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Scientific Reports volume 14, Article number: 25534 (2024 ) Cite this article Accelerator(Ns)
Drought stress is an abiotic stressor that impacts photosynthesis, plant growth, and development, leading to decreased crop yields. Sodium hydrosulfide (NaHS), an exogenous additive, has demonstrated potential regulatory effects on plant responses to polyethylene glycol-induced drought stress in tobacco seedlings. Compared to the control, drought stress induced by 15 g/L PEG-6000 significantly reduced several parameters in tobacco seedlings: shoot dry weight (22.83%), net photosynthesis (37.55%), stomatal conductance (33.56%), maximum quantum yield of PSII (Fv/Fm) (11.31%), photochemical quantum yield of PSII (ΦPSII) (25.51%), and photochemical quenching (qP) (18.17%). However, applying NaHS, an H2S donor, mitigated these effects, ultimately enhancing photosynthetic performance in tobacco seedlings. Furthermore, optimal NaHS concentration (0.4 mM) effectively increased leaf stomatal aperture, relative water content (RWC) and root activity, as well as facilitated the absorption of N, K, Mg and S. It also enhanced the accumulation of soluble sugar and proline content to maintain osmotic pressure balance under drought stress. Compared to drought alone, pretreatment with NaHS also bolstered the antioxidant defense system in leaves, leading to 22.93% decrease in hydrogen peroxide (H2O2) content, a 22.19% decrease in malondialdehyde (MDA) content and increased activities of ascorbate peroxidase (APX) by 28.13%, superoxide dismutase (SOD) by 17.07%, peroxidase (POD) by 46.99%, and catalase (CAT) by 65.27%. Consequently, NaHS protected chloroplast structure and attenuated chlorophyll degradation, thus mitigating severe oxidative damage. Moreover, NaHS elevated endogenous H2S levels, influencing abscisic acid (ABA) synthesis and the expression of receptor-related genes, collaboratively participating in the response to drought stress. Overall, our findings provide valuable insights into exogenous NaHS’s role in enhancing tobacco drought tolerance. These results lay the foundation for further research utilizing H2S-based treatments to improve crop resilience to water deficit conditions.
Environmental stresses profoundly influence plant growth and development in both natural and agricultural conditions. Among these stressors, drought stands out as a significant constraint on crop yield and growth, leading to food shortages and compromising food security on a global scale1,2. Drought stress elicits a cascade of plant changes, spanning various levels of organization, including physiological responses, alterations in gene transcription, and modifications in epigenetic regulation, all which impact plant growth and development. For example, drought stress can severely inhibit plant growth and even result in plant mortality. Excessive production of reactive oxygen species (ROS) under drought conditions can cause cellular and membrane damage3. Additionally, reduced transpiration due to decreased stomatal conductance or stomatal closure, along with impaired photosynthetic performance, contributes to the overall decline in plant vigor4. Drought stress also profoundly affects plant organelles and ultrastructures, including chloroplasts, which may undergo expansion, rupture, disintegration, and alterations in plastid and starch grain number and size5. Furthermore, changes in antioxidant and enzyme activities occur under drought stress6, alongside regulating the expression of drought-resistance-related genes7,8. Moreover, drought stress has been documented to affect the accumulation of various osmolytes or metabolites, including glycine betaine, polyols, polyamines, and proline. Furthermore, it alters the levels and distributions of phytohormone2,9. Consequently, plants typically undergo multiple changes in their morphology, cells, physiology, biochemistry, transcription, epigenetics, and metabolism when they are exposed to drought stress. This multifaceted response highlights the intricacy of plant adaptation to drought stress. It underscores the vitality of understanding these processes for developing strategies to enhance crop resilience and ensure food security in changing environmental conditions.
Hydrogen sulfide (H2S) is recognized as the third significant gasotransmitter, following carbon monoxide (CO) and nitric oxide (NO). In higher plants, H2S modulates various physiological functions such as seed germination, root organogenesis, and photosynthesis. It also coordinates physiological processes and defensive mechanisms. Endogenous H2S can maintain potassium (K+) and sodium (Na+) homeostasis under salt stress in cucumber, suppress the generation of reactive oxygen species (ROS), and prevent membrane lipid peroxidation10. Additionally, H2S enhance the activity of antioxidant enzymes by strengthening the ascorbate-glutathione (AsA-GSH) cycle to mitigate oxidative damage11. Increasing evidence suggests that exogenous H2S can enhance plants’ antioxidant capacity under various, including drought12, salt13, cold and heat14, and heavy metal exposure15, thereby mediating plant defense responses. For example, the application of exogenous sodium hydrosulfide (NaHS) has been found to improve crop drought resistance by reducing membrane lipid peroxidation, enhancing antioxidant enzyme activity, and promoting the accumulation of osmolytes such as proline and soluble sugars12,16. NaHS pretreatment inhibited chromium (Cr) toxicity in spinach by increasing antioxidant enzyme activity and non-enzymatic antioxidant content17. Furthermore, H2S interacts with other hormones and signal molecules to regulate plant responses to abiotic stresses18. It participates in alleviating salt stress damage in rice seedlings, enhancing photosynthetic capacity, and maintaining cell structure19. Moreover, exogenous NaHS has been found to improve drought tolerance by regulating stomata20.
Tobacco (Nicotiana tabacum), as a widely cultivated non-food crop worldwide, serves as a prominent biological model system. However, tobacco is highly sensitive to drought stress during the rosette and ripening stages, significantly restricting its growth and yield potential21, thereby resulting in substantial economic losses for tobacco growers and industries. Recently, the regulation of plant physiological metabolism by H2S under adversity stress has emerged as a focal point of research. However, the precise physiological mechanisms through which exogenous H₂S enhances drought tolerance in tobacco remain unclear. In this study, we used the Yuyan No. 10 cultivar and NaHS as an H₂S donor to separately investigate the optimal concentration and temporal effects of exogenous H₂S, aiming to investigate the effects of exogenous H₂S on the morphology, physiology, and biochemistry of tobacco seedlings subjected to drought stress, thereby offering a theoretical foundation for further exploration into its potential applications in augmenting tobacco drought resistance.
