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NRT2.1 can be phosphorylated at five different sites within N- and C-terminus. Here, we provide a systematic functional characterization of phosphorylation at S21 and S28 within the N-terminus of NRT2.1. We used existing phosphoproteomic data sets of nitrate starvation and nitrate resupply to construct a site-specific correlation network identifying kinase candidates to phosphorylate NRT2.1. By this approach, we identified NITRATE UPTAKE REGULATORY KINASE 1 (AT5G49770) which itself was regulated by phosphorylation at S839 and S870 within its kinase domain. In the active state, when S839 was dephosphorylated and S870 was phosphorylated, NURK1 was found to interact with NRT2.1 at dephosphorylated S28. Upon that interaction, NURK1 can phosphorylate NRT2.1 at S21. Phosphorylation of NRT2.1 at S21 resulted in low interaction of NRT2.1 with its activator protein NAR2.1. By contrast, phosphorylation of NRT2.1 at S28 by a yet unknown kinase enhanced the interaction with NAR2.1, but inhibited the interaction with NURK1. We propose that serines S21 and S28 are involved in a phospho-switch mechanism and by which the interaction of NRT2.1 with its activator NAR2.1, and thus NRT2.1 activity, is modulated. NURK1 here was identified as the kinase affecting this phospho-switch through phosphorylation of NRT2.1 at S21 leading to inactivation of NRT2.1.

While decades of research have considered redox metabolism as purely defensive, recent results show that ROS are necessary for growth and development. Strikingly, close relationships were found between the regulation of nitrogen (N) metabolism and ROS in response to both carbon (C) and N availability. Root NO3- uptake and N metabolism have been shown to be regulated by a signal coming from the Oxidative Pentose Pathway in response to C signaling. As a major source of NADP(H), OPPP is critical to maintain redox balance under stress situations. Furthermore, recent results suggest that at least part of the regulation of root NO3- transporter by N signaling is also linked to the redox status of the plant. This leads to the question of a more general role of redox metabolism in the regulation of N metabolism by C and N. This review will (i) highlight the role of OPPP in C signaling and redox metabolism and (ii) the interaction between redox and N metabolism. We will then discuss how redox metabolism could be an important player in the regulation of N metabolism in response to C/N interaction and the implication for plants' adaptation to extreme environment and the development of future crops.

Reactive oxygen species (ROS) can accumulate in cells at excessive levels, leading to unbalanced redox states and to potential oxidative stress, which can have damaging effects on the molecular components of plant cells. Several environmental conditions have been described as causing an elevation of ROS production in plants. Consequently, activation of detoxification responses is necessary to maintain ROS homeostasis at physiological levels. Misregulation of detoxification systems during oxidative stress can ultimately cause growth retardation and developmental defects. Here, we demonstrate that Arabidopsis (Arabidopsis thaliana) plants grown in a high nitrogen (N) environment express a set of genes involved in detoxification of ROS that maintain ROS at physiological levels. We show that the chromatin factor HIGH NITROGEN INSENSITIVE9 (HNI9) is an important mediator of this response and is required for the expression of detoxification genes. Mutation in HNI9 leads to elevated ROS levels and ROS-dependent phenotypic defects under high but not low N provision. In addition, we identify ELONGATED HYPOCOTYL5 as a major transcription factor required for activation of the detoxification program under high N. Our results demonstrate the requirement of a balance between N metabolism and ROS production, and our work establishes major regulators required to control ROS homeostasis under conditions of excess N.

In Arabidopsis thaliana, the High-Affinity Transport System (HATS) for root NO3 - uptake depends mainly on four NRT2 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 have been shown to 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 genes 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 highlighted the potential role of three putative transcription factors, TGA3, MYC1 and bHLH093. Functional analysis of mutants combined with yeast one hybrid experiments confirmed that all 3 transcription factors are regulators of NRT2.4 or NRT2.5 in response to N or C signals.

Membrane Transport in Plants

Understanding the plant response to nitrate availability is crucial for sustainable agriculture. In viticulture, there is an additional element to consider: the choice of scion–rootstock couple, which allows the management of environmental cues (including nitrate availability) and productivity. Using the two rootstocks 1103 Paulsen and Riparia Gloire de Montpellier, known to confer different vigour to grafted Cabernet Sauvignon scions, Cochetel et al. (2017) have now performed the first genome-wide transcriptome study indicating the genetic basis of the response to heterogeneous nitrate supply in this situation.

The nitrate signalling pathway now has a backbone. CPK calcium-dependent kinases are the long-awaited molecular link between major players in this pathway, the membrane-located nitrate transceptor NRT1.1 and the NLP transcription factors.

