Plant development and function are underpinned by redox reactions that depend on co-factors such as nicotinamide adenine dinucleotide (NAD). NAD has recently been shown to be involved in several signalling pathways that are associated with stress tolerance or defence responses. However, the mechanisms by which NAD influences plant gene regulation, metabolism and physiology still remain unclear. Here, we took advantage of Arabidopsis thaliana lines that overexpressed the nadC gene from E. coli, which encodes the NAD biosynthesis enzyme quinolinate phosphoribosyltransferase (QPT). Upon incubation with quinolinate, these lines accumulated NAD and were thus used as inducible systems to determine the consequences of an increased NAD content in leaves. Metabolic profiling showed clear changes in several metabolites such as aspartate-derived amino acids and NAD-derived nicotinic acid. Large-scale transcriptomic analyses indicated that NAD promoted the induction of various pathogen-related genes such as the salicylic acid (SA)-responsive defence marker PR1. Extensive comparison with transcriptomic databases further showed that gene expression under high NAD content was similar to that obtained under biotic stress, eliciting conditions or SA treatment. Upon inoculation with the avirulent strain of Pseudomonas syringae pv. tomato Pst-AvrRpm1, the nadC lines showed enhanced resistance to bacteria infection and exhibited an ICS1-dependent build-up of both conjugated and free SA pools. We therefore concluded that higher NAD contents are beneficial for plant immunity by stimulating SA-dependent signalling and pathogen resistance.

NAD and NADP are key brokers in cell redox and energy metabolism, but they also play important roles in signalling pathways. Several of these latter roles involve NAD cleavage, requiring continuous resynthesis of the molecule to maintain cellular pools. Mammals synthesize their cellular NAD from pyridine precursors that are obtained as niacin (vitamin B3) or produced from tryptophan. In many foods, the major source of niacin can be the pyridine ring of NAD and NADP. Plant NAD contents show considerable plasticity in response to environmental conditions, and recent advances have opened up new perspectives for rational manipulation of tissue NAD contents. In the model plant Arabidopsis, the de novo pathway of plant NAD synthesis has been identified, as have proteins involved in the subcellular transport of NAD and in NAD cleavage and recycling. Here, we discuss emerging concepts on the regulation of NAD contents in plants, and we report the use of transformation technology to enrich NAD several-fold in plant tissues through overexpression of the de novo synthesis pathway. Microarray analyses reveal that these increases trigger a characteristic transcriptomic signature, notably involving specific genes in defence phytohormone signalling. Thus, manipulation of plant NAD biosynthesis may offer interesting perspectives to (1) dissect redox-related signalling in stress responses, (2) enhance the nutritional value of plants, and (3) modify plant resistance to environmental stress.

Environmental controls on leaf NAD status remain poorly understood. Here, we analyzed the effects of two key environmental variables, CO2 and nitrogen, on leaf metabolite profiles, NAD status and the abundance of key transcripts involved in de novo NAD synthesis in wild-type (WT) Nicotiana sylvestris and the CMSII mutant that lacks respiratory complex I. High CO2 and increased N supply both significantly enhanced NAD(+) and NADH pools in WT leaves. In nitrogen-sufficient conditions, CMSII leaves were enriched in NAD(+) and NADH compared to the WT, but the differences in NADH were smaller at high CO2 than in air because high CO2 increased WT NADH/NAD(+). The CMSII-linked increases in NAD(+) and NADH status were abolished by growth with limited nitrogen, which also depleted the nicotine and nicotinic acid pools in the CMSII leaves. Few statistically significant genotype and N-dependent differences were detected in NAD synthesis transcripts, with effects only on aspartate oxidase and NAD synthetase mRNAs. Non-targeted metabolite profiling as well as quantitative amine analysis showed that NAD(+) and NADH contents correlated tightly with leaf amino acid contents across all samples. The results reveal considerable genotype- and condition-dependent plasticity in leaf NAD(+) and NADH contents that is not linked to modified expression of NAD synthesis genes at the transcript level and show that NAD(+) and NADH contents are tightly integrated with nitrogen metabolism. A regulatory two-way feedback circuit between nitrogen and NAD in the regulation of N assimilation is proposed that potentially links the nutritional status to NAD-dependent 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 (H(2)O(2)) responses through a combined genetic, transcriptomic, and redox profiling approach. To identify the potential role of changes in glutathione status in H(2)O(2) 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 H(2)O(2). 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 H(2)O(2)-glutathione interactions in cat2 gr1 double mutants established that GR1-dependent glutathione status is required for multiple responses to increased H(2)O(2) 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.

GENOPLANTE programme " Redoxome ". Financial support of postgraduate students from the following sources is gratefully acknowledged: the Tunisian Ministry of Higher Education Abstract Glutathione is a major cellular thiol that is maintained in the reduced state by glutathione reductases (GR), which is encoded by two genes in Arabidopsis thaliana (GR1 and GR2). This study addressed the role of GR1 in H 2 O 2 responses through a combined genetic, transcriptomic, and redox profiling approach. To identify the potential role of changes in glutathione status in H 2 O 2 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 H 2 O 2. 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 H 2 O 2-glutathione interactions in cat2 gr1 double mutants established that GR1-dependent glutathione status is required for multiple responses to increased H 2 O 2 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 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 glutathione reductase gene or by thiol systems such as the thioredoxin system. 5 Thiol-disulfide exchange plays crucial roles in protein structure, the regulation of enzymatic activity, and in redox signaling, and is principally mediated by thioredoxin (TRX) and glutathione reductase (GR)/glutathione systems (Buchanan and Balmer, 2005; Jacquot et al., 2008; Meyer et al., 2008). Arabidopsis lines identified in independent screens for alterations in heavy metal tolerance, meristem function, light signaling, and pathogen resistance have been shown to harbor mutations in the gene encoding the first enzyme of al., 2007). Studies on the rml1 mutant, which is severely deficient in glutathione synthesis, define a specific role for glutathione in root meristem function (Vernoux et al., 2000). However, shoot meristem function is regulated in a redundant manner by cytosolic glutathione and TRX (Reichheld …

Leaf metabolism produces H2O2 at high rates, but current concepts suggest that the potent signalling effects of this oxidant require that concentrations be controlled by a battery of antioxidative enzymes. The extent to which H2O2 is allowed to accumulate remains unclear. There is little consensus on leaf H2O2 values in the literature and measured concentrations in unstressed conditions range from 50-5000 nmol g(-1) fresh weight, a difference that probably reflects technical inaccuracies as much as biological variability. This article uses new experimental and literature data to examine some of the difficulties in accurately measuring H2O2 in leaf extracts. Potential problems relate to sensitivity, interference from other redox-active compounds, and H2O2 stability during sample preparation. Particular attention is drawn to the influence of tissue mass/extraction volume in the quantitative estimation of H2O2 contents, and the possibility that this factor could contribute to the variability of literature data.