Hydrogen Sulfide Inhibits Cadmium-Induced Cell Death of Cucumber Seedling Root Tips by Protecting Mitochondrial Physiological Function

Hydrogen sulfide (H2S) can alleviate Cd-induced cell death, but the molecular mechanisms are not clear. To shed light on these mechanisms, cell death induced by 200 μM cadmium chloride in cucumber seedlings root tips was used as a model system. Here, we report that the negative effect of Cd stress in mitochondrial physiological functions include changes in cytochrome c/a, mitochondrial membrane permeability transition pores, and adenosine triphosphatase (ATPase). Moreover, Cd stress led to the release of mitochondrial Ca2+ into the cytosol. Exogenous application of sodium hydrosulfide (NaHS, a donor of H2S) inhibited cell death and maintains mitochondrial function by reducing mitochondrial hydrogen peroxide accumulation, increasing ATPase activity and down-regulating CsVDAC and CsANT expression. In summary, H2S suppressed Cd-induced cell death by improving mitochondrial physiological properties.


Introduction
Calcium (Ca 2+ ) as a second messenger plays a vital role in plant activity, regulation of gene expression, cell cycle control (Berridge et al. 2003), control of light signaling (Harada et al. 2003), inducing hormone responses (Shintaro et al. 2007), controlling cell growth (Sabine et al. 2007) and trigger plan responses during plant-pathogen interactions (Zhi et al. 2010). The short-term increase of cytosolic calcium ([Ca 2+ ] Cyt ) concentration is an early event in a series of biological processes and stress responses (Jörg et al. 2010;Leng 2012). After receiving intracellular or extracellular stimulation, plants activate Ca 2+ channels and related transporters, induce extracellular Ca 2+ influx or stored Ca 2+ release to the cytosol, resulting in an instantaneous rise of Ca 2+ , which signals a multitude of downstream effects (Sanders et al. 2002). The Ca 2+ influx and release require different Ca 2+ ion channels, (i) voltage-dependent Ca 2+ -permeable channels (VDCCs) which are divided into depolarizationactivated Ca 2+ -permeable channels (DACCs) and hyperpolarization-activated Ca 2+ -permeable channels (HACCs); (ii) voltage-independent Ca 2+ -permeable channels (VICCs) . After the completion of Ca 2+ signal, in order to maintain a low concentration of Ca 2+ , plants need to return excess Ca 2+ to the calcium stores. H + /Ca 2+ antiporters and Ca 2+ -ATPase are the main transport and export protein, which transports Ca 2+ to extracellular domain or influxes to vacuoles and mitochondria (Boursiac and Harper 2007;Jörg et al. 2010). Mitochondria are not only the energy powerhouse of the cell but also a major hub for cellular Ca 2+ signaling crucial for cell life and death (Giorgi et al. 2012;Rimessi et al. 2008;Rosario et al. 2012). Mitochondria regulate cell activity by participating in dynamic regulation of Ca 2+ (Marchi et al. 2018). Mitochondria, as one of the organelles storing Ca 2+ , play a key role in the intracellular Ca 2+ signal transmission (Sisalli et al. 2012). Mitochondrial Ca 2+ is involved in the process of cell death. Jones et al. found that the release of mitochondrial Cyt c into the cytoplasm induced by Ca 2+ during the differentiation of tubular molecules leads to programmed cell death (PCD) (Jones 2002), and the increase of Ca 2+ content in tobacco protoplasts induced the opening of mitochondrial membrane permeability, which leaded to PCD (Lin et al. 2005).
