Physiologia Plantarum 156: 164–175. 2016
© 2015 Scandinavian Plant Physiology Society, ISSN 0031-9317
Over-expression of AtGSTU19 provides tolerance to salt, drought and methyl viologen stresses in Arabidopsis Jing Xu† , Yong-Sheng Tian† , Xiao-Juan Xing, Ri-He Peng, Bo Zhu, Jian-Jie Gao and Quan-Hong Yao* Shanghai Key Laboratory of Agricultural Genetics and Breeding, Biotechnology Research Institute of Shanghai Academy of Agricultural Sciences, Shanghai, 201106, China
Correspondence *Corresponding author, e-mail: [email protected] Received 28 January 2015; revised 24 March 2015 doi:10.1111/ppl.12347
The plant-specific tau class of glutathione S-transferases (GSTs) is often highly stress-inducible and expressed in a tissue-specific manner, thereby suggesting its important protective roles. Although activities associated with the binding and transport of reactive metabolites have been proposed, little is known about the regulatory functions of GSTs. Expression of AtGSTU19 is induced by several stimuli, but the function of this GST remains unknown. In this study, we demonstrated that transgenic over-expressing (OE) plants showed enhanced tolerance to different abiotic stresses and increased percentage of seed germination and cotyledon emergence. Transgenic plants exhibited an increased level of proline and activities of antioxidant enzymes, along with decreased malonyldialdehyde level under stress conditions. Real-time polymerase chain reaction (PCR) analyses revealed that the expression levels of several stress-regulated genes were altered in AtGSTU19 OE plants. These results indicate that AtGSTU19 plays an important role in tolerance to salt/drought/ methyl viologen stress in Arabidopsis.
Introduction Glutathione S-transferases (GSTs; EC 2.5.1.18) are a superfamily of multifunctional enzymes that catalyze the conjugation of electrophilic substrates to glutathione (GSH). In both plants and animals, GSTs are induced by diverse environmental stimuli and play direct roles in reducing oxidative damage or toxic products produced during xenobiotic metabolism (Dixon et al. 2002, Moons 2005, Frova 2006). Additionally, some plant GSTs are induced by phytohormones, such as ethylene, auxin, salicylic acid, cytokinin, abscisic acid (ABA) and methyl jasmonate, and are involved in the development (Gong et al. 2005, Moons 2005). †
Plant GSTs are classified into eight classes, termed phi (F), tau (U), lambda (L), theta (T), zeta (Z), DHAR (dehydroascorbate reductase), TCHQD (tetrachlorohydroquinone dehalogenase) and MAPEG (membraneassociated proteins in eicosanoid and glutathione metabolism) (Dixon et al. 2009, Dixon and Edwards 2010). Among these, the plant-specific phi (GSTF) and tau (GSTU) are the most abundant in plants and are involved mainly in xenobiotic metabolism (Frova 2003, 2006). Recently, Liu et al. studied the GST genes from Physcomitrella patens, a non-vascular representative of early land plants, and identified two new GST classes, hemerythrin and iota. The complex patterns of evolutionary divergence revealed the extensive
These authors contributed equally to this article.
Abbreviations – ABA, abscisic acid; AtAc2, Arabidopsis Actin2; CaMV 35S, cauliflower mosaic virus 35S; CDNB, 1-chloro-2,4-dinitrobenzene; GSTs, glutathione S-transferases; GSH, glutathione; IAA, indole acetic acid; MDA, malondialdehyde; MS, Murashige and Skoog; MV, methyl viologen; OE, over-expressing; POD, peroxidase; qRT-PCR, quantitative real-time polymerase chain reaction; ROS, reactive oxygen species; RT-PCR, real-time polymerase chain reaction; SOD, superoxide dismutase; WT, wild-type.
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functional divergence among the members of GST gene family in land plants (Liu et al. 2013). In both plants and animals, GST function has been closely linked to stress response. GST expression is induced under a wide variety of stress conditions. Increased GST levels occur to maintain cell redox homeostasis and to protect the organisms from oxidative stress (Chen et al. 2012). Abiotic stresses, such as salinity and drought stress, may lead to oxidative stress by increasing reactive oxygen species (ROS) production (Miller et al. 2010). Methyl viologen (MV, paraquat) may also cause oxidative stress by increasing the production of O2 − in both animal and plant systems (Hassan and Fridovich 1977). Arabidopsis GSTU19, (accession number: AT1G78380) a member of the third tau clade, is the best studied and characterized among major proteins. The expression of GSTU19 was enhanced in Arabidopsis cultures following exposure to herbicide safeners (DeRidder et al. 2002). However, information on the function of AtGSTU19 in fighting oxidative stress induced by salt, drought or MV stress in plants is limited. This study aimed to elucidate the function of AtGSTU19 genes in plant defense against these stresses. Transgenic over-expressing (OE) plants were used. These plants showed enhanced salt/drought/MV tolerance and increased activity of antioxidant enzymes under various stresses. The expression levels of several stress-regulated genes were altered in AtGSTU19 OE plants. The results suggest that AtGSTU19 plays a role in tolerance to these stresses in Arabidopsis.
Materials and methods Plant material and growth conditions Plants (Arabidopsis thaliana ecotype Columbia, Col) were surface-sterilized by treatment with 75% ethanol for 1 min, followed by commercial bleach (0.5% calcium hypochlorite) for 20 min and three washes with sterile distilled water. Seeds were stratified in the dark at 4∘ C for 2 days and then grown on Murashige and Skoog (MS) medium (Murashige and Skoog 1962) with 0.8% agar or pots filled with a 9:3:1 mixture of vermiculite/peat moss/perlite in a controlled environmental chamber at 22∘ C and maintained on a 16/8 h day/night cycle. Construction of plant transformation vector and transformation Full length AtGSTU19 cDNA was amplified by real-time polymerase chain reaction (RT-PCR) using total RNA obtained from wild-type (WT). Reactions were performed using the PCR primers 5′ -AAGGAT CCATGGCGAACGAGGTGATTCTTC-3′ and 5′ -AAGAG
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CTCTTACTCAGGTACAAATTTCTTC-3′ . The product was cloned into TA clone vector Simple pMD-18 (Takara Co. Ltd, Dalian, China) and the integrity of the construct was verified by sequencing. The AtGSTU19 cDNA was digested with BamHI and SacI, and inserted between the double cauliflower mosaic virus (CaMV 35S; D35S) promoter and nopaline synthase terminator (NOS-Ter) of plant expression vector pYM817 (Fig. 1A). The recombinant plasmid was introduced into Agrobacterium tumefaciens GV3101. The constructions were transformed by floral dip method as described previously (Zhang et al. 2006) into plants of Col. Transgenic plants were selected by hygromycin resistance and confirmed by PCR using the primers mentioned as described. Three Arabidopsis homozygous transgenic lines (lines 1, 4 and 6) expressing AtGSTU19 of T3 generation were used for further analysis. Total RNA extraction, reverse transcription and expression analysis using quantitative RT-PCR Total RNA was extracted from WT and transgenic plants using the multisource total RNA miniprep kit (Axygen Scientific, Inc., Union City, CA) according to the manufacturer’s instructions. First-strand cDNA synthesis was conducted with 5 μg of total RNA using a TransScript Fly First-Strand cDNA Synthesis SuperMix (Transgen Biotech, Shanghai, China). The synthesized cDNA was subjected to quantitative real-time polymerase chain reaction (qRT-PCR) analysis using SYBR Premix Ex Taq (TaKaRa) using a Mini Opticon Real Time PCR System (Bio-Rad Laboratories Inc., Hercules, CA). PCR amplification was performed with primers specific for AtGSTU19, forward: 5′ -CGAGTATGTTCGGGATG AGG-3′ and reverse: 5′ -GGAAGGATAGGGTTCTTGTGA G-3′ . Amplification of Arabidopsis Actin2 (AtAc2, AGI: At3g18780) was used as an internal control. Primers as following: AtAc2 (AtAc2F:5′ -AGTAAGGTCACGTCC AGCAAGG-3′ ; and AtAc2Z:5′ -GCACCCTGTTCTTCTTA CCGAG-3′ ). Relative quantity of the target gene expression level was measured as described (Xu et al. 2010). Three replicates were performed for each experiment. Stress tolerance analysis For germination and seedling growth assay, seeds of both transgenic and WT plants were stratified and planted on MS medium supplemented or not with different concentrations of NaCl, mannitol or MV. The seeds were incubated at 4∘ C for 2 days before being placed at 22∘ C under light conditions. Root length, germination rate and cotyledon emergence rate were scored. 165
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Fig. 1. Transgenic Arabidopsis plants over-expressing AtGSTU19. (A) The AtGSTU19 expression vector for Arabidopsis transformation. Nos-Ter, terminator sequence of the nopaline synthase; SAR, scaffold attached region. (B) Expression of the AtGSTU19 cDNA in transgenic and wild-type plants. Total RNA was isolated from 3-week-old plants grown under the normal conditions (WT as control). Values are mean ± SD (n = 3). (C) GST activity in Arabidopsis plants expressing AtGSTU19. GST activity was measured in leaves of 3-week-old plants grown under normal condition using CDNB as substrate. Values are means ± SD (n = 3) and different letters above bars indicate significant differences (P < 0.05) among different lines.
