Simultaneous expression of choline oxidase, superoxide dismutase and ascorbate peroxidase in potato plant chloroplasts provides synergistically enhanced protection against various abiotic stresses

Plant material and plasmid construction

Sterile transgenic potato plants (cv. Superior) expressing chloroplast-targeted CuZnSOD and APX genes under the control of the SWPA2 promoter (Tang et al. 2006) were used in transformation experiments. The plants were propagated via subculturing of shoot tips and stem nodal sections every 3–4 weeks on MS medium containing 3% sucrose (Murashige and Skoog 1962). The plant expression vector pScodA (Ahmad et al. 2008) containing a chloroplast-targeted codA gene under the control of the SWPA2 promoter was mobilized in the AGL0 strain of Agrobacterium and utilized for transformation.

Plant transformation and regeneration

For potato transformation, Agrobacterium harboring the pScodA vector was grown for 48 h at 28°C in 5 ml of yeast extract peptone medium (containing rifampicin 100 mg l⁻¹). The transformation was carried out as described by Ahmad et al. (2008). Briefly, stem internodes without axillary buds were cut into 5- to 10-mm-long sections. The explants were precultured for 2 days on MS basal medium containing 0.2 mg l⁻¹ 2, 4-D. These explants were inoculated with Agrobacterium (concentration 1 × 10⁶ cells ml⁻¹) for 10 min and cocultured for 2 more days in the dark on preculture medium (MS basal medium containing 0.2 mg l⁻¹ 2, 4-D). After coculture, explants were blot dried on sterile tissue papers and shifted to regeneration medium (MS medium containing 0.01 mg l⁻¹ naphthalene acetate (NAA), 0.1 mg l⁻¹ GA3 and 2 mg l⁻¹ zeatin) supplemented with 400 mg l⁻¹ cefotaxime and 1 mg l⁻¹ bialaphos. The first shoot regeneration was acquired after 1 month and was transferred to rooting medium (MS medium containing 3% sucrose, 400 mg l⁻¹ cefotaxime and 1 mg l⁻¹ bialaphos).

Methyl viologen treatment and tolerance assay

A leaf disc assay for oxidative stress tolerance was conducted as described by Kwon et al. (2002). In brief, leaf discs were punched from the fifth to sixth leaf from the top of 5- to 7-week-old plants and floated on solution containing 5 or 10 µM methyl viologen (MV) in 0.4% sorbitol. After incubation in the dark for 12 h , leaf discs were illuminated in continuous light (150 µmol photons m⁻² s⁻¹) at 25°C. Ion leakage of the solution was assessed using an ion conductivity meter (Model 455C, Isteck Co., Seoul, Korea). The H₂O₂ content was assessed with xylenol orange, in which H₂O₂ is reduced by ferrous ions in an acidic solution that forms a ferric product—xylenol orange complex, which was detected at 560 nm (Bindschedler et al. 2001). H₂O₂ measurements were expressed as relative values.

Salt stress treatment and tolerance assay

For the salt stress analysis at the whole-plant level, transgenic SSA, SSAC and non-transgenic (NT) plantlets were allowed to grow in a growth chamber with a 16/8 h photoperiod at 25°C and in 100 µmol photons m⁻² s⁻¹ light conditions. After 6 weeks of growth, plants under similar height and health conditions were selected for NaCl treatment. A volume of 200 mM NaCl solution was supplied via irrigation through the trays underneath the pots on alternate days for 2 weeks. Afterward, the plants were irrigated only with water for 2 weeks in order to allow the plants to recover. The fresh and dry weights of salt-treated plants were determined and compared with the plants grown under normal conditions. After harvesting and measuring the fresh weight, the plant samples were dried for 48 h at 80°C in order to measure the dry weight. Photosynthetic activity was recorded via chlorophyll fluorescence determinations of photochemical yield (Fv/Fm), which represented the maximum quantum yield of photosystem II, using a portable chlorophyll fluorescence meter (Handy PEA, Hansatech, England) after 30 min of dark adaptation. Chlorophyll content after salt stress was measured by a portable chlorophyll meter (SPAD-502, Konica Minolta, Tokyo, Japan) from intact fully expanded fifth leaves from the top of individual plants.

