An Arabidopsis mutant atcsr-2 exhibits high cadmium stress sensitivity involved in the restriction of H₂S emission

An Arabidopsis mutant atcsr-2 exhibits high cadmium stress sensitivity involved in the restriction of H 2 S emission 1 first-author 1 1 1 1 1 1 2 3 1 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:peiyanxi@sxu.edu.cn †E-mail:peiyanxi@sxu.edu.cn peiyanxi@sxu.edu.cn School of Life Science, Shanxi University, Taiyuan 030006, China School of Life Science, Shanxi University, Taiyuan 030006, China School of Chemical Engineering and Environment, North University of China, Taiyuan 030051, China School of Chemical Engineering and Environment, North University of China, Taiyuan 030051, China School of Environmental Science and Resources, Shanxi University, Taiyuan 030006, China School of Environmental Science and Resources, Shanxi University, Taiyuan 030006, China The gene AtCSR encodes peptidyl-prolyl cis/trans isomerases (PPIases) that accelerate energetically unfavorable cis/trans isomerization of the peptide bond preceding proline production. In our studies, we found that AtCSR was associated with cadmium (Cd)-sensitive response in Arabidopsis . Our results show that AtCSR expression was triggered by Cd-stress in wild type Arabidopsis . The expression of some genes responsible for Cd 2+ transportation into vacuoles was induced, and the expression of the iron-regulated transporter 1 ( IRT1 ) related to Cd 2+ absorption from the environment was not induced in wild type with Cd 2+ treatment. The expression of Cd-transportation related genes was not in response to Cd-stress, whereas IRT expression increased dramatically in atcsr-2 with Cd 2+ treatment. The expression of glutathione 1 ( GSH1 ) was consistent with GSH being much lower in atcsr-2 in comparison with the wild type with Cd 2+ treatment. Additionally, malondialdehyde (MDA), hydrogen peroxide, and Cd 2+ contents, and activities of some antioxidative enzymes, differed between the wild type and atcsr-2 . Hydrogen sulfide (H 2 S) has been confirmed as the third gas-transmitter over recent years. The findings revealed that the expression pattern of H 2 S-releasing related genes and that of Cd-induced chelation and transportation genes matched well in the wild type and atcsr-2 , and H 2 S could regulate the expression of the Cd-induced genes and alleviate Cd-triggered toxicity. Finally, one possible suggestion was given: down-regulation of atcsr-2 , depending on H 2 S gas-transmitter not only weakened Cd 2+ chelation, but also reduced Cd 2+ transportation into vacuoles, as well as enhancing the Cd 2+ assimilation, thus rendering atcsr-2 mutant sensitive to Cd-stress. AtCSR H 2 S gas-transmitter Glutathione Cadmium stress sensitivity Introduction 1 Cadmium (Cd 2+ ) is a heavy metal that exerts a detrimental effect on plants. It can be taken up by roots and transferred into aerial organs, where it accumulates preferentially in leaves (Salt et al., 1995 1995 ). Cd 2+ exposure in vivo or in vitro also leads to cellular energy disturbances due to limiting mitochondrial function (Byczkowski and Sorenson, 1984 1984 ; Dorta et al., 2003 2003 ; Sokolova, 2004 2004 ) and increased oxidative stress (Valko et al., 2005 2005 ; Cherkasov et al., 2007 2007 ). Cd 2+ also affects gene expression in multiple cellular pathways (Marie et al., 2006 2006 ; Sanni et al., 2008 2008 ). Recently, several studies have focused on plant response mechanisms to Cd 2+ . Plants may develop a host of mechanisms to protect themselves from Cd toxicity. One mechanism, detoxification, may transfer cytoplasmic