Overexpression of PbDHAR2 from Pyrus sinkiangensis in Transgenic Tomato Confers Enhanced Tolerance to Salt and Chilling Stresses

in HortScience

Ascorbic acid (AsA) is a major antioxidant and redox buffer in plants. Dehydroascorbate reductase (DHAR; EC 1.8.5.1) catalyzes the conversion of dehydroascorbate (DHA) to AsA and is crucial for AsA regeneration. In this study, we developed transgenic tomato plants that overexpressed PbDHAR2 to investigate whether PbDHAR2 could limit the deleterious effects of salt and chilling stresses. These transgenic plants contained significantly higher AsA levels than the wild-type (WT) plants. Overexpression of PbDHAR2 increased the expression of the AsA-glutathione (GSH) cycle genes in transgenic lines under salt and chilling stresses. In addition, the transgenic lines subjected to salt and chilling stresses showed higher levels of antioxidant enzyme activity, lower malondialdehyde (MDA) levels, and higher chlorophyll contents than the WT. Thus, our results demonstrate that the regulation of PbDHAR2 during AsA regeneration contributes to enhanced salt and chilling tolerance in tomato.

Abstract

Ascorbic acid (AsA) is a major antioxidant and redox buffer in plants. Dehydroascorbate reductase (DHAR; EC 1.8.5.1) catalyzes the conversion of dehydroascorbate (DHA) to AsA and is crucial for AsA regeneration. In this study, we developed transgenic tomato plants that overexpressed PbDHAR2 to investigate whether PbDHAR2 could limit the deleterious effects of salt and chilling stresses. These transgenic plants contained significantly higher AsA levels than the wild-type (WT) plants. Overexpression of PbDHAR2 increased the expression of the AsA-glutathione (GSH) cycle genes in transgenic lines under salt and chilling stresses. In addition, the transgenic lines subjected to salt and chilling stresses showed higher levels of antioxidant enzyme activity, lower malondialdehyde (MDA) levels, and higher chlorophyll contents than the WT. Thus, our results demonstrate that the regulation of PbDHAR2 during AsA regeneration contributes to enhanced salt and chilling tolerance in tomato.

When plants are subject to environmental stresses, including salt, chilling, heat, and drought, they are damaged because of oxidative stress caused by the accumulation of reactive oxygen species (ROS) in plant cells (Apel and Hirt, 2004). ROS affect plant growth, development, and crop productivity. Many cellular components, such as nucleic acids, carbohydrates, and proteins, are damaged oxidatively (Asada, 2006; Foyer and Noctor, 2005; Mittler et al., 2004). Therefore, plants have developed a set of complex adaptation mechanisms to reduce the deleterious effects of ROS. These mechanisms employ several enzymatic and nonenzymatic scavenging systems, such as antioxidant enzymes, including superoxide dismutase (SOD; EC 1.15.1.1), peroxidase (POD; EC 1.11.1.6), catalase (CAT; EC 1.11.1.6), and antioxidant compounds, such as AsA (Arrigoni, 1994; Conklin, 2001; Farooq et al., 2013).

AsA is a cofactor for many enzymes; it can control cell division and growth (Conklin, 2001; Gallie, 2013a; Gallie, 2013b; Potters et al., 2000); and it improves resistance to a number of environmental stresses, such as salt stress (Zhang et al., 2012), high temperature (Larkindale et al., 2005), oxidative stress (Bai et al., 2013), and inappropriate light levels (Pék et al., 2011). Most AsA are localized in the cytoplasm, whereas ≈10% are localized in the apoplast, and 12% to 30% are found in the chloroplasts (Horemans et al., 2000; Pignocchi and Foyer, 2003). It has been reported that an AsA-GSH pathway exists in cytosol, chloroplasts, mitochondria, and peroxisomes, and represents an important antioxidant defense system against H2O2 (Davey et al., 2000; Potters et al., 2000). In the AsA-GSH cycle, antioxidant enzymes, including ascorbate peroxidase (APX; EC 1.11.1.11), monodehydroascorbate reductase (MDHAR; EC 1.6.5.4), DHAR, and glutathione reductase (GR; EC 1.6.4.2), play an important role in protecting plants against environmental stresses. AsA is oxidized to monodehydroascorbate (MDHA) that reduces H2O2 to water. MDHA is unstable and rapidly disassociates into AsA and DHA. DHA returns to the cytosol for reduction to AsA by DHAR. Thus, DHAR allows the plant to recycle DHA before it is lost, which suggests that DHAR is indirectly involved in ROS scavenging by the AsA-GSH pathway (Giovannoni, 2007; Harb et al., 2010; Melino et al., 2009; Potters et al., 2000; Wang et al., 2010).

DHAR is a ubiquitous protein and DHAR genes form a distinct gene family. Shimaoka et al. (2000) reported that two major DHAR genes existed in spinach leaves. One DHAR gene (DHAR1) is in cytosol, and another DHAR gene (DHAR2) is in chloroplasts. Two major DHARs existed in the form of a gene family and occupied at least 90% of total DHAR activity. DHAR genes involved in abiotic stress are known, including DHAR1 involved in salt and temperature stresses (Eltayeb et al., 2006; Ushimaru et al., 2006; Wang et al., 2010), and DHAR2 involved in salt stress in tobacco (Kwon et al., 2003; Lee et al., 2007). Unraveling of these signalling factors offers a valuable approach for engineering salt and chillling tolerance.

