Hydrogen Sulfide Alleviates Cadmium Toxicity in Chrysanthemum indicum L. by Modulating Photosynthesis, Mineral Homeostasis and Antioxidant Capacity

doi:10.21203/rs.3.rs-5002660/v1

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Hydrogen Sulfide Alleviates Cadmium Toxicity in Chrysanthemum indicum L. by Modulating Photosynthesis, Mineral Homeostasis and Antioxidant Capacity

https://doi.org/10.21203/rs.3.rs-5002660/v1

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Hydrogen sulfide (H₂S), recognized as a third gaseous signaling molecule, plays a role in resistance to abiotic stress. However, the role of H₂S during cadmium (Cd) resistance in Chrysanthemum (Chrysanthemum indicum L.) remains uncharacterized. In this study, we aimed to investigate the regulatory role of H₂S in Chrysanthemum under Cd toxicity. Our objective is to provide a theoretical foundation for utilizing H₂S in combination with ornamental plants for the remediation of Cd pollution. We conducted hydroponic experiments to examine the effects of foliar H₂S spraying on the growth, photosynthesis, chloroplast ultrastructure, and physiological attributes of various C. indicum seedlings under Cd stress. The results demonstrated that Cd toxicity had a substantial impact on photosynthetic parameters and the structural integrity of chloroplasts, when compared to non-cadmium conditions. It also elevated the content of reactive oxygen species (ROS), causing disturbances in element uptake. However, the addition of H₂S played a pivotal role in preserving chloroplast integrity, consequently improving photosynthetic performance and avoiding cadmium-induced ultrastructural damage. Additionally, H₂S also positively promoted uptake of elements and regulated antioxidant enzyme activities on the one hand, and mitigated oxidative stress and reduced the accumulation of ROS on the other. In summary, our findings suggest that exogenously applied H₂S can effectively alleviate the detrimental effects of Cd toxicity.

Chrysanthemum

stress

cadmium

hydrogen sulfide

photosynthetic

With the rapid pace of urbanization, the escalation of industrial pollution, and the widespread use of pesticides and fertilizers, soil heavy metal pollution has become an increasingly pressing concern (Khan et al., 2008). China currently faces severe soil contamination by heavy metals, particularly cadmium, arsenic, mercury, lead, and copper. Cadmium pollution stands out as one of the most critical challenges within the realm of heavy metal contamination (Chi et al., 2023; Khan et al., 2021). Cadmium, known for its high mobility in soil, readily permeates plant root systems, accumulating in plant tissues and subsequently entering the food chain, posing a substantial threat to human health (Rizwan et al., 2018). Thus, addressing cadmium pollution has become a paramount issue in the domain of environmental protection.

Contemporary research suggests that phytoremediation techniques offer a cost-effective approach to combat heavy metal pollution. Nevertheless, cadmium, with its high biotoxicity, has the capacity to disrupt photosynthesis and cellular structures in plants, thus inhibiting their growth and nutrient absorption (You et al., 2023). In an effort to mitigate the adverse effects of heavy metals on plants, numerous innovative methods and technologies have emerged. For instance, the application of TiO₂ nanoparticles has been proven to significantly enhance plant tolerance to cadmium. Elemental sulfide has demonstrated its efficacy in reducing mercury accumulation in rice plants and alleviating growth inhibition. Furthermore, the symbiotic relationship between ectomycorrhizal fungi and the root systems of Pinus terrestris has shown promise in sequestering cadmium and copper within the mycelium's cell wall, consequently reducing the plant's cadmium and copper uptake and enhancing its heavy metal tolerance (Bakshi et al., 2023; Huang et al., 2024; Quan et al., 2023). Therefore, the utilization of soil amendments, foliar inhibitors, soilbio-remediation, and nanomaterials holds the potential to address the issue of heavy metal pollution, both by breeding cadmium-tolerant plants and by advancing phytoremediation techniques for the future.

Hydrogen sulfide (H₂S), an emerging gaseous signaling molecule in plants, plays a crucial role in regulating various physiological, biochemical, and developmental processes, including seed germination, photosynthesis, and stomatal movement. Additionally, H₂S enhances a plant's resistance to abiotic stresses (Cheng et al., 2019; Duan Huang et al., 2015; Fang et al., 2022). Recent studies have revealed that H₂S expedites seed germination by regulating glutathione (GSH) levels, thereby increasing cellular reducing capacity (Fang et al., 2022). Furthermore, it has been observed that H₂S can mitigate the adverse effects of nickel stress on plant photosynthesis capacity, reverse the impact of nickel ions on chloroplast structure, and maintain the orderly stacking of the matrix layer (Rizwan et al., 2019). Exogenous H₂S treatment also increases the activity of antioxidant enzymes in plants, reduces the content of ROS, and decreases the accumulation of heavy metals such as cadmium, chromium, and aluminum. This, in turn, alleviates the inhibition of plant growth caused by heavy metal stress and enhances the plant's ability to survive adverse conditions (Kharbech et al., 2017; Yu et al., 2023). However, the specific mechanism by which H₂S attenuates the toxic effects of cadmium remains poorly understood.

