Françoise Daniel-Vedele

Nitrogen uptake, assimilation and remobilization in plants: challenges for sustainable and productive agriculture

BACKGROUND Productive agriculture needs a large amount of expensive nitrogenous fertilizers. Improving nitrogen use efficiency (NUE) of crop plants is thus of key importance. NUE definitions differ depending on whether plants are cultivated to produce biomass or grain yields. However, for most plant species, NUE mainly depends on how plants extract inorganic nitrogen from the soil, assimilate nitrate and ammonium, and recycle organic nitrogen. Efforts have been made to study the genetic basis as well as the biochemical and enzymatic mechanisms involved in nitrogen uptake, assimilation, and remobilization in crops and model plants. The detection of the limiting factors that could be manipulated to increase NUE is the major goal of such research. SCOPE An overall examination of the physiological, metabolic, and genetic aspects of nitrogen uptake, assimilation and remobilization is presented in this review. The enzymes and regulatory processes manipulated to improve NUE components are presented. Results obtained from natural variation and quantitative trait loci studies are also discussed. CONCLUSIONS This review presents the complexity of NUE and supports the idea that the integration of the numerous data coming from transcriptome studies, functional genomics, quantitative genetics, ecophysiology and soil science into explanatory models of whole-plant behaviour will be promising.

Bay-0 × Shahdara recombinant inbred line population: a powerful tool for the genetic dissection of complex traits in Arabidopsis

Abstract.Natural genetic variation in Arabidopsis is considerable, but has not yet been used extensively as a source of variants to identify new genes of interest. From the cross between two genetically distant ecotypes, Bay-0 and Shahdara, we generated a Recombinant Inbred Line (RIL) population dedicated to Quantitative Trait Locus (QTL) mapping. A set of 38 physically anchored microsatellite markers was created to construct a robust genetic map from the 420 F6 lines. These markers, evenly distributed throughout the five chromosomes, revealed a remarkable equilibrium in the segregation of parental alleles in the genome. As a model character, we have analysed the genetic basis of variation in flowering time in two different environments. The simultaneous mapping of both large- and small-effect QTLs responsible for this variation explained 90% of the total genotypic variance. Two of the detected QTLs colocalize very precisely with FRIGIDA and FLOWERING LOCUS C genes; we provide information on the polymorphism of genes confirming this hypothesis. Another QTL maps in a region where no QTL had been found previously for this trait. This confirms the accuracy of QTL detection using the Bay-0 × Shahdara RIL population, which constitutes the largest in size available so far in Arabidopsis. As an alternative to mutant analysis, this population represents a powerful tool which is currently being used to undertake the genetic dissection of complex metabolic pathways.

A unified nomenclature of NITRATE TRANSPORTER 1/PEPTIDE TRANSPORTER family members in plants

Members of the plant NITRATE TRANSPORTER 1/PEPTIDE TRANSPORTER (NRT1/PTR) family display protein sequence homology with the SLC15/PepT/PTR/POT family of peptide transporters in animals. In comparison to their animal and bacterial counterparts, these plant proteins transport a wide variety of substrates: nitrate, peptides, amino acids, dicarboxylates, glucosinolates, IAA, and ABA. The phylogenetic relationship of the members of the NRT1/PTR family in 31 fully sequenced plant genomes allowed the identification of unambiguous clades, defining eight subfamilies. The phylogenetic tree was used to determine a unified nomenclature of this family named NPF, for NRT1/PTR FAMILY. We propose that the members should be named accordingly: NPFX.Y, where X denotes the subfamily and Y the individual member within the species.