Many studies suggested that hydrogen sulfide (H 2 S) is an important gas signal molecule in plant, just like carbon monoxide (CO) and nitric oxide (NO) (Hancock and Whiteman 2014). L-cysteine desulfhydrase (LCD) and D-cysteine desulfhydrase (DCD) have been reported to be the key enzymes, which generate endogenous H 2 S (Anja et al. 2005;Riemenschneider et al. 2010). Moreover, several researches showed that H 2 S is involved in plant growth and development, like root development and regulating stomatal closure (Kou et al. 2018;Li et al. 2018aLi et al. , 2018bMa et al. 2019;Chen et al. 2021), and H 2 S plays vital role in alleviating heavy metal stress, especially cadmium (Cd) stress (Ahmad et al. 2019;Guan et al. 2018;Zhang et al. 2015a). H 2 S has crosstalk with many molecules, like ethylene (Jia et al. 2018) and Ca 2+ . Fang et al. reported that Ca 2+ in a H 2 S-dependent manner resisted chromium stress in millet and Ca 2+ /calmo-dulin2 (CaM2)-mediated Ca 2+ signal regulated endogenous H 2 S content under Cr 6+ stress, Cr 6+ promoted the interaction between Ca 2+ /CAM2 and transcription factor TGA3 to enhance the expression of LCD in Arabidopsis thaliana (Fang et al. 2014a(Fang et al. , 2017. Ca 2+ activated endogenous H 2 S accumulation to improve thermo tolerance in tobacco suspension (Li et al. 2015). In addition, pretreatment with NaHS can improve thermo tolerance of tobacco suspension by regulating intracellular and extracellular Ca 2+ concentration (Li et al. 2012). Interestingly, H 2 S could reduce plant cell death, like hypoxia (Cheng et al. 2013), Cd stress (Zhang et al. 2015a), and GA-triggered PCD in wheat aleurone layers (Xie et al. 2014;Zhang et al. 2015b).
The components of MPTP include voltage-dependent anion channel (VDAC), adenine nucleotide translocator (ANT), and cyclophilin D (Cyp D). ANT is located in the inner membrane of mitochondrion. It catalyzes the energy conversion between ATP and ADP of cytoplasm and mitochondria, which is very important to cells (Javadov et al. 2000). ANT is a main role in cell death. The change of mitochondrial ANT induces the degree of MPTP opening. Such as salt stress induced PCD in tobacco protoplasts (Lin et al. 2006). The VDAC is mitochondrial outer membrane components, and it is a key player in mitochondria-mediated cell death (Kusano et al. 2009). Some studies have shown that the ROS produced by VDAC overexpression was the cause of apoptosis (Huang et al. 2014;Yuan et al. 2008). VDAC and ANT might also elicit mitochondrial Ca 2+ efflux since it is a part of the MPTP (Bernardi 1999;Crompton et al. 2002).
The change of intracellular Ca 2+ concentration affects the state of mitochondria and leads to cell death, while H 2 S alleviates cell death by reducing oxidative stress. It is noteworthy that it is unclear whether H 2 S reduces cell death by affecting the concentration of Ca 2+ , especially the concentration of mitochondrial Ca 2+ . In this study, we investigated H 2 S and mitochondrial Ca 2+ in terms of Cd-induced cell death, focusing on effect of H 2 S on mitochondrial Ca 2+ transport.

