Lipid peroxidation in cotton: Xanthomonas interactions and the role of lipoxygenases during the hypersensitive reaction.

Lipid peroxidation, often associated with hypersensitive cell death, may be initiated either by active oxygen species (AOS) or lipoxygenases (LOX). Here we report a detailed analysis of this oxidative process in both incompatible and compatible interactions between the cotton cultivar Reba B50 and Xanthomonas campestris pv. malvacearum (Xcm). The hypersensitive reaction (HR) was characterized by a massive production of polyunsaturated fatty acid (PUFA) hydroperoxides together with typical tissue dehydration. Among these, isomers peroxidized on carbon 9, largely predominant, were chiral, showing an excess in the S enantiomer. The HR process was accompanied by an increase in 9S-LOX activity and preceded by transcription of a LOX gene (GhKLox1). These results showed that: (i) AOS produced during the oxidative burst were not involved in PUFA peroxidation during HR; and (ii) as previously described in elicited leaves of tobacco, the massive enzymatic lipid peroxidation was closely associated with hypersensitive cell death. During disease development in this cotton cultivar, the 9-lipoxygenation of PUFAs was late, weak, preceded by a faint accumulation of GhKLox1 transcripts, and associated with chlorosis but not with necrosis. Consequently, the main difference between incompatible and compatible interactions was in the precocity and intensity of the oxidative process, rather than in its nature. These data provide the evidence for a correlation between lipid peroxidation and hypersensitive cell death induced by pathogens.

Repair of the main UV-induced thymine dimeric lesions within Arabidopsis thaliana DNA: evidence for the major involvement of photoreactivation pathways.

The UV-B induced formation of thymine cis-syn cyclobutane dimer and related (6-4) photoproduct was monitored within DNA of cultured cells and plants of Arabidopsis thaliana. This was achieved using a sensitive and accurate HPLC-tandem mass spectrometry assay. It was found that the cyclobutane pyrimidine dimer was formed in a ninefold higher yield than the (6-4) photoproduct. The removal of the lesions was then studied by incubating irradiated cells either in the darkness, under visible light or upon exposure to UV-A radiation. Dark repair of both cyclobutane dimers and (6-4) photoproducts was found to be very ineffective. In contrast, a rapid decrease in the level of photoproducts was observed when UV-B-irradiated cells were exposed to UV-A and, to a lesser extent, to visible light. The removal of (6-4) adducts was found to occur more efficiently. These results strongly suggest that repair of UV-induced photolesions in plants is mainly mediated by photolyases.

Evaluation of lipid peroxidation as a toxicity bioassay for plants exposed to copper.

Agricultural soils may contain toxic levels of copper (Cu) due to sewage sludge spreading or industrial pollution but chemical analyses may not be representative of Cu bioavailability, defined as the soil Cu fraction that plants can actually absorb (i.e. Cu fraction which is not strongly adsorbed to soil components). Lipid peroxidation caused by Cu in plants was investigated as a relevant bioassay of toxicity. Seven-day-old rapeseed plantlets were grown on Cu-supplemented medium in controlled conditions. Lipid-peroxidation was assessed by measuring: (1) the 2-thiobarbituric acid (TBA)-reactive substances; (2) the hydroperoxy acids by HPLC analysis; and (3) the alkane outputs by gas chromatography. We first verified the correlation between the results obtained by each method and then discussed their advantages and disadvantages within the context of a bioassay, showing that the volatile alkane output measurement is the most precise and easy to perform method for this purpose.

Involvement of lipoxygenase-dependent production of fatty acid hydroperoxides in the development of the hypersensitive cell death induced by cryptogein on tobacco leaves.

Lipid peroxidation was investigated in relation with the hypersensitive reaction in cryptogein-elicited tobacco leaves. A massive production of free polyunsaturated fatty acid (PUFA) hydroperoxides dependent on a 9-lipoxygenase (LOX) activity was characterized during the development of leaf necrosis. The process occurred after a lag phase of 12 h, was accompanied by the concomitant increase of 9-LOX activity, and preceded by a transient accumulation of LOX transcripts. Free radical-mediated lipid peroxidation represented 10% of the process. Inhibition and activation of the LOX pathway was shown to inhibit or to activate cell death, and evidence was provided that fatty acid hydroperoxides are able to mimic leaf necrotic symptoms. Within 24 h, about 50% of leaf PUFAs were consumed, chloroplast lipids being the major source of PUFAs. The results minimize the direct participation of active oxygen species from the oxidative burst in membrane lipid peroxidation. They suggest, furthermore, the involvement of lipase activity to provide the free PUFA substrates for LOX. The LOX-dependent peroxidative pathway, responsible for tissue necrosis, appears as being one of the features of hypersensitive programmed cell death.

Are elicitins cryptograms in plant-Oomycete communications?

