*3.7. Antioxidants Enzyme Activity*

The activity of antioxidants, SOD, APX, and GR was studied to investigate the regulatory role of ethylene, NO, or H2S in the alleviation of HS-induced oxidative stress (Tables 4 and 5). The increases in the activity of SOD (48.4% and 45.9%), APX (43.7% and 41.9%), and GR (42.1% and 31.2%) in Taipei-309 and Rasi, respectively, were noted under HS compared to control plants. Applying Eth to heat-treated plants showed a higher

increase in SOD, APX, and GR activity than SNP compared to control or heat-stressed plants in both cultivars. Similarly, NaHS stimulated SOD, APX, and GR activity in both cultivars compared to control or heat-stressed plants.

The combined application of (NBD and NaHS) and (cPTIO and NaHS) in heat-treated plants did not entirely counteract the beneficial effect of NaHS on antioxidative enzyme activity. In addition, HT application in heat-stressed plants completely reversed the positive effects of Eth and SNP on the antioxidant defense system. Thus, HT supplementation resulted in reduced antioxidative enzyme activity in Eth and SNP-treated heat-stressed plants.

#### *3.8. Hydrogen Sulfide and NO Content and Ethylene Production*

The endogenous content of H2S and NO in rice cultivar leaves was examined to evaluate the effects of HS on H2S and NO regulation. Figure 3a,b depicts increased NO and H2S contents in the leaves of rice cultivar plants subjected to HS. The treatment of HS increased NO levels by 49.2% and 46.7% and H2S levels by 36.3% and 34.0% in Taipei-309 and Rasi, respectively, compared to control plants. Exogenously-applied Eth, SNP, or NaHS enhanced the level of both NO and H2S in rice cultivar leaves compared to control and heat-stressed plants. The treatment of HT and Eth or SNP under HS reduced endogenous NO and H2S levels relative to heat-treated plants. Similarly, applying cPTIO and NaHS under HS reduced NO levels compared to stressed and controlled plants.

**Figure 3.** Content of (**a**) Nitric oxide (NO), (**b**) hydrogen sulfide (H2S), and (**c**) ethylene evolution in 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), 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.

The individual application of NBD and cPTIO and NaHS under HS did not affect H2S levels compared to control and heat-stressed plants.

Ethylene production in leaves of rice cultivars exposed to HS is depicted in Figure 3c. Compared to control plants, HS elevated ethylene levels by 200.5% and 222.1% in Taipei-309 and Rasi, respectively. Although SNP, NaHS, or Eth individually increased ethylene emission, the increase was less than that of plants that were subjected to high temperatures. Compared to heat-treated plants, plants that received Eth, SNP, or NaHS under stressful conditions showed a reduction in ethylene emission. The inhibition of H2S and NO using inhibitors HT and cPTIO, respectively, increased ethylene levels relative to control plants. Ethylene action inhibitor NBD application along with NaHS under HS decreased ethylene level compared to heat-stressed plants.
