In order to better understand the role of antioxidant enzymes in plant stress protection mechanisms, transgenic tobacco (Nicotiana tabacum cv. Xanthi) plants were developed that overexpress both superoxide dismutase (SOD) and ascorbate peroxidase (APX) in chloroplasts. These plants were evaluated for protection against methyl viologen (MV, paraquat)-mediated oxidative damage both in leaf discs and whole plants. Transgenic plants that express either chloroplast-targeted CuZnSOD (C) or MnSOD (M) and APX (A) were developed (referred to as CA plants and AM plants, respectively). These plant lines were crossed to produce plants that express all three transgenes (CMA plants and AMC plants). These plants had higher total APX and SOD activities than non-transgenic (NT) plants and exhibit novel APX and SOD isoenzymes not detected in NT plants. As expected, transgenic plants that expressed single SODs showed levels of protection from MV that were only slightly improved compared to NT plants. The expression of either SOD isoform along with APX led to increased protection while expression of both SODs and APX provided the highest levels of protection against membrane damage in leaf discs and visual symptoms in whole plants.
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Oxygen is essential for the existence of aerobic life, but toxic reactive oxygen species (ROS), which include the superoxide anion radical (O2–•), hydroxyl radical (OH•), and hydrogen peroxide (H2O2), are generated in all aerobic cells during metabolic processes (Foyer, Descourvieres & Kunert 1994; Asada 1999). Injury caused by these ROS, known as oxidative stress, is one of the major damaging factors in plants exposed to environmental stress. Chloroplasts are especially sensitive to damage by ROS because electrons that escape from the photosynthetic electron transfer system are able to react with relatively high concentration of O2 in chloroplasts (Foyer et al. 1994). This phenomenon can lower the rates of photosynthesis and diminish plant growth. Plants possess capabilities to cope with oxidative stress by using ROS-scavenging enzymes such as superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), and low molecular weight antioxidants including ascorbic acid, glutathione and phenolic compounds (Noctor & Foyer 1998; Asada 1999).
The water–water cycle has been proposed as an explanation of the role of these antioxidant mechanisms at the onset of oxidative stress in chloroplasts (Asada 1999). The most important function of the water–water cycle is the rapid scavenging of superoxide radicals and hydrogen peroxide at their site of generation prior to their interaction with target molecules. In addition to the ROS-scavenging enzymes SOD and APX (thylakoid-bound and stromal), enzymes such as monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), and glutathione reductase (GR) that are necessary for the regeneration of oxidized ascorbic acid and glutathione, are also involved.
To maintain the productivity of plants under the stress condition, it is possible to fortify the antioxidative mechanism in the chloroplasts by manipulating the antioxidant enzymes and small antioxidant molecules in the chloroplasts. Transgenic plants that contain single transgenes for expression of SOD, APX, and GR in chloroplasts or other compartment of plant cells have been developed and analysed. However, manipulation of a single antioxidant gene has provided only limited improvements in plant stress tolerance. The effects of elevated levels of SOD in transgenic plants against oxidative- or environmental-stress have been varied (Foyer et al. 1994; Allen 1995). For example, Pitcher et al. (1991), Tepperman & Dunsmuir (1990), and Payton et al. (1997) found no improvements to oxidative or environmental stress tolerances, whereas Sen Gupta et al. (1993a, b), Bowler et al. (1991), Van Camp et al. (1994, 1996), Perl et al. (1993), McKersie et al. (1993, 1996, 2000), and Mc-Kersie, Bowley & Jones (1999) found significant improvements. These differences have usually been attributed to the complexity of the ROS detoxification system, because changing one enzyme may not change the capacity of the pathway as a whole. In the case of APX, transgenic tobacco plants expressing gene constructs for either cytosolic APX or a chimeric chloroplast-targeted cytosolic APX from pea have increased protection against methyl viologen (MV)-mediated membrane damage compared with untransformed control plants, and have increased protection to photo-oxidative stress (Allen, Webb & Schake 1997).
Development of transgenic plants with optimized tolerance to environmental stress may necessitate the overexpression of multiple antioxidant enzymes in chloroplasts. In this report, we describe the development of transgenic tobacco plants that express SOD (CuZnSOD and MnSOD) and APX simultaneously in chloroplasts. Simultaneous expression of these ROS-scavenging enzymes provides larger increases in protection from MV-induced damage than expression of either of these enzymes alone, indicating that these SOD and APX can function synergistically to increase oxidative stress tolerance.
