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The Arabidopsis APR2 positively regulates cadmium tolerance through glutathione-dependent pathway
Ecotoxicology and Environmental Safety 187 (2020) 109819
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The Arabidopsis APR2 positively regulates cadmium tolerance through glutathione-dependent pathway
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Ziwei Xua, Meiping Wangb, Dongliang Xua, Zongliang Xiaa,∗ a b
College of Life Science, Henan Agricultural University, Zhengzhou, 450002, China Library of Henan Agricultural University, Zhengzhou, 450002, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Adenosine 5′-phosphosulfate reductase Cadmium stress Sulfate assimilation Glutathione Arabidopsis
Cadmium (Cd) is a dangerous environmental pollutant with high toxicity to plants. The adenosine 5′-phosphosulfate reductase 2 (APR2) is the dominant APRs in Arabidopsis and plays an important role in reductive sulfate assimilation pathway. However, whether the involvement of plant APRs in Cd stress response is largely unclear. Herein, we report that APR2 functions in Cd accumulation and tolerance in Arabidopsis. The transcript levels of APR2 were markedly induced by Cd exposure. Transgenic plants overexpressing APR2 improved Cd tolerance, whereas knockout of APR2 reduced Cd tolerance. APR2-overexpressing plants with increased Cd accumulation and tolerance showed higher glutathione (GSH) and phytochelatin (PC) levels than the wild type and apr2 mutant plants, but lower H2O2 and TBARS contents upon Cd exposure. Moreover, exogenous GSH application effectively rescued Cd hypersensitivity in APR2-knockout plants. Further analysis showed that buthionine sulfoximine (BSO, an inhibitor of GSH synthesis) treatment completely eliminated the enhanced Cd tolerance phenotypes of APR2-overexpressing plants, implying that APR2-mediated enhanced Cd tolerance is GSH dependent. In addition, over-expression of the APR2 led to elevated expressions of the GSH/PC synthesisrelated genes under Cd stress. Taken together, our results indicated that APR2 regulated Cd accumulation and tolerance possibly through modulating GSH-dependent antioxidant capability and Cd-chelation machinery in Arabidopsis. APR2 could be exploited for engineering heavy metal-tolerant plants in phytoremediation.
1. Introduction Cadmium (Cd) is one of the most dangerous environmental pollutants with high toxicity to plants (Lin and Aarts, 2012; Clemens et al., 2013; Åkesson et al., 2014). Cd causes cellular damage by inactivating some enzymes, cofactors, and regulatory proteins in plants (Lin and Aarts, 2012). In China, it is estimated that approximately 7% of the soil is Cd contaminated (Zhang et al., 2015). Thus, Cd toxicity has become an important factor restricting crop production. The most effective way to solve this problem is to breed Cd-tolerant varieties. However, this requires a comprehensive understanding of the underlying mechanisms of plant tolerance to Cd. Over the past two decades, several potential mechanisms of plant tolerance to Cd have been reported (Clemens, 2001; Lin and Aarts, 2012; Hou et al., 2019). First, detoxification of Cd in plants. This mechanism includes Cd-pump out at the plasma membrane, Cd-chelation by thiol compounds in the cytosol, and Cd-sequestration into vacuoles (Clemens, 2001; Kim et al., 2006). Second, antioxidant and signaling machinery. This involves roles of signal molecules nitric oxide (NO) and
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reactive oxygen species (ROS) in plant tolerance to Cd (Clemens, 2001). Third, a number of key genes encoding metal transporters and transcription factors are involved in Cd tolerance in plants (Lee et al., 2003; Kim et al., 2006; Lin and Aarts, 2012; Chen et al., 2016; Sheng et al., 2019). In higher plants, the sulfate assimilation pathway is carried out through highly coordinated mechanisms. Sulfate is first adsorbed by sulfate transporters, then activated by ATP sulfurylase to adenosine-5phosphosulfate (APS), and then reduced to sulfite by APS reductase (APR). The toxic intermediate sulfite is further reduced to sulfides, which are then incorporated into cysteine and other sulfur-containing amino acids, such as glutathione (GSH). GSH is a major sulfur-containing metabolite essential for Cd detoxification through the GSH- or phytochelatin (PC)-conjugated vacuolar sequestration mechanism in plants (Zhu et al., 1999; Cobbett and Goldsbrough, 2002; Lee et al., 2003; Lin and Aarts, 2012; Flores-Cáceres et al., 2015; Jozefczak et al., 2012, 2015; Hernández et al., 2015; Han et al., 2019). In Arabidopsis there are three isoenzymes of APR (APR1, 2, and 3), of which APR2 is the major one in the sulfate reduction, as revealed
Corresponding author. College of life science, Henan Agricultural University, Zhengzhou, 450002, China. E-mail address: [email protected] (Z. Xia).
https://doi.org/10.1016/j.ecoenv.2019.109819 Received 23 July 2019; Received in revised form 25 September 2019; Accepted 13 October 2019 0147-6513/ © 2019 Elsevier Inc. All rights reserved.
Ecotoxicology and Environmental Safety 187 (2020) 109819
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that knockout of the APR2 reduced by 80% of total APR activity (Martin et al., 2005; Loudet et al., 2007). Moreover, impairment of the APR2 did not show growth defects, indicating that both APR1 and APR3 are sufficient for growth and development of plants (Loudet et al., 2007). However, it was evidenced that transcripts of these three APR isoforms showed differential expression patterns under various stresses such as salinity, nitrogen status, and light (Kopriva et al., 1999; Koprivova et al., 2000, 2008), implying that each of the APR isoenzymes may have different role in development and stress responses (Kopriva et al., 2009). However, whether plant APR isoenzymes participate in Cd detoxification is unknown. In the present study, we have evidenced that the APR2 functions in Cd accumulation and tolerance in Arabidopsis. 2. Materials and methods 2.1. Plant cultivation The ecotype Columbia-0 (Col-0) of Arabidopsis thaliana was used as wild type (WT) in this study. Seeds were surface sterilized and sown on agar plates containing 1/2 Murashige and Skoog (MS) medium. After stratification in the dark at 4 °C for three days, the seeds were transferred to a growth chamber for germination (150 μmol m−2 s−1 PAR, 16 h light/8 h dark cycle, 22 °C). After seven days, the seedlings were transplanted into sterile, low-nutrient soils to obtain fully grown plants. Plants were cultivated in a growth room as described previously (Xia et al., 2016). 2.2. Quantitative real-time PCR analysis Quantitative real-time PCR (qPCR) was used to determine transcript levels of APR1, APR2, APR3, GSH1, GSH2, PCS1 and PCS2 genes. Total RNA extraction, first-strand cDNA synthesis and qPCR were carried out following our previous protocols (Xia et al., 2016). The primer sequences were listed in Table S1. The Arabidopsis Actin2 was used as an internal control to quantify the relative expression level of each target gene as described previously (Xia et al., 2016). Each sample was performed in three replicates. 2.3. Verification of APR2 insertion mutant The APR2 (At1g62180) T-DNA insertion mutant (apr2-1) seeds were obtained from the GABI-KAT (Rosso et al., 2003). Homozygous insertion mutants in the coding region of the gene were identified by PCR from genomic DNA using corresponding primers (P1, LBb1, and P2) (Table S1). The insertion site of the T-DNA in the gene was verified by DNA sequencing. The APR2 transcript abundance in the WT and apr2-1 mutant were checked by RT-PCR.
Fig. 1. The transcript profiles of APR genes in Arabidopsis plants under Cd stress. Changes in transcript levels of APR1 (A), APR2 (B), and APR3 (C) at various time points in response to Cd exposure in Arabidopsis plants. Two-weekold plants were exposed to 60 μM CdCl2 and sampled at 0, 6, 12, 24, 36, 48 and 60 h. Leaf samples were collected for qPCR analysis. In these assays, Actin2 was used as an internal control. For each experiment, three technical replicates were conducted. Data shown are Mean ± SE of three independent experiments. **ttest, with P < 0.01; *t-test, with P < 0.05.
2.4. Vector construction and development of over-expression transgenic lines
seven days, then the seedlings were transferred to grow vertically on 1/ 2 MS solid medium supplemented with 0 (Control), 30, or 60 μM CdCl2 for 10 days, and growth performance of each genotype was recorded. After 10 days of the stress, fresh weight and primary root length of different genotypes of Arabidopsis seedlings were documented. The whole experiment was conducted in triplicate.
The open reading frame (ORF) sequence of the APR2 was amplified using primers APR2-F and APR2-R (Table S1) and cloned into the binary vector pWM101 to generate pWM101-35S:APR2. The expression construct was introduced into Agrobacterium tumefaciens GV3101 strain and transformed into Arabidopsis via floral dipping (Clough and Bent, 1998). Transformed lines were screened by antibiotic resistance and confirmed by PCR. Homozygous lines with single-site insertion were identified and maintained until T3 generation. Homozygous T3 lines were used for further experiments.