Materials and Methods
Seeds of cucumber (Cucumis sativus 'Xinchun 4') were germinated in petri dishes linked with filter papers in darkness at 28 °C in illuminating incubation climate box; all the seeds are divided into four parts. The seeds of the first part germinated in darkness water for 48 h and then cultured in light for 48 h. The second part of the seeds germinated for 48 h and then transferred to 200 μM CdCl 2 solution for stress treatment for 48 h. The third part of the seeds germinated in distilled water for 24 h, then transferred them to petri dish containing 100 μM NaHS to pretreat for 24 h in the dark, and finally transferred seedlings to another petri dish containing 200 μM CdCl 2 for stress treatment for 48 h. The fourth part of seeds germinated in distilled water for 36 h and then transferred them to 400 μM hypotaurine (HT, a scavenger of H 2 S) to pretreat for 12 h in the dark and finally replaced with 200 μM CdCl 2 solution for cultivation for 48 h. Light culture was carried out during Cd stress treatment for 48 h, the condition of illuminating incubation climate box was set to 25 ± 1 °C, 12 h photoperiod and photosynthetically active radiation = 200 μmol m −2 s −1 . Then, the root length and fresh weight of seedling were measured.

Detection of Cell Death
Evans blue was used for cell death detection. The seedling root tips (2 cm) treated in different treatments were excised and stained with 0.1% (w/v) Evans blue for 15 min and washed them with water for 10 min and then taken pictures with fluorescence microscope (Revolve RVL-100-G, ECHO, USA). Finally, the stained samples were extracted with 1 mL 80% ethanol and bathed in water at 50 °C for 15 min and then centrifuged at 10,000 g for 10 min; the absorbance was measured at 600 nm (Zhang et al. 2015a).

Detection of Endogenous H 2 S Content and Mitochondrial H 2 O 2 Content
According to the method of determining H 2 S content by Fang et al. (Fang et al. 2014b), 0.2 g root sample was added with 5 mL 50 mM phosphate buffer solution (0.2 M ascorbic acid (AsA), 0.1 M EDTA, and 0.5 mL 1 M HCl, PH 6.8) was added to homogenate, the released H 2 S was caught by 1% (w/v) zinc acetate. After 30 min, 0.3 mL 5 mM dimethyl-pphenylenediamine dissolved in 3.5 mM H 2 SO 4 added to the mixing and then 0.3 mL of 50 mM ferric ammonium sulfate was added. After 15 min of reaction, the value of absorption at 667 nm was detected.
According to the method of He et al. (He et al. 2018), 1 mL of the isolated mitochondrial extract was taken and put into the test tube; 1 mL of 0.1 M phosphoric acid buffer (pH 7.0) and 2 mL KI were added, mixed, and shaken well and allowed to stand for 20 min at room temperature and then the change of absorption value at 390 nm was measured.

Mitochondrial Extraction from Root Tip
Mitochondrion was isolated from root tips of 4-d-old cucumber seedlings using a kit (Bestbio, BB-3611-1, China) and following the manufacture's instruction. 1 g root sample was added with 3 mL extract to grind on ice. The sample was homogenized at 4 °C, centrifuged at 100 g for 1 min, collected supernatant and discarded sediment. The filtrate was centrifuged at 700 g for 10 min and the supernatant was collected. Supernatant 11,000 g was centrifuged for 20 min and the supernatant was discarded. 1.5 mL of mitochondrial preservation solution was added to the precipitation and resuspended and then suspension at 11,000 g was centrifuged for 15 min, the supernatant was discarded, and the precipitation was mitochondria. The isolated mitochondria were resuspended with suspension and stored in refrigerator at 4 °C for further experiments.

Determination of Mitochondrial Membrane Permeability Transition Pore (MPTP) and Cyt c/a
The method of determination is referred to He et al. (He et al. 2018). The isolated mitochondria were suspended in buffer solution (220 mM mannitol, 70 mM sucrose, 5 mM HEPES, 5 mM sodium succinate, pH 7.2) with a protein concentration of 0.3 mg/mL at 20 °C for 2 min, and then, UV light photometer was used to detect the absorbance value of 540 nm.
The isolated mitochondria were suspended with 0.2% BSA with a protein concentration of 0.5 mg/mL, and then, the absorption values of 550 nm and 630 nm were detected. The absorption values of the two wavelengths were Cyt c/a.

Determination of Ca 2+ in Mitochondria
Mitochondrial extract (1.5 mL) added 5 mL concentrated nitric acid and mixed solution was cultured at 25 °C for 7 days, and then, 1% LaCl 3 was added to make the volume 10 mL. The absorption value was measured by flame atomic spectrophotometer, and the concentration was calculated according to the Ca 2+ standard curve (Li et al. 2018a(Li et al. , 2018b.
Ca 2+ -ATPase activity is determined according to H + -ATP. In the reaction system, the activity difference caused by adding or not adding 50 μL 3 mmol/L Ca(NO 3 ) 2 was taken as the Ca 2+ -ATPase activity. Similarly, the activity difference caused by adding or not adding 30 mmol/L MgSO 4 was taken as the Mg 2+ -ATPase activity.