An Arabidopsis T-DNA mutant affected in Nrt2 genes is impaired in nitrate uptake

Expression analyses of Nrt2 plant genes have shown a strict correlation with root nitrate influx mediated by the high‐affinity transport system (HATS). The precise assignment of NRT2 protein function has not yet been possible due to the absence of heterologous expression studies as well as loss of function mutants in higher plants. Using a reverse genetic approach, we isolated an Arabidopsis thaliana knock‐out mutant where the T‐DNA insertion led to the complete deletion of the AtNrt2.1 gene together with the deletion of the 3′ region of the AtNrt2.2 gene. This mutant is impaired in the HATS, without being modified in the low‐affinity system. Moreover, the de‐regulated expression of a Nicotiana plumbaginifolia Nrt2 gene restored the mutant nitrate influx to that of the wild‐type. These results demonstrate that plant NRT2 proteins do have a role in HATS.

Analysis of the NRT2 Nitrate Transporter Family in Arabidopsis. Structure and Gene Expression

Nitrate is an essential element for plant growth, both as a primary nutrient in the nitrogen assimilation pathway and as an important signal for plant development. The uptake of nitrate from the soil and its translocation throughout the plant has been the subject of intensive physiological and molecular studies. Using a reverse genetic approach, the AtNRT2.1 gene has been shown to be involved in the inducible component of the high-affinity nitrate transport system in Arabidopsis. The Arabidopsis Genome Initiative has released nearly the whole genome sequence of Arabidopsis, allowing the identification of a small NRT2 multigene family in this species. Thus, we investigated the phylogenetic relationship between NRT2 proteins belonging to several kingdoms and compared the structure of the different members of the Arabidopsis family. We analyzed, by semiquantitative reverse transcriptase-polymerase chain reaction, the expression pattern of each gene depending on plant organ and development or nutritional status, and compared the relative level of each gene by real-time polymerase chain reaction. We also evaluated the significance of each paralog on the basis of the relative levels of gene expression. The results are discussed in relation with distinct roles for the individual members of the AtNRT2 family.

The nodule inception‐like protein 7 modulates nitrate sensing and metabolism in Arabidopsis

Nitrate is an essential nutrient, and is involved in many adaptive responses of plants, such as localized proliferation of roots, flowering or stomatal movements. How such nitrate-specific mechanisms are regulated at the molecular level is poorly understood. Although the Arabidopsis ANR1 transcription factor appears to control stimulation of lateral root elongation in response to nitrate, no regulators of nitrate assimilation have so far been identified in higher plants. Legume-specific symbiotic nitrogen fixation is under the control of the putative transcription factor, NIN, in Lotus japonicus. Recently, the algal homologue NIT2 was found to regulate nitrate assimilation. Here we report that Arabidopsis thaliana NIN-like protein 7 (NLP7) knockout mutants constitutively show several features of nitrogen-starved plants, and that they are tolerant to drought stress. We show that nlp7 mutants are impaired in transduction of the nitrate signal, and that the NLP7 expression pattern is consistent with a function of NLP7 in the sensing of nitrogen. Translational fusions with GFP showed a nuclear localization for the NLP7 putative transcription factor. We propose NLP7 as an important element of the nitrate signal transduction pathway and as a new regulatory protein specific for nitrogen assimilation in non-nodulating plants.

Regulation of Root Ion Transporters by Photosynthesis: Functional Importance and Relation with Hexokinase

Coordination between the activity of ion transport systems in the root and photosynthesis in the shoot is a main feature of the integration of ion uptake in the whole plant. However, the mechanisms that ensure this coordination are largely unknown at the molecular level. Here, we show that the expression of five genes that encode root NO3−, NH4+, and SO42− transporters in Arabidopsis is regulated diurnally and stimulated by sugar supply. We also provide evidence that one Pi and one K+ transporter also are sugar inducible. Sucrose, glucose, and fructose are able to induce expression of the ion transporter genes but not of the carboxylic acids malate and 2-oxoglutarate. For most genes investigated, induction by light and induction by sucrose are strongly correlated, indicating that they reflect the same regulatory mechanism (i.e., stimulation by photosynthates). The functional importance of this control is highlighted by the phenotype of the atnrt2 mutant of Arabidopsis. In this mutant, the deletion of the sugar-inducible NO3− transporter gene AtNrt2.1 is associated with the loss of the regulation of high-affinity root NO3− influx by light and sugar. None of the sugar analogs used (3-O-methylglucose, 2-deoxyglucose, and mannose) is able to mimic the inducing effect of sugars. In addition, none of the sugar-sensing mutants investigated (rsr1-1, sun6, and gin1-1) is altered in the regulation of AtNrt2.1 expression. These results indicate that the induction of AtNrt2.1 expression by sugars is unrelated to the main signaling mechanisms documented for sugar sensing in plants, such as regulation by sucrose, hexose transport, and hexokinase (HXK) sensing activity. However, the stimulation of AtNrt2.1 transcript accumulation by sucrose and glucose is abolished in an antisense AtHXK1 line, suggesting that HXK catalytic activity and carbon metabolism downstream of the HXK step are crucial for the sugar regulation of AtNrt2.1 expression.