For drought treatment, watering of 3-week-old plants grown in pots was withheld for 2 weeks. The performance of WT and transgenic plants was compared when irrigation resumed. For salt treatment, 3-week-old plants grown in pots were watered with NaCl (250 mM). Two weeks later, the performance of WT and transgenic plants was compared. For MV treatment, seeds were surface-sterilized and grown on MS solid medium for 15 days. Twenty seedlings were then transferred aseptically into 50 ml conical flasks containing 20 ml MS liquid medium and grown with rotary shaking (60 rpm) for recovering. After 2 days, the liquid medium was refreshed with new one, which contained MV at different concentrations. The dry weight was scored after 10 days. Measurement of physiological parameters involved in stress tolerance To measure standardized water content, aerial parts from 3-week-old pot-grown plants were excised and placed on filter paper. The loss of fresh weight was monitored at indicated times. The method was described by Xu et al. (2010). Furthermore, 3-week-old plants were treated with 250 mM NaCl (salt stress) or 300 mM mannitol (drought stress) or 10 μM MV for 4 days, and the proline content, malondialdehyde (MDA) content, superoxide dismutase (SOD) and peroxidase (POD) activities were 166
measured. To measure proline and MDA levels, leaves from 3-week-old plants of the same genotype under the same treatment were mixed as sample. Proline was assayed on water-extracted seedlings using the ninhydrin assay (Bates et al. 1973). MDA content was determined by the reaction of thiobarbituric acid (TBA) as described previously (Cakmak and Horst 1991). To measure antioxidant enzyme activities, fresh leaf tissue (approximately 0.1 g) from 3-week-old plants of the same genotype under the same treatment was harvested and homogenized in 1.6 ml of chilled 50 mM phosphate buffer and kept in an ice bath. The homogenate was filtered through two layers of muslin cloth and centrifuged at 20 000 g for 10 min in a refrigerated centrifuge at 4∘ C. The supernatant was stored at 4∘ C and used for enzyme assays within 4 h. SOD (EC 1.15.1.1) activity was determined by monitoring the inhibition of photochemical reduction of nitro blue tetrazolium according to the method of Beyer and Fridovich (1987). POD (EC 1.11.1.7) activity was assayed according to the method of MacAdam et al. (1992). GST activity was measured according to Alfenito, with minor modifications (Alfenito et al. 1998). Enzyme activity was tested in the presence of 1 mM glutathione in 100 mM sodium phosphate buffer (pH 6.5). The reaction started with the addition of 1-chloro-2,4-dinitrobenzene (CDNB) to a final concentration of 1 mM. The changes in A340 were measured. The background levels of spontaneous CDNB decay were subtracted.
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Stress-related gene expression using qRT-PCR Three-week-old plants were given NaCl (250 mM) as salt stress, mannitol (300 mM) as drought stress or MV (10 μM) as oxidative stress. After 6 h, plants were harvested and frozen in liquid nitrogen. Total RNA isolation and reverse transcription were performed as described. PCR amplification was performed with oligonucleotides specific for various stress-responsive genes: desiccation responsive gene (RD29A) (At5g52310) forward: 5′ -TA ATCGGAAGACACGACAGG-3′ and reverse: 5′ -GATGTT TAGGAAAGTAAAGGCTAG-3′ ; RD29B (At5g52300): forward: 5′ -GGAATCCGAAAACCCCATAGTC-3′ and reverse: 5′ -GG AGTGAAGGAGACGCAACAAG-3′ ; RD22 (At5g25610) forward: 5′ -CATGAGTCTCCGGGAG GAAGTG-3′ and reverse: 5′ -CGGCTGGGGTAAAGAAG TTGTC-3′ ; cold regulated gene (COR47) (At1g20440) and forward: 5′ -GAAAAGCTTCACCGATCCAA-3′ ′ reverse: 5 -TACCGGGATGGTAGTGGAAA-3′ ; COR15A (At2g42540) forward: 5′ -AACTCTGCCGCCTTGTTTGC -3′ and reverse: 5′ -AGTCGTTGATCTACGCCGCTAA-3′ ; KIN1 (At5g15960) forward: 5′ -TGCCTTCCAAGCCGGT CAGA-3′ , and reverse: 5′ -AGGCCGGTCTTGTCCTTCAC-3′ ; peroxiredoxin gene (PRX) (AT3G49120) forward: 5′ -ACAAGCATTCCTGGAACTCG-3′ , and reverse: 5′ -G ATTGTGTCAGTGGCATTGG-3′ . Amplification of AtAc2 gene was used as an internal control, and qRT-PCR experimental procedures were performed as described. Relative amounts were calculated and normalized with respect to the expression of each gene in WT plants without treatment. Three replicates were performed for each experiment. Statistical analysis The SPSS software version 15.0J (SPSS Inc., Chicago, IL) was used for statistical analysis. ANOVA was performed followed by Duncan’s multiple comparison tests. Statistically significant differences (P < 0.05) are reported in the text and shown in the figures.
Results Transgenic Arabidopsis plants with higher expression levels of AtGSTU19 The full-length cDNA for AtGSTU19 was introduced into Arabidopsis cells in the sense orientation under the control of the double CaMV35S (D35S) promoter (Fig. 1A). Eleven independent lines of transgenic plants (T1 generation) were generated. Among these, three homozygous transgenic lines (OE1, OE4 and OE6) expressing AtGSTU19 in the T3 generation were chosen for further experiments.
