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Responses of wheat seedlings to cadmium, mercury and trichlorobenzene stresses
Journal of Environmental Sciences 21(2009) 806–813
Responses of wheat seedlings to cadmium, mercury and trichlorobenzene stresses GE Cailin1,2,∗, DING Yan1 , WANG Zegang2 , WAN Dingzhen2 , WANG Yulong1 , SHANG Qi2 , LUO Shishi2 1. Jiangsu Provencial Key Laboratory of Crop Genetics and Physiology, Yangzhou University, Yangzhou 225009, China. E-mail: [email protected] 2. College of Bioscience and Biotechnology, Yangzhou University, Yangzhou 225009, China Received 31 May 2008; revised 14 July 2008; accepted 13 August 2008
Abstract The molecular response of wheat (Triticum aestivum L., cv. Yangmai 13) seedlings to heavy metal (Cd, Hg) and 1,2,4trichlorobenzene (TCB) stresses were examined by two-dimensional gel electrophoresis, image analysis, and peptide mass fingerprinting. The results showed inhibitions of root and shoot growth by Cd, Hg, and TCB. These stresses led to water deficit and lipid phosphorylation in the seedling which also promoted protein phophorylation in the leaves. Hg stress inhibited protein synthesis while Cd and TCB stresses induced or up-regulated more proteins in the leaves. Most of these induced proteins played important roles in the biochemical reactions involved in tolerance of wheat to Cd and TCB stresses. The primary functions of Cd- and TCB-induced proteins included methionine metabolism, Rubisco modification, protein phosphorylation regulation, protein configuration protection, H+ transmembrane transportation and also the synthesis of ethylene, defense substances and cell wall compounds. Key words: wheat; proteomics; chemical pollutant; stress response DOI: 10.1016/S1001-0742(08)62345-1
Introduction Chemical pollution has been a worldwide problem as a result of rapid increase in chemical inputs in agricultural production in the past decades. Most of chemical pollutants in soil and water cause disturbances in crop growth and development, and subsequently decreased crop productivity. Recent reports on the toxic effects of heavy metals (especially Cd) of wheat indicate that heavy metals inhibit root and shoot growth (Liu and Zhang, 2007; Cao et al., 2007), and also induce oxidative stress and lipid peroxidation (Singh et al., 2008). Cavallini et al. (1999) also reported that certain heavy metal ions (such as Hg) tend to form covalent bonds with DNA. Various defense mechanisms adopted by wheat to avoid heavy metal toxicity have been reported by several researchers, these include alteration of antioxidant enzyme level (Liu et al., 2007; Lin et al., 2007; Yannarelli et al., 2007), increase in the content of phytochelatin (Sun et al., 2005; Lindberg et al., 2007), and also increased generation of polyamine and ethylene (Groppa et al., 2003). As compared to heavy metals, reports on the toxic effects of 1,2,4-trichlorobenzene (TCB) on crops are limited (Wang et al., 2006; Ge et al., 2007, 2008), and with no report on wheat. The present study reports the molecular responses of wheat to Cd, Hg, and TCB stresses, the * Corresponding author. E-mail: [email protected]
physiological toxicity of Cd, Hg, and TCB in wheat, and the Cd-, TCB-induced proteins in wheat leaves.
1 Materials and methods 1.1 Seedlings cultivation, treatment and toxicity assay Seeds of wheat (Triticum aestivum L.) variety Yangmai 13 were surface-sterilized in 3% (V/V) H2 O2 for 5 min and then rinsed with deionized water before germination on moist filter paper at 30°C for 3 d. Seedlings were transferred into colored vitreous pots containing 100-mL Hoagland nutrient solution, and grown in a growth chambers with regulated day/night temperatures 25/18°C and the light intensity range 250–300 μmol/(m2 ·s). Roots at three-leave stage seedlings were submerged in the test solutions containing 0.25, 0.5, 0.75, 1.0 mmol/L CdCl2 , 0.0125, 0.025, 0.0375, 0.05 mmol/L HgCl2 , and 0.014, 0.028, 0.042, 0.056 mmol/L TCB. Treatment combinations of the three chemicals were chosen based on preliminary studies in our laboratory (unpublished). Solutions were renewed daily within the 5 d treatment period to maintain their concentrations. The inhibitory effects of Cd, Hg, and TCB on wheat seedlings were determined by root and shoot dry weight after 5 d treatment period. The levels of lipid peroxidation in wheat leaves were measured in terms of malondialdehyde
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(MDA) content as reported by Dhindsa et al. (1980), and MDA content expressed in μmol/g fw (fresh weight). All measurements were in three replicates with data presented in means ± standard deviations (SD). Statistical analyses were performed using SPSS statistical package software (version 10.0). Comparisons between control and other treatments were evaluated by one-way ANOVA and the least-significant-differences (LSD) test. 1.2 In vitro protein phosphorylation analysis Fresh leaf tissues weighing 0.5 g each were sampled from seedlings treated with 0.5 mmol/L Cd and 0.056 mmol/L TCB for 5 d. These samples were homogenized in three volumes of extraction buffer (containing 50 mmol/L Tris-HCl pH 7.4, 150 mmol/L NaCl, 1% Triton X-100, 1 mmol/L EDTA, 5 μmol/L Na3 VO4 , 2 μmol/L Okadaic acid, 1 mmol/L PMSF). The homogenates were centrifuged at 12000 ×g, 4°C for 10 min. The soluble protein contents were determined by the Coomassie Brilliant Blue Method. About 20 μL soluble protein was added to the reaction mixture containing 20 mmol/L Tris-HCl pH 7.5, 20 mmol/L β-glycerol phosphate, 10 mmol/L MgCl2 , 10 μmol/L Na3 VO4 , 0.5 μCi [γ-32 P] ATP. The reaction mixture was incubated at 30°C for 30 min, and phosphorylation reactions were stopped by adding protein loading buffer with 3 min boiling. Proteins in the reaction mixture were separated by SDS-PAGE (12.5% polyacrylamide). The gels were stained in Bio-safe colloidal Coomassie Blue G-250 (Bio-Rad Company, USA), and dried over a filter paper. Phosphorylated protein bands were detected by autoradiography using Kodak medical X-ray film (Machado et al., 2002). 1.3 Proteomic analysis 1.3.1 Protein extraction Proteins from the leaves treated with 0.5 mmol/L Cd, 0.05 mmol/L Hg, and 0.056 mmol/L TCB for 5-d were extracted using polyethylene glycol (PEG) fractionation (Xi et al., 2006). All fractions of extracted proteins were rinsed with ice-cold acetone containing 0.07% (V/V) βmercaptoethanol, then air-dried and stored at –20°C. 1.3.2 Two-dimensional gel electrophoresis Above extracted proteins of F3 and F4 fractions were resuspended in rehydration buffer (Bio-Rad Company, USA) and incubated for 1 h at room temperature. After centrifugation at 12000 ×g at 4°C for 10 min, protein concentration in the supernatant was measured by Coomassie Brilliant Blue Method. IPG strip (Bio-Rad) of 11 cm (pH 4–7) was rehydrated in rehydration buffer (containing 400 μg protein sample) for 16 h to allow proteins to be uptaken. Iso-electric focusing (IEF) was performed using the PROTEAN IEF system (Bio-Rad Company, USA) at 20°C. Prior to second dimension analysis, the strips were equilibrated for 15 min in the equilibration buffer 1 (BioRad), and equilibrated for 15 min in the equilibration buffer 2 (Bio-Rad). Protein separation of the second dimension was carried out on a 12.5% SDS-PAGE. After 2-DE
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separation, the gels were washed twice (5 min each) with double distilled water, and transferred into Bio-safe colloidal Coomassie Blue G-250 staining for 8 h. After which the gels were washed three times (1 h each). 1.3.3 Image analysis Gels were scanned by Gel Doc XR system (Bio-Rad, USA). The protein spots were analyzed by PDQuest 2D analysis software (Bio-Rad). The molecular weight and isoelectric point of differential proteins were calculated. After analysis, the selected protein spots were manually excised from the gel and stored at –20°C. 1.4 Identification of Cd- and TCB-induced proteins The peptide mass fingerprints of Cd- and TCBinduced proteins were analyzed by mass spectrometer (MALDI-TOF MS, Applied Biosystem, USA) according to “Proteins and Proteomics: A Laboratory Manual” (Simpson, 2003). The peptide mass fingerprints were used for searching in MSDB, NCBInr or SwissProt database using MASCOT software search engine (http://www.matrixscience.com) to identify Cd- and TCBinduced proteins. In order to evaluate protein identification, we considered that the protein scores must be significant (P < 0.05).