Stimulation of plant natural defenses is an important challenge in phytoprotection prospects. In that context, elicitins, which are small proteins secreted by Phytophthora and Pythium species, have been shown to induce a hypersensitive-like reaction in tobacco plants. Moreover, these plants become resistant to their pathogens, and thus this interaction constitutes an excellent model to investigate the signaling pathways leading to plant resistance. However, most plants are not reactive to elicitins, although they possess the functional signaling pathways involved in tobacco responses to elicitin. The understanding of factors involved in this reactivity is needed to develop agronomic applications. In this review, it is proposed that elicitins could interact with regulating cell wall proteins before they reach the plasma membrane. Consequently, the plant reactivity or nonreactivity status could result from the equilibrium reached during this interaction. The possibility of overexpressing the elicitins directly from genomic DNA in Pichia pastoris allows site-directed mutagenesis experiments and structure/function studies. The recent discovery of the sterol carrier activity of elicitins brings a new insight on their molecular activity. This constitutes a crucial property, since the formation of a sterol-elicitin complex is required to trigger the biological responses of tobacco cells and plants. Only the elicitins loaded with a sterol are able to bind to their plasmalemma receptor, which is assumed to be an allosteric calcium channel. Moreover, Phytophthora and Pythium do not synthesize the sterols required for their growth and their fructification, and elicitins may act as shuttles trapping the sterols from the host plants. Sequence analysis of elicitin genes from several Phytophthora species sheds unexpected light on the phylogenetic relationships among the genus, and suggests that the expression of elicitins is under tight regulatory control. Finally, general involvement of these lipid transfer proteins in the biology of Pythiaceae, and in plant defense responses, is discussed. A possible scheme for the coevolution between Phytophthora and tobacco plants is approached.

Accumulation of small heat shock proteins, including mitochondrial HSP22, induced by oxidative stress and adaptive response in tomato cells.

Changes in gene expression, by application of H2O2, O2.- generating agents (methyl viologen, digitonin) and gamma irradiation to tomato suspension cultures, were investigated and compared to the well-described heat shock response. Two-dimensional gel protein mapping analyses gave the first indication that at least small heat shock proteins (smHSP) accumulated in response to application of H2O2 and gamma irradiation, but not to O2.- generating agents. While some proteins seemed to be induced specifically by each treatment, only part of the heat shock response was observed. On the basis of Northern hybridization experiments performed with four heterologous cDNA, corresponding to classes I-IV of pea smHSP, it could be concluded that significant amounts of class I and II smHSP mRNA are induced by H2O2 and by irradiation. Taken together, these results demonstrate that in plants some HSP genes are inducible by oxidative stresses, as in micro-organisms and other eukaryotic cells. HSP22, the main stress protein that accumulates following H2O2 action or gamma irradiation, was also purified. Sequence homology of amino terminal and internal sequences, and immunoreactivity with Chenopodium rubrum mitochondrial smHSP antibody, indicated that the protein belongs to the recently discovered class of plant mitochondrial smHSP. Heat shock or a mild H2O2 pretreatment was also shown to lead to plant cell protection against oxidative injury. Therefore, the synthesis of these stress proteins can be considered as an adaptive mechanism in which mitochondrial protection could be essential.

Measurement of thermally produced volatile alkanes: an assay for plant hydroperoxy fatty acid evaluation.

A new method designed to monitor lipid peroxidation in plants has been set up with soybean hypocotyl/radicles. The hydroperoxy fatty acids present in situ are converted by rapid thermal treatment (80 s and 210 J g-1) of the biological sample into ethane and n-pentane, which are analyzed by gas chromatography. The method has been directly calibrated by quantification of the hydroperoxy fatty acids by silica-phase HPLC analysis of their reduced hydroxy derivatives. Hypocotyl/radicles from the two soybean cultivars Argenta and Soriano were submitted to various chemical oxidative treatments and were analyzed for both thermally produced volatile alkanes and hydroperoxy fatty acid levels. Our results showed that ethane and n-pentane production are in both cases closely correlated with linolenic as well as linoleic acid hydroperoxide levels (P < 0.001). Within a given plant material, thermal conversion of both hydroperoxides into alkanes occurred with yields which were not dependent on the oxidative treatment. These yields are however functions of the biological material since in Soriano and Argenta cultivars they were around 6 and 25%, respectively. Taking into account the last point, the alkane test cannot be used to directly quantify the absolute lipid hydroperoxide levels of plant tissues but it is convenient to monitor the peroxidative phenomenon as it occurs. The assay is easy and rapid to perform (analysis of 50 samples per day) since no sample preparation is needed, and the low detection limit (20 pmol of alkane g-1) permits the analysis of small samples.

Involvement of Oxidative Processes in the Signaling Mechanisms Leading to the Activation of Glyceollin Synthesis in Soybean (Glycine max).