Seeds of the Yuyan No.10 cultivar of tobacco was sourced from the College of Tobacco Science, Henan Agricultural University, Henan Province. Sodium hydrosulfide (NaHS) from Sigma served as the donor of hydrogen sulfide (H2S), while polyethylene glycol-6000 (PEG-6000) from Sinopharm was employed to imitate drought stress.
The experiment started in April, 2017. The seeds were initially sterilized for 10 min using a 10% H2O2 solution, Subsequently, they were washed with distilled water 10 times. Following this, the seeds were immersed in distilled water at 25 °C in the dark for one day and had their seed coats removed. The seeds were then evenly distributed onto seeding sponges and transferred to a greenhouse under controlled conditions: a temperature of 28 ± 1 °C in light and 18 ± 1 °C in darkness, a 14-hour photoperiod, relative humidity of 70%, and illuminance of 2.2 klx at the leaf level.
Upon reaching the three-leaf stage, uniformly sized seedlings were selected and moved to vermiculite for further growth until the four-leaf stage. After that, they were transplanted into plastic containers (17 cm height, 15 cm length, and 15 cm breadth) containing half-strength Hoagland nutrient solution. The treatment phase commenced once the seedlings reached the five-leaf stage.
Prior to the formal experiment, preliminary trials were conducted using NaHS concentrations of 0.01, 0.1, 1, and 10 mmol/L. Results revealed lethality at the 1 mmol/L NaHS concentration. Therefore, a concentration gradient between 0.01 and 1 mmol/L NaHS was selected for alleviating drought stress.
We conducted two experiments: the NaHS concentration screening trial (Experiment 1) and the NaHS time effect trial (Experiment 2). Among them, Experiment 1 aims to screen out the optimal NaHS concentration that provides the best mitigation effect on drought stress, ensuring that the time effect evaluated in Experiment 2 is based on the optimal concentration.
This experiment included 7 treatments: Normal growth (CK); 15% PEG treatment (D); T1: 0.01 mM NaHS + 15% PEG treatment; T2: 0.1 mM NaHS + 15% PEG treatment; T3: 0.4 mM NaHS + 15% PEG; T4: 0.7mM NaHS + 15% PEG; T5: 1.0 mM NaHS + 15% PEG, all treatment were supplied with standard Hoagland nutrient solution.
Before subjecting the seedlings to drought stress treatment, the seedlings underwent pre-treatment with nutrient solutions containing various concentrations of NaHS for one day. Following the pre-treatment, the seedlings were placed to nutrient solutions supplemented with PEG-6000 to induce drought stress. After three days of exposure to drought conditions, photosynthetic and fluorescence parameters were measured to assess plant responses. Samples for analysis were collected from the second and third leaves from the top downwards, weighed, and promptly immersed in liquid nitrogen to preserve their physiological state for further analysis.
Based upon the findings of Experiment 1, wherein the concentration of NaHS demonstrating the most effective alleviation of drought stress was identified, experiment 2 aimed to investigate the temporal dynamics of this response. Specifically, the T3 treatment from Experiment 1, which exhibited promising results, was selected for further examination.
The experiment included 4 treatments: Normal growth (CK); normal treatment + 0.4 mmol/L NaHS (H); Drought treatment: 15%PEG-6000 (D); Drought treatment + 0.4 mM NaHS: 0.4 mM NaHS + 15%PEG-6000 (D + H), all treatment were supplied with Hoagland nutrient solution.
Before subjecting the seedlings to drought treatment, they underwent pre-treatment with nutrient solutions containing 0.4 mmol/L NaHS for one day. Subsequently, they were transferred to nutrient solutions supplemented with 15% PEG-6000 to induce drought stress. Samples were collected from the second and third leaves from the top downwards on the first, third, and fifth day following the initiation of drought treatment.
The tobacco seedlings were harvested after three days of treatment, and visual documentation was performed through photography. Subsequently, the samples were meticulously separated into shoots and roots. The roots underwent thorough washing before being subjected to oven-drying at a temperature of 80 °C for 12 h, and their resulting dry weight recorded. Similarly, the shoot samples were subjected to the same drying conditions and their dry weight was also recorded.
Photosynthetic parameters were determined using a Li-6000 portable photosynthesis system (LI-COR, USA). After subjecting the plants to 3 days of drought stress, between 9:00 AM and 11:00 AM, six randomly seedlings from each treatment were chosen. The third leaf from the top downward was selected for measurements. The instrument was configured with a leaf chamber temperature of 28 °C, light intensity of 800 µmol·m−2·s−1, and CO2 concentration of 400 µmol⋅ mol−1. Subsequently, the parameters were determined for each leaf: water use efficiency (WUE), intercellular CO2 concentration (Ci), net photosynthetic rate (Pn), and transpiration rate (Tr).
Parameters were assessed utilizing a PAM-2100 fluorometer (WALZ, Germany). Following three days of drought stress, from 9:00 AM to 11:00 AM, six randomly selected seedings from each treatment were chosen for measurement. The third leaf from the top downward was selected as the site for measurements. The instrument was configured with a light intensity of 1000 µmol·m−2·s−1, and the seedlings underwent a dark treatment for 20 min to ensure stabilization. Subsequently, chlorophyll fluorescence parameters were calculated, including PSII maximum photochemical efficiency (Fv/Fm), PSII potential activity (Fv/F0), photochemical quenching coefficient (qP), non-photochemical quenching coefficient (NPQ), and actual photochemical efficiency of PSII (ΦPSII). As follows are the formula to calculate the parameters:
Fresh weight of leaf samples from seedings were recorded. Subsequently, the samples were soaked in water for 4 h for saturation weight (FWsat) determination. The leaves were then dried for 12 h at 80 °C to determine the dry weight. The following formula was used to get the RWC:
For the root activity assay, fresh samples of clean roots weighing 0.5 g were immersed in a solution containing 2,3,5-triphenyl tetrazolium chloride (TTC) and incubated at 37 °C for 1 h. After incubation, the samples were ground using ethyl acetate to extract the material. The supernatant obtained was gathered and measured in accordance with the manufacturer’s guidelines. The root activity index was calculated based on the dehydrogenase activity reflected by the decrease in TTC measured at 485 nm.
