Exogenous Hydrogen Sulfide Enhances Photosynthesis Under Thiocyanate Stress by Regulating Rubisco Energy Metabolism and Activation in Rice Seedlings

Abstract

Thiocyanate (SCN⁻), a persistent inorganic contaminant widely present in industrial wastewater, poses severe risks to plant growth and photosynthesis. Hydrogen sulfide (H₂S) is an emerging gaseous signaling molecule involved in the regulation of plant stress responses; however, its role in modulating Rubisco energy metabolism and activation under SCN⁻ stress remains unclear. Here, we investigated the effects of exogenous H₂S on magnesium homeostasis, ATP/NADPH metabolism, Rubisco activation, and photosynthetic performance in rice seedlings exposed to SCN⁻ stress via physiological, biochemical, and transcriptional approaches. We found that exogenous H₂S significantly increased Mg²⁺ accumulation, enhanced H⁺-ATPase and Mg²⁺-ATPase activities, and promoted Rubisco activase (RCA) abundance and activity. These changes were accompanied by reduced steady-state ATP and NADPH contents, indicating that increased energy consumption was driven by accelerated Calvin cycle turnover. At the transcriptional level, H₂S regulated key genes involved in ATP hydrolysis, Mg²⁺ transport, Rubisco activation, and chlorophyll biosynthesis. Consequently, the chlorophyll content, stomatal conductance, and transpiration rate improved under SCN⁻ stress. Collectively, our results demonstrate that exogenous H₂S enhances photosynthetic efficiency and Rubisco carboxylation capacity by coordinating Rubisco energy metabolism and activation.

1. Introduction

Thiocyanate (SCN⁻) is an inorganic contaminant containing carbon, nitrogen, and sulfur and is widely present in industrial wastewater generated from the mining, chemical manufacturing, electroplating, and food processing industries. In particular, in coking wastewater, SCN⁻ concentrations can reach 50–400 mg·L⁻¹ [1]. Owing to its high water solubility and resistance to aerobic degradation, SCN⁻ is highly environmentally persistent and can readily migrate through soil–water systems [2]. SCN⁻ is commonly detected in wastewater from diverse industrial sectors, including pharmaceuticals and steel production, and is strictly regulated worldwide owing to its high toxicity [3]. Notably, SCN⁻ is not currently listed as a mandatory control indicator in China’s wastewater discharge standards, allowing it to be discharged from wastewater treatment plants and subsequently introduced into agricultural soils via irrigation systems, thereby posing increasing environmental and agronomic risks [4]. Accumulated SCN⁻ can enter the food chain through contaminated crops, threatening both ecosystem integrity and human health. SCN⁻ readily binds to proteins, inducing protein denaturation and strong biological toxicity [5]. In humans, exposure to SCN⁻ through drinking water has been associated with gastrointestinal, neurological, and cardiovascular toxicity [6]. In plants, SCN⁻ accumulation disrupts nutrient homeostasis and transpiration, degrades photosynthetic pigments, interferes with photosynthesis, alters free amino acid composition, and suppresses antioxidant enzyme activities, ultimately leading to impaired growth and development [7,8,9,10,11].

Photosynthesis is the fundamental process underlying plant growth and yield formation, converting light energy into chemical energy while assimilating inorganic carbon into organic compounds and releasing oxygen to maintain the global CO₂/O₂ balance [12,13]. Increasing both photosynthetic efficiency and light use efficiency has therefore been recognized as a key strategy for improving crop productivity [14,15]. Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), the central enzyme of the Calvin cycle, catalyzes the primary step of CO₂ fixation and directly determines photosynthetic carbon assimilation capacity [14,16]. Rubisco is a bifunctional enzyme capable of catalyzing both the carboxylation of ribulose-1,5-bisphosphate (RuBP) with CO₂ and the oxygenation of RuBP during photorespiration, making it a major determinant of the net photosynthetic rate [17,18]. Consequently, improving Rubisco efficiency and maintaining its activation state are critical for enhancing photosynthetic performance.

Rubisco activation is tightly regulated by Rubisco activase (RCA), which removes inhibitory sugar phosphates from the Rubisco active site and maintains the enzyme in its catalytically competent form [19]. RCAs belong to the AAA⁺ (ATPases associated with various cellular activities) protein family and utilize the energy derived from ATP hydrolysis to release phosphorylated inhibitors from inactive Rubisco complexes, thereby facilitating the binding of CO₂ and Mg²⁺ and restoring Rubisco activity [20,21]. Therefore, RCA-mediated Rubisco activation, which is influenced by ATP and Mg²⁺ levels, governs the Rubisco content, whereas Rubisco activity itself is further regulated by the stromal pH and free Mg²⁺ concentration, which are controlled by H⁺-ATPase and Mg²⁺-ATPase, respectively [21,22,23,24,25]. Under adverse environmental conditions, however, both Rubisco content and activity are frequently reduced, resulting in suppressed photosynthesis. For example, nitrogen deficiency leads to decreased Rubisco abundance and activity, accompanied by decreases in the net photosynthetic rate (Pn) and Fv/Fm efficiency [26]. Moreover, SCN⁻ has been shown to interact with the ASN322 residue in the α-helical region of the PSII D1 protein, disrupting PSII structure and function and simultaneously inhibiting Rubisco activity, thereby reducing carbon assimilation capacity in rice [8].