2.6. Determination of thiobarbituric acid reactive substances (TBARS) and H2O2 contents The Arabidopsis leaves from three to five individuals were used for the determination of TBARS and H2O2 contents. The TBARS contents were measured as described by us (Huo et al., 2019; Wang et al., 2016). The H2O2 content was assayed according to our previous method (Xia et al., 2012). For TBARS measurement, leaf samples (0.25 g) were homogenized with 1 mL of trichloroacetic acid (5%, w/v). After
2.5. Tolerance analysis of APR2 mutant, overexpression, and wild type Arabidopsis plants under Cd stress The WT, overexpression lines (OE-3, -8, and -23), and apr2-1 Arabidopsis seeds were germinated and grown in 1/2 MS medium for 2
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Fig. 2. Molecular identification and expression levels of Arabidopsis APR2 knockout mutant and overexpression lines. (A) Structure of the APR2 locus with the T-DNA insertion site in apr2-1 mutant. The insertion site is marked by a white arrow, exons are indicated as white boxes, and untranslated regions by black boxes. (B) Transcript levels of APR2 in the Col0 and apr2-1 mutant determined by RT-PCR. (C) Transcript levels of APR2 in the WT, apr2-1, and three overexpression lines (named OE-3, -8, and -23) determined by qPCR. (D) Total APR activity in leaf extracts from WT, apr2-1, and overexpression lines. In (C) and (D) histograms above, values are mean ± SE. The asterisks indicate significance of the difference from the corresponding control values determined by Student's t-test (**t-test, with P < 0.01; *t-test, with P < 0.05).
Fig. 3. Responses of APR2 mutant, overexpression, and wild type plants to Cd stress. (A) Effect of Cd stress on WT, apr2 mutant, APR2-OE seedlings growth. One-week-old WT, overexpression lines (OE-3, -8, and -23), and apr2-1 Arabidopsis seedlings were grown vertically on MS agar plates supplemented with 0 (Control), 30, or 60 μM CdCl2 for 10 days, and then the growth status of each genotype was documented. (B) Effect of Cd stress on primary root length of different genotypes of Arabidopsis seedlings after 10 days of stress. (C) Effect of Cd stress on fresh weight of different genotypes of Arabidopsis seedlings after 10 days of stress. In both Figures B and C, values are mean ± SE, n = 20. Error bars represent standard deviations. **t-test, with P < 0.01; *t-test, with P < 0.05.
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Fig. 4. Cd contents in the medium and different genotypes of Arabidopsis plants. (A) Cd contents in roots and shoots of the WT, apr2 mutant, APR2-OE lines upon Cd exposure. (B) Cd contents in the media for each genotype of plants. One-week-old seedlings from WT, apr2 mutant, APR2-OE lines were grown on MS media with 60 μM CdCl2 for 10 days, and roots and shoots of plants were collected respectively for Cd content measurements. Meanwhile, the media for each genotype of plants was also sampled for Cd content measurements. Data are presented as means ± SE of three replicate experiments. Values are mean ± SE. **t-test, with P < 0.01; *t-test, with P < 0.05.
Fig. 5. Changes of MDA and H2O2 in APR2 mutant, overexpression lines and wild type plants under Cd stress. (C) Determination of MDA accumulation in leaves of WT, apr2 mutant, APR2-OE lines upon Cd exposure. (D) Quantitative determination of H2O2 accumulation in leaves of WT, apr2 mutant, APR2-OE lines upon Cd exposure. In both (A) and (B), two-week-old WT, overexpression lines (OE-3, -8, and -23), and apr2-1 Arabidopsis seedlings were grown vertically on MS agar plates supplemented with 0 (Control), or 60 μM CdCl2 for 24 h, and then MDA and H2O2 contents were determined. The experiment was repeated twice with similar results. Bar indicates SE. Values are mean ± SE. **t-test, with P < 0.01; *t-test, with P < 0.05.
centrifugation at 10, 000 g for 15 min, the supernatant was mixed with the reaction solution, and the absorbance of the resulting solution was recorded at 530 nm. In the case of H2O2 assay, leaves (0.25 g) were homogenized with 1 mL of phosphate buffer (50 mM, pH 7.0). The absorbance of the reaction mixture was measured at 415 nm.
3. Results 3.1. Transcript profiles of three Arabidopsis APR genes during Cd stress
2.7. Determination of APR activity, Cd, reduced glutathione (GSH), and phytochelatin (PC) contents
Time-course analysis of three Arabidopsis APR (APR1, APR2, and APR3) transcript levels under Cd stress was conducted by qPCR (Fig. 1). Upon Cd stress, the transcripts of both APR1 and APR3 decreased significantly and kept lower levels until the end of the stress (Fig. 1A and C). In contrast, the APR2 expression increased significantly after 12 h, and reached a peak at 24 h (about 3-fold increase), and then gradually decreased, finally maintained higher levels during 60 h of the Cd treatment (Fig. 1B). These data suggest that the three APR genes showed differential response patterns under Cd stress. Moreover, the APR2 expression was induced by Cd stress.
Arabidopsis leaf proteins were extracted and protein concentration was determined as described previously (Wang et al., 2016). The assay solution contained 0.5 mM Sulfate, 100 mM EDTA, 10 mM APS, 50 mM GSH, and 100 mM Tris-acetate buffer. APR activity was determined in the presence of APS as a substrate and GSH as an electron donor according to the assay of Brychkova et al. (2012). Cd content in roots and shoots of Arabidopsis seedlings and media was measured using an atomic absorption spectrometer (Z5000, HITACHI) as described by Lee et al. (2003). GSH/PC were extracted from Arabidopsis seedlings and measured according to our previous methods (Chen et al., 2016; Huang et al., 2018).
3.2. Responses of APR2 knockout and overexpression Arabidopsis plants to Cd stress To explore the role of APR2 in Cd stress, the APR2 T-DNA insertion mutant (apr2-1) was obtained from the GABI-KAT (Rosso et al., 2003). The T-DNA insertion position is illustrated in Fig. 2A, and homozygous mutant in the coding region of the gene was identified by PCR, and further verified by DNA sequencing. Semi-quantitative PCR assays revealed that no APR2 mRNA abundance was detected in the apr2-1 compared to WT plants with the same age (Fig. 2B). In addition, several
2.8. Statistical analysis Statistical analysis was performed by SPSS and Excel, and data were plotted using GraphPad Prism (v. 5.01, GraphPad Software Inc., CA, USA).
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the apr2-1 mutant exhibited lower APR activity (30% of the WT level) (Fig. 2D). Thus, these three OE lines, along with the WT and the apr2-1, were used for phenotypic analyses. The APR2 OE lines (OE-3, -8, and -23), apr2-1 mutant, and WT seedlings were examined for their responses to Cd stress by investigating seedling growth. When grown vertically on the medium with 0 (Control), 30, or 60 μM CdCl2 for 10 days, the apr2-1 mutant seedlings showed much higher chlorosis than WT and OE lines (Fig. 3A). This was clearly seen upon the 60 μM Cd exposure (Fig. 3A). The root growth inhibition of the three OE seedlings was less than those of the WT and apr2-1 mutant either under 30 or 60 μM Cd stress (Fig. 3B). Accordingly, data from root length and fresh weight also exhibited significant differences among the apr2 mutant, WT, and OE lines under 30 or 60 μM of Cd stress (Fig. 3B and C). These results evidenced that APR2 overexpression significantly improved Cd tolerance, whereas impairment of APR2 decreases the stress tolerance in Arabidopsis. 3.3. Cd contents in the APR2 knockout and overexpression Arabidopsis plants upon Cd exposure To examine whether APR2-mediated Cd tolerance is associated with alternations in Cd accumulation, Cd contents in the WT, apr2-1, and APR2-OE seedlings upon Cd exposure was measured. As shown in Fig. 4A, compared to the WT, Cd content in roots significantly increased in the APR2-OE lines (~18% in OE-3, ~23% in OE-8 and ~20% in OE23), but decreased in the apr2-1 mutant plants upon Cd exposure (~35% in apr2-1) (Fig. 4A). Similarly, in shoots, compared to the WT, these three APR2-OE lines had significant increases in Cd content, whereas the apr2-1 mutant showed a marked reduction (Fig. 4A). Meanwhile, Cd content in the media was also measured. As shown in Fig. 4B, there is no significant difference in Cd content in the medium for all the genotypes of plants, except for the apr2 mutant (Fig. 4B). These results suggest that increased expression of APR2 accelerates Cd accumulation in plants.
Fig. 6. Glutathione and phytochelatin levels in the wild type, APR2 mutant, and overexpression plants upon Cd exposure. Contents of GSH (A) and phytochelatin (PC) (B) from two-week-old Arabidopsis plants were measured 24 h after Cd (60 μM) treatment. Each experiment was repeated three times. Bar indicates SE. Values are mean ± SE. **t-test, with P < 0.01; *t-test, with P < 0.05.
