Vitamin C (L-ascorbic acid, AsA) has important antioxidant and metabolic functions in both plants and animals. Once used, ascorbic acid can be regenerated from its oxidized form in a reaction catalyzed by dehydroascorbate reductase (DHAR, EC 184.108.40.206). To analyze the physiological role of DHAR catalyzing the reduction of DHA to ascorbate in environmental stress adaptation, we examined whether increasing the level of AsA through enhanced AsA recycling would limit the deleterious effects of oxidative stress. A chimeric construct consisting of the double CaMV35S promoter fused to the Myc-dhar gene was introduced into Arabidopsis thaliana. Transgenic plants were biochemically characterized and tested for responses to oxidative stress. Western blot indicated that the dhar-transgene was successfully expressed. In homozygous T4 transgenic seedlings, DHAR overexpression was increased up to 1.5 to 5.4 fold, which enhanced foliar ascorbic acid levels 2- to 4.25-fold and ratio of AsA/DHA about 3- to 16-fold relative to wild type. In addition, the level of glutathione, the reductant used by DHAR, also increased as did its redox state. When whole plants were treated with high light and high temperature stress or in vitro leaf discs were subjected to 10 μM paraquat, transgenic plants showed a larger AsA pool size, lower membrane damage, and a higher level of chlorophyll compared with controls. These data suggested that increasing the plant vitamin C content through enhanced ascorbate recycling could limit the deleterious effects of environmental oxidative stress.
Ascorbic acid (AsA) gains its name from its inherent property as vitamin C. It is required for cardiovascular functions, immune cell development, connective tissue, and iron utilization. The compound is also an important enzyme cofactor and it is involved in cell division, expansion and elongation (Smirnoff 1996; Arrigoni et al. 1997; Kato and Esaka 1999, 2000). AsA also plays an important role as an antioxidant or reductant in several pathways, including the xanthophyll cycle and regeneration of vitamin E in animals, and protects the plant during oxidative damage by scavenging free radicals and reactive oxygen species (Leowus 1999). Moreover, the AsA redox state, controlled by AO activity levels, is responsible for the apoplast capability of transmitting signals related with environmental changes or defense processes (Pignocchi and Foyer 2003).
Oxidation of AsA produces the short-lived radical mono-dehydroascorbate (MDHA), which is converted to AsA by MDHA reductase (MDHAR) or disproportionates nonenzymatically to AsA and dehydroascorbate (DHA) (Asada 1992). DHA undergoes irreversible hydrolysis to 2, 3-diketogulonic acid or is recycled to AsA by dehydroascorbate reductase (DHAR), which uses glutathione (GSH) as the reductant (Chen et al. 2003). Thus, DHAR allows the plant to recycle DHA, thereby recapturing AsA before it is lost (Figure 1). Plant DHAR cDNAs were cloned from rice (Urano et al. 2000), spinach (Shimaoka et al. 2000), Arabidopsis (Shimaoka et al. 2000), wheat (Chen et al. 2003) and tomato (Zou et al. 2006).
Chen et al. (2003) did an excellent study to confirm that overexpression of DHAR in plants would increase the level of ascorbic acid through improved ascorbate recycling. A DHAR cDNA from wheat was isolated and expressed in tobacco and maize, where DHAR expression was increased up to 32- and 100-fold, respectively. The increase in DHAR expression increased foliar and kernel endogenous ascorbic acid levels two- to fourfold and significantly increased the ascorbate redox state in both tobacco and maize. At the same time it did not exhibit any disadvantages such as growth impairment, chlorosis, necrosis, or premature senescence. These results demonstrate that the vitamin C content of plants can be elevated by increasing expression of the enzyme responsible for recycling ascorbate.
Naturally, various abiotic stresses, such as UV radiation, ozone, drought and chilling lead to the production of reactive oxygen species (ROS) in plant (Smirnoff 1998). The presence of ROS leads to the induction of antioxidative enzymes, which are ostensibly able to reduce the oxidizing environment created by ROS (Blokhina et al. 2003). Plants expressing these enzymes are more resistant to paraquat treatment (Van et al. 1994). Increasing the level of AsA through enhanced AsA recycling in tobacco (Nicotiana tabacum) provided greater protection against ozone (Chen and Gallie 2005) H2O2 and other abiotic stress (Kwon et al. 2003).