Article accepté; For microorganisms and plants, nitrate and ammonium are the main nitrogen sources and they are also important signaling molecules controlling several aspects of metabolism and development. Over the past decade, numerous studies revealed that nitrogen transporters are strongly regulated at the transcriptional level. However, more and more reports are now showing that nitrate and ammonium transporters are also subjected to post-translational regulations in response to nitrogen availability. Phosphorylation is so far the most well studied post-translational modification for these transporters and it affects both the regulation of nitrogen uptake and nitrogen sensing. For example, in Arabidopsis thaliana, phosphorylation was shown to activate the sensing function of the root nitrate transporter NRT1.1 and to switch the transport affinity. Also, for ammonium transporters, a phosphorylation-dependent activation/inactivation mechanism was elucidated in recent years in both plants and microorganisms. However, despite the fact that these regulatory mechanisms are starting to be thoroughly described, the signaling pathways involved and their action on nitrogen transporters remain largely unknown. In this review, we highlight the inorganic nitrogen transporters regulated at the post-translational level and we compare the known mechanisms in plants and microorganisms. We then discuss how these mechanisms could contribute to the regulation of nitrogen uptake and/or nitrogen sensing.

As a main feature of plant autotrophy, assimilation of inorganic nitrogen is not only of fundamental scientific interest, but also a crucial factor in crop productivity. In its broad sense – from root uptake of various forms of N in the soil to allocation of N assimilates to different organs – it involves a wide range of physiological processes whose mechanisms are far from being fully understood. The aim of this special issue is to provide a wide overview of recent progress in this field, and to draw an interdisciplinary picture of the prospects for future research.

N-fixing nodules are new organs formed on legume roots as result of the beneficial interaction with soil bacteria, rhizobia. The nodule functioning is still a poorly characterized step of the symbiotic interaction as only few of the genes induced in N-fixing nodules have been functionally characterized. We present here the characterization of the member of the Lotus japonicus nitrate transporter 1/peptide transporter family LjNPF8.6. The phenotypic characterization carried out in independent L. japonicus LORE1 insertion lines indicates a positive role of LjNPF8.6 on nodule functioning as knock out mutants display N-fixation deficiency (25%) and increased nodular superoxide content. The partially compromised nodule functioning induces two striking phenotypes: anthocyanin accumulation already displayed four weeks after inoculation and shoot biomass deficiency, which is detected by long-term phenotyping. LjNPF8.6 achieves nitrate uptake in Xenopus laevis oocytes both at 0.5 mM and 30 mM external concentrations and a possible role as nitrate transporter on the control of N-fixing nodule activity is discussed.

Most plants use nitrate (NO3-) as their major nitrogen (N) source. The NO3- uptake capacity of a plant is determined by three interdependent factors that are sensitive to NO3- availability: (i) the functional properties of the transporters in the roots that contribute to the acquisition of NO3- from the external medium, (ii) the density of functional transporters at the plasma membrane of root cells, and (iii) the surface and architecture of the root system. The identification of factors that regulate the NO3--sensing systems is important for both fundamental and applied science, because these factors control the capacity of plants to use the available NO3-, a process known as the “nitrate use efficiency”. The molecular component of the transporters involved in uptake and sensing mechanism in Arabidopsis roots are presented and their relative contribution discussed.

Economically viable and productive farming systems are required to meet the growing worldwide need for agricultural produce while at the same time reducing environmental impact. Within grazing systems of animal production, increasing concern exists as to the effect of intensive farming on potential N losses to ground and surface waters, which demands an appraisal of N flows within complete grass-based dairy farming systems. A 3-yr (2011 to 2013) whole-farm system study was conducted on a free-draining soil type that is highly susceptible to N loss under temperate maritime conditions. Soil solution concentrations of N from 3 spring-calving, grass-based systems designed to represent 3 alternative whole-farm stocking rate (SR) treatments in a post-milk quota situation in the European Union were compared: low (2.51 cows/ha), medium (2.92 cows/ha), and high SR (3.28 cows/ha). Each SR had its own farmlet containing 18 paddocks and 23 cows. Nitrogen loss from each treatment was measured using ceramic cups installed to a depth of 1 m to sample the soil water. The annual and monthly average nitrate, nitrite, ammonia, and total N concentrations in soil solution collected were analyzed for each year using a repeated measures analysis. Subsequently, and based on the biological data collated from each farm system treatment within each year, the efficiency of N use was evaluated using an N balance model. Based on similar N inputs, increasing SR resulted in increased grazing efficiency and milk production per hectare. Stocking rate had no significant effect on soil solution concentrations of nitrate, nitrite, ammonia, or total N (26.0, 0.2, 2.4, and 32.3 mg/L, respectively). An N balance model evaluation of each treatment incorporating input and output data indicated that the increased grass utilization and milk production per hectare at higher SR resulted in a reduction in N surplus and increased N use efficiency. The results highlight the possibility for the sustainable intensification of grass-based dairy systems and suggest that, at the same level of N inputs, increasing SR has little effect on N loss in pastoral systems with limited imported feed. These results suggest that greater emphasis should be attributed to increased grass production and utilization under grazing to further improve the environmental impact of grazing systems.