Drought stress treatment and tolerance assay

Plantlets rooted on MS medium were transferred to pots filled with equal quantities of soil and then grown in a growth chamber for 4 weeks under similar conditions as described earlier for the salt stress treatment. Drought stress was imposed by withholding the water supply to the plants, which were maintained at 25°C under light conditions of 100 µmol photons m⁻² s⁻¹. Before withholding the water supply, the plants were irrigated with similar quantities of water through trays placed underneath the pots for 1 week. After 2 weeks of water withholding, the plants were again irrigated and permitted to recover from the drought conditions. Fresh and dry weight gain was measured after 1 week of recovery, as described in the preceding section. The degree of drought stress was assessed by the relative water contents (RWCs) of leaves after 12 and 14 days of water withholding. The RWCs% were estimated as described by Ma et al. (2006). RWC% was measured from the first fully expanded leaf from the top.

Western blot analysis

Leaf discs treated with 10 µM MV solution for 72 h were used to detect the codA protein in selected SSAC lines. Primarily, western blots were performed as described by Sakamoto et al. (1998). Briefly, proteins were extracted in 20 mM sodium phosphate buffer (pH 7.0) by centrifuging the leaf homogenate at 12 000 g for 10 min to remove cell debris and insoluble fractions. Protein samples (40 µg) were subjected to electrophoresis on an 8% polyacrylamide gel containing 10% sodium dodecyl sulfate. Following electrophoresis, proteins were blotted onto polyvinyl difluoride membranes (Immobilin-N; Millipore, Bedford, MA). The resultant membrane was allowed to react with an antiserum raised against codA, as described previously (Deshnium et al. 1997).

GB analysis

GB was extracted and quantified as previously described by Park et al. (2004) and Ahmad et al. (2008). In brief, leaves frozen in liquid nitrogen were powdered with a ceramic mortar and pestle. This powder (2 g) was then suspended in 2 ml of ice-cold methanol:chloroform:water (60:25:15) and an equal volume of distilled water was added to the tubes. The resultant homogenate was shaken gently for 10 min and then centrifuged for 10 min at 570 g at room temperature. The upper methanol–water phase was transferred to clean tubes. The extracts were freeze-dried by speed vacuum and dissolved in distilled water (2 ml). These extracts were treated with the strong anion exchange resin, AG 1-X8 (Bio-Rad, Hercules, CA), as described by Bessieres et al. (1999). Micro Bio-spin chromatography columns (Bio-Rad) were packed with AG 1-X8 resin via the addition of 1 ml of resin slurry and were centrifuged at 1000 g at room temperature. Afterward, 1 ml of crude GB extract was loaded into the resin bed and centrifuged for 3 min at 1000 g. The resin was washed with 0.5 ml of distilled water and mixed with the previous flow-through fraction. GB was measured via high-performance liquid chromatography (HPLC) (SPD-10A VP, Shimadzu, Tokyo, Japan), as previously described by Bessieres et al. (1999). In brief, purified GB was detected on an Apollo C₁₈ column (250 × 4.6 mm) under isocratic conditions with 13 mM sodium 1-heptanesulphonate and 5 mM Na₂SO₄ solution (pH 3.7) as a mobile phase. The GB peak was monitored using a Ultraviolet detector at 200 nm and quantification was conducted via comparison of the peak surface areas with those obtained using a pure GB standard (Sigma, St Louis, MO).

