Nitrogen sensing and regulatory networks: it's about time and space.

A plant's response to external and internal nitrogen signals/status relies on sensing and signaling mechanisms that operate across spatial and temporal dimensions. From a comprehensive systems biology perspective, this involves integrating nitrogen responses in different cell types and over long distances to ensure organ coordination in real time and yield practical applications. In this prospective review, we focus on novel aspects of nitrogen (N) sensing/signaling uncovered using temporal and spatial systems biology approaches, largely in the model Arabidopsis. The temporal aspects span: transcriptional responses to N-dose mediated by Michaelis-Menten kinetics, the role of the master NLP7 transcription factor as a nitrate sensor, its nitrate-dependent TF nuclear retention, its "hit-and-run" mode of target gene regulation, and temporal transcriptional cascade identified by "network walking." Spatial aspects of N-sensing/signaling have been uncovered in cell type-specific studies in roots and in root-to-shoot communication. We explore new approaches using single-cell sequencing data, trajectory inference, and pseudotime analysis as well as machine learning and artificial intelligence approaches. Finally, unveiling the mechanisms underlying the spatial dynamics of nitrogen sensing/signaling networks across species from model to crop could pave the way for translational studies to improve nitrogen-use efficiency in crops. Such outcomes could potentially reduce the detrimental effects of excessive fertilizer usage on groundwater pollution and greenhouse gas emissions.

Distinct early transcriptional regulations by turgor and osmotic potential in the roots of Arabidopsis.

In a context of climate change, deciphering signaling pathways driving plant adaptation to drought, changes in water availability, and salt is key. A crossing point of these plant stresses is their impact on plant water potential (Ψ), a composite physico-chemical variable reflecting the availability of water for biological processes such as plant growth and stomatal aperture. The Ψ of plant cells is mainly driven by their turgor and osmotic pressures. Here we investigated the effect of a variety of osmotic treatments on the roots of Arabidopsis plants grown in hydroponics. We used, among others, a permeating solute as a way to differentiate variations on turgor from variations in osmotic pressure. Measurement of cortical cell turgor pressure with a cell pressure probe allowed us to monitor the intensity of the treatments and thereby preserve the cortex from plasmolysis. Transcriptome analyses at an early time point (15 min) showed specific and quantitative transcriptomic responses to both osmotic and turgor pressure variations. Our results highlight how water-related biophysical parameters can shape the transcriptome of roots under stress and provide putative candidates to explore further the early perception of water stress in plants.

Reduction in PLANT DEFENSIN 1 expression in Arabidopsis thaliana results in increased resistance to pathogens and zinc toxicity.

Ectopic expression of defensins in plants correlates with their increased capacity to withstand abiotic and biotic stresses. This applies to Arabidopsis thaliana, where some of the seven members of the PLANT DEFENSIN 1 family (AtPDF1) are recognised to improve plant responses to necrotrophic pathogens and increase seedling tolerance to excess zinc (Zn). However, few studies have explored the effects of decreased endogenous defensin expression on these stress responses. Here, we carried out an extensive physiological and biochemical comparative characterization of (i) novel artificial microRNA (amiRNA) lines silenced for the five most similar AtPDF1s, and (ii) a double null mutant for the two most distant AtPDF1s. Silencing of five AtPDF1 genes was specifically associated with increased aboveground dry mass production in mature plants under excess Zn conditions, and with increased plant tolerance to different pathogens - a fungus, an oomycete and a bacterium, while the double mutant behaved similarly to the wild type. These unexpected results challenge the current paradigm describing the role of PDFs in plant stress responses. Additional roles of endogenous plant defensins are discussed, opening new perspectives for their functions.

Nitrate in 2020: Thirty Years from Transport to Signaling Networks.