Cd 2+ into the vacuole, and thus lessen toxic action. Multiple genes have been reported to respond to Cd stress through this process. The structure, function, and biosynthesis of phytochelatins (PC) have been extensively reported (Ruegsegger et al., 1990 1990 ; Steffens, 1990 1990 ). PC synthesized from the substrate glutathione (GSH) (Thangavel et al., 2007 2007 ) can integrate with cytoplasmic Cd 2+ and are both transferred into the vacuole (Clemens, 2001 2001 ). Additionally, GSH can also facilitate Cd 2+ sequestration into vacuoles (Guo et al., 2008 2008 ). The two adenosine triphosphate (ATP)-dependent enzymes, γ-glutamylcysteine synthetase (GSH1) and glutathione synthetase (GSH2), catalyze GSH synthesis (Yadav, 2010 2010 ). ATP-binding cassette (ABC) transporters may mediate transportation of glutamine synthetase-conjugated Cd 2+ into the mitochondria with Cd 2+ treatment (Li et al., 1997 1997 ). Multidrug resistance-associated protein 3 (MRP3) is an ABC transporter, and expression of MRP3 increases after Cd 2+ treatment (Bovet et al., 2003 2003 ; Zientara et al., 2009 2009 ). Plants over-expressing iron-regulated transporter 1 ( IRT1 ) also accumulate higher concentrations of Cd and zinc (Zn) in comparison with wild type under iron (Fe)-deficient conditions, showing an additional role in transporting these metals (Connolly et al., 2002 2002 ). Several Arabidopsis thaliana cation exchangers (CAXs) encode tonoplast-localized transporters that appear to be major contributors to vacuolar sequestration of Cd 2+ . CAX2 and CAX4 had high transportation and selection of Cd 2+ in tonoplast vesicles (Koren′kov et al., 2007 2007 ). It is well known that Cd-stress can generate cellular oxidative stress responses, and may explain cellular toxicity (Romero-Puertas et al., 2004 2004 ). Hydrogen peroxide (H 2 O 2 ) accumulation and lipid peroxidation can indicate oxidative stress caused by Cd 2+ exposure in plant cells (Wang et al., 2011 2011 ). Plants have developed effective defense systems against H 2 O 2 accumulation, which include limiting H 2 O 2 formation and development, and H 2 O 2 removal under conditions of environmental stress. Antioxidative enzymes, such as catalase (CAT), peroxidases (POD), and ascorbate peroxidase (APX) play a key role in those processes. CAT and POD function in detoxification of H 2 O 2 , thereby preventing the formation of ·OH radicals (Sandalio et al., 2001 2001 ). APX is an important component of the ascorbate-glutathione cycle responsible for the removal of H 2 O 2 in plants (Jimenez et al., 1997 1997 ). Nitric oxide (NO) and carbon monoxide as two major gas-transmitters are well known and much studied in animals and plants. NO is reportedly beneficial for Cd toxicity in Arabidopsis by up-regulating genes related to Fe uptake and promoting Cd 2+ accumulation in roots (Besson-Bard et al., 2009 2009 ). Hydrogen sulfide (H 2 S), regarded as the third gas-transmitter, has been studied in animals, but there are few related reports in plants. It has been illustrated that L -cysteine desulfhydrase (LCD) could catalyze the decomposition of cysteine to pyruvate, ammonia and H 2 S (Bloem et al., 2004 2004 ). In addition, D -cysteine desulfhydrase ( DCD ) encodes a DCD that decomposes D -cysteine into H 2 S (Riemenschneider et al., 2005 2005 ). In this paper, we screened a Cd-sensitive Arabidopsis mutant, identified the mutational site and found a new gene involved in Cd-sensitive response. We further studied the reason for Cd-sensitivity of atcsr-2 . Materials and methods 1 Plant materials and growth conditions 