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qRT-PCR was used to examine the transcript levels. In the T3 generation, the levels of AtGSTU19 mRNA were higher in the three OE lines than in the WT (Fig. 1B). GST activity levels were detected in both OE and WT plants. Compared with the WT, different transgenic lines showed significantly enhanced GST activity with CDNB as substrate (Fig. 1C). No obvious effects on growth and development were observed in AtGSTU19 transgenic plants under normal growth conditions. Increased root growth and germination rate in transgenic lines under salt/drought stress The role of AtGSTU19 in salt or drought stress was analyzed by germinating seeds on MS medium containing NaCl and mannitol. During seedling growth, increased root length was observed for the transgenic lines compared with the WT (Fig. 2A). Under salt stress, root length increased by 35 and 60% (mean values) in OE plants when seedlings were grown on medium containing different NaCl concentration (50 and 100 mM, respectively) in comparison with the WT. Under drought stress, the mean values were 29 and 52% in OE lines compared with WT, when the medium containing mannitol (150 and 300 mM), respectively. To analyze whether increased root length is due to early germination of transgenic lines, the germination rates measured as radical emergence were compared between transgenic lines and WT. Increased germination rate was observed for the transgenic plants under different stress conditions (Fig. 2B). As observed, the germination rate of transgenic seedlings was about twofold higher compared with the WT on medium containing 100 mM NaCl (48 h). A similar pattern was observed for plants under drought stress. Seeds from transgenic lines germinated faster than those from WT when the medium contained different mannitol concentrations. Salt/drought stress tolerance of transgenic AtGSTU19 Arabidopsis plants To analyze the salt/drought tolerance, 3-week-old T3 seedlings were watered with NaCl (250 mM) to induce salt stress, or watering was withheld to induce drought stress. Two weeks later, irrigation was resumed, and plants were photographed (Fig. 3A). Under high salt stress treatment, nearly all the WT plants died, whereas >80% of the transgenic OE plants survived. When drought stress was imposed on plants, nearly all WT plants died after 2 weeks, whereas >70% of the AtGSTU19 OE lines survived (Fig. 3B). To further demonstrate drought tolerance, the water content from 3-week-old pot-grown plants was determined (Fig. 3C). Leaves of transgenic OE lines showed 167
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Time after stratification (h) Fig. 2. Comparative analysis of transgenic lines over-expressing AtGSTU19 on salt/drought stress conditions. (A) Comparison of root length between transgenic lines and WT seedlings grown on MS medium containing different concentration of NaCl and mannitol. Each value is the mean ± SD of at least 50 seedlings (n = 3). Different letters above bars indicate significant difference (P < 0.05) among different genotypes in the same treatment. (B) Comparison of germination rate between transgenic lines and WT seeds growth on the medium as mentioned in (A). Each value is the mean ± SD of at least 50 seeds (n = 3)
higher capacity of conserving water than those of WT plants, starting from 1 h after harvest. These results showed that transgenic plants had significantly higher tolerance to salt and drought stresses than WT plants. The elevated tolerance of the AtGSTU19 transgenic plants was correlated with the changes in their proline 168
and MDA content (Fig. 3D). Generally, proline acts as a compatible solute and is accumulated by the plants to maintain the turgor pressure during osmotic stress challenge and to protect the plant cells against damages caused by 1 O2 or HO (Delauney and Verma 1993, Matysik et al. 2002). However, environmental stressors
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higher than that in the WT, whereas the MDA content of the former was lower than the latter. We also measured the activities of some antioxidant enzymes (Fig. 4) and found no significant difference between transgenic and WT plants under normal conditions. Under salt/drought stress condition, the SOD and POD activities increased in both transgenic and WT plants. However, SOD and POD activities in transgenic lines were significantly higher than in the WT plants during NaCl and mannitol stresses. These results suggest that AtGSTU19 strengthens the cellular ROS scavenging ability and maintains ROS homeostasis by increasing proline content and the activities of antioxidant enzymes.
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Given that GST is involved in anti-oxidation processes in plants and animals, we analyzed the anti-oxidation capacity of transgenic plants. Seeds were germinated on MS medium containing different MV concentrations, and the percentage of cotyledon emergence was compared between transgenic plants and WT. Increased cotyledon emergence was observed for transgenic plants when these plants were grown on MS medium containing MV. Photographs were taken at 7 days after stratification (Fig. 5A). In the medium containing 0.5 μM MV, the cotyledon emergence rate of WT was 71%, and the mean rate of transgenic lines was approximately 86%. When the MV concentration reached 1.5 μM, the cotyledon emergence rate decreased to 20% in WT, and the mean rate of transgenic lines was approximately 65% (Fig. 5B). To analyze the MV tolerance, 2-week-old T3 seedlings grown on MS solid medium were transferred aseptically into MS liquid medium and grown with rotary shaking (60 rpm) for recovery. After 2 days, the liquid medium was refreshed with new one supplemented with different MV concentration. Photographs were taken at 10 days
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cause a rapid and excessive accumulation of ROS in plant cells, which leads to a lipid peroxidation chain reaction further, and the result is the accumulation of MDA. MDA is the principal by-product of lipid oxidation (Bartels 2001, Zhu 2001). In our study, we found that the proline content in transgenic OE lines was significantly
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Fig. 3. Salt/drought stress tolerance of transgenic plants. (A) Three-week-old plants of OE lines and WT were given salt or drought stress treatment. Control, 3-week-old plants growing under normal conditions; salt stress, plants watered with NaCl (250 mM); drought stress, plants withheld watering for 2 weeks, and then irrigated again. The experiment was replicated three times with similar results and 48 plants per line for each experiment. (B) Survival rate of OE lines and WT under stress treatment as described in (A). Values are means ± SD (n = 3), each from 48 plants. (C) Standardized water content of OE lines and WT. Each data point is mean ± SD (n = 3), each from ≥10 plants. (D) Changes in the content of proline and MDA between OE lines and WT treated with salt stress (NaCl 250 mM) or drought stress (mannitol 300 mM) for 4 days. Values are means ± SD (n = 3), each from 10 plants. Different letters above bars indicate significant differences (P < 0.05) among different genotypes in the same treatment.
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Fig. 4. Antioxidant enzyme activities in Arabidopsis plants expressing AtGSTU19. Antioxidant enzyme activities were measured in leaves of 3-week-old plants. Salt stress was performed with NaCl (250 mM), drought stress was performed with mannitol (300 mM) and oxidative stress was performed with MV (10 μM) for 4 days. Values are means ± SD (n = 3) and different letters above bars indicate significant differences (P < 0.05) among different genotypes in the same treatment.
after exposure to stress (Fig. 6A). The seedlings of WT and OE lines grown in the liquid medium without MV appeared normal. This result indicated that toxic effects were caused by MV stress and not the result of submersion in the liquid medium. The dry weights of WT and OE lines were nearly the same without MV stress. However, the growth of WT plants was inhibited when MV stress was induced (Fig. 6B). When WT plants were treated with 1.5 μM MV, the growth of WT plants was significantly inhibited, the chlorophyll content decreased, and the leaves turned chlorotic. The relative dry weight rates of WT and OE lines were 32 and 52%, respectively. When MV concentration reached 2 μM, the growth of both WT and transgenic plants was inhibited, and the leaves turned 170
Fig. 5. Comparative analysis of transgenic lines over-expressing AtGSTU19 on MV stress condition. (A) Comparison of seedling growth between transgenic lines and WT grown on MS medium containing different concentration of MV. (B) Comparison of cotyledon emergency rate between transgenic lines and WT seeds growth on the medium as mentioned in (A). Each value is the mean ± SD of at least 50 seedlings (n = 3). Different letters above bars indicate significant differences (P < 0.05) among different genotypes in the same treatment.
