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.In plant-pathogen interactions, a typical feature of plant resistance is hypersensitive reaction (HR), 1 characterized by the induction of rapid cell death at the site of an attempted attack by either an avirulent strain of a pathogen or a nonpathogen. The collapse of challenged cells, occurring during incompatible interactions, was shown in most cases to be dependent on a gene for gene plant pathogen interaction (1, 2). HR is accompanied by a battery of defense mechanisms including de novo synthesis of antimicrobial enzymes and metabolites, strengthening of the cell wall, and the onset of systemic acquired resistance dependent on salicylic acid accumulation (3, 4). HR often leads to dry lesions that are supposed to limit pathogen growth. Other proposed roles is the release in apoplasm of defense-related proteins and toxic metabolites, as well as of signals that activate the defenses of both neighboring and distant cells. Hypersensitive cell death appears to not be the result of the direct action of released pathogenic factors but is rather under the genetic control of the host. Indeed, several observations underline that HR is an example of PCD in plants (1, 2). Furthermore, hypersensitive cell death has morphological and molecular features similar to the mammalian PCD, called apoptosis. These include cytoplasm and chromatin condensation followed by their fragmentation, activation of calciumdependent endonucleases (5-8) and of cysteine proteases (9 -11), and involvement of similar regulation factors (2). Some differences between HR and mammalian apoptosis were observed, however, such as changes in DNA laddering (5,8) and the lack in HR of the repressor role of Bcl-x L (12). One ultimate characteristic of HR is the loss of membrane integrity, and thus HR is often characterized by an associated electrolyte le...

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.

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Active oxygen species (AOS), especially hydrogen peroxide, play a critical role in the defence of plants against invading pathogens and in the hypersensitive response (HR). This is characterized by the induction of a massive production of AOS and the rapid appearance of necrotic lesions is considered as a programmed cell death (PCD) process during which a limited number of cells die at the site of infection. This work was aimed at investigating the mode of cell death observed in cultures of BY-2 tobacco cells exposed to H(2)O(2). It was shown that H(2)O(2) is able to induce various morphological cell death features in cultured tobacco BY-2 cells. The hallmarks of cell death observed with fluorescent and electron microscopy differed greatly with the amount of H(2)O(2) added to the cell culture. The appearance of nuclear fragmentation similar to 'apoptotic bodies' associated with a fragmentation of the nuclear DNA into small fragments appear for almost 18% of the cells treated with 12.5 mM H(2)O(2). The early stages of the induction of this PCD process consisted in cell shrinkage and chromatin condensation at the periphery of the nucleus. Above 50 mM, H(2)O(2) induces high necrotic cell death. These data suggest that H(2)O(2)-induced cell damage is associated with the induction of various cell death processes that could be involved differently in plant defence reactions.

Twelve alpha and beta 20S proteasome subunits cDNAs showing 70-82% identity with the corresponding genes in Arabidopsis or rice, and features of eukaryotic proteasome subunits were cloned in tobacco. Only beta1-tcI 7, alpha3 and alpha6, 20S proteasome subunits encoding genes were up-regulated by cryptogein, a proteinaceous elicitor of plant defence reactions. These results led to the hypothesis that the activation of beta1-tcI 7, alpha3 and alpha6 could induce a specific proteolysis involved in the hypersensitive response and systemic acquired resistance monitored by cryptogein.