The Beneficial Fungus Mortierella hyalina Modulates Amino Acid Homeostasis in Arabidopsis under Nitrogen Starvation.

Non-mycorrhizal but beneficial fungi often mitigate (a)biotic stress-related traits in host plants. The underlying molecular mechanisms are mostly still unknown, as in the interaction between the endophytic growth-promoting soil fungus Mortierella hyalina and Arabidopsis thaliana. Here, abiotic stress in the form of nitrogen (N) deficiency was used to investigate the effects of the fungus on colonized plants. In particular, the hypothesis was investigated that fungal infection could influence N deficiency via an interaction with the high-affinity nitrate transporter NRT2.4, which is induced by N deficiency. For this purpose, Arabidopsis wild-type nrt2.4 knock-out and NRT2.4 reporter lines were grown on media with different nitrate concentrations with or without M. hyalina colonization. We used chemical analysis methods to determine the amino acids and phytohormones. Experimental evidence suggests that the fungus does not modulate NRT2.4 expression under N starvation. Instead, M. hyalina alleviates N starvation in other ways: The fungus supplies nitrogen (15N) to the N-starved plant. The presence of the fungus restores the plants' amino acid homeostasis, which was out of balance due to N deficiency, and causes a strong accumulation of branched-chain amino acids. We conclude that the plant does not need to invest in defense and resources for growth are maintained, which in turn benefits the fungus, suggesting that this interaction should be considered a mutualistic symbiosis.

The Root-Colonizing Endophyte Piriformospora indica Supports Nitrogen-Starved Arabidopsis thaliana Seedlings with Nitrogen Metabolites.

The root-colonizing endophytic fungus Piriformospora indica promotes the root and shoot growth of its host plants. We show that the growth promotion of Arabidopsis thaliana leaves is abolished when the seedlings are grown on media with nitrogen (N) limitation. The fungus neither stimulated the total N content nor did it promote 15NO3- uptake from agar plates to the leaves of the host under N-sufficient or N-limiting conditions. However, when the roots were co-cultivated with 15N-labelled P. indica, more labels were detected in the leaves of N-starved host plants but not in plants supplied with sufficient N. Amino acid and primary metabolite profiles, as well as the expression analyses of N metabolite transporter genes suggest that the fungus alleviates the adaptation of its host from the N limitation condition. P. indica alters the expression of transporter genes, which participate in the relocation of NO3-, NH4+ and N metabolites from the roots to the leaves under N limitation. We propose that P. indica participates in the plant's metabolomic adaptation against N limitation by delivering reduced N metabolites to the host, thus alleviating metabolic N starvation responses and reprogramming the expression of N metabolism-related genes.

Interplay between NIN-LIKE PROTEINs 6 and 7 in nitrate signaling.

NLP7 (NIN-LIKE-PROTEIN 7) is the major transcriptional factor responsible for the primary nitrate response (PNR), but the role of its homolog, NLP6, in nitrogen signaling and the interplay between NLP6 and NLP7 remain to be elucidated. In this study, we show that, like NLP7, nuclear localization of NLP6 via a nuclear retention mechanism is nitrate dependent, but nucleocytosolic shuttling of both NLP6 and NLP7 is independent of each other. Compared with single mutants, the nlp6nlp7 double mutant displays a synergistic growth retardation phenotype in response to nitrate. The transcriptome analysis of the PNR showed that NLP6 and NLP7 govern ∼50% of nitrate-induced genes, with cluster analysis highlighting 2 distinct patterns. In the A1 cluster, NLP7 plays the major role, whereas in the A2 cluster, NLP6 and NLP7 are partially functionally redundant. Interestingly, comparing the growth phenotype and PNR under high- and low-nitrate conditions demonstrated that NLP6 and NLP7 exert a more dominant role in the response to high nitrate. Apart from nitrate signaling, NLP6 and NLP7 also participated in high ammonium conditions. Growth phenotypes and transcriptome data revealed that NLP6 and NLP7 are completely functionally redundant and may act as repressors in response to ammonium. Other NLP family members also participated in the PNR, with NLP2 and NLP7 acting as broader regulators and NLP4, -5, -6, and -8 regulating PNR in a gene-dependent manner. Thus, our findings indicate that multiple modes of interplay exist between NLP6 and NLP7 that differ depending on nitrogen sources and gene clusters.

