*3.4. Leaf RWC*

The HS decreased the leaf RWC of both the cultivars by (18.5%) in Taipei-309 and (20.1%) in Rasi compared to control plants (Figure 2a). The results revealed that Eth-, SNP-, or NaHS-spraying treatments improved the RWC in no stress and HS conditions. In heat-stressed plants, Eth recovered RWC by (17.5% and 44.2%), SNP by (15.7% and 42%) or NaHS by (13.1% and 38.8%) in Taipei-309; and by (15.7% and 44.8%), (12.4% and 40.7%) or by (11.1% and 39.1%) in Rasi compared to control and heat-stressed plants, respectively. Overall, maximum RWC was recorded in plants treated with Eth, followed by SNP or NaHS spraying treatments under control and heat-stressed conditions. Furthermore, in heat-exposed plants, the combined application of (NBD and NaHS) and (cPTIO and NaHS) reduced RWC, relative to control plants. The addition of HT along with Eth or SNP under HS more drastically declined RWC, reversing the beneficial effects of Eth or SNP treatments on RWC.

**Figure 2.** (**a**) Relative water content, (**b**) proline content, (**c**) glycine betaine content, (**d**) trehalose content, and (**e**) soluble sugars content of rice (*Oryza sativa* L.) cultivars Taipei-309 and Rasi under control and high temperature stress (HS) supplied with 200 μL L−<sup>1</sup> ethephon (Eth), 100 μM sodium nitroprusside (SNP), 200 μM sodium hydrosulfide (NaHS), or 100 μM hypotaurine (HT), 100 μM 2-4-carboxyphenyl-4,4,5,5 -tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) or 100 μM norbornadiene (NBD) scavengers of hydrogen sulfide (H2S), nitric oxide (NO), and ethylene action inhibitors, respectively. Data are presented as treatments mean ± SE (*n* = 4). The values followed by the same letters did not differ significantly by LSD test at *p* < 0.05.

#### *3.5. Accumulation of Osmolytes*

Plants accumulate osmolytes or compatible solutes to protect the cellular machinery from various environmental stresses. Glycine betaine, sugars (trehalose), and proline are the most well-known osmolytes. The treatment of HS increased the proline and GB content by 45.5% and 50% in Taipei-309 and 42.5% and 47.3% in Rasi, respectively, compared to control plants (Figure 2b,c). With Eth, SNP, or NaHS application under no stress, the levels of proline and GB increased appreciably. The application of Eth improved proline content (129.4% and 57.5%) in Taipei-309 and (122.2% and 55.8%) in Rasi, and GB content (156.5% and 71.0%) in Taipei-309 and (150% and 69.6%) in Rasi, respectively, under stressful condition compared to control and heat-stressed plants.

Improvements in proline and GB contents were also observed by SNP application under HS conditions (117.6% and 111.1%) in Taipei-309 and (141.3% and 134.2%) in Rasi, respectively, compared to control plants. Foliar-applied NaHS enhanced proline content (102.9% and 98.1%) and GB content (136.9% and 128.9%) in Taipei-309 and Rasi, respectively, compared to controls. Under HS stress, the treatment of HT completely suppressed the beneficial effects of Eth or SNP on proline and GB accumulation. At the same time, NBD or cPTIO could not considerably inhibit the effects induced by NaHS on proline and GB content in the leaves of heat-stressed plants. A considerable increase in trehalose and soluble sugar accumulation was recorded in heat-stressed plants of both rice cultivars, with Taipei-309 (67.7%) and (17.1%) with a higher accumulation than Rasi (61.4%) and (15.4%), respectively, compared to control plants (Figure 2d,e). Under no stress conditions, foliar treatment with Eth, SNP, or NaHS alone further increased the trehalose and soluble sugars content, although the increase was cultivar specific. The trehalose and soluble sugars content also increased considerably in both rice cultivars due to exogenously-applied Eth, SNP, or NaHS alone under HS.