Chrysanthemum (Chrysanthemum indicum L.) is a perennial herbaceous plant belonging to the Asteraceae family. It has a wide distribution and exhibits robust resistance to cadmium stress (Wu et al., 2021). Current research on C. indicum primarily focuses on breeding, cultivation, medicinal and culinary applications, with limited attention given to its cadmium resistance (Gu et al., 2022). While some studies have suggested that H₂S can mitigate the adverse effects of cadmium toxicity in plants, there is limited research on the physiological and biochemical responses of H₂S in C. indicum under cadmium stress and its potential contribution to Cd pollution remediation. In this study, we hypothesized that the addition of H₂S to hydroponically cultivated C. indicum seedlings could protect the stability of chloroplast structure by reducing cell membrane lipid peroxidation and increasing the accumulation of essential nutrients in the leaves. This, in turn, could enhance photosynthetic efficiency and alleviate the toxic effects of cadmium ions on the plants. Therefore, we investigated the impact of exogenous H₂S on photosynthetic parameters, chloroplast ultrastructure, and mineral element content in different C. indicum seedling varieties under cadmium stress, aiming to provide insights into the potential mitigation mechanism of exogenous H₂S on Cd-stressed C. indicum seedlings.

2.1. Plant materials and experimental design

The test materials comprised C. indicum varieties introduced from Liaoning Province to the Flower Research Institute of Northeast Forestry University. After a preliminary screening for cadmium tolerance, the cadmium-resistant variety NY5103 (N1), the common variety PY3111 (P1), and the cadmium-sensitive variety MY361 (M1) were selected as the test materials. Stem segments with a length of approximately 15 cm at the top of each variety were selected for propagation by cutting, and the experiment was conducted after one month of culture. Seedlings with uniform growth were carefully chosen and transplanted into containers equipped with 10 liters of 1/2 Hoagland solution, maintaining the pH value of the nutrient solution at 6.0. The seedlings were grown at a temperature of 25°C, with a light cycle of 14 hours of light and 10 hours of darkness. Following three days of seedling restoration, the C. indicum seedlings were exposed to five different treatments:1)A control group without cadmium treatment (CK).2༉A treatment group with 200µM NaHS (a donor of H₂S) (NaHS).3༉A treatment group with 200µM Cd (Cd).4༉A treatment group with 200µM Cd and 200µM NaHS (NaHS + Cd).5༉A treatment group with 200µM Cd and 100µM HT (a scavenger of H₂S) (HT + Cd). For each treatment, there were 5 replicates, each consisting of 15 seedlings. The nutrient solution was changed every three days. After 7 days of treatment, samples were collected, and measurements were taken, including the determination of the total plant height (aboveground and underground parts) and the drying of samples in an oven at 80°C until a constant weight was achieved to measure dry weight (DW).

2.2. Determination of chlorophyll content and photosynthetic parameters

The third to fifth fully developed leaves from the top of the stem were selected. Chlorophyll a (Chla), chlorophyll b (Chlb), and the total chlorophyll content (chlorophyll a + b) were determined using Fan's method of acetone-ethanol mixture extraction (Fan et al., 2018). After 7 days of seedling stress, between 9:00 and 11:00, three plants with consistent growth conditions were randomly chosen from each treatment. The functional leaf of the seedling (the third leaf counting down from the top) was selected, and the leaf chamber area was set to 2 cm² to measure the following parameters: net photosynthetic rate (Pn) of the leaves, transpiration rate (Tr), stomatal conductance (Gs), and intercellular CO₂ concentration (Ci). The measurements were conducted using the Li-6400XT portable photosynthesis system (LI-COR, Lincoln, NE). The measurements were taken under controlled conditions with a systematic light intensity of 1000 µmol/(m²·s), CO₂ concentration set at 430 µmol/mol, and a leaf chamber temperature of 25°C.