Materials and methods
Plants and transformation
Nicotiana tabacum cv. Xanthi was used as plant materials. Chimeric gene constructs for overexpression of the chloroplastic CuZnSOD (referred to as C), MnSOD (M), and APX (A) were inserted into plant expression vectors, pBIN19, pCGN1578, and pGPTV-Bar (Becker et al. 1992) under the control of a CaMV 35S promoter, respectively. Double transgenic lines expressing both CuZnSOD and APX (referred to as CA plants) were generated by re-transformation of the previously described transgenic plants containing the chimeric chloroplast-targeted CuZnSOD (Sen Gupta et al. 1993a) with a chloroplast-targeted APX construct (Allen et al. 1997) using the pGPTV-Bar vector and bialaphos as a selection agent. Transgenic plants expressing APX and MnSOD (AM plants) were developed by transformation of the transgenic plants containing the chimeric chloroplast-targeted APX (Allen et al. 1997) with chloroplast-targeted MnSOD construct (Schake 1995) using bialaphos as a selection agent. In addition, transgenic plants expressing both forms of SOD (CuZnSOD and MnSOD) and APX in the chloroplasts (referred to as CMA plants and AMC plants) were obtained by crossing the CA and AM plants. Transgenic plants expressing SOD and/or APX were self-pollinated to produce T1 seeds. After selection of T1 seeds using kanamycin (100 mg L−1) for CuZnSOD, MnSOD and APX plants or combination of kanamycin and bialaphos (2 mg L−1) for CA, AM, CMA and MCA plants, the transgenic plants were grown in a greenhouse (16 h days, 30 °C day and 22 °C night) with daily watering. The plants were grown in 10-cm-diameter pots containing commercial mineral-mixed soil and 6- to 8-week-old plants at the five-leaf stage were used for the analysis.
The third fully expanded leaf from the top of each transgenic plant was collected to assay SOD and APX activities. Leaf tissue was ground in liquid N2, suspended in the appropriate homogenization buffer. The homogenization buffer for SOD was 50 mm potassium phosphate (pH 7·0) containing 0·1 mm EDTA, whereas that for APX was 50 mm Hepes (pH 7·0) containing 1 mm ascorbate and 1% (v/v) Triton X-100. After centrifugation in a microcentrifuge at 4 °C, supernatants were used determine enzyme activity and protein concentration (Bradford 1976).
Analysis of SOD isoenzymes was carried out using a method of Beauchamp & Fridovich (1971) and the determination of SOD-specific activity was assayed using the xanthine/xathine oxidase/cytochrome c method according to McCord & Fridovich (1969). One unit of SOD is the amount of the enzyme that inhibits the rate of reduction of cytochrome c by 50% in the coupled reaction.
Ion leakage was analysed according to the method of Bowler et al. (1991) with some modifications. Thirty leaf disks (7 mm in diameter) from fully expanded leaves of T1 plants, grown in a greenhouse for 2 months, were floated on the 0·4 m sorbitol containing 2 or 5 µm methyl viologen (MV). The leaf discs were incubated in the dark for 12 h to allow diffusion of MV into leaf, after which they were placed under continuous white light (150 µmol photons m−2 s−1). Thereafter loss of cytoplasmic solutes based on electrical conductance of the solution at 1 and 2 d after treatment (DAT) were measured using conductivity meter (Model 162; Thermo Orion, Beverly, MA, USA). The relative ion leakages of leaf discs from transgenic plants that measured at 1 and 2 DAT were compared with that of non-transgenic control plants.
MV treatment on whole plants
In order to investigate whether the transgenic plants (T1) have tolerance against MV-induced oxidative stress at the whole plant level, we evaluated the visible damage that appeared on the leaves by spraying various concentrations of MV solution (0, 25, 50 and 100 µm) at 3 DAT. MV dissolved in 0·1% Tween-20 solution was sprayed on the leaves of plants that have 7–8 leaves using a spray booth (Model SB-6; DeVries Manufacturing, Hollandale, MN, USA). The MV solution (70 mL) was applied to five tobacco plants using a 8001 VS type nozzle, 0·5 inch s−1, 0·22 MPa. The percentage of leaf damage that appeared on the seventh or eighth leaves after MV spraying was evaluated (0% indicates no damage on the leaves; 100% means fully damaged leaves).
