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

Abstract

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 Cd²⁺ transportation into vacuoles was induced, and the expression of the iron-regulated transporter 1 (IRT1) related to Cd²⁺ absorption from the environment was not induced in wild type with Cd²⁺ treatment. The expression of Cd-transportation related genes was not in response to Cd-stress, whereas IRT expression increased dramatically in atcsr-2 with Cd²⁺ treatment. The expression of glutathione 1 (GSH1) was consistent with GSH being much lower in atcsr-2 in comparison with the wild type with Cd²⁺ treatment. Additionally, malondialdehyde (MDA), hydrogen peroxide, and Cd²⁺ contents, and activities of some antioxidative enzymes, differed between the wild type and atcsr-2. Hydrogen sulfide (H₂S) has been confirmed as the third gas-transmitter over recent years. The findings revealed that the expression pattern of H₂S-releasing related genes and that of Cd-induced chelation and transportation genes matched well in the wild type and atcsr-2, and H₂S 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 H₂S gas-transmitter not only weakened Cd²⁺ chelation, but also reduced Cd²⁺ transportation into vacuoles, as well as enhancing the Cd²⁺ assimilation, thus rendering atcsr-2 mutant sensitive to Cd-stress.

1. Introduction

Cadmium (Cd²⁺) 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). Cd²⁺ exposure in vivo or in vitro also leads to cellular energy disturbances due to limiting mitochondrial function (Byczkowski and Sorenson, 1984; Dorta et al., 2003; Sokolova, 2004) and increased oxidative stress (Valko et al., 2005; Cherkasov et al., 2007). Cd²⁺ also affects gene expression in multiple cellular pathways (Marie et al., 2006; Sanni et al., 2008). Recently, several studies have focused on plant response mechanisms to Cd²⁺. Plants may develop a host of mechanisms to protect themselves from Cd toxicity. One mechanism, detoxification, may transfer cytoplasmic Cd²⁺ 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; Steffens, 1990). PC synthesized from the substrate glutathione (GSH) (Thangavel et al., 2007) can integrate with cytoplasmic Cd²⁺ and are both transferred into the vacuole (Clemens, 2001). Additionally, GSH can also facilitate Cd²⁺ sequestration into vacuoles (Guo et al., 2008). The two adenosine triphosphate (ATP)-dependent enzymes, γ-glutamylcysteine synthetase (GSH1) and glutathione synthetase (GSH2), catalyze GSH synthesis (Yadav, 2010). ATP-binding cassette (ABC) transporters may mediate transportation of glutamine synthetase-conjugated Cd²⁺ into the mitochondria with Cd²⁺ treatment (Li et al., 1997). Multidrug resistance-associated protein 3 (MRP3) is an ABC transporter, and expression of MRP3 increases after Cd²⁺ treatment (Bovet et al., 2003; Zientara et al., 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). Several Arabidopsis thaliana cation exchangers (CAXs) encode tonoplast-localized transporters that appear to be major contributors to vacuolar sequestration of Cd²⁺. CAX2 and CAX4 had high transportation and selection of Cd²⁺ in tonoplast vesicles (Koren′kov et al., 2007). It is well known that Cd-stress can generate cellular oxidative stress responses, and may explain cellular toxicity (Romero-Puertas et al., 2004). Hydrogen peroxide (H₂O₂) accumulation and lipid peroxidation can indicate oxidative stress caused by Cd²⁺ exposure in plant cells (Wang et al., 2011). Plants have developed effective defense systems against H₂O₂ accumulation, which include limiting H₂O₂ formation and development, and H₂O₂ 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 H₂O₂, thereby preventing the formation of ·OH radicals (Sandalio et al., 2001). APX is an important component of the ascorbate-glutathione cycle responsible for the removal of H₂O₂ in plants (Jimenez et al., 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 Cd²⁺ accumulation in roots (Besson-Bard et al., 2009). Hydrogen sulfide (H₂S), 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 H₂S (Bloem et al., 2004). In addition, D-cysteine desulfhydrase (DCD) encodes a DCD that decomposes D-cysteine into H₂S (Riemenschneider et al., 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.

2. Materials and methods

2.1. Plant materials and growth conditions

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/(m²∙s) light intensity and standard long-day conditions (i.e., 16 h/8 h of light/dark).