The quantification of photosynthetic pigment content was determined using the mixed-solvent extraction method as described by Lichtenthaler and Wellburn22. 0.5 g of fresh samples were immersed in a solution of 80% acetone at room temperature in the dark until the leaves became decolorized. After centrifugation, the supernatant was analyzed for absorbance at 663 nm, 645 nm, and 470 nm. The chlorophyll and carotenoids content were determined using the following formula:
Harvested plants were segregated into shoots and roots, then dried at 80 °C until a consistent weight was achieved. The nitrogen content in the samples was ascertained using a Vario ELcube elemental analyzer (Elementar, Germany). Concentrations of K, Mg, and S were quantified via inductively coupled plasma mass spectrometry (ICP-MS) following Emiko’s method23.
The free proline content was measured following the method of Bates et al.24. The soluble sugar content was calculated using the BCA method following the experimental procedure described by Xu et al.25. Relative electrical conductivity (REC) was measured based on the methodology described by Cao et al.26.
The hydrogen peroxide (H2O2) content was measured using the method described by Song et al.27, while the malondialdehyde (MDA) content was measured in accordance with method by Feng et al.28. The activity of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) were determined utilizing the nitrogen blue tetrazole photoreduction method27, guaiacol method29and hydrogen peroxide decomposition method30. For APX (L-ascorbate peroxidase) activity we used the method of Amir et al.31. The method for determining endogenous H2S levels is in accordance with Shi et al.16. The content of ABA was ascertained utilizing an enzyme-linked immunosorbent assay (ELISA) kit, and the subsequent calculations were executed with the ELISA Calc software.
Electron microscopy of chloroplast ultrastructure: After 3 days of drought stress, the third leaf from the top downward was chosen, and small pieces measuring 1 × 2 mm were then cut down from the area between the main vein to the edge of the leaf. These segments were soaked in 2.5% (v/v) glutaraldehyde for 4 h and subsequently fixed with osmic acid at 4 °C for 4 h. Then the samples were dehydrated with increasing concentrations of alcohol and acetone and were embedded in Epon epoxide resin. Ultrathin sections for electron microscopy were prepared using the LeicaEMUC6 ultramicrotome, and stained with uranyl, acetate and lead citrate. The leaf samples were viewed and photographed with an electron microscope JEM 100B (Japan). Selecting the same leaf parts as previously mentioned were selected to measure stomatal aperture. Three leaves from tobacco seedlings were randomly chosen, and the epidermis was carefully removed using tweezers. The epidermis was then stained with a 1% mass-volume fraction solution of iodine-potassium iodide. Observations of the stomatal apertures before and after drought stress were made using a confocal microscope (Olympus, Japan). Three random visual fields were chosen, and within each field, 3 ~ 4 stomata were observed at random. The stomatal apertures were measured using IMAGEJ analysis software (NIH, Bethesda, MD, USA).
RNA was extracted from the third leaf position from the top down after 5 days of drought treatment. The extraction steps followed the instructions of the Plant Total RNA Extraction Kit (DP423, Tiangen Biochemical Technology (Beijing) Co., Ltd.). Reverse transcription was performed using the M-MLV Reverse Transcriptase Kit (Invitrogen, USA). The tobacco ribosomal protein coding gene NtL25 was used as an internal reference. Three individual plants were selected for each treatment, and each plant underwent three technical replicates. Primers are shown in Table S1. The reaction system consisted of: MonAmp® Fast SYBR® Green qPCR Mix 10.0 µL, upstream and downstream primers (10.0 µmol/L) each 0.4 µL, cDNA 1.0 µL, Nuclease-Free Water 8.2 µL. The reaction conditions were: 95 °C for 10 min; 40 cycles of 95 °C for 15 s, 60 °C for 15 s, 72 °C for 30 s. After amplification, the dissolution curve was set as follows: 95 °C for 15 s, 60 °C for 15 s, gradually heating to 95 °C within 20 min, and finally 95 °C for 15 s. The 2−ΔΔCt method was used to calculate the expression levels.
Data processing and graphing were conducted using Statistical Program for Social Science (SPSS26.0) and GraphPad Prism 9 software, multiple comparisons were conducted using Ducan`s new multiple-range test method.
The overground parts of plants, particularly the leaves, serve as the primary sites for photosynthesis and respiration. The normal development of shoot parts is essential for maintaining plant vitality. Under drought stress (D), leaf wilting was a notable phenotypic characteristic observed in tobacco seedlings, which significantly reduced shoot dry weight by 22.83% compared to the control. With application of exogenous NaHS, the shoot dry weight of T2, T3, and T4 decreased by 18.16%, 11.67%, and 24.65%, respectively, compared to the control (Fig. 1B). The distribution of the root system markedly influences the capacity of plant to uptake water and nutrients, playing a pivotal role in determining its absorption ability. Drought stress alone (D) significantly increased root dry weight by 7.35% compared to the CK. However, in treatments T2, T3, and T4, pretreated with NaHS, root dry weight increased by 2.33%, 0.78%, and 1.25%, respectively. Conversely, treatment T5 decreased by 4.66% (Fig. 1C). In response to prolonged drought stress, plants typically enhanced the growth of their root systems, thereby expanding the area available for water absorption to facilitate water uptake. Simultaneously, there was a reduction in the development of aboveground parts to minimize water and energy consumption.
