**2. Materials and Methods**

#### *2.1. Plant Material, Growth Conditions, and Experimental Design*

Rice (*Oryza sativa* L.) cultivars, Taipei-309 (HS-tolerant) and Rasi (HS-non tolerant), obtained from the Indian Agricultural Research Institute, New Delhi were selected for the study. Their tolerance to HS was determined after their screening for changes in photosynthesis, growth, and yield parameters relative to controls, according to our earlier work [4]. After sterilizing the seeds with HgCl2 (0.01%) for 2 min and rinsing repetitively with double distilled water, they were soaked in distilled water for 12–24 h and then incubated at 30 ◦C. Following incubation, the seeds were sown in 23 cm diameter presterilized earthen pots containing 4 kg of acid-washed sand. Ten seeds of each cultivar were initially sown per pot, and later three seedlings were maintained after thinning. The pots were placed in an environmental growth chamber (Khera Instruments, New Delhi, India) with a day/night regime of 16/8 h, photosynthetically active photon flux density (PPFD) of 200 μmol m−<sup>2</sup> s−<sup>1</sup> at plant level, the temperature of 28 ◦C in the light and 22 ◦C in the dark, and relative humidity of 65 ± 5%.

In the experimentation, the plants were subjected to HS by exposing them to 40 ◦C temperature for six h daily, and the heat treatment was administered ten days after sowing (DAS). The heat treatment was maintained for 15 days for the same duration. After that, the plants were allowed to grow for five days at 28 ◦C (optimum temperature). The experimentation continued for 30 days. Control plants were kept at 28 ◦C for the duration of the experiment (30 days). A concentration of 200 μL L−<sup>1</sup> 2-chloroethyl phosphonic acid (Eth; as an ethylene donor), 100 μM sodium nitroprusside (SNP; as a NO donor), and 200 μM NaHS was applied to the foliage of both HS-treated and non-treated plants with a hand sprayer at 15 DAS. Moreover, 100 μM hypotaurine (HT; H2S scavenger), 100 μM norbornadiene (NBD; ethylene action inhibitor) and 100 μM 2-4-carboxyphenyl-4,4,5,5 -tetramethylimidazoline-1-oxyl-3-oxide (cPTIO; NO scavenger) were also applied to heatstressed plants. The concentration of 100 μM cPTIO [7], 100 μM NBD [40], and 100 μM HT [11] used was based on our earlier studies. A surfactant teepol (0.5%) was added with the control and other treatment solutions. Our experimental design consisted of twelve treatments as follows: (i) control, (ii) HS, (iii) Eth, (iv) SNP, (v) NaHS, (vi) HS + Eth, (vii) HS + SNP, (viii) HS + NaHS, (ix) HS + Eth + HT, (x) HS + SNP + HT, (xi) HS + NaHS + NBD, and (xii) HS + NaHS + cPTIO. The hydrolysis of ethephon releases ethylene and phosphate, and the yield of phosphate is equivalent to ethylene [41,42]. Thus, the phosphate effect was nullified by adjusting the phosphate available from 200 μL L−<sup>1</sup> Eth as single super phosphate. The arrangement of the treatments was a complete randomized block design with four replicates for each treatment (*n* = 4). The sampling of the plants was performed at 30 DAS to record various parameters of interest.

#### *2.2. Measurement of Photosynthetic and Growth Characteristics*

The Infrared Gas Analyzer (CID-340, Photosynthesis System, Bio-science, Washington, WI, USA) was used to measure gas exchange photosynthetic parameters (net photosynthetic rate, stomatal conductance, and intercellular CO2 concentration) in the fully expanded upper leaves. At the time of measurements (between 11.00 and 12.00 h), the atmospheric CO2 concentration was 380 ± <sup>5</sup> <sup>μ</sup>mol mol<sup>−</sup>1, the relative humidity was 70%, the photosynthetic active radiation was 780 μmol m−<sup>2</sup> s<sup>−</sup>1, and the air temperature was 28 ◦C. The chlorophyll content was measured in intact upper leaves of the plants with a SPAD chlorophyll meter (SPAD 502 DL PLUS, Spectrum Technologies, Plainfield, IL, USA) in the early morning hours. The dry weight of shoots and roots was recorded after separating the plants into roots and shoots, washed with water, and blotted with a soft paper towel to remove excess moisture. The separated shoots and roots were dried in a hot air oven (80 ◦C) for 72 h until a constant weight was achieved.

#### *2.3. Chlorophyll Fluorescence Measurement*

Fully expanded leaves were allowed to adapt under dark for 30 min before chlorophyll fluorescence measurements using Junior-PAM chlorophyll fluorometer (Heinz Walz GmbH, Eichenring, Effeltrich, Germany) were taken. The actual efficiency of PS II (ΦPSII), maximal efficiency of PS II (Fv/Fm), intrinsic efficiency of PS II (Φesc), photochemical quenching (qP), non-photochemical quenching (NPQ), and electron transport rate (ETR) were calculated. The details of the procedure are given in Supplementary File S1.