2.3. Investigation of leaf ultrastructure by transmission electron microscope (TEM)

The chloroplast ultrastructure was determined with reference to the method of Li et al. (2018). For this study, the third functional leaf from healthy plants was selected, and rectangular sections measuring 1mm×3mm were obtained on either side of the leaf veins. Three sections were collected for each treatment group. These sections were double-fixed with a 2.5% glutaraldehyde solution and 1% osmium acid, followed by rinsing with phosphate buffer at pH 6.8 after fixation. Subsequently, they were dehydrated using a series of ethanol and acetone solutions at varying concentrations and embedded in epoxy resin. Ultra-thin sections were then prepared using an LKB-5 ultra-thin sectioning machine. The sections were double-stained with uranyl acetate and lead citrate, and observed and photographed using a H7650 transmission electron microscope. This process was conducted to enhance the quality of the obtained material for electron microscopy analysis.

2.4 Quantification of ROS levels and antioxidant enzyme assays

The assay for antioxidant enzyme activity in the sample was conducted using Gong's method with slight modifications (Gong et al., 2019). Peroxidase (POD) activity was determined using the guaiacol method. In this assay, a reaction solution was prepared by mixing 2.9 mL of 100 mM PBS (pH 7.0), 0.1 mL of enzyme solution, and 0.05 mL of guaiacol. The activity was quantified as one unit of POD viability for every 0.01 change in absorbance per minute. Catalase (CAT) activity was determined in a reaction solution containing 3.3 mL of 100 mM PBS (pH 7.4), 0.1 mL of enzyme solution, 0.5 mL of 65 µM hydrogen peroxide solution, and 0.5 mL of 32.7 mM ammonium molybdate solution. Enzyme activity was calculated by measuring one enzyme vitality unit for every minute of decomposition of 1 µM hydrogen peroxide per gram of fresh weight of the samples. Ascorbate peroxidase (APX) activity was measured in a reaction solution containing 0.1 mL of enzyme solution, 1.7 mL of 100 mM PBS (pH 7.8), 0.1 mL of 5 mM ascorbic acid (ASA) solution, and 0.1 mL of 20 mM hydrogen peroxide solution. The change in absorbance of 0.01 per minute was calculated as one POD viability unit. For the determination of superoxide dismutase (SOD) activity, a SOD assay kit (WST-8) from Suzhou Comin Biotechnology Co. was used.

The content of malondialdehyde (MDA) was determined using the thiobarbituric acid colorimetric method (Luo et al., 2020). 0.2g of sample was weighed, and 5 mL of 20% trichloroacetic acid solution was added. After thorough grinding, 5 mL of 0.5% thiobarbituric acid solution was added. The resulting homogenate was then centrifuged (3000 rpm, 20 min, 4°C), and the supernatant was collected for MDA content determination. The electrolyte leakage (EL) assay of the sample was performed using Kaya's method with slight modifications (Kaya et al., 2023). This involved weighing 0.2g of leaves, placing them in 25 mL of deionized water, allowing them to stand for 12 hours to determine the initial conductivity (C1), and then subjecting them to a 30-minute boiling water bath to determine the final conductivity (C2). The relative conductivity was then calculated.

Kaya's method was used to determine the proline content and make brief modifications (Kaya et al., 2023). 0.2g leaves were placed in 5mL of 3% sulfosalicylic acid solution, extracted in a boiling water bath for 10min, cooled and filtered, 2mL of extract was mixed with 2mL of glacial acetic acid and 2mL of acidic ninhydrin, heated in a boiling water bath for 30min, 4mL of toluene was added after cooling, shaken for 30s, and centrifuged with supernatant after standing for 5min.

For the determination of hydrogen peroxide (H₂O₂) and superoxide anion content, hydrogen peroxide content assay kits and superoxide anion content assay kits from Suzhou Comin Biotechnology Co. were used. Leaf H₂O₂ and superoxide anion (O₂.⁻) staining were performed following the method of Liu with some modifications (Liu et al., 2023b). This involved removing 3 ~ 5 mature and complete leaves from the top of the stem, immersing them in 1mg/mL 3, 3'-Diaminobenzidine (DAB) solution (pH 5.8) and 0.5 mg/mL nitroblue tetrazolium (NBT) solution (pH 5.8), storing them overnight at 28°C in the dark, and subsequently boiling them in 80% ethanol-water bath several times until the leaf color was completely removed. The results were documented using a camera.

