*3.11. Pearson Correlation*

The Pearson correlation heatmap of Taipei-309 and Rasi showed a strong linear correlation of NO, ethylene, and H2S with growth, leaf water status, osmolytes, antioxidants, and the photosynthesis of plants (Figure 7). The plant growth and photosynthesis variables showed a negative correlation with the high temperature-induced oxidative stress biomarkers like H2O2 and TBARS content. The content of NO, H2S, GB, Tre, SG, and Pro showed a significant (*p* ≤ 0.05, *p* ≤ 0.01, and *p* ≤ 0.001) positive correlation with plant growth (SDW and RDW), photosynthetic parameters (SPAD, PN, gs, Ci, Actual PSII, Maximum PSII, qP, and ETR), the expression of photosynthesis-related genes (PSB A and PSB B), and the level of antioxidant enzymes. On the other hand, endogenous Eth showed a strong correlation with oxidative stress, suggesting its potential role and generation during stress conditions. The photosynthesis-related genes showed a strong dependency upon the relative expression of *Mn-SOD*, *Cu-SOD*, and *Fe-SOD* and *APX*. Therefore, these connections portray a nearby association between NO, Eth, and H2S and plant response to thermo-tolerance in Taipei-309 and Rasi cultivars.

**Figure 6.** Biplots of principal component analysis (PCA) represents the relationship among different treatments and variables of two rice cultivars, Taipei-309 (**a**) and Rasi (**b**) grown under different conditions such as control, high temperature stress; HS (T1), Eth; Eth (T2), sodium nitroprusside; SNP (T3), sodium hydrosulfide; NaHS (T4), HS + Eth (T5), HS + SNP (T6), HS + NaHS (T7), HS + Eth + hypotaurine; HT (T8), HS + SNP + HT (T9), HS + NaHS + norbornadiene; NBD (T10), and HS + NaHS + 2-4-carboxyphenyl-4,4,5,5 -tetramethylimidazoline-1-oxyl-3-oxide; cPTIO (T11). The variables included H2O2 (hydrogen peroxide), TBARS (thiobarbituric acid reactive substances), nonphotochemical quenching (NPQ), ethylene evolution, SOD (superoxide dismutase), APX (ascorbate peroxidase), GR (glutathione reductase) activity, gene expression of (*Mn-SOD*, *Cu-SOD*, *Fe-SOD*, and *APX*), contents of H2S (hydrogen sulfide), NO (nitric oxide), proline, trehalose, SG (soluble sugar) and GB (glycine betaine), gene expression of *psbA* and *psbB*, *Pn* (net photosynthesis), *Gs* (stomatal conductance), *Ci* (intercellular CO2 concentration), SPAD value, Fv/Fm (maximum efficiency of PSII), ΦPSII (actual efficiency of PSII), Φesc (intrinsic efficiency of PSII), qP (photochemical quenching), NPQ (non-photochemical quenching), ETR (electron transport rate), RDW (root dry weight), SDW (shoot dry weight), and RWC% (relative water content).

**Figure 7.** Pearson correlation represents the relationship among different variables of two rice cultivars, Taipei-309 (**a**) and Rasi (**b**) grown under different conditions such as control, high temperature stress; HS (T1), Eth; Eth (T2), sodium nitroprusside; SNP (T3), sodium hydrosulfide; NaHS (T4), HS + Eth (T5), HS + SNP (T6), HS + NaHS (T7), HS + Eth + hypotaurine; HT (T8), HS + SNP + HT (T9), HS + NaHS + norbornadiene; NBD (T10) and HS + NaHS + 2-4-carboxyphenyl-4,4,5,5 tetramethylimidazoline-1-oxyl-3-oxide; cPTIO (T11). The variables included H2O2 (hydrogen peroxide), TBARS (thiobarbituric acid reactive substances), non-photochemical quenching (NPQ), ethylene evolution, SOD (superoxide dismutase), APX (ascorbate peroxidase), GR (glutathione reductase) activity, gene expression of (*Mn-SOD*, *Cu-SOD*, *Fe-SOD*, and *APX*), contents of H2S (hydrogen sulfide), NO (nitric oxide), proline, Tre (trehalose), SG (soluble sugar) and GB (glycine betaine), gene expression of *psbA* and *psbB*, *Pn* (net photosynthesis), *Gs* (stomatal conductance), *Ci* (intercellular CO2 concentration), SPAD value, maximum efficiency of PSII, actual efficiency of PSII, qP (photochemical quenching), NPQ (non-photochemical quenching), ETR (electron transport rate), RDW (root dry weight), SDW (shoot dry weight), and RWC (relative water content).

