Elsevier

Journal of Plant Physiology

Volume 164, Issue 12, 3 December 2007, Pages 1626-1638
Journal of Plant Physiology

Simultaneous overexpression of both CuZn superoxide dismutase and ascorbate peroxidase in transgenic tall fescue plants confers increased tolerance to a wide range of abiotic stresses

https://doi.org/10.1016/j.jplph.2007.01.003Get rights and content

Summary

To mitigate the oxidative damage inflicted by biotic or abiotic stresses, plants have evolved complex anti-oxidative defense mechanisms that involve induction of antioxidant and anti-oxidative enzymes, such as superoxide dismutase (SOD) and ascorbate peroxidase (APX). To determine whether overexpression of the genes encoding copper–zinc SOD (CuZnSOD) and APX in plants is capable of decreasing reactive oxygen species (ROS) produced in response to abiotic stresses, we generated transgenic tall fescue plants expressing the CuZnSOD and APX genes in chloroplasts under the control of the oxidative stress-inducible promoter, sweet potato peroxidase anionic 2 (SWPA2). Transgenic plants were generated by Agrobacterium-mediated genetic transformation, and genotypes were confirmed by DNA blot analysis. Transgenic plants were exposed to several ROS-generating abiotic stresses, such as methyl viologen (MV), H2O2, and the heavy metals copper, cadmium, and arsenic, and their tolerance was evaluated. High levels of CuZnSOD and APX gene transcripts in the transgenic plants under these treatments suggested that the transgenes were functionally expressed. Compared to transgenic plants, higher amounts of ROS were generated in the leaves of control plants exposed to abiotic stresses, resulting in increased thiobarbituric acid reactive substances (TBARS), ion leakage, and chlorophyll degradation. These parameters were significantly lower in transgenic plants. Enzyme activity assays and native polyacrylamide gel electrophoresis (PAGE) showed that total SOD and APX were highly active in transgenic plants under the abiotic stresses examined. We conclude that one of the mechanisms of increased anti-oxidative defense in transgenic tall fescue plants is overexpression of the CuZnSOD and APX genes, which are utilized in scavenging ROS and thus provide improved tolerance to abiotic stresses.

Introduction

Exposure to abiotic stresses such as drought, cold, heat, and pollutants, including herbicides and heavy metals, can give rise to excess accumulation of reactive oxygen species (ROS) in plant cells (Price et al., 1989; Bowler et al., 1992; Stohs and Bagchi, 1995; Noctor and Foyer, 1998; Schützendübel and Polle, 2002). ROS are potentially harmful to the cell, as they can raise the level of oxidative damage through loss of cellular structure and function. Cells possess antioxidants and antioxidative enzymes to interrupt the cascades of uncontrolled oxidation in cellular organelles. Among all the antioxidative enzymes, superoxide dismutase (SOD) and ascorbate peroxidase (APX) play key roles in ROS detoxification in cells. SOD, the first enzyme in the detoxifying process, converts superoxide anaion radicals (O2-) to hydrogen peroxide (H2O2), and APX reduces H2O2 to water using ascorbic acid as a specific electron donor (Asada, 1992; Foyer et al., 1994; Asada, 1999). Chloroplasts, the major component of photosynthetic tissue, is highly sensitive to damage by ROS, which are frequently generated by the reaction of chloroplast O2 and electrons that escape from the photosynthetic electron transfer system (Foyer et al., 1994). To cope with this unavoidable generation of ROS-induced oxidative damage, plants employ the choloroplastic ROS-scavenging enzymes copper–zinc SOD (CuZnSOD) and APX. The role of these antioxidative enzymes at the onset of oxidative stress in chloroplasts has been extensively characterized (Asada, 1999).

