*2.6. Measurement of Hydrogen Peroxide (H2O2) and Lipid Peroxidation*

The Okuda et al. [48] method was used for the H2O2 assay and is explained earlier [49]. The details of the method are given in Supplementary File S1. Fresh leaf tissues (500 mg) were macerated in ice-cold 200 mM perchloric acid before being spun at 1200× *g* for 10 min. Later, 4 M KOH was used to neutralize the supernatant. In order to remove the insoluble potassium perchlorate from the homogenate, it was further centrifuged at 500× *g* for three minutes. The reaction mixture (1.5 mL) included 20 μL of peroxidase (0.25 unit), 400 μL of 12.5 mM 3-(dimethylamino) benzoic acid in 0.375 M phosphate buffer (pH 6.5), 80 μL of 3-methyl-2-benzothiazoline hydrazone, and 1 mL of the eluate. Peroxidase was added and the reaction was started at 25 ◦C. On a spectrophotometer, the increase in absorbance was calculated at 590 nm. The content of thiobarbituric acid reactive substances (TBARS) was estimated to determine lipid peroxidation as described by Dhindsa et al. [50] and explained earlier [49].

### *2.7. Assay of Antioxidant Enzymes Activities*

The activity of superoxide dismutase (SOD), ascorbate peroxidase (APX), and glutathione reductase (GR) was measured using the methods of Beyer and Fridovich [51], Giannopolitis and Ries [52], Nakano and Asada [53], and Foyer and Halliwell [54], respectively. The details of the methods are given in Supplementary File S1.

#### *2.8. Determination of Nitric Oxide, Hydrogen Sulfide, and Ethylene*

The method of Zhou et al. [55] was used for determining NO content with a slight modification by estimating nitrite content. Using a pre-chilled mortar and pestle, 500 mg of healthy leaves were ground in 3.0 mL of 50 mM ice-cold acetic acid buffer (pH 3.6) containing 4% zinc acetate. The mixture was then centrifuged at 11,500× *g* for 15 min at 4 ◦C. The supernatant was collected, and the pellets were rinsed in extraction buffer (1.0 mL) before being centrifuged again. After adding 100 mg of charcoal, the supernatants from the two spins were neutralized. The filtrate was collected after a brief vortex. Greiss reagent (0.1% N-1-naphthyl ethylenediamine dihydrochloride and 1% sulphanilamide in 5% H2PO4 solution) was added to each 1.0 mL filtrate and mixed in a 1:1 ratio before incubation at room temperature for 30 min. The NO content was determined using a calibration curve with sodium nitrite as a standard, and the absorbance was measured at 540 nm. Methylene blue formation from dimethyl-p-phenylenediamine in HCl was used to estimate the content of leaf H2S as described by Xie et al. [56] with minor modifications. The fresh leaf samples (700 mg) were homogenized in 2.5 mL of Tris-HCl buffer (20 mM L−1, pH 6.8) containing 10 mM L−<sup>1</sup> ethylene diamine tetraacetic acid (EDTA). The homogenate was centrifuged for 15 min at 4 ◦C and 12,000× *g*. For H2S trapping, 0.2 mL of 1% (*w*/*v*) zinc acetate was added to the supernatant (0.75 mL). After 30 min of development, 0.1 mL of 30 mM L−<sup>1</sup> ferric chloride in 1.2 mol L−<sup>1</sup> of HCl and 0.1 mL of 20 mM L−<sup>1</sup> dimethyl-p-phenylenediamine dissolved in 7.2 mol L−<sup>1</sup> of HCl were added. At 670 nm, spectrophotometric analysis was used to determine the methylene blue formation. As a standard curve, different concentrations of NaHS were used, expressed as nmol g−<sup>1</sup> fresh weight (FW).

The ethylene evolution in leaves was measured using a gas chromatograph following the procedure as described earlier by Fatma et al. [12]. The details are given in Supplementary File S1.

#### *2.9. RNA Isolation and cDNA Synthesis*

Following the manufacturer's instructions, total RNA was extracted from rice leaves using the TRIzol reagent (Ambion, Life Technologies, Austin, TX, USA). With the help of a Nanodrop spectrophotometer (Thermo Scientific, Waltham, MA, USA), the extracted RNA was quantified. The details of the procedure are given in Gautam et al. [49] and presented in Supplementary File S1.

#### *2.10. Quantitative Real-Time PCR Analysis*

Real-time PCR (RT-PCR) was performed in a 96-well reaction plate (Roche, Mannheim, Germany) containing 20 μL reaction mixture of ×10 reaction buffer, 2 mM dNTPs, 1 mM MgCl2, 0.35 μM each of forward and reverse primers, 1 μL Sybr green (×10), 10 μg cDNA template, and 5 U Taq polymerase on a thermal cycler (Light cycler 480 II, Roche, Germany). The details of the procedure are given in Gautam et al. [49] and presented in Supplementary File S1. Primer pairs used for the quantitative RT-PCR are listed in Supplementary Table S1. The data was interpreted as the differential expression of the target gene in the treated sample versus the untreated control in relation to the internal control.