The recovery of photosynthetic activity from the MV-treatment was estimated by chlorophyll fluorescence determination of photochemical yield (Fv/Fm), which represents the maximal yield of the photochemical reaction on photosystem II, using a portable chlorophyll fluorescence meter (PAM 2000; Heinz Walz GmbH, Effeltrich, Germany).
The tests for enzyme activities, ion leakage analysis, and visible damage analysis were repeated several times, at least three times, under the same conditions with essentially the same results. All measurements were subjected to analysis of variance (anova) to discriminate the significant difference. The significance in this paper refers to statistical significance at the P ≤ 0·05 level.
Transgenic tobacco plants expressing both SOD and APX in chloroplasts
Transgenic tobacco plants expressing both SOD and APX were successfully developed by the introduction of the second transgene into transgenic plants containing SOD or APX transgenes using the Agrobacterium-mediated transformation method. There were no apparent differences between the growth characteristics of transgenic plants and non-transgenic (NT) plants. Analysis of SOD and APX isoenzymes from NT plants and transgenic plants expressing SOD and APX transgenes alone and in combinations are shown in Figs 1a and 2a, respectively. Novel SOD and APX isoenzyme bands are apparent in extracts from transgenic plants that are not seen in NT plants. These bands are identical in plants that express both SOD and APX transgenes. In comparison with NT plants, the specific activity of SOD was increased by 1·15-fold in CA plants, 2·0-fold in AM plants, 2·4-fold in CMA plants, and 2·8-fold in MCA plants (Fig. 1b). The SOD-specific activity of C/A and A/C plants, which overexpress both Cu/Zn SOD and Mn SOD, was much higher than the transgenic plants that express only a single SOD transgene. APX-specific activity increased by 7·6-fold, 3·9-fold, 3·6-fold, and 3·0-fold, respectively (Fig. 2b). The APX-specific activity of CA plants was higher than that of plants expressing APX alone, whereas the APX activity from CMA plants and MCA plants was lower than that of APX plants and CA plants. The lower APX activities of CMA and AMC plants than those of CA and AM plants were likely to be caused by the introduction of two copies of the same APX transgene in a plant by crossing.
Protection of membrane damage in transgenic plants
The treatment of MV, a superoxide-generating herbicide, on leaf discs has been used as the test of tolerance to oxidative stress (Bowler et al. 1991; Sen Gupta et al. 1993b; Yun et al. 2000). The extent of cellular damage was quantified by ion leakage, which is a measure of membrane disruption.
The conductivity of the solution containing the leaf discs of transgenic plants expressing both SOD and APX (CA plants, AM plants, CMA plants and AMC plants) were much lower than that of NT plants or plants expressing the SOD or APX transgenes alone (Fig. 3a). When tobacco leaf discs were subjected to 2 µm MV, AMC plant and CMA plant showed about 82 and 47% reduction in membrane damage, respectively, compared with the NT plants (Xanthi) at 1 DAT. Severe necrosis was seen in the NT leaf discs, but the leaf discs of CA plants, AM plants, CMA plants and AMC plants showed only partial necrosis at the boundary of the leaf discs (data not shown). In comparison with the NT plants, relative ion leakage was 92·0% in CuZnSOD plants, 81·6% in MnSOD plants, 75·4% in APX plants, 58·3% in CA plants, and 81·7% in AM plants at 1 DAT. Although the 5 µm MV treatment was nearly saturating with respect to membrane damage, reduced levels of ion leakage were still seen in leaf discs from MCA plants (Fig. 3b). At 2 DAT, relative ion leakage from leaf discs of transgenic plants was equal to that of leaf discs of NT plants except for CMA plants and AMC plants, which had reductions of 23 and 41% ion leakage, respectively. The reduction in ion leakage from the leaf discs of plants expressing both SOD and APX indicated that the ROS was more efficiently scavenged by the simultaneous overexpression of SOD and APX in chloroplasts.
MV treatment on whole plants
Comparisons of the level of visible damage that appeared on leaves of transgenic and NT plants treated with MV at various concentrations is shown in Figs 4 and 5. Plants were evaluated at 3 DAT after spraying with solutions containing 0, 25, 50 and 100 µm MV. NT plants and transgenic plants expressing SOD or APX (CuZnSOD plants, MnSOD plants and APX plants) alone showed damage on about 30% of their leaf area at 25 µm MV whereas transgenic plants that express both SOD and APX transgenes (CA plants, AM plants, CMA plants and MCA plants) showed much less leaf damage (1·3–3% leaf area damage). Visible leaf damage on NT plants increased to 62% when the 50 µm MV solution was used and leaves of SOD-overexpressing plants had similar levels of damage. Plants that express the APX transgene showed about 40% damage whereas leaves of transgenic plants expressing both SOD and APX transgenes showed only 5–12% damage.
