Oxidative stress induced expression of monodehydroascorbate reductase gene in Eleusine coracana

Abiotic stresses constitute a serious threats to the world food security as they cause significant economic losses in terms of reduction in crop productivity and also greatly limit the geographical locations where crops can be grown. Exposure to abiotic stress causes over-production of reactive oxygen species, leading to oxidative stress in plants. Induction of oxidative stress is primarily responsible for a variety of detrimental changes in the cellular physiology. However, plants have evolved intricate anti-oxidative defence machinery, for their survival under stress. Plant defence strategies for stress tolerance rely on the expression of anti-oxidative genes required for scavenging the toxic reactive oxygen species. Monodehydroascorbate reductase is one of the key anti-oxidant enzyme responsible for scavenging reactive oxygen species. In the present study, efforts have been made to understand the role of monodehydroascorbate reductase in finger millet under different abiotic stresses (drought, salt and UV radiation). The study establishes a differential link between mdar gene expression and enzyme activity under oxidative stress that is validated under different types of imposed stresses. Alteration in correlation between gene expression and enzyme activities under varying magnitude of oxidative stress is elucidated.     

Effect of localized reduction of gibberellins in different tobacco organs on drought stress tolerance and recovery

Drought resistance is increased in plants by the absence of the hormone gibberellic acid (GA) or by a lack of GA sensitivity. We studied the effects of tissue-specific reduction in GA levels on drought tolerance, on recovery from drought stress, and on primary and secondary growth using transgenic tobacco plants expressing the GA-inactivating gene PtGA2ox ₁ (GA 2-oxidase) specifically in leaves, stems, or roots. Localized reduction of bioactive GA₁ levels was achieved by tissue-specific expression of the PtGA2ox ₁ gene in leaves using the rbcs promoter (LD plants), in roots using the TobRB7 promoter (RD plants), and in stems using the LMX5 promoter (SD plants). In response to drought stress, all transgenic tobacco plants exhibited reduced primary and secondary growth and increased drought tolerance with a corresponding reduction in malondialdehyde levels, higher relative water content, increased proline and sugar content, and elevated peroxidase, superoxide dismutase, and catalase activities relative to wild-type plants. The highest level of drought tolerance and the most rapid recovery from stress was achieved by localized reduction of GA₁ in the roots of the RD transgenic plants. In addition, although the total bioactive GA₁ content in RD and LD plants was essentially identical, the heights of LD plants were significantly greater and drought tolerance was significantly less than in RD plants. It is possible that the site of gibberellin-related gene expression plays an important role in the balance between growth and drought tolerance.     

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.     

Overproduction of an Arabidopsis aldo–keto reductase increases barley tolerance to oxidative and cadmium stress by an in vivo reactive aldehyde detoxification

In this work, we expressed an Arabidopsis thaliana-coded protein (AKR4C9) in transgenic barley to study its enzymatic activity and to enhance the reactive aldehyde neutralizing capacity (part of the oxidative stress tolerance) of transgenic plants. Total leaf protein was extracted from transgenic plants expressing either C or N-terminally His-tagged aldo–keto reductase (AKR) enzyme and purified by affinity chromatography. The Arabidopsis-coded enzyme showed moderate activity against the synthetic reactive aldehyde, glutaraldehyde, and low but detectable enzyme activity against fructose with a low Michaelis–Menten constant (K_m value). Activity of the C and the N-terminally His-tagged AKRs were found to be in the same range. Glutaraldehyde was also tested in vivo by spraying onto the leaves of the plants. The reactive aldehyde tolerance of both wild type and transgenic plants, as well as the general physiological effects of this reactive aldehyde treatment were evaluated. The growth rate was found to decrease in all (both wild type and transgenic) plants. The high AKR-expressing transgenic plants showed a lower respiratory rate, and they also showed higher fresh weight, higher chlorophyll content and photosynthetic activity, indicating a higher reactive aldehyde tolerance. Cadmium (Cd) treatment was also performed to validate this result. Cd caused strong lipid peroxidation; however, the Arabidopsis enzyme lowered the reactive aldehyde content as expected. This is the first report in which kinetic parameters of the fructose reduction by the stress inducible plant AKR enzyme are presented. Furthermore, data on the effects of a reactive aldehyde treatment on intact plants are also provided.     