2 Results 2.1 Toxicity of Cd, Hg, and TCB to wheat seedlings Figure 1 shows the effects of Cd, Hg, and TCB on the dry weight of roots and leaves of wheat seedlings compared to the control. The dry weights of both root and shoot were generally lower under stress conditions than in the control. There were highly significant reduction (P < 0.01) of both root and shoot dry weight at 1.0, 0.05, and 0.056 mmol/L for Cd, Hg, and TCB, respectively. The result showed 5.1% and 13.7% reduction at 0.5 and 1.0 mmol/L Cd for shoot dry weight and by 6.1% and 18.4% for root dry weight at 0.5 and 1.0 mmol/L Cd after 5 d treatment. TCB decreased shoot (or root) dry weights from 0.014 (or 0.028) to 0.056 mmol/L, while a concentration as low as 0.0125 of Hg decreased the shoot and root dry weights significantly (P < 0.05). These results confirm the inhibitory effects of Cd, Hg, and TCB on the growth of wheat. The MDA contents were consistently higher in the treated leaves compared to treated roots. MDA from 0.196 μmol/L in the control roots increased to 0.338, 0.256 or 0.262 μmol/L in 1.0 mmol/L Cd, 0.05 mmol/L Hg or 0.056 mmol/L TCB treated roots (Fig. 2). Furthermore, MDA accumulation in the leaves was also significant (P < 0.05) at 0.5, 0.75 mmol/L Cd, 0.025, 0.0375, 0.05 mmol/L Hg and 0.028, 0.042, 0.056 mmol/L TCB. The results confirm that Cd, Hg, and TCB stresses caused lipid peroxidation in wheat seedlings. 2.2 Effect of Cd and TCB on protein phosphorylation in wheat leaves Five phosphorylated proteins from the leaf extracts of
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Fig. 1 Inhibitory effects of Cd, Hg and TCB on wheat seedlings after 5 d of treatments. Each value is the mean of three individual triplicates ± SD. * and ** indicate the difference significantly from the control at P < 0.05 and P < 0.01, respectively.
Fig. 2 Effects of Cd, Hg and TCB on MDA contents in wheat seedlings after 5 d of treatments. Each value is the mean of three individual triplicates ± SD. * indicates the difference significantly from the control at P < 0.05.
the treated seedlings were detected by SDS-PAGE and autoradiography (Fig. 3). It was detected that 5 d stress of Cd and TCB enhanced the phophorylations of all detected proteins in the leaves. For example, PP1 and PP4 phosphorylated proteins were not detected in the control leaves but were strongly induced by Cd and TCB stresses. The results demonstrated that protein phophorylation in wheat leaves is stimulated by Cd and TCB stresses.
Fig. 3 Effects of 0.5 mmol/L Cd and 0.056 mmol/L TCB on protein phosphorylation in wheat leaves after 5 d of treatments. In vitro protein phosphorylation was performed at 30°C in 100 μL reaction mixture containing 20 mmol/L Tris-HCl pH 7.5, 20 mmol/L β-glycerol phosphate, 10 mmol/L MgCl2 , 10 μmol/L Na3 VO4 , 0.5 μCi [γ-32 P] ATP, and 20 μL leaf extracts. ck and TCB, Cd represent control and the wheat seedlings being exposed to 0.056 mmol/L TCB, 0.5 mmol/L Cd for 5 d, respectively.
2.3 Effects of Cd, Hg and TCB on proteome in wheat leaves In order to investigate the changes of wheat proteome in response to Cd, Hg and TCB stresses, 2-DE analysis of the total proteins in Yangmai 13 leaves was carried out. Figures 4 and 5 present the Cd-, Hg- and TCB-induced proteins in the leaves. In the 2-DE analysis, 0.05 mmol/L Hg stress lowered the quantity of proteins in the PEG fractionation F3 in the leaves of Yangmai 13 (Fig. 4), this result suggests that Hg stress inhibits protein biosynthesis or causes proteins to be precipitated into fractionation F1 or F2. Comparing with the results from 0.05 mmol/L Hg stress, 0.5 mmol/L Cd and 0.056 mmol/L TCB stresses induced more proteins in the PEG fractionation F3. In this study, twelve TCBinduced proteins (F3 T1 to F3 T12 ) and ten Cd-induced proteins (F3 C1 to F3 C10 ) were excised from the gels by mass spectrometer analysis. The data in Fig. 5 also confirm the inhibition of Hg stress on protein synthesis. More TCB- and Cd-induced proteins occurred in the PEG F4 fractionation from the leaves of Yangmai 13. Twelve TCB-induced proteins (F4 T1 to F4 T12 ) and ten Cd-induced proteins (F4 C1 to F4 C10 ) were excised from the gels by MS analysis. Most of the proteins in the leaf tissues were induced by both the Cd and TCB stresses. Several proteins were induced by only Cd or TCB stress. For example, F3 C4 , F3 C8 , F3 C9 in the PEG fractionation F3 and F4 C7 , F4 C8 in F4 were induced by the Cd stress, whereas, F3 T1 , F3 T6 , F3 T11 in the F3 and F4 T4 , F4 T5 , F4 T7 , F4 T12 in the F4 were induced by the TCB stress.
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Fig. 4 The 2-DE analysis of proteins from the PEG fractionation F3 in wheat leaves. About 400 μg protein samples was loaded on an 11 cm IPG strip with a linear gradient of pH 4–7 for IEF, following electrophoresis of 12.5% SDS-PAGE and Bio-safe colloidal Coomassie Blue G-250 staining. The Cd, Hg, and TCB inducible protein spots are pointed with the arrows. The protein profiles ck and TCB, Cd, Hg represent control, 0.056 mmol/L TCB, 0.5 mmol/L Cd, 0.05 mmol/L Hg stress for 5 d, respectively.
2.4 Identification of Cd- and TCB-induced proteins in Yangmai 13 leaves For the identification of the Cd- and TCB-induced proteins, the peptide mass fingerprints of above excised proteins were determined by means of MALDI-TOF MS. Twenty three proteins were identified to be associated by a search performed in the NCBInr protein database (Tables 1 and 2). The identified proteins in Yangmai 13 leaves based on Cd and/or TCB stresses could be classified into different groups on the basis of their functions. These groups includes protein phosphorylation regulation related enzymes, protein configuration protection related proteins, H+ transmembrane transportation related enzymes, a protein SET domain group 40 and Rubisco activase, methionine metabolism related enzymes and finally ethylene, defense substance as well as cell wall compound synthesis related enzymes.
3 Discussion Dose-dependent inhibitory effects of Cd, Hg, and TCB on root and shoot growth were observed. The toxic effects of Cd, Hg, and TCB to wheat seedlings could arise at a physiological level leading to water deficit (data not shown) and causing lipid peroxidation.
3.1 Response of protein modification In this study, we tried to elucidate the molecular mechanism of wheat in response to Cd, Hg, and TCB stresses both at the levels of protein expression and posttranslational modification. Studies indicate that protein phosphorylation plays an important regulatory role in the delivery and response to different biotic and abiotic stress signals, and also induces a variety of stress signal-induced gene expressions (Manzanero et al., 2002; Gerber and Dubery, 2004). In this study, we found protein phophorylation in Yangmai 13 leaves to be promoted by both Cd and TCB. This corresponds with the results of Subramanian et al. (2004), that Cd and TCB induced the expressions of protein phosphorylation regulation related enzymes. As shown by the proteome analysis, the 14-3-3-like protein A in the leaves was induced by TCB and Cd. The 14-3-3 proteins regulated the activities of protein kinases, such as MAK and PAK. Similarly, a putative PAS/PAC sensor protein in Yangmai 13 leaves was significantly induced by Cd and TCB; this PAS/PAC sensor protein had several roles in regulating the kinase catalytic activity (Amezcua et al., 2002). A protein phosphatase 2A 59 kDa regulatory subunit B’ gamma isoform (PR59/B’) which was found to be induced by TCB catalyzes the de-phosphorylation specific for phosphoserine and phosphothreonine residues generally expressed in plant cells. Consequently, this suggest that, by inducing the expression of protein phosphorylation
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Fig. 5
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The 2-DE analysis of proteins in the PEG fractionation F4 from wheat leaves.
Table 1 Identification of 0.5 mmol/L Cd-induced proteins in wheat leaves Protein spot
Protein identification
Protein scores*
Matched peptide
Sequence coverage (%)
Theoretical Mr/pI
F 4 C3
METE MESCR: Cobalamin-independent methionine synthase isozyme (common ice plant) Q6BCT3 HORVU: methionine synthase (barley) T03581: dnaK-type molecular chaperone Bip (rice) PWWTB: H+ -transporting two-sector ATPase beta chain (wheat chloroplast) 1A1C SOYBN: 1-aminocyclopropane-1-carboxylate synthase (ACC synthase) (soybean) CB21 SINAL: chlorophyll a/b binding protein 1 (sinapis alba) gi|145216925: putative PAS/PAC sensor protein [Mycobacterium gilvum PYR-GCK] Magnesium chelatase subunit of protochlorophyllide reductase Q9ZR33 WHEAT: UDP-glucose protein transglucosylase (Triticum aestivum) SDG40 ARATH: protein SET DOMAIN GROUP 40 (Arabidopsis thaliana) ATP synthase beta subunit (Fragment). Entransia fimbriata Rubisco activase (Fragment) (barley)
54
12
20
84.8/5.90
152 86 91 57
17 16 13 8
27 28 26 13
84.4/5.68 73.5/5.30 53.8/5.06 54.7/5.84
59 81
7 8
31 18
26.2/5.17 41.8/5.21
60 100 58 82 89
8 14 10 13 8
21 34 20 28 21
39.5/5.28 41.47/5.82 55.8/5.35 44.4/5.10 47.5/5.64
F 4 C2 F 4 C1 F 4 C4 F 4 C5 F 4 C9 F4 C10 F 4 C8 F 4 C6 F 3 C2 F 3 C5 F 3 C6
* Protein scores greater than 54 or 78 are significant (P < 0.05). Mr: molecule weight; pI: isoelectric point.