#### **4. Discussion**

High temperature stress, one of the most common types of abiotic stresses that plants face in nature, has an independent mode of action on the physiology and metabolism of plant cells. Previous studies have documented the effects of the application of several signaling molecules and growth regulators on various plant species; however, there aren't many reports on the comparative actions of ethylene, NO, and H2S on rice cultivars under HS. In the current study, we evaluated the efficacy of ethylene, NO, and H2S in modulating photosynthesis, growth, osmolytes, antioxidant metabolism, and the potential to ameliorate oxidative stress-induced impairments in rice cultivars subjected to HS. Among the various treatments used, 200 μL L−<sup>1</sup> ethylene treatments were the most effective, followed by 100 μM SNP and 200 μM NaHS. Meanwhile, we also explored the influence of H2S in ethylene or NO-mediated tolerance of HS in rice cultivars.

The findings of the present study revealed that HS negatively impacts growth parameters and photosynthesis, which could be linked to an elevated level of oxidative stress indicators in rice plants, as evident from PCA and Pearson correlation. High temperature stress reduced the SPAD value, stomatal conductance, intercellular CO2 concentration, and net photosynthetic rate. Heat stress has been observed to cause changes in plant growth, pigment concentrations, and photosynthesis in various plants [3,4,11,49]. Furthermore, previous research has found that severe heat stress in plants can result in cellular injury, cell death, and a reduction in the total dry weight of plants [57,58]. To examine whether or not Eth, SNP, or NaHS can mitigate the detrimental effect of HS on plant growth attributes and photosynthetic activity, they were sprayed onto the foliage of rice plants. The result showed that Eth, SNP, or NaHS relatively relieved reduced plant growth and photosynthesis. Overall, the maximum improvement in plant growth and photosynthesis was recorded from the plants treated with Eth followed by SNP and NaHS treatments. In comparison to SNP and NaHS, Eth might be an active growth regulator involved in the heat tolerance of rice cultivars. To understand more about whether H2S plays a role in ethylene or NO-induced heat tolerance of rice cultivars, HT, an H2S scavenger, was given to the plants treated with Eth or SNP under HS stress. The results of this study showed that when HT was applied along with Eth or SNP, these treatments were ineffective in enhancing plant growth and photosynthesis when exposed to HS stress. HT reversed the availability of H2S, and Eth or NO were unable to efficaciously sustain heat tolerance in rice cultivars. Thus, the findings imply that ethylene or NO causes H2S to be produced in heat-stressed plants and that H2S increases heat tolerance in rice cultivars. Eth, NO, and H2S are important gaseous signaling molecules that regulate each other's behavior, and H2S might work as a downstream signaling agent of NO and Eth on photosynthetic and growth under heat stress. In the present study, the supplementation of Eth, NO, and H2S effectively alleviated heat stress, which was reversed by the supplementation of HT, a H2S scavenger, suggesting that H2S works as a downstream signaling agent in NO and Eth-mediated heat stress tolerance. However, in the signaling cascade, H2S may act either upstream or downstream of NO, and there are complex relationships between NO, Eth, and H2S that are engaged in a variety of physiological processes and pathways.

Exogenously-applied H2S has been shown to promote growth and reduce lead (Pb) accumulation in *Zea mays* plants under Pb stress [59], improve photosynthesis, protect chloroplast structure, and promote growth in *Oryza sativa* under Ni stress [60], and improve the content of photosynthetic pigments and seedling biomass in *Cucurbita pepo* under nickel (Ni) stress [61]. H2S and NO are known to increase plant resistance to a variety of stresses, and they might serve as secondary signals to activate signal pathways downstream. [62,63]. The H2S has previously been shown to act as a downstream signal in the NO-induced enhanced adaptability of heat in maize plants [64]. The interaction of ethylene and H2S in heat stress tolerance has been shown [65].

