Houda Gouia

Arabidopsis GLUTATHIONE REDUCTASE1 Plays a Crucial Role in Leaf Responses to Intracellular Hydrogen Peroxide and in Ensuring Appropriate Gene Expression through Both Salicylic Acid and Jasmonic Acid Signaling Pathways

Glutathione is a major cellular thiol that is maintained in the reduced state by glutathione reductase (GR), which is encoded by two genes in Arabidopsis (Arabidopsis thaliana; GR1 and GR2). This study addressed the role of GR1 in hydrogen peroxide (H2O2) responses through a combined genetic, transcriptomic, and redox profiling approach. To identify the potential role of changes in glutathione status in H2O2 signaling, gr1 mutants, which show a constitutive increase in oxidized glutathione (GSSG), were compared with a catalase-deficient background (cat2), in which GSSG accumulation is conditionally driven by H2O2. Parallel transcriptomics analysis of gr1 and cat2 identified overlapping gene expression profiles that in both lines were dependent on growth daylength. Overlapping genes included phytohormone-associated genes, in particular implicating glutathione oxidation state in the regulation of jasmonic acid signaling. Direct analysis of H2O2-glutathione interactions in cat2 gr1 double mutants established that GR1-dependent glutathione status is required for multiple responses to increased H2O2 availability, including limitation of lesion formation, accumulation of salicylic acid, induction of pathogenesis-related genes, and signaling through jasmonic acid pathways. Modulation of these responses in cat2 gr1 was linked to dramatic GSSG accumulation and modified expression of specific glutaredoxins and glutathione S-transferases, but there is little or no evidence of generalized oxidative stress or changes in thioredoxin-associated gene expression. We conclude that GR1 plays a crucial role in daylength-dependent redox signaling and that this function cannot be replaced by the second Arabidopsis GR gene or by thiol systems such as the thioredoxin system.

Effects of NaCl on Flows of N and Mineral Ions and on NO3- Reduction Rate within Whole Plants of Salt-Sensitive Bean and Salt-Tolerant Cotton

The effects of NaCl on the transport rates of cations, NO3-, and reduced N compounds between roots and shoot and on NO3- assimilation rate were examined on plants of two species differing in their sensitivity to salinity, bean (Phaseolus vulgare L. cv Gabriella) and cotton (Gossypium hirsutum L. cv Akala). Biomass production after 20 d in response to 50 and 100 mM NaCl decreased by 48 and 59% in bean, but only 6 and 14% in cotton. The comparison of the flow patterns obtained for control and NaCl-fed plants showed that salinity induced a general decrease in all the fluxes involved in partitioning of N and the various ions. This decrease was markedly higher in bean than in cotton. Within either species, the different flows (uptake, xylem flux, phloem flux) of a given element were affected by NaCl to the same extent with minor exceptions. No specific effect of salinity on any of the components of N partitioning were discerned. The greater sensitivity of nitrate reductase activity to NaCl in bean leaves compared to cotton leaves seems to be due to a decreased compartmentalization of ions rather than to a difference in salt tolerance of the enzyme itself. Overall, our data show that alteration of mineral nutrition is not solely the reflection of a decreased growth rate, but also is a general process that impairs uptake of all the minerals even at mild NaCl salinity.

Cytosolic NADP-dependent isocitrate dehydrogenase contributes to redox homeostasis and the regulation of pathogen responses in Arabidopsis leaves

Cytosolic NADP-dependent isocitrate dehydrogenase (cICDH) produces 2-oxoglutarate (2-OG) and NADPH, and is encoded by a single gene in Arabidopsis thaliana. Three allelic lines carrying T-DNA insertions in this gene showed less than 10% extractable leaf ICDH activity, but only relatively small decreases in growth compared to wild-type Col0. Metabolite profiling by gas chromatography-time of flight-mass spectrometry (GC-TOF-MS) and high-performance liquid chromatography (HPLC) revealed that loss of cICDH function produced only small effects on leaf compounds involved in carbon and nitrogen assimilation. To analyse whether cICDH contributes to NADPH production under conditions of oxidative stress, the icdh mutation was introduced into the cat2 background, in which increased availability of H(2)O(2) causes perturbed redox homeostasis and induction of stress-related genes. Accumulation of oxidized glutathione and pathogen-related responses were enhanced in double cat2 icdh mutants compared to cat2. Single icdh mutants presented constitutive induction of PR genes, and enhanced resistance to bacteria in icdh, cat2 and cat2 icdh was quantitatively correlated with PR gene expression. However, the effect of icdh in both Col0 and cat2 backgrounds was not associated with enhanced accumulation of salicylic acid (SA). The results suggest that cICDH, previously considered mainly as an enzyme involved in amino acid synthesis, plays a role in redox signalling linked to pathogen responses.