chlorotic. The relative dry weight rates were 9 and 20% in WT and transgenic lines, respectively. When MV concentration reached 3 μM, both WT and transgenic plants died (data not shown). These results showed that transgenic plants exhibited higher tolerance to MV stress than WT plants. The antioxidant enzymes were also detected under MV stress condition (Fig. 4). Under stress conditions, the SOD and POD activities increased in both transgenic and WT plants. However, SOD and POD activities in transgenic lines were significantly higher than in the WT plants under MV stress. The enzyme profiles under MV stress were similar to those observed under salt/drought stress. GST activity was also detected under stress treatment. Transgenic plants exhibited higher level of GST
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activity under salt, drought and MV stress conditions (Fig. 6). These results suggested that AtGSTU19 increases GST activity, thereby resulting in increased abiotic stress resistance. Together, the over-expression of AtGSTU19 enhanced the tolerance to salt, drought and MV stresses in Arabidopsis. AtGSTU19 was suggested to play an important role in the adaptation to at least three distinct stress agents (salt, drought and MV). Expression of stress-inducible genes in transgenic AtGSTU19 plants The hallmark of stress adaptation in plants is the induction of numerous stress-responsive genes (Shinozaki and Yamaguchi-Shinozaki 1997, Zhu et al. 1997). To further elucidate the role of AtGSTU19 in stress tolerance, we examined the effects of AtGSTU19 on the transcript levels of several stress-inducible genes, including RD29A and RD29B (Yamaguchi-Shinozaki and Shinozaki 1994), RD22 (Yamaguchi-Shinozaki and Shinozaki 1993), KIN1 (Wang and Cutler 1995), COR15A (Baker et al. 1994) and COR47 (Hajela et al. 1990). We analyzed the
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expression of these genes under salt stress and dehydration, given the similarities of mechanisms underlying plant adaptation to water deficit caused by high salinity and drought (Fig. 7A). Moreover, PRX (Sharma and Davis 1994, Ezaki et al. 2000), an oxidative stress marker induced by ozone or aluminum treatments, was analyzed under MV stress (Fig. 7B). Under normal conditions, the expression levels of RD29A, KIN1 and COR15A were significantly higher in a transgenic OE line than in WT (Fig. 7A). Under salt/drought stress treatments, the expression levels of all of these stress-responsive genes, except for COR47 and RD22, were upregulated in the WT. The expression of KIN1 was upregulated only under drought stress. Moreover, the expressions of all these stress-responsive genes, except for RD22 and COR47, were significantly higher in transgenic lines than in WT (Fig. 7A). Under MV stress treatment, the expression of PRX was upregulated in WT and was significantly higher in transgenic plants than in WT (Fig. 7B). Together, transgenic plants over-expressing AtGSTU19 significantly enhanced the expression levels of all stress-induced genes except for RD22 and COR47.
Discussion GSTs are known to protect plants against oxidative stress induced by biotic and abiotic agents through detoxification of endogenous cytotoxic compounds, which accumulate during oxidative stress. In plants, GST expression is induced in many different stress situations (Wagner et al. 2002). Previous studies have shown that AtGSTU19 (first cloned as GST8) transcripts were induced by drought stress, oxidative stress and high doses of auxin or cytokinin (Bianchi et al. 2002). In this study, we focused on the function of AtGSTU19 in abiotic stress and extend it as playing a positive role in drought, salt and oxidative stress tolerance. Functional analysis on the role of AtGSTU19 in stress tolerance of Arabidopsis was carried out using transgenic OE lines. The phenomena of increased germination, root growth and the percentage of cotyledon emergence were observed under stress treatment (Figs 2A, B and 5B). In plants, GST expression is induced by phytohormones. Jain et al. (2010) analyzed the expression pattern of 74 GST genes in response to different hormones and the results showed that a large number of GSTs are responsive to hormones. Gong et al. (2005) also demonstrated the role of GST in plant growth and development in vivo and shoot morphogenesis in vitro. The expression of AtGSTU19 was induced by indole acetic acid (IAA) and other hormones (Bianchi et al. 2002), and seed germination rate increased in transgenic plants under stress. 171
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Fig. 7. Stress-induced gene expression. (A) Salt and drought-induced gene expression in transgenic and wild-type plants assayed by real-time PCR. Control 3-week-old plants growing under normal conditions, salt stress plants watered with NaCl (250 mM), drought stress plants watered with mannitol (300 mM) for 6 h. Values are mean ± SD of three replicates. (B) Oxidative-induced gene expression in transgenic and wild-type plants assayed by real-time PCR. Three-week-old plants grown in pots were given MV (10 μM) for 6 h as oxidative stress. Different letters above bars indicate significant differences (P < 0.05) among different genotypes in the same treatment.
These observations showed that AtGSTU19 could participate in plant growth and development. The production of ROS is a common reaction when plants are exposed to abiotic stresses (Yang et al. 2014). Therefore, ROS homeostasis is important in protecting the normal metabolism of organisms, and plants can regulate ROS levels through ROS scavenging via antioxidant enzymes, such as SOD, POD and GST (Jiang et al. 2007). SODs catalyze the dismutation of superoxide into oxygen and H2 O2 and constitute the first line of defense against ROS in a cell (Alscher et al. 2002). In turn, various forms of peroxidases are responsible for the 172
removal of H2 O2 from biological systems (Sergio et al. 2012). Moreover, over-expression of some GST genes has been shown to improve oxidative stress tolerance in transgenic plants (Mittler 2002). Besides, proline in plant cells plays a protective role in adaptation to water deprivation (Handa et al. 1986) and protects plant cells against oxidative damage by quenching 1 O2 and directly scavenging HO (Matysik et al. 2002). In our study, the transgenic plants over-expressing AtGSTU19 have higher levels of antioxidant enzyme activities (SOD, POD and GST), higher proline content and lower MDA content than WT plants. These results suggested
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that AtGSTU19 may act as a stress regulator though increasing the activities of antioxidant enzymes to strengthen the ROS scavenging activity or maintain ROS homeostasis. Synthesis of various osmoprotectants, such as proline, detoxification enzymes and various proteases is one of the strategies for protecting plants against stresses directly (Shinozaki and Yamaguchi-Shinozaki 2007). Another one involves the regulation of gene expression and signal transduction pathways (Kaur and Gupta 2005). Many stress-inducible genes contain recognizing motifs in their promoters and function in response to abiotic stresses (Shinozaki et al. 2003). Thus, the expression of some stress-regulated genes was examined to investigate the stress resistance further. Transgenic plants over-expressing AtGSTU19 activated the expression of some late stress-response genes, such as RD29A, COR15A and KIN1, even under normal growth conditions. The three genes were sensitive to various abiotic stressors and were induced by dehydration, high salinity or low temperature (Wang et al. 1995, Msanne et al. 2011, Liu et al. 2014). Our studies showed that plants over-expressing AtGSTU19 may activate stress response pathways to a certain degree under normal conditions. Under stress conditions, the expression levels of some stress-responsive genes in transgenic OE plants increased more substantially than those in WT. RD29B was sensitively induced by dehydration, high salinity or low temperature (Msanne et al. 2011). The expression of PRX gene was dependent on light, ascorbate and oxidative stress (Horling et al. 2003). The results demonstrated that the stress-response pathway was activated further. However, further studies should be performed to understand the role of phytohormones in plant stress responses and how the presence of AtGSTU19 gene enhances the expression of other stress-responsive genes. Taken together, transgenic plants over-expressing AtGSTU19 enhanced the salt/drought/MV stress tolerance (Figs 3A, 5A and 6A). Another tau GST, AtGSTU17, was reported as a negative component of the stressmediated signal transduction pathway in adaptive responses to drought and salt stress through accumulating GSH and ABA and exhibiting hyposensitivity to ABA in atgstu17 (Chen et al. 2012). As a multigene family, GSTs share very little overall amino acid sequence identity, typically no more than 25–35% (Marrs 1996), which suggests that the function of GSTs is complicated and multiplexed. Both tau GSTs, AtGSTU17 and AtGSTU19, were grouped into different tau clades (Dixon and Edwards 2010), which suggested that they could have different functions. It partially explains the differences in results between Chen’s findings (Chen et al. 2012) and ours.
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Acknowledgements – The research study was sponsored by Shanghai Rising-Star Program (Grant No. 14QB140 3400), Shanghai Natural Science Foundation (Grant No. 13ZR1460600), National Natural Science Foundation (Grant Nos 31300237, 31401458) and Youth Talents Growth Plan of Shanghai Academy of Agricultural Sciences (Grant No. 2014-1-19, 2015-1-26).