The Activity of Caspase-3-Like
Caspase-3-like assay kit (solarbio, BC3830) was used according to the manufacture's instruction. To measure the activity of caspase-3-like, assays were performed on 96-well microtitre plates by incubating 35 μL extraction solution and 65 μL reaction buffer [5 μL caspase-3 substrate (DEVD-pNA, 2 mM)]. Lysates were incubated at 37 °C for 4 h. The mixture was measured with an ELISA reader (CMax Plus, Molecular Devices, USA) at 405 nm.

Determination of CsVDAC and CsANT Expression by Quantitative RT-PCR
Total RNA was extracted from peanut root tips after different treatments for 24 h with an RNAiso plus kit (TaKaRa Inc., Japan) according to the manufacturer's instructions. The cDNA was amplified using the following primers shown in Table 1.

Statistical Analysis
All the experiments in this study were repeated three times, and the results shown are the mean ± SE of three independent experiments. Data analysis was used for Duncan's multiple test (P < 0.05) using SPSS 19.0 software (IBM SPSS, Chicago, USA).

H 2 S Alleviated the Inhibition of Cd Stress on Root Length and Fresh Weight
Observations about the effect of different treatments on the root length and fresh weight are shown in Fig. 1A-B. Treatment with 200 μM Cd caused significant decrease of 48.85% and 19.32% in root length and fresh weight, respectively, reference to CK. Pretreatment with HT enhanced Cd toxicity and reduced root length by 51.86% and fresh weight by 22.67%, respectively. However, exogenously supplied NaHS alleviated toxic effect of Cd by increasing the root length and fresh weight by 28.32% and 14.78%, respectively, in comparison to Cd-alone-treated seedlings. In comparison to HT + Cd-treated plants, treatment of NaHS + Cd led to 36.34% and 19.76% increase in root length and fresh weight. These results showed that H 2 S could alleviate the inhibition of root length and fresh weight under Cd stress.

H 2 S Promoted Root Length and Fresh Weight by Reducing Root Tip Cell Death
To investigate whether the inhibition of Cd on root length and fresh weight was caused by the cell death of root tip, we measured degree of cell death by Evans blue. Fig. 2A shows that staining degree of root tips cell death under different treatments, roots treated with Cd and HT showed a deeper

Effect of H 2 S on Mitochondrial Ca 2+ Concentration and Mitochondrion Ca 2+ -ATPase, H + -ATPase, and Mg 2+ -ATPase Activities Under Cd Stress
To investigate whether increased cytosolic Ca 2+ levels in cucumber seedling root tips are due to the release of mitochondrial Ca 2+ into cytoplasm, mitochondrial Ca 2+ concentration was measured. Cd and HT + Cd significantly To investigate whether H 2 S could maintain mitochondrial chemical potential and stabilize membrane structure, we measured mitochondrion Ca 2+ -ATPase activity, H + -ATP activity, and mitochondrion Mg 2+ -ATP activity. Under Cd stress, both mitochondrion Ca 2+ -ATPase activity (Fig. 4B), H + -ATPase activity (Fig. 4C), and Mg 2+ -ATPase activity (Fig. 4D) decreased significantly, especially under HT + Cd. Compared with Cd alone, pretreatment with NaHS increases mitochondrion Ca 2+ -ATPase activity (32.12%), H + -ATPase activity (24.72%), and mitochondrion Mg 2+ -ATPase activity (18.25%). These data suggested that H 2 S could inhibit the destruction of mitochondrial chemical potential and membrane stability by Cd stress and reduce the efflux of mitochondrial Ca 2+ .

