Littérature scientifique sur le sujet « Monodehydroascorbate reductase »

Ascorbic acid is a potent antioxidant and a crucial nutrient for plants and animals. The accumulation of ascorbic acid in plants is controlled by its biosynthesis, recycling, and degradation. Monodehydroascorbate reductase is deeply involved in the ascorbic acid cycle; however, the mechanism of monodehydroascorbate reductase genes in regulating kiwifruit ascorbic acid accumulation remains unclear. Here, we identified seven monodehydroascorbate reductase genes in the genome of kiwifruit (Actinidia eriantha) and they were designated as AeMDHAR1 to AeMDHAR7, following their genome identifiers. We found that the relative expression level of AeMDHAR3 in fruit continued to decline during development. The over-expression of kiwifruit AeMDHAR3 in tomato plants improved monodehydroascorbate reductase activity, and, unexpectedly, ascorbic acid content decreased significantly in the fruit of the transgenic tomato lines. Ascorbate peroxidase activity also increased significantly in the transgenic lines. In addition, a total of 1781 differentially expressed genes were identified via transcriptomic analysis. Three kinds of ontologies were identified, and 106 KEGG pathways were significantly enriched for these differently expressed genes. Expression verification via quantitative real-time PCR analysis confirmed the reliability of the RNA-seq data. Furthermore, APX3, belonging to the ascorbate and aldarate metabolism pathway, was identified as a key candidate gene that may be primarily responsible for the decrease in ascorbic acid concentration in transgenic tomato fruits. The present study provides novel evidence to support the feedback regulation of ascorbic acid accumulation in the fruit of kiwifruit.

Abstract Arabidopsis (Arabidopsis thaliana) 12-oxophytodienoic acid reductase isoform 3 (OPR3) is involved in the synthesis of jasmonic acid (JA) by reducing the α,β-unsaturated double bond of the cyclopentenone moiety in 12-oxophytodienoic acid (12-OPDA). Recent research revealed that JA synthesis is not strictly dependent on the peroxisomal OPR3. The ability of OPR3 to reduce trinitrotoluene suggests that the old yellow enzyme homolog OPR3 has additional functions. Here, we show that OPR3 catalyzes the reduction of a wide spectrum of electrophilic species that share a reactivity toward the major redox buffers glutathione (GSH) and ascorbate (ASC). Furthermore, we show that 12-OPDA reacts with ASC to form an ASC-12-OPDA adduct, but in addition OPR3 has the ability to regenerate ASC from monodehydroascorbate. The presented data characterize OPR3 as a bifunctional enzyme with NADPH-dependent α,β-ketoalkene double-bond reductase and monodehydroascorbate reductase activities (MDHAR). opr3 mutants showed a slightly less-reduced ASC pool in leaves in line with the MDHAR activity of OPR3 in vitro. These functions link redox homeostasis as mediated by ASC and GSH with OPR3 activity and metabolism of reactive electrophilic species.

We analyse the effect of Cd and H₂O₂ short-term treatments on the activity of ascorbate-glutathione recycling enzymes in barley root tip. Even a short transient exposure of barley roots to low 15 µmol Cd concentration caused a marked approximately 70% root growth inhibition. Higher Cd concentrations caused root growth cessation during the first 6 h after short-term Cd treatment. Similarly, a marked root growth inhibition was also detected after the short-term exposure of barley seedlings to H₂O₂. Our results indicate that root ascorbate pool is more sensitive to Cd treatment than glutathione pool. Rapid activation of dehydroascorbate reductase and monodehydroascorbate reductase is the important component of stress response to the Cd-induced alterations in barley root tips. H₂O₂ is probably involved in the Cd-induced activation of monodehydroascorbate reductase, but it is not involved in the Cd-induced increase of dehydroascorbate reductase activity.

The response of plants to the peroxidizing herbicide oxyfluorfen was investigated. The action of this p-nitrodiphenyl ether is based on inhibition of plastidic protoporphyrinogen oxidase, which leads to accumulation of protoporphyrin IX in the cytosol yielding reactive oxygen species by light activation. The induction of activities of antioxidative enzymes was followed in Nicotiana tabacum plants, var. BelW3. Glutathione reductase activity was elevated by 75% compared to control, monodehydroascorbate reductase by 65% and glutathione 5-transferase by 110% . The mRNA of ascorbate peroxidase and catalase isoform 2 was induced, the catalase isoform 1 was reduced. These findings were confirmed and supported by measuring enzymatic activity changes in photoheterotrophically grown soybean (Glycine max) suspension cultures. To find a possible involvement of compounds regulating oxidative stress response, we investigated the influence of salicylic acid and BTH (benzo(1,2,3)thiadiazole- 7-carbothioic acid 5-methylester), both inducers of pathogen defense, on soybean cell suspension cultures. The specific activities of glutathione reductase, monodehydroascorbate reductase and glutathione 5-transferase increased strongly, comparable to oxyfluorfen treatment. Both compounds protected the cells against oxyfluorfen-induced lipid peroxidation and alleviated the accumulation of protoporphyrin IX