Nitrogen (N) is an essential macronutrient for plants and a major limiting factor for plant growth and crop production. Nitrate is the main source of N available to plants in agricultural soils and in many natural environments. Sustaining agricultural productivity is of paramount importance in the current scenario of increasing world population, diversification of crop uses, and climate change. Plant productivity for major crops around the world, however, is still supported by excess application of N-rich fertilizers with detrimental economic and environmental impacts. Thus, understanding how plants regulate nitrate uptake and metabolism is key for developing new crops with enhanced N use efficiency and to cope with future world food demands. The study of plant responses to nitrate has gained considerable interest over the last 30 years. This review provides an overview of key findings in nitrate research, spanning biochemistry, molecular genetics, genomics, and systems biology. We discuss how we have reached our current view of nitrate transport, local and systemic nitrate sensing/signaling, and the regulatory networks underlying nitrate-controlled outputs in plants. We hope this summary will serve not only as a timeline and information repository but also as a baseline to define outstanding questions for future research.

Genome-wide analysis in response to nitrogen and carbon identifies regulators for root AtNRT2 transporters.

In Arabidopsis (Arabidopsis thaliana), the High-Affinity Transport System (HATS) for root nitrate (NO3-) uptake depends mainly on four NRT2 NO3- transporters, namely NRT2.1, NRT2.2, NRT2.4, and NRT2.5. The HATS is the target of many regulations to coordinate nitrogen (N) acquisition with the N status of the plant and with carbon (C) assimilation through photosynthesis. At the molecular level, C and N signaling pathways control gene expression of the NRT2 transporters. Although several regulators of these transporters have been identified in response to either N or C signals, the response of NRT2 gene expression to the interaction of these signals has never been specifically investigated, and the underlying molecular mechanisms remain largely unknown. To address this question we used an original systems biology approach to model a regulatory gene network targeting NRT2.1, NRT2.2, NRT2.4, and NRT2.5 in response to N/C signals. Our systems analysis of the data identified three transcription factors, TGA3, MYC1, and bHLH093. Functional analysis of mutants combined with yeast one-hybrid experiments confirmed that all three transcription factors are regulators of NRT2.4 or NRT2.5 in response to N or C signals. These results reveal a role for TGA3, MYC1, and bHLH093 in controlling the expression of root NRT2 transporter genes.

Identification of Molecular Integrators Shows that Nitrogen Actively Controls the Phosphate Starvation Response in Plants.

Nitrogen (N) and phosphorus (P) are key macronutrients sustaining plant growth and crop yield and ensuring food security worldwide. Understanding how plants perceive and interpret the combinatorial nature of these signals thus has important agricultural implications within the context of (1) increased food demand, (2) limited P supply, and (3) environmental pollution due to N fertilizer usage. Here, we report the discovery of an active control of P starvation response (PSR) by a combination of local and long-distance N signaling pathways in plants. We show that, in Arabidopsis (Arabidopsis thaliana), the nitrate transceptor CHLORINA1/NITRATE TRANSPORTER1.1 (CHL1/NRT1.1) is a component of this signaling crosstalk. We also demonstrate that this crosstalk is dependent on the control of the accumulation and turnover by N of the transcription factor PHOSPHATE STARVATION RESPONSE1 (PHR1), a master regulator of P sensing and signaling. We further show an important role of PHOSPHATE2 (PHO2) as an integrator of the N availability into the PSR since the effect of N on PSR is strongly affected in pho2 mutants. We finally show that PHO2 and NRT1.1 influence each other's transcript levels. These observations are summarized in a model representing a framework with several entry points where N signal influence PSR. Finally, we demonstrate that this phenomenon is conserved in rice (Oryza sativa) and wheat (Triticum aestivum), opening biotechnological perspectives in crop plants.

SDG8-Mediated Histone Methylation and RNA Processing Function in the Response to Nitrate Signaling.

Chromatin modification has gained increased attention for its role in the regulation of plant responses to environmental changes, but the specific mechanisms and molecular players remain elusive. Here, we show that the Arabidopsis (Arabidopsis thaliana) histone methyltransferase SET DOMAIN GROUP8 (SDG8) mediates genome-wide changes in H3K36 methylation at specific genomic loci functionally relevant to nitrate treatments. Moreover, we show that the specific H3K36 methyltransferase encoded by SDG8 is required for canonical RNA processing, and that RNA isoform switching is more prominent in the sdg8-5 deletion mutant than in the wild type. To demonstrate that SDG8-mediated regulation of RNA isoform expression is functionally relevant, we examined a putative regulatory gene, CONSTANS, CO-like, and TOC1 101 (CCT101), whose nitrogen-responsive isoform-specific RNA expression is mediated by SDG8. We show by functional expression in shoot cells that the different RNA isoforms of CCT101 encode distinct regulatory proteins with different effects on genome-wide transcription. We conclude that SDG8 is involved in plant responses to environmental nitrogen supply, affecting multiple gene regulatory processes including genome-wide histone modification, transcriptional regulation, and RNA processing, and thereby mediating developmental and metabolic processes related to nitrogen use.