2 Arabidopsis (Col-0) seeds were surface sterilized by 75% ethanol for 45 s and 5% (50 g/L) sodium hypochlorite (NaClO) for 7 min under sterile conditions, and washed three times with sterile water. The plates were stored for 3 d in darkness at 4 °C. They were then grown on 1/2 Murashige and Skoog (MS) media containing 0.7% (7 g/L) agar and 1% (10 g/L) sucrose under 23 °C, 100 μmol/(m 2 ∙s) light intensity and standard long-day conditions (i.e., 16 h/8 h of light/dark). Cd-stress, exogenous H 2 S treatment 2 For Cd-stress treatment, atcsr-2 seeds were first surface sterilized, then scattered on 1/2 MS media containing 90 μmol/L cadmium chloride (CdCl 2 ) solution which was added into 1/2 MS media before solidifying. After 3 d in darkness at 4 °C, seeds were incubated at 23 °C, with 100 μmol/(m 2 ∙s) light intensity and standard long-day conditions; wild type seeds were treated similarly to the controls. For exogenous H 2 S treatment, disinfected seeds, after 3 d in darkness at 4 °C, were preincubated for 12 d on 1/2 MS media under 23 °C, 100 μmol/(m 2 ∙s) light intensity, and standard long-day conditions; under sterile condition, closed culture dishes were opened before being transferred carefully into closed containers to make seedlings in an airtight atmosphere. Lastly, solutions with 10, 30, or 50 μmol/L sodium hydrosulfide (NaHS) were introduced into the closed containers. MDA, H 2 O 2 , GSH, Cd 2+ contents, antioxidase activity, and H 2 S production rate assays 2 Malondialdehyde (MDA) content was determined according to published method of Heath and Packer ( 1968 1968 ). A total weight of 0.3 g of 12-d-old seedlings were grinded with 0.05 mol/L phosphate buffer (pH 7.8), and diluted with buffer to 3 ml; the solution was centrifuged at 5 000× g for 5 min under 4 °C, and the supernatant was used to determine MDA content. The detection of H 2 O 2 was carried out using 3,3′-diaminobenzidine (DAB) (Sigma, MO, USA) (Thordal-Christensen et al., 1997 1997 ). The 12-d-old seedlings were soaked by 1 mg/L DAB for 1 h in dark conditions, and transferred into light conditions for 12 h; after flushing with water three times, leaves were faded by 95% ethanol in order to locate H 2 O 2 . GSH content was assayed using a GSH assay kit (Nanjing Jicheng Bioengineering Institute, China). CAT, POD, and APX activities were assayed according to published methods (Chance and Maehly, 1955 1955 ; Gupta et al., 1993 1993 ). The extraction buffer for the CAT assay contained 100 mmol/L phosphate, 1 mmol/L phenylmethylsulfonyl fluoride (PMSF), 1% (10 g/L) polyvinylpolypyrrolidone (PVPP), and 1 mmol/L ethylenediaminetetraacetic acid (EDTA) (pH 7.8), and the assay mixture 50 mmol/L phosphate and 11.6 mmol/L H 2 O 2 (pH 6.9). The extraction buffer for POD contained 100 mmol/L phosphate, 1 mmol/L PMSF, 1% (10 g/L) PVPP, and 1 mmol/L EDTA (pH 7.8), and the assay mixture 50 mmol/L phosphate, 10 mmol/L guaiacol, and 100 mmol/L H 2 O 2 (pH 6.4). The extraction buffer for APX contained 50 mmol/L phosphate, 0.5 mmol/L ascorbate, 1% (10 g/L) PVPP, and 0.1 mmol/L EDTA (pH 7.0), and the assay mixture 50 mmol/L phosphate, 0.5 mmol/L ascorbate, 0.1 mmol/L EDTA, and 0.1 mmol/L H 2 O 2 (pH 7.0). Cd 2+ content was estimated by atomic absorption spectrophotometer (Perkin-Elmer, Foster City, CA, USA). A total weight of 1 g of dry seedlings nitrified by concentrated nitric acid was used for assay Cd 2+ content. Determination of H 2 S production rate was referred to published measure (Jin et al., 2011 2011 ). Twelve-day-old seedlings were used to extract total protein. Analysis