2.2. Cd-stress, exogenous H₂S treatment

For Cd-stress treatment, atcsr-2 seeds were first surface sterilized, then scattered on 1/2 MS media containing 90 μmol/L cadmium chloride (CdCl₂) 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/(m²∙s) light intensity and standard long-day conditions; wild type seeds were treated similarly to the controls. For exogenous H₂S 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/(m²∙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.

2.3. MDA, H₂O₂, GSH, Cd²⁺ contents, antioxidase activity, and H₂S production rate assays

Malondialdehyde (MDA) content was determined according to published method of Heath and Packer (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 H₂O₂ was carried out using 3,3′-diaminobenzidine (DAB) (Sigma, MO, USA) (Thordal-Christensen et al., 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 H₂O₂. 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; Gupta et al., 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 H₂O₂ (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 H₂O₂ (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 H₂O₂ (pH 7.0). Cd²⁺ 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 Cd²⁺ content. Determination of H₂S production rate was referred to published measure (Jin et al., 2011). Twelve-day-old seedlings were used to extract total protein.

2.4. Analysis of transcript levels

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′.

3. Results

3.1. Transfer (T)-DNA insertion site identification

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; Hunter, 1998). PPIases include four structurally distinct subfamilies: cyclophilins, FK506-binding proteins, parvulins, and PP2A phosphatase activator (Lu et al., 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).

Fig. 1.

Isolation of T-DNA insertional mutant

(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

3.2. Cd-sensitivity of atcsr-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). Furthermore, to genetically characterize atcsr-2, F₁ progenies from crosses between the wild type and atcsr-2 were generated. All F₁ plants did not display Cd-sensitive phenotypes. In the F₂ 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.

Fig. 2.

Identification of Cd-sensitive phenotype of atcsr-2

The seeds were scattered on 1/2 MS with 90 μmol/L CdCl₂ 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

3.3. More Cd²⁺ accumulation and less GSH generation by atcsr-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). Additionally, Cd²⁺ content was examined, and, as expected atcsr-2 accumulated much more Cd²⁺ in plant cells (Fig. 3b).

Fig. 3.

Determination of MDA, H₂O₂, GSH, and Cd²⁺ contents and antioxidative enzyme activities

Seeds of wild type (WT) and atcsr-2 were scattered on 1/2 MS with 30 μmol/L CdCl₂ for 12 d; (a) GSH content. (b) Total Cd²⁺ content; (c) MDA content; (d) H₂O₂ accumulation (reddish-brown polymer representing the H₂O₂ 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

3.4. Higher oxidative stress and lower antioxidative ability in atcsr-2

MDA is regarded as an indicator of lipid peroxidation caused by oxidative stress (Cho and Seo, 2005). MDA content and H₂O₂ 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). H₂O₂ scavenging is accomplished by various antioxidant enzymes, and thus the activities of the major enzymes involved in H₂O₂ metabolism in seedlings exposed to Cd²⁺ treatment were determined, and found to be lower in atcsr-2 compared to the wild type (Fig. 3e).

3.5. Expression detection of Cd-response related genes in the wild type and atcsr-2 with or without Cd²⁺ treatment

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 Cd²⁺ treatment. The AtCSR expression dramatically increased with Cd²⁺ treatment in the wild type; however, it sustained a relatively low level with Cd²⁺ treatment in atcsr-2 (Fig. 4a). To further determine the role of AtCSR in Cd²⁺ 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). Differing from GSH1, the GSH2 expression was not regulated by Cd²⁺ treatment. Similarly, expressions of some genes involved in Cd²⁺ transportation were studied in the wild type and mutant with or without Cd²⁺ 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).

Fig. 4.

Expression analyses of Cd-response and H₂S-emission related genes in wild type (WT) and atcsr-2

Seeds of WT and atcsr-2 were scattered on 1/2 MS with 30 μmol/L CdCl₂ or without CdCl₂ 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 Cd²⁺-transportation related genes; (d) Expression analysis of H₂S-emission related genes (actin was used as a loading control); (e) Determination of H₂S production rate (Values are expressed as mean±SE (n=3),^* P<0.05)

3.6. Expression analysis of genes associated with H₂S release and H₂S production rate assay in the wild type and atcsr-2 with or without Cd²⁺ treatment

Our laboratory is studying the function of H₂S 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 H₂S emission in plants. To determine whether H₂S is related to Cd-stress response in Arabidopsis, the expressions of H₂S-releasing related genes were determined in the wild type and atcsr-2 with or without Cd²⁺ treatment. The expression pattern of genes related to H₂S release was the same as that of genes induced by Cd-stress. The expressions of LCD and DCD markedly increased with Cd²⁺ treatment in the wild type, but were not triggered in atcsr-2 (Fig. 4d). It has been demonstrated that LCD plays a far more important role in H₂S release (Jin et al., 2011). We assayed H₂S production rate using LCD as substrate, and founded that H₂S 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).