The Arabidopsis transcription factor NLP2 regulates early nitrate responses and integrates nitrate assimilation with energy and carbon skeleton supply.

Nitrate signaling improves plant growth under limited nitrate availability and, hence, optimal resource use for crop production. Whereas several transcriptional regulators of nitrate signaling have been identified, including the Arabidopsis thaliana transcription factor NIN-LIKE PROTEIN7 (NLP7), additional regulators are expected to fine-tune this pivotal physiological response. Here, we characterized Arabidopsis NLP2 as a top-tier transcriptional regulator of the early nitrate response gene regulatory network. NLP2 interacts with NLP7 in vivo and shares key molecular features such as nitrate-dependent nuclear localization, DNA-binding motif, and some target genes with NLP7. Genetic, genomic, and metabolic approaches revealed a specific role for NLP2 in the nitrate-dependent regulation of carbon and energy-related processes that likely influence plant growth under distinct nitrogen environments. Our findings highlight the complementarity and specificity of NLP2 and NLP7 in orchestrating a multitiered nitrate regulatory network that links nitrate assimilation with carbon and energy metabolism for efficient nitrogen use and biomass production.

PlantACT! - how to tackle the climate crisis.

Greenhouse gas (GHG) emissions have created a global climate crisis which requires immediate interventions to mitigate the negative effects on all aspects of life on this planet. As current agriculture and land use contributes up to 25% of total GHG emissions, plant scientists take center stage in finding possible solutions for a transition to sustainable agriculture and land use. In this article, the PlantACT! (Plants for climate ACTion!) initiative of plant scientists lays out a road map of how and in which areas plant scientists can contribute to finding immediate, mid-term, and long-term solutions, and what changes are necessary to implement these solutions at the personal, institutional, and funding levels.

The calcium sensor CBL7 is required for Serendipita indica-induced growth stimulation in Arabidopsis thaliana, controlling defense against the endophyte and K+ homoeostasis in the symbiosis.

Calcium is an important second messenger in plants. The activation of Ca2+ signalling cascades is critical in the activation of adaptive processes in response to environmental stimuli. Root colonization by the growth promoting endophyte Serendipita indica involves the increase of cytosolic Ca2+ levels in Arabidopsis thaliana. Here, we investigated transcriptional changes in Arabidopsis roots during symbiosis with S. indica. RNA-seq profiling disclosed the induction of Calcineurin B-like 7 (CBL7) during early and later phases of the interaction. Consistently, reverse genetic evidence highlighted the functional relevance of CBL7 and tested the involvement of a CBL7-CBL-interacting protein kinase 13 signalling pathway. The loss-of-function of CBL7 abolished the growth promoting effect and affected root colonization. The transcriptomics analysis of cbl7 revealed the involvement of this Ca2+ sensor in activating plant defense responses. Furthermore, we report on the contribution of CBL7 to potassium transport in Arabidopsis. We analysed K+ contents in wild-type and cbl7 plants and observed a significant increase of K+ in roots of cbl7 plants, while shoot tissues demonstrated K+ depletion. Taken together, our work associates CBL7 with an important role in the mutual interaction between Arabidopsis and S. indica and links CBL7 to K+ transport.

Molecular framework integrating nitrate sensing in root and auxin-guided shoot adaptive responses.