International audience; Nitrogen (N) is one of the key mineral nutrients for plants and its availability has a major impact on their growth and development. The main N sources for terrestrial plants in soils is nitrate (NO3-) and most often it is a growth limiting factor. Plants are able to sense NO3- in their environment, allowing them to quickly respond to the dramatic fluctuations of its availability. Significant advances have been made during the recent period concerning the molecular mechanisms of NO3- sensing and signalling in higher plants. The striking action of NO3- as a signal molecule on genome expression has been unravelled. Note worthily, NO3- sensing systems have been identified. These unexpectedly correspond to membrane transporters also ensuring the uptake of NO3- into root cells, thus generalizing the nutrient ‘transceptor’ (transporter/receptor) concept defined in yeast. Furthermore, components of the downstream transduction cascades, such as transcription factors or kinases, have also been isolated. Recently, a major breakthrough arising from this improved knowledge is a better understanding of the integration of NO3- and hormone signalling pathways, that explains the extraordinary developmental plasticity of plants in response to NO3-.

The Arabidopsis NO3- efflux transporter NAXT2 is involved in root-to-shoot NO3- translocation and contributes to plant tolerance to salt stress. Salt & Water Stress in Plants

L'article original est publié par The American Society of Plant Biologists; International audience; In Arabidopsis (Arabidopsis thaliana), the NRT2.1 gene codes for the main component of the root nitrate (NO(3)(-)) high-affinity transport system (HATS). Due to the strong correlation generally found between high-affinity root NO(3)(-) influx and NRT2.1 mRNA level, it has been postulated that transcriptional regulation of NRT2.1 is a key mechanism for modulation of the HATS activity. However, this hypothesis has never been demonstrated, and is challenged by studies suggesting the occurrence of posttranscriptional regulation at the NRT2.1 protein level. To unambiguously clarify the respective roles of transcriptional and posttranscriptional regulations of NRT2.1, we generated transgenic lines expressing a functional 35S::NRT2.1 transgene in an atnrt2.1 mutant background. Despite a high and constitutive NRT2.1 transcript accumulation in the roots, the HATS activity was still down-regulated in the 35S::NRT2.1 transformants in response to repressive nitrogen or dark treatments that strongly reduce NRT2.1 transcription and NO(3)(-) HATS activity in the wild type. In some treatments, this was associated with a decline of NRT2.1 protein abundance, indicating posttranscriptional regulation of NRT2.1. However, in other instances, NRT2.1 protein level remained constant. Changes in abundance of NAR2.1, a partner protein of NRT2.1, closely followed those of NRT2.1, and thus could not explain the close-to-normal regulation of the HATS in the 35S::NRT2.1 transformants. Even if in certain conditions the transcriptional regulation of NRT2.1 contributes to a limited extent to the control of the HATS, we conclude from this study that posttranscriptional regulation of NRT2.1 and/or NAR2.1 plays a predominant role in the control of the NO(3)(-) HATS in Arabidopsis.

The relationship between nitrate influx, BnNrt2 nitrate transporter gene expression and amino acid composition of phloem exudate was investigated during N-deprivation (short-term experiment) and over a growth cycle (long-term experiment) in Brassica napus L. The data showed a positive correlation between γ-aminobutyric acid (GABA) in phloem exudate and nitrate uptake in the short- and the long-term experiments. The hypothesis that this non-protein amino acid could up-regulate nitrate uptake via a long-distance signalling pathway was tested by providing an exogenous GABA supply to the roots. The effect of GABA was compared with the effects of Gln, Glu and Asn, each known to be inhibitors of nitrate uptake. The results showed that GABA treatment induced a significant increase of BnNrt2 mRNA expression, but had less effect on nitrate influx. By contrast, Gln, Glu and Asn significantly reduced nitrate influx and BnNrt2 mRNA expression compared with the control plants. This study provides the first evidence that GABA may act as a putative long-distance inter-organ signal molecule in plants in conjunction with negative control exerted by Gln. The up-regulation effect of GABA on nitrate uptake is discussed in the context of its role in N metabolism, nutritional stress and the recent discovery of a putative role of GABA as a signal molecule in plant development.