Hydrogen sulfide - cysteine cycle system enhances cadmium tolerance through alleviating cadmium-induced oxidative stress and ion toxicity in Arabidopsis roots

Abstract

Cadmium (Cd²⁺) is a common toxic heavy metal ion. We investigated the roles of hydrogen sulfide (H₂S) and cysteine (Cys) in plant responses to Cd²⁺ stress. The expression of H₂S synthetic genes LCD and DES1 were induced by Cd²⁺ within 3 h, and endogenous H₂S was then rapidly released. H₂S promoted the expression of Cys synthesis-related genes SAT1 and OASA1, which led to endogenous Cys accumulation. The H₂S and Cys cycle system was stimulated by Cd²⁺ stress, and it maintained high levels in plant cells. H₂S inhibited the ROS burst by inducing alternative respiration capacity (AP) and antioxidase activity. H₂S weakened Cd²⁺ toxicity by inducing the metallothionein (MTs) genes expression. Cys promoted GSH accumulation and inhibited the ROS burst, and GSH induced the expression of phytochelatin (PCs) genes, counteracting Cd²⁺ toxicity. In summary, the H₂S and Cys cycle system played a key role in plant responses to Cd²⁺ stress. The Cd²⁺ tolerance was weakened when the cycle system was blocked in lcddes1-1 and oasa1 mutants. This paper is the first to describe the role of the H₂S and Cys cycle system in Cd²⁺ stress and to explore the relevant and specificity mechanisms of H₂S and Cys in mediating Cd²⁺ stress.

Introduction

Cadmium (Cd²⁺) is a common toxic heavy metal ion in the environment. It greatly affects the growth and development of plants and is harmful to human health through the food chain^1,2. Because of its carcinogenic properties and its detrimental effects on the growth of organisms, Cd²⁺ contamination of agricultural soil has become a critical concern. Preventing reduced growth and accumulation of Cd²⁺ in harvested organs of plants growing on Cd²⁺-contaminated soils has become an urgent task as it can contribute to food safety. Thus, it is important to explore plant stress defense mechanisms and to find ways to reduce the Cd²⁺ accumulation in grains.

As a heavy metal not participating in redox reactions, Cd²⁺ can easily dissolve in water and quickly be taken up by plant roots^3,4. The physiological consequences of Cd²⁺ toxicity in plants are chlorosis, stunted growth, and cell death, among others^5,6,7. At the cellular level, Cd²⁺ can alter protein structure and inhibit enzyme activity by binding to sulfhydryl and carbonyl groups and replacing essential co-factors of enzymes^7,8,9. The overproduction of reactive oxygen species (ROS) is the primary response of plants to Cd²⁺ with negative impact on cell function¹⁰. Further damage can be caused by ROS-independent, secondary mechanisms. Lipid peroxidation is the most deleterious effect caused by Cd²⁺-induced ROS⁴. Malondialdehyde (MDA), one of the decomposition products of lipid peroxidation, can modify active substrates in plant cells, including nucleic acids, proteins and saccharides¹¹. To become resistant to Cd²⁺ toxicity, plants have developed several strategies, such as inducing the alternative respiratory pathway¹¹, activating antioxidants and glutathione (GSH)¹², and regulating the influx and efflux of heavy metals^13,14, as well as regulating the levels of heavy metal chelators, phytochelatins (PCs)¹⁵ and metallothioneins (MTs)¹⁶.

Hydrogen sulfide (H₂S) has been considered toxic gas for many years, which inhibits cytochrome oxidase activity in animal cell¹⁷. Recently, it has emerged as the third endogenous gasotransmitter, following the discovery of nitric oxide and carbon monoxide¹⁷. In plant systems, endogenous H₂S is generated through enzymatic pathways. Cysteine (Cys) desulfhydrases (CDes) are key enzymes involved in H₂S generation¹⁸. Cys synthesis occurs via two sequential phases catalyzed by serine acetyltransferase (SAT) and O-acetylserine(thiol)lyase (OAS-TL), both of which are encoded by multigene families¹⁹. L-Cys desulfhydrase (LCD) is the most understood CDes in Arabidopsis; it regulates L-Cys degradation into pyruvate, ammonia and H₂S²⁰. OAS-TL regulates that H₂S and O-acetylserine (OAS) synthetise L-Cys²⁰. These physiological processes form H₂S - Cys cycle system in cell. Recently, based on the sequence characteristics of DES1, it has been classified as an OAS-TL²¹. However, functional analysis of this enzyme revealed that DES1 has a higher affinity for L-Cys and degrades it to generate H₂S²¹.

The alternative respiratory pathway is a unique pathway in the mitochondrial electron transport chain in higher plants that is regulated by alternative oxidase (AOX)²². A large body of evidence suggests that the enhanced alternative pathway could improve stress tolerance through limiting the ROS burst^22,23. Recent research indicated that respiratory activity is regulated by endogenous H₂S in Escherichia coli²⁴. However, the relationship between H₂S and the alternative respiratory pathway in plant responses to Cd²⁺ stress is unclear.