The root-shoot ratio serves as an indicator of the relationship between the biomass of aboveground and underground parts of plants. In Fig. 1D, it is observed that D treatment led to a significant increase in root-shoot ratio, showing a rise of 39.32% compared to the control. Meanwhile, in treatments T2, T3, and T4, the root-shoot ratio increased by 33.76%, 25.23%, and 14.08%, respectively. These findings suggested that drought stress inhibited shoot growth while slightly enhancing root growth. Interestingly, pretreatment with NaHS facilitated the coordinated development of both shoot and root parts, as evidenced by the more balanced root-shoot ratio observed in treatments with exogenous NaHS. This indicated that NaHS treatment may mitigate the adverse effects of drought stress on plant growth, promoting more balanced growth between aboveground and underground biomass.
Performance of tobacco seedlings at different treatments. CK, normal growth; D, treatment with 15% PEG-6000; T1, treatment with 0.01 mmol/L NaHS and 15% PEG-6000; T2, treatment with 0.1 mmol/L NaHS and 15% PEG-6000; T3, treatment with 0.4 mmol/L NaHS and 15% PEG-6000; T4, treatment with 0.7 mmol/L NaHS and 15% PEG-6000; T5, treatment with 1 mmol/L NaHS and 15% PEG-6000. Error bars represent ± SD of three independent replicates. According to Duncan’s test, statistical differences at p < 0.05 are indicated by different letters above the error bars.
To assess the impact of exogenous NaHS on the photosynthesis of tobacco seedlings under drought stress, several parameters including Pn, Gs, Tr, Ci, and WUE were measured (Table 1). These parameters exhibited notable changes in response to the imposed drought stress. As compared to the CK, drought stress alone caused a significant decrease in Pn, Gs, Tr, and WUE by 37.55%, 33.56%, 33.56%, and 21.94%, respectively, while Ci increased by 24.18%. However, NaHS pretreatment at lower concentrations (0.1, 0.4, and 0.7 mmol/L) exhibited higher values of Pn, Gs, Tr, and WUE, along with lower Ci, compared to the drought treatment. Among these, pretreatment with 0.4 mmol/L NaHS notably increased Pn, Gs, Tr, and WUE by 25.85%, 12.76%, 36.73%, and 13.41%, respectively, while Ci decreased by 10.22%, compared to the drought alone. Exogenous NaHS pretreatment, particularly in T2 and T3, demonstrated positive effects on photosynthesis, while T5 showed inhibitory effects, indicating potential drawbacks for enhancing tobacco drought tolerance. These results underscored the importance of optimizing NaHS concentration to achieve maximum benefits in mitigating the adverse effects of drought stress on tobacco photosynthesis.
In adverse conditions, parameters such as Fv/Fm and Fv/F0 reflect the maximum photochemical efficiency and potential photochemical efficiency of PSII reaction centers, respectively. Similarly, ΦPSII, qP, and NPQ parameters provide insights into efficiency of photosynthetic and the openness of PSII reaction centers. As depicted in Table 2, drought treatment significantly reduced Fv/Fm, Fv/F0, ΦPSII, and qP by 11.31%, 43.72%, 25.51%, and 18.17%, respectively, compared to the CK. Conversely, NPQ exhibited a significant increase of 48.24%. However, NaHS pretreatment at lower concentrations (0.01, 0.1, 0.4, 0.7 mmol/L) partially mitigated the decrease induced by drought stress. Compared to the drought treatment alone, parameters such as Fv/Fm, Fv/F0, ΦPSII, and qP in treatments T1, T2, T3, and T4 were enhanced, while NPQ showed a corresponding decrease. Thus, pretreatment with NaHS proved beneficial in stabilizing the photosynthetic mechanism, enhancing reaction center activity, and improving the energy supply capacity of photosynthetic reactions. Furthermore, the inhibited plant growth and reduced photosynthetic efficiency caused by drought stress were effectively ameliorated through the application of 0.4 mmol/L exogenous NaHS. Consequently, 0.4 mmol/L NaHS was selected for further subsequent experiments to investigate its effects on tobacco seedlings under drought stress.
Changes in the living environment of plants can induce alterations in cellular structures, and drought stress often leads to modifications in organelle positioning, quantity, and structure. As depicted in Fig. 2, organelles within tissue cells were typically situated close to the cell wall in normally growing tobacco leaves. Osmiophilic granules exhibited minimal volume and quantity with neatly arranged regular, and structurally transparent thylakoid lamellae. Mitochondria and cell nuclei maintained their integrity under these conditions. However, under drought stress, severe plasmolysis occurred in tissue cells, dissociating some organelles into the cell cavity. Osmiophilic particles increased in size and volume, with many thylakoid lamellae showing signs of degradation. Both mitochondria and cell nuclei began to degrade as well. In contrast, after pretreated with 0.4 mmol/L exogenous hydrogen sulfide, most organelles within the tissue cells redistribute closed to the cell wall, with some still presented within the large central vacuole. Although the volume of osmiophilic particles slightly increased, there was no significant rise in their quantity. Thylakoid lamellae remained abundant, but some still exhibit signs of dissolution compared to the control conditions.
Effect of exogenous H2S on cell morphology and ultrastructure of tobacco seedling leaves under drought stress. A (3000×), D (10000×), G (50000×) represent tobacco seedlings under control conditions. B (3000×), E (10000×), H (50000×) represent tobacco seedlings under drought stress. C (3000×), F (10000×), I (50000×) represent tobacco seedlings under T3 treatment. Chl: chloroplasts; V: vacuoles; Op: osmiophilic granules; Pl: plasmolysis.