Although DHAR genes have been isolated from several plants (Chen et al., 2003; Shimaoka et al., 2000; Urano et al., 2000; Wang et al., 2010; Zou et al., 2006), there is no report of the function of the PbDHAR2 gene in pear. It is noticeable that knowledge of the DHAR cascade of fruit crops under abiotic stress is scarce compared with other plants, including Arabidopsis, tobacco, and tomato. Pear (Pyrus spp.) is one of the most important fruits in the world and the content of AsA in pears is quite low compared with other plants, such as kiwifruit (Koutinas et al., 2010) and pepper (Aloni et al., 2008). We wanted to know whether overexpression of PbDHAR2 had contributed to increased AsA content. On the other hand, the pear is a perennial ligneous plant and only slow progress has been made in the improvement of salt and chilling stresses via traditional cross-hybridization procedures. As a first step toward creation of pear transgenic plants with enhanced stress tolerance, efforts have been made to isolate and characterize PbDHAR2 gene in this study. Moreover, to gain a better understanding of the PbDHAR2 gene, transgenic tomato lines and WT plants were used to investigate the effects of increased AsA accumulation and any consequent changes in physiology under different stress conditions. We examined some chemical changes that took place after the plants were subjected to salt or chilling stress to evaluate the mechanism controlling PbDHAR2 functions and its potential application in Pyrus plant biotechnology.

Materials and Methods

Gene isolation and construction of the plant expression vector.

Total RNA was extracted from the flesh of pear, Pyrus sinkiangensis Yu ‘Kuerlexiangli’, using the cetyltrimethylammonium bromide (CTAB) method as described by Gambino et al. (2008). The cDNA synthesis was carried out according to the manufacturer’s protocol using a ReverTra Ace qPCR RT Kit (ToYoBo, Osaka, Japan). The primers for the PbDHAR2 gene coding sequence (Pbr 016672.1) were constructed using the National Center for Biotechnology Information (NCBI) database (http://www.ncbi.nlm.nih.gov/tools/primer-blast/) (Wu et al., 2013). The full length of the PbDHAR2 gene was amplified using a D2-1 primer and polymerase chain reaction (PCR) (Table 1). To construct a vector for plant transformation, the PbDHAR2 gene was amplified by PCR with the D2-2 primer including NcoI and BstEII restriction sites on their respective 5′-ends (Table 1). Then the PbDHAR2 PCR fragment was digested with NcoI and BstEII and inserted into the pCAMBIA1301 binary vector to replace the GUS gene. The process was controlled by the CaMV 35S promoter. The vector containing the PbDHAR2 gene sequence was introduced into Agrobacterium tumefaciens strain EHA105 and used to transform Micro-Tom tomato plants.

Table 1.

Primer sequences used for cloning, vector construction, transgenic confirmation, and expression analysis.

Table 1.

Plant transformation and generation of transgenic plants.

Agrobacterium-mediated transformation of leaf discs was carried out according to Huang et al. (2011) to generate transgenic tomato plants. The leaf discs were selected in Murashige and Skoog (MS) medium containing 2.0 mg·L−1 zeatin (ZT), 10 mg·L−1 hygromycin, and 400 mg·L−1 cefotaxime (sodium salt) for adventitious bud regeneration. Regenerated shoots were transferred to 0.5 MS medium supplemented with 0.2 mg·L−1 indole-3-butytric acid to promote rooting. Genomic DNA was isolated from the leaves of the WT and the transgenic plants. The presence of the PbDHAR2 transgenic lines in hygromycin-resistant seedlings (T0) were identified by PCR using the D2-1 primer (Table 1). Transgenic tomatoes were allowed to self pollinate. The genomic DNA of the transgenic plants (T1) was confirmed by PCR using the specific HYG primer (accession number AAF65341.1) to verify the presence of the PbDHAR2 transgene. Overexpression of the PbDHAR2 gene was examined by semiquantitative PCR [reverse transcription (RT)-PCR] using the D2-3 primer and three selected putative transgenic plants (Table 1). The Actin gene was used as the internal control (Table 1). RT-PCR amplification of the PbDHAR2 was performed using 100 ng of cDNA as a template with the following program: 3 min denaturing at 94 °C, followed by 35 cycles of 30 s at 94 °C, 1 min at 60 °C, 30 s at 72 °C, and a 10 min extension at 72 °C. T2 seeds of the overexpressing lines were used in the stress tolerance analysis.

Abiotic stress treatments.

The stress tolerance capacity of the WT and the transgenic plants were subjected to NaCl stress or 4 °C stress. At least 17 seedlings of each lines were transplanted into pots (7 × 7 cm square pots, 8 cm high) containing vermiculite and turf soil (1:1) at 14 h light cycle, 22 ± 4 °C and 60% relative humidity (RH). The WT and the transgenic plants were selected for the NaCl and the chilling (4 °C) treatments when the plants were 40 d old. Salt stress was applied by irrigating the plants with 0.5 Hoagland nutrient solution containing 50 mm NaCl every 5 d for 4 weeks. Plants (40 d old) grown in normal conditions were subjected to 4 °C (14 h light/10 h darkness) for 1 week as in the chilling stress. The samples were then harvested and stored at −80 °C until needed.

AsA and DHA content assay.

AsA and DHA contents were measured essentially as described by Li et al. (2009) but with minor modifications. Two hundred milligrams of fresh leaves from the same position of plants was homogenized in 8 mL 6% (w/v) metaphosphoric acid (HPO3) on ice to prevent AsA oxidation. The homogenate was centrifuged for 20 min at 12,000 g and the supernatant was analyzed for AsA and total ascorbate (AsA + DHA, T-AsA) content. Total AsA content was determined by initially incubating the samples for 15 min at 42 °C in 200 mm phosphate buffer solution (pH 7.4) and 1.5 mm dithiothreitol (DTT). After incubation, 0.2 mL of 0.5% (w/v) N-ethylmaleimide (NEM) was added for 2 min at room temperature to remove excess DTT. AsA content was determined in a similar manner except that 0.4 mL deionized H2O was substituted for DTT and NEM. Color was developed with the addition of 1 mL 10% trichloroacetic acid (TCA), 0.8 mL 42% o-phosphoric acid, 0.8 mL 2% 2, 2-dipyridyl, and 0.4 mL 3% (w/v) FeCl3. The reaction mixtures were then incubated at 42 °C for 1 h and quantified at 525 nm. A standard curve of AsA was established and used for quantification.