Sulfur is an essential element that is taken up by plants in its oxidized state, reduced to H₂S, and first incorporated into Cys before it is used in metabolic processes. The products of sulfur metabolism, such as Cys, GSH, PCs, MTs and H₂S, have biological functions in plant responses to heavy metal stress and oxidative stress²⁵. Recently, positive effects of H₂S in response to several types of abiotic stress in plants have been found, such as osmotic stress, salt stress, heat shock stress and heavy metal stress^26,27,28,29. It has been reported that a cross-talk between H₂S and nitric oxide is responsible for the increased Cd²⁺ tolerance in alfalfa and Bermuda grass plants^30,31. In addition, H₂S alleviates Cd²⁺ toxicity by regulating cadmium transport in Populus euphratica cells³². H₂S is also involved in the growth and development of plants through its effects on stomatal closure and seed germination and by increasing the growth rate^33,34,35,36. Cys acts as a functional precursor for many important biologic activators, such as PCs and GSH, which can enhance the tolerance of plants to heavy metal stress^25,37. In addition, Cd²⁺ tolerance significantly decreases when Cys biosynthesis is blocked in oasa1-1 and oasa1-2 mutants³⁷.

Compelling evidence has suggested that H₂S and Cys are involved in plant tolerance to heavy metal stress. The biosynthesis of H₂S and Cys are interrelated. H₂S is involved in the uptake of SO₄²⁻ and in the biosynthesis of Cys. Cys is also involved in the generation of H₂S. Nevertheless, the relation and interaction between H₂S and Cys are yet unknown under Cd²⁺ stress. In this study, we aimed to clarify the common and independent functions of H₂S and Cys in Cd²⁺ stress. Additionally, we sought to demonstrate the working mechanisms of the H₂S and Cys cycle system response to Cd²⁺ stress in Arabidopsis.

Results

Effect of Cd²⁺ on root elongation, MDA and EL in Arabidopsis roots

Arabidopsis seedlings (7-d-old) were transferred aseptically to 1/2 MS medium-containing CdCl₂, and the lengths of the primary roots were measured 5 d later. Cd²⁺ stress led to toxicity symptoms and inhibited the elongation of Arabidopsis roots in a dose-dependent manner. As shown in Fig. 1a and b, root growth was slightly inhibited under 25 μM Cd²⁺, but root elongation was significantly inhibited under 50 to 150 μM Cd²⁺, exhibiting 53.5% to 34.9% inhibition, respectively. Malondialdehyde (MDA) and electrolyte leakage (EL) are considered as good indicators of stress-induced cell damage. Cd²⁺ stress caused lipid peroxidation and MDA accumulation. When plants were treated with 50 to 150 μM Cd²⁺, the MDA content of the roots increased by 117% to 200%, respectively (Fig. 1c). In the presence of 50 to 150 μM Cd²⁺, EL increased by 131% to 217%, respectively (Fig. 1d). The 50 to 150 μM Cd²⁺ treatment had significant effects on Arabidopsis roots. 100 and 150 μM Cd²⁺ concentrations were too violent for plant growth and 150 μM Cd²⁺ concentrations lead to the seedling etiolation in Fig. 1a. Thus, 50 μM Cd²⁺ was chosen for further study of Cd²⁺ stress.

Figure 1

The effect of Cd²⁺ on root length, MDA and EL in Arabidopsis roots.

(a) Phenotype of root growth in WT seedlings. Bar = 1 cm. (b) The root lengths of WT seedlings (n > 25). (c) MDA contents in WT roots stressed by various concentrations of Cd²⁺. (d) EL in WT roots stressed by various concentrations of Cd²⁺. 7-d-old Arabidopsis seedlings were grown on 1/2 MS agar plates supplied with 0–150 μM Cd²⁺ for 5 d, and the lengths of the primary roots, MDA contents and EL were recorded. Mean values and SE were calculated from three replicates. Within each set of experiments, bars with different letters are significantly different (P < 0.05, Duncan’s multiple range tests).

Effect of Cd²⁺, NaHS and Cys on the H₂S and Cys cycle system

To explore the working mechanisms of the H₂S and Cys cycle system’s response to Cd²⁺ stress in Arabidopsis roots, a time-course analysis of endogenous H₂S and Cys contents was performed. Endogenous H₂S and Cys contents undulated along with the time of Cd²⁺ stress. H₂S content was rapidly induced after treatment with Cd²⁺ for 3 h, reached the highest level at 9 h, and then decreased at 12 h, but had another increase at 36 h (Fig. 2a). Treatment with Cys could enhance the H₂S level and maintain H₂S content at a high level in Cd²⁺ stress (Fig. 2a). The Cys level slightly decreased in the initial stage under Cd²⁺ treatment, but it increased after treatment with Cd²⁺ for 12 h and maintained a high level from 24 to 36 h (Fig. 2b). Treatment with NaHS promoted Cys accumulation and a high level of Cys was maintained during Cd²⁺ stress (Fig. 2b). When plants were treated with 50 or 100 μM Cd²⁺ for a long time, both H₂S and Cys levels were enhanced in Arabidopsis roots (Fig. 2c and d). As mentioned above, H₂S and Cys contents were elevated by Cd²⁺ stress, and H₂S appeared to be an important mediator in the Cd²⁺-induced increase of Cys, and the H₂S and Cys cycle system was enhanced.

Figure 2

Analysis of endogenous H₂S and Cys contents in WT roots of Arabidopsis.