Effects of cadmium on the co-ordination of nitrogen and carbon metabolism in bean seedlings.

The effect of cadmium (Cd) was investigated on the in vitro activities of leaf and root enzymes involved in carbon (C) and nitrogen (N) metabolism of bean (Phaseolus vulgaris L. cv. Morgane). Cd induced a high increase in maximal extractable activity of glutamate dehydrogenase (NADH-GDH, EC 1.4.1.2). Cd promoted ammonium accumulation in leaves and roots, and a tight correlation was observed between ammonium amount and GDH activity. Changes in GDH activity appear to be mediated by the increase in ammonium levels by Cd treatment. Cd stress also enhanced the activities of phosphoenolypyruvate carboxylase (PEPC, EC 4.1.1.31) and NADP(+)-isocitrate dehydrogenase (NADP(+)-ICDH, EC 1.1.1.42) in leaves while they were inhibited in roots. Immuno-titration, the PEPC sensitivity to malate and PEPC response to pH indicated that the increase in PEPC activity by Cd was due to de novo synthesis of the enzyme polypeptide and also modification of the phosphorylation state of the enzyme. Cd may have modified, via a modulation of PEPC activity, the C flow towards the amino acid biosynthesis. In leaves, Cd treatments markedly modified specific amino acid contents. Glutamate and proline significantly accumulated compared to those of the control plants. This study suggests that Cd stress is a part of the syndrome of metal toxicity, and that a readjustment of the co-ordination between N and C metabolism via the modulation of GDH, PEPC and ICDH activities avoided the accumulation of toxic levels of ammonium.

Redox and nitric oxide homeostasis are affected in tomato (Solanum lycopersicum) roots under salinity-induced oxidative stress

The nicotinamide adenine dinucleotide phosphate (NADPH) and reduced glutathione (GSH) molecules play important roles in the redox homeostasis of plant cells. Using tomato (Solanum lycopersicum) plants grown with 120mM NaCl, we studied the redox state of NADPH and GSH as well as ascorbate, nitric oxide (NO) and S-nitrosoglutathione (GSNO) content and the activity of the principal enzymes involved in the metabolism of these molecules in roots. Salinity caused a significant reduction in growth parameters and an increase in oxidative parameters such as lipid peroxidation and protein oxidation. Salinity also led to an overall decrease in the content of these redox molecules and in the enzymatic activities of the main NADPH-generating dehydrogenases, S-nitrosoglutathione reductase and catalase. However, NO content as well as gluthahione reductase and glutathione peroxidase activity increased under salinity stress. These findings indicate that salinity drastically affects redox and NO homeostasis in tomato roots. In our view, these molecules, which show the interaction between ROS and RNS metabolisms, could be excellent parameters for evaluating the physiological conditions of plants under adverse stress conditions.

Exogenous nitric oxide (NO) ameliorates salinity-induced oxidative stress in tomato (Solanum lycopersicum) plants

Abstract Nitric oxide (NO) is involved in numerous physiological and stress responses in higher plants. Tomato is one of the most important vegetable crops in the world and previously it has been reported that salinity induced an oxidative stress affecting its redox and NO homeostasis. Using tomato plant exposed to 120 mM NaCl, it was studied whether the exogenous application of NO could ameliorate the negative effects provoked by salinity. Thus, nitric oxide provoked a significantly increase in the main antioxidative enzymes including superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione reductase (GR) and peroxidase (POD) activities, and also a raise of some enzymes involved in nitrogen metabolism including nitrate reductase (NR) and nitrite reductase (NiR) activities. Moreover, NO-treated plants showed a higher content in both proline and ascorbate but lower content of H 2 O 2 . These data indicate that the exogenous NO application is useful to mitigate the salinity-induced oxidative stress in tomato plants.

Tissue-specific cadmium accumulation and its effects on nitrogen metabolism in tobacco (Nicotiana tabaccum, Bureley v. Fb9).