GARP transcription factors repress Arabidopsis nitrogen starvation response via ROS-dependent and -independent pathways.

Plants need to cope with strong variations of nitrogen availability in the soil. Although many molecular players are being discovered concerning how plants perceive NO3- provision, it is less clear how plants recognize a lack of nitrogen. Following nitrogen removal, plants activate their nitrogen starvation response (NSR), which is characterized by the activation of very high-affinity nitrate transport systems (NRT2.4 and NRT2.5) and other sentinel genes involved in N remobilization such as GDH3. Using a combination of functional genomics via transcription factor perturbation and molecular physiology studies, we show that the transcription factors belonging to the HHO subfamily are important regulators of NSR through two potential mechanisms. First, HHOs directly repress the high-affinity nitrate transporters, NRT2.4 and NRT2.5. hho mutants display increased high-affinity nitrate transport activity, opening up promising perspectives for biotechnological applications. Second, we show that reactive oxygen species (ROS) are important to control NSR in wild-type plants and that HRS1 and HHO1 overexpressors and mutants are affected in their ROS content, defining a potential feed-forward branch of the signaling pathway. Taken together, our results define the relationships of two types of molecular players controlling the NSR, namely ROS and the HHO transcription factors. This work (i) up opens perspectives on a poorly understood nutrient-related signaling pathway and (ii) defines targets for molecular breeding of plants with enhanced NO3- uptake.

Responses to Systemic Nitrogen Signaling in Arabidopsis Roots Involve trans-Zeatin in Shoots.

Plants face temporal and spatial variation in nitrogen (N) availability. This includes heterogeneity in soil nitrate (NO3-) content. To overcome these constraints, plants modify their gene expression and physiological processes to optimize N acquisition. This plasticity relies on a complex long-distance root-shoot-root signaling network that remains poorly understood. We previously showed that cytokinin (CK) biosynthesis is required to trigger systemic N signaling. Here, we performed split-root experiments and used a combination of CK-related mutant analyses, hormone profiling, transcriptomic analysis, NO3- uptake assays, and root growth measurements to gain insight into systemic N signaling in Arabidopsis thaliana By comparing wild-type plants and mutants affected in CK biosynthesis and ABCG14-dependent root-to-shoot translocation of CK, we revealed an important role for active trans-zeatin (tZ) in systemic N signaling. Both rapid sentinel gene regulation and long-term functional acclimation to heterogeneous NO3- supply, including NO3- transport and root growth regulation, are likely mediated by the integration of tZ content in shoots. Furthermore, shoot transcriptome profiling revealed that glutamate/glutamine metabolism is likely a target of tZ root-to-shoot translocation, prompting an interesting hypothesis regarding shoot-to-root communication. Finally, this study highlights tZ-independent pathways regulating gene expression in shoots as well as NO3- uptake activity in response to total N deprivation.

Temporal transcriptional logic of dynamic regulatory networks underlying nitrogen signaling and use in plants.

