Edited by C. H. Foyer
Enhanced tolerance to ozone and drought stresses in transgenic tobacco overexpressing dehydroascorbate reductase in cytosol
Article first published online: 24 FEB 2006
Volume 127, Issue 1, pages 57–65, May 2006
How to Cite
Eltayeb, A. E., Kawano, N., Badawi, G. H., Kaminaka, H., Sanekata, T., Morishima, I., Shibahara, T., Inanaga, S. and Tanaka, K. (2006), Enhanced tolerance to ozone and drought stresses in transgenic tobacco overexpressing dehydroascorbate reductase in cytosol. Physiologia Plantarum, 127: 57–65. doi: 10.1111/j.1399-3054.2006.00624.x
- Issue published online: 18 APR 2006
- Article first published online: 24 FEB 2006
- Received 16 August 2005; revised 20 September 2005
Ascorbate (vitamin C) is a potent antioxidant protecting plants against oxidative damage imposed by environmental stresses such as ozone and drought. Dehydroascorbate reductase (DHAR; EC 18.104.22.168) is one of the two important enzymes functioning in the regeneration of ascorbate (AsA). To examine the protective role of DHAR against oxidative stress, we developed transgenic tobacco plants overexpressing cytosolic DHAR gene from Arabidopsis thaliana. Incorporation of the transgene in the genome of tobacco plants was confirmed by polymerase chain reaction and Southern blot analysis, and its expression was confirmed by Northern and Western blot analyses. These transgenic plants exhibited 2.3–3.1 folds higher DHAR activity and 1.9–2.1 folds higher level of reduced AsA compared with non-transformed control plants. The transgenic plants showed maintained redox status of AsA and exhibited an enhanced tolerance to ozone, drought, salt, and polyethylene glycol stresses in terms of higher net photosynthesis. In this study, we report for the first time that the elevation of AsA level by targeting DHAR overexpression in cytosol properly provides a significantly enhanced oxidative stress tolerance imposed by drought and salt.
active oxygen species
Under stressful environment, plants are severely damaged due to oxidative stress derived by the accumulated active oxygen species (AOS) in plant cells. Active oxygen species, such as singlet oxygen (O21), superoxide radical (O2–), hydrogen peroxide (H2O2), and hydroxyl radical (OH·), are capable of unrestricted oxidation of many cellular components and can lead to oxidative destruction of the cell (Asada and Takahashi 1987, Mittler 2002). Main cellular components such as lipids, proteins, carbohydrates, and nucleic acids are candidates to be oxidatively damaged. Higher plants developed several enzymatic and non-enzymatic scavenging systems to reduce the deleterious effects of AOS. Plants detoxify AOS by the combination of antioxidants, such as ascorbate (AsA), glutathione (GSH), and α-tocopherol, and antioxidative enzymes, such as superoxide dismutase (SOD), ascorbate peroxidase (APX), and catalase (CAT). Antioxidative enzymes involved in the ascorbate–glutathione (AsA–GSH) pathway, mainly monodehydroascorbate reductase (MDAR), dehydroascorbate reductase (DHAR), and glutathione reductase (GR), are considered of a paramount importance in plant antioxidant defense mechanism. Components of AsA–GSH pathway have been reported to exist in chloroplast, cytosol, mitochondria, and peroxisomes representing an important antioxidant defense system against H2O2 generated in these organelles (Potters et al. 2002).