RT-PCR analysis

For expression analysis of foreign genes, total RNA was extracted using the cetyltrimethylammonium bromide method (Kim and Hamada 2005) and treated extensively with RNase-free DNase I in order to remove any contaminating genomic DNA. Two micrograms of RNA was utilized for reverse transcription in accordance with the manufacturer's instructions (Reverse Transcription System, Promega, Madison, WI). Gene-specific primers used for PCR reactions were as follows: the CuZnSOD primer set (5′-ATGGTGAAGGCTGAAGCTGTTCTT-3′, 5′-CTATCCTCGCAAACCAATACCG-3′), the APX primer set (5′-ATGGGAAAATCTTACCCAACTGTTA-3′, 5′-TTA GGCTTCAGCAAATCCAAGCTC-3′) and the codA gene primer set (5′-GCTGCTGGAATCGGGATA-3′, 5′- TGGG CTTATCGCGGAAGT-3′) were used to amplify products for CuZnSOD, APX and codA, respectively, from cDNA. The total synthesized cDNA was also used to amplify the actin gene as an internal standard using actin gene-specific primers (5′-TGGACTCTGGTGATGGTGTC-3′, 5′-CCTCCAATCCAAACACTGTA-3′).

Analysis of SOD, APX and CAT activity

Potato leaves were homogenized on ice with a mortar in a 0.1 M potassium phosphate buffer (pH 7). The homogenate was centrifuged at 12 000 g for 15 min at 4°C. The supernatant was used immediately for enzyme assays. Protein concentration was determined according to the Bradford (1976) method using BioRad's protein assay reagents and bovine serum albumin as a standard. SOD activity was measured according to the method of McCord and Fridovich (1969) using xanthine, xanthine oxidase and cytochrome c. One unit of SOD was defined as the amount of enzyme that inhibits the rate of ferricytochrome c reduction by 50%. APX activity was assayed according to the method described by Nakano and Asada (1981) using ascorbic acid as a substrate. The oxidation of ascorbate was initiated by H₂O₂, and the decrease at 290 nm for 1 min 30 s was monitored. One unit of APX was defined as the amount of enzyme oxidizing 1 µM of ascorbate per minute. CAT activity was assayed according to the method described by Aebi (1984). The activity was determined by the decrease at 240 nm for 1 min because of H₂O₂ consumption.

Statistical analysis

Data were statistically analyzed with Duncan's multiple range tests using Statistical Package for the Social Sciences (spss 12) and Student's t-test using Microsoft Excel 2003. The significance refers to statistical significance at the P≤ 0.05 level.

Generation of transgenic potato plants and line selection for further characterization

To evaluate the synergistic effects of multiple genes on stress tolerance in transgenic plants, we retransformed SSA transgenic potato plants (Fig. 1A) by using pScodA that harbored a chloroplast-targeted codA gene under the control of the SWPA2 promoter in an expression vector (Fig. 1B). In order to target codA to chloroplasts, a transit peptide (TP) from the small subunit of tobacco Rubisco was utilized (Hayashi et al. 1997). The putative transgenic potato plants were selected on MS medium containing bialaphos. Fifteen transgenic lines were obtained, and the codA gene integration was verified primarily via PCR of genomic DNA; the expected amplification profiles were acquired from all transgenic lines (data not shown). The transgenic lines harboring the codA, CuZnSOD and APX genes under the control of the SWPA2 promoter were referred to as ‘SSAC’ plants. All 15 of the transgenic lines were grown in a growth chamber for 6 weeks and were utilized to evaluate enhanced tolerance against MV-mediated oxidative stress using leaf discs. MV is a non-selective herbicide that produces massive bursts of ROS, which upon overaccumulation disrupt the membrane stability and lead to cell death (Bowler et al. 1991). Leaf discs were punched from the fifth to sixth leaves from the shoot tip and floated on a 0.4% sorbitol solution containing 5 µM MV (data not shown). Ion leakage was assessed over a time course; among these 15 lines, 2 transgenic lines with the highest observed tolerance (referred to as SSAC1 and SSAC2 plants) were selected for further characterization.