This study exploits time, the relatively unexplored fourth dimension of gene regulatory networks (GRNs), to learn the temporal transcriptional logic underlying dynamic nitrogen (N) signaling in plants. Our "just-in-time" analysis of time-series transcriptome data uncovered a temporal cascade of cis elements underlying dynamic N signaling. To infer transcription factor (TF)-target edges in a GRN, we applied a time-based machine learning method to 2,174 dynamic N-responsive genes. We experimentally determined a network precision cutoff, using TF-regulated genome-wide targets of three TF hubs (CRF4, SNZ, and CDF1), used to "prune" the network to 155 TFs and 608 targets. This network precision was reconfirmed using genome-wide TF-target regulation data for four additional TFs (TGA1, HHO5/6, and PHL1) not used in network pruning. These higher-confidence edges in the GRN were further filtered by independent TF-target binding data, used to calculate a TF "N-specificity" index. This refined GRN identifies the temporal relationship of known/validated regulators of N signaling (NLP7/8, TGA1/4, NAC4, HRS1, and LBD37/38/39) and 146 additional regulators. Six TFs-CRF4, SNZ, CDF1, HHO5/6, and PHL1-validated herein regulate a significant number of genes in the dynamic N response, targeting 54% of N-uptake/assimilation pathway genes. Phenotypically, inducible overexpression of CRF4 in planta regulates genes resulting in altered biomass, root development, and 15NO3- uptake, specifically under low-N conditions. This dynamic N-signaling GRN now provides the temporal "transcriptional logic" for 155 candidate TFs to improve nitrogen use efficiency with potential agricultural applications. Broadly, these time-based approaches can uncover the temporal transcriptional logic for any biological response system in biology, agriculture, or medicine.

Nutrient-Related Long-Distance Signals: Common Players and Possible Cross-Talk.

Nutrient fluctuations are more a rule rather than an exception in the life of sessile organisms such as plants. Despite this constraint that adds up to abiotic and biotic stresses, plants are able to accomplish their life cycle thanks to an efficient signaling network that reciprocally controls nutrient acquisition and use with growth and development. The majority of nutrients are acquired by the root system where multiple local signaling pathways that rely on nutrient-sensing systems are implemented to direct root growth toward soil resources. Moreover, long-distance signaling plays an essential role in integrating nutrient availability at the whole-plant level and adjusting nutrient acquisition to plant growth requirements. By studying the signaling network for single mineral nutrients, several long-distance signals traveling between roots and shoots and taking a diversity of forms have been identified and are summarized here. However, the nutritional environment is multifactorial, adding a tremendous complexity for our understanding of the nutrient signaling network as a unique system. For instance, long-distance signals are expected to support this nutrient cross-talk in part, but the mechanisms are still largely unknown. Therefore, the involvement of possible long-distance signals as conveyers of nutrient cross-talk is discussed here together with approaches and strategies that are now considered to build a picture from the nutrient signaling puzzle.

Long-distance nitrate signaling displays cytokinin dependent and independent branches.

The long-distance signaling network allowing a plant to properly develop its root system is crucial to optimize root foraging in areas where nutrients are available. Cytokinin is an essential element of the systemic signaling network leading to the enhancement of lateral root proliferation in areas where nitrate is available. Here, we explore more precisely: (i) which particular traits of lateral root growth (density and length of emerged lateral roots) are the targets of systemic signaling in a context of heterogeneous nitrate supply; and (ii) if the systemic signaling depends only on cytokinin or on a combination of several signalings.

Systems approach identifies TGA1 and TGA4 transcription factors as important regulatory components of the nitrate response of Arabidopsis thaliana roots.

Nitrate acts as a potent signal to control global gene expression in Arabidopsis. Using an integrative bioinformatics approach we identified TGA1 and TGA4 as putative regulatory factors that mediate nitrate responses in Arabidopsis roots. We showed that both TGA1 and TGA4 mRNAs accumulate strongly after nitrate treatments in roots. Global gene expression analysis revealed 97% of the genes with altered expression in tga1 tga4 double mutant plants respond to nitrate treatments, indicating that these transcription factors have a specific role in nitrate responses in Arabidopsis root organs. We found TGA1 and TGA4 regulate the expression of nitrate transporter genes NRT2.1 and NRT2.2. Specific binding of TGA1 to its cognate DNA sequence on NRT2.1 and NRT2.2 promoters was confirmed by chromatin immunoprecipitation assays. The tga1 tga4 double mutant plants exhibit nitrate-dependent lateral and primary root phenotypes. Lateral root initiation is affected in both tga1 tga4 and nrt1.2 nrt2.2 double mutants, suggesting TGA1 and TGA4 regulate lateral root development at least partly via NRT2.1 and NRT2.2. Additional root phenotypes of tga1 tga4 double mutants indicate that these transcription factors play an important role in root developmental responses to nitrate. These results identify TGA1 and TGA4 as important regulatory factors of the nitrate response in Arabidopsis roots.