AsA is a major antioxidant reacting directly with hydroxyl radicals, superoxide anion, and singlet oxygen (Noctor and Foyer 1998). Moreover, AsA is the major redox buffer in plants and is present in cytosol, chloroplast, vacuoles, mitochondria, and apoplast (Pignocchi and Foyer 2003, Potters et al. 2002). In addition to AsA importance as an antioxidant, it is also a cofactor of many enzymes (Smirnoff and Wheeler 2000), a regulator of cell division and growth (Kerk and Feldman 1995), and a molecule for signal transduction in plants (Noctor et al. 2000). AsA is synthesized in the mitochondria and consequently transported to other compartments of plant cells (Horemans et al. 2000a). Most of AsA is reported to be localized in cytoplasm (Pignocchi et al. 2003), up to 10% is localized in the apoplast (Noctor and Foyer 1998) and 12–30% could accumulate in chloroplasts (Horemans et al. 2000a). AsA serves as an electron donor for H2O2 detoxification. APX uses two molecules of AsA to reduce H2O2 to water with two molecules of monodehydroascorbate (MDHA) being generated in this reaction. In the chloroplast stroma, MDHA is reduced enzymatically to AsA by MDAR using both NADH and NADPH as electron donors. Being an unstable radical due to its short lifetime, MDHA spontaneously disproportionates to AsA and dehydroascorbate (DHA) if not rapidly reduces to AsA (Noctor and Foyer 1998). DHAR catalyzes the reduction of DHA to AsA using reduced GSH. Because DHA is also an unstable molecule, if not rapidly reduced to AsA, it undergoes spontaneous and irreversible hydrolysis to 2,3-diketogulonic acid (Deutsch 2000). Many lines of evidence suggest that apoplastic DHA has to be returned to the cytosol for the reduction to AsA (Horemans et al. 2000b, Pignocchi et al. 2003). DHAR was purified and characterized from spinach leaves (Hossain and Asada 1984), potato tubers (Dipierro and Borraccino 1991), and rice (Kato et al. 1997). A mammalian cDNA encoding DHAR was isolated from a rat liver cDNA library (Ishikawa et al. 1998). DHAR cDNAs were recently cloned from plants such as spinach (Shimaoka et al. 2000) and rice (Urano et al. 2000). Transgenic plants with manipulated expression levels of antioxidant enzymes provided a significant tool to study the defense mechanism against oxidative stress and have provided new insights into the role of antioxidant enzymes in scavenging AOS (Allen et al. 1997, Foyer et al. 1994). Many reports have focused on chloroplast-targeted antioxidant enzymes such as GR (Aono et al. 1993), CAT (Mohamed et al. 2003), chloroplast-targeted DHAR (Kwon et al. 2003), SOD (Badawi et al. 2004a), and APX (Badawi et al. 2004b). Overexpression of DHAR in cytosol remains an attractive area to investigate its significance in regenerating AsA and in protecting plants against oxidative stress. Therefore, in this study, we report the development of transgenic tobacco plants overexpressing DHAR in cytosol and its physiological importance in protecting plants against various stresses such as ozone, drought, salt, and polyethylene glycol (PEG).
Materials and methods
Construction of plant expression vector and tobacco transformation
The cDNA encoding Arabidopsis thaliana cytosolic DHAR (accession number AY140019) was amplified by reverse transcription polymerase chain reaction (RT-PCR) from A. thaliana total RNA using primers (5′-ATGGCTCTAGATATCTG-3′) and (5′-TCACGCAT- TCACCTTC-3′). The ends of the amplified fragment were modified by PCR to introduce SmaI and SacI sites using AtDHSm primer (5′-CGTCTAGACTCCACCCG- GGCTATGGCTCTAGATATCTG-3′) and AtDHSc primer (5′-ACGAGCTCGTCACGCATTCACC-3′). The end-modified fragment was digested with SmaI and SacI and cloned in the corresponding sites of pBE2113-GUS plant high-expression vector (Mitsuhara et al. 1996) downstream of CaMV35S (cauliflower mosaic virus promoter) and upstream of Tnos (polyadenylation signal of nopaline synthase) terminator (Fig. 1). This construct (pBE-DHAR) was introduced into Agrobacterium tumefaciens strain C58C1 by electroporation. Sterile leaf discs from Nicotiana tabacum (SR-1) plants grown on MS (Murashige and Skoog 1962) hormone-free medium under sterile growth conditions were used for Agrobacterium-mediated gene transfer as described by Badawi et al. (2004a). Genomic DNA was isolated from transgenic and non-transformed (SR-1) control plant leaves using ISOPLANT II kit (Nippon gene, Co., Ltd, Toyama, Japan). The presence of DHAR transgene in the genome of transgenic plants was verified by PCR using the isolated DNA, and AtDHSm and AtDHSc primers.