(a) Time-course of H₂S content. (b) Time-course of Cys content. 7-d-old WT seedlings were treated with 50 μM Cd²⁺, 50 μM Cd²⁺ + 1 mM Cys and 50 μM Cd²⁺ + 50 μM NaHS for 0 to 48 h. (c) Changes of H₂S content in various Cd²⁺ concentrations. (d) Changes of Cys content in various Cd²⁺ concentrations. 7-d-old WT seedlings were supplied with 0–150 μM Cd²⁺ for 5 d. Mean values and SE are calculated from three replicates. Within each set of experiments, bars with different letters are significant different (P < 0.05, Duncan’s multiple range tests).

The effects of Cd²⁺, NaHS and Cys on synthetic genes of H₂S and Cys

To study the direct effects of Cd²⁺, NaHS and Cys on genes regulating the synthesis of H₂S and Cys, the Arabidopsis seedlings were exposed to various treatments for 3 h. The expressions of H₂S synthetic genes LCD and DES1 were markedly induced by Cd²⁺ and Cys (Fig. 3a and b), but the expression of D-CDES was not significantly affected by Cd²⁺ and Cys treatments (Fig. 3c). SATs and OASs are the important synthetic genes of Cys, but they had different responses to Cd²⁺, NaHS and Cys. The expression levels of SAT1 and OASA1 were slightly increased in Cd²⁺ stress, but they were markedly induced by treatment with NaHS (Fig. 3d and g). Additionally, the expression of SAT5 was inhibited by Cys (Fig. 3f). SAT3, OASB and OASC did not respond to Cd²⁺, NaHS or Cys (Fig. 3e,h and i).

Figure 3

qRT-PCR analysis the synthesis genes of H₂S and Cys in Arabidopsis WT roots.

Relative expression levels were normalized with the internal standard EF1a. 7-d-old WT seedlings were grown on agar plates supplemented with 50 μM Cd²⁺, 50 μM NaHS, 1 mM Cys, 50 μM Cd²⁺ plus 50 μM NaHS, and 50 μM Cd²⁺ plus 1 mM Cys for 3 h, respectively. Mean values and SE were calculated from three replicates. Within each set of experiments, bars with different letters are significant different (P < 0.05, Duncan’s multiple range tests).

The effects of Cd²⁺, NaHS and Cys on root elongation, MDA and EL in lcddes1-1 and oasa1 mutants

Five-day-old Arabidopsis seedlings were transferred aseptically to Cd²⁺-containing 1/2 MS medium, and the lengths of the primary roots were measured one week later. The root elongation of lcddes1-1 mutant was shorter compared to WT root elongation under control conditions, but the root elongation of oasa1 was the same as WT (Fig. 4a and b). The lcddes1-1 and oasa1 mutants were more sensitive to Cd²⁺ stress. Application of NaHS or Cys recovered the Cd²⁺-induced growth inhibition in WT. NaHS markedly recovered the effect of Cd²⁺ in lcddes1-1, but it only partly recovered the effect of Cd²⁺ in oasa1 (Fig. 4a and b). On the contrary, treatment with Cys slightly recovered the effect of Cd²⁺ in lcddes1-1, but it significantly recovered the effect of Cd²⁺ in oasa1 (Fig. 4a and b). Cys-mediated partial recovery the root length may be due to an independent physiological action of Cys in lcddes1-1 because H₂S production was blocked in the double mutant. NaHS or Cys treatment markedly decreased the EL level and the content of MDA under Cd²⁺ stress in WT (Fig. 4c and d). NaHS strongly reduced the MDA content and the EL in lcddes1-1 and oasa1 (Fig. 4c and d). Cys prevented the effects of Cd²⁺ on the MDA content and EL in oasa1 but partly weakened the effects of Cd²⁺ on the MDA content and EL in lcddes1-1 (Fig. 4c and d).

Figure 4

The effect of H₂S and Cys on root length, MDA and EL in lcddes1-1 and oasa1 mutant plants under Cd²⁺ stress.

(a) Phenotype of Arabidopsis root growth. Bar = 1 cm. (b) The root lengths of Arabidopsis seedlings (n > 25). (c) MDA contents in Arabidopsis roots. (d) EL in Arabidopsis roots. 7-d-old Arabidopsis seedlings were grown on 1/2 MS agar plates supplied with 50 μM Cd²⁺, 50 μM Cd²⁺ plus 50 μM NaHS and 50 μM Cd²⁺ plus 1 mM Cys for 5 d respectively, and the lengths of the primary roots, MDA contents and EL were recorded. Mean values and SE were calculated from three replicates. Within each set of experiments, bars with different letters are significantly different (P < 0.05, Duncan’s multiple range tests).

