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      Clones of FeSOD, MDHAR, DHAR Genes from White Clover and Gene Expression Analysis of ROS-Scavenging Enzymes during Abiotic Stress and Hormone Treatments

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          Abstract

          Increased transcriptional levels of genes encoding antioxidant enzymes play important protective roles in coping with excessive accumulation of reactive oxygen species (ROS) in plants exposed to various abiotic stresses. To fully elucidate different evolutions and functions of ROS-scavenging enzymatic genes, we isolated iron superoxide dismutase ( FeSOD), dehydroascorbate reductase ( DHAR) and monodehydroascorbate reductase ( MDHAR) from white clover for the first time and subsequently tested dynamic expression profiles of these genes together with previously identified other antioxidant enzyme genes including copper zinc superoxide dismutase ( Cu/ZnSOD), manganese superoxide dismutase ( MnSOD), glutathione reductase ( GR), peroxidase ( POD), catalase ( CAT), and ascorbate peroxidase ( APX) in response to cold, drought, salinity, cadmium stress and exogenous abscisic acid (ABA) or spermidine (Spd) treatment. The cloned fragments of FeSOD, DHAR and MDHAR genes were 630, 471 and 669 bp nucleotide sequences encoding 210, 157 and 223 amino acids, respectively. Phylogenetic analysis indicated that both amino acid and nucleotide sequences of these three genes are highly conservative. In addition, the analysis of genes expression showed the transcription of GR, POD, MDHAR, DHAR and Cu/ZnSOD were rapidly activated with relatively high abundance during cold stress. Differently, CAT, APX, FeSOD, Cu/ZnSOD and MnSOD exhibited more abundant transcripts compared to others under drought stress. Under salt stress, CAT was induced preferentially (3–12 h) compared to GR which was induced later (12–72 h). Cadmium stress mainly up-regulated Cu/ZnSOD, DHAR and MDHAR. Interestingly, most of genes expression induced by ABA or Spd happened prior to various abiotic stresses. The particular expression patterns and different response time of these genes indicated that white clover differentially activates genes encoding antioxidant enzymes to mitigate the damage of ROS during various environmental stresses.

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          Overexpression of monodehydroascorbate reductase in transgenic tobacco confers enhanced tolerance to ozone, salt and polyethylene glycol stresses.

          Ascorbate (AsA) is a major antioxidant and free-radical scavenger in plants. Monodehydroascorbate reductase (MDAR; EC 1.6.5.4) is crucial for AsA regeneration and essential for maintaining a reduced pool of AsA. To examine whether an overexpressed level of MDAR could minimize the deleterious effects of environmental stresses, we developed transgenic tobacco plants overexpressing Arabidopsis thaliana MDAR gene (AtMDAR1) in the cytosol. Incorporation of the transgene in the genome of tobacco plants was confirmed by PCR and Southern-blot analysis and its expression was confirmed by Northern- and Western-blot analyses. These transgenic plants exhibited up to 2.1-fold higher MDAR activity and 2.2-fold higher level of reduced AsA compared to non-transformed control plants. The transgenic plants showed enhanced stress tolerance in term of significantly higher net photosynthesis rates under ozone, salt and polyethylene glycol (PEG) stresses and greater PSII effective quantum yield under ozone and salt stresses. Furthermore, these transgenic plants exhibited significantly lower hydrogen peroxide level when tested under salt stress. These results demonstrate that an overexpressed level of MDAR properly confers enhanced tolerance against ozone, salt and PEG stress.
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            Scavenging of reactive oxygen species in NaCl-stressed rice (Oryza sativa L.)—differential response in salt-tolerant and sensitive varieties

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              Enhanced tolerance to oxidative stress in transgenic tobacco plants expressing three antioxidant enzymes in chloroplasts.

              The effect of simultaneous expression of genes encoding three antioxidant enzymes, copper zinc superoxide dismutase (CuZnSOD, EC 1.15.1.1), ascorbate peroxidase (APX, EC 1.11.1.11), and dehydroascorbate (DHA) reductase (DHAR, EC 1.8.5.1), in the chloroplasts of tobacco plants was investigated under oxidative stress conditions. In previous studies, transgenic tobacco plants expressing both CuZnSOD and APX in chloroplast (CA plants), or DHAR in chloroplast showed enhanced tolerance to oxidative stresses, such as paraquat and salt. In this study, in order to develop transgenic plants that were more resistant to oxidative stress, we introduced the gene encoding DHAR into CA transgenic plants. Mature leaves of transgenic plants expressing all three antioxidant genes (CAD plants) had approximately 1.6-2.1 times higher DHAR activity, and higher ratios of reduced ascorbate (AsA) to DHA, and oxidized glutathione (GSSG) to reduced glutathione (GSH) compared to CA plants. CAD plants were more resistant to paraquat-induced stress, exhibiting only 18.1% reduction in membrane damage relative to CA plants. In addition, seedlings of CAD plants had enhanced tolerance to NaCI (100 mM) compared to CA plants. These results indicate that the simultaneous expression of multiple antioxidant enzymes, such as CuZnSOD, APX, and DHAR, in chloroplasts is more effective than single or double expression for developing transgenic plants with enhanced tolerance to multiple environmental stresses.
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                Author and article information

                Contributors
                Role: Academic Editor
                Journal
                Molecules
                Molecules
                molecules
                Molecules
                MDPI
                1420-3049
                24 November 2015
                November 2015
                : 20
                : 11
                : 20939-20954
                Affiliations
                Author notes
                [* ]Correspondence: pengyanlee@ 123456163.com (Y.P.); zhangxq@ 123456sicau.edu.cn (X.Z.); Tel.: +86-835-288-2422 (Y.P.); +86-835-288-2270 (X.Z.); Fax: +86-835-288-6080 (Y.P. & X.Z.)
                Article
                molecules-20-19741
                10.3390/molecules201119741
                6332117
                26610459
                fce041c5-2375-4f3d-b7b5-e6a993ee7078
                © 2015 by the authors.

                Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons by Attribution (CC-BY) license ( http://creativecommons.org/licenses/by/4.0/).

                History
                : 16 October 2015
                : 18 November 2015
                Categories
                Article

                white clover (trifolium repens l.),ros-scavenging,expression analysis,abiotic stress

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