Expression analysis by quantitative real-time PCR.

The genes involved in the tomato AsA-GSH cycle from the WT and transgenic line plants before and after salt and chilling treatment were analyzed by quantitative real-time PCR (qPCR) using the SYBR green dye method. Total RNA was extracted from the WT and the transgenic lines leaves using the CTAB method (Gambino et al., 2008) and then treated with DNase I (Invitrogen, Carlsbad, CA). RNA integrity was assessed by 1% agarose gel electrophoresis, and the concentration of extracted RNA was determined by NanoDrop (Thermo). The cDNA synthesis was carried out according to the manufacturer’s protocol using a ReverTra Ace qPCR RT Kit (ToYoBo, Japan). The relative expressions of DHAR1 (accession number AY971873.1), MDHAR (accession number NM_001247084.1) and APX (accession number NM_001247702.1) genes were determined by qPCR using the LightCycler® 480II Real-Time PCR System (Roche, Basel, Switzerland). A 20 μL total reaction volume, containing 1 μL prediluted cDNA, 10 μL LightCycler® 480 SYBR Green I Master (Roche, Mannheim, Germany), and 200 nm forward and reverse primers (Table 1) were used. Actin was used as an internal control to normalize the relative expression levels of the analyzed genes in tomato. Relative expression of a gene was calculated using the 2−ΔΔCT method (Livak and Schmittgen, 2001).

Analysis of antioxidant enzyme activity, and measurement of the MDA and chlorophyll contents.

The activities of three antioxidant enzymes, SOD, POD, and CAT, were measured as in previous reports (Huang et al., 2010; Mittova et al., 2000).

MDA and chlorophyll (chlorophyll a + chlorophyll b) content were evaluated in leaves taken from the WT and the transgenic lines. MDA content was measured as described in previous studies (Huang et al., 2010; Zhang et al., 2012). A sample (0.1 g) of leaf tissue was extracted with 2 mL of 5% (w/v) TCA and the homogenate was centrifuged at 3000 rpm for 10 min. Then, 2 mL of 10% TCA containing 0.67% barbituric acid (TBA) was added to the supernatant. The mixture was boiled at 100 °C for 30 min and centrifuged again after the mixture had been cooled on ice. Finally, the MDA content was determined by measuring the optical density at 532, 450, and 600 nm. Chlorophyll content was measured as described by Huang et al. (2010) and Liu et al. (2008) with minor modifications. Briefly, the chlorophyll content was extracted from ≈0.1 g of leaves directly into a 10 mL extract (ethanol:acetone:H2O = 4.5:4.5:1) at room temperature in the dark. The crude extraction was centrifuged for 2 min at 3000 rpm, which left the leaves completely white. The resultant supernatant was used for assessing absorbance at 663, 645, and 440 nm.

Statistical analysis.

The SPSS 17.0 statistical software package was used for all statistical analyses. Each sample was replicated three times and the results expressed as means with standard deviations (±sd), at a significance level of P < 0.05 and P < 0.01.

Results

Identification and regeneration of transgenic plants.

The 1048 base pair (bp) long PbDHAR2 gene was isolated and cloned from P. sinkiangensis. The gene was transformed into the pCAMBIA1301 binary vector. After sequence confirmation, the construct was introduced into A. tumefaciens strain EHA105. A specific 1kb amplification corresponding to PbDHAR2 was detected with 16 putative transgenic lines (T0), and no expected band was observed in WT plants (Fig. 1A). In total, three transgenic lines (T1) were confirmed by PCR using specific HYG primer (Fig. 1B). Furthermore, RT-PCR analysis using the D2-3 primer showed that PbDHAR2 was detected in transgenic lines, but it was not detected in WT plants (Fig. 1C). Two of the lines (L1 and L5), had higher PbDHAR2 gene expression levels, so these lines (T2) were selected for further experiment analysis.

Fig. 1.
Fig. 1.

Identification of transgenic lines containing PbDHAR2. (A) Polymerase chain reaction (PCR) analysis for the presence of the PbDHAR2 gene. WT, wild-type; L1–L16, transgenic lines. (B) PCR analysis using the HYG primer in the WT and the transgenic lines. L1, L5, and L6 transgenic lines contained the PbDHAR2. (C) Gene expression analysis of PbDHAR2 in the WT and the transgenic lines by reverse transcription-PCR analysis.

Citation: HortScience horts 50, 6; 10.21273/HORTSCI.50.6.789

Assessment of stress tolerance in WT and transgenic lines.

The WT and the transgenic lines were subjected to salt and chilling stresses to allow the role of PbDHAR2 to be studied in detail. There were no apparent differences between the WT and transgenic lines before they were subjected to salt and chilling stresses. The morphological appearances of L1 and L5 were better than that of WT, as more leaves remained green and grew strongly in the transgenic lines (Fig. 2A). Similarly, improved phenotypes were observed after the transgenic plants were subjected to chilling treatment compared with the WT plants (Fig. 2B). This finding implied that the overexpression of the PbDHAR2 gene may enhance tolerance to salt and chilling stresses.

Fig. 2.
Fig. 2.

Overexpression of the PbDHAR2 gene increased stress tolerance following salt and chilling treatment in the transgenic lines. (A) The phenotypes of the wild-type (WT) and the transgenic lines after salt stress. (B) The phenotypes of the WT and the transgenic lines after chilling stress. Asterisks show that values are significantly different between the WT and the transgenic lines under the same growth conditions.