The comparative effects of cold treatments upon the activities of five enzymes responsible for the elimination or reduction of toxic oxygen species were analyzed in two ecotypes of the C4 grass weed species Echinochloa crus-galli (L.) Beauv. from sites of contrasting climates in Quebec and Mississippi. Specific activities of the enzymes extracted from 4-week-old plants were measured daily for 10 consecutive days upon exposure to 14 °C light (L): 8 °C dark (D) and compared with those of corresponding control plants acclimated at 26 °C L: 20 °C D. Activities of superoxide dismutase were not substantially modified by the cold treatment. Activities of monodehydroascorbate reductase, expressed as percentages of the activities of control plants, increased significantly during the cold-treatment period and were significantly higher in Mississippi plants. Activities of glutathione reductase from Mississippi plants increased up to 200% during the cold-treatment period, while those from Quebec plants remained similar to those of corresponding control plants. The pattern of activity of ascorbate peroxidase in Mississippi plants was erratic but was reduced to about 50% that of Quebec plants during the last 2 days of the cold treatment, while in Quebec plants, ascorbate peroxidase activity was more constant over time and remained similar to control plants over the entire cold-treatment period. Dehydroascorbate reductase was the enzyme most affected by the cold treatment but, while the enzyme extracted from Mississippi plants was completely deactivated by day 4, residual activities were still recorded for the Quebec enzyme by day 9 of the cold-treatment period. The ascorbate contents of cold-acclimated Quebec plants were significantly higher than those of Mississippi plants with higher and similar values, respectively, when compared with control plants throughout the cold-temperature treatment. The glutathione content and reduced glutathione to oxidized glutathione ratios were significantly higher in cold-treated Mississippi plants compared with Quebec plants, although values in Quebec plants were never below those of control plants. The complex pattern of modifications in the activities of the oxygen-scavenging enzymes extracted from Quebec and Mississippi plants suggests that both weak adaptive and acclimatory processes are at play to counter, at least in part, the potential photoinhibitory effects imposed by the cold temperature treatment. Modifications of an acclimatory nature, which may benefit the enzyme performance of both Quebec and Mississippi plants, are shown by monodehydroascorbate reductase and, in particular, glutathione reductase, while higher ascorbate reductase and dehydroascorbate reductase activities in Quebec plants subjected to the cold-photoinhibitory treatment at the end of the treatment period would suggest that these enzymes may have been modified by natural selection to perform under cooler climatic conditions more likely to be associated with this cold-adapted ecotype. Key words: photoinhibition, cold temperatures, enzyme activities, superoxide dismutase, ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase, glutathione reductase, Echinochloa crus-galli, barnyard grass.

Le peroxyde d'hydrogène (H₂O₂) est une molécule de signalisation importante chez les plantes, et son contenu est contrôlé par divers systèmes, notamment les catalases (CAT) et les ascorbate peroxydases (APX). Le fonctionnement continu de l'APX nécessite la régénération de l'ascorbate, pour laquelle plusieurs possibilités existent. Un type d'enzyme régénérant l'ascorbate est la monodéshydroascorbate réductase (MDHAR), une protéine dépendante du NAD(P)H qui est codée par cinq MDAR gènes chez Arabidopsis. Ce travail visait à examiner l'importance de ces gènes en utilisant des mutants à perte de fonction spécifiques alliés à d'autres techniques. L'analyse des transcrits a révélé que des mutants knock-out pouvaient être obtenus pour tous les gènes. Les dosages de l'activité de MDHAR dans des extraits de feuilles ont révélé que la MDAR1 peroxysomale codait la majeure partie de l'activité dépendante du NADH, tandis que l'activité dépendante du NADPH était principalement codée par MDAR1, aux côtés de la MDAR2 cytosolique. À l'exception du mutant mdar4, qui nécessitait du sucre pour la germination et la croissance post-germinative précoce, tous les mutants montraient un phénotype de type sauvage lorsqu'ils étaient cultivés dans des conditions standard. Pour explorer si l'absence d'impact phénotypique des mutations était due à la redondance des gènes, deux approches ont été utilisées. Dans la première, les mutants ont été croisés avec le mutant cat2, qui présente une activité de catalase foliaire fortement réduite, afin de tester les interactions entre différents systèmes d'élimination du H₂O₂. Dans la seconde, nous avons cherché à explorer une redondance entre les deux gènes MDAR codant des isoformes peroxysomales en produisant des mutants doubles mdar1 mdar4. Sur la base de l'analyse moléculaire et biochimique de toutes ces lignées, il peut être conclu que l'expression d'au moins une isoforme peroxysomale pourrait être nécessaire à la viabilité des plantes et que la MDAR2 cytosolique semble coder la MDHAR la plus importante dans les conditions de stress oxydatif. Il est intéressant de noter que, l'introduction du mutant mdar2 dans le fond génétique cat2 a affaibli, plutôt que renforcé, certaines réponses au stress oxydatif, ce qui suggère des mécanismes de signalisation inédits liés à l'activité de la MDHAR
Hydrogen peroxide (H₂O₂) is an important signalling molecule in plants, and contents are controlled by various systems, notably catalases (CAT) and ascorbate peroxidases (APX). Continued function of APX requires regeneration of ascorbate, for which several possibilities exist. One type of ascorbate-regenerating enzyme is monodehydroascorbate reductase (MDHAR), an NAD(P)H-dependent protein which is encoded by five MDAR genes in Arabidopsis. This work aimed to examine the importance of these genes using specific loss-of-function mutants allied to other techniques. Transcript analysis showed that knockout mutants could be obtained for all the genes. Assays of MDHAR activity in leaf extracts revealed that the peroxisomal MDAR1 encoded most of the NADH-dependent activity while NADPH-dependent activity was chiefly encoded by MDAR1 alongside cytosolic MDAR2. Apart from mdar4, which required sugar for germination and early post-germinative growth, all mutants showed a wild-type phenotype when grown in standard conditions. To explore whether the lack of phenotypic impact of the mutations was caused by gene redundancy, two approaches were undertaken. In the first, mutants were crossed with the cat2 mutant, which has greatly decreased leaf catalase activity, to test for interactions between different H₂O₂-removing systems. In the second, we sought to examine redundancy between the two MDAR genes encoding peroxisomal isoforms by producing double mdar1 mdar4 mutants. Based on molecular and biochemical analysis of all these lines, it can be concluded that expression of at least one peroxisomal isoform might be required for plant viability and that the cytosolic MDAR2 seems to encode the most important MDHAR in oxidative stress conditions. Intriguingly, introduction of mdar2 into the cat2 background weakened rather than enforced some responses to oxidative stress, pointing to novel signalling mechanisms related to MDHAR activity