RootScape: a landmark-based system for rapid screening of root architecture in Arabidopsis.

The architecture of plant roots affects essential functions including nutrient and water uptake, soil anchorage, and symbiotic interactions. Root architecture comprises many features that arise from the growth of the primary and lateral roots. These root features are dictated by the genetic background but are also highly responsive to the environment. Thus, root system architecture (RSA) represents an important and complex trait that is highly variable, affected by genotype × environment interactions, and relevant to survival/performance. Quantification of RSA in Arabidopsis (Arabidopsis thaliana) using plate-based tissue culture is a very common and relatively rapid assay, but quantifying RSA represents an experimental bottleneck when it comes to medium- or high-throughput approaches used in mutant or genotype screens. Here, we present RootScape, a landmark-based allometric method for rapid phenotyping of RSA using Arabidopsis as a case study. Using the software AAMToolbox, we created a 20-point landmark model that captures RSA as one integrated trait and used this model to quantify changes in the RSA of Arabidopsis (Columbia) wild-type plants grown under different hormone treatments. Principal component analysis was used to compare RootScape with conventional methods designed to measure root architecture. This analysis showed that RootScape efficiently captured nearly all the variation in root architecture detected by measuring individual root traits and is 5 to 10 times faster than conventional scoring. We validated RootScape by quantifying the plasticity of RSA in several mutant lines affected in hormone signaling. The RootScape analysis recapitulated previous results that described complex phenotypes in the mutants and identified novel gene × environment interactions.

Signal interactions in the regulation of root nitrate uptake.

In most aerobic soils, nitrate (NO3(-)) is the main nitrogen source for plants and is often limiting for plant growth and development. To adapt to a changing environment, plants have developed complex regulatory mechanisms that involve short and long-range signalling pathways in response to both NO3(-) availability in the soil and other physiological processes like growth or nitrogen (N) and carbon (C) metabolisms. Over the past decade, transcriptomic approaches largely contributed to the identification of molecular elements involved in these regulatory mechanisms, especially at the level of root NO3(-)uptake. Most strikingly, the data obtained revealed the high level of interaction between N and both hormone and C signalling pathways, suggesting a strong dependence on growth, development, and C metabolism to adapt root NO3(-) uptake to both external NO3(-) availability and the N status of the plant. However, the signalling mechanisms involved in the cross-talk between N, C, and hormones for the regulation of root NO3(-) uptake remain largely obscure. The aim of this review is to discuss the recent advances concerning the regulatory pathways controlling NO3(-) uptake in response to N signalling, hormones, and C in the model plant Arabidopsis thaliana. Then, to further characterize the level of interaction between these signalling pathways we built on publicly available transcriptome data to determine how hormones and C treatments modify the gene network connecting root NO3(-) transporters and their regulators.

Finding a nitrogen niche: a systems integration of local and systemic nitrogen signalling in plants.

The ability of plants to sense their nitrogen (N) microenvironment in the soil and deploy strategic root growth in N-rich patches requires exquisite systems integration. Remarkably, this new paradigm for systems biology research has intrigued plant biologists for more than a century, when a split-root framework was first used to study how plants sense and respond to heterogeneous soil nutrient environments. This systemic N-signalling mechanism, allowing plants to sense and forage for mineral nutrients in resource-rich patches, has important implications for agriculture. In this review, we will focus on how advances in the post-genomic era have uncovered the gene regulatory networks underlying systemic N-signalling. After defining how local and systemic N-signalling can be experimentally distinguished for molecular study using a split-root system, the genetic factors that have been shown to mediate local and/or systemic N-signalling are reviewed. Second, the genetic mechanism of this regulatory system is broadened to the whole genome level. To do this, publicly available N-related transcriptomic datasets are compared with genes that have previously been identified as local and systemic N responders in a split-root transcriptome dataset. Specifically, (i) it was found that transcriptional reprogramming triggered by homogeneous N-treatments is composed of both local and systemic responses, (ii) the spatio-temporal signature of local versus systemic responsive genes is defined, and (iii) the conservation of systemic N-signalling between Arabidopsis and Medicago is assessed. Finally, the potential mediators, i.e. metabolites and phytohormones, of the N-related long-distance signals, are discussed.

