Sodium Hydrosulfide Mitigates Cadmium Toxicity by Promoting

Dec 20, 2018 - The association between hydrogen sulfide (H2S) and cell wall composition with regard to the mitigation of cadmium (Cd) toxicity in Bras...
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Article Cite This: J. Agric. Food Chem. 2019, 67, 433−440

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Sodium Hydrosulfide Mitigates Cadmium Toxicity by Promoting Cadmium Retention and Inhibiting Its Translocation from Roots to Shoots in Brassica napus Yan Yu, Xiangyu Zhou, Zonghe Zhu, and Kejin Zhou* School of Agronomy, Anhui Agricultural University, Hefei 230036, People’s Republic of China

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ABSTRACT: The association between hydrogen sulfide (H2S) and cell wall composition with regard to the mitigation of cadmium (Cd) toxicity in Brassica napus L. was investigated. Cd caused growth retardation, leaf chlorosis, and decreased endogenous H2S content in Brassica napus roots. Stimulating L-cysteine desulfhydrase (LCD)-mediated H2S production with H2S releaser (NaHS) markedly improved plant growth, reduced Cd content in stems and leaves, and rescued Cd-induced chlorosis. Furthermore, increased Cd retention was observed in root cell walls, indicating that NaHS reduced Cd movement from the roots to upper-plant parts. Exogenous NaHS also significantly increased the content of pectin and the activity of pectin methylesterase in cell walls of roots, thereby increasing Cd retention in pectin fractions. However, intensification of H2S barely affected hemicellulose content under Cd stress. Intensified H2S signal, therefore, alleviates Cd toxicity in Brassica napus by increasing pectin content and its demethylation, increasing Cd fixation in cell walls, and reducing root-to-shoot Cd translocation. KEYWORDS: hydrogen sulfide, cadmium toxicity, cell wall, pectin, PME, oilseed rape

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−COOH, and −SH), which have a high affinity for Cd.8,14 The binding of Cd to cell wall will in turn affect the wall’s properties.14 For example, Cd increased polysaccharide content, especially low-methylesterified pectin, as demonstrated in Linum usitatissimum,15 Arabidopsis thaliana,12 and Douglas firs.16 These modifications of cell wall properties could increase its capacity to retain Cd and decrease Cd translocation.6,17 Although substantial efforts have been done to decipher the function and modification of the cell wall in Cd-stressed plant roots, the signaling molecules that modulate cell wall properties remain largely unknown. Hydrogen sulfide (H2S) is an emerging gaseous signaling molecule involved in multiple plant physiological and biochemical processes, including cell wall synthesis.18−22 For example, an exogenous supply of H2S affected the expression of genes that encode the expansion (CsExp) and pectin methylesterase (CsPME) in Cucumis sativus L. roots,22 and H2S pretreatment decreased pectin and hemicellulose content in Oryza sativa L. roots.20 Interestingly, recent studies found that H2S can also interfere with Cd behavior and its accumulation in plants.23,24 Taking the important function of cell wall in plant Cd stress response into account, it is possible that H2S may affect Cd retention and its subsequent translocation in plants through mechanisms that modulate cell wall properties. In this study, the above hypothesis was investigated using oilseed rape plants and pharmacological agents. The results

INTRODUCTION Cadmium (Cd) is a hazardous environmental stress, and it is principally dispersed into agro-ecosystems through anthropogenic activities, which include industrial mining, waste incineration, and the utilization of phosphatic fertilizers, sewage sludge, and pesticides.1 With the nature of high mobility and persistence, Cd, although not required, can be readily assimilated by plants and accumulated through the food chain.2,3 The phytotoxicity of Cd stress has been associated with leaf chlorosis, growth retardation, and the disturbance of various metabolic processes, including photosynthesis.4 For human beings, excessive Cd intake from food is poisonous and may cause renal failure and osteodystrophy, cancer, and even mortality.1 Therefore, urgent decipher of the mechanisms of Cd phytotoxicity and tolerance are needed to develop strategies that reduce Cd accumulation, thus alleviating Cd toxicity and minimizing potential health risks. Three steps likely mediate the accumulation of Cd in edible plant parts: (1) root uptake; (2) translocation to shoots via xylem; and (3) phloem-mediated translocation to seeds.5 Recently, significant efforts have been made to mediate the processes involved in Cd accumulation in plants. As the first contact point when plants are subjected to Cd-stressed conditions, the root cell wall plays a pivotal role in controlling Cd accumulation and its subsequent translocation to shoots.6,7 The cell wall can act as a site that immobilizes and restricts the entrance of Cd into the cell.8,9 For instance, approximately 70−90%, 64−90%, 75%, and 80% of total root Cd was distributed in the cell wall of Avena strigosa Schreb.,10 Athyrium yokoscense,11 Arabidopsis thaliana,12 and Boehmeria nivea L. Gaud.,13 respectively. Root cell wall’s capacity to trap Cd attributes mainly to its available functional groups (e.g., −OH, © 2018 American Chemical Society

Received: Revised: Accepted: Published: 433

August 26, 2018 December 12, 2018 December 13, 2018 December 20, 2018 DOI: 10.1021/acs.jafc.8b04622 J. Agric. Food Chem. 2019, 67, 433−440