In the present study, for the first time, homogenous DHAR was introduced into Arabidopsis to investigate whether DHAR overexpression led to increased AsA content and a substantial change in the intracellular redox state, a stimulation of protective effects in environmental stress adaption under various stresses, including paraquat and high temperature treatment. In particular, we examined some of the chemical details of these enhancements to evaluate in-depth the mechanism of DHAR functions and its application potential in plant biotechnologies.
Plant transformation and molecular characterization of transgenic Arabidopsis
We transformed Arabidopsis with the pHB::dhar binary construct that contained both dhar and two selected marker genes (hygromycin and bar) on the same T-DNA fragment. We then selected primary transformants (T0) on the basis of resistance to the antibiotic hygromycin (50 μg/mL) and 1% phosphinothricin (PPT) and established transgenic lines by single seed descent. The accumulation of the Myc-DHAR fusion protein in transgenic plants was examined by western blot analysis using an antibody against Myc. A strong cross-reaction signal corresponding to a polypeptide with an expected molecular weight of 33 kDa for the Myc-DHAR fusion protein was present in all the six tested plant lines (Figure 2B). As expected, no signal was observed using total protein extracted from wild type (WT).
To investigate details on whether the dhar overexpression in Arabidopsis regulated endogenous genes involved in AsA metabolism at a transcriptional level, we designed specific primers and carried out quantitative reverse transcription-polymerase chain reaction (QRT-PCR) using total RNA from the same age and standard cultured WT and dhar transgenic plants (Figure 2C). Densitometry indicated that the expression of most candidate genes was not affected by dhar transgene whereas there was marked (range from 2 to 3.5 folds) increase of mdar and dhar expression observed in transgenic lines compared with WT expression. On the contrary, only the ao gene expression in dhar transgenic plants represented roughly a 60% decrease as compared with the WT expression.
Homozygous T4 plant seeds were analyzed in order to characterize the global effects of dhar overexpression and physiological differences. WT and transgenic seedlings showed similar phenotype and life cycle (Figure 3A, B). No statistically significant differences (P > 0.05) were observed for plant height and number of rosette leaves after tallow and at the end of the culture, respectively (data not shown). Overexpression of DHAR did not lead to chlorosis and necrosis or premature senescence.
Assessment of in vivo/in vitro oxidative stress tolerance
Morphological differences in response to the in vitro/in vivo oxidative stress between transgenic and WT plants were evident. VHb transgene has been shown to improve cellular protection against high light stress and high temperature. The difference in physical appearance was obvious in the whole plants where the WT paradoxically, exhibited evident anthocyanin accumulation (Figure 3C). However, the growth defect was not observed in dhar-ovx lines (Figure 3D). Under in vitro oxidative stress conditions, WT seedling turned yellow and shrinkage, displayed a profound reduction in chlorophyll (Figure 3E) while the dhar transgenic seedling was greener, healthier, and showed less leaf necrosis (Figure 3F).
Resistance to oxidative stress was assessed by measuring electrolyte leakage and the loss of chlorophyll (Chl) before and after oxidative stress. Before treatments, there were no significant differences in the ion leakage and chlorophyll content between transgenic and WT plants. DHAR transgenic plants showed approximately 46.2% less membrane damage caused by high temperature (40 °C) relative to WT. Transgenic leaf discs exposure to 10 μM paraquat for 2 h showed 23.3% less damage than WT (Figure 4A).
High temperature and exposure to paraquat resulted in a reduction in chlorophyll content. DHAR-OX leaves contained greater chlorophyll (both Chl a and b) content than control leaves (Figure 4B). Despite a substantial decrease in Chl a and b content, the Chl a/b ratio remained unaffected by the two kinds of stress treatments (data not shown).
Prior to the paraquat treatment, the stomata of DHAR-ovx plants opened wider than those of the WT (Figure 4C) and the average stomatal aperture as measured in the morning was 40% larger in transgenic leaves compared with the WT (Figure 4D). Although exposure to 10 μM paraquat for 2h was sufficient to induce stomatal closure in both WT and transgenic lines (Figure 4C), there was no significant difference in the responsive diminished stomatal aperture (Figure 4D).