A root proteomics-based insight reveals dynamic regulation of root proteins under progressive drought stress and recovery in <Emphasis Type="Italic">Vigna radiata</Emphasis> (L.) Wilczek

To understand the complex drought response mechanism in crop plants, a systematic root proteomics approach was adopted to identify and analyze the expression patterns of differentially expressed major root proteins of Vigna radiata during short-term (3 days) and consecutive long-term water-deficit (6 days) as well as during recovery (6 days after re-watering). Photosynthetic gas exchange parameters of the plant were measured simultaneously during the stress treatment and recovery period. A total of 26 major protein spots were successfully identified by mass spectrometry, which were grouped according to their expression pattern during short-term stress as significantly up-regulated (9), down-regulated (10), highly down-regulated, beyond detection level of the software (2) and unchanged (5). The subsequent changes in the expression patterns of these proteins during long-term stress treatment and recovery period was analyzed to focus on the dynamic regulation of these functionally important proteins during progressive drought and recovery period. Cytoskeleton-related proteins were down-regulated initially (3d) but regained their expression levels during subsequent water-deficit (6d) while glycoprotein like lectins, which were primarily known to be involved in legume–rhizobia symbiosis, maintained their enhanced expression levels during both short and long-term drought treatment indicating their possible role in drought stress response of legumes. Oxidative stress-related proteins including Cu/Zn superoxide dismutase, oxidoreductase and aldehyde reductase were also up-regulated. The analyses of the dynamic regulation of these root proteins during short- and long-term water-deficit as well as recovery period may prove crucial for further understanding of drought response mechanisms in food legumes.     

Improved reactive aldehyde,salt and cadmium tolerance of transgenic barley due to the expression of aldo–keto reductase genes

Under various stress conditions, plant cells are exposed to oxidative damage which triggers lipid peroxidation. Lipid peroxide breakdown products include protein crosslinking reactive aldehydes. These are highly damaging to living cells. Stress-protective aldo–keto reductase (AKR) enzymes are able to recognise and modify these molecules, reducing their toxicity. AKRs not only modify reactive aldehydes but may synthesize osmoprotective sugar alcohols as well. The role of these mixed function enzymes was investigated under direct reactive aldehyde, heavy metal and salt stress conditions. Transgenic barley (Hordeum vulgare L.) plants constitutively expressing AKR enzymes derived from either thale cress (Arabidopsis thaliana) (AKR4C9) or alfalfa (Medicago sativa) (MsALR) were studied. Not only AKR4C9 but MsALR expressing plants were also found to produce more sorbitol than the non-transgenic (WT) barley. Salinity tolerance of genetically modified (GM) plants improved, presumably as a consequence of the enhanced sorbitol content. The MsALR enzyme expressing line (called 51) exhibited almost no symptoms of salt stress. Furthermore, both transgenes were shown to increase reactive aldehyde (glutaraldehyde) tolerance. Transgenic plants also exhibited better cadmium tolerance compared to WT, which was considered to be an effect of the reduction of reactive aldehyde molecules. Transgenic barley expressing either thale cress or alfalfa derived enzyme showed improved heavy metal and salt tolerance. Both can be explained by higher detoxifying and sugar alcohol producing activity. Based on the presented data, we consider AKRs as very effective stress-protective enzymes and their genes provide promising tools in the improvement of crops through gene technology.     

Regulatory function of AMP1 in ABA biosynthesis and drought resistance in arabidopsis

In this study we reported the isolation of a mutant in which the reporter pVP14-LUC was highly expressed in Arabidopsis. The gene expression of maize VP14 is closely correlated with the endogenous ABA levels, and the Arabidopsis homolog of VP14, AtNCED1, encoding an enzyme of ABA biosynthesis, was up-regulated, and high ABA level was detected in the mutant. Map-based cloning revealed that the mutated gene is a novel allele of the AMP1 (Altered Meristem Program 1) which encodes a glutamate carboxypeptidase that plays an important role in shoot apical meristem development and phytohormone homeostasis. We found that the mutant displayed obvious drought tolerance, being with more lateral roots, high seed germination under mannitol, increased ABA accumulation, and highly induced gene expression of RD29A. Using the approaches of artificial microRNA gene silencing in transgenic plants, three AMP1 down-regulated lines were obtained. The AMP1 down-regulated plants exhibited a low rate of water loss, decreased stomatal aperture, and enhanced drought tolerance. These results provide evidence demonstrating the regulatory function of AMP1 in plant drought tolerance and stress responsive gene expression.     