Mineral nutrition is one of the key environmental factors determining plant development and growth. Nitrate is the major form of macronutrient nitrogen that plants take up from the soil. Fluctuating availability or deficiency of this element severely limits plant growth and negatively affects crop production in the agricultural system. To cope with the heterogeneity of nitrate distribution in soil, plants evolved a complex regulatory mechanism that allows rapid adjustment of physiological and developmental processes to the status of this nutrient. The root, as a major exploitation organ that controls the uptake of nitrate to the plant body, acts as a regulatory hub that, according to nitrate availability, coordinates the growth and development of other plant organs. Here, we identified a regulatory framework, where cytokinin response factors (CRFs) play a central role as a molecular readout of the nitrate status in roots to guide shoot adaptive developmental response. We show that nitrate-driven activation of NLP7, a master regulator of nitrate response in plants, fine tunes biosynthesis of cytokinin in roots and its translocation to shoots where it enhances expression of CRFs. CRFs, through direct transcriptional regulation of PIN auxin transporters, promote the flow of auxin and thereby stimulate the development of shoot organs.

Wounding and Insect Feeding Trigger Two Independent MAPK Pathways with Distinct Regulation and Kinetics.

Abiotic and biotic factors cause plant wounding and trigger complex short- and long-term responses at the local and systemic levels. These responses are under the control of complex signaling pathways, which are still poorly understood. Here, we show that the rapid activation of clade-A mitogen-activated protein kinases (MAPKs) MPK3 and MPK6 by wounding depends on the upstream MAPK kinases MKK4 and MKK5 but is independent of jasmonic acid (JA) signaling. In addition, this fast module does not control wound-triggered JA accumulation in Arabidopsis (Arabidopsis thaliana), unlike its orthologs in tobacco. We also demonstrate that a second MAPK module, composed of MKK3 and the clade-C MAPKs MPK1/2/7, is activated by wounding in a MKK4/5-independent manner. We provide evidence that the activation of this MKK3-MPK1/2/7 module occurs mainly through wound-induced JA production via the transcriptional regulation of upstream clade-III MAP3Ks, particularly MAP3K14. We show that mkk3 mutant plants are more susceptible to herbivory from larvae of the generalist lepidopteran herbivore Spodoptera littoralis, indicating that the MKK3-MPK1/2/7 module is involved in counteracting insect feeding.

Transient genome-wide interactions of the master transcription factor NLP7 initiate a rapid nitrogen-response cascade.

Dynamic reprogramming of gene regulatory networks (GRNs) enables organisms to rapidly respond to environmental perturbation. However, the underlying transient interactions between transcription factors (TFs) and genome-wide targets typically elude biochemical detection. Here, we capture both stable and transient TF-target interactions genome-wide within minutes after controlled TF nuclear import using time-series chromatin immunoprecipitation (ChIP-seq) and/or DNA adenine methyltransferase identification (DamID-seq). The transient TF-target interactions captured uncover the early mode-of-action of NIN-LIKE PROTEIN 7 (NLP7), a master regulator of the nitrogen signaling pathway in plants. These transient NLP7 targets captured in root cells using temporal TF perturbation account for 50% of NLP7-regulated genes not detectably bound by NLP7 in planta. Rapid and transient NLP7 binding activates early nitrogen response TFs, which we validate to amplify the NLP7-initiated transcriptional cascade. Our approaches to capture transient TF-target interactions genome-wide can be applied to validate dynamic GRN models for any pathway or organism of interest.

Harnessing symbiotic plant-fungus interactions to unleash hidden forces from extreme plant ecosystems.