Enhanced tolerance of transgenic plants to MV-mediated oxidative stress

To evaluate the synergistically enhanced tolerance of SSAC transgenic plants over SSA and NT plants, leaf discs from same-age plants were prepared and treated with 10 µM MV solutions. The differential visible damage on the leaf discs demonstrates the enhanced tolerance of SSAC plants as compared with SSA and NT plants (Fig. 2A). After 72 h of MV treatment, ion leakage of SSAC1 and SSAC2 plants was 42 and 37%, respectively, while it was 63% in SSA plants and 90% in NT plants (Fig. 2B). H₂O₂ is produced in the plants during stressful conditions; if it remains unscavenged, it can cause severe damage to the plants. We evaluated the relative H₂O₂ content of SSAC, SSA and NT plants after 10 µM MV treatment for 72 h. SSAC plants showed lower H₂O₂ content than NT and SSA plants. The order of relative H₂O₂ content was NT > SSA > SSAC (Fig. 2C).

Enhanced tolerance of SSAC potato to oxidative stress by an increase in GB content and antioxidant enzyme activities

To understand the tolerance mechanism that is active in SSAC plants following MV-mediated oxidative stress, we performed reverse transcription-polymerase chain reaction (RT-PCR) to determine time-course changes in the gene expression of the introduced CuZnSOD, APX and codA genes following the 10 µM MV treatments. Transcripts of chimeric genes were not detected in NT plants, while SSA and SSAC plants did show transcripts of chimeric genes (Fig. 3A). Additionally, SSAC plants maintained higher expression levels of the CuZnSOD and APX genes than did SSA transgenic plants. To further confirm the expressions of codA protein in SSAC plants, we performed western blot analysis. Leaf discs of NT, SSA and SSAC plants were treated with a 10 µM MV solution for 72 h, and then proteins were extracted and used for western blot analysis, which revealed that the SSAC1 and SSAC2 lines accumulated a 64-kDa protein corresponding to codA, while no such protein was detected in NT and SSA plants (Fig. 3B). In order to determine whether the expression of the codA gene resulted in GB synthesis in the transgenic SSAC plants, GB content was analyzed quantitatively with HPLC. Generally, higher MV concentrations are required for the imposition of stress at the whole-plant level, and thus transgenic and NT plants were subjected to 400 µM MV prior to sampling in order to induce the expression of the GB-synthesizing codA gene. The potato plant is naturally a non-accumulator of GB; we could not find GB in NT plants, whereas transgenic SSAC plants accumulated GB. The GB content in SSAC1 and SSAC2 plants was 1.26 and 1.65 µmol g⁻¹ FW after 72 h of MV treatment, respectively, while under non-stressed conditions, the GB content in SSAC1 and SSAC2 plants was 0.26 and 0.35 µmol g⁻¹ FW, respectively (Fig. 3C).

We also investigated the enzyme activities of SOD, APX and CAT in the total protein isolated from leaf discs of NT and transgenic plants over time following the 10 µM MV treatments. SSAC transgenic plants maintained higher SOD and APX activities during MV treatments than did SSA and NT plants (Fig. 4A, B). We also determined the CAT activity in the leaves of SSAC, SSA and NT plants after MV treatment. The CAT activity of SSAC plants was higher than SSA and NT plants during MV treatment (Fig. 4C).