The leaves of plants that express both SOD and APX transgenes appeared to have greater resistance against MV-induced oxidative stress than NT plants and SOD-overexpressing plants (Fig. 6). Photosynthetic efficiency (Fv/Fm) was significantly reduced in NT plants and SOD-overexpressing plants. There were no differences in photosynthetic efficiencies between untreated NT plants and transgenic plants (data not shown). Less damage was apparent in plants that express the APX transgene whereas plants that simultaneously express both SOD and APX showed little loss of variable chlorophyll fluorescence following MV treatment. These results indicate that transgenic plants that simultaneously express transgenes for both SOD and APX in chloroplasts are significantly more tolerant to the ROS-generating compound MV, suggesting that they have higher ROS scavenging activity than transgenic plants that express either SOD or APX alone.
Transgenic tobacco plants that express transgenes for chloroplastic SOD and APX were developed using Agrobacterium-mediated methods. The increased activities of SOD and APX in these transgenic plants led us to elevate protection against MV-mediated oxidative stress in the whole plant level as well as in vitro leaf disc assay. Transgenic tobacco plants expressing the same CuZnSOD and MnSOD transgenes used in this study showed elevated levels of tolerance against oxidative stress mediated by MV and photo-oxidative damage (Sen Gupta et al. 1993a; Allen et al. 1997). However, it should be noted that the Cu/ZnSOD expressing transgenic plants also had elevated levels of APX activity (Sen Gupta et al. 1993b). Tobacco plants overexpressing chloroplast-targeted APX in chloroplasts also showed enhanced tolerance against oxidative stresses (Allen et al. 1997). Therefore, we predicted that transgenic plants that express transgenes for both SOD and APX could show synergistic effects leading to increased stress tolerance.
The increase in total APX activity in plants that expressed the APX transgene was more substantial than the increase in SOD in the either the CuZn- or MnSOD-expressing plants (Figs 1 and 2) and this is consistent with previously reported results from plants with these transgenes (Sen Gupta et al. 1993b; Allen et al. 1997). It is also important to note that the plants that expressed the CuZn SOD transgene did not have increased levels of native APX activity, unlike the plants described by Sen Gupta et al. (1993b). Our unpublished results indicate that the increased APX expression seen in plants that overexpress CuZnSOD is unstable and APX activities returned to normal levels within two or three sexual generations (S.A. Schake and R.D. Allen, unpublished results). In general, increased SOD activity in transgenic plants correlated with an increased resistance against membrane damage caused by MV-treatment of leaf discs (Fig. 3). However, a simple relationship between APX activity and MV tolerance was not as apparent. Although elevated APX activity did correlate with increased MV tolerance in plants that expressed the APX transgene alone, plants that expressed the APX transgene in combinations that included the MnSOD transgene (AM, CMA, AMC) had APX activities that were approximately one-half that of APX plants or the CA plants. Yet, these plants had the highest levels of protection against MV-induced damage both in leaf discs and at the whole plant level using both visible symptoms (Figs 4 and 5) and chlorophyll fluorescence (Fv/Fm) assays (Fig. 6). These results could indicate that there is a threshold level of APX activity necessary for protective effects to be achieved but higher levels do not lead to further increases in protection. However, combined overexpression of SOD and APX is synergistic, and provides protective effects not seen with either transgene alone.
A likely mechanism for this synergistic effect takes into account that the dismutation of superoxide anion radicals produces H2O2 that can inactivate CuZnSOD unless it is quickly scavenged by APX or other H2O2 scavenging systems (Sen Gupta et al. 1993b). Therefore, coincident increases in SOD and APX are complementary not only because they increase the capacity for superoxide and H2O2 scavenging, but also because both the native and transgene-derived CuZnSODs are protected from deactivation during stress. MnSODs are not naturally found in chloroplasts and they are not sensitive to deactivation by H2O2. Although transgenic tobacco plants that overexpress chloroplast-targeted MnSOD show increased protection from MV (Bowler et al. 1991; Slooten et al. 1995; Allen et al. 1997), our results clearly show that coexpression of MnSOD and APX (as in AM, CMA, and AMC plants) provided substantially more oxidative stress protection than expression of the MnSOD transgene alone.