Article

Journal of Agricultural and Food Chemistry

Figure 1. Effects of NaHS on cadmium (Cd) toxicity in oilseed rape. Nineteen-day-old seedlings were first pretreated with or without 50 μM NaHS for 6 h and then exposed to 20 μM CaCl2 for 7 d. After that, plant leaf (A), stem (B), and root (C) biomass, leaf chlorosis phenotype (D), and chlorophyll content (E) were measured. Data are reported as means ± SD (n = 6 for A−C and n = 3 for E). Pictures were taken using a digital camera. Bars with different letters indicate significant differences among treatments at P < 0.05. CK, control. supernatant was collected. The reaction mixture contained 0.3 mL of 50 mM FeCl3, 0.3 mL of 50 mM N,N-dimethyl-p-phenylenediamine, and 1.4 mL of extraction buffer. H2S content was calculated from A670 after 15 min incubation at 25 °C. The calibration curve was made the same way as described above. Enzyme Preparation and Analysis. Plant materials were powdered using liquid N2, and the enzymes were extracted by 20 mM Tris-HCl buffer (pH 8.0), which contains 0.1% (w/v) dithiothreitol (DTT) and 0.2% (w/v) sodium ascorbate. The homogenates then were centrifuged, and the supernatants were collected for enzyme measurements. L-Cysteine desulfhydrase (LCD) activity was quantified by trapping H2S released from L-cysteine as catalyzed by LCD, and determining the formed methylene blue according to the procedure of Bloem et al.28 Cyanoalanine synthase (CAS) activity was assayed on the basis of H2S emission from L-cysteine in the participation of cyanide according to Meyer et al.29 O-Acetyl-L-serine (thiol) lyase (OAS-TL) activity was assayed according to Bloem et al.28 The reactions were initiated by adding Na2S, and terminated by an acidic ninhydrin reagent. After being heated at 100 °C for 10 min, the resulting color complex was stabilized by ethanol and measured at 560 nm. The calibration curve was made using L-cysteine as described above. Proteins were determined using the Bradford method. Cd Determination. After harvest, the roots were first immersed in 50 mM CaCl2, followed by washing with Milli-Q water. The stems and leaves were directly washed with Milli-Q water. After blot-drying, the plant samples were dried, weighed, and digested with 4 mL of HNO3 + 1 mL of HClO4 at 120 °C. The resulting solutions were diluted with Milli-Q water for determination. For cell wall Cd determination, cell wall as obtained below was extracted using 2 M HCl with occasional shaking for at least 2 d. For pectin Cd content determination, we first immersed the pellet (cell wall without pectin) in 2 M HCl with occasional vortexing for at least 2 d. Pectin Cd was obtained by subtracting the Cd in the cell wall without pectin from the Cd in the cell wall. Cd content assays were performed using an atomic absorption spectrometer (ZEEnit 700P, Analytic Jena, Germany). Determination of Uronic Acid of Cell Wall Polysaccharides. Cell wall polysaccharides were prepared according to the procedures

indicated that enhanced H2S levels augmented root cell wall Cd retention by increasing the content of low-methylesterified pectin, thus preventing Cd translation from root to shoot.

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MATERIALS AND METHODS

Plant Culture. Oilseed rape (Brassica napus L. “Xikou huazi”) seeds were sterilized in NaClO (1%, v/v) for 30 min, washed thoroughly, and immersed in distilled water overnight. After germination, the seeds were grown on floating nets in 4 L plastic containers with 1/4 strength nutrient solution. After growing for 4 d, rape seedlings were transplanted to pots filled with 1/2 strength and complete nutrient solution for 1 wk, respectively. The complete nutrient solution consists of 1 mM CaCl2, 3 mM KNO3, 1 mM NaH2PO4, 0.5 mM MgSO4, 0.1 μM (NH4)6Mo7O24, 0.5 μM ZnSO4, 0.1 μM CuSO4, 0.5 μM MnSO4, 50 μM Fe-EDTA, and 10 μM H3BO3. The solutions’ pH values were maintained at 5.5 by adjusting with 1 M NaOH. The solutions were freshed every 4 d. The growth conditions for the seedlings were maintained at temperatures of 24/ 18 °C, and a 16/8 h light/dark rhythm photoperiod at 180 μmol m−2 s−1. NaHS was applied as exogenous H2S donor as previously reported.19,20,25 Preliminary experiments indicated that the concentration of 50 μM NaHS pretreatment was required to alleviate growth inhibition and leaf chlorosis under Cd stress conditions (data not shown). Uniform seedlings then were pretreated with 0 or 50 μM NaHS (in complete nutrient solution, pH 5.5) for 6 h before transfer to 0 or 20 μM CdCl2 (in complete nutrient solution, pH 5.5) for 1 wk. Solutions were freshed every other day. After that, plant leaves, stems, and roots were collected and cleaned, respectively. The fresh weights (FW) were measured. Chlorophyll Quantification. Oilseed rape leaves were cleaned and ground in liquid N2 to fine powder. Pigments were completely extracted using acetone (80%, v/v) for at least two times. The absorbance of the extract at 663, 645, and 470 nm was determined for the calculation of total chlorophyll content.26 Determination of H2S. Endogenous H2S was determined as described by Qiao et al.27 with several alterations. Plant materials were extracted with phosphate buffer (100 mM, pH 7.4), which contains 10 mM EDTA and 0.25% Zn-acetate trap. After centrifugation, the 434