Global climate change is arguably one of the biggest threats of modern times and has already led to a wide range of impacts on the environment, economy, and society. Owing to past emissions and climate system inertia, global climate change is predicted to continue for decades even if anthropogenic greenhouse gas emissions were to stop immediately. In many regions, such as central Europe and the Mediterranean region, the temperature is likely to rise by 2-5 °C and annual precipitation is predicted to decrease. Expected heat and drought periods followed by floods, and unpredictable growing seasons, are predicted to have detrimental effects on agricultural production systems, causing immense economic losses and food supply problems. To mitigate the risks of climate change, agricultural innovations counteracting these effects need to be embraced and accelerated. To achieve maximum improvement, the required agricultural innovations should not focus only on crops but rather pursue a holistic approach including the entire ecosystem. Over millions of years, plants have evolved in close association with other organisms, particularly soil microbes that have shaped their evolution and contemporary ecology. Many studies have already highlighted beneficial interactions among plants and the communities of microorganisms with which they coexist. Questions arising from these discoveries are whether it will be possible to decipher a common molecular pattern and the underlying biochemical framework of interspecies communication, and whether such knowledge can be used to improve agricultural performance under environmental stress conditions. In this review, we summarize the current knowledge of plant interactions with fungal endosymbionts found in extreme ecosystems. Special attention will be paid to the interaction of plants with the symbiotic root-colonizing endophytic fungus Serendipita indica, which has been developed as a model system for beneficial plant-fungus interactions.

Nitrate acts at the Arabidopsis thaliana shoot apical meristem to regulate flowering time.

Optimal timing of flowering, a major determinant for crop productivity, is controlled by environmental and endogenous cues. Nutrients are known to modify flowering time; however, our understanding of how nutrients interact with the known pathways, especially at the shoot apical meristem (SAM), is still incomplete. Given the negative side-effects of nitrogen fertilization, it is essential to understand its mode of action for sustainable crop production. We investigated how a moderate restriction by nitrate is integrated into the flowering network at the SAM, to which plants can adapt without stress symptoms. This condition delays flowering by decreasing expression of SUPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) at the SAM. Measurements of nitrate and the responses of nitrate-responsive genes suggest that nitrate functions as a signal at the SAM. The transcription factors NIN-LIKE PROTEIN 7 (NLP7) and NLP6, which act as master regulators of nitrate signaling by binding to nitrate-responsive elements (NREs), are expressed at the SAM and flowering is delayed in single and double mutants. Two upstream regulators of SOC1 (SQUAMOSA PROMOTER BINDING PROTEIN-LIKE3 (SPL3) and SPL5) contain functional NREs in their promoters. Our results point at a tissue-specific, nitrate-mediated flowering time control in Arabidopsis thaliana.

Nitrogen Limitation Alters the Response of Specific Genes to Biotic Stress.

In their natural environment, plants are generally confronted with multiple co-occurring stresses. However, the interaction between stresses is not well known and transcriptomic data in response to combined stresses remain scarce. This study aims at characterizing the interaction between transcriptomic responses to biotic stress and nitrogen (N) limitation. Plants were grown in low or full N, infected or not with Erwinia amylovora (Ea) and plant gene expression was analyzed through microarray and qRT-PCR. Most Ea-responsive genes had the same profile (induced/repressed) in response to Ea in low and full N. In response to stress combination, one third of modulated transcripts responded in a manner that could not be deduced from their response to each individual stress. Many defense-related genes showed a prioritization of their response to biotic stress over their response to N limitation, which was also observed using Pseudomonas syringae as a second pathosystem. Our results indicate an interaction between transcriptomic responses to N and biotic stress. A small fraction of transcripts was prioritized between antagonistic responses, reflecting a preservation of the plant defense program under N limitation. Furthermore, this interaction also led to a complex and specific response in terms of metabolism and cellular homeostasis-associated genes.

Review: Mitogen-Activated Protein Kinases in nutritional signaling in Arabidopsis.

Mitogen-Activated Protein Kinase (MAPK) cascades are functional modules widespread among eukaryotic organisms. In plants, these modules are encoded by large multigenic families and are involved in many biological processes ranging from stress responses to cellular differentiation and organ development. Furthermore, MAPK pathways are involved in the perception of environmental and physiological modifications. Interestingly, some MAPKs play a role in several signaling networks and could have an integrative function for the response of plants to their environment. In this review, we describe the classification of MAPKs and highlight some of their biochemical actions. We performed an in silico analysis of MAPK gene expression in response to nutrients supporting their involvement in nutritional signaling. While several MAPKs have been identified as players in sugar, nitrogen, phosphate, iron and potassium-related signaling pathways, their biochemical functions are yet mainly unknown. The integration of these regulatory cascades in the current understanding of nutrient signaling is discussed and potential new avenues for approaches toward plants with higher nutrient use efficiencies are evoked.