Southern and Northern blot analyses
Southern blot analysis was conducted to further confirm the incorporation of DHAR transgene in the genome of transgenic plants and to determine the independent transgenic lines. Northern blot analysis was conducted to determine transgenic plants that expressed DHAR transgene. Ten micrograms of DNA from DHAR transgenic and SR-1 control plants was digested with EcoRI restriction enzyme and transferred to nylon membrane (Hybond™-N+, Amersham, UK). Leaf tissues (0.1 g) from DHAR transgenic and SR-1 control plants were used to isolate RNA by phenol/SDS and LiCl method (Chirgowin et al. 1979). Total RNA (20 µg) was separated in formaldehyde gel and transferred to nylon membrane (Hybond™-N+, Amersham). Hybridization was carried out following standard procedures (Sambrook et al. 1989). DHAR cDNA probe was labelled by PCR using PCR DIG Probe Synthesis Kit (Roche Applied Bioscience, S.A., Mannheim, Germany), and the detection of hybridized DIG labelled probe was conducted using DIG Luminescent Detection Kit (Roche Applied Science) according to manufacturer's instructions.
Western blot analysis
The full-length DHAR cDNA was cloned downstream of 6xHis-tag sequence in SmaI site of the pQE-32 vector (Qiagen, Tokyo, Japan). Expression and purification of the His-tagged recombinant DHAR protein was conducted using Ni-NTA agrose system (QIAexpress®, Qiagen) as instructed by manufacturer. Antibodies against the purified recombinant DHAR protein were raised by injecting this protein into guinea pig, and the serum containing anti-DHAR antibodies was used as primary antibody for Western blot analysis. Proteins were extracted from DHAR transgenic and SR-1 control plants using TRIzol (Invitrogen, Carlsbad, CA). Protein samples (25 µg) were separated in 15% SDS-PAGE and transferred to Hybond ECL Nitrocellulose membrane (Amersham Biosciences, Little Chalfont, UK) by ATTO semidry transfer cell (ATTO Corporation, Tokyo, Japan). Immunodetection was performed using diluted (1 : 20 000) guinea-pig antibodies against His-tagged DHAR protein as the first antibody and a diluted (1 : 5000) horseradish peroxidase-conjugated anti- guinea-pig IgG (Sigma, St. Louis, MI) as the second antibody.
DHAR activity assay
DHAR activity was assayed spectrophotometrically according to the method of Nakano and Asada (1981) with slight modification. Fresh leaf tissues (0.2 g) from DHAR transgenic and SR-1 control plants were frozen in liquid nitrogen, grounded to powder in pre-cooled mortars, and homogenized with 2 ml of 0.1 M Tris–HCl (pH 7.8). The slurry was centrifuged (15 000 g, 4°C) for 20 min, and the supernatant (crude extract) was used immediately for enzyme assay. The assay was performed at 25°C with a reaction mixture containing 50 mM 2-Morpholinoethanesulfonic acid (MES)–NaOH (pH 6.3), 2 mM dehydroascorbate, 5 mM GSH, and crude extract. The increase in absorbance at 290 nm due to AsA was monitored, and the activity was calculated using absorbance coefficient of 2.8 mM−1 cm−1. Total protein was determined according to Bradford (1976).
Determination of reduced and oxidized AsA levels
Leaf tissues (0.2 g) from DHAR transgenic and SR-1 control plants were frozen in liquid nitrogen, grounded to fine powder in pre-cooled mortars, and then homogenized in 2 ml of an ice-cold 5% metaphosphoric acid. The homogenate was centrifuged (15 000 g, 4°C) for 20 min, and 0.1 ml of the supernatant was added to 0.9 ml of 5% metaphosphoric acid then filtered through 0.45 µm MILLEX-®HV filter unit (Millipore, S.A, Molsheim, France). Ten microliters of sample was used for determination of reduced AsA on CAPCELL PAK C18 120 (Shiseido Co. Ltd, Tokyo, Japan) column, with 80% acetonitrile and 20% of 0.01 M potassium phosphate (pH 3.0) as the mobile phase at rate of 0.5 ml min−1. Ascorbate was detected in TOSOH UV-8010 absorbance detector (Tosoh Co., Tokyo, Japan) set at 248 nm. Calibrations were linear in the range of 50–300 ng of ascorbic acid. (DHA) was reduced to AsA by neutralizing metaphosphoric acid in samples with 5 M KOH and adding Dithiothreitol (DTT) to final concentration of 10 mM then incubated for 30 min at room temperature. DHA was calculated as the difference between the total AsA (reduced plus oxidized) and reduced AsA.