related enzymes in wheat leaves under Cd and/or TCB stress, promotes the reversible protein phosphorylation. This conversely elicits appropriate responses through the regulation of stress signal-induced gene expression and defense-associated enzymatic activity. Protein methylation has been an important aspect of protein modifications in plants and animals. In the present study, we found several enzymes related to the methyl
cycle and transfer being induced by Cd and TCB. This is supported by the expressions of two different methionine synthases (Ms) in Yangmai 13 leaves enhanced by Cd and TCB stresses. Ms catalyze the formation of methionine (Met) and in activated methyl cycle further converted into S-adenosylmethionine (SAM). Since SAM provides the methyl group for protein and DNA methylation, the effects of Cd and TCB on the expression of SET domain-
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Table 2 Identification of 0.056 mmol/L TCB-induced proteins in wheat leaves Protein spot
Protein identification
Protein scores*
Matched peptide
Sequence coverage (%)
Theoretical Mr/pI
F 4 T1 F 4 T2 F 4 T3 F 4 T4 F 4 T7 F 4 T9 F 4 T5 F 4 T6 F4 T8 F4 T12
Q8GSN3 CUCMA: non-cell-autonomous heat shock cognate protein 70 Q5MGA8 MEDSA: heat shock protein 70–Medicago sativa (Alfalfa) Q1W681 WHEAT: vacuolar proton–ATPase subunit A (Triticum aestivum) BAB47031: H+ -transporting two-sector ATPase alpha chain (Triticum aestivum) Q6XXZ2 LOTJA: Gamma-glutamylcysteine synthetase (Lotus japonicus) Q1W2K1 CAMSI: 14-3-3-like protein A (barley) BAB47031: H+ -transporting two-sector ATPase alpha chain (Triticum aestivum) PWWTAM: H+ -transporting two-sector ATPase alpha chain (wheat mitochondrion) PWWTB : H+ -transporting two-sector ATPase beta chain (wheat chloroplast) 2A5G ARATH: serine/threonine protein phosphatase 2A 59 kDa regulatory subunit B’ gamma isoform (Arabidopsis thaliana) S23452: sedoheptulose-bisphosphatase precursor (wheat)
92 84 206 157 62 113 274 197 80 60
16 10 24 19 10 14 28 22 13 10
30 22 51 39 19 48 50 43 31 23
71.4/5.10 71.0/5.08 68.4/5.23 55.3/6.11 55.9/6.22 29.3/4.83 55.3/6.11 55.2/5.7 53.8/5.06 59.1/8.04
82
8
22
42.0/6.04
F 3 T6
* Protein scores greater than 54 or 78 are significant (P < 0.05). Mr: molecule weight. pI: isoelectric point.
containing proteins, responsible for protein methylation are important. As shown by proteomic data in the results, we found a protein SET domain group 40 (SDG40) to be induced by Cd and TCB in Yangmai 13 leaves. SDG40 can modify methionine residue to form an α Nmethylmethionine (Ng et al., 2007). Many types of stresses (such as osmotic or heavy metals) can generate active oxygen species (AOS) in the chloroplasts. In Rubisco, one of the amino acids most susceptible to oxidation is Met. Oxidation of Met leads to the formation of methionine sulfoxide residues (Stadtman et al., 2005), which often results in functional inactivation and targeting for proteolytic degradation (Brot and Weissbach, 2000). Although the functional significance of Rubisco methylation is unknown, the induction of SDG40 leads us to hypothesize that methylation of Met may be related to the stability through a reduction in the proteolytic susceptibility to Rubisco (Zheng et al., 1998). In addition, Rubisco activase appears to catalyze a rather unusual posttranslational modification of Rubisco, which results in the conformation and activity changes (Portis, 2003). In this study, we also found a Rubisco activase in Yangmai 13 leaves being induced by Cd and TCB. It is obvious that the induction of the Rubisco activase may be favorable for rapid activation of Rubisco in response to Cd and TCB stresses. 3.2 Responses of protein configuration protection Heat shock response in organisms can confer protection from damage due to exposure to a wide variety of stresses (Mayer and Bukau, 2005). The most abundant and widely studied group of stress proteins is the heat shock protein 70 (Hsp70 ) family (including molecular chaperone binding protein, Bip). Cellular functions of Hsp70 include refolding or degradation of denatured proteins (Bierkens et al., 1998). A dnaK-type molecular chaperone Bip and two Hsp70 were found to be induced by Cd and TCB in this study. We suggest that the induction of Hsp70 and Bip assist the damaged and misfolded proteins to reconstitute into normal three-dimensional structure, thereby reestablish their biological functions, or participating in pathways of protein degradation, thus contribute to the elimination of denatured proteins (Terlecky et al., 1992).
3.3 Responses of detoxification As shown in Fig. 3, an important aspect of Cd, Hg and TCB toxicity to wheat seedlings was their induction of lipid peroxidation, which was displayed as an increase in MDA content. Therefore, the synthesis of defense substances is necessary to counteract the effect of these stresses. Glutathione (GSH) plays a vital role in defending against toxins and free radicals (Moellering et al., 1998). The rate-limiting step of GSH synthesis was catalyzed by γ-glutamylcysteine synthetase (GCS) (Richman and Meister, 1975). In the experiment, the GCS in Yangmai 13 leaves was found to be up-regulated under TCB stress. This in conformity with the conclusion by Myhrstad et al. (2001) that induction of the GCS promotes the synthesis of GSH which provides wheat seedlings with multiple defenses not only against AOS but also against other toxicities. Furthermore, because the induction of Ms cellular SAM level was increased under Cd and TCB stresses. SAM also provides methyl for the production of compatible solutes, such as glycinebetaine and methylated polyols (Bohnert and Jensen, 1996). Their accumulation in the cytoplasm can regulate osmotic balance. Another benefit of SAM is for synthesis of polyamines as substrate for maintaining osmotic balance and eliminating AOS. 3.4 Responses of H+ transmembrane transportation In this study, the H+ -transporting two-sector ATPase alpha and beta chain in Yangmai 13 leaves was induced by Cd and TCB, which is essential to maintain the ATP high level required by the stressed cells. Furthermore, we also found the vacuolar proton-ATPase (V-ATPase) subunit A induced by Cd and TCB. Fukuda et al. (2004) demonstrated that V-ATPase plays an essential role in plant responses to environmental stresses. V-ATPase uses the energy derived from the hydrolysis ATP to establish the electrochemical gradient of H+ across vacuolar membrane (Sun-Wada et al., 2003), which is also the driving force for the accumulation of toxic ions and other solutes in the vacuole (Hamilton et al., 2001). Further studies are needed to elucidate whether the induction of V-ATPase promotes the accumulation of Cd ions or TCB molecules in the vacuoles.
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3.5 Responses of ethylene and cell wall component synthesis In this study, ACC synthase in Yangmai 13 leaves was induced by Cd and TCB. The induction of ACC synthase can stimulate ethylene production as approved by Wong et al. (2001). Ethylene, as a mediator of the stressful signal, plays an important defense role such as regulating plant growth and development, inducing the expression of various defensive genes in response to Cd and TCB stresses (O’Donnell et al., 1996). The induction of UDP-glucose protein transglucosylase (UPTG) was also observed in Yangmai 13 leaves under Cd and TCB stresses. Because UPTG and its homologous proteins play important roles in synthesizing cell wall components (Dhugga et al., 1997). This agrees with the statemnet of Ge et al. (2007) that the induction of UPTG promotes synthesis of cell wall components and stimulate cell wall thickening. Furthermore, it is worth notice that the most of the responses induced by both heavy metal (e.g., Cd) and organic pollutant (e.g., TCB) in this study imply that there are few defensive mechanisms in wheat in response to the stresses of chemical pollutants. However, it indicated that when wheat seedlings undergo different kinds of chemical pollution, the optimum choice may be to activate the welldeveloped mechanisms that have been evolved to cope with their ever-changing environmental factors.
4 Conclusions The present study indicates that Cd, Hg, and TCB have toxic effects on wheat seedlings, such as inhibiting root and shoot growth, causing lipid peroxidation. However, in response to Cd and TCB toxicity, different defenseassociated processes were induced in the seedlings of Yangmai 13 leaves, which included protein modification, protein configuration protection, detoxification, H+ across membrane transportation, and ethylene and cell wall component synthesis. These confirm the induction and expression of various defensive genes in response to Cd and TCB stresses in wheat seedlings. Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 30300026) and the Application of Nuclear Techniques in Agriculture from the Chinese Ministry of Agriculture (No. 200803034). We would like to thank Dr. Jun Li (Jiangsu University) for helping with MALDI-TOF MS analysis.