In experiment 2, Fig. 3 illustrated that NaHS pretreatment did not induce significant changes in the RWC and root activity of seedlings under normal conditions without drought stress. The root activity of drought-stressed-seedlings (D) initially exhibited an increase, followed by a subsequent decline. By the 5th day of drought alone, the root activity of seedlings significantly decreased by 26.64%. In contrast, the D + H treatment showed only a 4.70% decrease compared to the CK (Fig. 3C). On the first day, both the drought-alone and D + H treatment groups displayed prominent dehydration in RWC (Fig. 3B). On the third day, there was no significant change in RWC between the CK and D + H treatment. However, by the fifth day, leaf RWC in the drought-alone group decreased by 29.89% compared to the control, while the D + H treatment exhibited a lower decrease of 12.64%. These results suggested that leaves undergo dehydration after a brief time of drought stress, and as the duration of drought stress increased, dehydration intensified. However, NaHS pretreatment improved the water retention capacity of leaves, thereby mitigating prolonged drought-induced leaf dehydration.
Effects of exogenous NaHS on changes in leaf water content and root activity in tobacco seedings under continuous drought. CK, normal growth; D, treatment with 15% PEG-6000; H, treatment with 0.4 mmol/L NaHS; D + H, treatment with 0.4 mmol/L NaHS and 15% PEG-6000. Error bars represent ± SD of three independent replicates. According to Duncan’s test, statistical differences at p < 0.05 are indicated by different letters above the error bars.
The influence of hydrogen sulfide (H2S) on stomatal movement in tobacco was examined by measuring the stomatal apertures under normal and drought conditions. Figure 4A illustrated the morphology of leaf stomata subjected to drought stress (Fig. 4A). The stomatal aperture of leaves under D treatment was significantly reduced compared to the CK, showing a progressive decrease as the duration of drought increased. However, the D + H treatment increased the stomatal aperture compared to the D treatment. These results were consistent with the observations on stomatal conductance, indicating that the application of exogenous NaHS mitigated the closure of stomata under drought stress and enhanced the net photosynthetic rate (Fig. 4B).
Stomatal morphology observed under the microscope. Bar = 20 μm. Mean stomatal aperture of leaves under different treatments. Error bars represent the standard error of three biological replicates, and different letters indicate significant differences between treatments (P < 0.05).
The uptake and transport of nutrients are essential for plant growth and health under adverse conditions. In Table 3, compared to the CK, H treatment did not significantly alter the levels of N, K, and Mg in shoot and root, but facilitated the accumulation of S, which increased by 5.13% and 8.33% respectively (Table 3). Drought stress restricted the root’s ability to absorb nutrients and translocate them to the leaves, resulting in a significant reduction in the accumulation of N, S, and Mg in the roots by 33.27%, 38.50%, and 8.05% respectively, and in the shoots by 36.42%, 28.79%, and 45.11% compared to the CK. Conversely, exogenous NaHS application enhanced the roots’ absorption of elements under drought conditions, with accumulations of N, S, and Mg in the roots increasing by 27.29%, 34.49%, and 31.40% respectively, and in the shoots by 34.08%, 30.16%, and 59.23% compared to drought alone.
Figure 5A showed that the total chlorophyll content in the CK and H groups exhibited minimal variation over the 5-day treatment period. However, drought treatment alone significantly decreased chlorophyll content by 22.38% on the first day compared to the control, with this reduction intensifying as the duration of drought prolonged. Remarkably, in leaves pretreated with exogenous NaHS (D + H), the chlorophyll content showed a significant increase by 17.89%, 44.10%, and 34.78% on the first, third, and fifth day, respectively, compared to drought alone (D). In the H group, carotenoid content in seedling leaves significantly increased by 11.57% on the first day and 10.00% on the fifth day compared to the control. Conversely, drought treatment led to a substantial reduction in carotenoid content by 18.20%, 32.39%, and 30.90% on the first, third, and fifth days, respectively, compared to the control. Interestingly, in NaHS-pretreated seedlings (D + H), the carotenoid content significantly increased by 36.54% and 20.29% on the third and fifth day, respectively, compared to drought treatment alone (Fig. 5B).
Effects of exogenous NaHS on changes in pigment content in tobacco seedings under continuous drought. Error bars represent ± SD of three independent replicates. According to Duncan’s test, statistical differences at p < 0.05 are indicated by different letters above the error bars.
To assess whether the application of NaHS can enhance the osmotic adjustment capacity of tobacco seedlings exposed to drought stress, the free proline and soluble sugar content of tobacco leaves were studied. On the first day, drought alone treatment led to a notable 92.81% increase in proline content in seeding leaves, as compared to the control. However, this proline accumulation decreased with the duration of drought. Notably, compared to drought alone, the D + H treatment showed a tendency to improve proline content, with increases of 14.69% and 17.11% observed on the third and fifth day, respectively (Fig. 6A). Furthermore, drought stress induced soluble sugars to accumulate in seeding leaves, while seedlings pretreated with NaHS (D + H) exhibited an even more significant increase in soluble sugar content. Overall, exogenous NaHS treatment elevated osmoprotectant contents in tobacco seedlings, stimulating osmotic adjustment and improving resistance to drought stress (Fig. 6B).
Effects of exogenous NaHS on changes in osmotic regulator in tobacco seedings under continuous drought. Error bars represent ± SD of three independent replicates. According to Duncan’s test, statistical differences at p < 0.05 are indicated by different letters above the error bars.
As depicted in Fig. 7, the first day under drought stress (D) exhibited significant increases in hydrogen peroxide (H2O2), malondialdehyde (MDA), and leaf relative electrical conductivity (REC) levels by 142.80%, 91.19%, and 38.31%, respectively, compared to the CK. Furthermore, these parameters exhibited an ascending trend as the duration of drought stress was prolonged. However, in the D + H treatment, the levels of H2O2, MDA, and REC were lower than those observed in the D treatment. After five days of continuous drought stress, exogenous NaHS application significantly reduced the contents of H2O2, MDA, and REC by 22.93%, 22.19%, and 17.26%, respectively, compared to the D treatment. This indicated that NaHS application effectively mitigated oxidative stress and membrane damage caused by drought stress in tobacco seedlings.