Hydrogen sulfide (H₂S), an endogenously produced gaseous signaling molecule in plants, has emerged as an important regulator of diverse physiological processes, including seed germination [27], photosynthesis [28], stomatal movement [29], and tolerance to abiotic stresses [30]. Increasing evidence indicates that exogenous H₂S application alleviates growth inhibition and physiological damage in various plant species, such as rice, Arabidopsis, Chinese cabbage, pepper, and tomato, under metal, osmotic, drought, low-temperature, and salt stresses [31,32,33,34,35]. Our previous study demonstrated that exogenous H₂S enhances carbon assimilation in rice seedlings under SCN⁻ stress by regulating PSII D1 protein turnover and Rubisco activity [8]. Exogenous application of H₂S significantly increased the chlorophyll content, net photosynthetic rate, stomatal conductance, intercellular CO₂ concentration, transpiration rate, and maximum quantum yield of PSII photochemistry in tall fescue (Festuca arundinacea Schreb.). Under low light stress, high photochemical efficiency is maintained to regulate photosynthesis [36,37]. However, the mechanistic basis by which H₂S modulates Rubisco energy metabolism and activation, thereby improving Rubisco carboxylation efficiency and photosynthesis under SCN⁻ stress, remains largely unresolved. We hypothesized that H₂S pretreatment enhances Mg²⁺ accumulation in rice seedlings, thereby influencing ATPase activity and ATP availability to regulate Rubisco energy metabolism while simultaneously increasing RCA abundance and activity to promote Rubisco activation. To test this hypothesis, we investigated the effects of exogenous H₂S on (i) the Mg²⁺ content; (ii) the content and activity of ATPases, Rubisco, and RCA; (iii) the chlorophyll content and photosynthetic parameters; and (iv) the expression of genes associated with chlorophyll biosynthesis, ATPases, Rubisco, and RCA. This study aims to elucidate the regulatory mechanisms by which H₂S coordinates Rubisco energy metabolism and activation to increase photosynthetic efficiency under SCN⁻ stress, providing mechanistic insights and potential strategies for improving stress tolerance and photosynthetic performance in crops.

2. Results

In previous studies, under both SCN⁻ and NaHS + SCN⁻ treatments, the relative growth rates of rice plants significantly decreased (p < 0.05) with increasing stress concentrations compared with those of their respective control groups, accompanied by notable phenotypic changes [38].

2.1. Effects of SCN⁻ and H₂S on Mg Accumulation in Rice Seedlings

Compared with those in the respective control groups, the Mg content in the shoots of the rice seedlings in both the SCN⁻ treatment and the NaHS + SCN⁻ treatment presented a biphasic response, initially increasing and subsequently decreasing with increasing SCN⁻ concentration (p < 0.05; Figure 1A), indicating a stress-induced disturbance of Mg homeostasis.

Compared with the SCN⁻ treatment alone, NaHS pretreatment significantly increased the magnesium content in the shoots of rice seedlings at SCN⁻ concentrations of 24.0 and 96.0 mg SCN⁻·L⁻¹ (p < 0.05). The increases at 0.0 and 300.0 mg SCN⁻·L⁻¹ were not significant (p > 0.05). These results indicate that exogenous H₂S can increase the Mg content in the shoots of rice plants.

2.2. Effects of SCN⁻ and H₂S on Rubisco-Related Energy Metabolism

2.2.1. ATP and NADPH Contents

Under both SCN⁻ treatment and NaHS + SCN⁻ treatment, compared with the corresponding control conditions, SCN⁻ exposure induced a gradual increase in ATP content in shoots across the tested concentration range (24.0, 96.0 and 300.0 mg SCN⁻·L⁻¹) (Figure 1B), although no significant differences were detected among the individual concentrations (p > 0.05), indicating that SCN⁻ stress did not markedly disrupt the pools of cellular ATP. Compared with the SCN⁻ treatment, NaHS pretreatment slightly reduced the ATP content in the shoots, with a significant decrease observed only under nonstress conditions (0.0 mg SCN⁻·L⁻¹; p < 0.05). The decreases in ATP content at 24.0, 96.0 and 300.0 mg SCN⁻·L⁻¹ were not statistically significant (p > 0.05).

Similarly, the NADPH content in shoots increased with increasing SCN⁻ concentration (Figure 1C). Under SCN⁻ treatment, compared with that under Control 1, the shoot NADPH content significantly increased at 96.0 and 300.0 mg SCN⁻·L⁻¹ (p < 0.05). Compared with Control 2, NaHS pretreatment slightly attenuated SCN⁻-induced NADPH accumulation in the NaHS + SCN− treatment group, although a significant increase was still detected in the shoots of 300.0 mg SCN⁻·L⁻¹ (p < 0.05). Compared with the SCN⁻ treatment alone, the NaHS + SCN⁻ treatment significantly reduced the shoot NADPH content at 96.0 mg SCN⁻·L⁻¹ (p < 0.05). The decreases in the NADPH content at 24.0, 96.0 and 300.0 mg SCN⁻·L⁻¹ were not statistically significant (p > 0.05).

These results indicate that exogenous H₂S reduces the ATP and NADPH contents in the shoots of rice plants or, alternatively, that exogenous H₂S increases the consumption of ATP and NADPH.

2.2.2. ATPase Activity

SCN⁻ stress significantly stimulated H⁺-ATPase activity in shoots. Compared with their respective controls, both the SCN⁻ treatment and the NaHS + SCN⁻ treatment resulted in marked increases observed at 96.0 and 300.0 mg SCN⁻·L⁻¹. (p < 0.05; Figure 1D). Compared with the SCN⁻ treatment alone, NaHS pretreatment significantly increased H⁺-ATPase activity at 96.0 and 300.0 mg SCN⁻·L⁻¹ (p < 0.05), indicating that H₂S positively regulated the proton pumping capacity under SCN⁻ stress.