Effects of lanthanum on the ascorbate and glutathione metabolism of Vigna radiata seedlings under salt stress

In order to elucidate the role of lanthanum (La) in response of Vigna radiata to a salt stress, we investigated the effects of La on the ascorbate and glutathione metabolism. The results show that in comparison with a control, the salt stress increased the activities of ascorbate peroxidase (APX), glutathione reductase (GR), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), γ-glutamylcysteine synthetase (γ-ECS), and L-galactono-1,4-lactone dehydrogenase (GalLDH), and the content of ascorbic acid (AsA) and glutathione (GSH). It also increased the malondialdehyde content (MDA) and electrolyte leakage. The salt stress significantly decreased the ratios of AsA/dehydroascorbate (DHA) and GSH/glutathione disulphide (GSSG) compared with the control. The pretreatment with La not only significantly increased the activities of the above enzymes, the content of AsA, GSH, and the ratios of AsA/DHA and GSH/GSSG, but also significantly reduced the MDA content and electrolyte leakage compared with the salt stress alone. Our results suggest that La could up-regulate the ascorbate and glutathione metabolisms and could have an important role for acquisition of salt stress tolerance in Vigna radiata.     

GbMPK3, a mitogen-activated protein kinase from cotton,enhances drought and oxidative stress tolerance in tobacco

Mitogen-activated protein kinase (MAPK) cascades are highly conserved signaling modules found in all eukaryotes, and play significant roles in developmental and environmental signal transduction. In this study, a MAPK gene (GbMPK3), which showed homologous to AtMPK3 and NtWIPK, was isolated from sea-island cotton (Gossypium barbadense) and induced during multiple abiotic stress treatments including salt, cold, heat, dehydration and oxidative stress. Transgenic tobacco (Nicotiana benthamiana) with constitutively higher expression of GbMPK3 was conferred with enhanced drought tolerance, reduced water loss during drought treatment and improved plant height and survival rates after re-watering. Additionally, the gene expression levels and enzymatic activity of antioxidant enzymes were more strongly induced with depressed hydrogen peroxide accumulation in GbMPK3-overexpressing tobacco compared with wild-type under drought condition. Furthermore, observation of seed germination and leaf morphology showed that tolerance of transgenic plants to methyl viologen was improved due to increased antioxidant enzyme expression, suggesting that GbMPK3 may positively regulate drought tolerance through enhanced reactive oxygen species scavenging ability.     

A novel NADPH-dependent aldehyde reductase gene from Vigna radiata confers resistance to the grapevine fungal toxin eutypine

Eutypine, 4-hydroxy-3-(3-methyl-3-butene-1-ynyl) benzyl aldehyde, is a toxin produced by Eutypa lata, the causal agent of eutypa dieback of grapevines. It has previously been demonstrated that tolerance of some cultivars to this disease was correlated with their capacity to convert eutypine to the corresponding alcohol, eutypinol, which lacks phytotoxicity. We have thus purified to homogeneity a protein from Vigna radiata that exhibited eutypine-reducing activity and have isolated the corresponding cDNA. This encodes an NADPH-dependent reductase of 36 kDa that we have named Vigna radiata eutypine-reducing enzyme (VR-ERE), based on the capacity of a recombinant form of the protein to reduce eutypine into eutypinol. The strongest homologies (86.8%) of VR-ERE at the amino acid level were found with CPRD14, a drought-inducible gene of unknown function, isolated from Vigna unguiculata and with an aromatic alcohol dehydrogenase (71.7%) from Eucalyptus gunnii . Biochemical characterization of VR-ERE revealed that a variety of compounds containing an aldehyde group can act as substrates. However, the highest affinity was observed with 3-substituted benzaldehydes. Expression of a VR-ERE transgene in Vitis vinifera cells cultured in vitro conferred resistance to the toxin. This discovery opens up new biotechnological approaches for the generation of grapevines resistant to eutypa dieback.