Enhanced tolerance of transgenic plants to higher levels of salinity

We evaluated transgenic (SSA and SSAC) and NT plants against salt stress at the whole-plant level. After 2 weeks of salt (200 mM NaCl) administration, plants were supplied with water only. Changes in photosynthetic activities induced by salt stress were determined by measuring fluorescence parameters (Fv/Fm). Salt stress affected the photosynthetic machinery of all transgenic and NT plants; however, the transgenic lines maintained significantly higher levels of photosynthetic activity than NT plants (Fig. 5A, B). A continuous decrease was noticed in the Fv/Fm value of plants treated with salt until the end. The Fv/Fm value of NT plants was decreased by 76% after 4 weeks of stress treatment, while SSA plants exhibited a 38% decrease in photosynthetic activity. On the other hand, GB-synthesizing SSAC plants showed significantly higher Fv/Fm values than NT and SSA plants. The photosynthetic activity of SSAC1 and SSAC2 plants was attenuated by 26 and 20%, respectively. We also measured the changes in the chlorophyll content of NT, SSA and SSAC plants after salt stress (Fig. 5C). The chlorophyll content of NT plants started to decrease earlier than that of SSA and SSAC plants. NT plants exhibited an 80% decrease in chlorophyll content, while SSA plants showed a 43% reduction in chlorophyll content. Contrary to NT and SSA plants, the chlorophyll content of SSAC1 and SSC2 plants was decreased by 23 and 18%, respectively. In addition, the SSAC transgenic lines 1 and 2 exhibited higher levels of tolerance in the presence of 200 mM NaCl, as seen by assessments of plant dry weight (Fig. 5D).

Enhanced drought stress tolerance of transgenic plants

To evaluate the enhanced tolerance of transgenic plants against a multitude of stresses, we subjected the plants to drought stress. Four-week-old soil-grown plants were used to impose drought stress by withholding the water supply for 2 weeks. Severe wilting of NT plants was noticed along with SSA plants, while SSAC plants showed less wilting compared with NT and SSA plants. Plants were rewatered after 2 weeks of drought stress and allowed to grow for 1 more week to recover. NT plants could not recover from severe drought stress, while SSA and SSAC plants recovered successfully; however, the pace of recovery was slow in SSA plants (Fig. 6A). RWC gives a good indication of the turgidity maintained by plants. We determined the RWC of drought-stressed plants after 12 and 14 days of water withholding (Fig. 6B). Drastic water loss was observed in the NT plants after 14 days of drought stress: the RWC was only 34% in NT plants, while SSA plants maintained 52%. Contrary to both, SSAC1 and SSAC2 plants maintained significantly higher RWC: 68 and 73%, respectively. We also determined the vegetative biomass of drought-stressed plants and compared it with the biomass obtained in normal growth conditions (Fig. 6C). The dry weight of drought-stressed NT plants was adversely affected. There was a 77 and 66% decrease in dry weight gain of NT and SSA plants, respectively. Compared with NT and SSA plants, SSAC1 and SSAC2 plants maintained better growth, and there was significantly less loss (47–42%) in biomass accumulation, respectively.

Endogenous GB accumulation stabilizes gene expression and enzymatic activities during salt and drought stress

GB accumulation can stabilize endogenous gene exressions and protects the proteisn during stressful conditions. To evaluate the GB accumulation, CuZnSOD, APX and codA genes' expression and enzymatic activities of SOD, APX during salt and drought stress, 6-week-old NT, SSA and SSAC plants were used. The GB content in SSAC1 and SSAC2 plants after salt treatment was found to be 1.28 and 1.77 µmol g⁻¹ FW, respectively, whereas the GB content after 1 week of drought stress was 1.25 and 1.74, respectively (Fig. 8A, B). We used RT-PCR analysis to reveal the changes in expression levels of chimeric genes in transgenic and NT plants treated with 200 mM NaCl or drought stress. Transcripts of chimeric genes were not detected in NT plants, while SSA and SSAC plants showed transcripts of chimeric genes. Particularly, SSAC2 plants maintained the highest expression levels of the CuZnSOD and APX genes relative to SSAC1 and SSA transgenic plants under stress conditions (Fig. 7). SSAC transgenic plants also maintained higher SOD enzyme activities during salt and drought stresses than SSA and NT plants did (Fig. 8C, D). Similarly, SSAC plants also showed higher APX and CAT activities than SSA and NT plants after salt and drought stress treatments (Fig. 8E–H).