NIN-like protein 8 is a master regulator of nitrate-promoted seed germination in Arabidopsis.

Seeds respond to multiple different environmental stimuli that regulate germination. Nitrate stimulates germination in many plants but how it does so remains unclear. Here we show that the Arabidopsis NIN-like protein 8 (NLP8) is essential for nitrate-promoted seed germination. Seed germination in nlp8 loss-of-function mutants does not respond to nitrate. NLP8 functions even in a nitrate reductase-deficient mutant background, and the requirement for NLP8 is conserved among Arabidopsis accessions. NLP8 reduces abscisic acid levels in a nitrate-dependent manner and directly binds to the promoter of CYP707A2, encoding an abscisic acid catabolic enzyme. Genetic analysis shows that NLP8-mediated promotion of seed germination by nitrate requires CYP707A2. Finally, we show that NLP8 localizes to nuclei and unlike NLP7, does not appear to be activated by nitrate-dependent nuclear retention of NLP7, suggesting that seeds have a unique mechanism for nitrate signalling.

Plant Nitrogen Acquisition Under Low Availability: Regulation of Uptake and Root Architecture.

Nitrogen availability is a major factor determining plant growth and productivity. Plants acquire nitrogen nutrients from the soil through their roots mostly in the form of ammonium and nitrate. Since these nutrients are scarce in natural soils, plants have evolved adaptive responses to cope with the environment. One of the most important responses is the regulation of nitrogen acquisition efficiency. This review provides an update on the molecular determinants of two major drivers of the nitrogen acquisition efficiency: (i) uptake activity (e.g. high-affinity nitrogen transporters) and (ii) root architecture (e.g. low-nitrogen-availability-specific regulators of primary and lateral root growth). Major emphasis is laid on the regulation of these determinants by nitrogen supply at the transcriptional and post-transcriptional levels, which enables plants to optimize nitrogen acquisition efficiency under low nitrogen availability.

Plant nitrogen assimilation and its regulation: a complex puzzle with missing pieces.

Nitrogen (N) is an essential element for plants that is available in agricultural soils mainly as macronutrients in the form of nitrate and ammonium. Interplay between high-affinity and low-affinity transporters ensures efficient uptake from the soil even under highly fluctuating N availability. After uptake, N assimilation comprises the reduction of nitrate to ammonium and its subsequent incorporation into amino acids. Amino acids, but also nitrate, are transported from root to shoot and vice versa. Most steps of N transport and assimilation are tightly controlled by a regulatory network acting both cell-autonomously and systemically. N sensors, transcription factors and further regulatory players have been identified during recent years, elucidating parts of the huge puzzle that represents the efficient use of N by plants.

Brachypodium: a promising hub between model species and cereals.

Brachypodium distachyon was proposed as a model species for genetics and molecular genomics in cereals less than 10 years ago. It is now established as a standard for research on C3 cereals on a variety of topics, due to its close phylogenetic relationship with Triticeae crops such as wheat and barley, and to its simple genome, its minimal growth requirement, and its short life cycle. In this review, we first highlight the tools and resources for Brachypodium that are currently being developed and made available by the international community. We subsequently describe how this species has been used for comparative genomic studies together with cereal crops, before illustrating major research fields in which Brachypodium has been successfully used as a model: cell wall synthesis, plant-pathogen interactions, root architecture, and seed development. Finally, we discuss the usefulness of research on Brachypodium in order to improve nitrogen use efficiency in cereals, with the aim of reducing the amount of applied fertilizer while increasing the grain yield. Several paths are considered, namely an improvement of either nitrogen remobilization from the vegetative organs, nitrate uptake from the soil, or nitrate assimilation by the plant. Altogether, these examples position the research on Brachypodium as at an intermediate stage between basic research, carried out mainly in Arabidopsis, and applied research carried out on wheat and barley, enabling a complementarity of the studies and reciprocal benefits.

