotemporal resolution of the root response to nitrate and identifies important components of cell-specific gene regulatory networks.

Barley transcription factor HvNLP2 mediates nitrate signaling and affects nitrogen use efficiency.

Plants have evolved complex mechanisms to adapt to the changing nitrogen levels in the environment. In Arabidopsis, more than a dozen nitrate signaling regulatory genes have been characterized, including the NODULE INCEPTION-LIKE PROTEIN (AtNLP) genes, which play essential roles in nitrate signaling. However, whether NLP genes in the Triticeae crops are involved in nitrate regulation and nitrogen use efficiency (NUE) remains unknown. Here, we isolated a barley (Hordeum vulgare L.) mutant, hvnlp2-1, from a TILLING (Targeting Local Lesions IN Genomes) population and constructed two RNAi lines, hvnlp2-2 and hvnlp2-3, to study the function of HvNLP2. The expression of the nitrate-responsive genes was substantially inhibited after nitrate treatment in the hvnlp2 mutants, indicating that HvNLP2 controls nitrate signaling. Nitrate content was significantly higher in the hvnlp2 mutants, which may result from the decreased assimilation of nitrogen caused by reduced nitrate reductase activity and expression of nitrate assimilatory genes. HvNLP2 is localized to the nucleus in the presence of nitrate. Further investigation showed that HvNLP2 binds to and activates the nitrate-responsive cis-elements. Moreover, hvnlp2 exhibited reduced biomass, seed yield, and NUE. Therefore, HvNLP2 controls nitrate signaling and plays an important role in NUE.

Role of Auxin and Nitrate Signaling in the Development of Root System Architecture.

The plant root is an important storage organ that stores indole-3-acetic acid (IAA) from the apical meristem, as well as nitrogen, which is obtained from the external environment. IAA and nitrogen act as signaling molecules that promote root growth to obtain further resources. Fluctuations in the distribution of nitrogen in the soil environment induce plants to develop a set of strategies that effectively improve nitrogen use efficiency. Auxin integrates the information regarding the nitrate status inside and outside the plant body to reasonably distribute resources and sustainably construct the plant root system. In this review, we focus on the main factors involved in the process of nitrate- and auxin-mediated regulation of root structure to better understand how the root system integrates the internal and external information and how this information is utilized to modify the root system architecture.

Dynamic Nutrient Signaling Networks in Plants.

Nutrients are vital to life through intertwined sensing, signaling, and metabolic processes. Emerging research focuses on how distinct nutrient signaling networks integrate and coordinate gene expression, metabolism, growth, and survival. We review the multifaceted roles of sugars, nitrate, and phosphate as essential plant nutrients in controlling complex molecular and cellular mechanisms of dynamic signaling networks. Key advances in central sugar and energy signaling mechanisms mediated by the evolutionarily conserved master regulators HEXOKINASE1 (HXK1), TARGET OF RAPAMYCIN (TOR), and SNF1-RELATED PROTEIN KINASE1 (SNRK1) are discussed. Significant progress in primary nitrate sensing, calcium signaling, transcriptome analysis, and root-shoot communication to shape plant biomass and architecture are elaborated. Discoveries on intracellular and extracellular phosphate signaling and the intimate connections with nitrate and sugar signaling are examined. This review highlights the dynamic nutrient, energy, growth, and stress signaling networks that orchestrate systemwide transcriptional, translational, and metabolic reprogramming, modulate growth and developmental programs, and respond to environmental cues.

Nitrate triggered phosphoproteome changes and a PIN2 phosphosite modulating root system architecture.

Nitrate commands genome-wide gene expression changes that impact metabolism, physiology, plant growth, and development. In an effort to identify new components involved in nitrate responses in plants, we analyze the Arabidopsis thaliana root phosphoproteome in response to nitrate treatments via liquid chromatography coupled to tandem mass spectrometry. 176 phosphoproteins show significant changes at 5 or 20 min after nitrate treatments. Proteins identified by 5 min include signaling components such as kinases or transcription factors. In contrast, by 20 min, proteins identified were associated with transporter activity or hormone metabolism functions, among others. The phosphorylation profile of NITRATE TRANSPORTER 1.1 (NRT1.1) mutant plants was significantly altered as compared to wild-type plants, confirming its key role in nitrate signaling pathways that involves phosphorylation changes. Integrative bioinformatics analysis highlights auxin transport as an important mechanism modulated by nitrate signaling at the post-translational level. We validated a new phosphorylation site in PIN2 and provide evidence that it functions in primary and lateral root growth responses to nitrate.

Structural Modeling and in planta Complementation Studies Link Mutated Residues of the Medicago truncatula Nitrate Transporter NPF1.7 to Functionality in Root Nodules.

Symbiotic nitrogen fixation is a complex and regulated process that takes place in root nodules of legumes and allows legumes to grow in soils that lack nitrogen. Nitrogen is mostly acquired from the soil as nitrate and its level in the soil affects nodulation and nitrogen fixation. The mechanism(s) by which legumes modulate nitrate uptake to regulate nodule symbiosis remain unclear. In Medicago truncatula, the MtNPF1.7 transporter has been shown to control nodulation, symbiosis, and root architecture. MtNPF1.7 belongs to the nitrate/peptide transporter family and is a symporter with nitrate transport driven by proton(s). In this study we combined in silico structural predictions with in planta complementation of the severely defective mtnip-1 mutant plants to understand the role of a series of distinct amino acids in the transporter's function. Our results support hypotheses about the functional importance of the ExxE(R/K) motif including an essential role for the first glutamic acid of the motif in proton(s) and possibly substrate transport. Results reveal that Motif A, a motif conserved among major facilitator transport (MFS) proteins, is essential for function. We hypothesize that it participates in intradomain packing of transmembrane helices and stabilizing one conformation during transport. Our results also question the existence of a putative TMH4-TMH10 salt bridge. These results are discussed in the context of potential nutrient transport functions for MtNPF1.7. Our findings add to the knowledge of the mechanism of alternative conformational changes as well as symport transport in NPFs and enhance our knowledge of the mechanisms for nitrate signaling.