References Amezcua C A, Harper S M, Rutter J, Gardner K H, 2002. Structure and interactions of PAS kinase N-terminal PAS domain: model for intramolecular kinase regulation. Structure (Camb.), 10: 1349–1361. Bierkens J, Maes J, Plaetse F V, 1998. Dose-dependent induction of heat shock protein 70 synthesis in Raphidocelis subcapitata following exposure to different classes of envi-
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ronmental pollutants. Environmental Pollution, 101: 91–97. Bohnert H J, Jensen R G, 1996. Strategies for engineering waterstress tolerance in plants. Trends Biotechnology, 14: 89–97. Brot N, Weissbach H, 2000. Peptide methionine sulfoxide reductase: Biochemistry and physiological role. Biopolymers, 55: 288–296. Cao Q, Hu Q H, Khan S, Wang Z J, Lin A J, Du X, Zhu Y G, 2007. Wheat phytotoxicity from arsenic and cadmium separately and together in solution culture and in a calcareous soil. Journal of Hazardous Materials, 148: 377–382. Cavallini A, Natali L, Durante M, Masertib B, 1999. Mercury uptake, distribution and DNA affinity in durum wheat (Triticum durum Desf.) plants. The Science of the Total Environment, 243/244: 119–127 Dhindsa R S, Dhinsa P P, Thorpe T A, 1980. Leaf senescence correlated with increased levels of membrane permeability and lipidperoxidation and decreased levels of superoxide dismutase and catalase. Journal of Experimental Botany, 32: 127–132. Dhugga K S, Tiwari S C, Ray P M, 1997. A reversibly glycosylated polypeptide (RGP1) possibly involved in plant cell wall synthesis: Purification, gene cloning and transGolgi localization. Proceedings of the National Academy of Sciences of the United States of America, 94: 7679–7684. Fukuda A, Chiba K, Maeda M, Nakamura A, Maeshima M, Tanaka Y, 2004. Effect of salt and osmotic stresses on the expression of genes for the vacuolar H+ -pyrophosphatase, H+ -ATPase subunit A, and Na+ /H+ antiporter from barley. Journal of Experimental Botany, 55: 585–594. Ge C L, Wan D Z, Wang Z G, Ding Y, Wang Y L, Shang Q, Ma F, Luo S S, 2007. Response of rice root to 1,2,4trichlorobenzene stress. Acta Agronomica Sinica, 33(12): 1991–2000. Ge C L, Wan D Z, Wang Z G, Ding Y, Wang Y L, Shang Q, Ma F, Luo S S, 2008. A proteomic analysis of rice seedlings responding to 1,2,4-trichlorobenzene stress. Journal of Environmental Sciences, 20(3): 309–319. Gerber I B, Dubery I A, 2004. Protein phosphorylation in Nicotiana tabacum cells in response to perception of lipopolysaccharides from Burkholderia cepacia. Phytochemistry, 65: 2957–2966. Groppa M D, Benavides M P, Tomaro M L, 2003. Polyamine metabolism in sunflower and wheat leaf discs under cadmium or copper stress. Plant Science, 164: 293–299. Hamilton C A, Good A G, Taylor G J, 2001. Induction of vacuolar ATPase and mitochondrial ATP synthase by aluminium in aluminium-resistant cultivar of wheat. Plant Physiology, 125: 2068–2077. Lin R Z, Wang X R, Luo Y, Du W C, Guo H Y, Yin D Q, 2007. Effects of soil cadmium on growth, oxidative stress and antioxidant system in wheat seedlings (Triticum aestivum L.). Chemosphere, 69: 89–98. Lindberg S, Landberg T, Greger M, 2007. Cadmium uptake and interaction with phytochelatins in wheat protoplasts. Plant Physiology and Biochemistry, 45: 47–53. Liu X L, Zhang S Z, Shan X Q, Christie P, 2007. Combined toxicity of cadmium and arsenate to wheat seedlings and plant uptake and antioxidative enzyme responses to cadmium and arsenate co-contamination. Ecotoxicology and Environmental Safety, 68: 305–313. Liu X L, Zhang S Z, 2007. Intraspecific differences in effects of co-contamination of cadmium and arsenate on early seedling growth and metal uptake by wheat. Journal of Environmental Sciences, 19: 1221–1227.
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Machado E L, Da Silva A C, Da Silva M J, Leite A, Ottoboni L M M, 2002. Endogenous protein phosphorylation and casein kinase activity during seed development in Araucaria angustifolia. Phytochemistry, 61: 835–842. Manzanero S, Rutten T, Kotseruba V, Houben A, 2002. Alterations in the distribution of histone H3 phosphorylation in mitotic plant chromosomes in response to cold treatment and the protein phosphatase inhibitor cantharidin. Chromosome Research, 10: 467–476. Mayer M P, Bukau B, 2005. Hsp 70 chaperones: cellular functions and molecular mechanism. Cellular and Molecular Life Sciences, 62: 670–684. Moellering D, McAndrew J, Patel R P, Cornwell T, Lincoln T, Cao X et al., 1998. Nitric Oxide-dependent induction of glutathione synthesis through increased expression of γglutamylcysteine synthetase. Archives of Biochemistry and Biophysics, 358(1): 74–82. Myhrstad M C, Husberg C, Murphy P, Nordstrom O, Blomhoff R, Moskaug J O, Kolsto A B, 2001. TCF11/Nrf1 overexpression increases the intracellular glutathione level and can transactivate the gamma-glutamylcysteine synthetase (GCS) heavy subunit promoter. Biochimica et Biophysica Acta, 1517: 212–219. Ng D W K, Wang T, Chandrasekharan M B, Aramayo R, Kertbundit S, Hall T C, 2007. Plant SET domain-containing proteins: Structure, function and regulation. Biochimica et Biophysica Acta, 1769: 316–329. O’Donnell P J, Calvert C, Atzorn R, Wasternack C, Leyser H M O, Bowles D J, 1996. Ethylene as a signal mediating the wound response of tomato plants. Science, 274: 1914–1917. Portis A R, 2003. Rubisco activase – Rubisco’s catalytic chaperone. Photosynthesis Research, 75: 11–27. Richman P G, Meister A, 1975. Regulation of γ-glutamylcysteine synthetase by nonallosteric feedback inhibition by glutathione. The Journal of Biological Chemistry, 250: 1422– 1426. Simpson R J, 2003. Proteins and Proteomics: A Laboratory Manual. New York: Cold spring harbor laboratory press. Stadtman E R, Remmen H V, Richardson A, Wehr N B, Levine R L, 2005. Methionine oxidation and aging. Biochimica et Biophysica Acta, 1703: 135–140. Singh H P, Batish D R, Kaur G, Arora K, Kohli R K, 2008. Nitric oxide (as sodium nitroprusside) supplementation
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ameliorates Cd toxicity in hydroponically grown wheat roots. Environmental and Experimental Botany, 63: 158–167. Subramanian R R, Zhang H Y, Wang H N, Ichijo H, Miyashita T, Fu H A, 2004. Interaction of apoptosis signal-regulating kinase 1 with isoforms of 14-3-3 proteins. Experimental Cell Research, 294: 581– 591. Sun Q, Wang X R, Ding S M, Yuan X F, 2005. Effects of exogenous organic chelators on phytochelatins production and its relationship with cadmium toxicity in wheat (Triticum aestivum L.) under cadmium stress. Chemosphere, 60: 22– 31. Sun-Wada G H, Wada Y, Futai M, 2003. Vacuolar H+ pumping ATPases in luminal acidic organelles and extracellular compartments: common rotational mechanism and diverse physiological roles. Journal of Bioenergetics and Biomembranes, 35: 347–358. Terlecky S R, Chiang H L, Olson T J, Dice J F, 1992. Protein and peptide binding and stimulation of in vitro lysosomal proteolysis by the 73-kDa heat-shock cognate protein. The Journal of Biological Chemistry, 267: 9202–9209. Wang Z G, Wan D Z, Yang Y C, Ge C L, Ma F, Yang J C, 2006. Effects of 1,2,4-trichlorobenzene and naphthalene on grain yield and quality of rice. Journal of Rice Science, 20(3): 295–300. Wong W S, Li G G, Ning W, Xu Z F, Hsiao W L W, Zhang L Y, Li N, 2001. Repression of chilling-induced ACC accumulation in transgenic citrus by over-production of antisense 1-aminocyclopropane-1-carboxylate synthase RNA. Plant Science, 161: 969–977. Xi J H, Wang X, Li S Y, Zhou X, Yue L, Fan J, Hao D Y, 2006. Polyethylene glycol fractionation improved detection of low-abundant proteins by two-dimensional electrophoresis analysis of plant proteome. Phytochemistry, 67: 2341– 2348. Yannarelli G G, Fernandez-Alvarez A J, Santa-Cruz D M, Tomaro M L, 2007. Glutathione reductase activity and isoforms in leaves and roots of wheat plants subjected to cadmium stress. Phytochemistry, 68: 505–512. Zheng Q, Simel E J, Klein P E, Royer M T, Houtz R L, 1998. Expression, purification, and characterization of recombinant ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit Nε -Methyltransferase. Protein Expression and Purification, 14: 104–112.