The effects of Cd²⁺, NaHS and Cys on the alternative respiratory pathway

The alternative respiratory pathway plays an important role in plant stress resistance by limiting the ROS burst³⁸. In this study, we sought to elucidate the roles of NaHS and Cys in the alternative respiratory pathway under Cd²⁺ stress. In general, the alternative pathway operates at a low level under normal conditions, but it can be significantly induced when plants are stimulated by various environmental stresses²³. We first checked the expression of AOX genes after Cd²⁺, NaHS and Cys treatments for 3 h. The expression of AOX1A, AOX1C and AOX2 were increased in Cd²⁺ stress (Fig. 5a,b and c). Interestingly, NaHS and Cys treatments also markedly enhanced the expression levels of AOX1A, AOX1C and AOX2 in both control and Cd²⁺ stress conditions (Fig. 5a,b and c). Furthermore, the total respiration capacity (TP), cytochrome respiration capacity (CP) and alternative respiration capacity (AP) were analyzed in WT and mutant plants. TP was slightly enhanced by 25 or 50 μM Cd²⁺, but it was inhibited by 100 or 150 μM Cd²⁺ in WT (Fig. 5d). Under Cd²⁺ stress, CP was inhibited in a dose-dependent manner; however, AP was increased under Cd²⁺ stress, and AP achieved its maximum induction with the 50 μM Cd²⁺ treatment (Fig. 5e). Similar to the pattern of expression of the AOX genes in response to NaHS or Cys treatments under Cd²⁺ stress, AP was increased by NaHS or Cys under Cd²⁺ stress (Fig. 5f). However, it was different in the mutant plants. Under Cd²⁺ stress, the effects of Cys on AP were not observed in lcd and des1-1, and they were especially decreased in lcddes1-1 (Fig. 5g,h and i). In oasa1, the effects of NaHS and Cys were the same as in WT under Cd²⁺ stress (Fig. 5g).

Figure 5

Effect of H₂S and Cys on the expression of AOX genes and the activity of TP, CP and AP in Cd²⁺ stress.

(a–c) The expression of AOX1A, AOX1C and AOX2. 7-d-old WT seedlings were grown on 1/2 MS agar plates supplied with 50 μM Cd²⁺, 50 μM NaHS, 1 mM Cys, 50 μM Cd²⁺ plus 50 μM NaHS, and 50 μM Cd²⁺ plus 1 mM Cys for 3 h, respectively. (d,e) Changes in TP, CP and AP activity in various Cd²⁺ concentrations for 5 d in WT. (f–l) AP activity in WT and mutants. 7-d-old Arabidopsis seedlings were grown on 1/2 MS agar plates treated with 50 μM Cd²⁺, 50 μM Cd²⁺ plus 50 μM NaHS and 50 μM Cd²⁺ plus 1 mM Cys for 5 d. Mean values and SE were calculated from three replicates. Within each set of experiments, bars with different letters are significantly different (P < 0.05, Duncan’s multiple range tests).

The effects of Cd²⁺, NaHS and Cys on antioxidant enzyme activity and GSH level, and the relationship among AP, antioxidant enzyme activity, and GSH level in Cd²⁺ stress

Antioxidant enzymes depress the level of ROS. A previous study showed that H₂S could enhance antioxidase activity in rice³⁹. In addition, many studies suggested that AOX was important in maintaining the homeostasis of the redox state^22,38. Therefore, the effects of Cd²⁺, NaHS, Cys and AP on antioxidant enzyme activity were analyzed. As shown in Fig. 6a and c, after 12 h of 50 μM Cd²⁺ treatment, the activities of SOD and CAT in plants were significantly higher than in the control plants in WT. NaHS or Cys treatments could enhance the antioxidase activity under unstressed conditions (Fig. 6b and d), and this enhancement was further strengthened under Cd²⁺ stress in WT (Fig. 6a and c). However, treatment with n-propyl gallate (nPG) had no significant effect on the antioxidase activity of the plants either under Cd²⁺ stress or under unstressed conditions. Furthermore, nPG did not affect the elevated antioxidase activity of the NaHS- and Cys-treated plants under Cd²⁺ stress (Fig. 6a and c). The effects of Cd²⁺ and Cys were altered in lcddes1-1; treatment with Cd²⁺ or Cys did not enhance the antioxidase activity in lcddes1-1 (Fig. 6b and d). Additionally, the effects of Cd²⁺ on the SOD activity were also weakened, and CAT activity was negligible in oasa1 (Fig. 6d). Contrarily, treatment with NaHS still enhanced the antioxidase activity in mutant plants (Fig. 6b and d).

Figure 6

Effect of H₂S and Cys on antioxidant enzymes activity and GSH level in Cd²⁺ stress.

7-d-old Arabidopsis seedlings were grown on 1/2 MS agar plates supplemented with 50 μM Cd²⁺, 50 μM NaHS, 1 mM Cys, and 200 μM nPG for 6 h, and SOD activity (a,b), CAT activity (c,d) and GSH content (e,f) were recorded. Mean values and SE were calculated from three replicates. Within each set of experiments, bars with different letters are significantly different (P < 0.05, Duncan’s multiple range tests).

GSH is the product of sulfur metabolism, and it has positive biological functions in plant responses to heavy metal stress and oxidative stress²⁵. As shown in Fig. 6e, the GSH content was increased in Cd²⁺ stress. NaHS and Cys also enhanced the GSH level in WT (Fig. 6e and f). Specially, Cys had a significant promoting effect on GSH content. The oasa1 mutant did not respond to Cd²⁺ and NaHS, and even had a reduced GSH level, but Cys still increased the GSH content in oasa1 (Fig. 6f). Additionally, the effect of Cd²⁺, NaHS and Cys on the GSH content in lcddes1-1 was the same as WT plants (Fig. 6f).

