Bertrand Gakière

Why are literature data for H2O2 contents so variable? A discussion of potential difficulties in the quantitative assay of leaf extracts

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.

H2O2-Activated Up-Regulation of Glutathione in Arabidopsis Involves Induction of Genes Encoding Enzymes Involved in Cysteine Synthesis in the Chloroplast

Glutathione is a key player in cellular redox homeostasis and, therefore, in the response to H(2)O(2), but the factors regulating oxidation-activated glutathione synthesis are still unclear. We investigated H(2)O(2)-induced glutathione synthesis in a conditional Arabidopsis catalase-deficient mutant (cat2). Plants were grown from seed at elevated CO(2) for 5 weeks, then transferred to air in either short-day or long-day conditions. Compared to cat2 at elevated CO(2) or wild-type plants in any condition, transfer of cat2 to air in both photoperiods caused measurable oxidation of the leaf glutathione pool within hours. Oxidation continued on subsequent days and was accompanied by accumulation of glutathione. This effect was stronger in cat2 transferred to air in short days, and was not linked to appreciable increases in the extractable activities of or transcripts encoding enzymes involved in the committed pathway of glutathione synthesis. In contrast, it was accompanied by increases in serine, O-acetylserine, and cysteine. These changes in metabolites were accompanied by induction of genes encoding adenosine phosphosulfate reductase (APR), particularly APR3, as well as a specific serine acetyltransferase gene (SAT2.1) encoding a chloroplastic SAT. Marked induction of these genes was only observed in cat2 transferred to air in short-day conditions, where cysteine and glutathione accumulation was most dramatic. Unlike other SAT genes, which showed negligible induction in cat2, the relative abundance of APR and SAT2.1 transcripts was closely correlated with marker transcripts for H(2)O(2) signaling. Together, the data underline the importance of cysteine synthesis in oxidant-induced up-regulation of glutathione synthesis and suggest that the chloroplast makes an important contribution to cysteine production under these circumstances.

Lysine catabolism: a stress and development super-regulated metabolic pathway

Lysine is a nutritionally important essential amino acid whose level in plants is largely regulated by the rate of its synthesis. In some plant tissues and under some stress conditions, however, lysine is also efficiently catabolized into glutamate and several other stress-related metabolites by novel mechanisms of metabolic regulation. Lysine catabolism is important for mammalian brain function; it is possible that the generation of glutamate regulates nerve transmission signals via glutamate receptors. Plants also possess homologues of animal glutamate receptors. It is thus likely that lysine catabolism also regulates various plant processes via these receptors.

Effect of sulfur availability on the integrity of amino acid biosynthesis in plants

Summary.Amino acid levels in plants are regulated by a complex interplay of regulatory circuits at the level of enzyme activities and gene expression. Despite the diversity of precursors involved in amino acid biosynthesis as providing the carbon backbones, the amino groups and, for the amino acids methionine and cysteine, the sulfhydryl group and despite the involvement of amino acids as substrates in various downstream metabolic processes, the plant usually manages to provide relatively constant levels of all amino acids. Here we collate data on how amino acid homeostasis is shifted upon depletion of one of the major biosynthetic constituents, i.e., sulfur. Arabidopsis thaliana seedlings exposed to sulfate starvation respond with a set of adaptation processes to achieve a new balance of amino acid metabolism. First, metabolites containing reduced sulfur (cysteine, glutathione, S-adenosylmethionine) are reduced leading to a number of downstream effects. Second, the relative excess accumulation of N over S triggers processes to dump nitrogen in asparagine, glutamine and further N-rich compounds like ureides. Third, the depletion of glutathione affects the redox and stress response system of the glutathione-ascorbate cycle. Thus, biosynthesis of aromatic compounds is triggered to compensate for this loss, leading to an increased flux and accumulation of aromatic amino acids, especially tryptophan. Despite sulfate starvation, the homeostasis is kept, though shifted to a new state. This adaptation process keeps the plant viable even under an adverse nutritional status.