2.5 Determination of the content nutrition elements

The mineral content of the samples was assessed following Zeng's method (Zeng et al., 2023). After the cadmium treatment, the leaves of C. indicum were washed with deionized water, oven-dried at 105°C for 0.5 hours, and further dried at 80°C until a constant weight was achieved. Subsequently, they were ground with a mortar and pestle, sieved through a 100-mesh sieve, and 0.05 grams of plant samples were transferred to a digestion tube. Digestion was carried out using the HNO₃-HClO₄ digestion method until the resulting solution became colorless and transparent or slightly yellow. The digested solution was then quantitatively transferred into a 25 mL volumetric flask. Ion concentration measurements were performed using an MP-AES flame atomic photometer after constant.

2.6 Statistical analysis

Data processing was conducted using Microsoft Excel 2016 software (Microsoft Office, USA). One-way analysis of variance (One-way ANOVA) was performed using SPSS 22.0, with the significance level set at P < 0.05. The Shapiro-Wilk test was performed before ANOVA, and when data were analysed as abnormally distributed (P < 0.05), we converted them to normal distribution by Box-Cox transformation. Multiple comparisons were executed using the Duncan method. Finally, data visualization was carried out using Prism 8.0 software (La Jolla, CA, USA).

3.1 Growth parameters of the plant

The impact of H₂S on the growth parameters of C. indicum seedlings under cadmium stress was investigated, with a focus on plant height and dry weight. In the presence of cadmium stress, both plant height and dry weight were significantly reduced compared to the control group (CK), with plant height decreasing by 11.87%, 10.5%, and 6.67%, respectively, and dry weight decreasing by 17%, 15.06%, and 12.85%, respectively. These results indicate that cadmium significantly inhibited the growth of C. indicum seedlings. The introduction of H₂S under cadmium stress conditions resulted in a notable increase in plant height and dry weight, as plant height increased by 8.61%, 6.9%, and 4.85%, respectively, and dry weight increased by 9.91%, 11.31%, and 8.5%, respectively, compared to the group exposed to cadmium stress alone. However, the addition of HT exacerbated the inhibitory effect of cadmium on the growth of C. indicum seedlings. These findings suggest that H₂S increased the tolerance of C. indicum to cadmium by mitigating the negative impact of cadmium on the growth parameters of C. indicum.

Table 1

Effects of Cd (200M) and NaHS (200M) on the growth of C. indicum seedlings.
µµ
Varieties	Treatments	Dry weight (g)	Plant geight (cm)
M1	CK	1.03 ± 0.02a	23.97 ± 0.33ab
	NaHS	1.07 ± 0.04a	24.79 ± 0.41a
	Cd	0.85 ± 0.02c	21.13 ± 0.46c
	NaHS + Cd	0.94 ± 0.02b	22.95 ± 0.09b
	HT + Cd	0.78 ± 0.07c	20.67 ± 0.75c
P1	CK	0.99 ± 0.08ab	24.57 ± 0.66ab
	NaHS	1.07 ± 0.03a	24.93 ± 0.82a
	Cd	0.85 ± 0.02c	21.99 ± 0.49c
	NaHS + Cd	0.95 ± 0.03b	23.51 ± 0.86b
	HT + Cd	0.80 ± 0.02c	21.56 ± 0.61c
N1	CK	1.03 ± 0.02ab	24.88 ± 0.12ab
	NaHS	1.07 ± 0.06a	25.21 ± 0.55a
	Cd	0.90 ± 0.03c	23.22 ± 0.30c
	NaHS + Cd	0.97 ± 0.01b	24.35 ± 0.07b
	HT + Cd	0.88 ± 0.04c	22.38 ± 0.58d
Note: Different letters indicate significant differences P < 0.05 between treatments.

3.2 Photosynthetic pigments and photosynthetic parameters

Photosynthesis is the physiological foundation of plant growth, and any factor affecting growth is likely to have a direct or indirect impact on photosynthesis. In comparison to the control group (CK), the Pn, Gs, and Tr were significantly reduced under Cd stress. Pn decreased by 39.79%, 33.59%, and 30.8%; Gs by 64.92%, 60.61%, and 57.38%; and Tr by 54.55%, 57.51%, and 55.98%, respectively (Fig. 1A, B, D). The addition of H₂S led to varying degrees of increase in Pn, Gs, and Tr. However, when H₂S was added alone, it did not have a significant effect on Pn, Gs, and Tr in the case of C. indicum. In the mixed treatment of Cd and HT, Pn, Gs, and Tr decreased more significantly. Leaf Ci increased under Cd treatment by 40.64%, 33.05%, and 27.51% and decreased with the addition of H₂S (Fig. 1C). These results suggest that H₂S may enhance the utilization of Ci by photosynthesis, thereby increasing Pn.