Nitrogen economics of root foraging: transitive closure of the nitrate-cytokinin relay and distinct systemic signaling for N supply vs. demand.

As sessile organisms, root plasticity enables plants to forage for and acquire nutrients in a fluctuating underground environment. Here, we use genetic and genomic approaches in a "split-root" framework--in which physically isolated root systems of the same plant are challenged with different nitrogen (N) environments--to investigate how systemic signaling affects genome-wide reprogramming and root development. The integration of transcriptome and root phenotypes enables us to identify distinct mechanisms underlying "N economy" (i.e., N supply and demand) of plants as a system. Under nitrate-limited conditions, plant roots adopt an "active-foraging strategy", characterized by lateral root outgrowth and a shared pattern of transcriptome reprogramming, in response to either local or distal nitrate deprivation. By contrast, in nitrate-replete conditions, plant roots adopt a "dormant strategy", characterized by a repression of lateral root outgrowth and a shared pattern of transcriptome reprogramming, in response to either local or distal nitrate supply. Sentinel genes responding to systemic N signaling identified by genome-wide comparisons of heterogeneous vs. homogeneous split-root N treatments were used to probe systemic N responses in Arabidopsis mutants impaired in nitrate reduction and hormone synthesis and also in decapitated plants. This combined analysis identified genetically distinct systemic signaling underlying plant N economy: (i) N supply, corresponding to a long-distance systemic signaling triggered by nitrate sensing; and (ii) N demand, experimental support for the transitive closure of a previously inferred nitrate-cytokinin shoot-root relay system that reports the nitrate demand of the whole plant, promoting a compensatory root growth in nitrate-rich patches of heterogeneous soil.

High nitrogen insensitive 9 (HNI9)-mediated systemic repression of root NO3- uptake is associated with changes in histone methylation.

In plants, root nitrate uptake systems are under systemic feedback repression by the N satiety of the whole organism, thus adjusting the N acquisition capacity to the N demand for growth; however, the underlying molecular mechanisms are largely unknown. We previously isolated the Arabidopsis high nitrogen-insensitive 9-1 (hni9-1) mutant, impaired in the systemic feedback repression of the root nitrate transporter NRT2.1 by high N supply. Here, we show that HNI9 encodes Arabidopsis INTERACT WITH SPT6 (AtIWS1), an evolutionary conserved component of the RNA polymerase II complex. HNI9/AtIWS1 acts in roots to repress NRT2.1 transcription in response to high N supply. At a genomic level, HNI9/AtIWS1 is shown to play a broader role in N signaling by regulating several hundred N-responsive genes in roots. Repression of NRT2.1 transcription by high N supply is associated with an HNI9/AtIWS1-dependent increase in histone H3 lysine 27 trimethylation at the NRT2.1 locus. Our findings highlight the hypothesis that posttranslational chromatin modifications control nutrient acquisition in plants.

Recent advances in local and systemic nitrate signaling in Arabidopsisthaliana.

Nitrate is the most abundant form of inorganic nitrogen in aerobic soils, serving both as a nutrient and a signaling molecule. Central to nitrate signaling in higher plants is the intricate balance between local and systemic signaling and response pathways. The interplay between local and systemic responses allows plants to regulate their global gene expression, metabolism, physiology, growth, and development under fluctuating nitrate availability. This review offers an overview of recent discoveries regarding new players on nitrate sensing and signaling, in local and systemic contexts in Arabidopsis thaliana. Additionally, it addresses unanswered questions that warrant further investigation for a better understanding of nitrate signaling and responses in plants.

Unleashing the potential of peptides in agriculture and beyond.

Peptides display a broad range of regulatory functions. Ormancey et al. recently identified an important new mechanism - complementary peptides (cPEPs) - that provide a versatile means to control cell functions. We draw a parallel between RNA and peptide biology, and discuss new routes of investigation and industrial applications opened by this work.