APR2-overexpressing Arabidopsis were generated. A few homozygous overexpression (OE) lines (T3 generation) were obtained, of which three lines (OE-3, -8, and -23) exhibited high levels of transgene expression as determined by qPCR (Fig. 2C). Accordingly, these OE lines showed higher levels of APR activity (1.5–1.6 folds of the WT level), whereas
3.4. TBARS and H2O2 accumulations in APR2 knockout and overexpression Arabidopsis plants under Cd stress The lipid peroxidation product, TBARS were measured among the Fig. 7. Effect of exogenous GSH on response of the wild type and APR2 mutant plants to Cd stress. (A) Representative growth phenotypes of WT and mutant plants when exposed to Cd and Cd + GSH. Seven days old seedlings of WT and apr2-1 were vertically growing on MS medium supplemented with 0 (Control), Cd (60 μM), and Cd (60 μM) plus GSH (10 mg/L) for 10 days. (B) Primary root length of WT and mutant seedlings after 10-day Cd stress. Values are mean ± SE, n = 15. *t-test, with P < 0.05. (C) Relative residual chlorophyll (%) in the WT and mutant plants after Cd and Cd plus GSH treatments. Values are mean ± SE, n = 10. *t-test, with P < 0.05.
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Fig. 8. Effect of BSO on response of the wild type and APR2 overexpression plants to Cd stress. (A) Representative growth phenotypes of WT and overexpression plants when exposed to Cd and Cd + BSO. Nine-day-old seedlings of WT and APR2-OE lines (OE-3 and OE-8) were vertically growing on MS medium supplemented with 0 (Control), Cd (60 μM), and Cd (60 μM) plus BSO (a GSH synthesis inhibitor, 0.3 mM) for seven days. (B) Primary root length of WT and APR2-OE seedlings after 7-day Cd stress. Values are mean ± SE, n = 10. *t-test, with P < 0.05. (C) Relative residual chlorophyll (%) in the WT and APR2-OE plants after Cd and Cd plus BSO treatments. Values are mean ± SE, n = 9. *t-test, with P < 0.05.
apr2 mutant, OE lines and WT plants under 60 μM of Cd stress. After 24h Cd stress, the TBARS levels in the mutant, OE lines, or WT plants markedly increased compared with their corresponding controls (Fig. 5A). However, there were differential magnitudes of increase in these types of plants. For example, levels of the TBARS in the three OE lines (109%, 83%, and 128% increases for OE-3, -8, and -23, respectively) were significantly lower than in the WT (192% increase) and the apr2 mutant (228% increase), indicating that knockout of the APR2 plants suffered more membrane damage than the WT, but APR2 OE plants significantly alleviated the damage (Fig. 5A). Under Cd stress the APR2 knockout plants accumulated higher TBARS, indicating that their oxidation damage may be more serious than the WT. Thus, hydrogen peroxide (H2O2) levels were detected in the mutant, OE and WT plants under Cd stress. As shown in Fig. 5B, H2O2 content clearly increased in the mutant, OE, and WT plants after 24-h Cd stress. However, the OE lines accumulated lower levels of H2O2 (95% increase on average) compared to WT (160% increase) and apr2 mutant (180% increase) after Cd stress (Fig. 5B). These physiological indices indicated that the different levels of ROS accumulation and lipid peroxidation in the apr2 mutant and OE lines could be related to their different tolerance to Cd stress.
reduction) compare to the WT (Fig. 6B). Additionally, it was worth mentioning that GSH levels in these OE lines had significant increases compared to those in the WT under control conditions, but not in the apr2 mutant (Fig. 6A), indicating that increased APR2 expression led to more GSH production. These data demonstrate that APR2 positively regulates Cd tolerance through the GSH-dependent PC synthesis pathway that confers Cd tolerate of plants.
3.6. APR2 mediated Cd tolerance through GSH-dependent pathway In the apr2 mutant plants, Cd-induced chlorophyll loss and cellular damage may be largely due to insufficient GSH levels (Figs. 3 and 6). To test this notion, 7-day-old seedlings from WT and apr2-1 mutant plants were treated with Cd or Cd plus GSH for 10 days. As shown in Fig. 7A, Cd exposure caused marked chlorosis and root growth inhibition that were clearly alleviated in the application of exogenous GSH in both WT and apr2-1 mutant plants (Fig. 7A). Correspondingly, determinations of root length and remaining chlorophyll confirmed these observations (Fig. 7B and C). This demonstrated that Cd hypersensitivity of APR2 knockout mutant was reversed by exogenous GSH. We next examined the growth phenotypes of the WT and APR2-OE plants in MS media supplemented with buthionine sulfoximine (BSO), an inhibitor of GSH synthesis (Kim et al., 2006). As shown in Fig. 8A, when grown in the Cd-containing media, both APR2-OE lines showed increased Cd tolerance than the WT (Fig. 8A, middle lane); however, when BSO was added to the Cd-containing media, the improved growth phenotypes of the APR2-OE plants over WT plants to Cd stress disappeared, and both WT and OE lines showed severe chlorosis and root growth inhibition (Fig. 8A, right lane). This phenomenon was also evidenced as parameters of root length and remaining chlorophyll of these genotypes of plants (Fig. 8B and C). These results supported the view that APR2 mediated Cd tolerance through the GSH-dependent pathway.
3.5. Glutathione and phytochelatin levels in APR2 knockout and overexpression Arabidopsis plants under Cd stress To understand effects of APR2 knockout or overexpression on glutathione and phytochelatin (PC) during Cd stress, reduced gluthione (GSH) and PC contents were measured under Cd stress. As shown in Fig. 5, Cd stress resulted in significant increases in either GSH or PC levels in the WT, apr2 mutant, and OE lines (Fig. 6A and B). For changes in the GSH levels, the three OE lines showed more increases (70%, 90%, and 75% increases for OE-3, OE-8, and OE-23, respectively) than WT (48% increase) and apr2 mutant (40% increase) plants under Cd stress (Fig. 6A). Correspondingly, there were significant increases in total PC content were detected among these three OE lines (95% increase on average), while there was a decrease in PC content in apr2 mutant (27% 6
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Fig. 9. Expression of GSH/PC synthesis-related genes in the APR2 mutant and overexpression lines in response to Cd exposure. (A) Scheme of GSH/PC synthesis and genes involved in this process. (B) Quantitative analysis of transcription of genes involved in GSH/PC synthesis in the apr2 mutant and APR2-OE lines, including GSH1, GSH2, PCS1, and PCS2. The wild type, apr2 mutant, and APR2-OE lines were grown on MS media for 2 weeks, and treated with 60 μM CdCl2 for 24 h, and then their RNAs were isolated for RT-qPCR analysis. Actin2 was used as internal control. Data are presented as means ± SE of three replicate experiments. **t-test, with P < 0.01; *t-test, with P < 0.05.
4. Discussion
3.7. Expression of GSH/PC synthesis-related genes in the APR2 mutant and overexpression lines upon Cd exposure
Plants can convert inorganic sulfate to reduced sulfur via the reductive sulfate assimilation pathway, in which several key enzymes such as APRs can eventually incorporate sulfate into cysteine, a precusor of glutathione (Kopriva and Koprivova, 2004). In our study, our genetic evidence suggests that the major APR isoform APR2 confers Cd tolerance through the GSH-dependent Cd-chelation mechanism in Arabidopsis. The three APR genes showed differential response patterns in Arabidopsis plants under Cd stress, in which APR2 transcripts were intensely induced by Cd stress (Fig. 1). APR2-overexpressing plants improved Cd tolerance but apr2 knockout mutant increased sensitivity to Cd stress upon Cd exposure (Fig. 3), implying that APR2 plays a pivotal part in Cd tolerance. Furthermore, less accumulations in TBARS and
The transcripts of glutathione synthesis-related enzymes GSH synthetase (GSH1 and GSH2) (Noctor et al., 2002; Jozefczak et al., 2012), phytochelatin synthase (PCS1 and PCS2) were examined by qPCR in the WT, APR2 mutant and overexpression lines upon Cd exposure (Fig. 9A). After Cd stress for 24 h, the transcripts of the four genes were elevated among the WT, apr2 mutant and OE lines (Fig. 9B). Particularly, increased transcripts of both GSH2 and PCS2 were quite evident in the OE lines compared to those in the WT upon Cd exposure (Fig. 9B). This indicates that APR2 coordinately regulates the transcription of GSH and PC synthesis genes under Cd stress.
