Purification and Characterization of a NADPH-Dependent Aldehyde Reductase from Mung Bean That Detoxifies Eutypine, a Toxin from Eutypa lata

Eutypine (4-hydroxy-3-[3-methyl-3-butene-1-ynyl] benzaldehyde) is a toxin produced by Eutypa lata, the causal agent of eutypa dieback in the grapevine (Vitis vinifera). Eutypine is enzymatically converted by numerous plant tissues into eutypinol (4-hydroxy-3-[3-methyl-3-butene-1-ynyl] benzyl alcohol), a metabolite that is nontoxic to grapevine. We report a four-step procedure for the purification to apparent electrophoretic homogeneity of a eutypine-reducing enzyme (ERE) from etiolated mung bean (Vigna radiata) hypocotyls. The purified protein is a monomer of 36 kD, uses NADPH as a cofactor, and exhibits a K_m value of 6.3 μm for eutypine and a high affinity for 3- and 4-nitro-benzaldehyde. The enzyme failed to catalyze the reverse reaction using eutypinol as a substrate. ERE detoxifies eutypine efficiently over a pH range from 6.2 to 7.5. These data strongly suggest that ERE is an aldehyde reductase that could probably be classified into the aldo-keto reductase superfamily. We discuss the possible role of this enzyme in eutypine detoxification.Many pathogenic bacteria and fungi produce toxins that interfere with various functions of plant cells and may affect plant defense mechanisms (Durbin, 1981). Toxin production is commonly associated with disease severity and can be involved in colonization or systemic invasion by the pathogen (Schäfer, 1994). Toxin resistance has been shown in most cases to be based on the ability of the plant to metabolically detoxify pathogen toxins (Meeley and Walton, 1991; Zhang and Birch, 1997; Zweimuller et al., 1997). Few cloned toxin-resistance genes that encode proteins involved in detoxification mechanisms have been described (Utsumi et al., 1988; Johal and Briggs, 1992; Zhang and Birch, 1997). In many cases a relationship exists between toxin tolerance and resistance to the disease (Anzai et al., 1989; Meeley et al., 1992). The availability of toxin-resistance genes will permit a greater understanding of the mechanisms causing plant disease and will also set the stage for engineering resistance to plant disease (Keen, 1993).Eutypine (4-hydroxy-3-[3-methyl-3-butene-1-ynyl] benzaldehyde) is a toxin produced by the ascomycete fungus Eutypa lata (Pers.: Fr.) Tul., the causal agent of eutypa dieback (Tey-Rulh et al., 1991). This disease is responsible for considerable loss in yield and is the most devastating disease of grapevine (Vitis vinifera) in many countries (Moller and Kasamitis, 1981; Munkvold et al., 1994). The fungus infects the stock through pruning wounds and is present in the xylem and phloem of the vine trunk and branches (Moller and Kasamitis, 1978; Duthie et al., 1991). After a long incubation period, a canker forms around the infected wound. The toxin synthesized by the fungus in the trunk is believed to be transported by the sap to the herbaceous parts of the vine (Fallot et al., 1997). Eutypine penetrates grapevine cells through passive diffusion and its accumulation in the cytoplasm has been explained by an ion-trapping mechanism related to the ionization state of the molecule (Deswarte et al., 1996b). In the cell the effects of eutypine include reduction of adenylated nucleotide content, inhibition of succinate dehydrogenase, uncoupling of oxidative phosphorylation, and mitochondrial swelling (Deswarte et al., 1996a).Symptoms of eutypa dieback in the herbaceous part of the plant lead to dwarfed and withered new growth of branches, marginal necrosis of the leaves, dryness of the inflorescence, and, finally, death of one or more branches (Moller and Kasamitis, 1981). The toxin appears to be an important virulence factor involved in symptom development of the disease (Deswarte et al., 1996a). However, the absence of toxin-deficient mutants of the fungus and its long incubation period in the trunk before symptom development have prevented a critical study of the toxin in vine plants. Determining the gene responsible for eutypine resistance would therefore be an important critical tool in determining the role of eutypine toxin in symptom development in the disease; and it has the potential to confer resistance to transgenic grapevines.Recently, Colrat et al. (1998) found detoxification to occur in grapevine cells through the enzymatic reduction of eutypine into its corresponding alcohol, eutypinol (4-hydroxy-3-[3-methyl-3-butene-1-ynyl] benzyl alcohol). We have determined that this derivative of the toxin is nontoxic for grapevine tissues. Furthermore, we have established a relationship between the susceptibility of grapevine to eutypa dieback and the ability of tissues to inactivate eutypine, suggesting that the detoxification mechanism plays an important role in defense reactions. Eutypine is enzymatically detoxified in numerous plant species and, among them, we found that the tissues of mung bean (Vigna radiata), a nonhost plant for the pathogen, exhibit an efficient detoxification activity. As a prerequisite for demonstrating the involvement of eutypine toxin in eutypa dieback, we report here the purification to homogeneity and the characterization of an ERE from etiolated mung bean hypocotyls.     