Tobacco (Nicotiana Tabaccum, Bureley v. Fb9) seedlings were grown for 30 days on control medium, and then treated for seven days with different concentrations (0, 10, 20, 50 and 100 muM) of CdCl(2). Cadmium (Cd) was mostly accumulated in the leaves. However, nitrate reductase and nitrite reductase activities (NR, EC 1.6.1.6 and NiR, EC 1.7.7.1) were more inhibited by Cd stress in the roots than in leaves. Glutamine synthetase activity (GS, EC 6.3.1.2) was inhibited by Cd treatment in roots and leaves. In both organs, aminating activity of glutamate dehydrogenase (GDH, EC 1.4.1.2) and protease activity were significantly stimulated in the leaves and roots of stressed plants. The lesser extents of Cd stress effects on leaves, despite their high Cd accumulation, suggest that: (i) tobacco leaves may evolve adaptive process to partially inactivate Cd ions; and (ii) tobacco is useful for phytoremediation.

Expression pattern of genes encoding nitrate and ammonium assimilating enzymes in Arabidopsis thaliana exposed to short term NaCl stress

Key steps in nitrate nutrition and assimilation were assessed over two weeks in control and 100mM NaCl-exposed Arabidopsis thaliana (Columbia) plants. The data showed that NaCl stress lowered nitrate contents in both leaves and roots. While NaCl stress decreased ammonium contents in leaves, it increased the contents in roots at the end of treatment. A survey of transcript levels of NIA1 (At1g77760) and NIA2 (At1g37130) and nitrate reductase (NR, EC 1.6.1.6) activity in the leaves and roots suggested a major role of NIA2 rather than NIA1 in the regulation of NR by salt stress. A drop in mRNA levels for GLN2 (At5g35630) and GLN1;2 (At1g66200) by salt was associated with a similar inhibition of glutamine synthetase (GS, EC 6.3.1.2) activity in the leaves. In the roots, NaCl stress was found to enhance mRNA levels of GLN2 and cytosolic-encoding genes (GLN1;1 (At5g37600) and GLN1;2).

An Arabidopsis mutant disrupted in ASN2 encoding asparagine synthetase 2 exhibits low salt stress tolerance

Salt tolerance of Arabidopsis knockout mutant with T-DNA insertion in ASN2 gene encoding asparagine synthetase (AS, EC 6.3.5.4) (asn2-1) was investigated. Wild-type Arabidopsis Col0 and asn2-1 mutant were grown for one month by hydroponic culture and subjected to 100 mM NaCl stress for a short time from 6 to 24 h. The salt treatment decreased chlorophyll and soluble protein contents, and increased ammonium level in the asn2-1 leaves. The salinity induced ASN1 mRNA level in the wild-type and asn2-1 leaves. By contrast, the salt treatment inhibited the transcript and protein levels of chloroplastic glutamine synthetase 2 (GS2, EC 6.3.1.2) in the wild-type and asn2-1 leaves. Increase in asparagine and proline contents in response to the salt treatment provides evidence for the role of asparagine as a prevailing stress responding amino acid. Glutamate dehydrogenase (NADH-GDH, EC 1.4.1.2) exhibited a slight increase in the α-subunit and β-subunit in the wild-type line and the asn2-1 line, respectively under the salinity, whereas its in vitro aminating activity in the wild-type leaves was not affected. The results indicate that the asn2-1 mutant was impaired in nitrogen assimilation and translocation under salt treatment.

Differential response of NADP-dehydrogenases and carbon metabolism in leaves and roots of two durum wheat (Triticum durum Desf.) cultivars (Karim and Azizi) with different sensitivities to salt stress.

Wheat (Triticum durum Desf.) is a common Mediterranean species of considerable agronomic importance. Salinity is one of the major threats to sustainable agricultural production mainly because it limits plant productivity. After exposing the Karim and Azizi durum wheat cultivars, which are of agronomic significance in Tunisia, to 100mM NaCl salinity, growth parameters (dry weight and length), proline content and chlorophylls were evaluated in their leaves and roots. In addition, we analyzed glutathione content and key enzymatic activities, including phosphoenolpyruvate carboxylase (PEPC), NADP-isocitrate dehydrogenase (NADP-ICDH), NADP-malic enzyme (NADP-ME), glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH), involved in the carbon metabolism and NADPH-generating system. The sensitivity index indicates that cv Karim was more tolerant to salinity than cv Azizi. This higher tolerance was corroborated at the biochemical level, as cv Karim showed a greater capacity to accumulate proline, especially in leaves, while the enzyme activities studied were differentially regulated in both organs, with NADP-ICDH being the only activity to be unaffected in all organs. In summary, the data indicate that higher levels of proline accumulation and the differential responses of some key enzymes involved in the carbon metabolism and NADPH regeneration contribute to the salinity tolerance mechanism and lead to increased biomass accumulation in cv Karim.