Responses of wheat seedlings to cadmium, mercury and trichlorobenzene stresses GE Cailin1,2,∗, DING Yan1 , WANG Zegang2 , WAN Dingzhen2 , WANG Yulong1 , SHANG Qi2 , LUO Shishi2 1. Jiangsu Provencial Key Laboratory of Crop Genetics and Physiology, Yangzhou University, Yangzhou 225009, China. E-mail: [email protected] 2. College of Bioscience and Biotechnology, Yangzhou University, Yangzhou 225009, China Received 31 May 2008; revised 14 July 2008; accepted 13 August 2008
Abstract The molecular response of wheat (Triticum aestivum L., cv. Yangmai 13) seedlings to heavy metal (Cd, Hg) and 1,2,4trichlorobenzene (TCB) stresses were examined by two-dimensional gel electrophoresis, image analysis, and peptide mass fingerprinting. The results showed inhibitions of root and shoot growth by Cd, Hg, and TCB. These stresses led to water deficit and lipid phosphorylation in the seedling which also promoted protein phophorylation in the leaves. Hg stress inhibited protein synthesis while Cd and TCB stresses induced or up-regulated more proteins in the leaves. Most of these induced proteins played important roles in the biochemical reactions involved in tolerance of wheat to Cd and TCB stresses. The primary functions of Cd- and TCB-induced proteins included methionine metabolism, Rubisco modification, protein phosphorylation regulation, protein configuration protection, H+ transmembrane transportation and also the synthesis of ethylene, defense substances and cell wall compounds. Key words: wheat; proteomics; chemical pollutant; stress response DOI: 10.1016/S1001-0742(08)62345-1
Introduction Chemical pollution has been a worldwide problem as a result of rapid increase in chemical inputs in agricultural production in the past decades. Most of chemical pollutants in soil and water cause disturbances in crop growth and development, and subsequently decreased crop productivity. Recent reports on the toxic effects of heavy metals (especially Cd) of wheat indicate that heavy metals inhibit root and shoot growth (Liu and Zhang, 2007; Cao et al., 2007), and also induce oxidative stress and lipid peroxidation (Singh et al., 2008). Cavallini et al. (1999) also reported that certain heavy metal ions (such as Hg) tend to form covalent bonds with DNA. Various defense mechanisms adopted by wheat to avoid heavy metal toxicity have been reported by several researchers, these include alteration of antioxidant enzyme level (Liu et al., 2007; Lin et al., 2007; Yannarelli et al., 2007), increase in the content of phytochelatin (Sun et al., 2005; Lindberg et al., 2007), and also increased generation of polyamine and ethylene (Groppa et al., 2003). As compared to heavy metals, reports on the toxic effects of 1,2,4-trichlorobenzene (TCB) on crops are limited (Wang et al., 2006; Ge et al., 2007, 2008), and with no report on wheat. The present study reports the molecular responses of wheat to Cd, Hg, and TCB stresses, the * Corresponding author. E-mail: [email protected]
physiological toxicity of Cd, Hg, and TCB in wheat, and the Cd-, TCB-induced proteins in wheat leaves.
1 Materials and methods 1.1 Seedlings cultivation, treatment and toxicity assay Seeds of wheat (Triticum aestivum L.) variety Yangmai 13 were surface-sterilized in 3% (V/V) H2 O2 for 5 min and then rinsed with deionized water before germination on moist filter paper at 30°C for 3 d. Seedlings were transferred into colored vitreous pots containing 100-mL Hoagland nutrient solution, and grown in a growth chambers with regulated day/night temperatures 25/18°C and the light intensity range 250–300 μmol/(m2 ·s). Roots at three-leave stage seedlings were submerged in the test solutions containing 0.25, 0.5, 0.75, 1.0 mmol/L CdCl2 , 0.0125, 0.025, 0.0375, 0.05 mmol/L HgCl2 , and 0.014, 0.028, 0.042, 0.056 mmol/L TCB. Treatment combinations of the three chemicals were chosen based on preliminary studies in our laboratory (unpublished). Solutions were renewed daily within the 5 d treatment period to maintain their concentrations. The inhibitory effects of Cd, Hg, and TCB on wheat seedlings were determined by root and shoot dry weight after 5 d treatment period. The levels of lipid peroxidation in wheat leaves were measured in terms of malondialdehyde
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(MDA) content as reported by Dhindsa et al. (1980), and MDA content expressed in μmol/g fw (fresh weight). All measurements were in three replicates with data presented in means ± standard deviations (SD). Statistical analyses were performed using SPSS statistical package software (version 10.0). Comparisons between control and other treatments were evaluated by one-way ANOVA and the least-significant-differences (LSD) test. 1.2 In vitro protein phosphorylation analysis Fresh leaf tissues weighing 0.5 g each were sampled from seedlings treated with 0.5 mmol/L Cd and 0.056 mmol/L TCB for 5 d. These samples were homogenized in three volumes of extraction buffer (containing 50 mmol/L Tris-HCl pH 7.4, 150 mmol/L NaCl, 1% Triton X-100, 1 mmol/L EDTA, 5 μmol/L Na3 VO4 , 2 μmol/L Okadaic acid, 1 mmol/L PMSF). The homogenates were centrifuged at 12000 ×g, 4°C for 10 min. The soluble protein contents were determined by the Coomassie Brilliant Blue Method. About 20 μL soluble protein was added to the reaction mixture containing 20 mmol/L Tris-HCl pH 7.5, 20 mmol/L β-glycerol phosphate, 10 mmol/L MgCl2 , 10 μmol/L Na3 VO4 , 0.5 μCi [γ-32 P] ATP. The reaction mixture was incubated at 30°C for 30 min, and phosphorylation reactions were stopped by adding protein loading buffer with 3 min boiling. Proteins in the reaction mixture were separated by SDS-PAGE (12.5% polyacrylamide). The gels were stained in Bio-safe colloidal Coomassie Blue G-250 (Bio-Rad Company, USA), and dried over a filter paper. Phosphorylated protein bands were detected by autoradiography using Kodak medical X-ray film (Machado et al., 2002). 1.3 Proteomic analysis 1.3.1 Protein extraction Proteins from the leaves treated with 0.5 mmol/L Cd, 0.05 mmol/L Hg, and 0.056 mmol/L TCB for 5-d were extracted using polyethylene glycol (PEG) fractionation (Xi et al., 2006). All fractions of extracted proteins were rinsed with ice-cold acetone containing 0.07% (V/V) βmercaptoethanol, then air-dried and stored at –20°C. 1.3.2 Two-dimensional gel electrophoresis Above extracted proteins of F3 and F4 fractions were resuspended in rehydration buffer (Bio-Rad Company, USA) and incubated for 1 h at room temperature. After centrifugation at 12000 ×g at 4°C for 10 min, protein concentration in the supernatant was measured by Coomassie Brilliant Blue Method. IPG strip (Bio-Rad) of 11 cm (pH 4–7) was rehydrated in rehydration buffer (containing 400 μg protein sample) for 16 h to allow proteins to be uptaken. Iso-electric focusing (IEF) was performed using the PROTEAN IEF system (Bio-Rad Company, USA) at 20°C. Prior to second dimension analysis, the strips were equilibrated for 15 min in the equilibration buffer 1 (BioRad), and equilibrated for 15 min in the equilibration buffer 2 (Bio-Rad). Protein separation of the second dimension was carried out on a 12.5% SDS-PAGE. After 2-DE
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separation, the gels were washed twice (5 min each) with double distilled water, and transferred into Bio-safe colloidal Coomassie Blue G-250 staining for 8 h. After which the gels were washed three times (1 h each). 1.3.3 Image analysis Gels were scanned by Gel Doc XR system (Bio-Rad, USA). The protein spots were analyzed by PDQuest 2D analysis software (Bio-Rad). The molecular weight and isoelectric point of differential proteins were calculated. After analysis, the selected protein spots were manually excised from the gel and stored at –20°C. 1.4 Identification of Cd- and TCB-induced proteins The peptide mass fingerprints of Cd- and TCBinduced proteins were analyzed by mass spectrometer (MALDI-TOF MS, Applied Biosystem, USA) according to “Proteins and Proteomics: A Laboratory Manual” (Simpson, 2003). The peptide mass fingerprints were used for searching in MSDB, NCBInr or SwissProt database using MASCOT software search engine (http://www.matrixscience.com) to identify Cd- and TCBinduced proteins. In order to evaluate protein identification, we considered that the protein scores must be significant (P < 0.05).