Mg²⁺-ATPase activity in shoots increased progressively with increasing SCN⁻ concentration (Figure 1E). Compared with those in the control treatment, SCN⁻ exposure significantly elevated Mg²⁺-ATPase activity in the shoots of the SCN⁻-treated plants at concentrations of 24.0, 96.0, and 300.0 mg SCN⁻·L⁻¹ (p < 0.05). Compared with that in Control 2, a significant increase in Mg²⁺-ATPase activity was observed in the NaHS + SCN⁻ treatment at 96.0 and 300.0 mg SCN⁻·L⁻¹ (p < 0.05). Compared with the SCN⁻ treatment alone, NaHS pretreatment further promoted Mg²⁺-ATPase activity, particularly in shoots at 0.0 and 96.0 mg SCN⁻·L⁻¹, with significant increases detected in shoots (p < 0.05), suggesting H₂S-mediated enhancement of Mg-dependent energy metabolism.

These results indicate that exogenous H₂S enhances proton pumping capacity and Mg²⁺-dependent ion transport in rice seedlings under SCN⁻ stress by increasing H⁺-ATPase and Mg²⁺-ATPase activities, thereby optimizing the energetic and ionic microenvironments required for Rubisco catalysis.

2.2.3. Rubisco and RCA Contents and Activity

SCN⁻ stress caused a concentration-dependent reduction in Rubisco content in shoots (Figure 2A). In both the SCN⁻ treatment and the NaHS + SCN⁻ treatment, compared with that in the corresponding control groups, the Rubisco content was significantly lower at all SCN⁻ levels (24.0, 96.0, and 300.0 mg SCN⁻·L⁻¹; p < 0.05). Under both SCN⁻ treatment and NaHS + SCN⁻ treatment, with increasing stress concentration, Rubisco activity also tended to decrease under SCN⁻ stress (Figure 2B), although the differences among the concentrations were not statistically significant. Compared with the SCN⁻ treatment alone, the NaHS pretreatment significantly alleviated the SCN⁻-induced reduction in the Rubisco content, with marked increases observed at 0.0, 24.0, and 96.0 mg SCN⁻·L⁻¹ (Figure 2A; p < 0.05). Compared with that in the SCN⁻ treatment alone, Rubisco activity in shoots was slightly increased by NaHS pretreatment across all SCN⁻ concentrations (Figure 2B), although the changes were not statistically significant. These results indicate a protective effect of H₂S on Rubisco function.

Under SCN⁻ treatment and NaHS+SCN⁻ treatment, compared with their respective control groups, SCN⁻ stress also stimulated the RCA content in shoots, with marked increases observed at 96.0 and 300.0 mg SCN⁻·L⁻¹ (p < 0.05; Figure 2C). Under SCN⁻ treatment and NaHS + SCN⁻ treatment, compared with their respective control groups, RCA activity decreased significantly with increasing SCN⁻ concentration (p < 0.05; Figure 2D), suggesting impaired Rubisco activation under severe SCN⁻ stress. Additionally, compared with the SCN⁻ treatment alone, NaHS pretreatment significantly increased both the RCA content and activity under low SCN⁻ stress (0.0 and 24.0 mg SCN⁻·L⁻¹; p < 0.05), partially restoring the Rubisco activation capacity under SCN⁻ exposure.

These results indicate that exogenous H₂S alleviates the inhibitory effects of SCN⁻ stress on Rubisco content and activity by increasing the contents and activities of both Rubisco and RCA, thereby maintaining Rubisco activation capacity and carbon fixation function in rice seedlings.

2.3. Effects of SCN⁻ and H₂S on Chlorophyll Content and Photosynthetic Performance

2.3.1. Chlorophyll Content

Under SCN⁻ treatment and NaHS + SCN⁻ treatment, compared with the corresponding control conditions, SCN⁻ stress led to a significant decline in the chlorophyll content in shoots, with marked reductions observed at 96.0 and 300.0 mg SCN⁻·L⁻¹ (p < 0.05; Figure 3A). Compared with the SCN⁻ treatment alone, NaHS pretreatment partially mitigated chlorophyll loss, resulting in a significantly greater chlorophyll content under nonstress conditions (0.0 mg SCN⁻·L⁻¹; p < 0.05) and slightly greater levels under SCN⁻ stress, although these increases were not statistically significant. Compared with that in the control treatment, the chlorophyll content in the SCN− treatment group at 24.0 mg SCN⁻·L⁻¹ also slightly increased, although this change did not reach statistical significance.