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Author contributions
H2O2, hallmarks of oxidative stress, were observed in the OE lines compared to the WT or apr2 mutant plants upon Cd stress (Fig. 5A and B). Further analysis demonstrated that a greater increase in GSH level was detected in the OE lines, but not in the WT and apr2 mutant plants either under normal conditions or Cd stress (Fig. 6A). This result suggested that APR2 expression directly affected GSH levels. As we know, GSH is a major thiol-containing metabolite and has a role in maintaining redox homeostasis during Cd stress (Jozefczak et al., 2015; Hernández et al., 2015). Our present result suggested that amounts of the GSH were influenced by the APR2 levels and in return, the GSH levels affected Cd stress responses of the APR2 OE and knockout mutant plants (Figs. 6A, 7 and 8). Compared with WT or apr2 mutant, the less accumulations of H2O2 in the OE plants may be a consequence of the higher GSH levels, which enhanced the ROS scavenging capacity. In support of our observation, the Arabidopsis apr2 knockout mutant (apr2-1) decreased tolerance to selenite toxicity due to glutathione deficiency, and thereby resulting in enhanced ROS levels (Grant et al., 2011). Thus, APR2 likely regulates Cd stress tolerance by affecting GSH-dependent antioxidant system that scavenged ROS and decreased membrane damage. As a precursor of the PCs, GSH functions in heavy metal detoxification in plants (Cobbett and Goldsbrough, 2002). It was reported that Arabidopsis GSH- deficient cad2-1 mutant was hypersensitive to Cd (Cobbett et al., 1998), whereas GSH1-overexpression plants with higher GSH levels enhanced Cd tolerance (Zhu et al., 1999). Therefore, it is concluded that the GSH-dependent Cd-chelation is the major mechanism contributing to APR2-mediated Cd tolerance in our study. PCs are synthesized by GSH1, GSH2, PCS1, and PCS2, and act in Cd detoxification in Arabidopsis (May et al., 1998; Cobbett and Goldsbrough, 2002; Noctor et al., 2002; Semane et al., 2007; Jozefczak et al., 2012; Lin and Aarts, 2012; Wang et al., 2018). Our data have demonstrated that the APR2 OE lines showed much more amounts of GSH and PC than the WT and apr2 mutant plants under Cd stress (Fig. 5A and B). Correspondingly, over-expression of the APR2 results in elevated transcripts of GSH1, GSH2, PCS1, and PCS2 (Fig. 9B), indicating that the increased levels of GSH and PC in the APR2 OE lines could be due to the increased APR2 coupled to elevated GSH/PC synthesis-related gene expression under Cd stress, and thereby effectively improving Cd tolerance. In support of this viewpoint, glutathione metabolic genes have been found to be involved in response to heavy metals (Xiang and Oliver, 1998). For example, overexpression of GSH1, PCS1, or PCS2 effectively improved plant tolerance to Cd stress (Zhu et al., 1999; Cazalé and Clemens, 2001; Pomponi et al., 2006; Wawrzynski et al., 2006; Gasic and Korban, 2007; Brunetti et al., 2011). However, the exact mechanism underlying APR2-regulated expression of the GSH/PC synthesis-related genes upon Cd exposure needs further study. In summary, we have identified a major Arabidopsis APR isoenzyme (APR2), whose expression was induced by Cd stress. Transgenic plants overexpressing APR2 improved Cd tolerance, whereas knockout of APR2 reduced Cd tolerance. APR2-overexpressing plants with increased Cd accumulation and tolerance showed higher glutathione (GSH) and phytochelatin (PC) levels than the wild type and apr2 mutant plants, but lower H2O2 and TBARS contents upon Cd exposure. Moreover, APR2-mediated enhanced Cd tolerance is GSH dependent. In addition, over-expression of the APR2 led to elevated expressions of the GSH/PC synthesis-related genes under Cd stress. Thus, we concluded that APR2 regulated Cd accumulation and tolerance possibly through affecting GSH-dependent antioxidant capability and Cd-chelation machinery in Arabidopsis. This study may facilitate our understanding of the molecular mechanism underlying APR2-mediated Cd detoxification in higher plants. In future work, it is necessary to dissect the exact mechanism by which the GSH-dependent pathway is involved in APR2mediated Cd stress response in Arabidopsis.
ZXia designed the research, ZXu, MW, DX, and ZXia performed research and conducted data analyses. ZXia wrote the manuscript. Acknowledgments This work was financially supported by the project of Innovative Talents of Science and Technology of He'nan Educational Committee (16HASTIT021). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ecoenv.2019.109819. References Åkesson, A., Barregard, L., Bergdahl, I.A., Nordberg, G.F., Nordberg, M., Skerfving, S., 2014. Non-renal effects and the risk assessment of environmental cadmium exposure. Environ. Health Perspect. 122, 431–438. Brunetti, P., Zanella, L., Proia, A., De Paolis, A., Falasca, G., Altamura, M.M., Sanità di Toppi, L., Costantino, P., Cardarelli, M., 2011. Cadmium tolerance and phytochelatin content of Arabidopsis seedlings overexpressing the phytochelatin synthase gene AtPCS1. J. Exp. Bot. 62, 5509–5519. Brychkova, G., Yarmolinsky, D., Sagi, M., 2012. Kinetic assays for determining in vitro APS reductase activity in plants without the use of radioactive substances. Plant Cell Physiol. 53, 1648–1658. Cazalé, A.C., Clemens, S., 2001. Arabidopsis thaliana expresses a second functional phytochelatin synthase. FEBS Lett. 507, 215–219. Chen, J., Yang, L., Yan, X., Liu, Y., Wang, R., Fan, T., Ren, Y., Tang, X., Xiao, F., Liu, Y., Cao, S., 2016. Zinc-finger transcription factor ZAT6 positively regulates cadmium tolerance through the glutathione-dependent pathway in Arabidopsis. Plant Physiol 171, 707–719. Clemens, S., 2001. Molecular mechanisms of plant metal tolerance and homeostasis. Planta 212, 475–486. Clemens, S., Aarts, M.G.M., Thomine, S., Verbruggen, N., 2013. Plant science: the key to preventing slow cadmium poisoning. Trends Plant Sci. 18, 92–99. Clough, S.J., Bent, A.F., 1998. Floral dip: a simplified method for Agrobaterium -mediated transformation of Arabidopsis thaliana. Plant J. 16, 735–743. Cobbett, C., Goldsbrough, P., 2002. Phytochelatins and metallothioneins: roles in heavy metal detoxification and homeostasis. Annu. Rev. Plant Biol. 53, 159–182. Cobbett, C.S., MayMJ, Howden R., Rolls, B., 1998. The glutathione-deficient, cadmiumsensitive mutant, cad2-1, of Arabidopsis thaliana is deficient in gamma-glutamylcysteine synthetase. Plant J. 16, 73–78. Flores-Cáceres, M.L., Hattab, S., Hattab, S., Boussetta, H., Banni, M., Hernández, L.E., 2015. Specific mechanisms of tolerance to copper and cadmium are compromised by a limited concentration of glutathione in alfalfa plants. Plant Sci. 233, 165–173. Gasic, K., Korban, S.S., 2007. Transgenic Indian mustard (Brassica juncea) plants expressing an Arabidopsis phytochelatin synthase (AtPCS1) exhibit enhanced as and Cd tolerance. Plant Mol. Biol. 64, 361–369. Grant, K., Carey, N.M., Mendoza, M., Schulze, J., Pilon, M., Pilon-Smits, E.A., van Hoewyk, D., 2011. Adenosine 5'-phosphosulfate reductase (APR2) mutation in Arabidopsis implicates glutathione deficiency in selenate toxicity. Biochem. J. 438, 325–335. Han, Y., Fan, T., Zhu, X., Wu, X., Ouyang, J., Jiang, L., Cao, S., 2019. WRKY12 represses GSH1 expression to negatively regulate cadmium tolerance in Arabidopsis. Plant Mol. Biol. 99, 149–159. Hernández, L.E., Sobrino-Plata, J., Montero-Palmero, M.B., Carrasco-Gil, S., FloresCáceres, M.L., Ortega-Villasante, C., Escobar, C., 2015. Contribution of glutathione to the control of cellular redox homeostasis under toxic metal and metalloid stress. J. Exp. Bot. 66, 2901–2911. Hou, X., Tan, L., Tang, S.F., 2019. Molecular mechanism study on the interactions of cadmium (II) ions with Arabidopsis thaliana glutathione transferase Phi8. Spectrochim. Acta A Mol. Biomol. Spectrosc. 216, 411–417. Huang, Q., Wang, M., Xia, Z., 2018. The SULTR gene family in maize (Zea mays L.): gene cloning and expression analyses under sulfate starvation and abiotic stress. J. Plant Physiol. 220, 24–33. Jozefczak, M., Bohler, S., Schat, H., Horemans, N., Guisez, Y., Remans, T., Vangronsveld, J., Cuypers, A., 2015. Both the concentration and redox state of glutathione and ascorbate influence the sensitivity of Arabidopsis to cadmium. Ann Bot (Lond). 116, 601–612. Jozefczak, M., Remans, T., Vangronsveld, J., Cuypers, A., 2012. Glutathione is a key player in metal-induced oxidative stress defenses. Int. J. Mol. Sci. 13, 3145–3175. Kim, D.Y., Bovet, L., Kushnir, S., Noh, E.W., Martinoia, E., Lee, Y., 2006. AtATM3 is involved in heavy metal resistance in Arabidopsis. Plant Physiol 140, 922–932. Kopriva, S., Koprivova, A., 2004. Plant adenosine 5'-phosphosulphate reductase: the past, the present, and the future. J. Exp. Bot. 55, 1775–1783. Kopriva, S., Mugford, S.G., Matthewman, C., Koprivova, A., 2009. Plant sulfate assimilation genes: redundancy versus specialization. Plant Cell Rep. 28, 1769–1780.