Citation: HortScience horts 50, 6; 10.21273/HORTSCI.50.6.789

The AsA content in the leaves of the WT plants and the transgenic lines were measured to determine the metabolic consequences of PbDHAR2 overexpression. We found that the AsA content in the two transgenic lines (6.03 mg·g−1 FW and 6.28 mg·g−1 FW for L1 and L5, respectively) were 1.44- and 1.50-fold higher than in the WT (4.13 mg·g−1 FW) (Fig. 3A), and DHA content had slightly increased in the L1 and L5 (Fig. 3B). The AsA:DHA ratio in the L1 (16.46) and L5 (17.54) were higher than that of the WT (12.84) (Fig. 3C). Following salt and chilling stresses, both AsA content and AsA:DHA ratio of leaves increased in the WT and the transgenic lines. The AsA content had slightly increased by 1.46- and 1.54-fold after salt stress and by 1.51- and 1.49-fold after chilling stress in the L1 and L5 compared with the WT, respectively (Fig. 3A). The AsA:DHA ratio in the L1 and L5 were 2.36- and 1.61-fold higher than in the WT under salt treatment (Fig. 3C). This indicated that PbDHAR2 was an important positive regulator of AsA content.

Fig. 3.
Fig. 3.

Analysis of ascorbic acid (AsA) content, dehydroascorbate (DHA) content, and AsA:DHA ratio of the wild-type (WT) and the transgenic lines before and after salt and chilling stresses. (A) AsA content in the WT and transgenic lines under normal, salt, and chilling conditions. (B) DHA content in the WT and transgenic lines under normal, salt, and chilling conditions. (C) AsA/DHA ratio in the WT and transgenic lines under normal, salt, and chilling conditions. Asterisks show that values are significantly different between the WT and the transgenic lines under the same growth conditions (*P < 0.05; **P < 0.01).

Citation: HortScience horts 50, 6; 10.21273/HORTSCI.50.6.789

Expression analysis of AsA-GSH cycle genes in the WT and the transgenic lines when under stress.

qPCR analysis was performed on the WT and the transgenic lines before and after they were subjected to the two stresses to investigate the effect of the stresses on the expression of AsA-GSH cycle genes (Fig. 4). These genes involved in AsA recycling (DHAR1, MDHAR, and APX). The increased tolerance of salt in plants overexpressing PbDHAR2 was associated with increased levels of expression of genes involved in AsA recycling compared with WT plants (Fig. 4). A similar effect on gene expression was observed under chilling stress. However, the transgenic lines response to salt stress were more pronounced than their response to chilling stress. These results suggested that overexpression of PbDHAR2 enhanced the expression levels of AsA recycling genes to regulate the AsA content, which led to reduced stress damage in transgenic plants.

Fig. 4.
Fig. 4.

Quantitative real-time polymerase chain reaction analysis of ascorbic acid-glutathione cycle genes expression levels in the wild-type (WT) and the transgenic lines under normal, salt, and chilling conditions. (A) DHAR1, (B) MDHAR, and (C) APX expression levels. Asterisks show that values are significantly different between the WT and the transgenic lines under the same growth conditions (*P < 0.05; **P < 0.01).

Citation: HortScience horts 50, 6; 10.21273/HORTSCI.50.6.789

Analysis of antioxidant enzyme activity and metabolite levels.

ROS can be scavenged by enzymatic and nonenzymatic pathways. Three antioxidative enzymes (SOD, POD, and CAT) play significant roles in the response to stress. Under normal growth conditions, the activities of the three enzymes showed no significant differences in the WT and the transgenic lines, except for POD activity in L1 (Fig. 5). Under salt and chilling stresses, the SOD, POD, and CAT activities were enhanced in the transgenic lines (Fig. 5A–C). This result indicated that the transgenic lines can reduce ROS accumulation and enhance stress tolerance.

Fig. 5.
Fig. 5.

Analysis of antioxidant activity in the wild-type (WT) and the transgenic lines under normal, salt, and chilling conditions. (A) Superoxide dismutase (SOD), (B) peroxidase (POD), and (C) catalase (CAT) activities. Asterisks show that values are significantly different between the WT and the transgenic lines under the same growth conditions (*P < 0.05; **P < 0.01).

Citation: HortScience horts 50, 6; 10.21273/HORTSCI.50.6.789

Under normal conditions, the MDA contents in the WT and the transgenic lines were no significantly difference. However, after salt treatment, the MDA content in the WT increased 2.65-fold, whereas it only increased 2.54-fold and 2.03-fold in L1 and L5, respectively (Fig. 6A). Changes to MDA under chilling treatment were correlated with changes in the salt treatment in the WT and the transgenic lines. Similarly, the chlorophyll content in the WT and the transgenic lines was not significantly different under normal conditions, but under salt and chilling stresses the chlorophyll content fell in both the WT plants and the transgenic lines (Fig. 6B). However, chlorophyll content in the WT was significantly lower than in the transgenic lines. These results supported the hypothesis that AsA recycling is an essential part of the enhanced salt and chilling tolerance conferred by PbDHAR2.

Fig. 6.
Fig. 6.

Analysis of metabolite levels in the wild-type (WT) and the transgenic lines under normal, salt, and chilling conditions. (A) Malondialdehyde (MDA) and (B) chlorophyll contents. Asterisks show that values are significantly different between the WT and the transgenic lines under the same growth conditions (*P < 0.05; **P < 0.01).