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Over-expression of AtGSTU19 provides tolerance to salt, drought and methyl viologen stresses in Arabidopsis Jing Xu† , Yong-Sheng Tian† , Xiao-Juan Xing, Ri-He Peng, Bo Zhu, Jian-Jie Gao and Quan-Hong Yao* Shanghai Key Laboratory of Agricultural Genetics and Breeding, Biotechnology Research Institute of Shanghai Academy of Agricultural Sciences, Shanghai, 201106, China
Correspondence *Corresponding author, e-mail: [email protected] Received 28 January 2015; revised 24 March 2015 doi:10.1111/ppl.12347
The plant-specific tau class of glutathione S-transferases (GSTs) is often highly stress-inducible and expressed in a tissue-specific manner, thereby suggesting its important protective roles. Although activities associated with the binding and transport of reactive metabolites have been proposed, little is known about the regulatory functions of GSTs. Expression of AtGSTU19 is induced by several stimuli, but the function of this GST remains unknown. In this study, we demonstrated that transgenic over-expressing (OE) plants showed enhanced tolerance to different abiotic stresses and increased percentage of seed germination and cotyledon emergence. Transgenic plants exhibited an increased level of proline and activities of antioxidant enzymes, along with decreased malonyldialdehyde level under stress conditions. Real-time polymerase chain reaction (PCR) analyses revealed that the expression levels of several stress-regulated genes were altered in AtGSTU19 OE plants. These results indicate that AtGSTU19 plays an important role in tolerance to salt/drought/ methyl viologen stress in Arabidopsis.
Introduction Glutathione S-transferases (GSTs; EC 2.5.1.18) are a superfamily of multifunctional enzymes that catalyze the conjugation of electrophilic substrates to glutathione (GSH). In both plants and animals, GSTs are induced by diverse environmental stimuli and play direct roles in reducing oxidative damage or toxic products produced during xenobiotic metabolism (Dixon et al. 2002, Moons 2005, Frova 2006). Additionally, some plant GSTs are induced by phytohormones, such as ethylene, auxin, salicylic acid, cytokinin, abscisic acid (ABA) and methyl jasmonate, and are involved in the development (Gong et al. 2005, Moons 2005). †
Plant GSTs are classified into eight classes, termed phi (F), tau (U), lambda (L), theta (T), zeta (Z), DHAR (dehydroascorbate reductase), TCHQD (tetrachlorohydroquinone dehalogenase) and MAPEG (membraneassociated proteins in eicosanoid and glutathione metabolism) (Dixon et al. 2009, Dixon and Edwards 2010). Among these, the plant-specific phi (GSTF) and tau (GSTU) are the most abundant in plants and are involved mainly in xenobiotic metabolism (Frova 2003, 2006). Recently, Liu et al. studied the GST genes from Physcomitrella patens, a non-vascular representative of early land plants, and identified two new GST classes, hemerythrin and iota. The complex patterns of evolutionary divergence revealed the extensive
These authors contributed equally to this article.
Abbreviations – ABA, abscisic acid; AtAc2, Arabidopsis Actin2; CaMV 35S, cauliflower mosaic virus 35S; CDNB, 1-chloro-2,4-dinitrobenzene; GSTs, glutathione S-transferases; GSH, glutathione; IAA, indole acetic acid; MDA, malondialdehyde; MS, Murashige and Skoog; MV, methyl viologen; OE, over-expressing; POD, peroxidase; qRT-PCR, quantitative real-time polymerase chain reaction; ROS, reactive oxygen species; RT-PCR, real-time polymerase chain reaction; SOD, superoxide dismutase; WT, wild-type.
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functional divergence among the members of GST gene family in land plants (Liu et al. 2013). In both plants and animals, GST function has been closely linked to stress response. GST expression is induced under a wide variety of stress conditions. Increased GST levels occur to maintain cell redox homeostasis and to protect the organisms from oxidative stress (Chen et al. 2012). Abiotic stresses, such as salinity and drought stress, may lead to oxidative stress by increasing reactive oxygen species (ROS) production (Miller et al. 2010). Methyl viologen (MV, paraquat) may also cause oxidative stress by increasing the production of O2 − in both animal and plant systems (Hassan and Fridovich 1977). Arabidopsis GSTU19, (accession number: AT1G78380) a member of the third tau clade, is the best studied and characterized among major proteins. The expression of GSTU19 was enhanced in Arabidopsis cultures following exposure to herbicide safeners (DeRidder et al. 2002). However, information on the function of AtGSTU19 in fighting oxidative stress induced by salt, drought or MV stress in plants is limited. This study aimed to elucidate the function of AtGSTU19 genes in plant defense against these stresses. Transgenic over-expressing (OE) plants were used. These plants showed enhanced salt/drought/MV tolerance and increased activity of antioxidant enzymes under various stresses. The expression levels of several stress-regulated genes were altered in AtGSTU19 OE plants. The results suggest that AtGSTU19 plays a role in tolerance to these stresses in Arabidopsis.
Materials and methods Plant material and growth conditions Plants (Arabidopsis thaliana ecotype Columbia, Col) were surface-sterilized by treatment with 75% ethanol for 1 min, followed by commercial bleach (0.5% calcium hypochlorite) for 20 min and three washes with sterile distilled water. Seeds were stratified in the dark at 4∘ C for 2 days and then grown on Murashige and Skoog (MS) medium (Murashige and Skoog 1962) with 0.8% agar or pots filled with a 9:3:1 mixture of vermiculite/peat moss/perlite in a controlled environmental chamber at 22∘ C and maintained on a 16/8 h day/night cycle. Construction of plant transformation vector and transformation Full length AtGSTU19 cDNA was amplified by real-time polymerase chain reaction (RT-PCR) using total RNA obtained from wild-type (WT). Reactions were performed using the PCR primers 5′ -AAGGAT CCATGGCGAACGAGGTGATTCTTC-3′ and 5′ -AAGAG
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CTCTTACTCAGGTACAAATTTCTTC-3′ . The product was cloned into TA clone vector Simple pMD-18 (Takara Co. Ltd, Dalian, China) and the integrity of the construct was verified by sequencing. The AtGSTU19 cDNA was digested with BamHI and SacI, and inserted between the double cauliflower mosaic virus (CaMV 35S; D35S) promoter and nopaline synthase terminator (NOS-Ter) of plant expression vector pYM817 (Fig. 1A). The recombinant plasmid was introduced into Agrobacterium tumefaciens GV3101. The constructions were transformed by floral dip method as described previously (Zhang et al. 2006) into plants of Col. Transgenic plants were selected by hygromycin resistance and confirmed by PCR using the primers mentioned as described. Three Arabidopsis homozygous transgenic lines (lines 1, 4 and 6) expressing AtGSTU19 of T3 generation were used for further analysis. Total RNA extraction, reverse transcription and expression analysis using quantitative RT-PCR Total RNA was extracted from WT and transgenic plants using the multisource total RNA miniprep kit (Axygen Scientific, Inc., Union City, CA) according to the manufacturer’s instructions. First-strand cDNA synthesis was conducted with 5 μg of total RNA using a TransScript Fly First-Strand cDNA Synthesis SuperMix (Transgen Biotech, Shanghai, China). The synthesized cDNA was subjected to quantitative real-time polymerase chain reaction (qRT-PCR) analysis using SYBR Premix Ex Taq (TaKaRa) using a Mini Opticon Real Time PCR System (Bio-Rad Laboratories Inc., Hercules, CA). PCR amplification was performed with primers specific for AtGSTU19, forward: 5′ -CGAGTATGTTCGGGATG AGG-3′ and reverse: 5′ -GGAAGGATAGGGTTCTTGTGA G-3′ . Amplification of Arabidopsis Actin2 (AtAc2, AGI: At3g18780) was used as an internal control. Primers as following: AtAc2 (AtAc2F:5′ -AGTAAGGTCACGTCC AGCAAGG-3′ ; and AtAc2Z:5′ -GCACCCTGTTCTTCTTA CCGAG-3′ ). Relative quantity of the target gene expression level was measured as described (Xu et al. 2010). Three replicates were performed for each experiment. Stress tolerance analysis For germination and seedling growth assay, seeds of both transgenic and WT plants were stratified and planted on MS medium supplemented or not with different concentrations of NaCl, mannitol or MV. The seeds were incubated at 4∘ C for 2 days before being placed at 22∘ C under light conditions. Root length, germination rate and cotyledon emergence rate were scored. 165
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Fig. 1. Transgenic Arabidopsis plants over-expressing AtGSTU19. (A) The AtGSTU19 expression vector for Arabidopsis transformation. Nos-Ter, terminator sequence of the nopaline synthase; SAR, scaffold attached region. (B) Expression of the AtGSTU19 cDNA in transgenic and wild-type plants. Total RNA was isolated from 3-week-old plants grown under the normal conditions (WT as control). Values are mean ± SD (n = 3). (C) GST activity in Arabidopsis plants expressing AtGSTU19. GST activity was measured in leaves of 3-week-old plants grown under normal condition using CDNB as substrate. Values are means ± SD (n = 3) and different letters above bars indicate significant differences (P < 0.05) among different lines.