2 Results 2.1 Toxicity of Cd, Hg, and TCB to wheat seedlings Figure 1 shows the effects of Cd, Hg, and TCB on the dry weight of roots and leaves of wheat seedlings compared to the control. The dry weights of both root and shoot were generally lower under stress conditions than in the control. There were highly significant reduction (P < 0.01) of both root and shoot dry weight at 1.0, 0.05, and 0.056 mmol/L for Cd, Hg, and TCB, respectively. The result showed 5.1% and 13.7% reduction at 0.5 and 1.0 mmol/L Cd for shoot dry weight and by 6.1% and 18.4% for root dry weight at 0.5 and 1.0 mmol/L Cd after 5 d treatment. TCB decreased shoot (or root) dry weights from 0.014 (or 0.028) to 0.056 mmol/L, while a concentration as low as 0.0125 of Hg decreased the shoot and root dry weights significantly (P < 0.05). These results confirm the inhibitory effects of Cd, Hg, and TCB on the growth of wheat. The MDA contents were consistently higher in the treated leaves compared to treated roots. MDA from 0.196 μmol/L in the control roots increased to 0.338, 0.256 or 0.262 μmol/L in 1.0 mmol/L Cd, 0.05 mmol/L Hg or 0.056 mmol/L TCB treated roots (Fig. 2). Furthermore, MDA accumulation in the leaves was also significant (P < 0.05) at 0.5, 0.75 mmol/L Cd, 0.025, 0.0375, 0.05 mmol/L Hg and 0.028, 0.042, 0.056 mmol/L TCB. The results confirm that Cd, Hg, and TCB stresses caused lipid peroxidation in wheat seedlings. 2.2 Effect of Cd and TCB on protein phosphorylation in wheat leaves Five phosphorylated proteins from the leaf extracts of
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Fig. 1 Inhibitory effects of Cd, Hg and TCB on wheat seedlings after 5 d of treatments. Each value is the mean of three individual triplicates ± SD. * and ** indicate the difference significantly from the control at P < 0.05 and P < 0.01, respectively.
Fig. 2 Effects of Cd, Hg and TCB on MDA contents in wheat seedlings after 5 d of treatments. Each value is the mean of three individual triplicates ± SD. * indicates the difference significantly from the control at P < 0.05.
the treated seedlings were detected by SDS-PAGE and autoradiography (Fig. 3). It was detected that 5 d stress of Cd and TCB enhanced the phophorylations of all detected proteins in the leaves. For example, PP1 and PP4 phosphorylated proteins were not detected in the control leaves but were strongly induced by Cd and TCB stresses. The results demonstrated that protein phophorylation in wheat leaves is stimulated by Cd and TCB stresses.
Fig. 3 Effects of 0.5 mmol/L Cd and 0.056 mmol/L TCB on protein phosphorylation in wheat leaves after 5 d of treatments. In vitro protein phosphorylation was performed at 30°C in 100 μL reaction mixture containing 20 mmol/L Tris-HCl pH 7.5, 20 mmol/L β-glycerol phosphate, 10 mmol/L MgCl2 , 10 μmol/L Na3 VO4 , 0.5 μCi [γ-32 P] ATP, and 20 μL leaf extracts. ck and TCB, Cd represent control and the wheat seedlings being exposed to 0.056 mmol/L TCB, 0.5 mmol/L Cd for 5 d, respectively.
2.3 Effects of Cd, Hg and TCB on proteome in wheat leaves In order to investigate the changes of wheat proteome in response to Cd, Hg and TCB stresses, 2-DE analysis of the total proteins in Yangmai 13 leaves was carried out. Figures 4 and 5 present the Cd-, Hg- and TCB-induced proteins in the leaves. In the 2-DE analysis, 0.05 mmol/L Hg stress lowered the quantity of proteins in the PEG fractionation F3 in the leaves of Yangmai 13 (Fig. 4), this result suggests that Hg stress inhibits protein biosynthesis or causes proteins to be precipitated into fractionation F1 or F2. Comparing with the results from 0.05 mmol/L Hg stress, 0.5 mmol/L Cd and 0.056 mmol/L TCB stresses induced more proteins in the PEG fractionation F3. In this study, twelve TCBinduced proteins (F3 T1 to F3 T12 ) and ten Cd-induced proteins (F3 C1 to F3 C10 ) were excised from the gels by mass spectrometer analysis. The data in Fig. 5 also confirm the inhibition of Hg stress on protein synthesis. More TCB- and Cd-induced proteins occurred in the PEG F4 fractionation from the leaves of Yangmai 13. Twelve TCB-induced proteins (F4 T1 to F4 T12 ) and ten Cd-induced proteins (F4 C1 to F4 C10 ) were excised from the gels by MS analysis. Most of the proteins in the leaf tissues were induced by both the Cd and TCB stresses. Several proteins were induced by only Cd or TCB stress. For example, F3 C4 , F3 C8 , F3 C9 in the PEG fractionation F3 and F4 C7 , F4 C8 in F4 were induced by the Cd stress, whereas, F3 T1 , F3 T6 , F3 T11 in the F3 and F4 T4 , F4 T5 , F4 T7 , F4 T12 in the F4 were induced by the TCB stress.
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Fig. 4 The 2-DE analysis of proteins from the PEG fractionation F3 in wheat leaves. About 400 μg protein samples was loaded on an 11 cm IPG strip with a linear gradient of pH 4–7 for IEF, following electrophoresis of 12.5% SDS-PAGE and Bio-safe colloidal Coomassie Blue G-250 staining. The Cd, Hg, and TCB inducible protein spots are pointed with the arrows. The protein profiles ck and TCB, Cd, Hg represent control, 0.056 mmol/L TCB, 0.5 mmol/L Cd, 0.05 mmol/L Hg stress for 5 d, respectively.
2.4 Identification of Cd- and TCB-induced proteins in Yangmai 13 leaves For the identification of the Cd- and TCB-induced proteins, the peptide mass fingerprints of above excised proteins were determined by means of MALDI-TOF MS. Twenty three proteins were identified to be associated by a search performed in the NCBInr protein database (Tables 1 and 2). The identified proteins in Yangmai 13 leaves based on Cd and/or TCB stresses could be classified into different groups on the basis of their functions. These groups includes protein phosphorylation regulation related enzymes, protein configuration protection related proteins, H+ transmembrane transportation related enzymes, a protein SET domain group 40 and Rubisco activase, methionine metabolism related enzymes and finally ethylene, defense substance as well as cell wall compound synthesis related enzymes.
3 Discussion Dose-dependent inhibitory effects of Cd, Hg, and TCB on root and shoot growth were observed. The toxic effects of Cd, Hg, and TCB to wheat seedlings could arise at a physiological level leading to water deficit (data not shown) and causing lipid peroxidation.
3.1 Response of protein modification In this study, we tried to elucidate the molecular mechanism of wheat in response to Cd, Hg, and TCB stresses both at the levels of protein expression and posttranslational modification. Studies indicate that protein phosphorylation plays an important regulatory role in the delivery and response to different biotic and abiotic stress signals, and also induces a variety of stress signal-induced gene expressions (Manzanero et al., 2002; Gerber and Dubery, 2004). In this study, we found protein phophorylation in Yangmai 13 leaves to be promoted by both Cd and TCB. This corresponds with the results of Subramanian et al. (2004), that Cd and TCB induced the expressions of protein phosphorylation regulation related enzymes. As shown by the proteome analysis, the 14-3-3-like protein A in the leaves was induced by TCB and Cd. The 14-3-3 proteins regulated the activities of protein kinases, such as MAK and PAK. Similarly, a putative PAS/PAC sensor protein in Yangmai 13 leaves was significantly induced by Cd and TCB; this PAS/PAC sensor protein had several roles in regulating the kinase catalytic activity (Amezcua et al., 2002). A protein phosphatase 2A 59 kDa regulatory subunit B’ gamma isoform (PR59/B’) which was found to be induced by TCB catalyzes the de-phosphorylation specific for phosphoserine and phosphothreonine residues generally expressed in plant cells. Consequently, this suggest that, by inducing the expression of protein phosphorylation
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Fig. 5
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The 2-DE analysis of proteins in the PEG fractionation F4 from wheat leaves.
Table 1 Identification of 0.5 mmol/L Cd-induced proteins in wheat leaves Protein spot
Protein identification
Protein scores*
Matched peptide
Sequence coverage (%)
Theoretical Mr/pI
F 4 C3
METE MESCR: Cobalamin-independent methionine synthase isozyme (common ice plant) Q6BCT3 HORVU: methionine synthase (barley) T03581: dnaK-type molecular chaperone Bip (rice) PWWTB: H+ -transporting two-sector ATPase beta chain (wheat chloroplast) 1A1C SOYBN: 1-aminocyclopropane-1-carboxylate synthase (ACC synthase) (soybean) CB21 SINAL: chlorophyll a/b binding protein 1 (sinapis alba) gi|145216925: putative PAS/PAC sensor protein [Mycobacterium gilvum PYR-GCK] Magnesium chelatase subunit of protochlorophyllide reductase Q9ZR33 WHEAT: UDP-glucose protein transglucosylase (Triticum aestivum) SDG40 ARATH: protein SET DOMAIN GROUP 40 (Arabidopsis thaliana) ATP synthase beta subunit (Fragment). Entransia fimbriata Rubisco activase (Fragment) (barley)
54
12
20
84.8/5.90
152 86 91 57
17 16 13 8
27 28 26 13
84.4/5.68 73.5/5.30 53.8/5.06 54.7/5.84
59 81
7 8
31 18
26.2/5.17 41.8/5.21
60 100 58 82 89
8 14 10 13 8
21 34 20 28 21
39.5/5.28 41.47/5.82 55.8/5.35 44.4/5.10 47.5/5.64
F 4 C2 F 4 C1 F 4 C4 F 4 C5 F 4 C9 F4 C10 F 4 C8 F 4 C6 F 3 C2 F 3 C5 F 3 C6
* Protein scores greater than 54 or 78 are significant (P < 0.05). Mr: molecule weight; pI: isoelectric point.