Citation: HortScience horts 50, 6; 10.21273/HORTSCI.50.6.789

Discussion

The role of AsA in plant growth, development and the response to various stresses, including salt, chilling, and oxidative stresses, has been well documented (Arrigoni, 1994; Conklin, 2001; Giovannoni, 2007; Horemans et al., 2000; Linster and Clarke, 2008). The DHAR protein catalyzes DHA to AsA in AsA-GSH cycle (Chen and Gallie, 2005; Gallie, 2013b). Thus, the PbDHAR2 gene may contribute to regulating AsA levels when a plant is subjected to abiotic stresses. The overexpression of DHAR1 significantly increased DHAR activities and AsA contents in potato (Qin et al., 2011). It has been reported that the DHAR protein regulated AsA synthesis by elevating the expression of the DHAR1 gene in tobacco (Chen et al., 2003), but the regulatory mechanism for the PbDHAR2 gene from P. sinkiangensis is still unclear.

In this study, the vector containing the PbDHAR2 gene was transformed into tomato plants. The PbDHAR2 gene was successfully introduced and was overexpressed in the L1 and L5 (Fig. 1C). The AsA content significantly rose in the transgenic lines, which indicated that PbDHAR2 overexpression could regulate AsA content (Fig. 3A). The AsA:DHA ratio in the transgenic lines were higher than in the WT (Fig. 3B). It was suggested that PbDHAR2 overexpression in the transgenic lines would increase the likelihood that DHA is converted to AsA before decaying, that would lead to the increase in AsA in the transgenic plants. Furthermore, AsA content in plants can be elevated by increasing the expression of DHAR1, which affects AsA recycling (Chen et al., 2003). Ushimaru et al. (2006) found that the overexpression of DHAR1 did not significantly increase the AsA content, but it did enhance Arabidopsis plant tolerance to high temperature stress. In contrast, a study by Haroldsen et al. (2011) supported our conclusion that the overexpression of DHAR1 produced a 1.6-fold increase in AsA content in tomato compared with the WT. Therefore, PbDHAR2 overexpression was able to increase the content of AsA, which provided us a new direction to improve the AsA content of pear flesh in the future work.

According to the phenotype analysis, the PbDHAR2 overexpression lines showed stronger plant growth when subjected to salt and chilling stresses than the WT plants (Fig. 2). These results further confirmed that PbDHAR2 overexpression could enhance plant resistance to abiotic stresses.

ROS production is induced in response to various stresses (Noctor and Foyer, 1998). In plant cells, ROS can be transformed into H2O2, and the AsA-GSH recycling pathway, including DHAR1, MDHAR, and APX, can scavenge H2O2 by converting it into H2O using AsA (Mittova et al., 2000). We used biochemical and physiological analysis to determine whether ROS accumulation in the transgenic lines was less than in the WT. Our results showed the expression levels of the AsA-GSH cycle genes were upregulated significantly in the transgenic lines under salt and chilling stresses (Fig. 4), implying these regulatory genes participate in the AsA cycle and lead to greater accumulation of AsA. Haroldsen et al. (2011) reported plants overexpressing DHAR1 and MDHAR had higher levels of total AsA compared with plants grown in growth chambers. Overexpression of APX increased salt tolerance in tobacco (Wu et al., 2014). Therefore, the increased levels of DHAR1, MDHAR, and APX expression influenced the levels of AsA, thereby resulting in the reduced damage to the plant cells, which might explain the lower levels of ROS in the transgenic lines.

Plants have several enzymatic scavenging systems for ROS detoxification, such as SOD, POD, and CAT (Miller et al., 2010). In our study, the transgenic lines showed higher SOD, POD, and CAT activities under salt and chilling stresses compared with the WT plants (Fig. 5). Abiotic stresses can improve the MDA content (Del Rio et al., 2005). We found the L1 and L5 had a lower content of MDA and a greater content of chlorophyll compared with the WT plants when under salt and chilling stresses (Fig. 6). The transgenic lines lowered their ROS levels by increasing the activities of their enzymatic and nonenzymatic scavenging systems, which explains why the transgenic lines have better phenotypes compared with WT. Interestingly, an increasing number of reports have shown that AsA content rise when plants are under stress (Eltayeb et al., 2007; Fotopoulos et al., 2006; Lee et al., 2007). Wang et al. (2010) found that the expression of Arabidopsis DHAR1 protected against oxidative stress. These findings are in agreement with a previous report by Chen and Gallie (2006).

In conclusion, PbDHAR2 is an important positive regulator of AsA content. The AsA-GSH cycle genes were upregulated by PbDHAR2 overexpression under salt and chilling stresses, and the enhanced stress tolerance was also correlated with a rise in antioxidant enzymes and antioxidants, which led to more efficient ROS scavenging. These results indicated that overexpression of PbDHAR2 improved stress tolerance in the L1 and L5. Therefore, the PbDHAR2 gene has the potential to be a candidate gene for enhancing salt and chilling stresses tolerance in the future.

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  • HoremansN.FoyerH.PottersC.G.AsardH.2000Ascorbate function and associated transport systems in plantsPlant Physiol. Biochem.38531540

  • HuangX.S.LiuJ.H.ChenX.J.2010Overexpression of PtrABF gene, a bZIP transcription factor isolated from Poncirus trifoliata, enhances dehydration and drought tolerance in tobacco via scavenging ROS and modulating expression of stress-responsive genesBMC Plant Biol.10230

    • Search Google Scholar
    • Export Citation
  • HuangX.S.LuoT.FuX.Z.FanQ.J.LiuJ.H.2011Cloning and molecular characterization of a mitogen-activated protein kinase gene from Poncirus trifoliata whose ectopic expression confers dehydration/drought tolerance in transgenic tobaccoJ. Expt. Bot.6251915206

    • Search Google Scholar
    • Export Citation
  • KoutinasN.SotiropoulosT.PetridisA.AlmaliotisD.DeligeorgisE.TheriosI.VoulgarakisN.2010Effects of preharvest calcium foliar sprays on several fruit quality attributes and nutritional status of the kiwifruit cultivar TsechelidisHortScience45984987