DOI: 10.1021/acs.jafc.8b04622 J. Agric. Food Chem. 2019, 67, 433−440

Article

Journal of Agricultural and Food Chemistry

Figure 2. Effects of other sulfur- or sodium-containing compounds on cadmium (Cd) toxicity in oilseed rape. Seedlings exposed to 20 μM CdCl2 were pretreated with either 50 μM Na2S, Na2SO4, Na2SO3, NaHSO4, NaHSO3, NaAC, cysteine (Cys), or methionine (Met). Plant biomass (A) and chlorophyll content (B) were measured. Data are presented as means ± SD (n = 6 for A and n = 3 for B). Bars with different letters indicate significant differences among treatments at P < 0.05. CK, control. as described by Yu et al.30 Roots of the seedlings were cut and ground under liquid N2, homogenized with 75% ethanol on ice, and centrifuged at 8000g for 10 min. The obtained pellets were successively rinsed with acetone, methanol:chloroform (v:v = 1:1), and methanol, respectively. The remaining pellets (cell wall material) then were freeze-dried. Fractions of pectin, hemicellulose 1 (HC1), and hemicellulose 2 (HC2) were obtained by extracting the cell wall material with three kinds of solution containing 0.1% NaBH4, which were 0.5% ammonium oxalate buffer (pH 4.0), 4% KOH, and 24% KOH, respectively. The contents of uronic acid in each polysaccharide fractions were assayed as previously described.31 The calibration curve was made with galacturonic acid (GalA; Sigma-Aldrich). Analysis of Pectin Methylation Degree. Pectin methylation degree was analyzed by calculating the ratio of the content of methanol to GalA.32 Pectin fractions were extracted following the same protocol used for uronic acid determination. First, the pectin methyl ester was hydrolyzed by adding 10 mL of 1 M potassium hydroxide to an aliquot of 5 mL pectin fraction for 40 min, and neutralized with H3PO4 following by diluting to 20 mL. Alcohol oxidase (AO; 1 UN/mL) then was added, following by 15 min of incubation at 25 °C. After that, fluoral-P was added and kept at 60 °C for 15 min before being cooled to room temperature and measured at 412 nm. GalA content was measured as described above, and ethanol was used for the calibration curve. Measurement of Pectin Methylesterase Activity. The enzyme proteins were extracted and analyzed using the method by Zhu et al.12 First, cell wall materials were homogenized in 1 M sodium chloride for 1 h on ice with repeated vortexing, and centrifuged at 15 000g for 10 min at 4 °C. The extracts were added to the PME assay buffer (100 μL of 200 mM phosphate buffer, 0.64 mg mL−1 pectin, and 10 μL AO), followed by 10 min of incubation at 30 °C. Sodium hydroxide solution (0.5 N sodium hydroxide containing 5 mg mL−1 Purpald) then was added with 30 min of incubation at 30 °C to form the color complex of formaldehyde−Purpald. The mixture was

ultimately adjusted to 1 mL by adding distilled water, and measured at 550 nm. Statistical Analysis. The data were examined using the SPSS package (version 11.0, U.S.) by one-way ANOVA, and the differences at P < 0.05 were compared using the least significant difference (LSD) test.

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RESULTS

H2S Alleviates Cd Toxicity in Oilseed Rape. To verify the role H2S played in oilseed rape’s response to Cd toxicity, plants were first pretreated with NaHS, a well-known H2S releaser, before being exposed to Cd-stressed conditions. Because growth inhibition and leaf yellowing are typical symptoms of Cd phytotoxicity, the following four parameters were evaluated: chlorophyll content and leaf, stem, and root biomass. The results indicated that Cd alone decreased leaf, stem, root biomass, and young leaf chlorophyll content by 53% (Figure 1A), 38% (Figure 1B), 44% (Figure 1C), and 77% (Figure 1E), respectively, when compared to the control. NaHS pretreatment alone slightly decreased the fresh weight (FW) of both leaf and root, and there was no visible effect on stem FW or leaf chlorophyll content (Figure 1). However, the combined treatment (NaHS + Cd) clearly rescued the plants from Cd-induced chlorosis (Figure 1D), increased chlorophyll content (Figure 1E), and increased leaf (Figure 1A), stem (Figure 1B), and root growth (Figure 1C). Treatment with other sulfur- or sodium-containing reagents, including Na2S, Na2SO4, NaHSO4, Na2SO3, NaHSO3, NaAC, cysteine (Cys), and methionine (Met), barely alleviated Cd-induced growth retardation (Figure 2A) and leaf chlorosis (Figure 2B) in oilseed rape seedlings. Taken together, these results confirmed that H2S, not sodium nor other components derived from 435

DOI: 10.1021/acs.jafc.8b04622 J. Agric. Food Chem. 2019, 67, 433−440

Article

Journal of Agricultural and Food Chemistry

Figure 3. Effects of NaHS treatment on H2S content and the biosynthetic enzyme activities in oilseed rape roots. Nineteen-day-old seedlings were pretreated with or without 50 μM NaHS for 6 h, and were then exposed to 20 μM CdCl2 for 7 days. Roots were collected to determine H2S content (A) and L-cysteine desulfhydrase (L-CDS) (C), cyanoalanine synthase (CAS) (D), and O-acetyl-L-serine (thiol) lyase (OAS-TL) (E) activities. The biosynthetic pathway of H2S in plants (B). Data are reported as means ± SD (n = 3). Columns with different letters are significantly different at P < 0.05. CK, control.

Figure 4. Effects of NaHS on cadmium (Cd) accumulation in oilseed rape. Nineteen-day-old seedlings exposed to 20 μM CdCl2 were pretreated with 50 μM NaHS for 6 h. Plant leaves (A), stems (B), and roots (C) were collected for Cd content measurement. Data are reported as means ± SD (n = 3). Bars with different letters indicate significant differences among treatments at P < 0.05.