Effect of NaHS and Cys on ROS, and the relationship between AP and ROS in Cd²⁺ stress

To estimate the potential role of the H₂S and Cys cycle in ROS homeostasis, we visualized the production of H₂O₂ in the roots under Cd²⁺ stress. Over-accumulation of H₂O₂ was visualized by fluorescence labeling in the roots subjected to Cd²⁺ stress (Fig. 7a). Conversely, NaHS or Cys treatment considerably diminished the accumulation of H₂O₂ in Cd²⁺ stress (Fig. 7a and b). Additionally, inhibiting the alternative respiratory pathway with nPG caused an over-accumulation of H₂O₂ under Cd²⁺ stress. The effects of NaHS and Cys were partly averted and slightly weakened by nPG in Cd²⁺ stress, respectively (Fig. 7a and b). As shown in the time-course of H₂O₂, the ROS burst occurred during the early phase of Cd²⁺ stress. Then, high levels of ROS were maintained from 4 to 8 h and declined after 12 h. H₂S supplementation could maintain H₂O₂ at a low level during Cd²⁺ stress. Treatment with Cys did not alter the burst of H₂O₂ in the early phase, but it prevented the over-accumulation of H₂O₂ after 6 h (Fig. 7c).

Figure 7

The changes of the endogenous H₂O₂ level in Arabidopsis root.

(a) H₂O₂ H₂DCF-DA fluorescence in WT roots (bar = 100 μm). (b) Quantification of H₂O₂ H₂DCF-DA fluorescence intensity. (c) Time-course of H₂O₂ level. 7-d-old Arabidopsis seedlings were grown on 1/2 MS agar plates supplemented with 50 μM Cd²⁺, 50 μM NaHS, 1 mM Cys, and 200 μM nPG for 6 h (a,b), 0–24 h (c). H₂DCF-DA fluorescence intensity data represents mean grey values. SE was calculated from measurements of at least five roots for each treatment, and the experiments were repeated three times. Within each set of experiments, bars with different letters are significantly different (P < 0.05, Duncan’s multiple range tests).

Effect of NaHS and Cys on Cd²⁺ accumulation

The role of H₂S and Cys in Cd²⁺ homeostasis was investigated by measuring the percentage of Cd²⁺ in the root. The results in Fig. 8a show that Cd²⁺ accumulation increased in roots under Cd²⁺ stress in WT and in the mutants, but the mutants accumulated more Cd²⁺ than the WT. NaHS or Cys supplementation had inhibitory effects on Cd²⁺ uptake and accumulation in WT and oasa1. Nevertheless, lcddes1-1 did not respond to the effect of NaHS under Cd²⁺ stress, but the effect of Cys on Cd²⁺ uptake and accumulation was only partially reduced in lcddes1-1.

Figure 8

Effect of NaHS and Cys on Cd²⁺ percentage and qRT-PCR analysis the expression of heavy metal chelators genes in Arabidopsis roots.

(a) Cd²⁺ accumulation in Arabidopsis roots. (b) The expression of PCS1, PCS2, MT1A, MT1B and MT2B in WT. (c) Time-course of PCS1 gene expression. (d) Time-course of MT1A gene expression. 7-d-old Arabidopsis seedlings were grown on 1/2 MS agar plates supplied with 50 μM Cd²⁺, 50 μM NaHS and 1 mM Cys for 5 d (a), 3 h (b), 0–12 (c,d). Mean values and SE were calculated from three replicates. Within each set of experiments, bars with different letters are significantly different (P < 0.05, Duncan’s multiple range tests).

Effect of NaHS and Cys on the expression of heavy metal chelator genes

When plants were treated with Cd²⁺ for 3 h, the expression of the heavy metal chelator genes PCS1, PCS2, MT1A, MT1B and MT2B was significantly up-regulated in WT. Cys supplementation promoted the expression of PCS1 and PCS2, and NaHS promoted the expression of MT1A, MT1B and MT2B (Fig. 8b). To further study the effect of the H₂S and Cys cycle system on the heavy metal chelator genes, the time-course of PCS1 and MT1A gene expression was investigated. Cd²⁺ was found to up-regulate the expression of PCS1 and MT1A genes at 0.5 h, which then remained at a high expression level. Cys enhanced the expression of the PCS1 gene at 0.5 h, which reached a maximum by 1 h, but NaHS enhanced the expression of the PCS1 gene at 6 to 12 h. The expression of MT1A was different from PCS1. After 3 h of Cys supplementation, the expression of MT1A started to increase, reaching a maximum at 6 h, and NaHS enhanced the expression of MT1A gene at 0.5 h (Fig. 8c and d).