Effects of exogenous NaHS on changes in H2O2, MDA and electric conductivity in tobacco seedings under continuous drought. Error bars represent ± SD of three independent replicates. According to Duncan’s test, statistical differences at p < 0.05 are indicated by different letters above the error bars.
In Fig. 8, it is evident that NaHS pretreatment alone (H) did not induce significant alternations in the activity of various antioxidant enzymes in seedling leaves over the entire experiment. On the first day, drought alone treatment significantly increased the activity of SOD, POD, CAT, and APX enzymes by 4.14%, 4.56%, 3.37%, and 3.12%, respectively, compared to the CK. These activities showed slight improvements in the subsequent third and fifth days under drought alone. Remarkably, on the first day, the D + H treatment significantly elevated the activity of SOD, POD, CAT, and APX enzymes by 17.07%, 46.99%, 65.27%, and 28.13%, respectively, compared to the drought alone (D). These elevated levels were maintained in the later stages of the experiment. These findings revealed that the enhanced drought resistance of tobacco seedlings was manifested after encountering drought stress, as evidenced by the elevated activity of antioxidant enzymes to counteract drought stress-induced damage. Moreover, pretreatment with exogenous NaHS prompted higher activity of antioxidant enzymes in the leaves, improving their ability to resist drought stress and facilitating the removal of excessive ROS in the seedlings.
Effects of exogenous NaHS on changes in antioxidant enzyme activities in tobacco seedings under continuous drought. Error bars represent ± SD of three independent replicates. According to Duncan’s test, statistical differences at p < 0.05 are indicated by different letters above the error bars.
Figure 9 illustrated the effect of exogenous NaHS on changes in H2S and ABA levels. The H treatment significantly increased the H2S content by 64.90%, 57.57% and 46.79% compared to the CK on the days 1, 3 and 5, respectively (Fig. 9A). Drought alone also led to an accumulation of H2S in leaves, with significant increases of 22.91%, 62.87% and 67.34% compared to the CK on the first, third, and fifth day, respectively. Notably, the D + H treatment significantly inhanced H2S content compared to drought alone, with further increases as the duration of drought extended. The abscisic acid (ABA) content under drought stress showed a progressive increase, rising by 180.74%, 204.58% and 248.84% on the days 1, 3 and 5, respectively, compared to the CK. Whereas, D + H treatment resulted in smaller increases in leaf ABA content, with elevations of 100.85%, 105.92%, and 128.45% on the same days (Fig. 9B).
Effect of exogenous NaHS on changes of endogenous hormone in tobacco seedlings under sustained drought conditions. Error bars represent ± SD of three independent replicates. According to Duncan’s test, statistical differences at p < 0.05 are indicated by different letters above the error bars.
ABA (abscisic acid) is pivotal in amplifying plant drought resistance by modulating stomatal activity via an intricate signaling network. Studies have shown that H2S can act in stomatal movements regulated by ABA32. To further explore the function role of H2S in drought resistance, we analyzed the gene expression related to ABA synthesis and signal transduction in tobacco seedling leaves using qRT-PCR technology (Fig. 10). The results showed that under drought alone, the expression levels of genes related to ABA synthesis (NtNCED1, NtNCED2, NtNCED3b, and NtNCED5a) and ABA signal transduction-related genes (NtAREB1, NtAREB3and NtLTP1) were significantly higher than those in the CK. In leaves pretreated with NaHS (D + H), the expression levels of the four ABA synthesis-related genes were significantly higher than the control group but lower than those under drought alone. The three ABA transduction-related genes also demonstrated increased expression compared to the CK, with NtAREB3 and NtLTP1 showing notably higher expression than other treatments. Interestingly, H treatment did not induce significant alterations in the gene expression related to ABA synthesis and transduction in the leaves, aligning with the ABA content measurements in the leaves.
Exogenous H2S on changes of the expression levels of ABA synthesis and receptor genes in tobacco seedings. A, B, C, D represents ABA synthesis-related genes. E, F, G represents ABA receptor-related genes. The data represent the average of three replicates per treatment ± SD. The relative expression of related genes was calculated using the CK as the calibration According to Duncan’s test, statistical differences at p < 0.05 are indicated by different letters above the error bars.
This study investigates the protective effects of exogenous NaHS on tobacco under drought stress, focusing on the effects of concentration and duration. The isolated application of NaHS did not significantly impact the growth or physiological responses of tobacco seedlings. However, under drought stress, external application of NaHS treatment notably enhanced the resilience of tobacco.
Drought stress significantly impedes plant growth, impacting various aspects including shoot and root development, leaf area, and root length, as evidenced by previous studies33,34. Notably, indicators such as fresh and dry weights are crucial for assessing plant dry matter accumulation, providing insights into the plant water absorption capabilities (Fig. 1A). Previous research has demonstrated that moderate concentrations of NaHS can stimulate plant growth, whereas higher concentrations may have inhibitory effects35. In our study, drought stress visibly hampered the development of shoot components in tobacco seedlings, leading to a reduction in their dry weight (Fig. 1B,C). Conversely, pretreatment with NaHS led to notable enhancements in plant height and shoot length in tobacco seedlings under drought conditions, alongside enhanced shoot dry weight. These findings validate that the application of NaHS can indeed bolster plant tolerance to drought, indicating its potential as a valuable approach to alleviate the detrimental impacts of drought stress on plant growth and development.