2.3.2. Photosynthesis Parameters

Under both SCN⁻ treatment and NaHS+SCN⁻ treatment, compared with the corresponding control conditions, SCN⁻ exposure caused a gradual decrease in stomatal conductance and transpiration rate in shoots (Figure 3B,C), with significant reductions in transpiration observed at 300.0 mg SCN⁻·L⁻¹ (p < 0.05). Compared with that in Control 2, stomatal conductance in the NaHS + SCN− treatment group slightly but non-significantly decreased, whereas the transpiration rate significantly decreased at 24.0 and 96.0 mg SCN⁻·L⁻¹ (p < 0.05). Compared with the SCN⁻ treatment alone, the NaHS pretreatment group presented significantly greater stomatal conductance at 300.0 mg SCN⁻·L⁻¹ (p < 0.05) and a significantly greater transpiration rate at 0.0 mg SCN⁻·L⁻¹ (p < 0.05). In contrast, the intercellular CO₂ concentration and net photosynthetic rate were relatively insensitive to SCN⁻ stress (p > 0.05; Figure 3D,E). Compared with those in the corresponding control groups, the changes in the intercellular CO₂ concentration and net photosynthetic rate across all concentrations were not significant (p > 0.05) under either the SCN⁻ treatment or the NaHS + SCN⁻ treatment. Compared with those in the SCN⁻ treatment alone, the increases in the intercellular CO₂ concentration and net photosynthetic rate induced by NaHS pretreatment were also not statistically significant (p > 0.05). Compared with SCN⁻ treatment alone, NaHS pretreatment increased the stomatal conductance and transpiration rate but also slightly increased the intercellular CO₂ concentration and net photosynthetic rate across all SCN⁻ levels, indicating an overall improvement in photosynthetic performance.

Exogenous H₂S improves the overall photosynthetic performance of rice seedlings under SCN⁻ stress by alleviating chlorophyll loss, increasing stomatal conductance and the transpiration rate, and moderately increasing the intercellular CO₂ concentration and net photosynthetic rate.

2.4. Transcriptional Responses of Genes Related to Photosynthesis and Energy Metabolism

2.4.1. Responses of Chlorophyll Biosynthesis-Related Genes

SCN⁻ stress markedly altered the expression patterns of chlorophyll biosynthesis-related genes in rice shoots (Figure 4A). The expression levels of OsDVR, OsCAO1 and OsCAO2 were significantly upregulated under SCN⁻ treatment (p < 0.05), indicating the activation of chlorophyll synthesis pathways in response to SCN⁻ exposure. Moreover, OsPORB was significantly induced at 24.0 and 96.0 mg SCN⁻·L⁻¹ (p < 0.05). In contrast, the expression of OsPORA exhibited a biphasic pattern, with significant upregulation at 24.0 mg SCN⁻·L⁻¹ followed by downregulation at 300.0 mg SCN⁻·L⁻¹ (p < 0.05), suggesting a stress intensity-dependent regulatory response.

Additional photosystem-related genes also presented pronounced transcriptional responses to SCN⁻ stress (Figure 4B). The expression of OsPsbO, OsPsbP, OsPsbR1, OsPsbR2, and OsPsbR3 was significantly upregulated at concentrations of 24.0, 96.0, and 300.0 mg SCN⁻·L⁻¹ (p < 0.05). OsPS1-F expression was significantly elevated at 24.0 and 300.0 mg SCN⁻·L⁻¹ (p < 0.05), whereas OsPsbS2 expression barely changed at all SCN⁻ concentrations (p > 0.05), indicating the selective sensitivity of photoprotective components to SCN⁻ stress.

NaHS pretreatment substantially reshaped the transcriptional responses of chlorophyll biosynthesis genes under SCN⁻ stress (Figure 4A). The expression of OsDVR, OsPORB, OsCAO1 and OsCAO2 was significantly upregulated following NaHS pretreatment, whereas that of OsPORA was significantly upregulated at 24.0 and 96.0 mg SCN⁻·L⁻¹ (p < 0.05).

Moreover, NaHS pretreatment also significantly altered the expression of genes related to the photosynthetic system (Figure 4B). NaHS significantly induced the expression of OsPsbO, OsPsbS2, OsPsbP, OsPsbR1 and OsPsbR2 (p < 0.05). Notably, OsPS1-F and OsPsbR3 were significantly upregulated only at concentrations of 24.0 and 96.0 mg SCN⁻·L⁻¹ (p < 0.05), suggesting that H₂S modulated photoprotective gene expression in a concentration-dependent manner.

2.4.2. Responses of Fd-NADPH-Related Genes

SCN⁻ stress significantly affected the expression of genes involved in ferredoxin (Fd)-NADP⁺ oxidoreductase-mediated redox metabolism in shoots (Figure 4C). The transcript level of OsLFNR1 was significantly upregulated under SCN⁻ treatment (p < 0.05), indicating increased photosynthetic electron transport demand. In contrast, OsPRRFNR14 exhibited a dual response, with significant upregulation at 24.0 mg SCN⁻·L⁻¹ followed by significant downregulation at 96.0 and 300.0 mg SCN⁻·L⁻¹ (p < 0.05). Similarly, OsLFNR2 expression was significantly induced at 24.0 mg SCN⁻·L⁻¹ but significantly suppressed at the highest SCN⁻ concentration (300.0 mg SCN⁻·L⁻¹; p < 0.05).

NaHS pretreatment enhanced the transcriptional activation of Fd-NADPH-related genes under SCN⁻ stress (Figure 4C). The expression of OsLFNR1 was significantly upregulated across all SCN⁻ concentrations, whereas that of OsPRRFNR14 and OsLFNR2 was significantly induced only at 24.0 mg SCN⁻·L⁻¹ (p < 0.05). Overall, compared with SCN⁻ treatment alone, NaHS pretreatment resulted in more sustained and coordinated induction of Fd-NADPH genes.