of transcript levels 2 The total RNA was extracted from 12-d-old seedlings. The complementary DNA (cDNA) was synthesized using a reverse transcription system kit (TaKaRa, Japan) and oligo(dT) primers. The cDNA products were standardized for reverse transcription polymerase chain reaction (RT-PCR) using β-actin as the internal control. The sequence-specific primers were designed as follows. AtCSR : 5′-AACTGCAGATGTCGGCAAGACCTGAAGGAAGTC-3′, 5′-CCCAAGCTTTCAATCGATCACTTTGGTCCTTAGA-3′; GSH1 : 5′-GGGTCAGTTCGAGCTTAGTGG-3′, 5′-GTGCGGTCCTTGTCAGTGTCT-3′; GSH2 : 5′-TTCCCTCACATCATCTCCATA-3′, 5′-AAGCAGTCGCAGTGGTTTATT-3′; MRP3 : 5′-TACAATCTTCCCGACACTATCTC-3′, 5′-ATCACCGAATGAAAGTATCTCCA-3′; IRT3 : 5′-CAGCTAGAAGATCCACGAGTGCC-3′, 5′-CCCGCAAATGATGTTACCTTACC-3′; CAX1 : 5′-CGAAGAAACAGATGGCAATAATGAA-3′, 5′-TGATGACGATGTGGAGCAAGA-3′; CAX2 : 5′-GACCCATCATCCATCCAATCA-3′, 5′-CCCTGTCAAGGTTCAGCAGTT-3′; CAX4 : 5′-GGTTTCGGTTGAGGGTGAGTT-3′, 5′-CCGTGATTGGAATGTGGAGTG-3′; CAX11 : 5′-ACAAGAACAAAGCAGCAATCA-3′, 5′-AATGGCGGCGTCATCAACTAT-3′; heavy metal ATPases ( HMA2 ): 5′-CATTGGATGGCTTGACTTGCT-3′, 5′-GATTGCGTGGTTGCGAAGATG-3′; HMA4 : 5′-CTCACAGTTTCCATCCACCAC-3′, 5′-CGGCTTACTTCTCCTCCTATC-3′; LCD : 5′-CATGCCATGGCAATGGAGGCGGGAGAGCGGCGCAATG-3′, 5′-CGGGATCCCTACAATGCAGGAAGGTTTTGACAAG-3′; DCD : 5′-CATGCCATGGCAATGAGAGGACGAAGCTTGACACTCTC-3′, 5′-CGGGATCCCTAGAACATTTTCCCAACACCATCTT-3′. Results 1 Transfer (T)-DNA insertion site identification 2 In previous studies, our laboratory established the Arabidopsis mutant pool using ethyl methanesulfonate mutagenic methods. In the selfed progenies, we screened a Cd-sensitive mutant. Tail-PCR results show that the mutant site was located in At2g36130 . To validate that AtCSR was related to Cd-sensitive stress response, we obtained atcsr-2 (SALK_051078C). AtCSR encodes peptidyl-prolyl cis/trans isomerases (PPIases), which are enzymes that energetically accelerate unfavorable cis/trans isomerization of the peptide bond preceding proline production (Kiefhaber et al., 1990 1990 ; Hunter, 1998 1998 ). PPIases include four structurally distinct subfamilies: cyclophilins, FK506-binding proteins, parvulins, and PP2A phosphatase activator (Lu et al., 2007 2007 ). We further detected the transcription expression level of AtCSR and, in contrast to wild type, it was significantly lower in atcsr-2 (Fig. 1 1 ). (a) Schematic representation of T-DNA insertion site in the knock-down mutant. The thick lines and thin lines represent exons and introns, respectively. The position of the T-DNA insertion in atcsr-2 is indicated by a triangle (not to scale). (b) RT-PCR analysis of expression of AtCSR transcripts in wild type (WT) and atcsr-2 . Expression of actin was analyzed as a loading control Cd-sensitivity of atcsr-2 2 To examine the degree of Cd-sensitivity, the root length was determined in the wild type and atcsr-2 : this was about 2.2 cm in the wild type and 1.1 cm in atcsr-2 (Fig. 2 2 ). Furthermore, to genetically characterize atcsr-2 , F 1 progenies from crosses between the wild type and atcsr-2 were generated. All F 1 plants did not display Cd-sensitive phenotypes. In the F 2 generation, plants exhibited a 1:3 segregation ratio of mutant to the wild type phenotypes and all plants showing the mutant phenotype were atcsr-2 homozygous. The seeds were scattered on 1/2 MS with 90 μmol/L CdCl 2 for 12 d. (a) Representative images of both wild type (WT) and atcsr-2 seedlings; (b) Root length of WT and atcsr-2 . Values are expressed as mean±standard error (SE) ( n =10), * P < 0.05 More Cd 2+ accumulation and less GSH generation by atcsr-2 2 GSH also plays a