NaHS, contributed to the amelioration of Cd toxicity in oilseed rape. NaHS Treatment Enhances Endogenous H2S Levels and Boosts LCD Activity. Endogenous H2S content was analyzed to ascertain the function of H2S played in plant Cd stress. Here, we found that 7 d of Cd stress significantly decreased root H2S content by 30% as compared to control (Figure 3A). Application of NaHS restored the internal levels to approximately those of the control (Figure 3A). To elucidate the possible mechanisms underlying NaHS-enhanced H2S production in oilseed rape roots, we measured the activities of LCD, CAS, and OAS-TL, which are the key biosynthetic enzymes for H2S in plants (Figure 3B). Exposure to Cd for 7 d resulted in a significant decrease in LCD activity

(Figure 3C) and a slight increase in CAS activity (Figure 3D), but barely changed the activity of OAS-TL (Figure 3E). Application of NaHS significantly increased LCD activity by 48%, but it had little effect on CAS and OAS-TL activities (Figure 3). This result suggested that NaHS may induce H2S production by activating LCD in the roots of Cd-stressed oilseed rape seedlings. H2S Detains Cd in Root Cell Walls. To elucidate the mechanisms underlying H2S-alleviated Cd phytotoxicity, we investigated the impacts of NaHS on Cd distribution in Cdstressed oilseed rape seedlings. In Cd stressed seedlings, more than 73% of the Cd was accumulated in oilseed rape roots (Figure 4). Application of NaHS augmented Cd accumulation 436

DOI: 10.1021/acs.jafc.8b04622 J. Agric. Food Chem. 2019, 67, 433−440

Article

Journal of Agricultural and Food Chemistry

Figure 5. Effects of NaHS on cadmium (Cd) distribution in roots of oilseed rape seedlings. Nineteen-day-old seedlings exposed to 20 μM CdCl2 were pretreated with 50 μM NaHS for 6 h. The roots then were collected for Cd content measurement (A) and root cell wall extrations (A,B). Cd in root cell wall was extracted using 2 M HCl. Data are reported as means ± SD (n = 3). Bars with different letters indicate significant differences among the treatments at P < 0.05. CW, cell wall.

enzyme PME, we compared the degree of pectin methylation and PME activity between Cd and NaHS + Cd treatments. As shown in Figure 7A, Cd exposure conspicuously amplified the methyl-esterification degree of pectin, while application of NaHS markedly reduced this increase. As shown in Figure 7C, Cd treatment decreased PME activity, while the NaHS + Cd treatment reversed this decrease. These results propose that H2S may facilitate the Cd binding capacity of root cell walls by elevating both content and demethylation of pectin.

in roots (Figure 4C), while decreased its content in leaves (Figure 4A) and stems (Figure 4B). Because cell well is an important site binding Cd in plant roots,14 we measured cell wall Cd content in oilseed rape roots. As shown in Figure 5A, approximately 72% of the root Cd was stored in cell wall. The pretreatment of seedlings with NaHS in the presence of Cd increased cell wall Cd content by 52% (Figure 5B). H2S Increases the Cd Binding Capacity of Root Cell Walls. The uronic acid content of polysaccharides, comprising pectin and HCs, critically affected the binding capacity of cell wall to metal ions.12,14 As shown in Figure 6A, NaHS

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DISCUSSION Cd is detrimental to plant growth and development.1,4,5 However, the employment of chemical compounds has been recently regarded as a cogent strategy that can help plants adapt to multiple environmental stresses.17,33,34 Here, we detected that the toxic effects of Cd on oilseed rape seedlings can be mitigated by the application of NaHS, a widely used H2S releaser. The results indicated that 20 μM CdCl2 significantly decreased plant biomass and chlorophyll content (Figure 1) in oilseed rape seedlings. The exogenous application of NaHS, instead of other sulfur- or sodium-containing compounds, markedly restored chlorosis symptoms and improved growth in Cd-stressed oilseed rape seedlings (Figures 1 and 2). Moreover, application of NaHS significantly increased internal H2S levels in oilseed rape roots under Cd stress (Figure 3), thus confirming that the ameliorative role of NaHS should be attributed to H2S. This result was consistent with previous studies of various plant stress responses, including studies of wheat,35 strawberry,36 maize,25 and cauliflower37 plants. Furthermore, we demonstrated that the intensified H2S signal acts to enhance plant Cd tolerance, and this likely occurs when Cd fixation in root cell walls is increased, thus reducing root-to-shoot Cd translocation. Endogenous H2S levels may be altered, and these levels could play differential roles in plant responses to Cd stress.21 For instance, Cd-induced H2S production has been correlated with higher Cd tolerance, as reported in Medicago sativa,38 Arabidopsis thaliana,39 Oryza sativa,40 and Brassica rapa.41 However, in other species, it has been demonstrated that increased H2S generation underpins Cd toxicity, for example, in the roots of Brassica rape42 and Nicotiana tabacum.43 This discrepancy could be attributed to different plant species, Cd concentrations, and treatment durations used. Therefore, it is possible that H2S acts as “double-edged sword”, either being

Figure 6. Effects of NaHS on uronic acid content in the cell wall fractions of oilseed rape roots. Seedlings were pretreated with or without 50 μM NaHS for 6 h, and were then exposed to 20 μM CdCl2 for 7 d. Roots were cut, and cell wall polysaccharides were fractioned into pectin (A), hemicellulose (HC1) (B), and hemicellulose 2 (HC2) for uronic acid measurements. Data are reported as means ± SD (n = 3). Bars with different letters indicate significant differences among treatments at P < 0.05. CW, cell wall.

application markedly elevated the uronic acid content of pectin as compared to Cd treatment alone. However, there was no obvious difference in the uronic acid content of HC1 and HC2 between the oilseed rape roots treated with Cd and those treated with NaHS + Cd (Figure 6A). Consistently, the NaHS + Cd treatment significantly increased Cd accumulation in cell wall pectin (Figure 7B). Because the binding capacity of pectin depends on the process of pectin demethylation, which is catalyzed by the 437

DOI: 10.1021/acs.jafc.8b04622 J. Agric. Food Chem. 2019, 67, 433−440

Article

Journal of Agricultural and Food Chemistry

Figure 7. Effects of NaHS on the degree of pectin methylation, cell wall pectin cadmium (Cd) content, and pectin methylesterase (PME) activity in oilseed rape. Nineteen-day-old seedlings were then pretreated with 50 μM NaHS for 6 h, followed by exposure to 20 μM CaCl2 for 7 d. The degree of pectin methylation in roots (A), Cd content in pectin (B), and PME activity (C) were analyzed. Data are reported as means ± SD (n = 3). Bars with different letters indicate significant differences among treatments at P < 0.05. CK, control; CW, cell wall.

