The Defensive Role of Endogenous H2S in Brassica rapa against Mercury-Selenium Combined Stress

Plants are always exposed to the environment, polluted by multiple trace elements. Hydrogen sulfide (H2S), an endogenous gaseous transmitter in plant cells, can help plant combat single elements with excess concentration. Until now, little has been known about the regulatory role of H2S in response to combined stress of multiple elements. Here we found that combined exposure of mercury (Hg) and selenium (Se) triggered endogenous H2S signal in the roots of Brasscia rapa. However, neither Hg nor Se alone worked on it. In roots upon Hg + Se exposure, the defensive role of endogenous H2S was associated to the decrease in reactive oxygen species (ROS) level, followed by alleviating cell death and recovering root growth. Such findings extend our knowledge of plant H2S in response to multiple stress conditions.


Introduction
Heavy metal pollution poses a threat to biological systems, which is a serious problem worldwide. Heavy metal exposure impacts plant growth and development [1]. Hg (mercury) is one of the toxic metals even at low levels. Hg content increases continuously in the environment because of natural release and anthropogenic activities. The average content of soil Hg is 1.1 mg/kg worldwide. The polluted soils contain Hg at various levels even more than 10 mg/kg or higher [2]. Hg 2+ is the most toxic form of Hg. Hg 2+ can be readily taken up by plants to induce phytotoxicity. In hydroponic studies, plants exposed to levels of Hg 2+ (even at 1-5 µM) begin to exhibit toxic symptoms [3,4]. Hg-induced phytotoxicity is associated with the accumulation of ROS (reactive oxygen species), followed by oxidative stress, physiological disturbance, and cell death [3,5]. ROS accumulation is considered as bio-indicator of Hg-induced phytotoxicity [4].
Se (selenium) is a beneficial micronutrient for plant growth. Se at proper concentrations, e.g., 0.5-6.0 mg/L SeO 3 2− (about 3.9-47 µM Se) can rescue plant from the toxicity of heavy metals including Hg [6]. However, Se application with excess concentration shows synergistic toxic effect with Hg [7]. Excessive Se is an emerging environmental problem [8]. Se and Hg frequently coexist in the environment [9]. Our previous study also found synergistic toxic effect of Se and Hg on Brassica rapa, showing enhanced ROS accumulation and aggravated growth inhibition as compared to Se or Hg alone [10]. However, we still rarely know the plant signaling regulation in response to Hg + Se combined stress.
H 2 S (hydrogen sulfide) is an endogenous signaling molecule in plant cells. H 2 S can be generated during sulfate assimilation. DES ( L -cysteine desulfhydrase, EC4.4.1.1) is 2 of 9 considered to be the main enzyme to produce H 2 S, regulating various plant physiological processes [11]. H 2 S plays important roles in the modulation of plant development and stress responses [12]. Few studies focus on the function of H 2 S in plants upon multiple stress conditions at the same time. H 2 S regulates plant tolerance against heavy metal stress, such as Cd (cadmium) [13], Al (aluminum) [14], Cr (chromium) [15], Pb (lead) [16], and Hg [17], etc. These reports suggest that H 2 S protects plants from metal toxicity by alleviating oxidative injury. We previously found that H 2 S was essential for plant combating excessive Se stress [18]. However, whether and how endogenous H 2 S regulates plant response upon Se + Hg combination remains elusive.
In this study, we identified the intensified H 2 S signaling in the root of B. rapa under Hg + Se stress conditions. The results not only help reveal plant signaling regulation of synergistic toxicity of Hg and Se, but also extend our knowledge of H 2 S in plant cells in response to multiple stress conditions.