Various C. indicum varieties exhibited a consistent decrease in chlorophyll a and chlorophyll b levels when subjected to cadmium stress. Specifically, chlorophyll a decreased by 31.12%, 22.81%, and 23.35%, while chlorophyll b decreased by 23.26%, 28.13%, and 26.77%, respectively (Fig. E-G). This reduction resulted in a total leaf chlorophyll content lower than that of the control group, indicating that cadmium stress has an inhibitory effect on the chlorophyll content in C. indicum leaves. The introduction of H₂S proved to be effective in mitigating the negative impact of cadmium on chlorophyll levels, resulting in a significant increase in total chlorophyll content by 15.46%, 16.28%, and 17.09%. Notably, when H₂S was added independently, it led to a significant difference in chlorophyll a level compared to the control group. On the other hand, the addition of HT further exacerbated the adverse effects of cadmium on chlorophyll levels under cadmium stress conditions. These findings indicate that the inclusion of H₂S alleviates the inhibitory effect of cadmium on C. indicum leaf chlorophyll content to some extent.

3.3 Chloroplast ultrastructure

Under cadmium stress, the ultrastructure of chloroplasts in the leaf blades of C. indicum seedlings displayed noticeable changes. In the control and H₂S treatment groups, the chloroplasts appeared mostly pike-shaped, with closely stacked stroma thylakoids, neatly arranged granum thylakoids, few osmium granules or starch granules, and intact chloroplast membrane envelopes. N1 contained fewer osmium granules than M1 and P1 and had no starch granules (Fig. 2). Under cadmium stress, chloroplast structures were damaged to varying degrees. In N1, chloroplasts became rounded, with partial loss of the chloroplast membrane envelope, appearance of starch granules, and a decrease in both stroma and granum thylakoids. P1 chloroplasts retained a small portion of the membrane envelope, saw an enlargement in starch granules, an increase in the number of osmium granules, and exhibited disordered stroma thylakoids. M1 chloroplasts were completely disintegrated, showing enlarged and numerous starch grains, along with diffuse and vague stroma thylakoids. Under the combined treatment of cadmium and H₂S, M1 exhibited a clear chloroplast membrane envelope structure, complete granum thylakoids, and a more regular arrangement of stroma thylakoids. Osmium granules decreased in P1, the membrane envelope remained intact, and the granum thylakoids and stroma thylakoids were more orderly. In N1, the membrane envelope was more complete, and both the granum thylakoids and stroma thylakoids were clearly visible. When subjected to HT and cadmium treatment, which exacerbated the damage caused by cadmium to chloroplasts, the membrane envelope of N1 and P1 was compromised, and stroma thylakoids were also affected. In M1, chloroplasts were completely disintegrated, making it impossible to identify their inner components. Recognition of the inner components of the chloroplasts was hindered in this case.

3.4 Plant mineral content

To investigate the effect of H₂S on the Cd content in the leaves of seedlings, the Cd content in the leaves was determined. As shown in Fig. 3A, the addition of H₂S significantly decreased the Cd content in the leaves under Cd stress. The addition of HT significantly increased the Cd content in N1 but had no significant effect on the Cd content in M1 and P1.

Nutrient elements play a pivotal role in the process of photosynthesis. To comprehend the impact of H₂S on the absorption of copper, iron, zinc, manganese, and magnesium in the leaves of C. indicum seedlings under cadmium-induced stress, we analyzed the ion content in the leaves. Under the influence of cadmium stress, the iron, zinc, manganese, and magnesium levels in the leaves of various varieties significantly decreased when compared to the CK (Fig. 3B-F). Iron content decreased by 34.92%, 33.25%, and 26.07%, zinc content decreased by 39.26%, 37.03%, and 34.91%, manganese content decreased by 28.95%, 27.2%, and 21.92%, and magnesium content decreased by 21.26%, 19.47%, and 18.92%, respectively. Notably, the impact on copper content was less pronounced. The application of H₂S led to an increase in the levels of iron, zinc, manganese, magnesium, and copper compared to the Cd stress group. Iron content increased by 18.8%, 19.19%, and 15.45%, zinc content increased by 22.63%, 16.82%, and 16.97%, manganese content increased by 8.59%, 4.51%, and 12.79%, magnesium content increased by 7.11%, 6.53%, and 5.17%, and copper content increased by 18.49%, 10.01%, and 18.52%, respectively. Notably, the addition of HT resulted in a significant reduction in the levels of iron, zinc, manganese, magnesium, and copper in C. indicum seedlings. These results collectively suggest that H₂S mitigates the toxicity of cadmium on leaves by increasing the nutrient content in the leaves.