The efficiency of hydroperoxides (tert-butyl hydroperoxide, hydrogen peroxide) and sulfhydryl reagents (iodoacetamide, p-chloromercuribenzene sulfonic acid) as glyceollin elicitors was examined in relation to sulfhydryl oxidation, or alteration, and to lipid peroxidation, in 3-d-old soybean hypocotyl/radicle, Glycine max. These oxidative events were investigated as possible early steps in the transduction mechanisms leading to phytoalexin synthesis. Free protein sulfhydryl groups were not modified after any of the eliciting treatments, thus indicating that immediate massive protein oxidation or modification cannot be considered a signal transduction step. Unlike sulfhydryl reagents, which led to a decrease of the free nonprotein sulfhydryl group (free np-SH) pool under all of the eliciting conditions, the results obtained with hydroperoxides indicated that immediate oxidation of the np-SH is not required for the signal transduction. Moreover, elicitation with 10 mM tertbutyl hydroperoxide did not lead to further oxidation or to changes in np-SH level during the critical phase of phenylalanine ammonialyase activation (the first 20 h), suggesting that np-SH modifications are probably not involved in hydroperoxide-induced elicitation. On the other hand, all treatments leading to significant glyceollin accumulation were able to trigger a rapid (within 2 h) lipid peroxidation process, whereas noneliciting treatments did not. In addition, transition metals, such as Fe2+ and Cu+, were shown to stimulate both hydrogen peroxide-induced lipid peroxidation and glyceollin accumulation, again emphasizing that the two processes are at least closely linked in soybean. Among the oxidative processes triggered by activated oxygen species, oxidation of sulfhydryl compounds, or lipid peroxidation, our results suggest that lipid peroxidation is sufficient to initiate glyceollin accumulation in soybean. This further supports the hypothesis that lipid peroxidation could be involved as a step in the signal cascade that leads to induction of plant defenses.

Short-Term Effects of gamma-Irradiation on 1-Aminocyclopropane-1-Carboxylic Acid Metabolism in Early Climacteric Cherry Tomatoes : Comparison with Wounding.

gamma-Irradiation of early climacteric (breaker) cherry tomatoes (Lycopersicon pimpinellifollium L.) caused a sharp burst in ethylene production during the first hour. The extent of ethylene production was dose dependent and was maximum at about 3 kilograys. The content of 1-aminocyclopropane-1-carboxylic acid (ACC), followed the same evolution as ethylene production, while malonyl ACC increased steadily with time in irradiated fruits. The burst in ethylene production was accompanied by a sharp stimulation of ACC synthase activity which began 15 minutes after irradiation. The stimulation was completely prevented by cycloheximide, but not by actinomycin d or cordycepin. In contrast with irradiation, mechanical wounding continuously stimulated ethylene production over several hours. gamma-Irradiation and cordycepin applied to wounded tissues both caused the cessation of this continuous increase, but the initial burst was still persisting. These data suggest that gamma-irradiation, like wounding, stimulates the translation of preexisting mRNAs. It also reduces, at least temporarily, the subsequent transcription-dependent stimulation of ethylene production. gamma-Irradiation greatly inhibited the activity of ethylene-forming enzyme at doses higher than 1 kilogray. Such sensitivity is in accordance with a highly integrated membranebound enzyme.

Multi-competitive enzymatic reactions in organic media: a simple test for the determination of lipase fatty acid specificity.

Multiple substrate competition was kinetically analyzed to study lipase-catalyzed reactions in organic media. For each substrate, a competitive factor (the ratio of the specificity constants kcat/Km) was measured by reference to the best substrate using a mixture of fatty acid ethyl esters submitted to a solvolysis reaction by n-propanol. A scale of competitive factors was established which quantitatively described the lipase specificity. This principle was applied to the determination of the specificity of four commercial lipase preparations towards fatty acid chain length and degree of unsaturation. The results were not affected by changes in the physicochemical conditions of the reaction (water content, substrate concentration, nature of nucleophile, etc.). The simple test will be a useful tool to characterize lipase specificity.

Lipase-catalyzed reactions in organic media: competition and applications.

Lipases (triacylglycerol acylhydrolase, EC 3.1.1.3) have been used in organic media for the catalysis of reactions such as hydrolysis, esterification and transesterification. In these conditions it was confirmed that all reactions proceed through an acyl enzyme intermediate in two successive steps: acyl enzyme formation and solvolysis. The competition between two acyl acceptors (acyl donors) for reaction with a donor (acceptor) is described for the first time. A kinetic model is proposed using a competitive factor which is in good accordance with experimental results. The model was used successfully for the prediction of alcohol (acid) separations and resolutions by lipases.

Determination of glycolic acid level in higher plants during photorespiration by stable isotope dilution mass spectrometry with double-labeling experiments.

Determination of glycolic acid by stable isotope dilution was applied to the measurement of the glycolic acid pool size in tomato and maize leaves during photorespiration. Detached leaves were maintained in the presence of 18O2; [13C]glycolate was added to the foliar extract as an internal standard and the mixture of biological glycolate and [13C]glycolate was analyzed by combined gas chromatography-mass spectrometry. The level of foliar glycolate pool was measured via the 13C label, and 18O incorporation was determined.