Therefore, our results suggest that intensified H2S enhances Cd tolerance in oilseed rape seedlings by promoting Cd retention in root cell walls and reducing its transport to shoots. Analysis of cell wall modifications provided further insight into the exact mode of action used by H2S to increase root Cd retention. The plant cell wall mainly consists of cellulose that is decorated with matrixes of pectins, HCs, and elements such as lignin.46 Among those components, pectins and hemicelluloses are recently identified as the two major sites binding Cd in the cell wall.12,14,17 For instance, higher content of pectins and/or HCs led to more Cd detained in the cell walls of Arabidopsis,12 rice,17 Sedum alfredii,6 and tomato cell suspensions.47 Thus, factors that can change cell wall components may affect the binding capacity of the cell wall. Our results found NaHS + Cd treatment led to marked increases in the content of pectins, with little effect on HC1 and HC2 (Figure 6), implying that H2S likely affects the Cd-binding capacity by interfering with pectins (but not HCs). The increased accumulation of Cd in pectin fractions (Figure 7B), but not hemicellulose fractions (Figure S1), following the application of exogenous NaHS to Cd-treated roots further supported this conclusion. Pectins are usually secreted from the Golgi in highly methylated forms, and they then undergo demethylation processes catalyzed by PME in the cell wall.46 Differences in the accumulation of cell wall metals (e.g., aluminum and zinc) were reported as negatively related to the degree of pectin methylation.48,49 Therefore, besides content, the methylation degree of pectin could also be determinable to the binding capacity of cell wall to Cd. Indeed, we observed a clear decrease of the methylation degree of pectin in the present study (Figure 7A), which could release more negatively charged carboxyl groups associated with Cd binding. Moreover, a significant increase of PME activity was consistently observed under the treatment of NaHS combined with Cd as compared to Cd alone (Figure 7B). These results suggest that the intensified H2S production augmented the negatively charged sites in the cell wall by increasing pectin content and stimulating PME activity, which in turn increased Cd retention. However, further efforts at a transcriptional level are still needed to validate how H2S regulates the biosynthesis and remodeling of pectins. As enzyme activity activation by H2S-induced persulfidation,50 an important process of post-translational modification (PTM) of proteins, has been reported recently,50 it was possible that H2S modulates this process through protein persulfidation. This assumption needs to be validated in future studies.

toxic or being protective, depending on the intensity and duration of H2S production. Results from the present study show that Cd treatment for 7 d significantly reduced H2S production in roots of oilseed rape seedlings, which was restored following the application of exogenous NaHS (Figure 3). In addition, there was an obvious alleviation of Cd-induced growth retardation and leaf chlorosis (Figure 1), which are typical symptoms of Cd toxicity in plants. Further investigation of the possible sources of elevated H2S manifested that the mitigated Cd toxicity might be attributed to activated LCD because NaHS significantly enhanced the activity of LCD but had little effect on those of CAS and OAS-TL (Figure 3). As H2S is a signal molecule that can freely permeate lipid membranes,44 it is proposed that the diffusion of H2S may also increase internal H2S content. However, this may not contribute to internal H2S increase here as DL-propargylglycine (an LCD inhibitor) supply can abolish the NaHS-induced H2S elevation (data not shown). The proper reason for this may be that internal H2S was determined 7 days after NaHS pretreatment, and the earlier diffused H2S may have been transferred to other tissues for its high lipophilic character19 or metabolized through its rapid oxidation in mitochondria or methylation in the cytosol.44 Altogether, our findings show that NaHS initiates a tolerance response in oilseed rape under Cd stress, and this primarily occurs via the induction of LCDmediated H2S production. Enhanced Cd stress tolerance in NaHS-pretreated oilseed rape seedlings could be partially attributed to attenuated Cd translocation, which serves as a crucial process that determines Cd accumulation in upper plant parts.5 A relationship between the higher root Cd accumulations versus lower leaf Cd accumulations has been previously established in cabbage.45 Similarly, we found that exogenous NaHS promoted Cd storage in the roots (Figure 4A), whereas it decreased Cd shuttling to both the leaves (Figure 4A) and the stems (Figure 4B) of Cd-stressed oilseed rape plants. The cell wall plays pivotal roles in controlling the process of Cd transport from roots to upper plant parts.6,17 As the first barrier in contact with Cd, the root cell wall can reduce the amount of Cd2+ ions entering cells via binding.9,14 The results from subcellular Cd distribution analyses confirmed cell wall as the main spot binding Cd in oilseed rape roots. As shown in Figure 5, a high percentage (>70%) of the root Cd was stored in cell wall, and significantly more Cd accumulated in root cell wall when the stressed oilseed rape seedlings were pretreated with NaHS. 438