Results and Discussion
The roots of B. rapa were exposed to HgCl 2 (mercury chloride) (1 µM), Na 2 SeO 3 (sodium selenite) (4 µM), or their combination for 3 days. Treatment with Hg alone slightly inhibited root growth ( Figure 1A). This confirms the phytotoxic effect of Hg even at low levels [19]. Se induces phytotoxicity only at high concentrations [20]. Here we found that treatment with Se alone failed to affect root growth ( Figure 1A), suggesting that Se at 4 µM was safe for the growth of B. rapa seedlings. However, simultaneous treatment with Hg and Se resulted in remarkable decrease in root length by 48.2% as compared to the control ( Figure 1A). Since Hg + Se, but not Hg or Se alone, impacted root growth, we further performed time-course experiment to monitor root length upon Hg + Se exposure. Root growth was significantly inhibited after treatment with Hg + Se for 6 h. Then root growth speed declined with prolonged Hg + Se treatment ( Figure 1B). Either Hg or Se was relative safe for the growth of B. rapa (Hg showed slight toxicity), but Hg + Se induced severe phytotoxicity. Treatment with Hg and Se together may overwhelm toxic threshold of single element, which further impacting plant growth. Therefore, Se showed synergistic but not antagonistic effect on Hg stress. and Hg [17], etc. These reports suggest that H2S protects plant alleviating oxidative injury. We previously found that H2S was es ing excessive Se stress [18]. However, whether and how endoge response upon Se + Hg combination remains elusive.
In this study, we identified the intensified H2S signaling in Hg + Se stress conditions. The results not only help reveal plan synergistic toxicity of Hg and Se, but also extend our knowledg response to multiple stress conditions.

Results and Discussion
The roots of B. rapa were exposed to HgCl2 (mercury chlori dium selenite) (4 µM), or their combination for 3 days. Treatmen inhibited root growth ( Figure 1A). This confirms the phytotoxic levels [19]. Se induces phytotoxicity only at high concentrations treatment with Se alone failed to affect root growth ( Figure 1A) µM was safe for the growth of B. rapa seedlings. However, simu Hg and Se resulted in remarkable decrease in root length by 48 control ( Figure 1A). Since Hg + Se, but not Hg or Se alone, impac ther performed time-course experiment to monitor root length Root growth was significantly inhibited after treatment with Hg growth speed declined with prolonged Hg + Se treatment (Figure relative safe for the growth of B. rapa (Hg showed slight toxicit severe phytotoxicity. Treatment with Hg and Se together may ov of single element, which further impacting plant growth. Therefo but not antagonistic effect on Hg stress. The elongation of root tip determines root growth in res stress [21]. H2S plays defensive roles in plants against environme the response of H2S in root tip, we used specific probe WSP-1 spiro[isobenzofuran-1, 9'-xanthen]-6'-yl 2-(pyridin-2-yldisulfany The elongation of root tip determines root growth in response to environmental stress [21]. H 2 S plays defensive roles in plants against environmental stress [12]. To study the response of H 2 S in root tip, we used specific probe WSP-1 (3 -methoxy-3-oxo-3H-spiro[isobenzofuran-1, 9 -xanthen]-6 -yl 2-(pyridin-2-yldisulfanyl)benzoate) to label endogenous H 2 S in situ. Neither Hg nor Se treatment alone affected endogenous H 2 S level in root tip ( Figure 2). Hg + Se treatment enhanced endogenous H 2 S remarkably ( Figure 2A). WSP-1 fluorescent density increased significantly by 7.71 fold in root tip upon Hg + Se as compared to control ( Figure 2B). The changing pattern of endogenous H 2 S was associated with root growth inhibition upon stress conditions. Endogenous H 2 S were stimulated by Hg + Se (inhibiting growth remarkably), but not Hg or Se alone (without remarkable growth inhibition) ( Figures 1A and 2B). The increase in endogenous H 2 S level has been found in plant cells in response to growth-inhibited stimuli, such as salinity [22], cadmium (Cd) [23], and chromium (Cr) [24]. Thus, the roots of B. rapa may enhance endogenous H 2 S to sense Hg + Se stress.