2.4.3. Responses of H⁺-ATPase-Related Genes

SCN⁻ exposure triggered substantial transcriptional reprogramming of H⁺-ATPase genes in shoots (Figure 4D). The expression of OsA6, OsA7, OsA9 and OsA10 was significantly upregulated (p < 0.05), whereas that of OsA1 and OsA3 was significantly downregulated (p < 0.05), indicating isoform-specific regulation of proton transport under SCN⁻ stress. Additionally, OsA2 expression was significantly suppressed only at the highest SCN⁻ concentration (300.0 mg·L⁻¹). OsA5 and OsA8 expression remained largely unchanged under all SCN⁻ concentrations.

NaHS pretreatment further modified H⁺-ATPase gene expression patterns in the shoots (Figure 4D). The expression of OsA5, OsA6, OsA7, OsA9 and OsA10 significantly increased, particularly under moderate to high SCN⁻ stress (p < 0.05). OsA8 was significantly upregulated at 24.0 mg SCN⁻·L⁻¹, whereas OsA2 was significantly induced at 24.0 and 96.0 mg SCN⁻·L⁻¹. However, the expression of OsA1 and OsA3 was significantly downregulated (p < 0.05). These results indicate that H₂S modulates proton transport capacity by selectively regulating H⁺-ATPase isoforms.

2.4.4. Responses of RCA Gene

Under SCN⁻ and NaHS + SCN⁻ treatments, gene expression of RCA exhibited a trend of initial increase followed by a decrease with increasing stress concentrations (Figure 4E). In both treatments, compared with the control group, RCA expression was significantly upregulated at 24.0, 96.0, and 300.0 mg SCN⁻·L⁻¹ (p < 0.05). Compared with SCN⁻ treatment alone, NaHS pretreatment significantly upregulated RCA expression at 24.0 and 300.0 mg SCN⁻·L⁻¹. These results indicate that exogenous H₂S can promote RCA gene expression.

2.4.5. Responses of Mg²⁺-ATPase-Related Genes

SCN⁻ stress significantly influenced the expression of Mg²⁺-ATPase-related genes in the shoots (Figure 4F). The transcript levels of three Mg²⁺-ATPase genes (LOC_Os05g02940, LOC_Os07g43040, and LOC_Os10g27220) significantly increased at all SCN⁻ concentrations (p < 0.05), whereas the transcript level of LOC_Os03g27040 significantly increased at 96.0 and 300.0 mg SCN⁻·L⁻¹. In contrast, LOC_Os08g29150 presented biphasic expression pattern (24.0 mg SCN⁻·L⁻¹), with initial upregulation followed by significant downregulation at 300.0 mg SCN⁻·L⁻¹ (p < 0.05).

NaHS pretreatment markedly enhanced the transcriptional activation of Mg²⁺-ATPase genes under SCN⁻ stress (Figure 4F). The expression of LOC_Os08g29150 was significantly upregulated at 0.0, 24.0 and 96.0 mg SCN⁻·L⁻¹, whereas the expression of LOC_Os05g02940, LOC_Os03g27040, and LOC_Os07g43040 was significantly increased at 24.0–300.0 mg SCN⁻·L⁻¹ (p < 0.05). In contrast, LOC_Os10g27220 expression remained relatively stable under the NaHS + SCN⁻ treatment, showing significant upregulation only at 24.0 mg SCN⁻·L⁻¹ (p < 0.05). These transcriptional patterns suggest that H₂S preferentially enhances Mg²⁺ transport and Mg-dependent energy metabolism at the molecular level.

These gene expression results indicate that exogenous H₂S enhances photosynthetic electron transport, proton pumping capacity, RCA activity, and Mg²⁺-dependent ion transport in rice seedlings under SCN⁻ stress by significantly increasing the expression of chlorophyll biosynthesis genes (OsPORA, OsCAO1), photosystem stability genes (OsPS1-F, OsPsbP), and H⁺-ATPase genes (OsA5, OsA10), as well as subsequently increasing Fd-NADPH-related genes (OsLFNR1, OsPRRFNR14), the RCA gene (RCA), and Mg²⁺-ATPase genes (LOC_Os08g29150, LOC_Os05g02940, LOC_Os03g27040). These transcriptional regulations collectively optimize the microenvironments for chlorophyll synthesis, photosystem stability, Rubisco activation, and energy metabolism, thereby synergistically improving Rubisco catalytic efficiency and overall photosynthetic performance.

3. Discussion

Thiocyanate, a typical nitrogen-containing pollutant widely present in industrial wastewater, has been well documented to exert severe inhibitory effects on the growth and yield of rice and other staple crops [8]. Previous studies have demonstrated that SCN⁻ stress disrupts the structural and functional integrity of the photosynthetic apparatus and suppresses Rubisco activity, thereby markedly reducing photosynthetic carbon assimilation [8]. Our previous work revealed that exogenous H₂S can alleviate SCN⁻-induced damage to the photosynthetic system and increase Rubisco activity and carbon assimilation efficiency [8,30]. However, how H₂S coordinates Rubisco energy metabolism and enzyme activation under SCN⁻ stress to systematically improve photosynthetic performance has remained largely unclear. In this study, we provide an integrated mechanistic interpretation of how exogenous H₂S regulates Rubisco energy metabolism and activation processes, thereby increasing Rubisco carboxylation efficiency and photosynthesis in rice under SCN⁻ stress.

In the Calvin cycle, Rubisco catalyzes the carboxylation of RuBP to produce 3-phosphoglycerate (3-PGA), which is subsequently reduced to glyceraldehyde-3-phosphate (G3P) (Figure 5). This process is highly dependent on ATP and NADPH generated by light reactions [17,39]. An insufficient ATP or NADPH supply or metabolic constraints leading to the accumulation of PGA can lead to feedback inhibition of Rubisco activity. Rubisco activity is not merely governed by substrate availability; it is tightly coupled to the stromal energetic state and ionic microenvironment through complex regulatory mechanisms [40].