critical role in detoxification of heavy metals because of its capacity to chelate heavy metals and act as a substrate of PC. To contrast differences in GSH content, we measured levels in the wild type and atcsr-2 following Cd treatment; and found levels were lower in the latter (Fig. 3a 3a ). Additionally, Cd 2+ content was examined, and, as expected atcsr-2 accumulated much more Cd 2+ in plant cells (Fig. 3b 3b ). Seeds of wild type (WT) and atcsr-2 were scattered on 1/2 MS with 30 μmol/L CdCl 2 for 12 d; (a) GSH content. (b) Total Cd 2+ content; (c) MDA content; (d) H 2 O 2 accumulation (reddish-brown polymer representing the H 2 O 2 distribution is shown by full arrows); (e) Antioxidative enzyme activities (Values are means±SE ( n =3), * P < 0.05). FW: fresh weight; DW: dry weight; GSH: glutathione; MDA: malondialdehyde; CAT: catalase; POD: peroxidases; APX: ascorbate peroxidase Higher oxidative stress and lower antioxidative ability in atcsr-2 2 MDA is regarded as an indicator of lipid peroxidation caused by oxidative stress (Cho and Seo, 2005 2005 ). MDA content and H 2 O 2 accumulation were monitored to show Cd-induced oxidative stress in atcsr-2 and the wild type—they were both higher in atcsr-2 (Figs. 3c and 3d 3c and 3d ). H 2 O 2 scavenging is accomplished by various antioxidant enzymes, and thus the activities of the major enzymes involved in H 2 O 2 metabolism in seedlings exposed to Cd 2+ treatment were determined, and found to be lower in atcsr-2 compared to the wild type (Fig. 3e 3e ). Expression detection of Cd-response related genes in the wild type and atcsr-2 with or without Cd 2+ treatment 2 To investigate the role of AtCSR in responding to Cd-stress, we determined the AtCSR expression in the wild type and mutant with or without Cd 2+ treatment. The AtCSR expression dramatically increased with Cd 2+ treatment in the wild type; however, it sustained a relatively low level with Cd 2+ treatment in atcsr-2 (Fig. 4a 4a ). To further determine the role of AtCSR in Cd 2+ chelation, we focused on expressions of GSH1 and GSH2 . With Cd-stress, GSH1 expression increased in the wild type; however, it was not induced in atcsr-2 (Fig. 4b 4b ). Differing from GSH1 , the GSH2 expression was not regulated by Cd 2+ treatment. Similarly, expressions of some genes involved in Cd 2+ transportation were studied in the wild type and mutant with or without Cd 2+ treatment. The expressions of MRP3 , CAX1 , CAX2 , and CAX4 were intensely up-regulated, whereas there were no strong inductions in atcsr-2 . IRT1 expression did not change in the wild type with or without Cd-stress, but greatly increased in atcsr-2 with Cd-stress. The expression levels of CAX11 and HMA2 were not induced by Cd-stress and HM4 was not detected; these gene expression levels and trends were the same in atcsr-2 as in the wild type (Fig. 4c 4c ). Seeds of WT and atcsr-2 were scattered on 1/2 MS with 30 μmol/L CdCl 2 or without CdCl 2 treatment for 12 d, and then seedlings were used in this experiment. (a) Expression analysis of AtCSR ; (b) Expression analysis of GSH-synthesis related genes; (c) Expression analysis of Cd 2+ -transportation related genes; (d) Expression analysis of H 2 S-emission related genes ( actin was used as a loading control); (e) Determination of H 2 S production rate (Values are expressed as mean±SE ( n =3), * P < 0.05) Expression analysis of genes associated with H 2 S release and H 2 S production rate assay in the wild type and atcsr-2 with or without Cd 2+ treatment 2 Our laboratory is studying the function of H 2 S as the third gas-transmitter