Several reports have shown that drought, salt, freezing and heavy metal stresses are also accompanied by the formation of ROS (Holmberg & Bülow 1998; Dat et al. 2000). The expressions of a CuZnSOD gene from cassava and peroxidase (POD) genes from sweet potato were increased by various stresses such as MV, ozone and chilling treatment (Huh et al. 1997; Lee et al. 1999; Kim et al. 1999). Transgenic tobacco plants expressing a sweet potato anionic POD (swpa1) showed about a 25% reduction in membrane damage relative to NT plants (Yun et al. 2000).
Development of plants with tolerance to more extreme conditions may be achieved by introducing genes involved in different stress resistance mechanisms into a single plant. That is, introduction of genes for production of osmoprotectants, heat-shock protein, and ROS scavenging enzyme in a plant by multiple transformation or by cross-pollination of plants containing different stress-tolerant genes, could contribute to overcoming various stresses. Furthermore, it is critical that protective gene products are targeted to appropriate locations and the level and time of expression are controlled to ensure precursor availability for each enzyme in order to avoid negative effects.
We propose that plants that overexpress both SOD and APX are able to more rapidly scavenge superoxide anion radicals and hydrogen peroxide at the site of generation, prior to their interaction with target molecules. In order to further improve protection from oxidative stress in chloroplasts, manipulation of enzymes such as DHAR, MDHR and GR may be required to increase the regeneration of reduced ascorbate and glutathione (Kwon et al. 2001). Aono et al. (1995) showed that coexpression of GR from Escherichia coli and CuZnSOD from rice together in the cytosol of transgenic tobacco plants increased oxidative stress tolerance. Plants expressing both GR and CuZnSOD exhibited less damage following MV treatment than plants expressing individual GR or CuZnSOD transgenes. Recently, Payton et al. (2001) showed increased activities of APX and GR in chloroplasts of cotton could improve the recovery of photosynthesis following exposures to low temperature and high photon flux density. These results indicate that the expression of combinations of transgenes for ROS-scavenging enzymes and antioxidant-regenerating enzymes in transgenic plants could have synergistic effects on stress tolerance.
ROS, particularly hydrogen peroxide, have been shown to act as critical signals for plant adaptation to biotic and abiotic stresses (Mittler et al. 1999; Karpinski et al. 1999). Therefore, under the stress conditions, ROS may play two very different roles: damaging the cellular components or signalling the activation of defence responses (Dat et al. 2000; Grant & Loake 2000). To allow for these different roles, cellular levels of ROS must be tightly controlled. A strong constitutive promoter such as CaMV 35S promoter is typically used for expression of foreign genes in plants. However, a more precise regulation of expression using an inducible promoter, especially a stress-inducible promoter, might be useful for development of stress-tolerant plants and production of proteins that have deleterious effects on plant growth (Yoshida & Shinmyo 2000). For example, constitutive expression of a stress-inducible transcription factor caused retardation of the plant growth but this effect could be negated by using the stress responsive rd29A promoter (Kasuga et al. 1999). Recently, we developed an oxidative stress-inducible POD promoter (SWPA2 promoter) from sweet potato (Kim 2000). The SWPA2 promoter will be useful for the development of an efficient plant with enhanced tolerance to environmental stresses.
In conclusion, we developed transgenic tobacco plants expressing both SOD and APX in chloroplasts under the control of CaMV 35S promoter for enhanced protection against oxidative stress. Our results indicate that the simultaneous expression of SOD and APX in chloroplasts provided much better protection from MV-mediated oxidative stress than single expression of SOD or APX, showing the additive effect of two enzymes in ROS-scavenging activity. Now transgenic plants expressing multiple antioxidant enzymes under the control of a stress-inducible promoter (Kim 2000) are under investigation.
This work was supported by a research grant (CGM0300111) from Crop Functional Genomics Center, Korea Ministry of Science and Technology, the USDA/NRI, the Human Frontiers in Sciences Program and the Texas Advanced Technology Program. Partial support for S.Y.K. was provided through an overseas postdoctoral fellowship from Korea Science and Engineering Foundation (KOSEF). The authors gratefully acknowledge Dr Robert Webb, Dr Sheryl Schake and Ms Young-Sook You for developing the APX- and MnSOD-expressing transgenic plants.
Received 2 October 2001;received inrevised form 18 February 2002;accepted for publication 25 February 2002