Anne Krapp

An evaluation of direct and indirect mechanisms for the "sink-regulation" of photosynthesis in spinach: Changes in gas exchange, carbohydrates, metabolites, enzyme activities and steady-state transcript levels after cold-girdling source leaves

Mature source leaves of spinach (Spinacia oleracea L.) plants growing hydroponically in a 9 h light (350 μmol photons·m−2 · s−1)/15 h dark cycle at 20° C in a climate chamber were fitted with a cold girdle around the petiole, 2 h into the light period. Samples were taken 1, 3 and 7 h later, and at the end of the photoperiod for the following 4 d. Control samples were taken from ungirdled leaves. In the first 7 h after fitting the cold girdle there was (compared to the control leaves) a two to five-fold accumulation of sucrose, glucose, fructose and starch, a 40–50% increase of hexose-phosphates and ribulose-1,5-bisphosphate, a decrease of glycerate-3-phosphate, a small decrease in sucrose-phosphate synthase activation, an increase of fructose-2,6-bisphosphate, increased activation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), but no significant change in photosynthetic rate or stomatal conductance. Steady-state transcript levels for rbcS (small subunit of Rubisco) and atp-D (D-subunit of the thylakoid ATP synthase) decreased 30%, cab (chlorophyll-a-binding protein) decreased by 15% and agp-S (S-isoenzyme of ADP-glucose pyrophosphorylase) and nra (nitrate reductase) rose twofold. On the following days, levels of carbohydrates continued to rise and the changes of metabolites were maintained. Transcripts for rbcS, cab and atpD declined to 20, 70 and 25% of the control values. From day 3 onward the maximum activity of Rubisco declined. This was accompanied by a further increase of Rubisco activation to over 90% and, from day 4 onwards, an inhibition of photosynthesis which was associated with high internal CO2 concentration (ci), high ribulose-1,5-bisphosphate, and low glycerate-3-phosphate. When the cold-girdle was removed on day 5 there was a gradual recovery of photosynthesis and decline of ci over the next 2 d. Hexose-phosphates levels and transcripts for rbcS, cab and atp-D completely recovered within 2 d, even though the levels of carbohydrates had not fully recovered. Activity of Rubisco only reverted partly after 2 d, and Rubisco activation state and the ribulose-1,5-bisphosphate/glycerate-3-phosphate ratio were still higher than in control leaves. Transcripts for nra and agp-S were also still higher than in control leaves. It is concluded (i) that a reversible modulation of gene expression in response to the export rate plays a central role in the mid-term feedback “sink” regulation of photosynthesis, and (ii) that feedback regulation of CO2 fixation by changes of Pi are of little importance in spinach under these conditions. Further (iii) the rapid and reciprocal changes in nra and agpS transcripts, compared to rbcS, provide evidence that gene expression could also contribute to the modulation of nitrate assimilation and carbohydrate storage in conditions of decreased sink demand.

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.

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.

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.

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.

Sucrose-feeding leads to increased rates of nitrate assimilation, increased rates of alpha-oxoglutarate synthesis, and increased synthesis of a wide spectrum of amino acids in tobacco leaves

Abstract. To investigate the importance of the sugar supply for the regulation of nitrogen and organic acid metabolism, various sugars and nitrogenous compounds were supplied for 8 h to detached tobacco leaves in low light. (i) In control leaves supplied with water, there was a large decrease of the Nia transcript level, a 50% decline of nitrate reductase (NR) activity, starch increased and sugars remained low, nitrate decreased by 50%, and amino acids increased only slightly during the 8 h incubation. About half of the nitrogen accumulating in amino acids was present in glutamine (Gln). (ii) When 25 mM sucrose was supplied, the in-vivo rate of nitrate assimilation (estimated from the accumulation of ammonium and amino acids) increased 2-fold. The Nia transcript level still decreased, but the decline of NR activity was less pronounced and NR activation was increased. The in-vivo net rate of ammonium assimilation (estimated from the accumulation of amino acids) also doubled after feeding sucrose. Ammonium and glutamate (Glu) decreased and Gln rose markedly, showing that in-vivo activity of glutamine synthetase had been stimulated. Glutamine still accounted for about half of the nitrogen, indicating that sucrose does not selectively stimulate glutamine synthase. Glutamate and aspartate decreased and all the minor amino acids increased, showing that the amino acid biosynthesis pathways are activated by sucrose. There was a decrease of 3-phosphoglycerate (3PGA) and phosphoenolpyruvate (PEP) and a large increase of α-oxoglutarate, showing that the flow of carbon from glycolysis into organic acids has been stimulated by sucrose. (iii) The changes of 3PGA, PEP, α-oxoglutarate, Glu, aspartate and the minor amino acids were smaller when 50 mM glucose was supplied, even though the internal levels of sugars at the end of the incubation resembled those found after feeding 25 mM sucrose. This indicates that the signals that regulate nitrogen and respiratory metabolism are derived from the uptake or metabolism of sucrose, rather than glucose. (iv) A different spectrum of changes was found when 20 mM nitrate was supplied. The estimated rate of nitrate assimilation increased 2-fold, and this was accompanied by an increase of NR activity but not of NR activation. Nitrate-feeding did not lead to a decrease of Glu, and the increase of minor amino acids was slightly smaller than with sucrose. There was a decrease of sugars, starch, and hexose phosphates, but 3PGA and PEP were not significantly decreased and isocitrate increased instead of α-oxoglutarate. (v) A different spectrum of changes was also found when 10 mM Gln was supplied. The estimated rate of nitrate assimilation decreased, and this was accompanied by a decrease of NR activity and NR activation. Glutamate did not decrease, and the increase of minor amino acids was smaller than with sucrose. Starch and sugars remained high and, although hexose phosphates decreased, 3PGA and PEP were not significantly decreased. Isocitrate remained unaltered and the increase of α-oxoglutarate was smaller than after supplying sucrose. (vi) When 25 mM sucrose was added together with 20 mM nitrate or 10 mM Gln, the effect on NR activity, NR activation and the estimated rate of nitrate assimilation was additive to the effect of nitrate, and antagonistic to the effect of Gln. Sucrose still led to a decrease of Glu, an increase of the minor amino acids, a decrease of 3PGA and PEP, and an increase of α-oxoglutarate when it was supplied together with nitrate or Gln. (vii) It is concluded that sucrose initiates a co-ordinate activation of nitrate assimilation, ammonium assimilation, amino acid biosynthesis, and α-oxoglutarate synthesis. Sucrose acts in concert with nitrate and antagonistically to Gln to increase NR activity and nitrate assimilation, and complements the action of nitrate and Gln to increase the flow of nitrogen from ammonium into amino acids, and to increase α-oxoglutarate formation.