Plant growth condition
The transgenic progeny (T1) from the self-pollinated primary transformed lines (T0) were germinated on kanamycin-containing MS medium, whereas SR-1 seeds were germinated on antibiotic-free MS medium and maintained in a growth chamber for 6 weeks at 12 h light cycle, 25°C, and 23% RH. Seedlings were transplanted in vermiculite, maintained in controlled conditions (25°C, 45–55% RH, and 14 h light cycle), and irrigated with water supplied with 1 ml l−1 nutrient solution (Hyponex 5-10-5, Hyponex, Osaka, Japan). Unless mentioned elsewhere, three replications of 8–10-week-old plants that were uniform in height and number of leaves were used in all stress evaluation experiments.
Applying ozone, salt, drought, and PEG stress
SR-1 control plants and two DHAR transgenic lines (Dha4 and Dha7) were selected for ozone stress experiment, transferred to growth chamber (Koito Co. Ltd, Tokyo, Japan) and fumigated with 0.2 p.p.m. ozone. Ozone was generated using OES-10A ozone generator (Dylec, Inc., Osaka, Japan), and the accurate fumigation level was continuously monitored by ozone monitor (OZONE MONITOR, Model 1200, Dylec, Inc.). SR-1 control plants and three DHAR transgenic lines (Dha3, Dha4, and Dha7) were selected for drought, salt, and PEG stress experiments. Salt stress was applied by irrigating plants with 0.3 M NaCl solution supplemented with 1 ml l−1 Hyponex nutrient solution. Drought stress was applied by withholding water from the plants. PEG stress was applied by irrigating plants with 10% (w/v) PEG solution supplied with 1 ml l−1 Hyponex nutrient solution.
Net photosynthesis measurements
Net photosynthesis (µmol CO2 m−2 s−1) was measured by portable photosynthesis system (LI-6400; Li-Cor Inc., Lincoln, NE) starting from zero time which was immediately before applying stress and continued for a designated time. Measurements were taken in enclosed chamber under controlled growth conditions as described above.
Data points represent the mean of three replications. Data were analyzed using Student's t-test at 95% confidence limit.
Generation of DHAR-overexpressing transgenic tobacco
The cDNA of A. thaliana cytosolic DHAR under CaMV35S promoter and upstream of Tnos terminator of the plant's higher expression vector pBE2113-GUS was mobilized in A. tumefaciens strain C58C1 and used for Agrobacterium-mediated gene transfer of N. tabacum cultivar SR-1. Analysis by PCR using genomic DNA isolated from transgenic and SR-1 control plants confirmed the presence of the transgene in eight DHAR transgenic lines (Fig. 2A). Southern blot analysis using genomic DNA isolated from transgenic and SR-1 control plants confirmed that DHAR transgenic plants had independently incorporated the DHAR transgene (Fig. 2B). Northern blot analysis using RNA isolated from DHAR transgenic and SR-1 control plants indicated that DHAR transgenic plants had expressed DHAR transgene (Fig. 2C). Western blotting using antibodies raised against DHAR detected high levels of DHAR protein (23.4 kDa) derived from DHAR transgene in the extracts prepared from transgenic plants but not from SR-1 control plants (Fig. 3).
Higher DHAR activity
DHAR enzyme assay was carried out using leaf crude extracts from three DHAR transgenic lines and SR-1 control plants grown under normal conditions. Compared with SR-1 control plants, DHAR transgenic lines Dha3, Dha4, and Dha7 exhibited increased DHAR activity up to 2.4, 3.1, and 2.3 fold, respectively (Fig. 4A).