Mechanisms to account for maintenance of the soluble methionine pool in transgenic Arabidopsis plants expressing antisense cystathionine γ-synthase cDNA

To investigate the role of cystathionine gamma-synthase (CGS) in the regulation of methionine synthesis Arabidopsis plants were transformed with a full-length antisense CGS cDNA and transformants analysed. Plants that were heterozygous for the transgene showed a 20-fold reduction of CGS activity that was accompanied by severe growth retardation and morphological abnormalities, from germination to flowering. Application of exogenous methionine to the transgenic lines restored normal growth. Surprisingly, transformed Arabidopsis plants exhibited a modest decrease in methionine content (35% reduction of the wild-type level) but a seven-fold decrease in the soluble pool of S-methylmethionine (SMM), a compound that plays a major role in storage and transport of reduced sulphur and labile methyl moieties. Several mechanisms can account for the maintenance of the soluble pool of methionine. First, the observed 20-fold increase in O-phosphohomoserine, a substrate of CGS, could compensate for the depressed level of CGS polypeptide by increasing the net rate of catalysis supported by the remaining enzyme. Second, the transgenic plants exhibited a two-fold increased level of cystathionine beta-lyase, the second enzyme in the methionine biosynthetic pathway. This indicates that enzymes other than CGS are subjected to a regulatory control by methionine or one of its metabolites. In addition to these mechanisms affecting de novo methionine synthesis, the recruitment of SMM to produce methionine may account for the small change of methionine levels in transgenic lines.

Uracil salvage is necessary for early Arabidopsis development

Uridine nucleotides can be formed by energy-consuming de novo synthesis or by the energy-saving recycling of nucleobases resulting from nucleotide catabolism. Uracil phosphoribosyltransferases (UPRTs; EC 2.4.2.9) are involved in the salvage of pyrimidines by catalyzing the formation of uridine monophosphate (UMP) from uracil and phosphoribosylpyrophosphate. To date, UPRTs are described as non-essential, energy-saving enzymes. In the present work, the six genes annotated as UPRTs in the Arabidopsis genome are examined through phylogenetic and functional complementation approaches and the available T-DNA insertion mutants are characterized. We show that a single nuclear gene encoding a protein targeted to plastids, UPP, is responsible for almost all UPRT activity in Arabidopsis. The inability to salvage uracil caused a light-dependent dramatic pale-green to albino phenotype, dwarfism and the inability to produce viable progeny in loss-of-function mutants. Plastid biogenesis and starch accumulation were affected in all analysed tissues, with the exception of stomata. Therefore we propose that uracil salvage is of major importance for plant development.

The Bifunctional LKR/SDH Locus of Plants Also Encodes a Highly Active Monofunctional Lysine-Ketoglutarate Reductase Using a Polyadenylation Signal Located within an Intron

Both plants and animals catabolize lysine (Lys) via two consecutive enzymes, Lys-ketoglutarate reductase (LKR) and saccharopine dehydrogenase (SDH), which are linked on a single polypeptide encoded by a single LKR/SDHgene. We have previously shown that the ArabidopsisLKR/SDH gene also encodes a monofunctional SDH that is transcribed from an internal promoter. In the present report, we have identified two cDNAs derived from cotton (Gossypium hirsutum) boll abscission zone that encode a novel enzymatic form of Lys catabolism, i.e. a catabolic monofunctional LKR. The monofunctional LKR mRNA is also encoded by theLKR/SDH gene, using two weak polyadenylation sites located within an intron. In situ mRNA hybridization and quantitative reverse transcriptase-polymerase chain reaction analyses also suggest that the cotton monofunctional LKR is relatively abundantly expressed in parenchyma cells of the abscission zone. DNA sequence analysis of theLKR/SDH genes of Arabidopsis, maize (Zea mays), and tomato (Lycopersicon esculentum) suggests that these genes can also encode a monofunctional LKR mRNA by a similar mechanism. To test whether the LKR/SDH and monofunctional LKR enzymes possess different biochemical properties, we used recombinant Arabidopsis LKR/SDH and monofunctional LKR enzymes expressed in yeast (Saccharomyces cerevisiae) cells. The K m of the monofunctional LKR to Lys was nearly 10-fold lower than its counterpart that is linked to SDH. Taken together, our results suggest that theLKR/SDH locus of plants is a super-composite locus that can encode three related but distinct enzymes of Lys catabolism. These three enzymes apparently operate in concert to finely regulate Lys catabolism during plant development.