Our results revealed that exogenous H₂S significantly decreased ATP levels in rice shoots under SCN⁻ stress and markedly reduced the NADPH content at high SCN⁻ concentrations (Figure 1B,C). Rather than indicating an impairment of energy supply, these reductions more likely reflect enhanced consumption of ATP and NADPH driven by increased Rubisco carboxylation (Figure 5). This interpretation is supported by concurrent changes in Mg content and ATPase activity. Specifically, exogenous H₂S significantly increased the shoot Mg content under relatively high SCN⁻ concentrations and markedly increased the activities of Mg²⁺-ATPase and H⁺-ATPase (Figure 1A,D,E).

Light-driven electron transport induces proton flux from the stroma into the thylakoid lumen, leading to stromal alkalization (pH increases from ~7.0 to ~8.0) and a concomitant influx of Mg²⁺ from the thylakoid lumen into the stroma (from ~1–3 mM to ~3–6 mM) [40]. These ionic changes are essential for Rubisco activation, as carbamylation of the catalytic Lys201 residue requires alkaline pH, and the stability of the active enzyme-CO₂-Mg²⁺ (ECM) complex depends on high stromal Mg²⁺ concentrations [40,41]. Importantly, these light-driven changes are mediated by H⁺-ATPase and Mg²⁺-ATPase.

H⁺-ATPase and Mg²⁺-ATPase utilize the energy released from ATP hydrolysis to regulate the stromal pH and Mg²⁺ concentration, respectively. H⁺-ATPase promotes proton extrusion from the stroma, thereby increasing the stromal pH, whereas Mg²⁺-ATPase facilitates the transport of Mg²⁺ from the cytosol or thylakoid lumen into the stroma, increasing stromal Mg²⁺ levels [25] (Figure 5). Previous studies have demonstrated that an elevated stromal pH and Mg²⁺ concentration favor the maintenance of Rubisco in its activated conformation and increase its carboxylation efficiency [40]. Thus, exogenous H₂S appears to optimize the ionic and energetic microenvironment required for Rubisco catalysis by activating ATP-driven ion transport systems, thereby indirectly promoting Rubisco carboxylation. However, we did not directly measure the stromal pH or Mg²⁺ content; our inference is supported by the observed correlation with ATPase activity and Mg content.

Before catalysis, Rubisco must undergo an activation process. The lysine residue near the active site of the Rubisco large subunit (Lys201) is first carbamylated by CO₂ and subsequently binds Mg²⁺ to form a catalytically competent ternary complex (enzyme-CO₂-Mg²⁺, ECM) [24,42] (Figure 5). However, carbamylated Rubisco readily forms stable inactive complexes with RuBP and various sugar-phosphate inhibitors, such as 2-carboxyarabinitol-1-phosphate (CA1P) and 2-carboxytetritol-1,4-bisphosphate (CTBP), thereby reverting to a nonactivated state [43]. Rubisco activase (RCA) utilizes the energy derived from ATP hydrolysis to exert mechanical force on these complexes, promoting inhibitor dissociation and restoring Rubisco catalytic activity [24] (Figure 5). The activity of RCA and its effects on Rubisco activation and photosynthesis are regulated by changes in the chloroplast environment, including changes in redox status and adenosine diphosphate (ADP)/adenosine triphosphate (ATP) ratios, which are induced by varying light levels reaching the leaf [44].

In the present study, exogenous H₂S significantly increased the Rubisco content, RCA content, and RCA activity under low to moderate SCN⁻ stress and moderately increased Rubisco activity (Figure 1D,E and Figure 2D). These results support the role of RCA in the H₂S-mediated response, indicating that H₂S not only improves the energetic environment for Rubisco but also enhances processes associated with RCA activation. Exogenous H₂S promoted increases in the RCA content and activity, which not only helped maintain Rubisco function under stress conditions but also increased RCA-mediated Rubisco activation, leading to increased ATP consumption. This further supports our aforementioned inference that the decrease in ATP content does not reflect an impaired energy supply but rather likely results from increased energy consumption driven by increased Rubisco carboxylation activity. Our results showed that exogenous H₂S treatment promoted RCA gene expression, with significant differences observed at concentrations of 24.0 and 300.0 mg SCN⁻·L⁻¹ (Figure 4E). Combined with previous reports that H₂S upregulates the expression of Rubisco genes and enhances carboxylation efficiency [8,45,46], our data are consistent with a model where H₂S may mitigate SCN⁻-induced Rubisco dysfunction by increasing RCA abundance and activation, thereby sustaining enzymatic activity.

Metabolite levels in the regeneration phase of the Calvin cycle are widely regarded as sensitive indicators of changes in carbon flux. On the basis of prior nontargeted metabolomic analyses, exogenous H₂S treatment significantly reduced the relative abundances of erythrose-4-phosphate, sedoheptulose-7-phosphate, and ribulose-5-phosphate (unpublished data; see Table S2 for methodological details, although the precision of this nontargeted approach was limited). These metabolites are key intermediates in the RuBP regeneration phase, and their decreased abundance suggests accelerated turnover and enhanced conversion into RuBP under H₂S treatment (Figure 5). When considered alongside the observed increases in Rubisco and RCA activities and changes in energy metabolism, these metabolomic data indicate that exogenous H₂S enhances RuBP supply capacity by accelerating carbon flux through the regeneration phase of the Calvin cycle. This metabolic evidence provides further support, at the whole-carbon-flow level, for the role of H₂S in promoting carbon assimilation efficiency under SCN⁻ stress.

