Mathilde Orsel

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.

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.

The Arabidopsis ATNRT2.7 Nitrate Transporter Controls Nitrate Content in Seeds

In higher plants, nitrate is taken up by root cells where Arabidopsis thaliana NITRATE TRANSPORTER2.1 (ATNRT2.1) chiefly acts as the high-affinity nitrate uptake system. Nitrate taken up by the roots can then be translocated from the root to the leaves and the seeds. In this work, the function of the ATNRT2.7 gene, one of the seven members of the NRT2 family in Arabidopsis, was investigated. High expression of the gene was detected in reproductive organs and peaked in dry seeds. β-Glucuronidase or green fluorescent protein reporter gene expression driven by the ATNRT2.7 promoter confirmed this organ specificity. We assessed the capacity of ATNRT2.7 to transport nitrate in Xenopus laevis oocytes or when it is expressed ectopically in mutant plants deficient in nitrate transport. We measured the impact of an ATNRT2.7 mutation and found no difference from the wild type during vegetative development. By contrast, seed nitrate content was affected by overexpression of ATNRT2.7 or a mutation in the gene. Finally, we showed that this nitrate transporter protein was localized to the vacuolar membrane. Our results demonstrate that ATNRT2.7 plays a specific role in nitrate accumulation in the seed.

The Arabidopsis Nitrate Transporter NRT2.4 Plays a Double Role in Roots and Shoots of Nitrogen-Starved Plants

The NITRATE TRANSPORTER2.4 (NRT2.4) is a member of the NRT2 gene family in Arabidopsis thaliana and is preferentially expressed under nitrogen starvation. This study shows that NRT2.4 is a nitrate transporter involved both in root nitrate uptake at very low nitrate concentration and in delivering nitrate to the shoot phloem under nitrogen starvation. Plants have evolved a variety of mechanisms to adapt to N starvation. NITRATE TRANSPORTER2.4 (NRT2.4) is one of seven NRT2 family genes in Arabidopsis thaliana, and NRT2.4 expression is induced under N starvation. Green fluorescent protein and β-glucuronidase reporter analyses revealed that NRT2.4 is a plasma membrane transporter expressed in the epidermis of lateral roots and in or close to the shoot phloem. The spatiotemporal expression pattern of NRT2.4 in roots is complementary with that of the major high-affinity nitrate transporter NTR2.1. Functional analysis in Xenopus laevis oocytes and in planta showed that NRT2.4 is a nitrate transporter functioning in the high-affinity range. In N-starved nrt2.4 mutants, nitrate uptake under low external supply and nitrate content in shoot phloem exudates was decreased. In the absence of NRT2.1 and NRT2.2, loss of function of NRT2.4 (triple mutants) has an impact on biomass production under low nitrate supply. Together, our results demonstrate that NRT2.4 is a nitrate transporter that has a role in both roots and shoots under N starvation.

Plant homologs of the Plasmodium falciparum chloroquine-resistance transporter, PfCRT, are required for glutathione homeostasis and stress responses

In Arabidopsis thaliana, biosynthesis of the essential thiol antioxidant, glutathione (GSH), is plastid-regulated, but many GSH functions, including heavy metal detoxification and plant defense activation, depend on cytosolic GSH. This finding suggests that plastid and cytosol thiol pools are closely integrated and we show that in Arabidopsis this integration requires a family of three plastid thiol transporters homologous to the Plasmodium falciparum chloroquine-resistance transporter, PfCRT. Arabidopsis mutants lacking these transporters are heavy metal-sensitive, GSH-deficient, and hypersensitive to Phytophthora infection, confirming a direct requirement for correct GSH homeostasis in defense responses. Compartment-specific measurements of the glutathione redox potential using redox-sensitive GFP showed that knockout of the entire transporter family resulted in a more oxidized glutathione redox potential in the cytosol, but not in the plastids, indicating the GSH-deficient phenotype is restricted to the cytosolic compartment. Expression of the transporters in Xenopus oocytes confirmed that each can mediate GSH uptake. We conclude that these transporters play a significant role in regulating GSH levels and the redox potential of the cytosol.