DOI: 10.1021/acs.jafc.8b04622 J. Agric. Food Chem. 2019, 67, 433−440

Article

Journal of Agricultural and Food Chemistry

(2) Li, S. W.; Sun, H. J.; Li, H. B.; Luo, J.; Ma, L. Q. Assessment of cadmium bioaccessibility to predict its bioavailability in contaminated soils. Environ. Int. 2016, 94, 600−606. (3) Aziz, R.; Rafiq, M. T.; Li, T. Q.; Liu, D.; He, Z. L.; Stoffella, P. J.; Sun, K.; Xiaoe, Y. Uptake of cadmium by rice grown on contaminated soils and its bioavailability/toxicity in human cell lines (Caco-2/HL7702). J. Agric. Food Chem. 2015, 63 (13), 3599−3608. (4) Sandalio, L. M.; Dalurzo, H. C.; Gomez, M.; Romero-Puertas, M. C.; del Rio, L. A. Cadmium-induced changes in the growth and oxidative metabolism of pea plants. J. Exp. Bot. 2001, 52 (364), 2115−2126. (5) Uraguchi, S.; Mori, S.; Kuramata, M.; Kawasaki, A.; Arao, T.; Ishikawa, S. Root-to-shoot Cd translocation via the xylem is the major process determining shoot and grain cadmium accumulation in rice. J. Exp. Bot. 2009, 60 (9), 2677−2688. (6) Li, T. Q.; Tao, Q.; Shohag, M. J. I.; Yang, X. E.; Sparks, D. L.; Liang, Y. C. Root cell wall polysaccharides are involved in cadmium hyperaccumulation in Sedum alf redii. Plant Soil 2015, 389 (1−2), 387−399. (7) Peng, J. S.; Wang, Y. J.; Ding, G.; Ma, H. L.; Zhang, Y. J.; Gong, J. M. A pivotal role of cell wall in cadmium accumulation in the Crassulaceae hyperaccumulator Sedum plumbizincicola. Mol. Plant 2017, 10 (5), 771−774. (8) Krzeslowska, M. The cell wall in plant cell response to trace metals: polysaccharide remodeling and its role in defense strategy. Acta Physiol. Plant. 2011, 33 (1), 35−51. (9) Fernandez, R.; Fernandez-Fuego, D.; Bertrand, A.; Gonzalez, A. Strategies for Cd accumulation in Dittrichia viscosa (L.) Greuter: Role of the cell wall, non-protein thiols and organic acids. Plant Physiol. Biochem. 2014, 78, 63−70. (10) Uraguchi, S.; Kiyono, M.; Sakamoto, T.; Watanabe, I.; Kuno, K. Contributions of apoplasmic cadmium accumulation, antioxidative enzymes and induction of phytochelatins in cadmium tolerance of the cadmium-accumulating cultivar of black oat (Avena strigosa Schreb.). Planta 2009, 230 (2), 267−276. (11) Nishizono, H.; Ichikawa, H.; Suziki, S.; Ishii, F. The role of the root cell-wall in the heavy-metal tolerance of Athyrium-Yokoscence. Plant Soil 1987, 101 (1), 15−20. (12) Zhu, X. F.; Lei, G. J.; Jiang, T.; Liu, Y.; Li, G. X.; Zheng, S. J. Cell wall polysaccharides are involved in P-deficiency-induced Cd exclusion in Arabidopsis thaliana. Planta 2012, 236 (4), 989−997. (13) Zhu, Q. H.; Huang, D. Y.; Liu, S. L.; Luo, Z. C.; Rao, Z. X.; Cao, X. L.; Ren, X. F. Accumulation and subcellular distribution of cadmium in ramie (Boehmeria nivea L. Gaud.) planted on elevated soil cadmium contents. Plant, Soil Environ. 2013, 59, 57−61. (14) Parrotta, L.; Guerriero, G.; Sergeant, K.; Cal, G.; Hausman, J. F. Target or barrier? The cell wall of early- and later-diverging plants vs cadmium toxicity: differences in the response mechanisms. Front. Plant Sci. 2015, 6, 16. (15) Douchiche, O.; Driouich, A.; Morvan, C. Spatial regulation of cell-wall structure in response to heavy metal stress: cadmiuminduced alteration of the methyl-esterification pattern of homogalacturonans. Ann. Bot. 2010, 105 (3), 481−491. (16) Astier, C.; Gloaguen, V.; Faugeron, C. Phytoremediation of cadmium-contaminated soils by young Douglas fir trees: effects of cadmium exposure on cell wall composition. Int. J. Phytorem. 2014, 16 (7−8), 790−803. (17) Xiong, J.; An, L. Y.; Lu, H.; Zhu, C. Exogenous nitric oxide enhances cadmium tolerance of rice by increasing pectin and hemicellulose contents in root cell wall. Planta 2009, 230 (4), 755−765. (18) Shi, H. T.; Ye, T. T.; Han, N.; Bian, H. W.; Liu, X. D.; Chan, Z. L. Hydrogen sulfide regulates abiotic stress tolerance and biotic stress resistance in Arabidopsis. J. Integr. Plant Biol. 2015, 57 (7), 628−640. (19) Li, Z. G.; Min, X.; Zhou, Z. H. Hydrogen sulfide: A signal molecule in plant cross-adaptation. Front. Plant Sci. 2016, 7, 12. (20) Zhu, C. Q.; Zhang, J. H.; Sun, L. M.; Zhu, L. F.; Abliz, B.; Hu, W. J.; Zhong, C.; Bai, Z. G.; Sajid, H.; Cao, X. C.; Jin, Q. Y. Hydrogen