. Mol. Sci. 2022, 23, x FOR PEER REVIEW 2A). WSP-1 fluorescent density increased significantly by 7.71 fold Se as compared to control ( Figure 2B). The changing pattern of end sociated with root growth inhibition upon stress conditions. Endog ulated by Hg + Se (inhibiting growth remarkably), but not Hg or markable growth inhibition) ( Figures 1A and 2B). The increase in e has been found in plant cells in response to growth-inhibited stimul cadmium (Cd) [23], and chromium (Cr) [24]. Thus, the roots of B. dogenous H2S to sense Hg + Se stress. Stress-induced endogenous H2S production is involved in plant otic stress [25,26]. Intensified endogenous H2S signal help sweet po osmotic stress [27]. To further confirm the defensive role of H2S up applied pharmacological experiments to alter endogenous H2S leve pretreated with H2S donor NaHS (sodium hydrosulfide) for 6 h fol posure. Applying NaHS promoted root growth under Hg + Se stres manner, with maximal effect occurring at 2 mM NaHS ( Figure 3A). P scavenger HT (hypotaurine) or DES inhibitor PAG (DL-proparg growth retardation upon Hg + Se stress ( Figure 3B, C). In addition, H µM) counteracted the promoting effect of NaHS (2 mM) on root g stress ( Figure 3D). In situ fluorescent detection showed that NaHS dogenous H2S level in root tip treated with Hg + Se, an effect rever PAG ( Figure 3E,F). Hg + Se exposure increased root endogenous H ficient to help plants combat stress condition. Further enhancing en ered root growth from subsequent Hg + Se stress. An adverse effect w ing endogenous H2S through either scavenging already generated biosynthesis. These results suggested a protective role of plant end Hg + Se stress. Stress-induced endogenous H 2 S production is involved in plant tolerance against abiotic stress [25,26]. Intensified endogenous H 2 S signal help sweet potato seedlings against osmotic stress [27]. To further confirm the defensive role of H 2 S upon Hg + Se stress, we applied pharmacological experiments to alter endogenous H 2 S level in roots. Roots were pretreated with H 2 S donor NaHS (sodium hydrosulfide) for 6 h followed by Hg + Se exposure. Applying NaHS promoted root growth under Hg + Se stress in a dosedependent manner, with maximal effect occurring at 2 mM NaHS ( Figure 3A). Pretreatment with H 2 S scavenger HT (hypotaurine) or DES inhibitor PAG (DL-propargylglicine) aggravated growth retardation upon Hg + Se stress ( Figure 3B,C). In addition, HT (4 µM) or PAG (0.5 µM) counteracted the promoting effect of NaHS (2 mM) on root growth upon Hg + Se stress ( Figure 3D). In situ fluorescent detection showed that NaHS further enhanced endogenous H 2 S level in root tip treated with Hg + Se, an effect reversed by adding HT or PAG ( Figure 3E,F). Hg + Se exposure increased root endogenous H 2 S, but it was not sufficient to help plants combat stress condition. Further enhancing endogenous H 2 S recovered root growth from subsequent Hg + Se stress. An adverse effect was found by decreasing endogenous H 2 S through either scavenging already generated H 2 S or impairing H 2 S biosynthesis. These results suggested a protective role of plant endogenous H 2 S against Hg + Se stress.  The next step was to study how endogenous H2S help plants detoxify Hg + Se. Our previous study found that Hg + Se induced more severe oxidative injury than that of Hg or Se alone in B. rapa [10]. We studied the effect of altering endogenous H2S on oxidative injury in roots upon Hg + Se stress. Plant development needs to maintain the homeostasis of ROS at proper levels as it is an important signaling molecule. ROS overaccumulation is harmful to plants [28]. Total ROS in root tip were detected in vivo with specific fluorescent probe DCFH-DA (2′,7′-dichlorofluorescein diacetate) [29]. Hg + Se exposure led to remarkable increase in ROS level in root tip as compared to the low level of ROS in control. Pretreatment with NaHS significantly decreased ROS level in root tips under Hg + Se exposure. Further adding PAG or HT resulted in ROS accumulation ( Figure 4A,B). These results suggested that enhancing endogenous H2S was able to inhibit ROS accumulation induced by Hg + Se stress.