Photosystem II is a pigment-protein complex with many components that are important for water splitting, oxygen evolution, and plastoquinone reduction. The photosystem PS II is more sensitive to environmental stress than PS I in chloroplasts [66,67]. Chlorophyll

fluorescence parameters have been proven to be an effective measure of stress intensity [68]. The result of the present study demonstrated that chlorophyll fluorescence parameters were reduced in heat-exposed rice plants, which contributed to the decrease in net photosynthesis. Under HS stress, there was a decrease in ΦPS II, Fv/Fm, Φesc, ETR, and qP, as well as an increase in NPQ. This indicates that heat stress-induced ROS production causes a decrease in PS II reaction center activity and renders the reaction center unable to use light energy efficiently. Havaux [69] observed an irreversible decline in the photochemical efficiency after 90 min of exposure of *Solanum tuberosum* plants to 39.5 ◦C, whereas Camejo et al. [70] showed that heat-sensitive tomato (*Lycopersicon esculentum* Mill. cv. Campbell-28) plants exhibited a decline in Rubisco activity and PS II performance after exposure of plants to 45 ◦C for 2 h. Heat stress not only enhances thylakoid membrane fluidity but also causes protein complexes and photosystems to reorganize and even dissociate [71,72]. Extreme heat stress causes structural changes in protein complexes, photosystem degradation, and a loss of oxygen-evolving activity, all of which impair the photosystem's ability to transfer electrons [73]. The application of Eth, SNP, or NaHS reversed photo-inhibition and the impairment of photosynthetic characteristics caused by HS. Furthermore, the application of HT along with Eth or SNP enhanced the detrimental effects of HS and reversed the mitigation effects of Eth or SNP on chlorophyll fluorescence attributes, implying that H2S plays an important role in regulating the impact of HS on photosynthetic attributes. As a result, H2S is implicated in the augmentation of PS II reaction center activity via ethylene or NO in rice cultivars subjected to heat stress, and it participated with ethylene or NO to improve light energy utilization efficiency. It has previously been demonstrated that ethylene application contributes to waterlogging stress reduction by strengthening photosynthetic pigment or improving electron transport [74]. According to Shi et al. [75], the Fv/Fm and ETR were higher in the presence of SNP, indicating that NO partially alleviated photodamage in UV-B-stressed bean leaves. In a previous study on the mung bean cultivars, it was reported that ethylene and H2S can protect photosynthesis against hexavalent chromium stress [37].

In this study, we assessed the RWC of rice cultivars under various treatments. Under HS stress, RWC declined significantly in both rice cultivars, with Rasi experiencing a greater decrease. Heat stress may reduce the water status of the leaves by reducing the hydraulic conductance, resulting in a decrease in water absorption, or by lowering stomatal conductance [76]. In this study, the heat-sensitive cultivar Rasi was shown to lose more water than Taipei-309, the heat-tolerant cultivar. Individual applications of Eth, SNP, or NaHS significantly altered the RWC of rice cultivars in which Eth showed better results than SNP and NaHS treatments. Furthermore, the applications of Eth, SNP, or NaHS enhanced the RWC of leaves under HS, thereby reducing heat-related plant damage. Intriguingly, the treatment of HT with Eth or SNP reversed this effect, suggesting that H2S was involved in ethylene or NO-induced changes in RWC in rice cultivars under HS stress. NO treatment, according to Khan et al. [77], benefitted mustard plants in retaining more water when subjected to salt stress. Similarly, Li et al. [78] observed that H2S maintained leaf RWC in cadmium (Cd)-stressed seedlings of *Brassica rapa*. Higher leaf RWC may have enhanced stomatal conductivity and, as a result, photosynthetic activity and biomass production [79]. Tomato plants treated with NaHS or Eth showed no decrease in RWC in response to low osmotic stress but did show a slight decrease in response to severe osmotic stress [38].

Many organisms counteract the environmental challenges by accumulating low molecular weight water-soluble substances known as osmolytes. Under heat stress, the accumulation of osmolytes aids in osmotic adjustment increases the concentration of cell protoplasm to maintain proper membrane function and quenches ROS in plants. [80,81]. The reason for higher proline levels is related to the synthesis and accumulation of free amino acids under stressed conditions [82]. Furthermore, GB accumulated in transgenic tobacco plants improved PS II thermo-tolerance from heat stress [83]. Osmolytes serve as stress markers and hence play an important role in stress reduction. *Zea mays* L. plants subjected to copper (Cu) and Pb exhibited higher levels of proline, which protected them from an oxidative burst and helped to maintain cell structures [84]. Heat tolerance, a crucial physiological trait

for heat resistance, necessitates the accumulation of sugars in plants and the availability of carbohydrates [85]. A non-reducing disaccharide known as trehalose accumulated in Arabidopsis plants exposed to heat stress and served as a ROS scavenger in heat-exposed wheat plants [86,87]. Trehalose is essential for maintaining growth under adverse conditions because it controls how efficiently most plants use water and stomatal movement [88]. According to Li et al. [89] trehalose serves as an osmoprotectant during water deficit, which aids in stabilizing dehydrated enzymes, proteins, and membrane lipids and guards against damage to biological structures.