In addition to modulating cell wall properties, H2S may also function through cooperating with proline, which can function as ROS quencher, osmoprotectant, or chelator in alleviating heavy metal stress.51,52 Thus, the relationships between H2S and proline in modulating Cd translocation and whether proline is involved in reducing Cd toxicity in oilseed rape need further investigation. In conclusion, our study revealed that Cd stress decreases H2S production in the roots of B. napus, and that exogenously applied NaHS could intensify the H2S signal via the activation of the LCD pathway. Furthermore, we demonstrated that LCD-mediated H2S accumulation increased the content of pectin with a lower degree of methylation, which consequently facilitated the binding of Cd to cell wall. This resulted in less Cd shuttling from roots to shoots, and thus diminished Cdinduced toxicity in B. napus. These results suggest the maintenance of H2S production via the mediation of LCD activity to be an effective strategy to control Cd contamination in B. napus. Nevertheless, further studies on the biosynthesis and remodeling of pectins at a transcriptional level are needed to better illustrate the cell wall retention process.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.8b04622. Figure S1: Effects of NaHS on cadmium (Cd) content in the hemicellulose of oilseed rape roots (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +86 551-5786951. Fax: +86 551-5786869. E-mail: [email protected]. ORCID

Kejin Zhou: 0000-0001-7115-0290 Funding

This research was supported by the Youth Foundation of Anhui Agricultural University (2016ZR016), the 13th FiveYear Plan for Rape-Cotton Industry System of Anhui Province (AHCYJSTX-04), and the National Key R&D Project (2018YFD0100600602-4). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Prof. Dr. Ying Feng for providing Brassica napus L. “Xikou huazi” seeds. ABBREVIATIONS USED H2S, hydrogen sulfide; Cd, cadmium; NaHS, sodium hydrosulfide; CAS, cyanoalanine synthase; LCD, L -cysteine desulfhydrase; OAS-TL, O-acetyl-L-serine (thiol) lyase; DTT, dithiothreitol; Met, methionine; Cys, cysteine; PME, pectin methylesterase; HC1, hemicellulose 1; HC2, hemicellulose 2; GalA, galacturonic acid; FW, fresh weight

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REFERENCES

(1) Clemens, S.; Ma, J. F. Toxic heavy metal and metalloid accumulation in crop plants and foods. In Annual Review of Plant Biology; Merchant, S. S., Ed.; Annual Reviews: Palo Alto, CA, 2016; Vol. 67, pp 489−512. 439

DOI: 10.1021/acs.jafc.8b04622 J. Agric. Food Chem. 2019, 67, 433−440

Article

Journal of Agricultural and Food Chemistry sulfide alleviates aluminum toxicity via decreasing apoplast and symplast Al contents in rice. Front. Plant Sci. 2018, 9, 14. (21) He, H. Y.; Li, Y. Q.; He, L. F. The central role of hydrogen sulfide in plant responses to toxic metal stress. Ecotoxicol. Environ. Saf. 2018, 157, 403−408. (22) Wang, B. L.; Shi, L.; Li, Y. X.; Zhang, W. H. Boron toxicity is alleviated by hydrogen sulfide in cucumber (Cucumis sativus L.) seedlings. Planta 2010, 231 (6), 1301−1309. (23) Li, L.; Wang, Y. Q.; Shen, W. B. Roles of hydrogen sulfide and nitric oxide in the alleviation of cadmium-induced oxidative damage in alfalfa seedling roots. BioMetals 2012, 25 (3), 617−631. (24) Sun, J.; Wang, R. G.; Zhang, X.; Yu, Y. C.; Zhao, R.; Li, Z. Y.; Chen, S. L. Hydrogen sulfide alleviates cadmium toxicity through regulations of cadmium transport across the plasma and vacuolar membranes in Populus euphratica cells. Plant Physiol. Biochem. 2013, 65, 67−74. (25) Chen, J.; Wu, F. H.; Shang, Y. T.; Wang, W. H.; Hu, W. J.; Simon, M.; Liu, X.; Shangguan, Z. P.; Zheng, H. L. Hydrogen sulphide improves adaptation of Zea mays seedlings to iron deficiency. J. Exp. Bot. 2015, 66 (21), 6605−6622. (26) Arnon, D. I. Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physiol. 1949, 24 (1), 1−15. (27) Qiao, Z. J.; Jing, T.; Jin, Z. P.; Liang, Y. L.; Zhang, L. P.; Liu, Z. Q.; Liu, D. M.; Pei, Y. X. CDPKs enhance Cd tolerance through intensifying H2S signal in Arabidopsis thaliana. Plant Soil 2016, 398 (1−2), 99−110. (28) Bloem, E.; Riemenschneider, A.; Volker, J.; Papenbrock, J.; Schmidt, A.; Salac, I.; Haneklaus, S.; Schnug, E. Sulphur supply and infection with Pyrenopeziza brassicae influence L-cysteine desulphydrase activity in Brassica napus L. J. Exp. Bot. 2004, 55 (406), 2305− 2312. (29) Meyer, T.; Burow, M.; Bauer, M.; Papenbrock, J. Arabidopsis sulfurtransferases: investigation of their function during senescence and in cyanide detoxification. Planta 2003, 217, 1−10. (30) Yu, Y.; Jin, C. W.; Sun, C. L.; Wang, J. H.; Ye, Y. Q.; Lu, L. L.; Lin, X. Y. Elevation of arginine decarboxylase-dependent putrescine production enhances aluminum tolerance by decreasing aluminum retention in root cell walls of wheat. J. Hazard. Mater. 2015, 299, 280−288. (31) Blumenkr, N.; Asboehan, G. New method for quantitative determination of uronic acids. Anal. Biochem. 1973, 54 (2), 484−489. (32) Klavons, J. A.; Bennett, R. D. Determination of methanol using alcohol oxidase and its application to methyl-ester content of pectins. J. Agric. Food Chem. 1986, 34 (4), 597−599. (33) Savvides, A.; Ali, S.; Tester, M.; Fotopoulos, V. Chemical priming of plants against multiple abiotic stresses: mission possible? Trends Plant Sci. 2016, 21 (4), 329−340. (34) Wang, T. T.; Shi, Z. Q.; Hu, L. B.; Xu, X. F.; Han, F. X. X.; Zhou, L. G.; Chen, J. Thymol ameliorates cadmium-induced phytotoxicity in the root of rice (Oryza sativa) seedling by decreasing endogenous nitric oxide generation. J. Agric. Food Chem. 2017, 65 (34), 7396−7405. (35) Zhang, H.; Tan, Z. Q.; Hu, L. Y.; Wang, S. H.; Luo, J. P.; Jones, R. L. Hydrogen sulfide alleviates aluminum toxicity in germinating wheat seedlings. J. Integr. Plant Biol. 2010, 52 (6), 556−567. (36) Christou, A.; Manganaris, G. A.; Papadopoulos, I.; Fotopoulos, V. Hydrogen sulfide induces systemic tolerance to salinity and nonionic osmotic stress in strawberry plants through modification of reactive species biosynthesis and transcriptional regulation of multiple defence pathways. J. Exp. Bot. 2013, 64 (7), 1953−1966. (37) Chen, Z.; Yang, B. F.; Hao, Z. K.; Zhu, J. Q.; Zhang, Y.; Xu, T. T. Exogenous hydrogen sulfide ameliorates seed germination and seedling growth of cauliflower under lead stress and its antioxidant role. J. Plant Growth Regul. 2018, 37 (1), 5−15. (38) Cui, W. T.; Chen, H. P.; Zhu, K. K.; Jin, Q. J.; Xie, Y. J.; Cui, J.; Xia, Y.; Zhang, J.; Shen, W. B. Cadmium-induced hydrogen sulfide synthesis is involved in cadmium tolerance in Medicago sativa by reestablishment of reuced (Homo) glutathione and ractive oygen secies homeostasis. PLoS One 2014, 9 (10), 12.