related enzymes in wheat leaves under Cd and/or TCB stress, promotes the reversible protein phosphorylation. This conversely elicits appropriate responses through the regulation of stress signal-induced gene expression and defense-associated enzymatic activity. Protein methylation has been an important aspect of protein modifications in plants and animals. In the present study, we found several enzymes related to the methyl
cycle and transfer being induced by Cd and TCB. This is supported by the expressions of two different methionine synthases (Ms) in Yangmai 13 leaves enhanced by Cd and TCB stresses. Ms catalyze the formation of methionine (Met) and in activated methyl cycle further converted into S-adenosylmethionine (SAM). Since SAM provides the methyl group for protein and DNA methylation, the effects of Cd and TCB on the expression of SET domain-
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Table 2 Identification of 0.056 mmol/L TCB-induced proteins in wheat leaves Protein spot
Protein identification
Protein scores*
Matched peptide
Sequence coverage (%)
Theoretical Mr/pI
F 4 T1 F 4 T2 F 4 T3 F 4 T4 F 4 T7 F 4 T9 F 4 T5 F 4 T6 F4 T8 F4 T12
Q8GSN3 CUCMA: non-cell-autonomous heat shock cognate protein 70 Q5MGA8 MEDSA: heat shock protein 70–Medicago sativa (Alfalfa) Q1W681 WHEAT: vacuolar proton–ATPase subunit A (Triticum aestivum) BAB47031: H+ -transporting two-sector ATPase alpha chain (Triticum aestivum) Q6XXZ2 LOTJA: Gamma-glutamylcysteine synthetase (Lotus japonicus) Q1W2K1 CAMSI: 14-3-3-like protein A (barley) BAB47031: H+ -transporting two-sector ATPase alpha chain (Triticum aestivum) PWWTAM: H+ -transporting two-sector ATPase alpha chain (wheat mitochondrion) PWWTB : H+ -transporting two-sector ATPase beta chain (wheat chloroplast) 2A5G ARATH: serine/threonine protein phosphatase 2A 59 kDa regulatory subunit B’ gamma isoform (Arabidopsis thaliana) S23452: sedoheptulose-bisphosphatase precursor (wheat)
92 84 206 157 62 113 274 197 80 60
16 10 24 19 10 14 28 22 13 10
30 22 51 39 19 48 50 43 31 23
71.4/5.10 71.0/5.08 68.4/5.23 55.3/6.11 55.9/6.22 29.3/4.83 55.3/6.11 55.2/5.7 53.8/5.06 59.1/8.04
82
8
22
42.0/6.04
F 3 T6
* Protein scores greater than 54 or 78 are significant (P < 0.05). Mr: molecule weight. pI: isoelectric point.
containing proteins, responsible for protein methylation are important. As shown by proteomic data in the results, we found a protein SET domain group 40 (SDG40) to be induced by Cd and TCB in Yangmai 13 leaves. SDG40 can modify methionine residue to form an α Nmethylmethionine (Ng et al., 2007). Many types of stresses (such as osmotic or heavy metals) can generate active oxygen species (AOS) in the chloroplasts. In Rubisco, one of the amino acids most susceptible to oxidation is Met. Oxidation of Met leads to the formation of methionine sulfoxide residues (Stadtman et al., 2005), which often results in functional inactivation and targeting for proteolytic degradation (Brot and Weissbach, 2000). Although the functional significance of Rubisco methylation is unknown, the induction of SDG40 leads us to hypothesize that methylation of Met may be related to the stability through a reduction in the proteolytic susceptibility to Rubisco (Zheng et al., 1998). In addition, Rubisco activase appears to catalyze a rather unusual posttranslational modification of Rubisco, which results in the conformation and activity changes (Portis, 2003). In this study, we also found a Rubisco activase in Yangmai 13 leaves being induced by Cd and TCB. It is obvious that the induction of the Rubisco activase may be favorable for rapid activation of Rubisco in response to Cd and TCB stresses. 3.2 Responses of protein configuration protection Heat shock response in organisms can confer protection from damage due to exposure to a wide variety of stresses (Mayer and Bukau, 2005). The most abundant and widely studied group of stress proteins is the heat shock protein 70 (Hsp70 ) family (including molecular chaperone binding protein, Bip). Cellular functions of Hsp70 include refolding or degradation of denatured proteins (Bierkens et al., 1998). A dnaK-type molecular chaperone Bip and two Hsp70 were found to be induced by Cd and TCB in this study. We suggest that the induction of Hsp70 and Bip assist the damaged and misfolded proteins to reconstitute into normal three-dimensional structure, thereby reestablish their biological functions, or participating in pathways of protein degradation, thus contribute to the elimination of denatured proteins (Terlecky et al., 1992).
3.3 Responses of detoxification As shown in Fig. 3, an important aspect of Cd, Hg and TCB toxicity to wheat seedlings was their induction of lipid peroxidation, which was displayed as an increase in MDA content. Therefore, the synthesis of defense substances is necessary to counteract the effect of these stresses. Glutathione (GSH) plays a vital role in defending against toxins and free radicals (Moellering et al., 1998). The rate-limiting step of GSH synthesis was catalyzed by γ-glutamylcysteine synthetase (GCS) (Richman and Meister, 1975). In the experiment, the GCS in Yangmai 13 leaves was found to be up-regulated under TCB stress. This in conformity with the conclusion by Myhrstad et al. (2001) that induction of the GCS promotes the synthesis of GSH which provides wheat seedlings with multiple defenses not only against AOS but also against other toxicities. Furthermore, because the induction of Ms cellular SAM level was increased under Cd and TCB stresses. SAM also provides methyl for the production of compatible solutes, such as glycinebetaine and methylated polyols (Bohnert and Jensen, 1996). Their accumulation in the cytoplasm can regulate osmotic balance. Another benefit of SAM is for synthesis of polyamines as substrate for maintaining osmotic balance and eliminating AOS. 3.4 Responses of H+ transmembrane transportation In this study, the H+ -transporting two-sector ATPase alpha and beta chain in Yangmai 13 leaves was induced by Cd and TCB, which is essential to maintain the ATP high level required by the stressed cells. Furthermore, we also found the vacuolar proton-ATPase (V-ATPase) subunit A induced by Cd and TCB. Fukuda et al. (2004) demonstrated that V-ATPase plays an essential role in plant responses to environmental stresses. V-ATPase uses the energy derived from the hydrolysis ATP to establish the electrochemical gradient of H+ across vacuolar membrane (Sun-Wada et al., 2003), which is also the driving force for the accumulation of toxic ions and other solutes in the vacuole (Hamilton et al., 2001). Further studies are needed to elucidate whether the induction of V-ATPase promotes the accumulation of Cd ions or TCB molecules in the vacuoles.
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3.5 Responses of ethylene and cell wall component synthesis In this study, ACC synthase in Yangmai 13 leaves was induced by Cd and TCB. The induction of ACC synthase can stimulate ethylene production as approved by Wong et al. (2001). Ethylene, as a mediator of the stressful signal, plays an important defense role such as regulating plant growth and development, inducing the expression of various defensive genes in response to Cd and TCB stresses (O’Donnell et al., 1996). The induction of UDP-glucose protein transglucosylase (UPTG) was also observed in Yangmai 13 leaves under Cd and TCB stresses. Because UPTG and its homologous proteins play important roles in synthesizing cell wall components (Dhugga et al., 1997). This agrees with the statemnet of Ge et al. (2007) that the induction of UPTG promotes synthesis of cell wall components and stimulate cell wall thickening. Furthermore, it is worth notice that the most of the responses induced by both heavy metal (e.g., Cd) and organic pollutant (e.g., TCB) in this study imply that there are few defensive mechanisms in wheat in response to the stresses of chemical pollutants. However, it indicated that when wheat seedlings undergo different kinds of chemical pollution, the optimum choice may be to activate the welldeveloped mechanisms that have been evolved to cope with their ever-changing environmental factors.
4 Conclusions The present study indicates that Cd, Hg, and TCB have toxic effects on wheat seedlings, such as inhibiting root and shoot growth, causing lipid peroxidation. However, in response to Cd and TCB toxicity, different defenseassociated processes were induced in the seedlings of Yangmai 13 leaves, which included protein modification, protein configuration protection, detoxification, H+ across membrane transportation, and ethylene and cell wall component synthesis. These confirm the induction and expression of various defensive genes in response to Cd and TCB stresses in wheat seedlings. Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 30300026) and the Application of Nuclear Techniques in Agriculture from the Chinese Ministry of Agriculture (No. 200803034). We would like to thank Dr. Jun Li (Jiangsu University) for helping with MALDI-TOF MS analysis.