Over-expression of cystathionine γ-synthase in Arabidopsis thaliana leads to increased levels of methionine and S-methylmethionine

Cystathionine γ-synthase (CGS, EC 4.2.99.9), the first committed enzyme in methionine biosynthesis, was over-expressed in Arabidopsis thaliana by introducing in the genome of this plant the coding sequence of the Arabidopsis enzyme under the control of the cauliflower mosaic virus 35S promoter. In order to target the recombinant protein to the chloroplast, the transgene included the sequence encoding the N-terminal transit peptide of Arabidopsis CGS. CGS activity and polypeptide were increased several fold in these plants. There was a markedly increased level of soluble methionine in the leaves of the transformed plants, up to 15-fold, indicating that CGS is a rate-limiting enzyme in this metabolic pathway. In addition, the transformed plants strongly over-accumulated S-methylmethionine, but not S-adenosylmethionine, in agreement with the view that S-methylmethionine corresponds to a storage form of labile methyl groups in plants and/or plays a role in preventing S-adenosylmethionine accumulation. The same strategy was used to increase the level of cystathionine � -lyase (CBL, EC 4.4.1.8), the second committed enzyme in methionine biosynthesis, in transformed A. thaliana. Despite an increase in both CBL activity and polypeptide in transformed Arabidopsis plants over-expressing Arabidopsis CBL, there was very little change in the contents of soluble methionine and S-methylmethionine, suggesting strongly that CBL is not rate limiting in the methionine biosynthetic pathway.

NAD: Not just a pawn on the board of plant-pathogen interactions

Many metabolic processes that occur in living cells involve oxido-reduction (redox) chemistry underpinned by redox compounds such as glutathione, ascorbate and/or pyridine nucleotides. Among these redox carriers, nicotinamide adenine dinucleotide (NAD) is the cornerstone of cellular oxidations along catabolism and is therefore essential for plant growth and development. In addition to its redox role, there is now compelling evidence that NAD is a signal molecule controlling crucial functions like primary and secondary carbon metabolism. Recent studies using integrative -omics approaches combined with molecular pathology have shown that manipulating NAD biosynthesis and recycling lead to an alteration of metabolites pools and developmental processes, and changes in the resistance to various pathogens. NAD levels should now be viewed as a potential target to improve tolerance to biotic stress and crop improvement. In this paper, we review the current knowledge on the key role of NAD (and its metabolism) in plant responses to pathogen infections.Many metabolic processes that occur in living cells involve oxido-reduction (redox) chemistry underpinned by redox compounds such as glutathione, ascorbate and/or pyridine nucleotides. Among these redox carriers, nicotinamide adenine dinucleotide (NAD) is the cornerstone of cellular oxidations along catabolism and is therefore essential for plant growth and development. In addition to its redox role, there is now compelling evidence that NAD is a signal molecule controlling crucial functions like primary and secondary carbon metabolism. Recent studies using integrative -omics approaches combined with molecular pathology have shown that manipulating NAD biosynthesis and recycling lead to an alteration of metabolites pools and developmental processes, and changes in the resistance to various pathogens. NAD levels should now be viewed as a potential target to improve tolerance to biotic stress and crop improvement. In this paper, we review the current knowledge on the key role of NAD (and its metabolism) in plant responses to pathogen infections.

NAD acts as an integral regulator of multiple defense layers

NAD-mediated defense responses stimulate PAMP-triggered immunity in Arabidopsis by stimulating redox signaling and modulating the hormonal balance. Pyridine nucleotides, such as NAD, are crucial redox carriers and have emerged as important signaling molecules in stress responses. Previously, we have demonstrated in Arabidopsis (Arabidopsis thaliana) that the inducible NAD-overproducing nadC lines are more resistant to an avirulent strain of Pseudomonas syringae pv tomato (Pst-AvrRpm1), which was associated with salicylic acid-dependent defense. Here, we have further characterized the NAD-dependent immune response in Arabidopsis. Quinolinate-induced stimulation of intracellular NAD in transgenic nadC plants enhanced resistance against a diverse range of (a)virulent pathogens, including Pst-AvrRpt2, Dickeya dadantii, and Botrytis cinerea. Characterization of the redox status demonstrated that elevated NAD levels induce reactive oxygen species (ROS) production and the expression of redox marker genes of the cytosol and mitochondrion. Using pharmacological and reverse genetics approaches, we show that NAD-induced ROS production functions independently of NADPH oxidase activity and light metabolism but depends on mitochondrial respiration, which was increased at higher NAD. We further demonstrate that NAD primes pathogen-induced callose deposition and cell death. Mass spectrometry analysis reveals that NAD simultaneously induces different defense hormones and that the NAD-induced metabolic profiles are similar to those of defense-expressing plants after treatment with pathogen-associated molecular patterns. We thus conclude that NAD triggers metabolic profiles rather similar to that of pathogen-associated molecular patterns and discuss how signaling cross talk between defense hormones, ROS, and NAD explains the observed resistance to pathogens.