Arabidopsis Roots and Shoots Show Distinct Temporal Adaptation Patterns toward Nitrogen Starvation

Nitrogen (N) is an essential macronutrient for plants. N levels in soil vary widely, and plants have developed strategies to cope with N deficiency. However, the regulation of these adaptive responses and the coordinating signals that underlie them are still poorly understood. The aim of this study was to characterize N starvation in adult Arabidopsis (Arabidopsis thaliana) plants in a spatiotemporal manner by an integrative, multilevel global approach analyzing growth, metabolites, enzyme activities, and transcript levels. We determined that the remobilization of N and carbon compounds to the growing roots occurred long before the internal N stores became depleted. A global metabolite analysis by gas chromatography-mass spectrometry revealed organ-specific differences in the metabolic adaptation to complete N starvation, for example, for several tricarboxylic acid cycle intermediates, but also for carbohydrates, secondary products, and phosphate. The activities of central N metabolism enzymes and the capacity for nitrate uptake adapted to N starvation by favoring N remobilization and by increasing the high-affinity nitrate uptake capacity after long-term starvation. Changes in the transcriptome confirmed earlier studies and added a new dimension by revealing specific spatiotemporal patterns and several unknown N starvation-regulated genes, including new predicted small RNA genes. No global correlation between metabolites, enzyme activities, and transcripts was evident. However, this multilevel spatiotemporal global study revealed numerous new patterns of adaptation mechanisms to N starvation. In the context of a sustainable agriculture, this work will give new insight for the production of crops with increased N use efficiency.

Disruption of the nitrate transporter genes AtNRT2.1 and AtNRT2.2 restricts growth at low external nitrate concentration

The high-affinity transport systems in Arabidopsis thaliana (L.) Heynh. involve potentially seven genes. Among these, the AtNRT2.1 and/or AtNRT2.2 genes have been shown to play a major role in the inducible component of this transport system. The physiological impact of a disruption of AtNRT2.1 and AtNRT2.2 on plant growth and N-metabolism was investigated. The reduced nitrate uptake in the mutant under a limiting N-regime was found to correlate with a significant difference in shoot/root ratio between wild type and mutant and a drastically reduced nitrate level in the shoot of the mutant. Carbohydrate analyses of plants under a low nitrate supply revealed a slight increase in glucose and fructose in the mutant shoots as well as an increase in sucrose and starch contents in mutant shoots. Interestingly, the AtNRT2.4 and AtNRT2.5 genes were over-expressed in the mutant growing in reduced N-conditions, without any compensation by root nitrate influx. These results are discussed in the context of the putative role of the different NRT2 genes.

Sixteen cytosolic glutamine synthetase genes identified in the Brassica napus L. genome are differentially regulated depending on nitrogen regimes and leaf senescence

Summary BnaGLN1 coding sequences and expression profiles in response to nitrogen availability and ageing are essentially conserved compared with A. thaliana, suggesting that the roles of GLN1 families are conserved among the Brassiceae tribe.

Multiscale investigation of mealiness in apple: an atypical role for a pectin methylesterase during fruit maturation

BackgroundApple fruit mealiness is one of the most important textural problems that results from an undesirable ripening process during storage. This phenotype is characterized by textural deterioration described as soft, grainy and dry fruit. Despite several studies, little is known about mealiness development and the associated molecular events. In this study, we integrated phenotypic, microscopic, transcriptomic and biochemical analyses to gain insights into the molecular basis of mealiness development.ResultsInstrumental texture characterization allowed the refinement of the definition of apple mealiness. In parallel, a new and simple quantitative test to assess this phenotype was developed.Six individuals with contrasting mealiness were selected among a progeny and used to perform a global transcriptome analysis during fruit development and cold storage. Potential candidate genes associated with the initiation of mealiness were identified. Amongst these, the expression profile of an early down-regulated transcript similar to an Arabidopsis thaliana pectin methylesterase gene (AtPME2) matched with mealiness development. In silico analyses of this Malus x domestica PME gene (MdPME2) confirmed its specific pattern compared with all other identified MdPME genes. Protein fusion experiments showed that MdPME2 is secreted into the apoplast in accordance with a possible activity on pectin structure. Further microscopic analysis indicated a progressive loss of cell to cell adhesion in mealy apple fruits. Biochemical analysis revealed specific modifications of pectin residues associated with mealiness, without global changes in the degree of methylesterification of pectins.ConclusionsThese data support the role of PME in cell wall remodelling during apple fruit development and ripening and suggest a local action of these enzymes. Mealiness may partially result from qualitative and spatial variations of pectin microarchitecture rather than quantitative pectin differences, and these changes may occur early in fruit development. The specific MdPME2 gene highlighted in this study could be a good early marker of texture unfavourable trait in apple.