ROS can cause irreversible cell death in plants in response to stress [30]. Root tip cell death indicated by PI (propidium iodide) showed similar changing pattern to ROS under same treatment conditions ( Figure 4C,D), suggesting that enhancing endogenous H2S attenuated cell death in root treated with (Hg + Se). Root length was negatively correlated to ROS and cell death, respectively, under the condition of altering endogenous H2S level ( Figure 4E,F). Therefore, H2S-supressed ROS accumulation and cell death may contribute to the recovery of root elongation from Hg + Se stress. The next step was to study how endogenous H 2 S help plants detoxify Hg + Se. Our previous study found that Hg + Se induced more severe oxidative injury than that of Hg or Se alone in B. rapa [10]. We studied the effect of altering endogenous H 2 S on oxidative injury in roots upon Hg + Se stress. Plant development needs to maintain the homeostasis of ROS at proper levels as it is an important signaling molecule. ROS overaccumulation is harmful to plants [28]. Total ROS in root tip were detected in vivo with specific fluorescent probe DCFH-DA (2 ,7 -dichlorofluorescein diacetate) [29]. Hg + Se exposure led to remarkable increase in ROS level in root tip as compared to the low level of ROS in control. Pretreatment with NaHS significantly decreased ROS level in root tips under Hg + Se exposure. Further adding PAG or HT resulted in ROS accumulation ( Figure 4A,B). These results suggested that enhancing endogenous H 2 S was able to inhibit ROS accumulation induced by Hg + Se stress.
ROS can cause irreversible cell death in plants in response to stress [30]. Root tip cell death indicated by PI (propidium iodide) showed similar changing pattern to ROS under same treatment conditions ( Figure 4C,D), suggesting that enhancing endogenous H 2 S attenuated cell death in root treated with (Hg + Se). Root length was negatively correlated to ROS and cell death, respectively, under the condition of altering endogenous H 2 S level ( Figure 4E,F). Therefore, H 2 S-supressed ROS accumulation and cell death may contribute to the recovery of root elongation from Hg + Se stress.  ROS can attack cell membrane lipid, leading to loss of membrane integrity. This is one of the typical consequences of oxidative injury [31]. We applied histochemical staining to evaluate oxidative injury in roots. Lipid peroxidation (indicated by Schiff's reagent) and loss of membrane integrity (indicated by Evans blue) showed pink and blue, respectively ( Figure 5). Hg + Se resulted in extensive staining as compared to light staining in control. Pretreatment with NaHS let to lighter staining in roots treated with Hg + Se, an effect reversed by further adding PAG or HT ( Figure 5). These results suggested that endogenous H2S was important for plants to combat oxidative induced by Hg + Se stress. Linking the above results provides an important clue that endogenous H2S protects plant from Hg + Se stress by suppressing ROS accumulation followed by the alleviation of oxidative injury and cell death.  ROS can attack cell membrane lipid, leading to loss of membrane integrity. This is one of the typical consequences of oxidative injury [31]. We applied histochemical staining to evaluate oxidative injury in roots. Lipid peroxidation (indicated by Schiff's reagent) and loss of membrane integrity (indicated by Evans blue) showed pink and blue, respectively ( Figure 5). Hg + Se resulted in extensive staining as compared to light staining in control. Pretreatment with NaHS let to lighter staining in roots treated with Hg + Se, an effect reversed by further adding PAG or HT ( Figure 5). These results suggested that endogenous H 2 S was important for plants to combat oxidative induced by Hg + Se stress. Linking the above results provides an important clue that endogenous H 2 S protects plant from Hg + Se stress by suppressing ROS accumulation followed by the alleviation of oxidative injury and cell death. ROS can attack cell membrane lipid, leading to loss of membrane integrity. This is one of the typical consequences of oxidative injury [31]. We applied histochemical staining to evaluate oxidative injury in roots. Lipid peroxidation (indicated by Schiff's reagent) and loss of membrane integrity (indicated by Evans blue) showed pink and blue, respectively ( Figure 5). Hg + Se resulted in extensive staining as compared to light staining in control. Pretreatment with NaHS let to lighter staining in roots treated with Hg + Se, an effect reversed by further adding PAG or HT ( Figure 5). These results suggested that endogenous H2S was important for plants to combat oxidative induced by Hg + Se stress. Linking the above results provides an important clue that endogenous H2S protects plant from Hg + Se stress by suppressing ROS accumulation followed by the alleviation of oxidative injury and cell death. The antioxidant role of H2S has been associated with its ability of limiting ROS accumulation in response to abiotic stress [32]. H2S limits ROS accumulation probably through The antioxidant role of H 2 S has been associated with its ability of limiting ROS accumulation in response to abiotic stress [32]. H 2 S limits ROS accumulation probably through two ways. First, H 2 S can scavenge already generated ROS by activating both antioxidative enzymes (enzymatic system) and antioxidants (non-enzymatic system) [33]. Second, H 2 S is able to suppress ROS generation. Superoxide radical and hydrogen peroxide are two typical ROS. NADPH oxidase and PAO (polyamine oxidase) generate superoxide radical and hydrogen peroxide, respectively, in plants upon abiotic stress [34,35]. Excessive Se alone induces PAO-and NADPH oxidase-dependent ROS accumulation in plants [36,37]. Hg stress alone can induce NADPH oxidase-dependent ROS burst in plants as well [38]. It has been reported that H 2 S can protect both plant and mammalian cells from oxidative stress by repressing NADPH oxidase-dependent ROS generation [39,40]. Further studies are needed to understand whether H 2 S decreases ROS accumulation in plants against Hg + Se stress through the similar mechanisms mentioned above.