Nitrogen metabolism meets phytopathology.

Nitrogen (N) is essential for life and is a major limiting factor of plant growth. Because soils frequently lack sufficient N, large quantities of inorganic N fertilizers are added to soils for crop production. However, nitrate, urea, and ammonium are a major source of global pollution, because much of the N that is not taken up by plants enters streams, groundwater, and lakes, where it affects algal production and causes an imbalance in aquatic food webs. Many agronomical data indicate that the higher use of N fertilizers during the green revolution had an impact on the incidence of crop diseases. In contrast, examples in which a decrease in N fertilization increases disease severity are also reported, indicating that there is a complex relationship linking N uptake and metabolism and the disease infection processes. Thus, although it is clear that N availability affects disease, the underlying mechanisms remain unclear. The aim of this review is to describe current knowledge of the mechanisms that link plant N status to the plant's response to pathogen infection and to the virulence and nutritional status of phytopathogens.

The plant RWP-RK transcription factors: key regulators of nitrogen responses and of gametophyte development.

The plant specific RWP-RK family of transcription factors, initially identified in legumes and Chlamydomonas, are found in all vascular plants, green algae, and slime molds. These proteins possess a characteristic RWP-RK motif, which mediates DNA binding. Based on phylogenetic and domain analyses, we classified the RWP-RK proteins of six different species in two subfamilies: the NIN-like proteins (NLPs), which carry an additional PB1 domain at their C-terminus, and the RWP-RK domain proteins (RKDs), which are divided into three subgroups. Although, the functional analysis of this family is still in its infancy, several RWP-RK proteins have a key role in regulating responses to nitrogen availability. The nodulation-specific NIN proteins are involved in nodule organogenesis and rhizobial infection under nitrogen starvation conditions. Arabidopsis NLP7 in particular is a major player in the primary nitrate response. Several RKDs act as transcription factors involved in egg cell specification and differentiation or gametogenesis in algae, the latter modulated by nitrogen availability. Further studies are required to extend the general picture of the functional role of these exciting transcription factors.

The Arabidopsis nitrate transporter NRT2.5 plays a role in nitrate acquisition and remobilization in nitrogen-starved plants.

Nitrogen is a key mineral nutrient playing a crucial role in plant growth and development. Understanding the mechanisms of nitrate uptake from the soil and distribution through the plant in response to nitrogen starvation is an important step on the way to improve nitrogen uptake and utilization efficiency for better growth and productivity of plants, and to prevent negative effects of nitrogen fertilizers on the environment and human health. In this study, we show that Arabidopsis NITRATE TRANSPORTER 2.5 (NRT2.5) is a plasma membrane-localized high-affinity nitrate transporter playing an essential role in adult plants under severe nitrogen starvation. NRT2.5 expression is induced under nitrogen starvation and NRT2.5 becomes the most abundant transcript amongst the seven NRT2 family members in shoots and roots of adult plants after long-term starvation. GUS reporter analyses showed that NRT2.5 is expressed in the epidermis and the cortex of roots at the root hair zone and in minor veins of mature leaves. Reduction of NRT2.5 expression resulted in a decrease in high-affinity nitrate uptake without impacting low-affinity uptake. In the background of the high-affinity nitrate transporter mutant nrt2.4, an nrt2.5 mutation reduced nitrate levels in the phloem of N-starved plants further than in the single nrt2.4 mutants. Growth analyses of multiple mutants between NRT2.1, NRT2.2, NRT2.4, and NRT2.5 revealed that NRT2.5 is required to support growth of nitrogen-starved adult plants by ensuring the efficient uptake of nitrate collectively with NRT2.1, NRT2.2 and NRT2.4 and by taking part in nitrate loading into the phloem during nitrate remobilization.