Effect of H 2 S on Mitochondrial Cyt c/a, MPTP, and Caspase-3-Like Activity Under Cd Stress
As shown in Fig. 5A-B, Cd stress markedly decreased Cyt c content and the OD value of MPTP by 28.42% and 37.17% compared with CK. Moreover, after application of H 2 S scavenger HT, Cyt c/a and MPTP showed lower than CK and Cd alone. Under NaHS pretreatment, Cyt c/a and MPTP were 25.20% and 34.90% higher than Cd stress. Likewise, pretreatment with NaHS increased by 48.16% and 67.93% compared with HT + Cd. We measured caspase-3-like activity (Fig. 5C). The results showed that caspase-3-like activity of Cd stress and HT + Cd were 2.10 and 2.76 times higher than CK, respectively. Pretreatment with 0.1 mM NaHS significantly decreased caspase-3-like activity compared with Cd and HT + Cd treatments. The above results suggested that H 2 S inhibited the opening of MPTP, release of mitochondrial Cyt c and reduced caspase-3-like activity under Cd stress.

H 2 S Downregulates Expression of MPTP-Related Genes to Prevent Loss of Ca 2+
Furthermore, CsVDAC2, CsVDAC4, CsANT1, and CsANT2 gene expression was as a molecular probe to investigate the molecular mechanism of H 2 S regulating MPTP. Compared with CK, Cd and HT + Cd treatments were able to induce high expression of CsVDAC2, CsVDAC4, CsANT1, and CsANT2. These gene expressions were matched with the opening of MPTP. It was worth noting that NaHS pretreatment down-regulated these gene expressions by 24.63%, 32.15%, 27.87%, and 21.65%, respectively. These results showed that H 2 S down-regulated CsVDAC and CsANT expression to inhibit the opening of MPTP.