Overexpressing dhar increased DHAR activity and maintained a larger AsA pool
Before the stress treatment, the DHAR activity of transgenic lines was (12.8 ± 2.92) × 10−5 mol/min per g fresh weight (FW), compared with the WT (4.1 ± 0.8) × 10−5 mol/min per g FW (Figure 5A). Transgenic leaves, in which DHAR expression had increased up to 3.1-fold, exhibited a 3.3-fold increase in the level of AsA and a 60% reduction in the level of DHA respectively, thus resulting in an increase in the AsA redox state (AsA/DHA) from 2.8 in WT to 13.6 in transgenic leaves (Figure 5B). In addition to AsA, changes in DHAR expression affected the GSH pool size and redox state. GSH content was 1.25 ± 0.17 μmol/g FW in transgenic plants and 0.46 ± 0.07 μmol/g FW in WT plants. Transgenic leaves exhibited a 2.71-fold increase in the level of GSH. Small differences in the level of oxidized glutathione (GSSG) were observed in WT and transgenic leaves (Figure 5B).
Following oxidative stress treatment, the DHAR activity was not significantly different in WT lines, but it increased approximately 11.7% under high temperature (40 °C) culture condition and decreased 15.6% after an acute exposure (2 h) to 10 μM paraquat treatment in transgenic plants (Figure 5A). The foliar level of both AsA and GSH, including their oxides (DHA and GSSG) increased slightly in WT and dhar transgenic plants after two kinds of oxidative stress treatment (Figure 5B). Although total ascorbate content was not found to be significantly different between the genotypes, the AsA redox state in transgenic leaves was 8.47 times higher than in the WT plants. As with AsA, oxidative stress treatment resulted in an altered ratio of reduced GHS redox state in both DHAR transgenic and WT plants (Figure 5B).
Metabolic engineering is a new approach to understand and use metabolic processes. Advances of plant biochemical pathways and availability of new gene transfer techniques have led to a growing interest in using this technique to redirect metabolic fluxes in plants for industrial purposes. AsA provides major nutritional value, both for plants and humans, and is also an important factor of stress tolerance in agricultural species. The information gained in recent years about the AsA biosynthesis pathway in plants offers a solid foundation for modern breeding aimed at changing the AsA content in crop species.
Our results have demonstrated that the vitamin C content of A. rabidopsis thaliana can be increased by overexpressing DHAR. The increase in ascorbate is exciting, as it suggests that although AsA is the major antioxidant contributing to the redox status of the cell and that its synthesis is subject to feedback inhibition by the pool size of ascorbate (Pallanca and Smirnoff 2000), its content can be increased by genetic engineering. Accompanied by a 3.1-fold increase in DHAR activity, a 3.3-fold increase in the level of AsA and a 60% reduction in the level of DHA were observed after introduction of homogeneous DHAR cDNA into A. rabidopsis thaliana. Because GSH is used as the reductant by DHAR, the levels of reduced (GSH) and oxidized (GSSG) glutathione were also determined. An increase in the level and redox state of glutathione was observed in DHAR-overexpressing plants. Increased glutathione redox state plays an important role in maintaining their viability during storage (Chen and Gallie 2006).
The cellular concentration of AsA is determined by the rate of its synthesis and decay. If not salvaged by DHAR, DHA is rapidly hydrolyzed into 2, 3-diketogulonic acid, finally decayed into CO2, H2O2 and L-threarate (Green and Fry 2005). The overexpression of DHAR in transgenic plants would increase the likelihood that DHA is converted to AsA before decaying, that would lead to the increase in AsA in transgenic plants. Therefore, the enhanced rescue of DHA from its decay pathway may be the reason that the AsA concentration in DHAR-overexpressing plants increased. The increase in AsA was accompanied by an increase in DHAR activity, which suggests that the amount of endogenous DHAR may be a limiting factor. It might enable plants to control their intracellular redox state, particularly in response to developmental cues or changes in their external environment (Chen et al. 2003).