The results of the present study showed that heat stress treatment increased the levels of osmolytes such as proline, GB, trehalose, and soluble sugars; however, these increased levels of osmolytes were unable to counteract heat stress and settled the stressed rice cultivars with decreased water status. However, the application of Eth, SNP, and NaHS to stressed rice cultivars augmented proline, GB, and trehalose levels, which reduced heat stress and enabled plant cells with increased osmotic pressure to take in more water as evidenced by enhanced RWC. On the other hand, the application of HT with Eth or SNP under heat stress lowered the amount of these osmolytes, confirming the role of H2S in the ethylene- or NO-mediated osmotic adjustment of plants. Previously, it was shown that NO-induced H2S generated an increase in proline and GB, which protected wheat plants from osmotic stress-induced oxidative stress [90]. The observed augmentation of proline, GB, total soluble sugars, and total soluble proteins in response to NO application potentially improved salt tolerance through osmotic regulation [77]. Under heat stress and after applying ABA and NO, trehalose accumulation increased even more [91]. In *P. eryngiivar. tuoliensis* under heat stress, trehalose accumulation increased with NO [92]. Heat-stressed rice cultivar leaves accumulated higher levels of H2O2 and TBARS contents. Increased levels of ROS may be attributed to the altered photosynthetic process in rice cultivars under heat stress. The enhanced ROS levels were accompanied by increased lipid peroxidation. However, Eth, SNP, or NaHS application resulted in a reduction in the levels of H2O2 and TBARS in both heat-stressed cultivars, more effectively in Taipei-309. Therefore, the application of Eth, SNP, or NaHS could be a useful strategy to prevent plants from oxidative damage brought on by HS. These results show that Eth treatment alleviated heat-induced oxidative damage more effectively than SNP and NaHS in rice cultivars by lowering H2O2 and TBARS contents. An excess in ROS could cause severe damage to lipids and proteins, which is a major cause of plant growth reduction [93]. However, using HT with Eth or SNP dramatically reversed this effect and resulted in considerable cell membrane damage exhibited as a significant increase in TBARS and H2O2 levels. The elimination of H2S via scavenging resulted in the production of ROS again. It reveals that ethylene or NO-induced H2S were involved in reducing stress intensity in plants by scavenging ROS and decreasing lipid peroxidation, hence minimizing oxidative damage. Moreover, investigations have shown that individual applications of ethylene or H2S have the capacity to reduce the levels of ROS in plants under abiotic stress [94,95]. The treatment of NO has an important role in enhancing endogenous H2S production, which helps plants resist abiotic stress-induced oxidative stress by reducing ion leakage, H2O2, O2 −, and TBARS levels [96–98]. NO has been found to reduce lipid peroxidation and ROS production in plants grown in Cd and Cu-enriched environments [23,99].

The result showed that HS resulted in oxidative stress as observed by the excessive production of ROS. Overproduction of ROS occurs in stressed cells when the cellular antioxidant defense mechanism is slower than the ROS synthesis that causes oxidative stress. In the present study, under HS, the activities of antioxidant enzymes, APX, GR, and SOD were enhanced; simultaneously, the H2O2 and TBARS content also increased in both the cultivars. As a result, increased levels of antioxidant enzymes in heat-stressed plants were insufficient to detoxify ROS, resulting in an excess of H2O2 and TBARS accumulation. Meanwhile, the application of Eth, SNP, or NaHS to heat-stressed plants increased antioxidant enzyme activity to the point where it was capable of detoxifying ROS by significantly lowering the levels of H2O2 and TBARS. Furthermore, in heat-challenged plants, the treatment of HT