Eth application increased trehalose and soluble sugars content (216.1% and 200.0%) and (45.7% and 43.6%) while SNP increased (203.2% and 194.7%) and (44.5% and 39.8%), and NaHS (196.7% and 184.2%) and (42.6% and 37.8%) in Taipei-309 and Rasi, respectively, under heat-stressed conditions, compared to control plants. However, even NBD and cPTIO did not completely inhibit NaHS from accumulating trehalose and soluble sugars. Furthermore, trehalose and soluble sugars accumulation by Eth and SNP was not sustained on the addition of HT to Eth and SNP-supplemented heat-stressed plants, which supported the role of H2S in ethylene and NO-induced osmolytes accumulation under HS.

#### *3.6. Oxidative Stress*

The oxidative stress was measured as H2O2 and TBARS. The plants subjected to HS showed a rise in the content of H2O2 by 3.0 fold in Taipei-309 and by 3.2 fold in Rasi, as well as TBARS content by 2.0 fold in Taipei-309 and by 2.1 fold in Rasi, compared to control plants (Tables 4 and 5). However, the spraying of Eth, SNP, or NaHS reduced the contents of H2O2 and TBARS in both heat-stressed and non-stressed conditions. The Eth application reduced heat-induced oxidative stress, as evidenced by the observed reductions in the levels of H2O2 (66.0% and 67.5%) and TBARS (50.6% and 50.9%) in Taipei-309 and Rasi, respectively, compared to heat-stressed plants. Under HS, the application of SNP or NaHS decreased the contents of H2O2 by 65.1% and 64.3% in Taipei-309 and by 66.3% and 66.0% in Rasi, as well as TBARS content by 49.3% and 47.9% in Taipei-309 and by 49.0% and 47.1% in Rasi, respectively, relative to the values of heat-treated plants. These findings suggest that the individual treatment of Eth, SNP, or NaHS mitigated heat-induced oxidative stress by lowering the accumulation of H2O2 and TBARS. However, the addition of HT further stimulated H2O2 and TBARS in both rice cultivars under HS. Furthermore, Eth and SNP did not rescue the negative effects of HT on H2O2 and TBARS accumulation. Therefore, the application of HT completely reversed the alleviating effects of both Eth and SNP. The application of NBD or cPTIO reversed the reduced oxidative stress induced by NaHS.

**Table 4.** Hydrogen peroxide (H2O2), thiobarbituric acid reactive substances (TBARS) content, and activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), and glutathione reductase (GR) in the leaves of rice (*Oryza sativa* L.) cultivar Taipei-309 after foliar treatment of plants with 200 μL L−<sup>1</sup> ethephon (Eth), 100 μM sodium nitroprusside (SNP) or 200 μM sodium hydrosulfide (NaHS) grown with or without high temperature stress (HS; 40 ◦C) or 100 μM hypotaurine (HT), 100 μM 2-4 carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) or 100 μM norbornadiene (NBD) scavengers of hydrogen sulfide (H2S), nitric oxide (NO), and ethylene action inhibitors, respectively, with HS at 15 days after sowing. Data are presented as treatments mean ± SE (*n* = 4). The values followed by the same letters did not differ significantly by LSD test at *p* < 0.05. FW, fresh weight.


**Table 5.** Hydrogen peroxide (H2O2), thiobarbituric acid reactive substances (TBARS) content, and activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), and glutathione reductase (GR) in the leaves of rice (*Oryza sativa* L.) cultivar Rasi after foliar treatment of plants with 200 μL L−<sup>1</sup> ethephon (Eth), 100 μM sodium nitroprusside (SNP) or 200 μM sodium hydrosulfide (NaHS) grown with or without high temperature stress (HS; 40 ◦C) or 100 μM hypotaurine (HT), 100 μM 2-4 carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), or 100 μM norbornadiene (NBD) scavengers of hydrogen sulfide (H2S), nitric oxide (NO), and ethylene action inhibitors, respectively, with HS at 15 days after sowing. Data are presented as treatments mean ± SE (*n* = 4). The values followed by the same letters did not differ significantly by LSD test at *p* < 0.05. FW, fresh weight.