3.5 Plant antioxidant enzymes and oxidative stress response

Antioxidant enzymes are pivotal components of the reactive oxygen radical scavenging mechanism in plants, and they play a crucial role in enhancing a plant's resilience to adverse conditions. To comprehend the impact of H₂S on the plant antioxidant system under cadmium stress, we investigated the activities of antioxidant enzymes. Cadmium stress significantly elevated the activities of SOD by 32.69%, 42.96%, and 51.48%, APX by 51.26%, 59.71%, and 75.47%, CAT by 34.01%, 44.7%, and 61.79%, and POD by 61.31%, 60.89%, and 65.36% in various C. indicum varieties compared to the control (Fig. 4A-D). Additionally, the application of NaHS solution also augmented the activities of these enzymes in C. indicum under cadmium stress. However, simultaneous exposure to cadmium and HT led to a decrease in antioxidant enzyme activities when compared to cadmium stress alone. This suggests that H₂S significantly enhances the activities of antioxidant enzymes under cadmium stress, thereby mitigating the toxicity of cadmium.

The most prominent indication of heavy metal toxicity in plants is the oxidative stress response. To comprehend the impact of H₂S on the oxidative stress induced by cadmium stress, we analyzed relevant indicators of oxidative stress and conducted visual leaf staining experiments using C. indicum seedlings. Cadmium stress resulted in a significant increase in H₂O₂ (90.38%, 85.24%, 77.78%) and O₂.⁻ (88.69%, 91.51%, 88.85%) content compared to the CK (Fig. 4F-G). However, the addition of NaHS reduced H₂O₂ (16.16%, 19.44%, 18.75%) and O₂.⁻ (15.11%, 19.29%, 25.16%) levels. Furthermore, the simultaneous addition of HT and Cd intensified the oxidative stress response. Leaf coloration deepened and expanded in all three varieties under Cd stress but lightened and narrowed with the addition of H₂S (Fig. 5). However, the application of HT deepened the oxidative damage caused by cadmium to the leaves. This provides a more intuitive indication that H₂S effectively alleviates oxidative damage caused by cadmium by reducing H₂O₂ and superoxide anion levels in the leaves.

Cadmium stress increased EL (96.61%, 96.78%, 80.06%) and MDA (171.82%, 156.08%, 124.89%) levels, along with Pro (62.97%, 68.84%, 82.63%) content compared to the control (Fig. 4E, H, I). However, when cadmium treatments were combined with the addition of NaHS, EL (17.58%, 24.69%, 19.72%) and MDA (25.79%, 31.84%, 34.07%) levels decreased, while Pro (30.83%, 35.29%, 39.57%) content further increased. The opposite results were observed with the addition of HT. These findings suggest that H₂S mitigates the damage caused by Cd in plants by reducing ROS accumulation and decreasing membrane peroxidation.

Cadmium (Cd), chromium (Cr), lead (Pb), and nickel (Ni) are non-essential elements for plants that significantly inhibit plant growth (Dad et al., 2024; Mumtaz et al., 2022; Oloumi et al., 2024;Wang et al.,2023a). Hydrogen sulfide (H₂S), as a novel gaseous signaling molecule, plays an important role in counteracting heavy metal stress (de Bont et al., 2022; Fang et al., 2014). Studies have found that under cadmium stress, hydrogen sulfide significantly promotes the growth of plants such as Brassica rapa L., Cucumis sativus L., and barley (Fu et al., 2019; Li et al., 2021; Luo et al., 2020). In this study, cadmium stress inhibited the growth indices of C. indicum seedlings, but the application of hydrogen sulfide alleviated the inhibitory effects of cadmium on the growth of C. indicum. Additionally, under the toxic effects of heavy metals such as chromium (Cr), aluminum (Al), and lead (Pb), the application of hydrogen sulfide significantly enhanced the biomass of plant seedlings (Haghi et al., 2022; kumar Singh et al., 2022; Yu et al., 2023). Therefore, this study supports the positive impact of hydrogen sulfide application on improving plant growth and development under cadmium toxicity.