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reverse genetics. Plant Mol. Biol. 53, 247–259. Semane, B., Cuypers, A., Smeets, K., Opdenakker, K., Keunen, E., Remans, T., Horemans, N., Vanhoudt, N., et al., 2007. Cadmium responses in Arabidopsis thaliana: glutathione metabolism and antioxidative defence system. Physiol. Plant. 129, 519–528. Sheng, Y., Yan, X., Huang, Y., Han, Y., Zhang, C., Ren, Y., Fan, T., Xiao, F., Liu, Y., Cao, S., 2019. The WRKY transcription factor, WRKY13, activates PDR8 expression to positively regulate cadmium tolerance in Arabidopsis. Plant Cell Environ. 42, 891–903. Wang, K., Tang, S.F., Hou, X., 2018. Molecular mechanism investigation on the interactions of copper (II) ions with glutathione peroxidase 6 from Arabidopsis thaliana. Spectrochim. Acta A Mol. Biomol. Spectrosc. 203, 428–433. Wang, M., Jia, Y., Xu, Z., Xia, Z., 2016. Impairment of sulfite reductase decreases oxidative stress tolerance in Arabidopsis thaliana. Front. Plant Sci. 7, 1843. Wawrzynski, A., Kopera, E., Wawrzy_nska, A., Kaminska, J., Bal, W., Sirko, A., 2006. Effects of simultaneous expression of heterologous genes involved in phytochelatin biosynthesis on thiol content and cadmium accumulation in tobacco plants. J. Exp. Bot. 57, 2173–2182. Xia, Z., Huo, Y., Wei, Y., Chen, Q., Xu, Z., Zhang, W., 2016. The Arabidopsis LYST INTERACTING PROTEIN 5 acts in regulating abscisic acid signaling and drought response. Front. Plant Sci. 7, 758. Xia, Z., Sun, K., Wang, M., Wu, K., Zhang, H., 2012. Overexpression of a maize sulfite oxidase gene in tobacco enhances tolerance to sulfite stress via sulfite oxidation and CAT-mediated H2O2 scavenging. PLoS One 7, e37383. Xiang, C., Oliver, D.J., 1998. Glutathione metabolic genes coordinately respond to heavy metals and jasmonic acid in Arabidopsis. Plant Cell 10, 1539–1550. Zhang, X.Y., Zhong, T.Y., Liu, L., Ouyang, X.Y., 2015. Impact of soil heavy metal pollution on food safety in China. PLoS One 8, e0135182. Zhu, Y.L., Pilon-Smits, E.A., Tarun, A.S., Weber, S.U., Jouanin, L., Terry, N., 1999. Cadmium tolerance and accumulation in Indian mustard is enhanced by overexpressing Ƴ-glutamylcysteine synthetase. Plant Physiol 121, 1169–1178.
Kopriva, S., Muheim, R., Koprivova, A., Trachsel, N., Catalano, C., Suter, M., Brunold, C., 1999. Light regulation of assimilatory sulfate reduction in Arabidopsis thaliana. Plant J. 20, 37–44. Koprivova, A., North, K.A., Kopriva, S., 2008. Complex signaling network in regulation of adenosine 5-phosphosulfate reductase by salt stress in Arabidopsis roots. Plant Physiol 146, 1408–1420. Koprivova, A., Suter, M., Op den Camp, R., Brunold, C., Kopriva, S., 2000. Regulation of sulfate assimilation by nitrogen in Arabidopsis. Plant Physiol 122, 737–746. Lee, S., Moon, J.S., Ko, T.S., Petros, D., Goldsbrough, P.B., Korban, S.S., 2003. Overexpression of Arabidopsis phytochelatin synthase paradoxically leads to hypersensitivity to cadmium stress. Plant Physiol 131, 656–663. Lin, Y.F., Aarts, M.G., 2012. The molecular mechanism of zinc and cadmium stress response in plants. Cell. Mol. Life Sci. 69, 3187–3206. Loudet, O., Saliba-Colombani, V., Camilleri, C., Calenge, F., Gaudon, V., Koprivova, A., North, K.A., Kopriva, S., Daniel-Vedele, F., 2007. Natural variation for sulfate content in Arabidopsis thaliana is highly controlled by APR2. Nat. Genet. 39, 896–900. Martin, M.N., Tarczynski, M.C., Shen, B., Leustek, T., 2005. The role of 5-adenylylsulfate reductase in controlling sulfate reduction in plants. Photosynth. Res. 86, 1–15. May, M.J., Vernoux, T., Leaver, C.J., Van Montagu, M., Inzé, D., 1998. Glutathione homeostasis in plants: implications for environmental sensing and plant development. J. Exp. Bot. 49, 649–667. Noctor, G., Gomez, L., Vanacker, H., Foyer, C.H., 2002. Interactions between biosynthesis, compartmentation and transport in the control of glutathione homeostasis and signaling. J. Exp. Bot. 53, 1283–1304. Pomponi, M., Censi, V., Di Girolamo, V., Paolis, A.D., Toppi, L.S., Aromolo, R., Costantino, P., Cardarelli, M., 2006. Overexpression of Arabidopsis phytochelatin synthase in tobacco plants enhances Cd21 tolerance and accumulation but not translocation to the shoot. Planta 223, 180–190. Rosso, M.G., Li, Y., Strizhov, N., Reiss, B., Dekker, K., Weisshaar, B., 2003. An Arabidopsis thaliana T-DNA mutagenized population (GABI-KAT) for flanking sequence tag-based
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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
The Arabidopsis APR2 positively regulates cadmium tolerance through glutathione-dependent pathway
T
Ziwei Xua, Meiping Wangb, Dongliang Xua, Zongliang Xiaa,∗ a b
College of Life Science, Henan Agricultural University, Zhengzhou, 450002, China Library of Henan Agricultural University, Zhengzhou, 450002, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Adenosine 5′-phosphosulfate reductase Cadmium stress Sulfate assimilation Glutathione Arabidopsis
Cadmium (Cd) is a dangerous environmental pollutant with high toxicity to plants. The adenosine 5′-phosphosulfate reductase 2 (APR2) is the dominant APRs in Arabidopsis and plays an important role in reductive sulfate assimilation pathway. However, whether the involvement of plant APRs in Cd stress response is largely unclear. Herein, we report that APR2 functions in Cd accumulation and tolerance in Arabidopsis. The transcript levels of APR2 were markedly induced by Cd exposure. Transgenic plants overexpressing APR2 improved Cd tolerance, whereas knockout of APR2 reduced Cd tolerance. APR2-overexpressing plants with increased Cd accumulation and tolerance showed higher glutathione (GSH) and phytochelatin (PC) levels than the wild type and apr2 mutant plants, but lower H2O2 and TBARS contents upon Cd exposure. Moreover, exogenous GSH application effectively rescued Cd hypersensitivity in APR2-knockout plants. Further analysis showed that buthionine sulfoximine (BSO, an inhibitor of GSH synthesis) treatment completely eliminated the enhanced Cd tolerance phenotypes of APR2-overexpressing plants, implying that APR2-mediated enhanced Cd tolerance is GSH dependent. In addition, over-expression of the APR2 led to elevated expressions of the GSH/PC synthesisrelated genes under Cd stress. Taken together, our results indicated that APR2 regulated Cd accumulation and tolerance possibly through modulating GSH-dependent antioxidant capability and Cd-chelation machinery in Arabidopsis. APR2 could be exploited for engineering heavy metal-tolerant plants in phytoremediation.