3.7. Effects of H₂S on expression of Cd-induced genes and Cd-triggered toxicity

Previous studies demonstrated that Cd-induced genes sufficiently matched with genes involved in H₂S generation in the wild type and atcsr-2. We proposed that H₂S as a gas-transmitter regulated the expression of Cd-induced genes, and to test this hypothesis we treated Arabidopsis with different H₂S 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 H₂S physiological concentrations (Fig. 5). Since H₂S 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 H₂S fumigation was longer than that of atcsr-2 without H₂S fumigation under conditions of Cd stress, but was shorter than that of atcsr-2 without Cd stress (Fig. 6).

Fig. 5.

Effect of H₂S on the expression of GSH-synthesis and Cd-transportation related genes

Wild type seeds were scattered on 1/2 MS for 12 d, and then fumigated with different concentrations of H₂S (10, 30, or 50 μmol/L) for 5 h. The seedlings were then used for RT-PCR analysis of gene expression

Fig. 6.

Alleviation of Cd-triggered toxicity in atcsr-2 by H₂S fumigation

atcsr-2 seeds were scattered on 1/2 MS for 12 d with or without 30 μmol/L CdCl₂, and then fumigated or not with 30 μmol/L H₂S for 4 d. (a) Representative images of atcsr-2 seedlings with H₂S fumigation alone and with both Cd-stress and H₂S fumigation as well as with Cd-stress alone. The atcsr-2 was cultivated with neither CdCl₂ nor H₂S fumigation as control group. (b) Root length corresponded to the seedlings of Fig. 6a. Values are expressed as mean±SE (n=10). Values with different letters indicate significant differences (P<0.05) among them

4. Discussion

PC can chelate heavy metals and then PC-metal complexes are transported across the tonoplast into vacuoles (Cobbett, 2000). PC can be synthesized from reduced GSH by the enzyme PC synthase through a transpeptidation reaction (Rea et al., 2004). GSH plays a vital role in protecting plants against Cd-triggered toxicity by providing a sequestration system (Sanità di Toppi and Gabbrielli, 1999). GSH1 reportedly acts as a major control point under conditions of increasing demand for GSH (Yadav, 2010). In the present study, Cd²⁺ 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). Therefore, the synthesis of GSH was significantly restricted in atcsr-2 (Fig. 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 Cd²⁺ transportation. All were sharply elevated in the wild type with Cd-stress compared to atcsr-2 (Fig. 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 Cd²⁺ into the vacuole, thus protecting plants from Cd²⁺ 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 Cd²⁺ 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 Cd²⁺ into plant cells. IRT1 expression was induced by Cd-stress in atcsr-2 (Fig. 4c). Consequently, atcsr-2 transported more Cd²⁺ into the cells, accumulating superfluous Cd²⁺ 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 Cd²⁺ in cytoplasm, which could lead to increased MDA and H₂O₂, and inhibition of antioxidative enzymes’ activities (Gonçalves et al., 2009), as demonstrated in atcsr-2 (Figs. 3c–3e).

The expression pattern of most H₂S-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). This suggests that genes involved in H₂S 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). Similarly, the expression of genes related to Cd detoxification and transportation was not detected in atcsr-2 (Fig. 4c). Other results indicated that H₂S functions in regulating the expression of genes associated with Cd²⁺ chelation and transportation (Fig. 5), and alleviates the Cd-triggered toxicity (Fig. 6). Together the results suggest that AtCSR expression affects the emission of H₂S, which as a gas-transmitter could regulate expression of a host of genes involved in Cd²⁺ 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 H₂S-emission related genes and, as a result, H₂S production decreased. Finally, the decreased H₂S emission possibly led to increased assimilation of Cd²⁺ from the environment, and weakened Cd²⁺ chelation and transportation. Thus the normal functions of Cd²⁺ chelation and transportation in atcsr-2 were all destroyed. At the posttranscriptional level, the underlying mechanism of how the PPIase impacts the emission rate of H₂S remains unclear. We propose that PPIase could energetically accelerate isomerization of protein related to H₂S emission, promoting H₂S-release protein folding accurately, and as a result, H₂S gas-transmitters could be triggered rapidly.