Photosynthetic performance is ultimately reflected in changes in chlorophyll content and gas exchange parameters [12]. Previous studies have reported that SCN⁻ stress significantly suppresses transpiration and photosynthetic capacity in rice [47]. In this study, exogenous H₂S significantly increased the chlorophyll content under low SCN⁻ concentrations and markedly improved stomatal conductance under high SCN⁻ stress while increasing the transpiration rate under low-SCN⁻ conditions and moderately increasing the intercellular CO₂ concentration and net photosynthetic rate (Figure 3). These physiological responses indicate that H₂S contributes to the maintenance of stomatal function and CO₂ diffusion into leaves under SCN⁻ stress.

At the molecular level, qRT–PCR analysis revealed that exogenous H₂S significantly upregulated the expression of multiple genes involved in chlorophyll biosynthesis and photosystem stability, including OsPORB, OsCAO1, OsCAO2, OsPS1-F, OsPsbP, and OsPsbR1 (Figure 4A,B). These transcriptional changes are consistent with increased chlorophyll accumulation and improved photosynthetic apparatus stability. Together with previous reports showing that H₂S increases the net photosynthetic rate, electron transport efficiency, chlorophyll content, and Rubisco activity [45], our findings indicate that exogenous H₂S enhances photosynthesis under SCN⁻ stress by synergistically strengthening light reactions and carbon assimilation processes.

Overall, exogenous H₂S alleviates SCN⁻-induced inhibition of photosynthesis in rice through a multilevel regulatory mechanism. H₂S optimizes the energetic and ionic microenvironment for Rubisco by increasing Mg²⁺ and H⁺ transport and ATPase activity, reinforces Rubisco activation by increasing RCA abundance and activity, accelerates carbon flux through the RuBP regeneration phase of the Calvin cycle, and promotes chlorophyll biosynthesis and stomatal function. These coordinated processes collectively increase Rubisco carboxylation efficiency and overall photosynthetic performance under SCN⁻ stress (Figure 5).

4. Methods and Materials

4.1. Plant Material and Experimental Treatments

Rice seeds (Oryza sativa L. XZX 45) were soaked in deionized water for 12 h and then evenly sown on plastic cups filled with sandy soil. The seedlings were cultivated in an artificial climate chamber under controlled conditions (light intensity: 20,000 lux; temperature: 25 ± 0.5 °C; relative humidity: 60 ± 2%) and irrigated regularly with a modified 8692 nutrient mixture [48]. After 16 days of growth, rice seedlings with a uniform growth status (similar biomass and plant height) were selected according to our previous method for subsequent experiments [4]. The exposure concentrations of SCN⁻ were selected according to previous work [4], which indicated effective concentrations for the inhibition of plant growth by EC₂₀ (24.0 mg/L), EC₅₀ (96.0 mg/L), and EC₇₅ (300.0 mg/L) for 3 days of exposure. According to previous studies, a NaHS pretreatment concentration of 100 μM and a pretreatment duration of 6 h had the most significant effects on plant growth [4,49]. Each experimental setup for a particular concentration involved ten seedlings, and four replicates of each test were carried out for the present study. The experimental treatments were designed as follows:

(1)

SCN⁻ treatment: Rice seedlings were exposed to nutrient solutions containing 0.0 (Control 1), 24.0, 96.0, or 300.0 mg SCN⁻·L⁻¹ for 3 days [8].

(2)

NaHS + SCN⁻ treatment: Rice seedlings were pretreated with 100 μM NaHS solution for 6 h and then exposed to nutrient solutions containing 0.0 (Control 2), 24.0, 96.0, or 300.0 mg SCN⁻·L⁻¹ for 3 days. NaHS was used as a donor of H₂S [8].

All stress solutions were prepared using nutrient solution as the solvent. The exposure conditions were consistent with those described in our previous study [50].

4.2. Determination of Mg Content

After 3 days of exposure, the belowground parts of the rice seedlings were separated and dried in an oven at 90 °C for 48 h. Prior to digestion, the dry samples were pretreated overnight with 10 mL of a nitric acid–perchloric acid mixture (HNO₃:HClO₄ = 4:1), and the digestion bottles were sealed with paraffin film to prevent excessive acid volatilization. The samples were then digested on an electric heating plate at 200 °C until the solution became clear. After cooling, the digested residue was diluted with 2 mL of 1% HNO₃ and brought to a final volume of 50 mL. The magnesium (Mg) content in the shoots was determined via inductively coupled plasma–atomic emission spectroscopy (ICP–AES, Optima 7000 DV, PerkinElmer, Waltham, MA, USA) following the method described by Yu et al. (2013) [7]. The detection limit, determined as the mean of the blank plus 3 times the standard deviation of 10 blanks, was 0.07 μg/L Mg [48].

4.3. Determination of Rubisco, RCA, and ATPase Activities and Related Metabolite Contents

After 3 days of exposure, fresh rice seedling tissues (0.1–0.4 g) were homogenized in precooled grinding buffer (0.01 mol/L PBS, pH 7.2–7.4) at 4 °C. The homogenate was centrifuged at 2000–3000 r·min⁻¹ for 20 min at 4 °C, and the supernatant was collected for subsequent analyses. The activities of Rubisco, RCA, H⁺-ATPase, and Mg²⁺-ATPase, as well as the contents of Rubisco, RCA, ATP, and NADH, were determined via commercial assay kits purchased from BioOK (Fujian) Technology Co., Ltd. (Fuzhou, China), following the manufacturer’s instructions. Detailed information and operating instructions for the kit are provided in the Supplementary Materials.