with Eth or SNP caused an alteration in the antioxidant enzyme activities induced by Eth or SNP. According to the present study, the exogenous application of SNP or Eth induced H2S production and improved heat stress tolerance, which could be altered by treatment with an HT, suggesting that NO or ethylene-activated H2S might be required for heat stress response in rice plants. Under stress, the levels of ROS are tightly regulated by enzymatic and non-enzymatic antioxidants, determining the stressed plant's fate. The enzyme SOD is well-known for dismutating superoxide (O2 − radicals to hydrogen peroxide (H2O2), whereas APX and GR transform H2O2 to water and oxygen. An increase in H2O2 levels due to an inhibition in APX activity could damage lipids and proteins [98]. Exogenous NO may stimulate the synthesis of endogenous NO, that can function as a signaling molecule or ROS scavenger under intense stress circumstances by controlling and improving the activities of antioxidant enzymes [100,101]. Through explorations into the effects of exogenous H2S in wheat during flooding-induced hypoxic stress, it has been reported that NaHS application positively enhanced the activity of certain enzymes, including APX and GR [102]. In arsenate-only treated seedlings, the addition of NaHS raised NO levels, implying that both (H2S and NO) cause the upregulation of the ascorbate-glutathione (AsA and GSH) cycle to counterbalance ROS-mediated damage, resulting in enhanced pea seedling growth according to Singh et al. [98]. Additionally, NO plays a crucial role in encouraging endogenous H2S production, which increases the activity of antioxidant enzymes and aids wheat plants in tolerating oxidative stress brought on by osmotic stress [92]. Furthermore, NO and H2S promote protein post-translational modifications via S-nitrosylation and tyrosine nitration. The altered protein function and activity caused by such Pb modifications may have given plants greater tolerance to abiotic stress [103,104]. Consistent with the accumulation of antioxidant enzymes in rice cultivars subjected to heat stress, either alone or in combination with Eth, SNP, or NaHS, the expression of SOD isoforms (*Mn-SOD*, *Fe-SOD*, *Cu-SOD*) and *APX* genes were also upregulated in treated rice cultivars. This suggested that up-regulation of *SOD* isoforms and *APX* genes could improve the activities of the SOD and APX enzymes, hence protecting cells from oxidative damage caused by HS stress. The activity of SOD isoenzymes is increased by ethylene in Arabidopsis plants under Cd stress, which affects root morphology [105]. In EIN2-1 mutant plants, higher transcription levels of Cu/Zn SOD2 and CAT3 resulted in higher SOD and CAT enzyme activity when compared to control plants [106,107]. In contrast, other investigations have revealed that inducing ethylene under abiotic stress could be harmful to plants, decreasing the activity of antioxidative enzymes and increasing the accumulation of ROS [108,109]. The hormone, NO's antioxidant property may be due to its direct interaction with ROS, which is then neutralized by several cellular processes, or NO could boost the antioxidant potential of cells by enhancing antioxidant enzyme activities [110]. At the post-translational level, NO modulates APX through S-nitrosation of cysteine residues, which enhances its activity, and metal nitrosation and tyrosine nitrosation, which both decrease its activity [111,112]. In a prior study, NaHS root pretreatment boosted the gene expression of antioxidant enzymes (cAPX, CAT, Mn-SOD, and GR), heat shock proteins (HSP70, HSP80, and HSP90), and aquaporins (PIP) [113]. Furthermore, we found that HT treatment with Eth or SNP under HS stress reversed Eth- or SNP-induced upregulation of *SOD* isoforms and *APX* genes. In *Solanum lycopersicum*, ethylene and H2S fumigation sustained higher levels of *SlAPX1*, *SlAPX2*, and *SlCAT3* expression [39].

A previous study indicated that HS stress may enhance NO synthesis in tobacco [114] and higher plants [115]. Similarly, in this study, HS stress increased NO levels in rice cultivar leaves. Furthermore, plants under HS stress had higher levels of H2S in their leaves. Similar to these results, increased H2S generation was seen in wheat [36] and maize [116] exposed to heat stress, as well as bermudagrass exposed to cold, salt, and osmotic stresses [87]. In our experimental conditions, a rise in both NO and H2S levels under HS stress was detected, which is in good agreement with these reports. In the present study, donors and inhibitors of H2S and NO were applied to rice cultivars subjected to heat stress in order to better understand the interaction between H2S and NO. In this study, NaHS, SNP, and Eth treatment raised NO content in rice cultivar leaves in both heat-stressed and no-stress conditions; such an increase has previously been documented in barley and wheat plants [117,118]. Under HS stress, the decrease in NO content was greatest when HT was combined with Eth or SNP, which affected stress alleviation. Research indicates an interaction between NO and H2S and it was recently reviewed [119]. Similarly, SNP, Eth, or NaHS treatment improved the H2S content in leaves of rice cultivars in control and stressed plants, but significantly with NaHS treatment. Furthermore, the suppression of ethylene and NO in the presence of NaHS under HS stress using their inhibitors NBD and cPTIO, respectively, had no significant influence on H2S levels.