The synergistic toxicity of Hg and Se may directly result from the protein disfunction. The -SH in cystine (Cys) play important roles to maintain functional structure of the proteins. Hg 2+ has strong affinity to -SH groups in functional proteins [41]. Cys in proteins can be easily replaced by SeCys [8]. Both actions are detrimental to the proteins. The combination of Hg and Se stress may aggravate the disturbance of the structure of functional proteins, further leading to negative physiological responses in plants. Recent studies have revealed that H 2 S improves protein function via persulfidation of Cys [42,43]. Whether H 2 S-mediated protein modification may help overcome the toxicity of Hg + Se needs further studies.
In sum, we found that endogenous H 2 S could be triggered as a defensive signal in response to synergistic toxicity of Hg and Se in B. rapa. Neither Hg nor Se alone worked on it. Further studies are needed to reveal the molecular mechanism for H 2 S-mediated detoxification of Hg + Se, our current results propose a novel role of endogenous H 2 S in regulating plant adaption upon multiple stress conditions at the same time. PAG + NaHS or HT + NaHS, respectively, for 6 h. Then roots were exposed to Hg + Se for another 72 h, followed by measuring root length, root tip endogenous H 2 S level, root tip endogenous ROS level, and root tip cell death.

Evaluation of Total ROS Level in Root Tip
Fluorescent probe DCFH-DA (2 ,7 -dichlorofluorescein diacetate) was applied to detect root tip ROS in situ [29]. Roots were incubated in DCFH-DA solution (10 µM) at 25 • C for 10 min followed by rinsing with distilled water. Then root tips were observed and captured with a fluorescent microscope (ECLIPSE, TE2000-S) with setting of excitation 480-490 nm/emission 515-525 nm.

Evaluation of Cell Death in Root Tip
Fluorescent probe PI was used to detect root tip cell death in situ [47]. Roots were incubated in PI (propidium iodide) solution (20 µM) at 25 • C for 20 min followed by rinsing with distilled water. Then the root tips were observed and captured with a fluorescent microscope (ECLIPSE, TE2000-S) with setting of excitation 530-540 nm/emission 610-620 nm.

Evaluation of Oxidative Injury in Root Tip
Schiff's reagent was used to detect root lipid peroxidation in situ [48]. Roots were stained with Schiff's reagent for 20 min followed by rinsing with 0.5% (w/v) K 2 S 2 O 5 (prepared in 0.05 M of HCl) until the color became light red. Then roots were photographed with a digital camera.
Evans blue was used to detect loss of membrane integrity in root cells in situ [49]. Roots were stained with Schiff's reagent (0.025%, w/v) for 20 min followed by rinsing with distilled water. Then roots were photographed with a digital camera.

Statistical Analysis
Each result was presented as mean ± SD (standard deviation) of at 3-10 replicates. Least significant difference test (LSD) was performed on data following ANOVA tests to test for significant (p < 0.05) differences among treatments. The significant difference at p < 0.05 level between two designated treatments was compared using ANOVA with F test.