Abstract: The interest in reactive electrophile species (RES) stems largely from the fact that they can have powerful biological activities. RES stimulate the expression of cell survival genes as well many other genes commonly upregulated in environmental stress and pathogenesis. RES levels must be carefully controlled in healthy cells but their formation and destruction during stress is of great interest. Unlike many 'classical' signals and hormones, RES can potentially affect gene expression at all levels by chemically reacting with nucleic acids, proteins and small molecules as well as by indirectly lowering pools of cellular reductants. Recent works involving genetic approaches have begun to provide compelling evidence that, although excess RES production can lead to cell damage, lower levels of RES may modulate the expression of cell survival genes and may actually contribute to survival during severe stress.

Abstract: Malondialdehyde (MDA) is a small, ubiquitous, and potentially toxic aldehyde that is produced in vivo by lipid oxidation and that is able to affect gene expression. Tocopherol deficiency in the vitamin E2 mutant vte2-1 of Arabidopsis thaliana leads to massive lipid oxidation and MDA accumulation shortly after germination. MDA accumulation correlates with a strong visual phenotype (growth reduction, cotyledon bleaching) and aberrant GST1 (glutathione S-transferase 1) expression. We suppressed MDA accumulation in the vte2-1 background by genetically removing tri-unsaturated fatty acids. The resulting quadruple mutant, fad3-2 fad7-2 fad8 vte2-1, did not display the visual phenotype or the aberrant GST1 expression observed in vte2-1. Moreover, cotyledon bleaching in vte2-1 was chemically phenocopied by treatment of wild-type plants with MDA. These data suggest that products of tri-unsaturated fatty acid oxidation underlie the vte2-1 seedling phenotype, including cellular toxicity and gene regulation properties. Generation of the quadruple mutant facilitated the development of an in situ fluorescence assay based on the formation of adducts of MDA with 2-thiobarbituric acid at 37 degrees C. Specificity was verified by measuring pentafluorophenylhydrazine derivatives of MDA and by liquid chromatography analysis of MDA-2-thiobarbituric acid adducts. Potentially applicable to other organisms, this method allowed the localization of MDA pools throughout the body of Arabidopsis and revealed an undiscovered pool of the compound unlikely to be derived from trienoic fatty acids in the vicinity of the root tip quiescent center.

Abstract: The response to reactive electrophile species (RES) is now considered as part of the plant response to pathogen and insect attacks. Thanks to a previously established high-performance liquid chromatography tandem mass spectrometry methodology, we have investigated the production of oxylipin RES adducts to glutathione (GSH) during the hypersensitive response (HR) of plants. We have observed that RES conjugation to GSH in tobacco (Nicotiana tabacum) leaves is facile and nonspecific. In cryptogein-elicited tobacco leaves, we show that the oxylipin RES adducts to GSH are produced in correlation with GSH consumption, increase in glutathione S-transferase activity, and the appearance of the cell death symptoms. In this model, the adducts arise mainly from the downstream 13 lipoxygenase (LOX) metabolism, although the induced 9 LOX pathway leads massively to the accumulation of upstream metabolites. The main adducts were obtained from 2-hexenal and 12-oxo-phytodienoic acid. They accumulate transiently as 1-hexanol-3-GSH, a reduced adduct, and 12-oxo-phytodienoic acid-GSH, respectively. RES conjugation does not initiate cell death but explains part of the GSH depletion that accompanies HR cell death. The nature of these GSH conjugates shows the key role played by the 13 LOX pathway in RES signaling in the tobacco HR.

Abstract: Both biotic and abiotic stress activate the oxylipin pathway in plants. As reactive electrophile species (RES), some oxylipins are expected to bind cellular nucleophiles in a Michaël-type addition reaction. Using the HPLC-tandem mass spectrometry techniques, we have established the analytical basis for the investigation of oxylipin conjugation to glutathione (GSH) in plant extracts. The GSH adducts to the four keto fatty acid isomers issued from both linoleic and linolenic acids were first produced and their mass spectrometric features analyzed in the positive electrospray ionization mode. In all cases, the main fragmentation (MS2 mode) of the pseudomolecular ion leads to the neutral loss of a glutamyl moiety (-129 Da), affording an ion that gives structural information upon an additional fragmentation (MS3 mode). The glutamyl loss was confirmed by the analysis of other GSH adducts to oxylipin RES and appeared as being characteristic of GSH adducts. It is thus proposed to search GSH adducts in plant extracts by HPLC-MS/MS, using initially the neutral loss mode and then the MS2 mode to further characterize the identified compounds. This methodology was successfully applied to the analysis of GSH adducts upon infiltration into leaves of the four previous keto fatty acids at 5 mM, a concentration inducing cell death. The production of GSH adducts to oxylipin RES was observed for the first time in plant tissues. Furthermore, the levels of adduct production explain in part the observed GSH depletion. These results support the role of RES in altering protein activities and cellular redox balance of plant cells, via addition reactions to cellular nucleophiles.

Abstract: Two bursts of H(2)O(2) production have been detected by in situ 3,3'-diaminobenzidine (DAB) staining after cutting of Lolium perenne L. leaf blades. The first burst, which occurred immediately after wounding was inhibited by Na-diethydithiocarbamate (DIECA), a Cu/Zn-superoxide dismutase (SOD) inhibitor. The second burst, which was initiated several hours later, coincided with the induction of oxalate oxidase (G-OXO) activity detected in vitro or visualized in situ by the alpha-chloronaphtol assay. Four genes encoding G-OXO have been identified from cDNA obtained from wounded L. perenne L. leaf blades. Comparison of protein sequences revealed more than 91% homology in the coding region between G-OXOs of the true cereals and G-OXOs of ryegrass, which is a Gramineae belonging to the tribe of Festucaceae. The wound-dependent increase of G-OXO activity in floated cut leaf blades was the result of differential induction of the four g-oxo genes. The involvement of G-OXOs in wound-induced H(2)O(2) production coincided with the presence in leaf tissues of oxalate throughout the period of increase of G-OXO synthesis. Moreover, expression of g-oxo genes was enhanced by an exogenous supply of H(2)O(2) or methyljasmonate (MeJa). Expression of the four g-oxo genes was also induced after in planta stinging of leaf blades. The pattern of their expression in planta was identical to that occuring in senescing leaf sheaths. These results emphasize the importance of G-OXOs in H(2)O(2) production in oxalate-producing plant species such as ryegrass. G-OXOs might be crucial during critical events in the life of plants such as cutting and senescence by initiating H(2)O(2)-mediated defences against pathogens and foraging animals.