Drought stress induces notable shifts in biomass production and allocation patterns within plants36. Under drought conditions, plants often adjust their biomass allocation strategies to enhance water productivity and nutrient utilization efficiency. This typically involves a decrease in shoot biomass production coupled with a greater allocation of biomass to roots, leading in an elevated root–shoot ratio. Consequently, the adaptive capacity of plants to drought stress is intricately linked to their biomass allocation between their shoot and root components25,37. In this study, we observed a significant reduction in aboveground biomass due to drought stress, accompanied by an increase in root biomass and the root–to–shoot ratio of tobacco seedlings. This indicates that the root growth exhibited lower sensitivity to water deficit, and maintaining a higher root-to-shoot ratio can enhance tobacco’s resilience to drought. However, the application of NaHS mitigated this effect, resulting in a reduction in the root–shoot ratio and promoting balanced development between above and underground plant parts under drought stress (Fig. 1D). This suggests that NaHS treatment can optimize biomass allocation patterns, potentially enhancing the overall drought tolerance of tobacco plants.
Photosynthesis is a crucial metabolic process in plants that highly susceptible to water deficit38. Drought stress triggers stomatal closure in plant leaves, impacting gas exchange and thereby carbon assimilation39. In this study, drought stress significantly reduced stomatal aperture of tobacco leaves, resulting in a decrease in Pn, accompanied by an increase in Ci. It is initially speculated that the decrease in Pn caused by non-stomatal limitations20. On the contrary, D + H effectively alleviated the negative effects of drought stress on stomatal aperture and photosynthetic efficiency. Notably, within a specific concentration range, increasing the concentration of NaHS further alleviated the adverse effects of drought stress, as evidenced by increased Pn, Tr, Gs, and WUE, which align with findings from previous studies40.
Drought stress disrupts the ultrastructure of chloroplasts and interferes with pigment synthesis, thereby affecting photosynthesis in plants41. Under drought stress, plasmolysis transpires between the cytoplasm and cell wall, with chloroplasts detaching from the cell wall and some structures being disrupted, leading to leakage of their contents. Moreover, certain granal lamellae underwent degradation, with their structures becoming indistinct, presumably due to oxidative damage incited by drought (Fig. 2). Similar findings have been reported in Arabidopsis42, rice43and tomato44. Complementing the pigment measurements from Experiment 2 (Fig. 5), drought stress also caused a significant reduction in chlorophyll and carotenoid content possibly due to Mg deficiency or the toxic effects of ROS inhibiting chlorophyll synthesis45. Additionally, drought stress led to a decrease in the maximum photochemical efficiency of PSII (Fv/Fm) in maize (Table 2), indicating an inhibition of PSII reaction center activity. In this study, degradation of the thylakoid membranes may result in a reduction in Fv and Fv/Fm, thereby decreasing the activity of the PSII reaction center46. Nevertheless, the application of exogenous NaHS maintained the chloroplast ultrastructure and enhanced the content of photosynthetic pigments, suggesting that H₂S, acting as a signaling molecule, stimulated chloroplast synthesis and increased the stacking of thylakoid membranes47. Similarly, Liu et al48. reported that exogenous NaHS promoted the expression of genes related to chlorophyll and carotenoid synthesis, thereby improving photosynthetic capacity. Furthermore, H2S maintained photochemical efficiency and suppressed the dissipation of light energy, enhancing the primary light energy conversion efficiency and electron transport of PSII15. This study illustrated that under drought conditions, the addition of NaHS improved Fv/Fm, ETR, qP, and ΦPSII, while reducing NPQ. Such changes were instrumental in maintaining the activity of the PSII reaction center, showcasing the potential of NaHS as a protective agent against drought-induced photosynthetic impairments49.
In Experiment 2, we found that increasing drought stress duration resulted in decreased root activity and relative water content (RWC) in tobacco seedlings, accompanied by reduced accumulation of mineral elements. This phenomenon is likely due to prolonged drought exposure decreasing stomatal conductance, which in turn reduces effective transpiration flux, ultimately impairing nutrient absorption by the roots and vertical transport to the leaves50. Elements such as Nitrogen (N), Magnesium (Mg), and Sulfur (S) are essential for chlorophyll biosynthesis and participate in various physiological reactions related to plant development51. Potassium (K) plays a critical role in photosynthesis, maintaining stomatal regulation, osmotic adjustment, and enzyme activity52. Pretreatment with NaHS (D + H) significantly improved the RWC in tobacco leaves under drought stress and increased the accumulation of N, K, S, and Mg in tobacco seedings, promoting plant growth and photosynthetic activity (Table 3).
Water deficit impacts leaf water potential and intracellular water balance, resulting in osmotic stress53. Plants directly respond to drought stress by accumulating osmolytes such as soluble sugars, amino acids, and soluble proteins for osmotic adjustment31. Soluble sugars and proline help maintain intracellular osmotic pressure54, while proline can also mitigate photosynthetic damage by scavenging ROS in the thylakoids55. In this study, the accumulation of proline and soluble sugars in leaves under drought stress was higher than in the CK, which supported osmotic balance across cell membranes and enhancing the plant’s drought tolerance (Fig. 6). Following NaHS pretreatment (D + H), tobacco seedlings subjected to drought stress further increased their accumulation of soluble sugars and proline, aiding in the clearance of intracellular ROS. Similarly, exogenous application of NaHS also increased the accumulation of osmolytes in spinach leaves under drought stress31.