Increased AsA levels in DHAR transgenic plants
To determine the metabolic consequences of DHAR overexpression, we measured the content of the reduced AsA present in the leaves of DHAR transgenic and SR-1 control plants. Compared with SR-1 control plants, the level of AsA in DHAR transgenic lines Dha3, Dha4, and Dha7 increased to 1.9, 2.1, and 1.9 folds, respectively (Fig. 4B), and the level of DHA decreased by 23.3, 16.4, and 20.4%, respectively. The redox status of ascorbate (AsA : DHA) increased from a ratio of 1.9 in SR-1 control plants to 4.8, 4.9, and 4.5 in transgenic lines Dha3, Dha4, and Dha7, respectively.
Enhanced tolerance to ozone stress in DHAR transgenic plants
Exposure of DHAR transgenic (Dha4 and Dha7) and SR-1 control plants to 0.2 p.p.m. ozone resulted in a steady decrease in their net photosynthesis rate. The net photosynthesis of DHAR transgenic lines was significantly (P < 0.05) higher than in SR-1 control plants during the entire period of ozone stress (Fig. 5). After 30 h of ozone stress, DHAR transgenic lines Dha4 and Dha7 maintained 48.9 and 55.3% of their original photosynthesis, respectively, compared with 38.5% for SR-1 control plants.
Enhanced tolerance to drought, salt, and PEG-induced stress
We investigated the effect of drought stress on net photosynthesis of DHAR transgenic and SR-1 control plants during 8 days. Although the net photosynthesis decreased in both transgenic and control plants, the net photosynthesis rate of DHAR transgenic plants was significantly (P < 0.05) higher than that for SR-1 control plants during the entire period of the stress (Fig. 6). After 8 days of drought stress, DHAR transgenic lines Dha3, Dha4, and Dha7 maintained 20.6, 20.3, and 25.6% of their original photosynthesis, respectively, compared with only 10.1% for SR-1 control plants.
Under salt stress, a decrease in the net photosynthesis rate was observed in both transgenic and SR-1 control plants. Net photosynthesis of DHAR transgenic plants was higher than that for SR-1 control plants during the entire period of the stress (Fig. 7). After 8 days of salt stress, DHAR transgenic lines Dha3, Dha4, and Dha7 maintained 20.3, 21.7, and 24.7% of their original photosynthesis, respectively, compared with only 13.4% for SR-1 control plants.
In this study, compared with other type of stresses, the decrease in net photosynthesis of plants caused by 10% (w/v) PEG-induced stress was relatively lower. DHAR transgenic plants showed significantly (P < 0.05) higher net photosynthesis rate (Fig. 8). After 8 days of PEG-induced stress, DHAR transgenic lines Dha3, Dha4, and Dha7 maintained 74.0, 79.7, and 80.1% of their original photosynthesis, respectively, compared with only 63.3% for SR-1 control plants.
In plant cells, the regeneration of AsA occurs via two ways: Mehler APX reaction that mainly reduces MDHA to AsA and Halliwell–Foyer–Asada cycle that is found in several compartments of plant cells and mainly reduces DHA to AsA (Horemans et al. 2000b). For efficient regeneration of AsA from DHA, we developed transgenic tobacco plants overexpressing DHAR in cytosol using a cytosolic DHAR cDNA from A. thaliana. DHAR transgenic plants showed significant increase in DHAR activity compared with SR-1 control plants (Fig. 4A). These results indicate that the enhanced DHAR activity in transgenic plants has resulted from the introduced DHAR transgene. Similar increase in DHAR activity was obtained in transgenic tobacco plants overexpressing human DHAR targeted to chloroplast (Kwon et al. 2001).
The level of AsA was markedly increased in DHAR transgenic plants compared with SR-1 control plants (Fig. 4B). This increase could be attributed to that transgenic plants were more efficient in converting DHA to AsA before being hydrolyzed into 2,3-diketogulonic acid. The importance of cytosolic DHAR in reducing DHA diffused from other cell compartments was reported (Horemans et al. 2000b). NADH or NADPH appears to be absent from apoplast to drive the reactions in AsA–GSH cycle, and consequently, apoplastic DHA has to be returned to cytosol for reduction to AsA (Pignocchi et al. 2003). Therefore, the significant increase in AsA level in the leaves of DHAR transgenic plants coupled with improved redox status of AsA could be mainly due to the enhanced activity of the cytosolic DHAR in reducing the oxidized DHA diffused from apoplast, chloroplast, and other compartments of the cell into cytosol. Similar increase in AsA due to an increased expression of DHAR was reported by Chen et al. (2003).