Through its AsA recycling function, DHAR affects the level of foliar ROS and photosynthetic activity during leaf development and as a consequence, influences the rate of plant growth and leaf aging (Creissen et al. 1999). Tobacco plants that overexpressed γ-glutamylcysteine synthetase (γ-ECS) exhibited chlorosis and necrosis. Although DHAR-overexpressing plants exhibit elevated levels of ascorbate and glutathione, they differ from γ-ECS-overexpressing plants and did not exhibit chlorosis and necrosis (Chen et al. 2003). In our study, the life cycle and the rate of plant growth was similar with the control. It suggested that these growth defects were not correlated with the increase in glutathione but rather with a decrease in the glutathione redox state.
Oxidative stresses are encountered in all living organisms. Oxidative damage to proteins, nucleic acid can occur when the concentration of reactive species exceeds the capacity of the cell's mechanisms for elimination (Farr and Kogoma 1991). Naturally, various abiotic stresses, such as UV radiation, ozone, drought and chilling lead to the production of ROS in plants (Smirnoff 1998). The presence of ROS leads to the induction of antioxidative enzymes, which are ostensibly able to reduce the oxidizing environment created by ROS (Blokhina et al. 2003). Plants expressing these enzymes are more resistant to paraquat treatment (Van et al. 1994). The positive effects of DHAR expression in plant genetic engineering have been previously described (Chen and Gallie 2005; Huang et al. 2005; Ratajczak and Pukacka 2006; Ushimaru et al. 2006; Yoshida et al. 2006), which leads to resistance to ozone, salt and desiccation tolerance. To evaluate the potential of DHAR function as concerning the response to oxidative stress with a genetic engineering based approach, we have tested DHAR transgenic Arabidopsis plants under in vitro and in vivo stress conditions.
In the present study we analyzed the effects of the DHAR transgene on the oxidative stress protection in the presence of ROS in Arabidopsis in vivo and in vitro. The DHAR transgene has been shown to improve cellular protection against high light and high temperature stress. The difference in physical appearance was obvious in the transgenic plants for the increased AsA played a key photoprotective role in protecting plant against light-dependent photooxidative cell damage (Smirnoff 1996).
We also found some significant differences in oxidative stress defense imposed by paraquat between DHAR transgenic lines and WT leaf discs. When exposed to the 10% paraquat for 2 h, although typical oxidative stress symptoms could be seen, DHAR transgene lines showed distinctive leaf morphology compared with the control. Cell leakage and total chl content were less affected in DHAR-expressing cultures compared with WT. To examine whether the level of AsA had a similar effect on guard cell responsiveness following exposure to paraquat, the stomatal aperture was measured before and immediately after paraquat treatment. Prior to the paraquat treatment, the average stomatal aperture was significantly larger in the DHAR transgenic leaves relative to the control. But little difference was observed between WT and transgenic lines following exposure to 2 h of 10% paraquat. These findings are in good agreement with previous measurements (Chen and Gallie 2005, 2006). Our results have clearly demonstrated that overexpressing DHAR properly works for the protection against oxidative stress in plants, as reflected in relieving oxidative stress and protecting it from oxidative damage both in vivo and in vitro.
Although chemical synthesis is the major method to produce vitamin C, the natural product is welcome in the market. Increasing AsA in plants gives convenience in industrial production. And also, such an increase would require decreased dietary emphasis of foodstuffs rich in ascorbate. Overexpressing DHAR to increase AsA represents a simple strategy of metabolic engineering.
For future development of the full potential of metabolic engineering it is thus necessary to increase our detailed knowledge about plant AsA metabolism on the level of intermediates, enzymes and genes. On the physiology of the pathway, the fluxes through the pathway are controlled to a greater extent at the level of enzymes and intracellular and intercellular transport. Fluxes are not only determined by gene expression, but also by post-translational regulation of enzyme activity and enzyme and metabolite compartmentation and transport. Classic biochemistry will continue to play an important role in determining cellular regulatory features and in measuring flux and intermediate concentrations to provide essential metabolic context. In addition, developmental processes, stress responses and environmental conditions are all known to markedly affect the AsA content of individual species. A better understanding of internal and external mechanisms of the plant that controls AsA accumulation could provide an opportunity for enhancing nutritional quality and stress tolerance of crop plants.