Discussion

The root is the primary organ that plants deploy to accumulate most of the heavy metals to which they are exposed^40,41. Sulfur metabolism is required for the growth and development of plants, and the production of sulfur metabolites also plays a critical role in plant responses to heavy metal-induced stress²⁵. H₂S and Cys are important sulfur metabolism products that participate in suppressing heavy metal stress in plants⁴⁰. In previous reports, H₂S and Cys were always studied separately in plant responses to abiotic stress^39,42. Recently, H₂S and H₂S-induced Cys accumulation were reported to be critical in imparting Cr⁶⁺ tolerance in Arabidopsis⁴³. Therefore, the H₂S and Cys cycle is an important system for regulating H₂S and Cys functions in heavy metal stress. In this study, we used the lcddes1-1 and oasa1 Arabidopsis mutants to block the H₂S and Cys cycle system. Then, we intensively researched the relevant and specificity roles of H₂S and Cys in Cd²⁺ stress. Our results indicated that Cd²⁺ can rapidly accumulate in Arabidopsis roots and inhibit the primary root growth in a Cd²⁺ concentration-dependent manner (Figs 1a and 7a), suggesting that Cd²⁺ is easily absorbed and highly toxic. We observed that endogenous H₂S and Cys levels undulate from 3 to 48 h under Cd²⁺ stress (Fig. 2). However, the endogenous patterns of change were different for H₂S and Cys levels. Endogenous H₂S was first induced by Cd²⁺ stress, and then Cys levels increased. On this account, we suppose that H₂S is produced rapidly under Cd²⁺ stress and that it acts as second messenger to activate the synthesis of Cys, implying that Cd²⁺ stress could be the direct cause of endogenous H₂S release but that Cys accumulation is a secondary effect of Cd²⁺ stress. Data for the expression of H₂S and Cys synthetic genes supports this hypothesis. The expression of H₂S synthetic genes was directly induced by Cd²⁺, and then, exogenous H₂S supplementation induced the upregulation of Cys synthetic-related genes (Fig. 3). Additionally, exogenous H₂S or Cys supplementation during Cd²⁺ stress could rapidly induce mutual endogenous levels of the other contents in Cd²⁺ stress. These results suggested that the H₂S - Cys cycle system was triggered by Cd²⁺ and that H₂S and Cys could promote the production of each other, forming a cycle of activation. Finally, treatment with Cd²⁺ for 5 d, H₂S and Cys contents increased significantly in Arabidopsis roots.

The expression levels of the Cys synthesis-related genes OASA1 and SAT1 were up-regulated significantly by H₂S treatment, and the H₂S synthesis genes LCD and DES1 were up-regulated significantly by Cys treatment (Fig. 3). OASA1 directly regulated Cys synthesis, and LCD and DES1 directly regulated H₂S synthesis; thus, lcd, des1-1, lcddes1-1 and oasa1 were used to study the H₂S and Cys cycle system in Cd²⁺ stress. The Cd²⁺-induced root shortening and increases in MAD and EL were markedly enhanced in mutant plants, suggesting that the Cd²⁺ resistance was weakened when the H₂S and Cys cycle was blocked. Exogenous H₂S or Cys supplementation only partly restored the root growth, MAD and EL levels, suggesting that H₂S or Cys alone could not replace the function of the H₂S and Cys cycle in plant cells. Additionally, the H₂S and Cys system is also important for stress caused by other heavy metals, such as Cr⁶⁺; it was reported that NaHS treatment increases the expression levels of the Cys synthesis-related genes⁴³. However, different heavy metal stress condition lead to the difference genes expression include MTs genes⁴³, thus Cd²⁺ and Cr⁶⁺ condition also could lead to the difference MTs genes expression. The details regarding the mechanism of H₂S in heavy metal resistance requires further study.

Excessive Cd²⁺ can induce the production of ROS, which is highly toxic to biomembranes, nucleic acids and proteins¹¹. The alternative respiratory pathway plays an important role in stress conditions by repressing the production of ROS^22,23,42. Our study also found that the CP and AP activities were altered by Cd²⁺ stress (Fig. 5e). Plant signaling molecules, such as nitric oxide, can regulate the alternative respiratory pathway in stress conditions⁴⁴. Whether an H₂S signal or Cys could affect AP activity was not previously known; our analysis found that exogenous H₂S or Cys supplementation could further induce the activity of AP in Cd²⁺ stress. However, in H₂S synthesis mutants, the effect of Cys was negligible, and in Cys synthesis mutants, the effect of H₂S was not altered. These data imply that H₂S is a direct trigger of AP activity and that Cys might play an indirect role in Cd²⁺ stress. The expression of AOX genes was also induced by H₂S within 3 h, but not by Cys.

Antioxidases are also one of the central elements in maintaining ROS levels in plant cells⁴⁵. We investigated the connection between the alternative respiratory pathway and antioxidases, but we found that the activities of SOD and CAT were not altered when the alternative respiratory pathway was inhibited by nPG (Fig. 6a and c), suggesting that the alternative respiratory pathway and antioxidases have independent functions in response to Cd²⁺ stress. The activities of SOD and CAT were induced by Cd²⁺ and increased Cd²⁺ resistance (Fig. 6). H₂S or Cys biosynthesis was necessary for the increase in SOD and CAT activities in response to Cd²⁺ stress because Cd²⁺-induced activities of SOD and CAT were weakened in H₂S and Cys synthesis mutants. We further studied the relationship of H₂S and Cys in this physiological process. H₂S supplementation could remedy the deficiency of Cys biosynthesis and increase the activities of SOD and CAT in oasa1 mutants, but Cys supplementation could not. These data suggest that the activities of SOD and CAT are directly regulated by H₂S and that Cys indirectly affects the activities of SOD and CAT by promoting the generation of H₂S.