Ethylene is produced in response to a variety of environmental stresses, implying that it acts as a connection between environmental change and developmental adaptability [120]. Ethylene increases photosynthesis and dry matter accumulation in plants under optimal and stressful environments. However, ethylene homeostasis is important for plant response and stress tolerance since excess ethylene formation under stress condition negatively impacts plant physiological and metabolic functions and plant growth. The involvement of ethylene in heat stress tolerance has been investigated earlier [17,49]. Previous studies have shown that heat stress, particularly in the 30–38 ◦C range, causes an increase in ethylene production in plants such as *Phaseolus vulgaris* [121] and *Triticum aestivum* [122]. Salt tolerance depends on ethylene production, and ethylene signaling is crucial for plants to self-correct quickly in response to salinity stress and to adapt better to the stress condition [123]. Under heat stress conditions, plants release stress ethylene by the same process that produces ethylene during normal development. In the present study, the ethylene level in heat-stressed plants was higher than control plants because of the burst of ethylene that occurred under stress. The application of ethephon following heat treatment resulted in ethylene release that showed protective functions at this stage and induced mechanisms of the activation of the antioxidant system to scavenge ROS and relieve plants from the stress. As the plants were relieved from the stress, the burst of stress ethylene was minimized resulting in a lower level of ethylene compared to heat-stressed plants. It has been shown that when plants are exposed to conditions that threaten their ability to survive, the same mechanism that produces ethylene for normal development instead functions to produce what is known as stress ethylene [124]. The paradoxical effects of stress ethylene on plants were shown emphasizing the fact that in stressed plant tissues, there is an initial small peak of ethylene close in time to the onset of stress and the second much larger peak some time later. The first small peak shows the protective response of plant. The second peak is so large that processes that are inhibitory to plant survival are initiated [124]. Thus, the modulation of ethylene production could reduce the stress-related injuries. According to studies, the signaling molecule NO modulates endogenous ethylene levels at different levels by altering a variety of pathways, leading to post-climacteric biochemical changes related to fruit quality [125]. In the current study, the application of SNP or NaHS resulted in lower ethylene levels more notably when there was no stress, compared to HS plants. It was recently proposed that H2S counteracts the effect of ethylene action in banana fruit ripening and senescence [126]. As a result, H2S might be able to resist ethylene function. Furthermore, H2S, which is similar to NO, inhibits 1-amino-cyclopropane carboxylic acid oxidase (ACO) activity in tomato leaves [127]. In this study, we used HT to investigate the mechanism of H2S and its effect on ethylene levels. Under HS stress, the application of HT with Eth or SNP increased the level of ethylene.

Furthermore, HS conditions have been shown to affect the light-harvesting complex, water-oxidizing complex, and PS II reaction center [128]. Chloroplast gene expression and responses to environmental stress may be related [31]. The chloroplast genes *psbA* and *psbB* encode the D1 protein of PS II and the PS II chlorophyll-binding protein (CP47), respectively [129,130]. In the present study, the qRT-PCR analysis revealed that heat stress downregulated the expression of the *psbA* and *psbB* genes, which were linked to PS II inactivation. Reduced photosynthetic pigments and organic solutes, such as soluble sugars, sucrose, and proline, were associated with the deleterious consequences of HS stress [131]. Salt stress was reported to cause the degradation of D1 protein (encoded by the *psbA* gene) in *Avena sativa* plants, as well as the downregulation of *psbA*, *psbB*, *psbC*, and *psbD* [132]. Meanwhile, Eth, SNP, or NaHS treatments upregulated the *psbA* and *psbB* gene expression of heat-stressed rice cultivar leaves, which may be responsible for PS II stability under heat stress. Notably, the expression of the investigated genes was significantly higher in Ethtreated leaves compared to SNP and NaHS leaves. Photosystem II tolerance to high light would be improved by enhanced *psbA* transcription and translation [31]. On the contrary, the application of HT along with Eth or SNP under HS resulted in the downregulation of *psbA* and *psbB* gene expression. It was investigated that H2S was involved in the ethyleneor NO-mediated protection of photosynthetic machinery during HS stress, hence enhancing photosynthetic efficiency and mitigating the negative consequences of HS stress.