Materials and Methods
Construction of expression cassette, transformation, and growth conditions of plants
Arabidopsis seeds were sterilized with 20% bleach and were put in a 4 °C refrigerator for 3–5 d, germinated on MS medium and then transferred to soil and grown in long-day light (16 h light/8 h dark) at 22 °C at a fluence rate of 100 μmol/s per m2 of white light produced by cool-white fluorescent lamps.
Full-length Arabidopsis (Columbia ecotype) DHAR cDNA was isolated from seedling total RNA by one-step RNA PCR. The 6×Myc tag was cloned into Bam HI and Spe I sites of pBluescript SK+ (pBS; Stratagene, Shanghai, China). The dhar gene was amplified from the pMD-dhar construct using PCR (forward primer, 5′-CCGAATTCAAGATGGCTCTGGAAATCTGT G-3′; reverse primer, 5′-AACTGCAGAGGGTTAACCTTGGGAGCCC-3′) with an introduced EcoR I site at the first ATG and was cloned into EcoR I and Pst I sites of pBS-6×Myc. After confirmation by sequencing, the fragment encoding dhar-myc was excised with EcoR I and Spe I and subsequently inserted into the PHB binary vector (supplied by Professor H. Yang, SIPPE, CAS) containing a hygromicin resistant gene and anti-herbicide gene (BAR) inside the T-DNA for the selection of transformants.
The pHB::dhar construct (Figure 2A) was transferred into Agrobacterium tumefaciens GV3101 (rifampicin resistant) by triparental mating and the floral-dip method was used to conduct transformation of Arabidopsis, Columbia ecotype (Clough and Bent 1998). T0 seeds were screened on 15 cm Murashige–Skoog (MS) basal plates supplemented with 50 μg/mL hygromycin. Then, 30 to 50 independent lines expressing transgene were transferred to soil and screened on 1% PPT. PCR and protein gel blot analysis were carried out on 8–16 T3 independent transgenic lines to check for homozygosity (T4). Six independent dhar-overexpressing homozygous T4 lines (4, 5, 12, 17, 26, 33) were randomly selected for final analysis. Chemical components were measured in plants 18–20-day-old plants.
Western blot analysis
Protein gel blot analysis was carried out as described previously with minor modifications (Yang et al. 2000). Aliquots of 50 μg total soluble proteins per sample, determined with the DC Protein Assay Kit (Bio-Rad, Hercules, CA, USA), was fractionated on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) mini-gel and blotted to a polyvinylidene difluoride membrane (Amersham, Stafford, UK). The blots were probed with the -Myc (α-Myc, Santa Cruz Biotechnology, Santa Cruz, CA, USA) (diluted in phosphate-buffered saline tween-20 [PBST][ primary antibody 80 mM Na2HPO4, 20 mM NaH2PO4, 100 mM NaCl, and 0.1% Tween 20]), washed in PBST three times, reacted with goat anti-mouse immunoglobulin G (IgG) conjugated with horseradish peroxidase (1:5 000, Amersham), washed, and exposed to X-ray film using the enhanced chemiluminescence method according to the manufacturer's instructions (Amersham).
Real-time quantitative analysis
Total RNA was extracted from 5-week-old both transgenic dhar leaf tissue and WT controls cultured in standard conditions with Plant RNA Mini Kit (Watson, Shanghai, China) and treated with RNase-free DNase (Promega, Madison, WI, USA). The quality and concentration of RNA samples were examined by agarose gel electrophoresis and spectrophotometer analysis. Total RNA was reversely transcribed by using AMV reserve transcriptase (Takara, Otsu, Japan) to generate cDNA. All of the gene-specific primers were designed according to the conserved region of the corresponding sequences of A. thaliana. Part of the polyubiquitin gene was amplified with primers (5′-ACCCTCACGGGGAAGACCATC-3′ and 5′-ACCACGGAGACGGAGGACAAG-3′) as a control. The real-time PCR was carried out according to manufacturer's instructions (Takara) under the following condition: 1 min pre-denaturation at 95 °C, 1 cycle; 10 s denaturation at 95 °C, 30 s annealing at 56 °C, 15 s collection fluorescence at 72 °C, 40 cycles. The products of real-time quantitative PCR were run on 1.5% agarose gel electrophoresis and showed an equal-sized band as predicted. Quantification of the gene expression was done with comparative computed tomography (CT) method. Data represents the average of three experiments.