Effects of Buthus occitanus tunetanus envenomation on an experimental murine model of gestation

Scorpion envenoming is less studied in pregnant victims. In this work, the effect of Buthus occitanus tunetanus on parturition in late pregnancy was studied in an animal model. Four groups of six primigravid female rats, each one at the 22nd day of pregnancy, were used. The first two groups had received an intra-peritoneal injection of 500 microg/kg of Buthus occitanus tunetanus crude venom or a physiological saline solution and left until foetal delivery. Then, the time elapsed until the first pup delivery and that separating the first and latest ones were measured. The other two groups served for the uterine electrophysiological activity exploration. Rats were anaesthetized, artificially ventilated and had received an intraperitoneal injection of 500 microg/kg of Buthus occitanus tunetanus crude venom or a physiological saline solution. Our results showed a significant increase of the latency to foetal delivery, labour time, and uterine contractile activity in envenomed rats compared to controls. Such signs are usually seen in dynamic dystocia. It was concluded that Buthus occitanus tunetanus envenoming might induce a dynamic dystocia, when it occurred in late pregnancy.     

TdERF1, an ethylene response factor associated with dehydration responses in durum wheat (Triticum turgidum L. subsp. durum)

Water deficit and increasing salinization reduce productivity of wheat, the leading crop for human diet. While the complete genome sequence of this crop has not been deciphered, a BAC library screening allowed the isolation of TdERF1, the first ethylene response factor gene from durum wheat. This gene is putatively involved in mediating salt stress tolerance and its characterization provides clues toward understanding the mechanisms underlying the adaptation/tolerance of durum wheat to suboptimal growth conditions. TdERF1 expression is differentially induced by high salt treatment in 2 durum wheat varieties, the salt-tolerant Grecale (GR) and the salt-sensitive Om Rabiaa (OR). To further extend these findings, we show here that the expression of this ERF is correlated with physiological parameters, such as the accumulation of osmo-regulators and membrane integrity, that discriminate between the 2 contrasted wheat genotypes. The data confirm that GR and OR are 2 contrasted wheat genotypes with regard to salt-stress and show that TdERF1 is also induced by water stress with an expression pattern clearly discriminating between the 2 genotypes. These findings suggest that TdERF1 might be involved in responses to salt and water stress providing a potential genetic marker discriminating between tolerant and sensitive wheat varieties.     

Er5, a tomato cDNA encoding an ethylene-responsive LEA-like protein: characterization and expression in response to drought, ABA and wounding

We report the isolation by differential display of a novel tomato ethylene-responsive cDNA, designated ER5. RT-PCR analysis of ER5 expression revealed an early (15 min) and transient induction by ethylene in tomato fruit, leaves and roots. ER5 mRNA accumulated during 2 h of ethylene treatment and thereafter underwent a dramatic decline leading to undetectable expression after 5 h of treatment. The full-length cDNA clone of 748 bp was obtained and DNA sequence analysis showed strong homologies to members of the atypical hydrophobic group of the LEA protein family. The predicted amino acid sequence shows 67%, 64%, 64%, and 61% sequence identity with the tomato Lemmi9, soybean D95-4, cotton Lea14-A, and resurrection plant pcC27-45 gene products, respectively. As with the other members of this group, ER5 encodes a predominantly hydrophobic protein. Prolonged drought stress stimulates ER5 expression in leaves and roots, while ABA induction of this ethylene-responsive clone is confined to the leaves. The use of 1-MCP, an inhibitor of ethylene action, indicates that the drought induction of ER5 is ethylene-mediated in tomato roots. Finally, wounding stimulates ER5 mRNA accumulation in leaves and roots. Among the Lea gene family this novel clone is the first to display an ethylene-regulated expression.     

α-l-Arabinofuranosidases: the potential applications in biotechnology

Recently, alpha-L-arabinofuranosidases (EC3.2.1.55) have received increased attention primarily due to their role in the degradation of lignocelluloses as well as their positive effect on the activity of other enzymes acting on lignocelluloses. As a result, these enzymes are used in many biotechnological applications including wine industry, clarification of fruit juices, digestion enhancement of animal feedstuffs and as a natural improver for bread. Moreover, these enzymes could be used to improve existing technologies and to develop new technologies. The production, mechanisms of action, classification, synergistic role, biochemical properties, substrate specificities, molecular biology and biotechnological applications of these enzymes have been reviewed in this article.     

Under-expression of the Auxin Response Factor Sl-ARF4 improves post-harvest behavior of tomato fruits

Auxin is one of the most prominent phytohormones regulating many aspects of fleshy fruit development including fruit set, fruit size through the control of cell division and cell expansion, and fruit ripening. To shed light on the role of auxin fruit ripening, we have previously shown that Sl-ARF4 is a major player in mediating the auxin control of sugar metabolism in tomato fruit (cv MicroTom). Further extending this study, we show here that down-regulation of Sl-ARF4 in tomato alters some ripening-related fruit quality traits including enhanced fruit density at mature stage, increased firmness, prolonged shelf-life and reduced water (weight) loss at red ripe stage. These findings suggest that Sl-ARF4 plays a role in determining fruit cell wall architecture and thus providing a potential genetic marker for improving post-harvest handling and shelf life of tomato fruits.