    • Search Google Scholar
    • Export Citation
  • KwonS.Y.ChoiS.M.AhnY.O.LeeH.S.LeeH.B.ParkY.M.KwakS.S.2003Enhanced stress-tolerance of transgenic tobacco plants expressing a human dehydroascorbate reductase geneJ. Plant Physiol.160347353

    • Search Google Scholar
    • Export Citation
  • LarkindaleJ.HallJ.D.KnightM.R.VierlingE.2005Heat stress phenotypes of Arabidopsis mutants implicate multiple signaling pathways in the acquisition of thermotolerancePlant Physiol.138882897

    • Search Google Scholar
    • Export Citation
  • LeeY.P.KimS.H.BangJ.W.LeeH.S.KwakS.S.KwonS.Y.2007Enhanced tolerance to oxidative stress in transgenic tobacco plants expressing three antioxidant enzymes in chloroplastsPlant Cell Rpt.26591598

    • Search Google Scholar
    • Export Citation
  • LiM.LiangD.PuF.MaF.HouC.LuT.2009Ascorbate levels and the activity of key enzymes in ascorbate biosynthesis and recycling in the leaves of 22 Chinese persimmon cultivarsSci. Hort.120250256

    • Search Google Scholar
    • Export Citation
  • LinsterC.L.ClarkeS.G.2008L-Ascorbate biosynthesis in higher plants: The role of VTC2Trends Plant Sci.13567573

  • LiuJ.-H.InoueH.MoriguchiT.2008Salt stress-mediated changes in free polyamine titers and expression of genes responsible for polyamine biosynthesis of apple shootsEnviron. Expt. Bot.622835

    • Search Google Scholar
    • Export Citation
  • LivakK.J.SchmittgenT.D.2001Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT methodMethods25402408

    • Search Google Scholar
    • Export Citation
  • MelinoV.J.SooleK.L.FordC.M.2009Ascorbate metabolism and the developmental demand for tartaric and oxalic acids in ripening grape berriesBMC Plant Biol.9145

    • Search Google Scholar
    • Export Citation
  • MillerG.SuzukiN.Ciftci-YilmazS.MittlerR.2010Reactive oxygen species homeostasis and signalling during drought and salinity stressesPlant Cell Environ.33453467

    • Search Google Scholar
    • Export Citation
  • MittlerR.VanderauweraS.GolleryM.Van BreusegemF.2004Reactive oxygen gene network of plantsTrends Plant Sci.9490498

  • MittovaV.VolokitaM.GuyM.TalM.2000Activities of SOD and the ascorbate-glutathione cycle enzymes in subcellular compartments in leaves and roots of the cultivated tomato and its wild salt-tolerant relative Lycopersicon pennelliiPhysiol. Plant.1104251

    • Search Google Scholar
    • Export Citation
  • NoctorG.FoyerC.H.1998Ascorbate and glutathione: Keeping active oxygen under controlAnnu. Rev. Plant Biol.49249279

  • PékZ.SzuvandzsievP.NemenyiA.HelyesL.LugasiA.2011The effect of natural light on changes in antioxidant content and color parameters of vine-ripened tomato (Solanum lycopersicum L.) fruitsHortScience46583585

    • Search Google Scholar
    • Export Citation
  • PignocchiC.FoyerC.H.2003Apoplastic ascorbate metabolism and its role in the regulation of cell signallingCurr. Opin. Plant Biol.6379389

    • Search Google Scholar
    • Export Citation
  • PottersG.HoremansN.CaubergsR.J.AsardH.2000Ascorbate and dehydroascorbate influence cell cycle progression in a tobacco cell suspensionPlant Physiol.1241720

    • Search Google Scholar
    • Export Citation
  • QinA.ShiQ.YuX.2011Ascorbic acid contents in transgenic potato plants overexpressing two dehydroascorbate reductase genesMol. Biol. Rpt.3815571566

    • Search Google Scholar
    • Export Citation
  • ShimaokaT.YokotaA.MiyakeC.2000Purification and characterization of chloroplast dehydroascorbate reductase from spinach leavesPlant Cell Physiol.4111101118

    • Search Google Scholar
    • Export Citation
  • UranoJ.NakagawaT.MakiY.MasumuraT.TanakaK.MurataN.UshimaruT.2000Molecular cloning and characterization of a rice dehydroascorbate reductaseFEBS Lett.466107111

    • Search Google Scholar
    • Export Citation
  • UshimaruT.NakagawaT.FujiokaY.DaichoK.NaitoM.YamauchiY.NonakaH.AmakoK.YamawakiK.MurataN.2006Transgenic Arabidopsis plants expressing the rice dehydroascorbate reductase gene are resistant to salt stressJ. Plant Physiol.16311791184

    • Search Google Scholar
    • Export Citation
  • WangZ.XiaoY.ChenW.TangK.ZhangL.2010Increased vitamin C content accompanied by an enhanced recycling pathway confers oxidative stress tolerance in ArabidopsisJ. Integr. Plant Biol.52400409

    • Search Google Scholar
    • Export Citation
  • WuG.WangG.JiJ.GaoH.GuanW.WuJ.GuanC.F.WangY.2014Cloning of a cytosolic ascorbate peroxidase gene from Lycium chinense Mill. and enhanced salt tolerance by overexpressing in tobaccoGene5438592

    • Search Google Scholar
    • Export Citation
  • WuJ.WangZ.ShiZ.ZhangS.MingR.ZhuS.KhanM.A.TaoS.KorbanS.S.WangH.ChenN.J.NishioT.XuX.CongL.QiK.HuangX.WangY.ZhaoX.WuJ.DengC.GouC.ZhouW.YinH.QinG.ShaY.TaoY.ChenH.YangY.SongY.ZhanD.WangJ.LiL.DaiM.GuC.WangY.ShiD.WangX.ZhangH.ZengL.ZhengD.WangC.ChenM.WangG.XieL.SoveroV.ShaS.HuangW.ZhangS.ZhangM.SunJ.XuL.LiY.LiuX.LiQ.ShenJ.WangJ.PaullR.E.BennetzenJ.L.WangJ.ZhangS.2013The genome of the pear (Pyrus bretschneideri Rehd.)Genome Res.23396408