GSH performs numerous physiological functions in the plant response to heavy metal stress⁴⁶. Cys is a precursor of GSH, which stores and transports GSH via the γ-glutamyl cycle⁴⁷. In this study, supplementation with exogenous H₂S or Cys strengthened Cd²⁺-mediated GSH elevation in WT plants (Fig. 6f). It is interesting that the effects of Cd²⁺ and H₂S were reversed in oasa1, but the effects of Cd²⁺ and H₂S were not altered in lcddes1-1 (Fig. 6f). These results suggest that Cys is a direct regulatory factor of GSH, and H₂S affects GSH levels indirectly. Additionally, the GSH content was not altered by nPG (Fig. 6e), suggesting that the alternative respiratory pathway and GSH are not related in their responses to Cd²⁺ stress.

Cd²⁺ enrichment was also observed in this study (Fig. 8a). Inhibiting Cd²⁺ uptake and enhancing Cd²⁺ efflux are the main defense strategies that plant cells use to prevent Cd²⁺ toxicity. Exogenous H₂S or Cys supplementation effectively inhibited the accumulation of Cd²⁺ (Fig. 8a). When endogenous H₂S or Cys synthesis was blocked, Cd²⁺ over-accumulation occurred (Fig. 8a), suggesting that the H₂S and Cys cycle system is important for inhibiting Cd²⁺ uptake or enhancing Cd²⁺ efflux. Additionally, the effect of Cys was partly inhibited in the lcddes1-1 mutant, implying that the role of H₂S in the H₂S and Cys cycle might be to directly regulate Cd²⁺ uptake or efflux.

The generation of chelators is also an effective pathway in plant cells for avoiding Cd²⁺ toxicity. PCS1, PCS2, MT1A, MT1B and MT2B are mainly expressed in roots and regulate PCs and MTs synthesis; the expression of these chelators is generally induced by numerous heavy metal ions^42,43,48. Interestingly, the expression of PCS1 and PCS2 was found to be induced by Cys in a very short time, and the expression of MT1A, MT1B and MT2B was induced by H₂S (Fig. 8b). However, only long-term supplementation of Cys or H₂S induced the expression of PCS1 and MT1A (Fig. 8c,d). These data suggest that the generation of chelators can be regulated differently in plant cells. Cys and H₂S played different roles in the physiological process, but when combined Cys and H₂S mutually promoted the expression of chelator synthesis genes to a level higher than when they were used as separate supplements.

Based on the data described above, a signal pathway model was developed and is depicted in Fig. 9. It shows the specific roles of H₂S and Cys in regulating plant responses to Cd²⁺ stress and their interaction. H₂S is activated much earlier than Cys in plant responses to Cd²⁺ stress, acting as a secondary messenger to increase Cys accumulation by regulating the transcription levels of SAT1 and OASA1. In addition, the production of H₂S might deplete the endogenous Cys pool, which might subsequently increase the expression of SAT1 or OASA1. Furthermore, once the H₂S and Cys cycle is initiated, it works to maintain elevated H₂S and Cys levels. H₂S inhibits the ROS burst by promoting CP and antioxidase activities, and it weakens Cd²⁺ ion toxicity by inducing the gene expression of MTs. Cys acts as a precursor of GSH to promote GSH accumulation, which then contributes to inhibiting the ROS burst. GSH also induces genes expression of PCs, leading to raised PC activity, which counteracts Cd²⁺ ion toxicity. In sum, the H₂S and Cys cycle system is a key regulator of the response to Cd²⁺ stress in plants that acts to induce and maintain levels of bioactive molecules (H₂S, Cys, GSH, PCs, and MTs) that improve plant resistance to Cd²⁺ stress.

Figure 9

A diagram representing Cd²⁺-induced toxicity and protective mechanism of the H₂S and Cys cycle system in Arabidopsis roots.

Arrows indicate enhanced effects and hyphens indicate suppressed effects.

Materials and Methods

Plant material and chemical treatments

This study was carried out on Arabidopsis thaliana, including wild ecotypes Columbia (Col-0) and the lcd (SALK_082099), des1-1 (SALK_103855), lcddes1-1 and oasa1 (SALK_074242c) mutants. Seeds were surface sterilized with 70% ethanol for 30 s and 15% sodium hypochlorite for 15 min and were washed at least five times with sterilized water before sowing on solid 1/2 Murashige and Skoog (MS) medium (pH 5.7), which contained 1% (w/v) sucrose, and 0.8% (w/v) agar. After that, the seeds were vernalized for 48 h at 4 °C. Then, the seedlings were grown in a growth room, which had the temperature set at 22 °C and a 14/10 h light/dark photoperiod under a photon flux density of 120 mmol m⁻²s⁻¹. The Arabidopsis plants used throughout this work were grown routinely in a growth chamber under 50–60% humidity.

Following 7 d growth, Arabidopsis seedlings were transferred to the following mediums: (1) 1/2 MS agar medium, (2) 1/2 MS agar medium containing 25–150 μM CdCl₂, 50 μM sodium hydrosulfide (NaHS), 1 mM Cys, or 200 μM n-propyl gallate (nPG), respectively. The H₂S donors NaHS, Cys and nPG were purchased from Sigma (USA).

Root elongation assays

Seven-day-old Arabidopsis seedlings grown on the vertical 1/2 MS agar plates were transferred to the 1/2 MS agar medium containing various chemicals for the different treatments. Root elongation was measured after 5 d of various treatments. All experiments were repeated at least three times, with photographs collected at 7 d from one representative experiment being shown. The root length was measured with ImageJ.