Plants often encounter the accumulation of reactive oxygen species (ROS), such as O2·− and H2O2, under conditions of drought stress56. This excessive accumulation of ROS can lead to oxidative damage and compromise membrane integrity. To counteract this, plants boost their antioxidant capabilities to eliminate the accumulated ROS57. Parameters like REC and MDA content serve as indicators of membrane integrity and lipid peroxidation. Our research showed that drought stress significantly elevated the levels of H2O2, REC, and MDA in tobacco seedlings (Fig. 7). This suggested that the imbalance in ROS metabolism, lipid peroxidation, and increased membrane permeability caused by drought stress disrupts intracellular homeostasis and inhibits the growth of tobacco seedlings. When exposed to abiotic stress, the antioxidant enzyme system is activated to maintain stable levels of ROS. SOD dismutates O2·− to H2O258, which is then eliminated by enzymes like POD, APX, and CAT to produce H2O and O259. In the case of D + H treatment, the levels of H2O2, MDA, and REC were significantly reduced compared to the drought alone, and the activity of the four studied antioxidant enzymes was increased, aligning with previous findings14,60 (Fig. 9). Moreover, studies have demonstrated that H2S in plants can react with ROS to form thiols, which react with H2O2to produce polysulfides, thereby aiding in suppressing the production of reactive oxygen species (ROS)61.
ABA plays a pivotal role in plant responses to drought stress, primarily by promoting stomatal closure to reduce water evaporation62. Studies have shown that H2S can further enhance ABA-induced stomatal closure and is involved in regulating the expression of genes related to ABA synthesis and signal transduction63. This study found that under drought stress, the expression of ABA synthesis-related gene NtNCED1, NtNCED2, NtNCED3b, and NtNCED5a64 significantly increased, while their expression levels under D + H treatment were lower than those under drought alone. The genes NtAREB1, NtLTP1 and NtDREB3 are involved in the ABA signaling transduction pathway in tobacco65 (Fig. 11). Under drought stress, the expression of these genes significantly higher than the CK, with D + H treatment further elevating their expression. The levels of ABA in D + H treatment was lower than those in D treatment for several potential reasons. Firstly, the balance of ABA content is crucial for certain physiological processes. Secondly, enhanced perception and signal transduction efficiency of ABA caused by increased receptor expression, enabling low levels of ABA to effectively contribute to drought resistance. Lastly, elevated H2S levels may play a role physiologically similar to ABA66 (Fig. 10). Unexpectedly, D + H treatment did not promote stomatal closure. We speculate that the decrease in photosynthetic rate of tobacco seedlings under drought stress could be attributed to non-stomatal factors, such as chloroplast structure damage or lack of essential nutrients, which impeded photosynthetic activity. Exogenous addition of NaHS increased the endogenous H2S content in tobacco, stimulated the increase of antioxidant enzyme activity, and reduced the damage of ROS to chloroplast structure, while increasing the accumulation of N, K, S, and Mg. These findings are consistent with the Abeer et al.67,68, who reported that H2S promotes stomatal opening by reducing ABA accumulation, Subsequetly observing the Pn in this study. Therefore, H2S affects the ability of ABA to induce stomatal closure and modulates the expression of ABA receptors. The regulatory relationship between these two deserves further study.
Our study revealed that exogenous NaHS treatment significantly enhanced the drought tolerance of tobacco seedlings. This enhancement was characterized by the elevation of endogenous H2S levels, which stimulated the antioxidant system, leading to heightened antioxidant enzyme activity, reduced ROS levels, mitigation of membrane damage, preservation of chloroplast and leaf structure integrity, elevated chlorophyll content, and enhanced photosynthetic and electron transfer rates. Furthermore, NaHS treatment promoted the synthesis of plant osmotic substances, thereby improving water metabolism balance (Fig. 11). The study also suggested that NaHS pretreatment equilibrated endogenous ABA levels and amplifies ABA receptor-related gene expression, with H2S and ABA collaboratively enhancing tobacco’s resilience to drought stress. The administration of NaHS effectively counteracted the negative impacts of drought on tobacco seedlings. Consequently, these results underscore the potential of exogenous NaHS as a viable strategy for augmenting tobacco drought resistance in water-scarce field environments.
Physiological mechanisms depicted in schematic form. Illustration outlining the physiological mechanisms by which exogenous NaHS enhances drought tolerance in tobacco seedings. The red line denotes NaHS + PEG-6000, while the blue line represents PEG-6000.
All data generated or analyzed during this study are included in this published article.
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This work is funded by the National Natural Science Foundation of China (32102230) and the Talents Project of Henan Agriculture University (30500846).
College of Tobacco Science, National Tobacco Cultivation and Physiology and Biochemistry Research Center, Key Laboratory for Tobacco Cultivation of Tobacco Industry, Henan Agricultural University, Zhengzhou, 450046, Henan, China
Hang Wang, Wuxing Huang, Dan Han, Bingjun Dang, Zicheng Xu & Wei Jia
International Center for Biosaline Agriculture, ICBA, P.O. Box 14660, Dubai, United Arab Emirates
Soil and Water Research Department, Nuclear Research Center, Egyptian Atomic Energy Authority, Cairo, 13759, Egypt
Zhumadian Branch of Henan Provincial Tobacco Company, Zhumadian, 463000, Henan, China
China Tobacco Jiangsu Industry Co., LTD, Nanjing, 210009, Jiangsu, China
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Hang Wang: Data curation, Investigation, Writing original draft. Mohamed G. Moussa: Writing review and editing, Investigation. Wuxing Huang: Validation, Data curation. Dan Han: Conceptualization, Formal analysis. Bingjun Dang: Investigation, Validation. Haohao Hao: Resources, Data curation. Li Zhang: Investigation, Supervision. Zicheng Xu: Conceptualization, Formal analysis, Project administration, Resources. Wei Jia: Conceptualization, Investigation, Supervision, Writing review and editing, Funding Acquisition.
Correspondence to Zicheng Xu or Wei Jia.
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Wang, H., Moussa, M.G., Huang, W. et al. Exogenous hydrogen sulfide increased Nicotiana tabacum L. resistance against drought by the improved photosynthesis and antioxidant system. Sci Rep 14, 25534 (2024). https://doi.org/10.1038/s41598-024-76284-2
DOI: https://doi.org/10.1038/s41598-024-76284-2
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