    • Search Google Scholar
    • Export Citation
  • ZhangZ.WangJ.ZhangR.HuangR.2012The ethylene response factor AtERF98 enhances tolerance to salt through the transcriptional activation of ascorbic acid synthesis in ArabidopsisPlant J.71273287

    • Search Google Scholar
    • Export Citation
  • ZouL.LiH.OuyangB.ZhangJ.YeZ.2006Cloning and mapping of genes involved in tomato ascorbic acid biosynthesis and metabolismPlant Sci.170120127

    • Search Google Scholar
    • Export Citation

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Contributor Notes

This work was supported by the Jiangsu Agriculture Science and Technology Innovation Fund [No. CX(14)2020].

To whom reprint requests should be addressed; e-mail slzhang@njau.edu.cn.

  • View in gallery

    Identification of transgenic lines containing PbDHAR2. (A) Polymerase chain reaction (PCR) analysis for the presence of the PbDHAR2 gene. WT, wild-type; L1–L16, transgenic lines. (B) PCR analysis using the HYG primer in the WT and the transgenic lines. L1, L5, and L6 transgenic lines contained the PbDHAR2. (C) Gene expression analysis of PbDHAR2 in the WT and the transgenic lines by reverse transcription-PCR analysis.

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    Overexpression of the PbDHAR2 gene increased stress tolerance following salt and chilling treatment in the transgenic lines. (A) The phenotypes of the wild-type (WT) and the transgenic lines after salt stress. (B) The phenotypes of the WT and the transgenic lines after chilling stress. Asterisks show that values are significantly different between the WT and the transgenic lines under the same growth conditions.

  • View in gallery

    Analysis of ascorbic acid (AsA) content, dehydroascorbate (DHA) content, and AsA:DHA ratio of the wild-type (WT) and the transgenic lines before and after salt and chilling stresses. (A) AsA content in the WT and transgenic lines under normal, salt, and chilling conditions. (B) DHA content in the WT and transgenic lines under normal, salt, and chilling conditions. (C) AsA/DHA ratio in the WT and transgenic lines under normal, salt, and chilling conditions. Asterisks show that values are significantly different between the WT and the transgenic lines under the same growth conditions (*P < 0.05; **P < 0.01).

  • View in gallery

    Quantitative real-time polymerase chain reaction analysis of ascorbic acid-glutathione cycle genes expression levels in the wild-type (WT) and the transgenic lines under normal, salt, and chilling conditions. (A) DHAR1, (B) MDHAR, and (C) APX expression levels. Asterisks show that values are significantly different between the WT and the transgenic lines under the same growth conditions (*P < 0.05; **P < 0.01).

  • View in gallery

    Analysis of antioxidant activity in the wild-type (WT) and the transgenic lines under normal, salt, and chilling conditions. (A) Superoxide dismutase (SOD), (B) peroxidase (POD), and (C) catalase (CAT) activities. Asterisks show that values are significantly different between the WT and the transgenic lines under the same growth conditions (*P < 0.05; **P < 0.01).

  • View in gallery

    Analysis of metabolite levels in the wild-type (WT) and the transgenic lines under normal, salt, and chilling conditions. (A) Malondialdehyde (MDA) and (B) chlorophyll contents. Asterisks show that values are significantly different between the WT and the transgenic lines under the same growth conditions (*P < 0.05; **P < 0.01).

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  • HoremansN.FoyerH.PottersC.G.AsardH.2000Ascorbate function and associated transport systems in plantsPlant Physiol. Biochem.38531540

  • HuangX.S.LiuJ.H.ChenX.J.2010Overexpression of PtrABF gene, a bZIP transcription factor isolated from Poncirus trifoliata, enhances dehydration and drought tolerance in tobacco via scavenging ROS and modulating expression of stress-responsive genesBMC Plant Biol.10230

    • Search Google Scholar
    • Export Citation
  • HuangX.S.LuoT.FuX.Z.FanQ.J.LiuJ.H.2011Cloning and molecular characterization of a mitogen-activated protein kinase gene from Poncirus trifoliata whose ectopic expression confers dehydration/drought tolerance in transgenic tobaccoJ. Expt. Bot.6251915206

    • Search Google Scholar
    • Export Citation
  • KoutinasN.SotiropoulosT.PetridisA.AlmaliotisD.DeligeorgisE.TheriosI.VoulgarakisN.2010Effects of preharvest calcium foliar sprays on several fruit quality attributes and nutritional status of the kiwifruit cultivar TsechelidisHortScience45984987

    • Search Google Scholar
    • Export Citation
  • KwonS.Y.ChoiS.M.AhnY.O.LeeH.S.LeeH.B.ParkY.M.KwakS.S.2003Enhanced stress-tolerance of transgenic tobacco plants expressing a human dehydroascorbate reductase geneJ. Plant Physiol.160347353

    • Search Google Scholar
    • Export Citation
  • LarkindaleJ.HallJ.D.KnightM.R.VierlingE.2005Heat stress phenotypes of Arabidopsis mutants implicate multiple signaling pathways in the acquisition of thermotolerancePlant Physiol.138882897

    • Search Google Scholar
    • Export Citation
  • LeeY.P.KimS.H.BangJ.W.LeeH.S.KwakS.S.KwonS.Y.2007Enhanced tolerance to oxidative stress in transgenic tobacco plants expressing three antioxidant enzymes in chloroplastsPlant Cell Rpt.26591598