In the present study, the data were also examined using PCA in order to identify and classify the enormous data set in terms of growth, physio-biochemical and molecular characteristics into a manageable set of dynamically interrelated variables [133–135]. The PCA explained 94.70% and 94.67% of the data variability in Taipei-309 and Rasi, respectively, which accords with Sneath and Sokal [136], who considered that data must account for at least 70% of the total variability. The lines originating from the central point of the biplot represent correlations between various variables, with the closeness of the lines indicating the strength of the correlation with a specific treatment. Treatments such as (HS + Eth + HT), (HS + SNP + HT), and control were distributed in left quadrant, i.e., in the negative direction. However, treatments such as Eth, SNP, and NaHS with no stress and HS stress were present away from the origin in a positive direction. Growth and physiological attributes clustered opposite to oxidative stress attributes and NPQ. Biplot categorized the traits into three groups. The first group provided three treatments, Eth, SNP, and NaHS, under control conditions. These treatments were correlated to the variables including the dry weight of root and shoot, chlorophyll fluorescence, photosystem II genes, photosynthetic traits, and leaf RWC. This suggests that under control conditions, these treatments significantly increased these traits. Afterward, Eth, SNP, and NaHS under HS were grouped together. These treatments have a significant association with antioxidant enzyme activity (SOD, APX and GR), antioxidant genes (*Mn-SOD*, *Cu-SOD*, *Fe-SOD*, and *APX*), and osmolytes (proline, GB, soluble sugar, and trehalose), NO, and H2S content. In addition, HS treatment shows a significant association with oxidative stress markers (H2O2 and TBARS), NPQ, and ethylene levels. However, treatments with inhibitors and scavengers such as HT (HS + Eth + HT) and (HS + SNP + HT), NBD (HS + NaHS + NBD) and cPTIO (HS + Eth + cPTIO) seemed to have no significant association with any parameter. This analysis confirmed our results that HS stress reduced the majority of morpho-physiological and biochemical traits while increasing H2O2 and TBARS content and NPQ in the two cultivars tested. On the other hand, Eth, SNP, and NaHS increased most of the traits under control and heat conditions. In contrast, the application of HT did not demonstrate any significant association with any parameters A proposed model to show the significance of ethylene, NO and H2S in high temperature stress tolerance is presented in Figure 8.

To further confirm the role of ethylene and NO in H2S-mediated thermotolerance, we run our dataset for Pearson correlation. The correlation between all paired attributes of antioxidant enzymes, their relative expression and osmolytes were positively significant with growth and photosynthesis parameters and negatively correlated with oxidative stress markers.

**Figure 8.** Proposed model for the role of ethylene, NO and H2S in the alleviation of high temperature stress in rice (*Oryza sativa* L.) NBD; norbornadiene, cPTIO; 2-4-carboxyphenyl-4,4,5,5 tetramethylimidazoline-1-oxyl-3-oxide, HT; hypotaurine.

#### **5. Conclusions**

In summary, signaling molecules such as ethylene, H2S, and NO have been found as promising for enhancing rice plants' thermo-tolerance, as well as growth and photosynthesis, particularly more in Taipei than Rasi. However, in both rice cultivars, Eth was more effective than SNP and NaHS in alleviating HS. The activation of antioxidant enzymes, the detoxification of ROS, and the higher accumulation of osmolytes caused by the use of signaling molecules equipped the plants in combating the negative effects of heat stress. In addition, the scavenging of H2S by HT subsequently damaged the rice plants in the presence of Eth or SNP, confirming that the beneficial action of Eth and SNP is, at least to some extent, reliant on H2S.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/antiox11081478/s1, File S1: Material and methods details, Table S1: Primer pairs used for quantitative RT-PCR.

**Author Contributions:** Conceptualization: N.A.K.; Investigation and data curation: H.G.; M.F.; Biochemical analysis: H.G., Z.S., I.R.M., and M.F.; Original draft preparation: H.G., M.F.; Editing and content improvement: N.A.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study and the Supplementary Materials are available in the graphs provided in the manuscript.

**Acknowledgments:** The research work on the mechanisms of heat stress tolerance in the laboratory of N.A.K. is supported by the Council of Scientific and Industrial Research, New Delhi (38(1473)/19/EMR-II).

**Conflicts of Interest:** The authors declare no conflict of interest.
