Plant adaptation to N availability : The NLP7-dependent nitrate signalling pathway

L’azote est un des macronutriments essentiels pour les plantes. Dans les sols, l’azote est présent sous différentes formes organiques et inorganiques. Les plantes utilisent préférentiellement le nitrate, qui n’est pas toujours disponible en quantités suffisantes dans les sols. Récemment, une étude a permis de montrer que le facteur de transcription NLP7 est un régulateur majeur de la réponse primaire au nitrate. La localisation subcellulaire de cette protéine est régulée par le nitrate : en son absence, elle est localisée dans le cytoplasme alors qu’après ajout de nitrate, une rétention nucléaire est activée. Les mécanismes moléculaires de cette rétention restent encore à comprendre ainsi que la transmission du signal nitrate, de l’extérieur de la plante à la protéine NLP7. Le transporteur de nitrate NPF6.3 a été montré comme jouant un rôle dans la perception du nitrate, c’est donc un transcepteur. Notre hypothèse était que NPF6.3 est le récepteur de nitrate en amont de NLP7. Pour tester cette hypothèse, nous avons étudié par des approches génétiques les liens d’épistasie entre les deux gènes. L’étude de la biomasse et de l’expression des gènes sentinelles en réponse au nitrate chez les simples et double mutants a permis d’observer des phénotypes additifs. Nous avons pu montrer que le mécanisme de relocalisation rapide de la protéine NLP7 dans le noyau est toujours actif dans le fond mutant npf6.3. Ces résultats ont donc permis de montrer que NLP7 et NPF6.3 n’appartiennent pas à la même voie de signalisation mais que ces deux voies pourraient être dépendantes selon les conditions. D’autre part, peu de régulateurs de la réponse au nitrate sont connus. De manière intéressante, les gènes cibles de NLP7 sont enrichis en protéines régulatrices comme par exemple d’autres facteurs de transcription ou encore des protéines kinases, ce qui place NLP7 à un haut niveau hiérarchique de régulation dans la voie de signalisation en réponse au nitrate. En effet, ces cibles directes de NLP7 pourraient elles-mêmes être impliquées dans des voies de signalisation en aval de NLP7. Dans le but de disséquer la voie de signalisation en aval de NLP7, nous avons étudié deux cibles directes de NLP7, des Mitogen-Activated Protein Kinase Kinase Kinases (MAPKKKs), MAPKKK13 et MAPKKK14. Les MAPKs sont connues pour leur mode d’action en cascades de phosphorylations. Par des approches biochimiques en protoplastes, nous avons montré que MAPKKK13/14 sont capables d’activer des MAPKs du groupe C via MKK3. De plus, nous avons obtenu de premières indications montrant que certaines réponses développementales au nitrate ainsi que la réponse primaire au nitrate seraient partiellement modifiées dans les simples mutants mapkkk13 et mapkk14, et dans le double mutant mapkkk13/14.Nitrogen is one of the most important macronutrients for plants. In the soils, nitrogen can be found under different organic and inorganic forms. Plants preferentially use nitrate, which is not always available for plant uptake and assimilation in soils. Recently, it was shown that the NLP7 transcription factor is a master regulator of the primary nitrate response. Its subcellular protein localization is regulated by nitrate: without nitrate, the protein is localised in the cytoplasm whereas after nitrate resupply, a nuclear retention is observed. Molecular mechanisms of this nuclear-cytosolic shuttling and of the nitrate signal transduction from the external medium to the NLP7 protein are still unknown. It has been shown that the NPF6.3 nitrate transporter plays a role in the nitrate signal sensing, which made this protein a transceptor. Our hypothesis was that NPF6.3 is the nitrate sensor upstream of NLP7. To test this hypothesis, we studied by genetic approaches the epistasis link between the two genes. By studying the simple and double mutant’s biomass and sentinel gene regulation in response to nitrate, we observed additive phenotypes. We showed that the nuclear relocation mechanism of NLP7 is still active in the npf6.3 mutant background. All together, these results showed that NLP7 and NPF6.3 are not in the same signalling pathway but there would be an interplay depending on the conditions. On the other hand, only a few regulators of the nitrate response are known. Interestingly the direct target genes of NLP7 are highly enriched for regulatory proteins such as other transcription factors or protein kinases, which places NLP7 at a high hierarchical place in the nitrate signalling pathway. Indeed these direct targets of NLP7 may themselves be involved in signalling cascades downstream of NLP7. In order to identify molecular events downstream of NLP7, we studies two NLP7 direct targets, Mitogen-Activated Protein Kinase Kinase Kinases (MAPKKKs), MAPKKK13 and MAPKKK14. MAPKs are known to act as phosphorylation cascades. Using biochemical approaches in protoplasts, we have shown that MAPKKKK13/14 are able to activate Group C MAPKs via MKK3. In addition we showed in planta that nitrate addition indeed triggered the activation of group C MAPKs and that this activation is dependent on NLP7 and MKK3. Furthermore we obtained first indications that nitrate-dependent developmental traits and the primary nitrate response are partially impaired in the single mutants mapkkk13 and mapkkk14, and the double mutant mpkkk13mpkkk14

Review: Mitogen-Activated protein kinases in nutritional signaling in Arabidopsis

International audienceMitogen-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

Brachypodium: a promising hub between model species and cereals

International audienceBrachypodium 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

Nitrate transport and signalling in [i]Arabidopsis[/i]

A large number of nitrate transporters ensure uptake and distribution of this main nutrient. Nitrate also acts as a signal, and elements of the signalling pathway have been identified.Plants have developed adaptive responses allowing them to cope with nitrogen (N) fluctuation in the soil and maintain growth despite changes in external N availability. Nitrate is the most important N form in temperate soils. Nitrate uptake by roots and its transport at the whole-plant level involves a large panoply of transporters and impacts plant performance. Four families of nitrate-transporting proteins have been identified so far: nitrate transporter 1/peptide transporter family (NPF), nitrate transporter 2 family (NRT2), the chloride channel family (CLC), and slow anion channel-associated homologues (SLAC/SLAH). Nitrate transporters are also involved in the sensing of nitrate. It is now well established that plants are able to sense external nitrate availability, and hence that nitrate also acts as a signal molecule that regulates many aspects of plant intake, metabolism, and gene expression. This review will focus on a global picture of the nitrate transporters so far identified and the recent advances in the molecular knowledge of the so-called primary nitrate response, the rapid regulation of gene expression in response to nitrate. The recent discovery of the NIN-like proteins as master regulators for nitrate signalling has led to a new understanding of the regulation cascade