    • Search Google Scholar
    • Export Citation
  • LiM.LiangD.PuF.MaF.HouC.LuT.2009Ascorbate levels and the activity of key enzymes in ascorbate biosynthesis and recycling in the leaves of 22 Chinese persimmon cultivarsSci. Hort.120250256

    • Search Google Scholar
    • Export Citation
  • LinsterC.L.ClarkeS.G.2008L-Ascorbate biosynthesis in higher plants: The role of VTC2Trends Plant Sci.13567573

  • LiuJ.-H.InoueH.MoriguchiT.2008Salt stress-mediated changes in free polyamine titers and expression of genes responsible for polyamine biosynthesis of apple shootsEnviron. Expt. Bot.622835

    • Search Google Scholar
    • Export Citation
  • LivakK.J.SchmittgenT.D.2001Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT methodMethods25402408

    • Search Google Scholar
    • Export Citation
  • MelinoV.J.SooleK.L.FordC.M.2009Ascorbate metabolism and the developmental demand for tartaric and oxalic acids in ripening grape berriesBMC Plant Biol.9145

    • Search Google Scholar
    • Export Citation
  • MillerG.SuzukiN.Ciftci-YilmazS.MittlerR.2010Reactive oxygen species homeostasis and signalling during drought and salinity stressesPlant Cell Environ.33453467

    • Search Google Scholar
    • Export Citation
  • MittlerR.VanderauweraS.GolleryM.Van BreusegemF.2004Reactive oxygen gene network of plantsTrends Plant Sci.9490498

  • MittovaV.VolokitaM.GuyM.TalM.2000Activities of SOD and the ascorbate-glutathione cycle enzymes in subcellular compartments in leaves and roots of the cultivated tomato and its wild salt-tolerant relative Lycopersicon pennelliiPhysiol. Plant.1104251

    • Search Google Scholar
    • Export Citation
  • NoctorG.FoyerC.H.1998Ascorbate and glutathione: Keeping active oxygen under controlAnnu. Rev. Plant Biol.49249279

  • PékZ.SzuvandzsievP.NemenyiA.HelyesL.LugasiA.2011The effect of natural light on changes in antioxidant content and color parameters of vine-ripened tomato (Solanum lycopersicum L.) fruitsHortScience46583585

    • Search Google Scholar
    • Export Citation
  • PignocchiC.FoyerC.H.2003Apoplastic ascorbate metabolism and its role in the regulation of cell signallingCurr. Opin. Plant Biol.6379389

    • Search Google Scholar
    • Export Citation
  • PottersG.HoremansN.CaubergsR.J.AsardH.2000Ascorbate and dehydroascorbate influence cell cycle progression in a tobacco cell suspensionPlant Physiol.1241720

    • Search Google Scholar
    • Export Citation
  • QinA.ShiQ.YuX.2011Ascorbic acid contents in transgenic potato plants overexpressing two dehydroascorbate reductase genesMol. Biol. Rpt.3815571566

    • Search Google Scholar
    • Export Citation
  • ShimaokaT.YokotaA.MiyakeC.2000Purification and characterization of chloroplast dehydroascorbate reductase from spinach leavesPlant Cell Physiol.4111101118

    • Search Google Scholar
    • Export Citation
  • UranoJ.NakagawaT.MakiY.MasumuraT.TanakaK.MurataN.UshimaruT.2000Molecular cloning and characterization of a rice dehydroascorbate reductaseFEBS Lett.466107111

    • Search Google Scholar
    • Export Citation
  • UshimaruT.NakagawaT.FujiokaY.DaichoK.NaitoM.YamauchiY.NonakaH.AmakoK.YamawakiK.MurataN.2006Transgenic Arabidopsis plants expressing the rice dehydroascorbate reductase gene are resistant to salt stressJ. Plant Physiol.16311791184

    • Search Google Scholar
    • Export Citation
  • WangZ.XiaoY.ChenW.TangK.ZhangL.2010Increased vitamin C content accompanied by an enhanced recycling pathway confers oxidative stress tolerance in ArabidopsisJ. Integr. Plant Biol.52400409

    • Search Google Scholar
    • Export Citation
  • WuG.WangG.JiJ.GaoH.GuanW.WuJ.GuanC.F.WangY.2014Cloning of a cytosolic ascorbate peroxidase gene from Lycium chinense Mill. and enhanced salt tolerance by overexpressing in tobaccoGene5438592

    • Search Google Scholar
    • Export Citation
  • WuJ.WangZ.ShiZ.ZhangS.MingR.ZhuS.KhanM.A.TaoS.KorbanS.S.WangH.ChenN.J.NishioT.XuX.CongL.QiK.HuangX.WangY.ZhaoX.WuJ.DengC.GouC.ZhouW.YinH.QinG.ShaY.TaoY.ChenH.YangY.SongY.ZhanD.WangJ.LiL.DaiM.GuC.WangY.ShiD.WangX.ZhangH.ZengL.ZhengD.WangC.ChenM.WangG.XieL.SoveroV.ShaS.HuangW.ZhangS.ZhangM.SunJ.XuL.LiY.LiuX.LiQ.ShenJ.WangJ.PaullR.E.BennetzenJ.L.WangJ.ZhangS.2013The genome of the pear (Pyrus bretschneideri Rehd.)Genome Res.23396408

    • Search Google Scholar
    • Export Citation
  • ZhangZ.WangJ.ZhangR.HuangR.2012The ethylene response factor AtERF98 enhances tolerance to salt through the transcriptional activation of ascorbic acid synthesis in ArabidopsisPlant J.71273287

    • Search Google Scholar
    • Export Citation
  • ZouL.LiH.OuyangB.ZhangJ.YeZ.2006Cloning and mapping of genes involved in tomato ascorbic acid biosynthesis and metabolismPlant Sci.170120127

    • Search Google Scholar
    • Export Citation
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