Oxidative stress tolerance assay and stomatal aperture measurements
Four-week-old plants grown in normal conditions were subjected to high light (1 000 μmol/s per m2) and high temperature (40 °C) for 1 week as in vivo oxidative stress. In vitro oxidative stress experiments were carried out as described previously (Wang et al. 2009) with minor modifications. Leaf discs from 3- to 4-week-old plants were incubated for 2 d in 10 μM paraquat (Sigma-Aldrich, CA, USA) solution or in water at 22 °C with constant light. Mature stomata of epidermal strips were used for stomatal aperture measurements as described (Mao et al. 2005).
Measurement of chlorophyll content and electrolyte leakage
After in vitro and in vivo oxidative stress treatment, 1 g of young leaves from transgenic and WT Arabidopsis lines were collected for the presence of chlorophyll a and b and total protein by spectral determination. Immediately after sampling, leaves were frozen in liquid N2 and stored at −80 °C. Subsequently, frozen leaf samples were ground in an ice-cold mortar and pigments were extracted with 5 mL 80% (v/v) acetone. The homogenates were centrifuged at 350 g for 10 min at 4°C. The extraction procedures were carried out under low light intensity. Chl a, b were determined by measuring the absorbance of the supernatant at 663 and 646 nm, respectively (Wellburn 1994). Electrolyte leakage was determined using an osmometer (DDS-11A, SuoShen, Shanghai, China). Values were given as a percentage: for each sample 100% was determined by boiling the sample for 3 min leading to the complete release of electrolytes. Electrolyte leakage was given as the ratio of the amounts of electrolyte with and without stress treatment. Each analysis was repeated with 10 plants per treatment and three times per plant.
Leaves were ground in extraction buffer (50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 2 mM ethylenediaminetetraacetic acid [EDTA], 1 mM MgCl2), after being centrifuged at 9 500g for 5 min, the soluble protein in the leaves was obtained. DHAR was assayed as described (Bradford 1976; Hossain and Asada 1984) in 50 mM K2HPO4/KH2PO4, pH 6.5, 0.5 mM DHA, 1 mM GSH (equal loading of total protein), and its activity was followed by an increase in absorbance at 265 nm.
Ascorbic acid, dehydroascorbate, glutathione, and oxidized glutathione measurements
Leaves were ground in 2.5 M HClO4 and after a 10 min centrifugation at 9 500g, two volumes of 1.25 M Na2CO3 were added to the supernatant. One hundred microliters of the mixture after centrifugation was added to 895 μL of 100 mM K2HPO4-KH2PO4, pH 5.6. After 0.25 units of ascorbate oxidase (AO) was added, AsA was determined by the change in absorbance at 265 nm. When DHA was reduced to AsA (100 mM K2HPO4/KH2PO4 at pH 6.5, 2 mM GSH, and 0.1 μg of AtDHAR protein, incubated at 25 °C for 20 min) before measuring AsA, the total amount of reduced and oxidized ascorbic acid (i.e., AsA and DHA) could be determined. The amount of DHA was determined as the difference between these two assays above. GSH and GSSG were determined as described (Griffith 1980).
The statistical significance of the genes expression level, responses to oxidative stress, chlorophylls, electrolyte leakage, enzyme activity and metabolites content among various transgenic plants and wild type controls were calculated using Statgraphics Plus 5.0 (Statistical Graphics Corp., Warrenton, VA, USA) and error bars on graphs indicate standard error (SE) calculated from at least 10 replicated samples.
(Co-Editor: Qiaoquan Qi)
The authors are very grateful to Professor HongquanYang (Shanghai JiaoTong University, China) for the generous gift of pBS-MYC vector and for assistance with Arabidopsis transformation. The authors also gratefully acknowledge Dr Peng Zou (National University of Singapore, Singapore) for assistance in high performance liquid chromatography analyses. This work was supported by grants from the State Key Basic research and development Plan of China (2007CB108805), the National Natural Science Foundation of China (30600807 and 30900786), the Modernization of Traditional Chinese Medicine Foundation (08DZ1971502) and Shanghai Western Development Cooperation Foundation (084358014), Shanghai Science and Technology Committee.