For drought treatment, watering of 3-week-old plants grown in pots was withheld for 2 weeks. The performance of WT and transgenic plants was compared when irrigation resumed. For salt treatment, 3-week-old plants grown in pots were watered with NaCl (250 mM). Two weeks later, the performance of WT and transgenic plants was compared. For MV treatment, seeds were surface-sterilized and grown on MS solid medium for 15 days. Twenty seedlings were then transferred aseptically into 50 ml conical flasks containing 20 ml MS liquid medium and grown with rotary shaking (60 rpm) for recovering. After 2 days, the liquid medium was refreshed with new one, which contained MV at different concentrations. The dry weight was scored after 10 days. Measurement of physiological parameters involved in stress tolerance To measure standardized water content, aerial parts from 3-week-old pot-grown plants were excised and placed on filter paper. The loss of fresh weight was monitored at indicated times. The method was described by Xu et al. (2010). Furthermore, 3-week-old plants were treated with 250 mM NaCl (salt stress) or 300 mM mannitol (drought stress) or 10 μM MV for 4 days, and the proline content, malondialdehyde (MDA) content, superoxide dismutase (SOD) and peroxidase (POD) activities were 166
measured. To measure proline and MDA levels, leaves from 3-week-old plants of the same genotype under the same treatment were mixed as sample. Proline was assayed on water-extracted seedlings using the ninhydrin assay (Bates et al. 1973). MDA content was determined by the reaction of thiobarbituric acid (TBA) as described previously (Cakmak and Horst 1991). To measure antioxidant enzyme activities, fresh leaf tissue (approximately 0.1 g) from 3-week-old plants of the same genotype under the same treatment was harvested and homogenized in 1.6 ml of chilled 50 mM phosphate buffer and kept in an ice bath. The homogenate was filtered through two layers of muslin cloth and centrifuged at 20 000 g for 10 min in a refrigerated centrifuge at 4∘ C. The supernatant was stored at 4∘ C and used for enzyme assays within 4 h. SOD (EC 1.15.1.1) activity was determined by monitoring the inhibition of photochemical reduction of nitro blue tetrazolium according to the method of Beyer and Fridovich (1987). POD (EC 1.11.1.7) activity was assayed according to the method of MacAdam et al. (1992). GST activity was measured according to Alfenito, with minor modifications (Alfenito et al. 1998). Enzyme activity was tested in the presence of 1 mM glutathione in 100 mM sodium phosphate buffer (pH 6.5). The reaction started with the addition of 1-chloro-2,4-dinitrobenzene (CDNB) to a final concentration of 1 mM. The changes in A340 were measured. The background levels of spontaneous CDNB decay were subtracted.
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Stress-related gene expression using qRT-PCR Three-week-old plants were given NaCl (250 mM) as salt stress, mannitol (300 mM) as drought stress or MV (10 μM) as oxidative stress. After 6 h, plants were harvested and frozen in liquid nitrogen. Total RNA isolation and reverse transcription were performed as described. PCR amplification was performed with oligonucleotides specific for various stress-responsive genes: desiccation responsive gene (RD29A) (At5g52310) forward: 5′ -TA ATCGGAAGACACGACAGG-3′ and reverse: 5′ -GATGTT TAGGAAAGTAAAGGCTAG-3′ ; RD29B (At5g52300): forward: 5′ -GGAATCCGAAAACCCCATAGTC-3′ and reverse: 5′ -GG AGTGAAGGAGACGCAACAAG-3′ ; RD22 (At5g25610) forward: 5′ -CATGAGTCTCCGGGAG GAAGTG-3′ and reverse: 5′ -CGGCTGGGGTAAAGAAG TTGTC-3′ ; cold regulated gene (COR47) (At1g20440) and forward: 5′ -GAAAAGCTTCACCGATCCAA-3′ ′ reverse: 5 -TACCGGGATGGTAGTGGAAA-3′ ; COR15A (At2g42540) forward: 5′ -AACTCTGCCGCCTTGTTTGC -3′ and reverse: 5′ -AGTCGTTGATCTACGCCGCTAA-3′ ; KIN1 (At5g15960) forward: 5′ -TGCCTTCCAAGCCGGT CAGA-3′ , and reverse: 5′ -AGGCCGGTCTTGTCCTTCAC-3′ ; peroxiredoxin gene (PRX) (AT3G49120) forward: 5′ -ACAAGCATTCCTGGAACTCG-3′ , and reverse: 5′ -G ATTGTGTCAGTGGCATTGG-3′ . Amplification of AtAc2 gene was used as an internal control, and qRT-PCR experimental procedures were performed as described. Relative amounts were calculated and normalized with respect to the expression of each gene in WT plants without treatment. Three replicates were performed for each experiment. Statistical analysis The SPSS software version 15.0J (SPSS Inc., Chicago, IL) was used for statistical analysis. ANOVA was performed followed by Duncan’s multiple comparison tests. Statistically significant differences (P < 0.05) are reported in the text and shown in the figures.
Results Transgenic Arabidopsis plants with higher expression levels of AtGSTU19 The full-length cDNA for AtGSTU19 was introduced into Arabidopsis cells in the sense orientation under the control of the double CaMV35S (D35S) promoter (Fig. 1A). Eleven independent lines of transgenic plants (T1 generation) were generated. Among these, three homozygous transgenic lines (OE1, OE4 and OE6) expressing AtGSTU19 in the T3 generation were chosen for further experiments.