Our DHAR transgenic plants exhibited an increased resistance to ozone stress (Fig. 5). Ozone sensitivity is generally correlated with the AsA status of the leaf tissues (Conklin and Barth 2004), and several reports indicated that higher activities of scavenger antioxidant enzymes may protect from oxidative stress (Asada 1997, Pasqualini et al. 2001). The enhanced tolerance to ozone in our transgenic plants could be due to the elevated levels of AsA (Fig. 4B), which mainly resulted from the enhanced activity of DHAR (Fig. 4A). Furthermore, AsA pool both inside and outside the cell is reported to be regulated by the level of cytosolic DHAR (Chen and Gallie 2005). Apoplastic DHA is translocated by specific carrier in cytosol in exchange of AsA (Horemans et al. 2000b). Because that apoplastic AsA represents the first line of defense against potentially damaging external oxidants such as ozone, SO2, and NO2 (Barnes et al. 2002, Plöchl et al. 2000), overexpressing DHAR in cytosol might maintain continuous flux of reduced AsA toward apoplast in the exchange of oxidized DHA and consequently provide better protection against ozone. These results are in general agreement with several reports indicating the importance of AsA in providing resistance against oxidative stress imposed by ozone (Chen and Gallie 2005, Sharma and Davis 1997, Tanaka et al. 1985).
AsA is important in photo-protection and the regulation of photosynthesis (Noctor and Foyer 1998). DHAR transgenic plants showed higher photosynthesis rates under drought stress (Fig. 6) and water stress induced by PEG (Fig. 8) compared with SR-1 control plants. These results could be attributed directly to the higher levels of AsA in these transgenic plants. Adriano et al. (2005) reported that the level of total AsA that could limit cellular damage caused by AOS is an important attribute linked to drought tolerance, and the activity of DHAR increased in relation to the severity of drought stress in four interspecific Prunus hybrids.
Chen and Gallie (2004) reported that suppressing DHAR expression and reducing AsA redox state on transgenic tobacco conferred increased drought tolerance. Plants with an increased guard cell AsA redox state exhibited lower CO2 assimilation after severe water stress that caused leaf wilting, whereas an increased net photosynthesis was monitored during the entire period of drought stresses in our transgenic plants. Therefore, it is difficult to compare the drought tolerance in terms of net photosynthesis during the overall period of stress.
Excess accumulation of H2O2 is one of the mechanisms by which plants are damaged under salt and drought stresses (Allen et al. 1997, Polle, 2001). H2O2 is able to pass through cell membranes and reach cell locations remote from its site of formation (Foyer et al. 1997). For the reductive detoxification of H2O2 and maintenance of the antioxidative activity of AsA, its regeneration from DHA to AsA is necessary (Hossain et al. 1984), as well as its biosynthesis (Wheeler et al. 1998). Therefore, the better performance of DHAR transgenic plants under drought (Fig. 6) and salt stresses (Fig. 7) could be mainly due to a fast removal of H2O2 during the stress that resulted from the elevated levels of AsA. Enhanced tolerance against salt stress and H2O2-induced stress was reported in transgenic plants overexpressing chloroplast-targeted DHAR (Kwon et al. 2003).
The reduction in net photosynthesis in both transgenic and controlled plants with the advancement of the stresses might be mainly due to the accumulation of AOS including H2O2, which exceeds AOS scavenging ability of antioxidant enzymes functioning on its removal. In conclusion, our results suggest that elevating AsA levels through overexpression of DHAR in cytosol would contribute significantly in enhancing plants' tolerance to oxidative stress. Further studies on the AsA transport capacity from cytosol to other cell compartments and determination of various AOS levels under stressing conditions are needed and will be valuable in answering any questions that might remain.
Acknowledgements – This work was supported by grant from Japan Ministry of Education, Culture, Sports, Science and Technology (MONBUSHO) and the Strategic International Cooperative Program, Japan Science and Technology Agency (JST). We thank Dr Yuko Ohashi (National Institute of Agrobiological Resources, Tsukuba, Ibaraki, Japan) for providing pBE2113-GUS plant transformation vector.
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