1. Introduction Cadmium (Cd) is one of the most dangerous environmental pollutants with high toxicity to plants (Lin and Aarts, 2012; Clemens et al., 2013; Åkesson et al., 2014). Cd causes cellular damage by inactivating some enzymes, cofactors, and regulatory proteins in plants (Lin and Aarts, 2012). In China, it is estimated that approximately 7% of the soil is Cd contaminated (Zhang et al., 2015). Thus, Cd toxicity has become an important factor restricting crop production. The most effective way to solve this problem is to breed Cd-tolerant varieties. However, this requires a comprehensive understanding of the underlying mechanisms of plant tolerance to Cd. Over the past two decades, several potential mechanisms of plant tolerance to Cd have been reported (Clemens, 2001; Lin and Aarts, 2012; Hou et al., 2019). First, detoxification of Cd in plants. This mechanism includes Cd-pump out at the plasma membrane, Cd-chelation by thiol compounds in the cytosol, and Cd-sequestration into vacuoles (Clemens, 2001; Kim et al., 2006). Second, antioxidant and signaling machinery. This involves roles of signal molecules nitric oxide (NO) and
∗
reactive oxygen species (ROS) in plant tolerance to Cd (Clemens, 2001). Third, a number of key genes encoding metal transporters and transcription factors are involved in Cd tolerance in plants (Lee et al., 2003; Kim et al., 2006; Lin and Aarts, 2012; Chen et al., 2016; Sheng et al., 2019). In higher plants, the sulfate assimilation pathway is carried out through highly coordinated mechanisms. Sulfate is first adsorbed by sulfate transporters, then activated by ATP sulfurylase to adenosine-5phosphosulfate (APS), and then reduced to sulfite by APS reductase (APR). The toxic intermediate sulfite is further reduced to sulfides, which are then incorporated into cysteine and other sulfur-containing amino acids, such as glutathione (GSH). GSH is a major sulfur-containing metabolite essential for Cd detoxification through the GSH- or phytochelatin (PC)-conjugated vacuolar sequestration mechanism in plants (Zhu et al., 1999; Cobbett and Goldsbrough, 2002; Lee et al., 2003; Lin and Aarts, 2012; Flores-Cáceres et al., 2015; Jozefczak et al., 2012, 2015; Hernández et al., 2015; Han et al., 2019). In Arabidopsis there are three isoenzymes of APR (APR1, 2, and 3), of which APR2 is the major one in the sulfate reduction, as revealed
Corresponding author. College of life science, Henan Agricultural University, Zhengzhou, 450002, China. E-mail address: [email protected] (Z. Xia).
https://doi.org/10.1016/j.ecoenv.2019.109819 Received 23 July 2019; Received in revised form 25 September 2019; Accepted 13 October 2019 0147-6513/ © 2019 Elsevier Inc. All rights reserved.
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that knockout of the APR2 reduced by 80% of total APR activity (Martin et al., 2005; Loudet et al., 2007). Moreover, impairment of the APR2 did not show growth defects, indicating that both APR1 and APR3 are sufficient for growth and development of plants (Loudet et al., 2007). However, it was evidenced that transcripts of these three APR isoforms showed differential expression patterns under various stresses such as salinity, nitrogen status, and light (Kopriva et al., 1999; Koprivova et al., 2000, 2008), implying that each of the APR isoenzymes may have different role in development and stress responses (Kopriva et al., 2009). However, whether plant APR isoenzymes participate in Cd detoxification is unknown. In the present study, we have evidenced that the APR2 functions in Cd accumulation and tolerance in Arabidopsis. 2. Materials and methods 2.1. Plant cultivation The ecotype Columbia-0 (Col-0) of Arabidopsis thaliana was used as wild type (WT) in this study. Seeds were surface sterilized and sown on agar plates containing 1/2 Murashige and Skoog (MS) medium. After stratification in the dark at 4 °C for three days, the seeds were transferred to a growth chamber for germination (150 μmol m−2 s−1 PAR, 16 h light/8 h dark cycle, 22 °C). After seven days, the seedlings were transplanted into sterile, low-nutrient soils to obtain fully grown plants. Plants were cultivated in a growth room as described previously (Xia et al., 2016). 2.2. Quantitative real-time PCR analysis Quantitative real-time PCR (qPCR) was used to determine transcript levels of APR1, APR2, APR3, GSH1, GSH2, PCS1 and PCS2 genes. Total RNA extraction, first-strand cDNA synthesis and qPCR were carried out following our previous protocols (Xia et al., 2016). The primer sequences were listed in Table S1. The Arabidopsis Actin2 was used as an internal control to quantify the relative expression level of each target gene as described previously (Xia et al., 2016). Each sample was performed in three replicates. 2.3. Verification of APR2 insertion mutant The APR2 (At1g62180) T-DNA insertion mutant (apr2-1) seeds were obtained from the GABI-KAT (Rosso et al., 2003). Homozygous insertion mutants in the coding region of the gene were identified by PCR from genomic DNA using corresponding primers (P1, LBb1, and P2) (Table S1). The insertion site of the T-DNA in the gene was verified by DNA sequencing. The APR2 transcript abundance in the WT and apr2-1 mutant were checked by RT-PCR.
Fig. 1. The transcript profiles of APR genes in Arabidopsis plants under Cd stress. Changes in transcript levels of APR1 (A), APR2 (B), and APR3 (C) at various time points in response to Cd exposure in Arabidopsis plants. Two-weekold plants were exposed to 60 μM CdCl2 and sampled at 0, 6, 12, 24, 36, 48 and 60 h. Leaf samples were collected for qPCR analysis. In these assays, Actin2 was used as an internal control. For each experiment, three technical replicates were conducted. Data shown are Mean ± SE of three independent experiments. **ttest, with P < 0.01; *t-test, with P < 0.05.
2.4. Vector construction and development of over-expression transgenic lines
seven days, then the seedlings were transferred to grow vertically on 1/ 2 MS solid medium supplemented with 0 (Control), 30, or 60 μM CdCl2 for 10 days, and growth performance of each genotype was recorded. After 10 days of the stress, fresh weight and primary root length of different genotypes of Arabidopsis seedlings were documented. The whole experiment was conducted in triplicate.
The open reading frame (ORF) sequence of the APR2 was amplified using primers APR2-F and APR2-R (Table S1) and cloned into the binary vector pWM101 to generate pWM101-35S:APR2. The expression construct was introduced into Agrobacterium tumefaciens GV3101 strain and transformed into Arabidopsis via floral dipping (Clough and Bent, 1998). Transformed lines were screened by antibiotic resistance and confirmed by PCR. Homozygous lines with single-site insertion were identified and maintained until T3 generation. Homozygous T3 lines were used for further experiments.
2.6. Determination of thiobarbituric acid reactive substances (TBARS) and H2O2 contents The Arabidopsis leaves from three to five individuals were used for the determination of TBARS and H2O2 contents. The TBARS contents were measured as described by us (Huo et al., 2019; Wang et al., 2016). The H2O2 content was assayed according to our previous method (Xia et al., 2012). For TBARS measurement, leaf samples (0.25 g) were homogenized with 1 mL of trichloroacetic acid (5%, w/v). After
2.5. Tolerance analysis of APR2 mutant, overexpression, and wild type Arabidopsis plants under Cd stress The WT, overexpression lines (OE-3, -8, and -23), and apr2-1 Arabidopsis seeds were germinated and grown in 1/2 MS medium for 2
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Fig. 2. Molecular identification and expression levels of Arabidopsis APR2 knockout mutant and overexpression lines. (A) Structure of the APR2 locus with the T-DNA insertion site in apr2-1 mutant. The insertion site is marked by a white arrow, exons are indicated as white boxes, and untranslated regions by black boxes. (B) Transcript levels of APR2 in the Col0 and apr2-1 mutant determined by RT-PCR. (C) Transcript levels of APR2 in the WT, apr2-1, and three overexpression lines (named OE-3, -8, and -23) determined by qPCR. (D) Total APR activity in leaf extracts from WT, apr2-1, and overexpression lines. In (C) and (D) histograms above, values are mean ± SE. The asterisks indicate significance of the difference from the corresponding control values determined by Student's t-test (**t-test, with P < 0.01; *t-test, with P < 0.05).
Fig. 3. Responses of APR2 mutant, overexpression, and wild type plants to Cd stress. (A) Effect of Cd stress on WT, apr2 mutant, APR2-OE seedlings growth. One-week-old WT, overexpression lines (OE-3, -8, and -23), and apr2-1 Arabidopsis seedlings were grown vertically on MS agar plates supplemented with 0 (Control), 30, or 60 μM CdCl2 for 10 days, and then the growth status of each genotype was documented. (B) Effect of Cd stress on primary root length of different genotypes of Arabidopsis seedlings after 10 days of stress. (C) Effect of Cd stress on fresh weight of different genotypes of Arabidopsis seedlings after 10 days of stress. In both Figures B and C, values are mean ± SE, n = 20. Error bars represent standard deviations. **t-test, with P < 0.01; *t-test, with P < 0.05.
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Fig. 4. Cd contents in the medium and different genotypes of Arabidopsis plants. (A) Cd contents in roots and shoots of the WT, apr2 mutant, APR2-OE lines upon Cd exposure. (B) Cd contents in the media for each genotype of plants. One-week-old seedlings from WT, apr2 mutant, APR2-OE lines were grown on MS media with 60 μM CdCl2 for 10 days, and roots and shoots of plants were collected respectively for Cd content measurements. Meanwhile, the media for each genotype of plants was also sampled for Cd content measurements. Data are presented as means ± SE of three replicate experiments. Values are mean ± SE. **t-test, with P < 0.01; *t-test, with P < 0.05.