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qRT-PCR was used to examine the transcript levels. In the T3 generation, the levels of AtGSTU19 mRNA were higher in the three OE lines than in the WT (Fig. 1B). GST activity levels were detected in both OE and WT plants. Compared with the WT, different transgenic lines showed significantly enhanced GST activity with CDNB as substrate (Fig. 1C). No obvious effects on growth and development were observed in AtGSTU19 transgenic plants under normal growth conditions. Increased root growth and germination rate in transgenic lines under salt/drought stress The role of AtGSTU19 in salt or drought stress was analyzed by germinating seeds on MS medium containing NaCl and mannitol. During seedling growth, increased root length was observed for the transgenic lines compared with the WT (Fig. 2A). Under salt stress, root length increased by 35 and 60% (mean values) in OE plants when seedlings were grown on medium containing different NaCl concentration (50 and 100 mM, respectively) in comparison with the WT. Under drought stress, the mean values were 29 and 52% in OE lines compared with WT, when the medium containing mannitol (150 and 300 mM), respectively. To analyze whether increased root length is due to early germination of transgenic lines, the germination rates measured as radical emergence were compared between transgenic lines and WT. Increased germination rate was observed for the transgenic plants under different stress conditions (Fig. 2B). As observed, the germination rate of transgenic seedlings was about twofold higher compared with the WT on medium containing 100 mM NaCl (48 h). A similar pattern was observed for plants under drought stress. Seeds from transgenic lines germinated faster than those from WT when the medium contained different mannitol concentrations. Salt/drought stress tolerance of transgenic AtGSTU19 Arabidopsis plants To analyze the salt/drought tolerance, 3-week-old T3 seedlings were watered with NaCl (250 mM) to induce salt stress, or watering was withheld to induce drought stress. Two weeks later, irrigation was resumed, and plants were photographed (Fig. 3A). Under high salt stress treatment, nearly all the WT plants died, whereas >80% of the transgenic OE plants survived. When drought stress was imposed on plants, nearly all WT plants died after 2 weeks, whereas >70% of the AtGSTU19 OE lines survived (Fig. 3B). To further demonstrate drought tolerance, the water content from 3-week-old pot-grown plants was determined (Fig. 3C). Leaves of transgenic OE lines showed 167
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Time after stratification (h) Fig. 2. Comparative analysis of transgenic lines over-expressing AtGSTU19 on salt/drought stress conditions. (A) Comparison of root length between transgenic lines and WT seedlings grown on MS medium containing different concentration of NaCl and mannitol. Each value is the mean ± SD of at least 50 seedlings (n = 3). Different letters above bars indicate significant difference (P < 0.05) among different genotypes in the same treatment. (B) Comparison of germination rate between transgenic lines and WT seeds growth on the medium as mentioned in (A). Each value is the mean ± SD of at least 50 seeds (n = 3)
higher capacity of conserving water than those of WT plants, starting from 1 h after harvest. These results showed that transgenic plants had significantly higher tolerance to salt and drought stresses than WT plants. The elevated tolerance of the AtGSTU19 transgenic plants was correlated with the changes in their proline 168
and MDA content (Fig. 3D). Generally, proline acts as a compatible solute and is accumulated by the plants to maintain the turgor pressure during osmotic stress challenge and to protect the plant cells against damages caused by 1 O2 or HO (Delauney and Verma 1993, Matysik et al. 2002). However, environmental stressors
Physiol. Plant. 156, 2016
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MV stress tolerance of transgenic AtGSTU19 Arabidopsis plants
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higher than that in the WT, whereas the MDA content of the former was lower than the latter. We also measured the activities of some antioxidant enzymes (Fig. 4) and found no significant difference between transgenic and WT plants under normal conditions. Under salt/drought stress condition, the SOD and POD activities increased in both transgenic and WT plants. However, SOD and POD activities in transgenic lines were significantly higher than in the WT plants during NaCl and mannitol stresses. These results suggest that AtGSTU19 strengthens the cellular ROS scavenging ability and maintains ROS homeostasis by increasing proline content and the activities of antioxidant enzymes.
salt stress
drought stress
a b b b
4
a
Given that GST is involved in anti-oxidation processes in plants and animals, we analyzed the anti-oxidation capacity of transgenic plants. Seeds were germinated on MS medium containing different MV concentrations, and the percentage of cotyledon emergence was compared between transgenic plants and WT. Increased cotyledon emergence was observed for transgenic plants when these plants were grown on MS medium containing MV. Photographs were taken at 7 days after stratification (Fig. 5A). In the medium containing 0.5 μM MV, the cotyledon emergence rate of WT was 71%, and the mean rate of transgenic lines was approximately 86%. When the MV concentration reached 1.5 μM, the cotyledon emergence rate decreased to 20% in WT, and the mean rate of transgenic lines was approximately 65% (Fig. 5B). To analyze the MV tolerance, 2-week-old T3 seedlings grown on MS solid medium were transferred aseptically into MS liquid medium and grown with rotary shaking (60 rpm) for recovery. After 2 days, the liquid medium was refreshed with new one supplemented with different MV concentration. Photographs were taken at 10 days
b b b
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Fig. 3. Legend in next column.
cause a rapid and excessive accumulation of ROS in plant cells, which leads to a lipid peroxidation chain reaction further, and the result is the accumulation of MDA. MDA is the principal by-product of lipid oxidation (Bartels 2001, Zhu 2001). In our study, we found that the proline content in transgenic OE lines was significantly
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Fig. 3. Salt/drought stress tolerance of transgenic plants. (A) Three-week-old plants of OE lines and WT were given salt or drought stress treatment. Control, 3-week-old plants growing under normal conditions; salt stress, plants watered with NaCl (250 mM); drought stress, plants withheld watering for 2 weeks, and then irrigated again. The experiment was replicated three times with similar results and 48 plants per line for each experiment. (B) Survival rate of OE lines and WT under stress treatment as described in (A). Values are means ± SD (n = 3), each from 48 plants. (C) Standardized water content of OE lines and WT. Each data point is mean ± SD (n = 3), each from ≥10 plants. (D) Changes in the content of proline and MDA between OE lines and WT treated with salt stress (NaCl 250 mM) or drought stress (mannitol 300 mM) for 4 days. Values are means ± SD (n = 3), each from 10 plants. Different letters above bars indicate significant differences (P < 0.05) among different genotypes in the same treatment.
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Fig. 4. Antioxidant enzyme activities in Arabidopsis plants expressing AtGSTU19. Antioxidant enzyme activities were measured in leaves of 3-week-old plants. Salt stress was performed with NaCl (250 mM), drought stress was performed with mannitol (300 mM) and oxidative stress was performed with MV (10 μM) for 4 days. Values are means ± SD (n = 3) and different letters above bars indicate significant differences (P < 0.05) among different genotypes in the same treatment.
after exposure to stress (Fig. 6A). The seedlings of WT and OE lines grown in the liquid medium without MV appeared normal. This result indicated that toxic effects were caused by MV stress and not the result of submersion in the liquid medium. The dry weights of WT and OE lines were nearly the same without MV stress. However, the growth of WT plants was inhibited when MV stress was induced (Fig. 6B). When WT plants were treated with 1.5 μM MV, the growth of WT plants was significantly inhibited, the chlorophyll content decreased, and the leaves turned chlorotic. The relative dry weight rates of WT and OE lines were 32 and 52%, respectively. When MV concentration reached 2 μM, the growth of both WT and transgenic plants was inhibited, and the leaves turned 170
Fig. 5. Comparative analysis of transgenic lines over-expressing AtGSTU19 on MV stress condition. (A) Comparison of seedling growth between transgenic lines and WT grown on MS medium containing different concentration of MV. (B) Comparison of cotyledon emergency rate between transgenic lines and WT seeds growth on the medium as mentioned in (A). Each value is the mean ± SD of at least 50 seedlings (n = 3). Different letters above bars indicate significant differences (P < 0.05) among different genotypes in the same treatment.
chlorotic. The relative dry weight rates were 9 and 20% in WT and transgenic lines, respectively. When MV concentration reached 3 μM, both WT and transgenic plants died (data not shown). These results showed that transgenic plants exhibited higher tolerance to MV stress than WT plants. The antioxidant enzymes were also detected under MV stress condition (Fig. 4). Under stress conditions, the SOD and POD activities increased in both transgenic and WT plants. However, SOD and POD activities in transgenic lines were significantly higher than in the WT plants under MV stress. The enzyme profiles under MV stress were similar to those observed under salt/drought stress. GST activity was also detected under stress treatment. Transgenic plants exhibited higher level of GST
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WT
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0.01
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Fig. 6. MV stress tolerance of transgenic plants. (A) Twenty seedlings grown in MS solid medium for 2 weeks were transferred to MS liquid medium in flasks containing 0, 1.5 and 2 μM MV. (B) Dry weights of seedlings in the individual flasks in the above treatment. Values are means ± SD (n = 3) and different letters above bars indicate significant differences (P < 0.05) among different genotypes in the same treatment.