Photosynthesis is essential for the growth and development of plants and is closely related to plant tolerance to abiotic stress(Kumari et al., 2023; Lal et al., 2023). Kaya et al. (2024) found that H2S significantly increased the photosynthesis of Capsicum annuum L., thereby enhancing the plant's tolerance to salt stress. In this study, it was found that under cadmium stress, the photosynthetic rate, stomatal conductance, and transpiration rate significantly decreased, whereas the addition of hydrogen sulfide significantly improved photosynthetic efficiency. Similar results were observed in the study by Wang et al. (2021b) Furthermore, H₂S also improved the photosynthetic efficiency of pepper, tall fescue (Festuca arundinacea Schreb.), and Brassica napus under abiotic stress (Ali et al., 2015; Liu et al., 2022; Wang et al., 2021a). Therefore, hydrogen sulfide may enhance the tolerance of C. indicum to cadmium by improving its photosynthetic efficiency.

Photosynthetic pigments are fundamental to photosynthesis, as they largely determine the photosynthetic capacity of plants (Wang et al., 2024b; Xu et al., 2024). The growth indices of plants also positively correlate with chlorophyll content. Studies have found that cadmium stress reduces chlorophyll content. This reduction may result from excessive accumulation of reactive oxygen species (ROS) in the leaves or cadmium replacing mineral elements in the chloroplast structure, thereby inhibiting chlorophyll synthesis (Parmar et al., 2013; Shomali et al., 2023). Notably, the addition of hydrogen sulfide mitigates the negative impact of cadmium on chlorophyll, consistent with previous findings on the alleviating effects of hydrogen sulfide on cadmium stress in Brassica rapa (Ali et al., 2014). Recent studies have revealed that under heavy metal stress, hydrogen sulfide enhances plant photosynthesis by promoting the synthesis of photosynthetic pigments and the expression of photosynthetic enzymes (Alamri et al., 2020; Bharwana et al., 2014).

Chloroplasts are the core components of photosynthesis and the most active organelles in higher plants. Adverse stress can easily affect the structure and function of chloroplasts(Kaur et al., 2017; Seifikalhor et al., 2020; Wang et al., 2023b; Wang et al., 2024a). In our study, we observed varying degrees of changes in the chloroplasts of C. indicum leaves following exposure to cadmium. These changes included the loss of the membrane envelope, the presence of larger starch granules, an increased number of osmium granules, and disorganized and diffuse stroma thylakoid. Interestingly, the addition of H₂S led to smaller starch granules and an increase in stroma thylakoid in chloroplasts, mitigating the toxic effects of cadmium. Experiments by Ali et al. (2014)demonstrated that hydrogen sulfide enhances photosynthesis by mitigating lead-induced ultrastructural damage to the chloroplasts of oilseed rape. Some studies have found that hydrogen sulfide maintains chloroplast structural integrity, thereby improving the negative effects of abiotic stress on chloroplast function and the internal physiological and biochemical processes (Rizwan et al., 2019; Younis et al., 2024).

Mineral elements are absorbed by plant roots and transported to the leaves via the xylem and phloem through the action of transport proteins (Liu et al., 2023a; Ning et al., 2022; Rascio et al., 2011; Zhou et al., 2019). However, due to similar ionic radii and cation competition, cadmium ions adversely affect the absorption and transport of divalent cations(Hänsch et al., 2009; Maathuis et al., 2009). The deficiency of mineral elements can impact enzyme synthesis, thereby affecting plant growth, development, and stress resistance. This study found that under cadmium stress, the content of iron, zinc, manganese, and magnesium decreased, while copper content was less affected. However, Zhu et al. (2022)found that cadmium stress significantly reduced copper content in wheat, possibly due to species-specific differences. In hydroponically grown Platycladus orientalis and Solanum lycopersicum L., a decrease in mineral elements was observed under cadmium stress(Ou et al., 2023; Qufan et al., 2023). After the application of hydrogen sulfide, the nutrient element content in C. indicum seedlings increased. This is consistent with Li et al.'s (2021)findings that exogenous hydrogen sulfide promotes the absorption of nutrient elements in Brassica rapa under cadmium stress. These results suggest that hydrogen sulfide may influence ionic competition, maintaining the balance of mineral elements in plants and thereby enhancing the tolerance of C. indicum to cadmium.

Plant growth and development is usually accompanied by ROS production in sites such as chloroplasts and mitochondria (Wu et al., 2023). However, ROS production is also regulated to a certain level by the plant's own defence system. It has been shown that moderate ROS content plays an important role in maintaining normal plant growth and improving plant stress tolerance (Ali et al., 2023; Ravi et al., 2023). However, under abiotic stress, the accumulation of large amounts of ROS disrupts the internal balance, leading to an increase in MDA, which accelerates peroxidation of cell and chloroplast membranes, and consequently destroys the structural integrity of chloroplasts (Ali et al., 2018; Huang et al., 2017). In the present study, we found that the ROS content in plants under Cd stress was significantly increased, resulting in the elevation of MDA and EL, which indicated that the integrity of cell membranes was severely damaged. The application of H₂S significantly reduced the content of ROS and MDA, and effectively alleviated the membrane lipid peroxidation induced by cadmium toxicity. A similar phenomenon has been found in studies of Brassica rapa, Populus euphratica and Phlox paniculata L (Li et al., 2021; Sun et al., 2013; Wang et al., 2020). This suggests that H₂S effectively alleviated the cadmium toxicity-induced oxidative stress response by regulating the content of ROS, thus improving the tolerance of plants.