Characterization of a Two-Component High-Affinity Nitrate Uptake System in Arabidopsis. Physiology and Protein-Protein Interaction

The identification of a family of NAR2-type genes in higher plants showed that there was a homolog in Arabidopsis (Arabidopsis thaliana), AtNAR2.1. These genes encode part of a two-component nitrate high-affinity transport system (HATS). As the Arabidopsis NRT2 gene family of nitrate transporters has been characterized, we tested the idea that AtNAR2.1 and AtNRT2.1 are partners in a two-component HATS. Results using the yeast split-ubiquitin system and Xenopus oocyte expression showed that the two proteins interacted to give a functional HATS. The growth and nitrogen (N) physiology of two Arabidopsis gene knockout mutants, atnrt2.1-1 and atnar2.1-1, one for each partner protein, were compared. Both types of plants had lost HATS activity at 0.2 mm nitrate, but the effect was more severe in atnar2.1-1 plants. The relationship between plant N status and nitrate transporter expression revealed a pattern that was characteristic of N deficiency that was again stronger in atnar2.1-1. Plants resulting from a cross between both mutants (atnrt2.1-1 × atnar2.1-1) showed a phenotype like that of the atnar2.1-1 mutant when grown in 0.5 mm nitrate. Lateral root assays also revealed growth differences between the two mutants, confirming that atnar2.1-1 had a stronger phenotype. To show that the impaired HATS did not result from the decreased expression of AtNRT2.1, we tested if constitutive root expression of a tobacco (Nicotiana plumbaginifolia) gene, NpNRT2.1, previously been shown to complement atnrt2.1-1, can restore HATS to the atnar2.1-1 mutant. These plants did not recover wild-type nitrate HATS. Taken together, these results show that AtNAR2.1 is essential for HATS of nitrate in Arabidopsis.

Quantitative Trait Loci Analysis of Nitrogen Use Efficiency in Arabidopsis

Improving plant nitrogen (N) use efficiency or controlling soil N requires a better knowledge of the regulation of plant N metabolism. This could be achieved using Arabidopsis as a model genetic system, taking advantage of the natural variation available among ecotypes. Here, we describe an extensive study of N metabolism variation in the Bay-0 × Shahdara recombinant inbred line population, using quantitative trait locus (QTL) mapping. We mapped QTL for traits such as shoot growth, total N, nitrate, and free-amino acid contents, measured in two contrasting N environments (contrasting nitrate availability in the soil), in controlled conditions. Genetic variation and transgression were observed for all traits, and most of the genetic variation was identified through QTL and QTL × QTL epistatic interactions. The 48 significant QTL represent at least 18 loci that are polymorphic between parents; some may correspond to known genes from the N metabolic pathway, but others represent new genes controlling or interacting with N physiology. The correlations between traits are dissected through QTL colocalizations: The identification of the individual factors contributing to the regulation of different traits sheds new light on the relations among these characters. We also point out that the regulation of our traits is mostly specific to the N environment (N availability). Finally, we describe four interesting loci at which positional cloning is feasible.

Nitrate transport and signalling in Arabidopsis

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.