activity under salt, drought and MV stress conditions (Fig. 6). These results suggested that AtGSTU19 increases GST activity, thereby resulting in increased abiotic stress resistance. Together, the over-expression of AtGSTU19 enhanced the tolerance to salt, drought and MV stresses in Arabidopsis. AtGSTU19 was suggested to play an important role in the adaptation to at least three distinct stress agents (salt, drought and MV). Expression of stress-inducible genes in transgenic AtGSTU19 plants The hallmark of stress adaptation in plants is the induction of numerous stress-responsive genes (Shinozaki and Yamaguchi-Shinozaki 1997, Zhu et al. 1997). To further elucidate the role of AtGSTU19 in stress tolerance, we examined the effects of AtGSTU19 on the transcript levels of several stress-inducible genes, including RD29A and RD29B (Yamaguchi-Shinozaki and Shinozaki 1994), RD22 (Yamaguchi-Shinozaki and Shinozaki 1993), KIN1 (Wang and Cutler 1995), COR15A (Baker et al. 1994) and COR47 (Hajela et al. 1990). We analyzed the
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expression of these genes under salt stress and dehydration, given the similarities of mechanisms underlying plant adaptation to water deficit caused by high salinity and drought (Fig. 7A). Moreover, PRX (Sharma and Davis 1994, Ezaki et al. 2000), an oxidative stress marker induced by ozone or aluminum treatments, was analyzed under MV stress (Fig. 7B). Under normal conditions, the expression levels of RD29A, KIN1 and COR15A were significantly higher in a transgenic OE line than in WT (Fig. 7A). Under salt/drought stress treatments, the expression levels of all of these stress-responsive genes, except for COR47 and RD22, were upregulated in the WT. The expression of KIN1 was upregulated only under drought stress. Moreover, the expressions of all these stress-responsive genes, except for RD22 and COR47, were significantly higher in transgenic lines than in WT (Fig. 7A). Under MV stress treatment, the expression of PRX was upregulated in WT and was significantly higher in transgenic plants than in WT (Fig. 7B). Together, transgenic plants over-expressing AtGSTU19 significantly enhanced the expression levels of all stress-induced genes except for RD22 and COR47.
Discussion GSTs are known to protect plants against oxidative stress induced by biotic and abiotic agents through detoxification of endogenous cytotoxic compounds, which accumulate during oxidative stress. In plants, GST expression is induced in many different stress situations (Wagner et al. 2002). Previous studies have shown that AtGSTU19 (first cloned as GST8) transcripts were induced by drought stress, oxidative stress and high doses of auxin or cytokinin (Bianchi et al. 2002). In this study, we focused on the function of AtGSTU19 in abiotic stress and extend it as playing a positive role in drought, salt and oxidative stress tolerance. Functional analysis on the role of AtGSTU19 in stress tolerance of Arabidopsis was carried out using transgenic OE lines. The phenomena of increased germination, root growth and the percentage of cotyledon emergence were observed under stress treatment (Figs 2A, B and 5B). In plants, GST expression is induced by phytohormones. Jain et al. (2010) analyzed the expression pattern of 74 GST genes in response to different hormones and the results showed that a large number of GSTs are responsive to hormones. Gong et al. (2005) also demonstrated the role of GST in plant growth and development in vivo and shoot morphogenesis in vitro. The expression of AtGSTU19 was induced by indole acetic acid (IAA) and other hormones (Bianchi et al. 2002), and seed germination rate increased in transgenic plants under stress. 171
A
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Relative gene expression
B 10
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6 b 4 2
a
a
a
a
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Fig. 7. Stress-induced gene expression. (A) Salt and drought-induced gene expression in transgenic and wild-type plants assayed by real-time PCR. Control 3-week-old plants growing under normal conditions, salt stress plants watered with NaCl (250 mM), drought stress plants watered with mannitol (300 mM) for 6 h. Values are mean ± SD of three replicates. (B) Oxidative-induced gene expression in transgenic and wild-type plants assayed by real-time PCR. Three-week-old plants grown in pots were given MV (10 μM) for 6 h as oxidative stress. Different letters above bars indicate significant differences (P < 0.05) among different genotypes in the same treatment.
These observations showed that AtGSTU19 could participate in plant growth and development. The production of ROS is a common reaction when plants are exposed to abiotic stresses (Yang et al. 2014). Therefore, ROS homeostasis is important in protecting the normal metabolism of organisms, and plants can regulate ROS levels through ROS scavenging via antioxidant enzymes, such as SOD, POD and GST (Jiang et al. 2007). SODs catalyze the dismutation of superoxide into oxygen and H2 O2 and constitute the first line of defense against ROS in a cell (Alscher et al. 2002). In turn, various forms of peroxidases are responsible for the 172
removal of H2 O2 from biological systems (Sergio et al. 2012). Moreover, over-expression of some GST genes has been shown to improve oxidative stress tolerance in transgenic plants (Mittler 2002). Besides, proline in plant cells plays a protective role in adaptation to water deprivation (Handa et al. 1986) and protects plant cells against oxidative damage by quenching 1 O2 and directly scavenging HO (Matysik et al. 2002). In our study, the transgenic plants over-expressing AtGSTU19 have higher levels of antioxidant enzyme activities (SOD, POD and GST), higher proline content and lower MDA content than WT plants. These results suggested
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that AtGSTU19 may act as a stress regulator though increasing the activities of antioxidant enzymes to strengthen the ROS scavenging activity or maintain ROS homeostasis. Synthesis of various osmoprotectants, such as proline, detoxification enzymes and various proteases is one of the strategies for protecting plants against stresses directly (Shinozaki and Yamaguchi-Shinozaki 2007). Another one involves the regulation of gene expression and signal transduction pathways (Kaur and Gupta 2005). Many stress-inducible genes contain recognizing motifs in their promoters and function in response to abiotic stresses (Shinozaki et al. 2003). Thus, the expression of some stress-regulated genes was examined to investigate the stress resistance further. Transgenic plants over-expressing AtGSTU19 activated the expression of some late stress-response genes, such as RD29A, COR15A and KIN1, even under normal growth conditions. The three genes were sensitive to various abiotic stressors and were induced by dehydration, high salinity or low temperature (Wang et al. 1995, Msanne et al. 2011, Liu et al. 2014). Our studies showed that plants over-expressing AtGSTU19 may activate stress response pathways to a certain degree under normal conditions. Under stress conditions, the expression levels of some stress-responsive genes in transgenic OE plants increased more substantially than those in WT. RD29B was sensitively induced by dehydration, high salinity or low temperature (Msanne et al. 2011). The expression of PRX gene was dependent on light, ascorbate and oxidative stress (Horling et al. 2003). The results demonstrated that the stress-response pathway was activated further. However, further studies should be performed to understand the role of phytohormones in plant stress responses and how the presence of AtGSTU19 gene enhances the expression of other stress-responsive genes. Taken together, transgenic plants over-expressing AtGSTU19 enhanced the salt/drought/MV stress tolerance (Figs 3A, 5A and 6A). Another tau GST, AtGSTU17, was reported as a negative component of the stressmediated signal transduction pathway in adaptive responses to drought and salt stress through accumulating GSH and ABA and exhibiting hyposensitivity to ABA in atgstu17 (Chen et al. 2012). As a multigene family, GSTs share very little overall amino acid sequence identity, typically no more than 25–35% (Marrs 1996), which suggests that the function of GSTs is complicated and multiplexed. Both tau GSTs, AtGSTU17 and AtGSTU19, were grouped into different tau clades (Dixon and Edwards 2010), which suggested that they could have different functions. It partially explains the differences in results between Chen’s findings (Chen et al. 2012) and ours.
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Acknowledgements – The research study was sponsored by Shanghai Rising-Star Program (Grant No. 14QB140 3400), Shanghai Natural Science Foundation (Grant No. 13ZR1460600), National Natural Science Foundation (Grant Nos 31300237, 31401458) and Youth Talents Growth Plan of Shanghai Academy of Agricultural Sciences (Grant No. 2014-1-19, 2015-1-26).
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