References Amezcua C A, Harper S M, Rutter J, Gardner K H, 2002. Structure and interactions of PAS kinase N-terminal PAS domain: model for intramolecular kinase regulation. Structure (Camb.), 10: 1349–1361. Bierkens J, Maes J, Plaetse F V, 1998. Dose-dependent induction of heat shock protein 70 synthesis in Raphidocelis subcapitata following exposure to different classes of envi-
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ronmental pollutants. Environmental Pollution, 101: 91–97. Bohnert H J, Jensen R G, 1996. Strategies for engineering waterstress tolerance in plants. Trends Biotechnology, 14: 89–97. Brot N, Weissbach H, 2000. Peptide methionine sulfoxide reductase: Biochemistry and physiological role. Biopolymers, 55: 288–296. Cao Q, Hu Q H, Khan S, Wang Z J, Lin A J, Du X, Zhu Y G, 2007. Wheat phytotoxicity from arsenic and cadmium separately and together in solution culture and in a calcareous soil. Journal of Hazardous Materials, 148: 377–382. Cavallini A, Natali L, Durante M, Masertib B, 1999. Mercury uptake, distribution and DNA affinity in durum wheat (Triticum durum Desf.) plants. The Science of the Total Environment, 243/244: 119–127 Dhindsa R S, Dhinsa P P, Thorpe T A, 1980. Leaf senescence correlated with increased levels of membrane permeability and lipidperoxidation and decreased levels of superoxide dismutase and catalase. Journal of Experimental Botany, 32: 127–132. Dhugga K S, Tiwari S C, Ray P M, 1997. A reversibly glycosylated polypeptide (RGP1) possibly involved in plant cell wall synthesis: Purification, gene cloning and transGolgi localization. Proceedings of the National Academy of Sciences of the United States of America, 94: 7679–7684. Fukuda A, Chiba K, Maeda M, Nakamura A, Maeshima M, Tanaka Y, 2004. Effect of salt and osmotic stresses on the expression of genes for the vacuolar H+ -pyrophosphatase, H+ -ATPase subunit A, and Na+ /H+ antiporter from barley. Journal of Experimental Botany, 55: 585–594. Ge C L, Wan D Z, Wang Z G, Ding Y, Wang Y L, Shang Q, Ma F, Luo S S, 2007. Response of rice root to 1,2,4trichlorobenzene stress. Acta Agronomica Sinica, 33(12): 1991–2000. Ge C L, Wan D Z, Wang Z G, Ding Y, Wang Y L, Shang Q, Ma F, Luo S S, 2008. A proteomic analysis of rice seedlings responding to 1,2,4-trichlorobenzene stress. Journal of Environmental Sciences, 20(3): 309–319. Gerber I B, Dubery I A, 2004. Protein phosphorylation in Nicotiana tabacum cells in response to perception of lipopolysaccharides from Burkholderia cepacia. Phytochemistry, 65: 2957–2966. Groppa M D, Benavides M P, Tomaro M L, 2003. Polyamine metabolism in sunflower and wheat leaf discs under cadmium or copper stress. Plant Science, 164: 293–299. Hamilton C A, Good A G, Taylor G J, 2001. Induction of vacuolar ATPase and mitochondrial ATP synthase by aluminium in aluminium-resistant cultivar of wheat. Plant Physiology, 125: 2068–2077. Lin R Z, Wang X R, Luo Y, Du W C, Guo H Y, Yin D Q, 2007. Effects of soil cadmium on growth, oxidative stress and antioxidant system in wheat seedlings (Triticum aestivum L.). Chemosphere, 69: 89–98. Lindberg S, Landberg T, Greger M, 2007. Cadmium uptake and interaction with phytochelatins in wheat protoplasts. Plant Physiology and Biochemistry, 45: 47–53. Liu X L, Zhang S Z, Shan X Q, Christie P, 2007. Combined toxicity of cadmium and arsenate to wheat seedlings and plant uptake and antioxidative enzyme responses to cadmium and arsenate co-contamination. Ecotoxicology and Environmental Safety, 68: 305–313. Liu X L, Zhang S Z, 2007. Intraspecific differences in effects of co-contamination of cadmium and arsenate on early seedling growth and metal uptake by wheat. Journal of Environmental Sciences, 19: 1221–1227.
No. 6
Responses of wheat seedlings to cadmium, mercury and trichlorobenzene stresses
Machado E L, Da Silva A C, Da Silva M J, Leite A, Ottoboni L M M, 2002. Endogenous protein phosphorylation and casein kinase activity during seed development in Araucaria angustifolia. Phytochemistry, 61: 835–842. Manzanero S, Rutten T, Kotseruba V, Houben A, 2002. Alterations in the distribution of histone H3 phosphorylation in mitotic plant chromosomes in response to cold treatment and the protein phosphatase inhibitor cantharidin. Chromosome Research, 10: 467–476. Mayer M P, Bukau B, 2005. Hsp 70 chaperones: cellular functions and molecular mechanism. Cellular and Molecular Life Sciences, 62: 670–684. Moellering D, McAndrew J, Patel R P, Cornwell T, Lincoln T, Cao X et al., 1998. Nitric Oxide-dependent induction of glutathione synthesis through increased expression of γglutamylcysteine synthetase. Archives of Biochemistry and Biophysics, 358(1): 74–82. Myhrstad M C, Husberg C, Murphy P, Nordstrom O, Blomhoff R, Moskaug J O, Kolsto A B, 2001. TCF11/Nrf1 overexpression increases the intracellular glutathione level and can transactivate the gamma-glutamylcysteine synthetase (GCS) heavy subunit promoter. Biochimica et Biophysica Acta, 1517: 212–219. Ng D W K, Wang T, Chandrasekharan M B, Aramayo R, Kertbundit S, Hall T C, 2007. Plant SET domain-containing proteins: Structure, function and regulation. Biochimica et Biophysica Acta, 1769: 316–329. O’Donnell P J, Calvert C, Atzorn R, Wasternack C, Leyser H M O, Bowles D J, 1996. Ethylene as a signal mediating the wound response of tomato plants. Science, 274: 1914–1917. Portis A R, 2003. Rubisco activase – Rubisco’s catalytic chaperone. Photosynthesis Research, 75: 11–27. Richman P G, Meister A, 1975. Regulation of γ-glutamylcysteine synthetase by nonallosteric feedback inhibition by glutathione. The Journal of Biological Chemistry, 250: 1422– 1426. Simpson R J, 2003. Proteins and Proteomics: A Laboratory Manual. New York: Cold spring harbor laboratory press. Stadtman E R, Remmen H V, Richardson A, Wehr N B, Levine R L, 2005. Methionine oxidation and aging. Biochimica et Biophysica Acta, 1703: 135–140. Singh H P, Batish D R, Kaur G, Arora K, Kohli R K, 2008. Nitric oxide (as sodium nitroprusside) supplementation
813
ameliorates Cd toxicity in hydroponically grown wheat roots. Environmental and Experimental Botany, 63: 158–167. Subramanian R R, Zhang H Y, Wang H N, Ichijo H, Miyashita T, Fu H A, 2004. Interaction of apoptosis signal-regulating kinase 1 with isoforms of 14-3-3 proteins. Experimental Cell Research, 294: 581– 591. Sun Q, Wang X R, Ding S M, Yuan X F, 2005. Effects of exogenous organic chelators on phytochelatins production and its relationship with cadmium toxicity in wheat (Triticum aestivum L.) under cadmium stress. Chemosphere, 60: 22– 31. Sun-Wada G H, Wada Y, Futai M, 2003. Vacuolar H+ pumping ATPases in luminal acidic organelles and extracellular compartments: common rotational mechanism and diverse physiological roles. Journal of Bioenergetics and Biomembranes, 35: 347–358. Terlecky S R, Chiang H L, Olson T J, Dice J F, 1992. Protein and peptide binding and stimulation of in vitro lysosomal proteolysis by the 73-kDa heat-shock cognate protein. The Journal of Biological Chemistry, 267: 9202–9209. Wang Z G, Wan D Z, Yang Y C, Ge C L, Ma F, Yang J C, 2006. Effects of 1,2,4-trichlorobenzene and naphthalene on grain yield and quality of rice. Journal of Rice Science, 20(3): 295–300. Wong W S, Li G G, Ning W, Xu Z F, Hsiao W L W, Zhang L Y, Li N, 2001. Repression of chilling-induced ACC accumulation in transgenic citrus by over-production of antisense 1-aminocyclopropane-1-carboxylate synthase RNA. Plant Science, 161: 969–977. Xi J H, Wang X, Li S Y, Zhou X, Yue L, Fan J, Hao D Y, 2006. Polyethylene glycol fractionation improved detection of low-abundant proteins by two-dimensional electrophoresis analysis of plant proteome. Phytochemistry, 67: 2341– 2348. Yannarelli G G, Fernandez-Alvarez A J, Santa-Cruz D M, Tomaro M L, 2007. Glutathione reductase activity and isoforms in leaves and roots of wheat plants subjected to cadmium stress. Phytochemistry, 68: 505–512. Zheng Q, Simel E J, Klein P E, Royer M T, Houtz R L, 1998. Expression, purification, and characterization of recombinant ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit Nε -Methyltransferase. Protein Expression and Purification, 14: 104–112.