Nuclear retention of the transcription factor NLP7 orchestrates the early response to nitrate in plants

Nitrate is both an important nutrient and a signalling molecule for plants. Although several components of the nitrate signalling pathway have been identified, their hierarchical organization remains unclear. Here we show that the localization of NLP7, a member of the RWP-RK transcription factor family, is regulated by nitrate via a nuclear retention mechanism. Genome-wide analyses revealed that NLP7 binds and modulates a majority of known nitrate signalling and assimilation genes. Our findings indicate that plants, like fungi and mammals, rely on similar nuclear retention mechanisms to instantaneously respond to the availability of key nutrients.

PCR-identification of a Nicotiana plumbaginifolia cDNA homologous to the high-affinity nitrate transporters of the crnA family.

A family of high-affinity nitrate transporters has been identified in Aspergillus nidulans and Chlamydomonas reinhardtii, and recently homologues of this family have been cloned from a higher plant (barley). Based on six of the peptide sequences most strongly conserved between the barley and C. reinhardtii polypeptides, a set of degenerate primers was designed to permit amplification of the corresponding genes from other plant species. The utility of these primers was demonstrated by RT-PCR with cDNA made from poly(A)+ RNA from barley, C. reinhardtii and Nicotiana plumbaginifolia. A PCR fragment amplified from N. plumbaginifolia was used as probe to isolate a full-length cDNA clone which encodes a protein, NRT2;1Np, that is closely related to the previously isolated crnA homologue from barley. Genomic Southern blots indicated that there are only 1 or 2 members of the Nrt2 gene family in N. plumbaginifolia. Northern blotting showed that the Nrt2 transcripts are most strongly expressed in roots. The effects of external treatments with different N sources showed that the regulation of the Nrt2 gene(s) is very similar to that reported for nitrate reductase and nitrite reductase genes: their expression was strongly induced by nitrate but was repressed when reduced forms of N were supplied to the roots.

Regulation of Root Nitrate Uptake at the NRT2.1 Protein Level in Arabidopsis thaliana

In Arabidopsis the NRT2.1 gene encodes a main component of the root high-affinity nitrate uptake system (HATS). Its regulation has been thoroughly studied showing a strong correlation between NRT2.1 expression and HATS activity. Despite its central role in plant nutrition, nothing is known concerning localization and regulation of NRT2.1 at the protein level. By combining a green fluorescent protein fusion strategy and an immunological approach, we show that NRT2.1 is mainly localized in the plasma membrane of root cortical and epidermal cells, and that several forms of the protein seems to co-exist in cell membranes (the monomer and at least one higher molecular weight complex). The monomer is the most abundant form of NRT2.1, and seems to be the one involved in \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{NO}_{3}^{-}\) \end{document} transport. It strictly requires the NAR2.1 protein to be expressed and addressed at the plasma membrane. No rapid changes in NRT2.1 abundance were observed in response to light, sucrose, or nitrogen treatments that strongly affect both NRT2.1 mRNA level and HATS activity. This suggests the occurrence of post-translational regulatory mechanisms. One such mechanism could correspond to the cleavage of NRT2.1 C terminus, which results in the presence of both intact and truncated proteins in the plasma membrane.