In order to adapt to the ever-changing living environment, plants have evolved their own antioxidant system against abiotic stresses during the long-term evolutionary process. When subjected to oxidative stress induced by abiotic factors, it leads to the production of large amounts of ROS. Once ROS start to accumulate, plants will remove them by activating their own antioxidant system, thus effectively resisting the effects of external stresses. SOD serves as the first line of defense against ROS, as it dismutates superoxide anions to generate hydrogen peroxide and reduce the formation of hydroxyl radicals. Additionally, CAT and POD work together to convert hydrogen peroxide into water and oxygen molecules. APX, in the presence of ASA, converts hydrogen peroxide into water and dehydroascorbate (DHA), which reduces the peroxidation of membrane lipids (Aborisade et al., 2023;Tan et al., 2022; Zhang et al., 2023). In our study, we found that cadmium treatment resulted in increased antioxidant enzyme activities, along with elevated levels of superoxide anions and hydrogen peroxide when compared to the control group. This suggests that cadmium stress activates the antioxidant system. Under cadmium stress, H₂S treatment increased the activity of antioxidant enzymes, which in turn scavenged excess ROS to maintain a moderate amount of ROS. These findings are consistent with the results of Li et al. (2021)study. It suggests that H₂S enhances plant tolerance to cadmium by increasing antioxidant enzyme activities and safeguarding the cell membrane and chloroplast structure of C. indicum.

In summary, H₂S alleviates the inhibitory effects of cadmium toxicity on plant growth. It does so by reducing the stress caused by cadmium in C. indicum through an increase in the activity of antioxidant enzymes, higher proline content, and effective scavenging of ROS within the plant. Furthermore, chloroplast ultrastructural analysis revealed that H₂S maintains the structural integrity of chloroplasts and mitigates the ultrastructural damage caused by cadmium ions. This reduced loss of mineral elements and chloroplast integrity leads to a higher chlorophyll content and increased photosynthetic efficiency in the plant, thereby enhancing its tolerance to Cd. In conclusion, H₂S enhances cadmium tolerance in C. indicum by modulating the antioxidant system, mineral element uptake, and photosynthesis. In addition, we used a laboratory approach to describe the ameliorative effect of H₂S on the growth of C. indicum when subjected to Cd stress. To investigate the ameliorative effects of H₂S in a cadmium-toxic environment, a soil environment-based approach is needed.

Author contribution statement

Shuguang Liu: Conceptualization, Data curation, analysis, Investigation, Methodology, Resources, Software, Writing – original draft. Liran Yue: Funding acquisition, Resources, Supervision, Writing – review & editing. Miao He: Data analysis, Supervision, Validation, Writing – review & editing. Shengyan Che: Data analysis, Supervision, Validation. Kaiyuan Zhang: Conceptualization, Data analysis. Xingyu Ni: Conceptualization,Data analysis.

Acknowledgements

This research was supported by Special Fund for Basic Scientific Research Business Expenses of Central Universities(2572014BA22).

Data availability

Data will be made available on request.

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Hydrogen Sulfide Alleviates Cadmium Toxicity in Chrysanthemum indicum L. by Modulating Photosynthesis, Mineral Homeostasis and Antioxidant Capacity

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Abstract

Figures

1. Introduction

2. Materials and methods

2.1. Plant materials and experimental design

2.2. Determination of chlorophyll content and photosynthetic parameters

2.3. Investigation of leaf ultrastructure by transmission electron microscope (TEM)

2.4 Quantification of ROS levels and antioxidant enzyme assays

2.5 Determination of the content nutrition elements

2.6 Statistical analysis

3. Results

3.1 Growth parameters of the plant

3.2 Photosynthetic pigments and photosynthetic parameters

3.3 Chloroplast ultrastructure

3.4 Plant mineral content

3.5 Plant antioxidant enzymes and oxidative stress response

4. Discussion

5. Conclusions

Declarations

Acknowledgements

Data availability

References

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