Fig. 5. Changes of MDA and H2O2 in APR2 mutant, overexpression lines and wild type plants under Cd stress. (C) Determination of MDA accumulation in leaves of WT, apr2 mutant, APR2-OE lines upon Cd exposure. (D) Quantitative determination of H2O2 accumulation in leaves of WT, apr2 mutant, APR2-OE lines upon Cd exposure. In both (A) and (B), two-week-old WT, overexpression lines (OE-3, -8, and -23), and apr2-1 Arabidopsis seedlings were grown vertically on MS agar plates supplemented with 0 (Control), or 60 μM CdCl2 for 24 h, and then MDA and H2O2 contents were determined. The experiment was repeated twice with similar results. Bar indicates SE. Values are mean ± SE. **t-test, with P < 0.01; *t-test, with P < 0.05.
centrifugation at 10, 000 g for 15 min, the supernatant was mixed with the reaction solution, and the absorbance of the resulting solution was recorded at 530 nm. In the case of H2O2 assay, leaves (0.25 g) were homogenized with 1 mL of phosphate buffer (50 mM, pH 7.0). The absorbance of the reaction mixture was measured at 415 nm.
3. Results 3.1. Transcript profiles of three Arabidopsis APR genes during Cd stress
2.7. Determination of APR activity, Cd, reduced glutathione (GSH), and phytochelatin (PC) contents
Time-course analysis of three Arabidopsis APR (APR1, APR2, and APR3) transcript levels under Cd stress was conducted by qPCR (Fig. 1). Upon Cd stress, the transcripts of both APR1 and APR3 decreased significantly and kept lower levels until the end of the stress (Fig. 1A and C). In contrast, the APR2 expression increased significantly after 12 h, and reached a peak at 24 h (about 3-fold increase), and then gradually decreased, finally maintained higher levels during 60 h of the Cd treatment (Fig. 1B). These data suggest that the three APR genes showed differential response patterns under Cd stress. Moreover, the APR2 expression was induced by Cd stress.
Arabidopsis leaf proteins were extracted and protein concentration was determined as described previously (Wang et al., 2016). The assay solution contained 0.5 mM Sulfate, 100 mM EDTA, 10 mM APS, 50 mM GSH, and 100 mM Tris-acetate buffer. APR activity was determined in the presence of APS as a substrate and GSH as an electron donor according to the assay of Brychkova et al. (2012). Cd content in roots and shoots of Arabidopsis seedlings and media was measured using an atomic absorption spectrometer (Z5000, HITACHI) as described by Lee et al. (2003). GSH/PC were extracted from Arabidopsis seedlings and measured according to our previous methods (Chen et al., 2016; Huang et al., 2018).
3.2. Responses of APR2 knockout and overexpression Arabidopsis plants to Cd stress To explore the role of APR2 in Cd stress, the APR2 T-DNA insertion mutant (apr2-1) was obtained from the GABI-KAT (Rosso et al., 2003). The T-DNA insertion position is illustrated in Fig. 2A, and homozygous mutant in the coding region of the gene was identified by PCR, and further verified by DNA sequencing. Semi-quantitative PCR assays revealed that no APR2 mRNA abundance was detected in the apr2-1 compared to WT plants with the same age (Fig. 2B). In addition, several
2.8. Statistical analysis Statistical analysis was performed by SPSS and Excel, and data were plotted using GraphPad Prism (v. 5.01, GraphPad Software Inc., CA, USA).
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the apr2-1 mutant exhibited lower APR activity (30% of the WT level) (Fig. 2D). Thus, these three OE lines, along with the WT and the apr2-1, were used for phenotypic analyses. The APR2 OE lines (OE-3, -8, and -23), apr2-1 mutant, and WT seedlings were examined for their responses to Cd stress by investigating seedling growth. When grown vertically on the medium with 0 (Control), 30, or 60 μM CdCl2 for 10 days, the apr2-1 mutant seedlings showed much higher chlorosis than WT and OE lines (Fig. 3A). This was clearly seen upon the 60 μM Cd exposure (Fig. 3A). The root growth inhibition of the three OE seedlings was less than those of the WT and apr2-1 mutant either under 30 or 60 μM Cd stress (Fig. 3B). Accordingly, data from root length and fresh weight also exhibited significant differences among the apr2 mutant, WT, and OE lines under 30 or 60 μM of Cd stress (Fig. 3B and C). These results evidenced that APR2 overexpression significantly improved Cd tolerance, whereas impairment of APR2 decreases the stress tolerance in Arabidopsis. 3.3. Cd contents in the APR2 knockout and overexpression Arabidopsis plants upon Cd exposure To examine whether APR2-mediated Cd tolerance is associated with alternations in Cd accumulation, Cd contents in the WT, apr2-1, and APR2-OE seedlings upon Cd exposure was measured. As shown in Fig. 4A, compared to the WT, Cd content in roots significantly increased in the APR2-OE lines (~18% in OE-3, ~23% in OE-8 and ~20% in OE23), but decreased in the apr2-1 mutant plants upon Cd exposure (~35% in apr2-1) (Fig. 4A). Similarly, in shoots, compared to the WT, these three APR2-OE lines had significant increases in Cd content, whereas the apr2-1 mutant showed a marked reduction (Fig. 4A). Meanwhile, Cd content in the media was also measured. As shown in Fig. 4B, there is no significant difference in Cd content in the medium for all the genotypes of plants, except for the apr2 mutant (Fig. 4B). These results suggest that increased expression of APR2 accelerates Cd accumulation in plants.
Fig. 6. Glutathione and phytochelatin levels in the wild type, APR2 mutant, and overexpression plants upon Cd exposure. Contents of GSH (A) and phytochelatin (PC) (B) from two-week-old Arabidopsis plants were measured 24 h after Cd (60 μM) treatment. Each experiment was repeated three times. Bar indicates SE. Values are mean ± SE. **t-test, with P < 0.01; *t-test, with P < 0.05.
APR2-overexpressing Arabidopsis were generated. A few homozygous overexpression (OE) lines (T3 generation) were obtained, of which three lines (OE-3, -8, and -23) exhibited high levels of transgene expression as determined by qPCR (Fig. 2C). Accordingly, these OE lines showed higher levels of APR activity (1.5–1.6 folds of the WT level), whereas
3.4. TBARS and H2O2 accumulations in APR2 knockout and overexpression Arabidopsis plants under Cd stress The lipid peroxidation product, TBARS were measured among the Fig. 7. Effect of exogenous GSH on response of the wild type and APR2 mutant plants to Cd stress. (A) Representative growth phenotypes of WT and mutant plants when exposed to Cd and Cd + GSH. Seven days old seedlings of WT and apr2-1 were vertically growing on MS medium supplemented with 0 (Control), Cd (60 μM), and Cd (60 μM) plus GSH (10 mg/L) for 10 days. (B) Primary root length of WT and mutant seedlings after 10-day Cd stress. Values are mean ± SE, n = 15. *t-test, with P < 0.05. (C) Relative residual chlorophyll (%) in the WT and mutant plants after Cd and Cd plus GSH treatments. Values are mean ± SE, n = 10. *t-test, with P < 0.05.
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Fig. 8. Effect of BSO on response of the wild type and APR2 overexpression plants to Cd stress. (A) Representative growth phenotypes of WT and overexpression plants when exposed to Cd and Cd + BSO. Nine-day-old seedlings of WT and APR2-OE lines (OE-3 and OE-8) were vertically growing on MS medium supplemented with 0 (Control), Cd (60 μM), and Cd (60 μM) plus BSO (a GSH synthesis inhibitor, 0.3 mM) for seven days. (B) Primary root length of WT and APR2-OE seedlings after 7-day Cd stress. Values are mean ± SE, n = 10. *t-test, with P < 0.05. (C) Relative residual chlorophyll (%) in the WT and APR2-OE plants after Cd and Cd plus BSO treatments. Values are mean ± SE, n = 9. *t-test, with P < 0.05.
apr2 mutant, OE lines and WT plants under 60 μM of Cd stress. After 24h Cd stress, the TBARS levels in the mutant, OE lines, or WT plants markedly increased compared with their corresponding controls (Fig. 5A). However, there were differential magnitudes of increase in these types of plants. For example, levels of the TBARS in the three OE lines (109%, 83%, and 128% increases for OE-3, -8, and -23, respectively) were significantly lower than in the WT (192% increase) and the apr2 mutant (228% increase), indicating that knockout of the APR2 plants suffered more membrane damage than the WT, but APR2 OE plants significantly alleviated the damage (Fig. 5A). Under Cd stress the APR2 knockout plants accumulated higher TBARS, indicating that their oxidation damage may be more serious than the WT. Thus, hydrogen peroxide (H2O2) levels were detected in the mutant, OE and WT plants under Cd stress. As shown in Fig. 5B, H2O2 content clearly increased in the mutant, OE, and WT plants after 24-h Cd stress. However, the OE lines accumulated lower levels of H2O2 (95% increase on average) compared to WT (160% increase) and apr2 mutant (180% increase) after Cd stress (Fig. 5B). These physiological indices indicated that the different levels of ROS accumulation and lipid peroxidation in the apr2 mutant and OE lines could be related to their different tolerance to Cd stress.
reduction) compare to the WT (Fig. 6B). Additionally, it was worth mentioning that GSH levels in these OE lines had significant increases compared to those in the WT under control conditions, but not in the apr2 mutant (Fig. 6A), indicating that increased APR2 expression led to more GSH production. These data demonstrate that APR2 positively regulates Cd tolerance through the GSH-dependent PC synthesis pathway that confers Cd tolerate of plants.