4.4. Determination of Chlorophyll Content and Photosynthetic Parameters

After stress treatments, the shoots of the rice seedlings were harvested, washed, blotted dry, cut into small segments, and weighed. The samples were placed into 25 mL stoppered colorimetric tubes and extracted with 80% acetone to a final volume of 25 mL. The extracts were shaken thoroughly and incubated in darkness for 24 h. Absorbance was measured at 663 nm and 645 nm via a UV–visible spectrophotometer, with 80% acetone used as the blank control [51]. The chlorophyll content (mg·g⁻¹ FW) was calculated via Equations (1) and (2):

Chlorophyll a = 0.025 × (12.7 × A663 − 2.69 × A645)/m

(1)

Chlorophyll b = 0.025 × (22.9 × A645 − 4.68 × A663)/m

(2)

where m represents the fresh weight of the sample (g).

After 3 days of exposure, the photosynthetic parameters of the rice seedlings were measured via a Yaxin-1105 portable photosynthesis and fluorescence system (Beijing Yaxin Liyi Technology Co., Ltd., Beijing, China). The net photosynthetic rate (Pn), transpiration rate (Tr), stomatal conductance (C_leaf), and intercellular CO₂ concentration (CO₂_int) were recorded. The detailed methods used to measure the photosynthetic parameters and instrument parameter settings are provided in the Supplementary Materials.

4.5. Quantitative Real-Time PCR (qRT–PCR) Analysis of Related Genes

We screened the target genes for this study via the China Rice Data Center (https://www.ricedata.cn/gene/, accessed on 24 May 2024), the Rice Annotation Project Database (RAP-DB, http://rapdb.dna.affrc.go.jp/, accessed on 24 May 2024) constructed by the National Institute of Agrobiological Sciences (NIAS), and the Rice Genome Annotation Project (MSU-RGAP, http://rice.plantbiology.msu.edu/, accessed on 24 May 2024) from Michigan State University.

After 3 days of exposure, the rice seedling tissues (approximately 0.2 g) were rapidly frozen in liquid nitrogen and ground into a fine powder. Total RNA was extracted via the UItrapure RNA Kit (Kangwei Century Biotechnology Co., Ltd., Beijng, China) and reverse-transcribed into cDNA via the HiFiScript cDNA Synthesis Kit. Quantitative real-time PCR was performed with OsGAPDH as the internal reference gene. Relative gene expression levels of chlorophyll biosynthesis genes, photosynthesis system-related genes, and Fd-NADPH, H⁺-ATPase, RCA and Mg²⁺-ATPase genes were calculated via the 2^−ΔΔCt method. These gene-specific primers are listed in Table S1. Also, the detailed criteria and literature support for selecting these 29 candidate genes are provided in Supplementary Table S1. The detailed measurement methods are provided in the Supplementary Materials.

4.6. Date Analysis

All of the experimental procedures were independently conducted in quadruplicate (n = 4), and the results are expressed as the means ± standard deviations in both the figures and the tables. All experimental data were subsequently subjected to analysis of variance (ANOVA) and Tukey’s multiple range test, with the significance level α set at 0.01 or 0.05, enabling comparisons between the treatment groups and the control. The distinct letters accompanying values within individual graphs indicate statistical significance (p < 0.05).

5. Conclusions

In this study, we demonstrated that exogenous H₂S effectively alleviates SCN⁻-induced inhibition of photosynthesis in rice by coordinately regulating Rubisco energy metabolism and activation processes. SCN⁻ stress disrupted photosynthetic performance by suppressing Rubisco function and carbon assimilation, whereas H₂S application significantly improved Rubisco carboxylation efficiency and overall photosynthetic capacity. Mechanistically, H₂S optimized the energetic and ionic microenvironment required for Rubisco catalysis by increasing Mg²⁺ and H⁺ transport and increasing the activities of Mg²⁺-ATPase and H⁺-ATPase, thereby increasing the stromal pH and Mg²⁺ availability. In parallel, H₂S reinforced Rubisco activation through increasing the abundance and activity of RCA, which facilitated the maintenance of Rubisco in its catalytically competent state under SCN⁻ stress. Furthermore, nontargeted metabolomic evidence indicated that H₂S accelerated carbon flux through the RuBP regeneration phase of the Calvin cycle, increasing the substrate supply for Rubisco carboxylation. At the physiological and molecular levels, H₂S promoted chlorophyll biosynthesis, improved stomatal function, and supported photosynthetic gas exchange. Collectively, these findings reveal a multilevel regulatory mechanism by which H₂S enhances photosynthetic carbon assimilation in rice under SCN⁻ stress, integrating energy metabolism, enzyme activation, carbon flux regulation, and photosynthetic apparatus stability. This study provides new mechanistic insights into the role of H₂S as a signaling molecule in plant stress tolerance and highlights its potential application in mitigating nitrogen-containing pollutant stress in crop production systems. This study has certain limitations: potential functional perturbations of ATPase/RCA remain to be clarified, and the stromal pH and free Mg²⁺ content were not directly measured, which also warrants further investigation in future studies.