(39) Qiao, Z. J.; Jing, T.; Liu, Z. Q.; Zhang, L. P.; Jin, Z. P.; Liu, D. M.; Pei, Y. X. H2S acting as a downstream signaling molecule of SA regulates Cd tolerance in Arabidopsis. Plant Soil 2015, 393 (1−2), 137−146. (40) Mostofa, M. G.; Rahman, A.; Ansary, M. M. U.; Watanabe, A.; Fujita, M.; Tran, L. S. P. Hydrogen sulfide modulates cadmiuminduced physiological and biochemical responses to alleviate cadmium toxicity in rice. Sci. Rep. 2015, 5, 17. (41) Zhang, L. P.; Pei, Y. X.; Wang, H. J.; Jin, Z. P.; Liu, Z. Q.; Qiao, Z. J.; Fang, H. H.; Zhang, Y. J. Hydrogen sulfide alleviates cadmiuminduced cell death through restraining ROS accumulation in roots of Brassica rapa L. ssp pekinensis. Oxid. Med. Cell. Longevity 2015, 2015, 11. (42) Lv, W.; Yang, L.; Xu, C.; Shi, Z.; Shao, J.; Xian, M.; Chen, J. Cadmium disrupts the balance between hydrogen peroxide and superoxide radical by regulating endogenous hydrogen sulfide in the root tip of Brassica rapa. Front. Plant Sci. 2017, 8, 1 DOI: 10.3389/ fpls.2017.00232. (43) Ye, X. F.; Xue, Y. F.; Ling, T. X.; Wang, Y.; Yu, X. N.; Cheng, C. X.; Feng, G. S.; Hu, L. B.; Shi, Z. Q.; Chen, J. Cinnamaldehyde ameliorates cadmium-inhibited root elongation in tobacco seedlings via decreasing endogenous hydrogen sulfide production. Molecules 2017, 22 (1), 17. (44) Wang, R. The gasotransmitter role of hydrogen sulfide. Antioxid. Redox Signaling 2003, 5 (4), 493−501. (45) Sun, J. Y.; Cui, J.; Luo, C. L.; Gao, L.; Chen, Y. H.; Shen, Z. G. Contribution of cell walls, nonprotein thiols, and organic acids to cadmium resistance in two cabbage varieties. Arch. Environ. Contam. Toxicol. 2013, 64 (2), 243−252. (46) Cosgrove, D. J. Growth of the plant cell wall. Nat. Rev. Mol. Cell Biol. 2005, 6 (11), 850−861. (47) Muschitz, A.; Riou, C.; Mollet, J. C.; Gloaguen, V.; Faugeron, C. Modifications of cell wall pectin in tomato cell suspension in response to cadmium and zinc. Acta Physiol. Plant. 2015, 37 (11), 11. (48) Schmohl, N.; Pilling, J.; Fisahn, J.; Horst, W. J. Pectin methylesterase modulates aluminium sensitivity in Zea mays and Solanum tuberosum. Physiol. Plant. 2000, 109 (4), 419−427. (49) Khotimchenko, M. Y.; Kolenchenko, E. A.; Khotimchenko, Y. S. Zinc-binding activity of different pectin compounds in aqueous solutions. J. Colloid Interface Sci. 2008, 323 (2), 216−222. (50) Aroca, A.; Gotor, C.; Romero, L. C. Hydrogen sulfide signaling in plants: Emerging roles of protein persulfidation. Front. Plant Sci. 2018, 9, 1 DOI: 10.3389/fpls.2018.01369. (51) Tian, B.; Qiao, Z.; Zhang, L.; Li, H.; Pei, Y. Hydrogen sulfide and proline cooperate to alleviate cadmium stress in foxtail millet seedlings. Plant Physiol. Biochem. 2016, 109, 293−299. (52) Szabados, L.; Savoure, A. Proline: a multifunctional amino acid. Trends Plant Sci. 2010, 15 (2), 89−97.

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DOI: 10.1021/acs.jafc.8b04622 J. Agric. Food Chem. 2019, 67, 433−440