3.6. APR2 mediated Cd tolerance through GSH-dependent pathway In the apr2 mutant plants, Cd-induced chlorophyll loss and cellular damage may be largely due to insufficient GSH levels (Figs. 3 and 6). To test this notion, 7-day-old seedlings from WT and apr2-1 mutant plants were treated with Cd or Cd plus GSH for 10 days. As shown in Fig. 7A, Cd exposure caused marked chlorosis and root growth inhibition that were clearly alleviated in the application of exogenous GSH in both WT and apr2-1 mutant plants (Fig. 7A). Correspondingly, determinations of root length and remaining chlorophyll confirmed these observations (Fig. 7B and C). This demonstrated that Cd hypersensitivity of APR2 knockout mutant was reversed by exogenous GSH. We next examined the growth phenotypes of the WT and APR2-OE plants in MS media supplemented with buthionine sulfoximine (BSO), an inhibitor of GSH synthesis (Kim et al., 2006). As shown in Fig. 8A, when grown in the Cd-containing media, both APR2-OE lines showed increased Cd tolerance than the WT (Fig. 8A, middle lane); however, when BSO was added to the Cd-containing media, the improved growth phenotypes of the APR2-OE plants over WT plants to Cd stress disappeared, and both WT and OE lines showed severe chlorosis and root growth inhibition (Fig. 8A, right lane). This phenomenon was also evidenced as parameters of root length and remaining chlorophyll of these genotypes of plants (Fig. 8B and C). These results supported the view that APR2 mediated Cd tolerance through the GSH-dependent pathway.
3.5. Glutathione and phytochelatin levels in APR2 knockout and overexpression Arabidopsis plants under Cd stress To understand effects of APR2 knockout or overexpression on glutathione and phytochelatin (PC) during Cd stress, reduced gluthione (GSH) and PC contents were measured under Cd stress. As shown in Fig. 5, Cd stress resulted in significant increases in either GSH or PC levels in the WT, apr2 mutant, and OE lines (Fig. 6A and B). For changes in the GSH levels, the three OE lines showed more increases (70%, 90%, and 75% increases for OE-3, OE-8, and OE-23, respectively) than WT (48% increase) and apr2 mutant (40% increase) plants under Cd stress (Fig. 6A). Correspondingly, there were significant increases in total PC content were detected among these three OE lines (95% increase on average), while there was a decrease in PC content in apr2 mutant (27% 6
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Fig. 9. Expression of GSH/PC synthesis-related genes in the APR2 mutant and overexpression lines in response to Cd exposure. (A) Scheme of GSH/PC synthesis and genes involved in this process. (B) Quantitative analysis of transcription of genes involved in GSH/PC synthesis in the apr2 mutant and APR2-OE lines, including GSH1, GSH2, PCS1, and PCS2. The wild type, apr2 mutant, and APR2-OE lines were grown on MS media for 2 weeks, and treated with 60 μM CdCl2 for 24 h, and then their RNAs were isolated for RT-qPCR analysis. Actin2 was used as internal control. Data are presented as means ± SE of three replicate experiments. **t-test, with P < 0.01; *t-test, with P < 0.05.
4. Discussion
3.7. Expression of GSH/PC synthesis-related genes in the APR2 mutant and overexpression lines upon Cd exposure
Plants can convert inorganic sulfate to reduced sulfur via the reductive sulfate assimilation pathway, in which several key enzymes such as APRs can eventually incorporate sulfate into cysteine, a precusor of glutathione (Kopriva and Koprivova, 2004). In our study, our genetic evidence suggests that the major APR isoform APR2 confers Cd tolerance through the GSH-dependent Cd-chelation mechanism in Arabidopsis. The three APR genes showed differential response patterns in Arabidopsis plants under Cd stress, in which APR2 transcripts were intensely induced by Cd stress (Fig. 1). APR2-overexpressing plants improved Cd tolerance but apr2 knockout mutant increased sensitivity to Cd stress upon Cd exposure (Fig. 3), implying that APR2 plays a pivotal part in Cd tolerance. Furthermore, less accumulations in TBARS and
The transcripts of glutathione synthesis-related enzymes GSH synthetase (GSH1 and GSH2) (Noctor et al., 2002; Jozefczak et al., 2012), phytochelatin synthase (PCS1 and PCS2) were examined by qPCR in the WT, APR2 mutant and overexpression lines upon Cd exposure (Fig. 9A). After Cd stress for 24 h, the transcripts of the four genes were elevated among the WT, apr2 mutant and OE lines (Fig. 9B). Particularly, increased transcripts of both GSH2 and PCS2 were quite evident in the OE lines compared to those in the WT upon Cd exposure (Fig. 9B). This indicates that APR2 coordinately regulates the transcription of GSH and PC synthesis genes under Cd stress.
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Author contributions
H2O2, hallmarks of oxidative stress, were observed in the OE lines compared to the WT or apr2 mutant plants upon Cd stress (Fig. 5A and B). Further analysis demonstrated that a greater increase in GSH level was detected in the OE lines, but not in the WT and apr2 mutant plants either under normal conditions or Cd stress (Fig. 6A). This result suggested that APR2 expression directly affected GSH levels. As we know, GSH is a major thiol-containing metabolite and has a role in maintaining redox homeostasis during Cd stress (Jozefczak et al., 2015; Hernández et al., 2015). Our present result suggested that amounts of the GSH were influenced by the APR2 levels and in return, the GSH levels affected Cd stress responses of the APR2 OE and knockout mutant plants (Figs. 6A, 7 and 8). Compared with WT or apr2 mutant, the less accumulations of H2O2 in the OE plants may be a consequence of the higher GSH levels, which enhanced the ROS scavenging capacity. In support of our observation, the Arabidopsis apr2 knockout mutant (apr2-1) decreased tolerance to selenite toxicity due to glutathione deficiency, and thereby resulting in enhanced ROS levels (Grant et al., 2011). Thus, APR2 likely regulates Cd stress tolerance by affecting GSH-dependent antioxidant system that scavenged ROS and decreased membrane damage. As a precursor of the PCs, GSH functions in heavy metal detoxification in plants (Cobbett and Goldsbrough, 2002). It was reported that Arabidopsis GSH- deficient cad2-1 mutant was hypersensitive to Cd (Cobbett et al., 1998), whereas GSH1-overexpression plants with higher GSH levels enhanced Cd tolerance (Zhu et al., 1999). Therefore, it is concluded that the GSH-dependent Cd-chelation is the major mechanism contributing to APR2-mediated Cd tolerance in our study. PCs are synthesized by GSH1, GSH2, PCS1, and PCS2, and act in Cd detoxification in Arabidopsis (May et al., 1998; Cobbett and Goldsbrough, 2002; Noctor et al., 2002; Semane et al., 2007; Jozefczak et al., 2012; Lin and Aarts, 2012; Wang et al., 2018). Our data have demonstrated that the APR2 OE lines showed much more amounts of GSH and PC than the WT and apr2 mutant plants under Cd stress (Fig. 5A and B). Correspondingly, over-expression of the APR2 results in elevated transcripts of GSH1, GSH2, PCS1, and PCS2 (Fig. 9B), indicating that the increased levels of GSH and PC in the APR2 OE lines could be due to the increased APR2 coupled to elevated GSH/PC synthesis-related gene expression under Cd stress, and thereby effectively improving Cd tolerance. In support of this viewpoint, glutathione metabolic genes have been found to be involved in response to heavy metals (Xiang and Oliver, 1998). For example, overexpression of GSH1, PCS1, or PCS2 effectively improved plant tolerance to Cd stress (Zhu et al., 1999; Cazalé and Clemens, 2001; Pomponi et al., 2006; Wawrzynski et al., 2006; Gasic and Korban, 2007; Brunetti et al., 2011). However, the exact mechanism underlying APR2-regulated expression of the GSH/PC synthesis-related genes upon Cd exposure needs further study. In summary, we have identified a major Arabidopsis APR isoenzyme (APR2), whose expression was induced by Cd stress. Transgenic plants overexpressing APR2 improved Cd tolerance, whereas knockout of APR2 reduced Cd tolerance. APR2-overexpressing plants with increased Cd accumulation and tolerance showed higher glutathione (GSH) and phytochelatin (PC) levels than the wild type and apr2 mutant plants, but lower H2O2 and TBARS contents upon Cd exposure. Moreover, APR2-mediated enhanced Cd tolerance is GSH dependent. In addition, over-expression of the APR2 led to elevated expressions of the GSH/PC synthesis-related genes under Cd stress. Thus, we concluded that APR2 regulated Cd accumulation and tolerance possibly through affecting GSH-dependent antioxidant capability and Cd-chelation machinery in Arabidopsis. This study may facilitate our understanding of the molecular mechanism underlying APR2-mediated Cd detoxification in higher plants. In future work, it is necessary to dissect the exact mechanism by which the GSH-dependent pathway is involved in APR2mediated Cd stress response in Arabidopsis.
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