碩士
國立中山大學
海洋生物科技暨資源學系研究所
107
Monodehydroascorbate reductase (MDAR) is an antioxidant enzyme that converts oxidized ascorbate (MDA) to ascorbate (ASA) to maintain high ASA concentration for algae against oxidative stress. In this study, whether the annotated Chlamydomonas reinhardtii P. A. Dang CrMDAR1 (Cre17.g712100.t1.2) showed MDAR activity was first identified. Using the recombinant protein, we identified that the gene Cre17.g712100.t1.2 was MDAR. Next, the role of CrMDAR1 in response to abiotic stress were performed on C. reinhardtii via overexpression and downregulation of CrMDAR1. We have obtained four CrMDAR1-overexpressing lines (MDAR1_’5, 49, 81 and ‘93) and two CrMDAR1-knockdown lines (MDAR1_ami 30 and 36). Overexpression lines can increase their survival, MADR activity, mRNA abundance, AsA concentration, and AsA/DHA (dehydroascorbate) ratio, and induced lower lipid peroxidation after high-intensity illumination (1,800 μmol m-2 s-1). The CrMDAR1-knockdown lines showed a contrast result in response to 1,400 μmol m-2 s-1 illumination. Cell viability in response to other stresses (NaCl, MV, and H2O2) appeared a similar result to that under high light stress. In conclusion, ASA conversion efficiency (AsA/DHA ratio) and the damage of oxidative stress of C. reinhardtii was affected by CrMDAR1 overexpression/downregulation in response to abiotic stress.

Most of the objectives that were outlined in the original proposal have been met with two exceptions. Briefly, our goals were to: (1) constract transgenic tobacco plants which overproduce one or more of the enzymatic oxyradical scavengers and associated ancillary enzymes, including superoxide dismutase, ascorbate peroxidase, glutathione peroxidase, glutathione reductase, and monodehydrascorbate reductase; (2) evaluate the tolerance of these transgenic plants to oxidative stress; and (3) extend these studies to an agronomically important crop such as citrus. As can be seen i the following pages, our objectives (1) and (2) have been achieved, although transgenic lines overexpressing phospholipid hydroperoxidase glutathione peroxidase (PHGPX) were not obtained and our evidence to date suggests that constitutive overexpressing of the enzyme is probably lethal. Howeever, transgenic tobacco expressing the antisense construct for PHGPX were obtained. Tobacco plants overexpressing ascorbate peroxidase and those sensesuppressing monodehydroascorbate reductase are more tolerant to oxidative stress, as mediated by the redox-cycling agent paraquant; in contrast, plants expressing the PHGPX-antisense construct are more sensitive to paraquat. Additional research is warranted on each of the six types of transgenic lines which we generated with regard to their tolerance to saline stress. Until recently, attempts to transform citrus were not very successful, and thus additional attention is currently being directed at objective (3). We are optimistic that use of the plant transformation vector, pBIN, will lead to stable transgenic citrus, as preliminary experiments demonstrate stable expression of the GUS reporter gene. Other important contributions resulting from this BARD project include the biochemical characterization of the first plant phospholipid glutathione peroxidase and the biochemical and molecular analysis of another key antioxidant enzyme, monodehydroascorbate reductase. Overall this BARD-supported project was quite successful, and the biological resource of numerous transgenic lines which have altered levels of antioxidant enzymes should be valuable for years to come.