Electrolyte leakage assay

Measurement of ion leakage was determined according to Sairam and Srivastava (2002) with some modifications⁴³. The 7-d-old Arabidopsis seedlings were treated for 5 d on the 1/2 MS agar medium containing different chemicals. Following the treatments, the roots were collected and washed in deionized water three times to remove surface-adhered electrolytes. Then, they were immersed in 10 ml deionized water for 3 h at 25 °C in test tubes. After the incubation, the conductivity in the bathing solution was determined (C₁), and the conductivity of deionized water was also determined (C₀). The samples were heated in boiling water for 1 h before the total conductivity was measured in the bathing solution (C₂). Relative ion leakage was expressed as a percentage of the total conductivity after heating in boiling water [relative ion leakage = (C₁ − C₀)/(C₂ − C₀) × 100].

MDA and GSH content assays

The chemical treatments were the same as the measurements of ion leakage. Following the treatments⁴⁹, Arabidopsis roots were collected. Lipid peroxidation of the roots was measured by estimating the MDA content according to the method of Heath and Packer. The GSH content was measured based on a previously described method⁴⁹.

Measurement of H₂S content

H₂S quantification was performed as described by Nashef et al.⁵⁰. The chemical treatments were the same as the methods of ion leakage. Following the treatments, the seedling roots were collected with liquid nitrogen and ground into fine powder with mortar and pestle; 0.3 g of frozen tissue was homogenized in 1 ml 100 mM potassium phosphate buffer (pH = 7), which contained 10 mM ethylenediaminetetra-acetic acid (EDTA). The homogenates were centrifuged at 15,000 × g for 20 min at 4 °C, and 100 μl of supernatant was used for the quantification of H₂S in an assay mixture containing 1,880 μl extraction buffer and 20 μl of 20 mM 5,5′-dithiobis (2-nitrobenzoic acid), for a total volume of 2 ml. The assay mixture was incubated at room temperature for 2 min, and the absorbance was read at 412 nm. H₂S was quantified based on a standard curve of known concentrations of NaHS.

Measurement of the Cys content

The chemical treatments were the same as the measurements of ion leakage. Following the treatments, Arabidopsis roots were collected. Cys can react specifically with acid ninhydrin, and the product was extracted by methylbenzene, which has a maximum absorbance at 560 nm. The reaction is highly sensitive for Cys determination. Thus, the Cys content could be determined as described previously⁵¹.

RNA isolation and qRT-PCR

Seven-day-old Arabidopsis seedlings were transferred to the 1/2 MS agar medium containing different chemicals and treated for 0–12 h. Following the treatments, roots of Arabidopsis were harvested to extract total RNA for real-time polymerase chain reaction (RT-PCR). Total RNA was extracted using an RNAprep pure plant kit (Tiangen, Beijing) and was treated with RNase-free DNase reagent (RNase-free DNase kit, Tiangen). The total RNA was reverse-transcribed into first-strand cDNA using PrimeScript™ Reverse Transcriptase (Takara, Japan) and Oligo (dT)₁₅ primer (Takara) following the manufacturer’s instructions. The samples were amplified using SYBR Green I (SYBR^® Premix Ex Taq™ Kit, Takara). The housekeeping gene EF1A was used as an internal control. The thermal cycle used was as follows: 95 °C for 10 s, and 40 cycles of 95 °C for 5 s and 59 °C for 25 s. This was followed by 80 cycles of 10 s during the time elapsed at 55–95 °C. The PCR amplifications for each gene were performed in triplicate. The results were analyzed by Rotor-Gene Real-Time Analysis Software 6.1 (Build 81).

Extraction and assay of antioxidant enzymes

Seven-day-old Arabidopsis seedlings were transferred to the 1/2 MS agar medium containing different chemicals and treated for 6 h. Following the treatments, Arabidopsis roots were collected and enzymes extracted according to the method of Mostofa et al.⁵¹. Activities of antioxidase and glyoxalase were determined by the standard methods reported in Mostofa and Fujita⁵² for SOD (EC 1.15.1.1) and CAT (EC 1.11.1.6). The protein standard was bovine serum albumin (BSA), which was employed to determine the protein content.

Determination of H₂O₂ contents

H₂O₂ was visualized using the specific H₂O₂ fluorescent probe dichlorofluorescein diacetate (H₂DCF-DA) according to the method described by Maffei et al.⁵³. Seven-day-old Arabidopsis seedlings were transferred to the 1/2 MS agar medium containing different chemicals and treated for 0–24 h. Following the treatments, Arabidopsis seedlings were incubated in the reaction buffer containing 10 mM 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid (HEPES)-NaOH (pH 7.5) and 10 μM H2DCF-DA for 15 min at 25 °C. Thereafter, the roots were washed three times with the HEPES-NaOH buffer (pH 7.4) prior to visualization using a laser confocal scanning microscope (Leica SM IRBE Multisync FE 1250). Excitation was at 480 nm and emission was at 520 nm. Images were processed and analyzed using the Leica Tcs SP2 software.

Statistical analysis

Each experiment was repeated at least three times and with three replications each time. Values were expressed as the mean ± SE. Experiments that required an analysis of variance were analyzed using SPSS 17.0 for one-way analysis of variance (ANOVA) followed by Dunnett’s post hoc multiple comparisons. The confidence coefficient was set at 0.05.