in a variety of physiological actions. Specifically, our attention has focused on some genes encoding cysteine desulfydrase that are related to H 2 S emission in plants. To determine whether H 2 S is related to Cd-stress response in Arabidopsis , the expressions of H 2 S-releasing related genes were determined in the wild type and atcsr-2 with or without Cd 2+ treatment. The expression pattern of genes related to H 2 S release was the same as that of genes induced by Cd-stress. The expressions of LCD and DCD markedly increased with Cd 2+ treatment in the wild type, but were not triggered in atcsr-2 (Fig. 4d 4d ). It has been demonstrated that LCD plays a far more important role in H 2 S release (Jin et al., 2011 2011 ). We assayed H 2 S production rate using LCD as substrate, and founded that H 2 S production rates in the wide type with Cd-stress increased dramatically in comparison to the wide type without Cd-stress, while in atcsr-2 there was no increase with Cd-stress (Fig. 4e 4e ). Effects of H 2 S on expression of Cd-induced genes and Cd-triggered toxicity 2 Previous studies demonstrated that Cd-induced genes sufficiently matched with genes involved in H 2 S generation in the wild type and atcsr-2 . We proposed that H 2 S as a gas-transmitter regulated the expression of Cd - induced genes, and to test this hypothesis we treated Arabidopsis with different H 2 S physiological concentrations and monitored expression of Cd-induced genes. The expressions of GSH1 , MRP3 , CAX1 , CAX2 , and CAX4 were all up-regulated and IRT1 was down-regulated by different H 2 S physiological concentrations (Fig. 5 5 ). Since H 2 S could regulate the expression of Cd-chelation and Cd-transportation related genes, we examined whether it could alleviate the Cd-triggered toxicity. The root length of atcsr-2 with H 2 S fumigation was longer than that of atcsr-2 without H 2 S fumigation under conditions of Cd stress, but was shorter than that of atcsr-2 without Cd stress (Fig. 6 6 ). Wild type seeds were scattered on 1/2 MS for 12 d, and then fumigated with different concentrations of H 2 S (10, 30, or 50 μmol/L) for 5 h. The seedlings were then used for RT-PCR analysis of gene expression atcsr-2 seeds were scattered on 1/2 MS for 12 d with or without 30 μmol/L CdCl 2 , and then fumigated or not with 30 μmol/L H 2 S for 4 d. (a) Representative images of atcsr-2 seedlings with H 2 S fumigation alone and with both Cd-stress and H 2 S fumigation as well as with Cd-stress alone. The atcsr-2 was cultivated with neither CdCl 2 nor H 2 S fumigation as control group. (b) Root length corresponded to the seedlings of Fig. 6a 6a . Values are expressed as mean±SE ( n =10). Values with different letters indicate significant differences ( P < 0.05) among them Discussion 1 PC can chelate heavy metals and then PC-metal complexes are transported across the tonoplast into vacuoles (Cobbett, 2000 2000 ). PC can be synthesized from reduced GSH by the enzyme PC synthase through a transpeptidation reaction (Rea et al., 2004 2004 ). GSH plays a vital role in protecting plants against Cd-triggered toxicity by providing a sequestration system (Sanità di Toppi and Gabbrielli, 1999 1999 ). GSH1 reportedly acts as a major control point under conditions of increasing demand for GSH (Yadav, 2010 2010 ). In the present study, Cd 2+ treatment did not trigger expression of GSH1 in atcsr-2 , in contrast to the wild type. The expression of GSH2 was not induced by Cd-stress in the wild type and atcsr-2 (Fig. 4b 4b ). Therefore, the synthesis of GSH was significantly restricted in atcsr-2 (Fig. 3a 3a ). We propose that one reason for the Cd-sensitive phenotype of atcsr-2 may be the decreased GSH content. We further studied the expression levels of MRP3 , CAX1 , CAX2 , and CAX4 , which are involved in Cd 2+ transportation. All were sharply elevated in the wild type with Cd-stress compared to atcsr-2 (Fig. 4c 4c ). This indicated that the increased expression levels of MRP3 , CAX1 , CAX2 , and CAX4 in the wild type were probably due to demand for plant cells to transport Cd 2+ into the vacuole, thus protecting plants from Cd 2+ toxicity. However, inducible expression of these genes by Cd-stress as for the wild type was not observed in atcsr-2 . We hypothesized that dysfunctional Cd 2+ transportation to the vacuole may be another aspect of the Cd-sensitive phenotype of atcsr-2 . Additionally, IRT is one Fe transporter that could transfer environmental Cd 2+ into plant cells. IRT1 expression was induced by Cd-stress in atcsr-2 (Fig. 4c 4c ). Consequently, atcsr-2 transported more Cd 2+ into the cells, accumulating superfluous Cd 2+ in cytoplasm. This may be the third reason for the Cd-sensitive phenotype of atcsr-2 . All of the discussed possibilities for the Cd-sensitive phenotype of atcsr-2 could result in mounting accumulation of dissociated Cd 2+ in cytoplasm, which could lead to increased MDA and H 2 O 2 , and inhibition of antioxidative enzymes’ activities (Gonçalves et al., 2009 2009 ), as demonstrated in atcsr-2 (Figs. 3c–3e 3c–3e ). The expression pattern of most H 2 S-release related genes (including LCD and DCD ) was very consistent with that of Cd-chelation and Cd-transportation related genes both in the wild type and atcsr-2 (Figs. 4c and 4d 4c and 4d ). This suggests that genes involved in H 2 S release may participate in the process of Cd-stress response in Arabidopsis . The expression of LCD and DCD induced by Cd-stress was not found in atcsr-2 (Fig. 4d 4d ). Similarly, the expression of genes related to Cd detoxification and transportation was not detected in atcsr-2 (Fig. 4c 4c ). Other results indicated that H 2 S functions in regulating the expression of genes associated with Cd 2+ chelation and transportation (Fig. 5 5 ), and alleviates the Cd-triggered toxicity (Fig. 6 6 ). Together the results suggest that AtCSR expression affects the emission of H 2 S, which as a gas-transmitter could regulate expression of a host of genes involved in Cd 2+ chelation and transportation. The possible reason of Cd-sensitive phenotype of atcsr-2 was due to down-regulation of AtCSR expression, which could suppress the expression of H 2 S-emission related genes and, as a result, H 2 S production decreased. Finally, the decreased H 2 S emission possibly led to increased assimilation of Cd 2+ from the environment, and weakened Cd 2+ chelation and transportation. Thus the normal functions of Cd 2+ chelation and transportation in atcsr-2 were all destroyed. 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Activity of the AtMRP3 promoter in transgenic Arabidopsis thaliana and Nicotiana tabacum plants is increased by cadmium, nickel, arsenic, cobalt and lead but not by zinc and iron 10.1631/jzus.B1200089 Project supported by the Research Fund for the Doctoral Program of Higher Education of China (No. 20091401110004), the Science and Technology Special Project of Shanxi Province, China (2012, to Qiang ZHANG), and the Shanxi Scholarship Council of China (No. 2011-007) 2012-03-21 2012-10-31 2012-07-29 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2012 2012 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 12 13