Efforts to fortify antioxidant defenses in chloroplasts or in the cytosol include overexpression of CuZnSOD or APX, which provides enhanced tolerance to oxidative stress in plants (Perl et al., 1993; Gupta et al., 1993a, Gupta et al., 1993b; Mittler et al., 1999; Murgia et al. 2004). In certain cases, however, overexpression of a single antioxidative enzyme did not provide protection against oxidative or abiotic stresses (Tepperman and Dunsmuir, 1990; Pitcher et al., 1991; Torsethaugen et al., 1997), suggesting that overexpression of one enzyme may not alter the function of the entire anti-oxidant pathway. Therefore, it has been suggested that combined expression of SOD and APX may provide better stress tolerance than overexpression of a single gene (Gupta et al., 1993a, Gupta et al., 1993b). In support of this hypothesis, Kwon et al. (2002) demonstrated that overexpression of both the CuZnSOD and APX genes in tobacco chloroplasts resulted in enhanced tolerance to methyl viologen (MV)-induced oxidative stress compared to expression of either of these genes alone. Recently, Tang et al. (2006) generated transgenic potato plants expressing the CuZnSOD and APX genes in chloroplasts, where instead of the CaMV 35S promoter, the both genes were independently controlled by the oxidative stress-induced SWPA2 promoter. Although, CaMV 35S, a strong constitutive promoter, is frequently used for foreign gene expression in plants, the use of an oxidative stress-inducible promoter, such as SWPA2 (Kim et al., 2003), might confer more precise regulation of abiotic stress tolerance in transgenic plants (Tang et al., 2006).

Various environmental conditions (cold, heat, drought, salt) and/or man-made secondary stress factors (wear, traffic, herbicides, irrigation scheduling, or other cultural practices) constantly interact with and either enhance or alleviate a wide range of primary abiotic and edaphic stresses on forage and turfgrass cultivars, which ultimately decreases their growth and production (Duncan and Carrow, 2001). Recently, efforts have been made to produce enhanced biotic or abiotic stress tolerance in transgenic forage grasses (Toyama et al. 2003; Hisano et al., 2004; Hu et al., 2005; Takahashi et al., 2006). To the best of our knowledge, however, there have been no reports of overexpression either SOD or APX in forage grasses, particularly in tall fescue. Previously, we established an Agrobacterium-mediated genetic transformation protocol for tall fescue (Lee et al., 2004) and orchardgrass (Lee et al., 2006). Consequence of our research, we introduced a chimeric gene construct, SWPA2::CuZnSOD::APX (Tang et al., 2006), into tall fescue plants to generate an abiotic stress-tolerant forage crop. It has been shown that transgenic potato plants expressing this construct had enhanced tolerance to MV or heat treatment (Tang et al., 2006), suggesting that overexpression of both SOD and APX provides better tolerance to abiotic stress-induced oxidative damage.

In the present study, targeted overexpression of the CuZnSOD and APX genes in chloroplasts of transgenic tall fescue plants conferred enhanced tolerance to MV, and protection against heavy metal-induced oxidative damage. Our results suggest that overexpression of both CuZnSOD and APX is a worthwhile approach to producing transgenic plants with enhanced tolerance to a wide range of abiotic stresses.

Section snippets

Plant materials and genetic transformation

Dehusked mature seeds of tall fescue (Festuca arundinacea Schreb. cv. Kentucky-31) were used as the primary source of explant material for genetic transformation. The binary vector pCAMBIA1300, containing the hygromycin phosphotransferase gene (hpt) under the control of the CaMV 35S promoter, was used as the control vector. A chimeric gene construct containing cassava CuZnSOD (Genbank accession no. AF170297; Lee et al., 1999) and pea APX (Genbank accession no. X62077; Allen et al., 1997)

Expression of transgenes in transgenic plants

Six independent transgenic plant lines were obtained by selection on hygromycin-containing selection medium. To confirm the stable integration of both the CuZnSOD and APX genes into the genome of tall fescue, genomic DNA was isolated, digested by restriction enzymes and hybridized with 32P-labelled probes specific for CuZnSOD or APX (Fig. 2A, B). DNA blot analysis of genomic DNA revealed that both the CuZnSOD and APX genes were successfully integrated in all six transgenic tall fescue lines (

Conclusion

In this study, we generated transgenic lines of the forage grass tall fescue with enhanced abiotic stress tolerance using Agrobacterium-mediated genetic transformation, and evaluated its tolerance to a wide range of oxidative stress-generating abiotic stresses. Transgenic plants overexpressed both CuZnSOD and APX under the control of the oxidative stress-inducible promoter, SWAP2, and expression was targeted to chloroplasts. Our results in this study showed that leaf-damage was paralleled with

Acknowledgements

This work was supported by a grant from the BioGreen 21 Program, Rural Development Administration, Korea. SH Lee, DG Lee, KW Lee and DH Kim are supported by scholarships from the BK21 program, Ministry of Education & Human Resources Development, Korea.

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    Both authors contributed equally to this work.

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