Discussion
H 2 S is a signal molecule involved in regulating physiological processes of animals and plants (García-Mata and Lamattina 2010; Li et al. 2011). In addition, H 2 S is involved in heavy metal stress (Ahmad et al. 2019;Mostofa et al. 2015;Qian et al. 2014). Our study showed that Cd treatment significantly inhabited root elongation and fresh weight of cucumber seedlings (Fig. 1). Moreover, Cd stress led to root tips cell death, while pretreatment with 0.1 mM NaHS increased root length and fresh weigh by reducing cell death (Figs. 1, 2). These findings were consistent with those reported in the literature. Zhang et al. reported that 5 mM CdCl 2 seriously affected the root growth of cabbage seedlings and markedly caused root tips cell death. When seedlings were treated with 5 μM for 24 h, the inhibition effect of Cd stress was significantly reduced (Zhang et al. 2015a). In rice seedlings, 0.5 mM CdCl 2 significantly decreased plant height, fresh weight, and dry weight; Evans blue staining showed that Cd stress caused cell death. NaHS treatment (0.1 mM) could alleviate Cd toxicity to maintain growth (Mostofa et al. 2015). Thus, all findings presented here indicate that NaHS appears to positively effect in plant growth.
The accumulation of endogenous H 2 S is the response of plants to stress. In this study, Cd stress led to the accumulation of endogenous H 2 S content that was higher than CK. Pretreatment with NaHS strengthened this accumulation effect (Fig. 3). High-concentration heavy metal ions can stimulate a ROS burst (Dai et al. 2016;Qian et al. 2014;Zhang et al. 2015a), which is one of the inducers of cell death (Jabs 1999). Figure 3 shows that mitochondria H 2 O 2 accumulation was stimulated by Cd stress. NaHS + Cd treatment significantly decreased this accumulation, which was consistent with other reports (Huang et al. 2016). Mitochondria are generally considered to be an important site of ROS-mediated PCD events; the release of Cyt c and collapse of MPTP are also reported to be one of the vital events in PCD (He et al. 2018;Yao et al. 2004). Mitochondria are involved in cell apoptosis of plants (Rurek 2014); because of the increased mitochondrial membrane permeability and decreased membrane potential, mitochondrial Ca 2+ and Cyt c were released into the cytoplasm, causing an increase in cytosolic Ca 2+ levels. We also confirmed that Cd induced the release of mitochondria Ca 2+ (Fig. 4A) and decreased mitochondria Cyt c/a (Fig. 5A). Li et al. also found that allelochemicals induced cell death in maize root tip, which was released from mitochondria with Cyt c and Ca 2+ (Li et al. 2018a(Li et al. , 2018b. Cyt c release has been seen also in plant cells under-going opening of MPTP and subsequent PCD under the influence of biotic and abiotic stress conditions (Panda et al. 2008). We noticed that the OD value of MPTP also decreased by Cd stress (Fig. 5B). This is consistent with He et al. who reported that aluminum caused Cyt c/a and OD value of MPTP in peanut root (He et al. 2018). In our study, Cd stress induced up-regulation of VDAC and ANT expression that indicated the opening of MPTP. NaHS pretreatment significantly down-regulated VDAC and ANT expression to inhibit the opening of MPTP compared with Cd, while scavenger HT treatment exacerbates this condition (Fig. 6). Similarly, in a rat model of ischemia-reperfusion, H 2 S prevents cardiomyocyte apoptosis by inhibiting the opening of MPTP (Yao et al. 2010;Zhang et al. 2013). These results suggested that H 2 S as an antioxidant protected mitochondrial function from Cd toxicity.
ATP is the energy to maintain plant life activities, and H + -ATPase, Ca 2+ -ATPase, and Mg 2+ -ATPase located on plasma membrane is an important ATPase. They play a key role in maintaining the chemical potential inside and outside the membrane and stabilizing the function of plasma membrane. Ca 2+ -ATPase is an important regulator of Ca 2+ transport in plants, which can maintain the gradient of Ca 2+ concentration intracellular and extracellular (Ferrol and Bennett 1996). Similar studies are reported (Ahn et al. 2001;Zhan et al. 2014;Zhao et al. 2008). Our study demonstrated that Cd stress decreased H + -ATPase, Ca 2+ -ATPase, and Mg 2+ -ATPase activity (Fig. 4B-D), but pretreatment with NaHS maintained mitochondrial ATPase activity and inhibited mitochondrial Ca 2+ efflux into cytoplasm (Fig. 4A). When Ca 2+ -ATPase was activated, mitochondrial Ca 2+ exchanged with cytoplasmic Ca 2+ to maintain balance. Cd stress decreased Ca 2+ -ATPase enzyme activity and increased mitochondrial membrane permeability, breaking this balance and led to [Ca 2+ ] cyt overload. Dawood and Li have similar results to ours (Dawood et al. 2012;Juan et al. 2018). These results showed that H 2 S could maintain mitochondria ATP activity to limit mitochondria Ca 2+ efflux and [Ca 2+ ] cyt overload.
Cd can cause plant chlorosis, growth inhibition, and cell death due to its toxicity; the typical PCD upon Cd treatment were observed in cell suspensions of tobacco (Kuthanova et al. 2008;Ma et al. 2010), tomato (Elena et al. 2005;Gallego et al. 2012), and Arabidopsis (De Michele et al. 2009). Our results confirmed that Cd induced the occurrence of cell death caspase-3-like enzyme activation (Fig. 5C). Caspase activity plays an important role in the process of PCD and caspase-3 is the executor of PCD (Li and Xing 2010). Cd treatment induced the increase of caspase-3-like activity significantly (Fig. 5C). Exogenous NaHS pretreatment reduced the occurrence of PCD, while HT (H 2 S scavenger) could accelerate the process of cell death.

Conclusion
Taken together, Cd-induced cell death in cucumber root tips was mitochondria-dependent cell death. Cd stress damaged mitochondrial physiological functions by mitochondrial H 2 O 2 accumulation and the opening of MPTP to release Cyt c and Ca 2+ . H 2 S prevented cell death of Cd-induced by improving mitochondrial physiological properties and a potential schematic diagram is shown in Fig. 7.

Conflict of interest
The authors declare that they have no conflict of interest.
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