Li 6400 40

The relative water content was measured on the 3rd leaf using the protocol in [83 ]. Free proline content was determined according to [84 ] with slight modifications. Homogenized leaves (100 mg) were incubated in 3 ml of 3% sulphosalicylic acid at 96 °C for 10 minutes. Samples were clarified by centrifugation and 1 ml of supernatant was mixed with 2 ml of 50% acetic acid, 2 ml of 2.5% acidic ninhydrin solution and boiled for another 30 min. The reaction product was liquid-liquid extracted by 5 ml of toluene and the absorbance of the toluene fraction was measured at 520 nm. The concentration of proline was determined using a standard curve (0-30 μg) and expressed as μg·mg^− 1 of protein. Estimation of protein concentration was according to [85 (link)] by spectrophotometric measurement of absorbance of PBS (100 mM, pH 7.8) buffered leaf extracts at 260 and 280 nm. The level of lipid peroxidation was determined by measuring the MDA concentration as described in [86 (link)]. Gas exchange measurements were made on the 2nd leaf using the LI-COR 6400-XT infrared gas analyzer with attached leaf chamber LI6400-40 (Li-COR, Biosciences). The measurements were performed with a CO₂ reference concentration of 400 μmol·mol^− 1, an air flow of 200 μmol·s^− 1, block temperature of 20 °C, photosynthetic photon flux density of 1800 μmols·m^− 2·s^− 1 and relative humidity between 55 and 65%.

Before Fv/Fm measurement, whole rice plant was dark-adapted for 20 min, and chlorophyll Fv/Fm was measured using a steady-state gas-exchange system with an integrated fluorescence chamber head (LI-6400-40, LI-COR, Lincoln, NE, USA). Fv/Fm value was calculated as Fv/Fm = (Fm−F₀)/Fm [55 (link)]. Pn was determined using the LI-6400 portable photosynthesis system (LI-COR, Lincoln, NE, USA) under a 2000 μmol·m⁻²·s⁻¹ of photosynthetic photon flux density (PPFD), 370 μmol·mol⁻¹ of CO₂ concentration and a 1.3–1.6 kPa leaf-to-air vapor-pressure difference. During measurement, leaf temperatures were maintained at 30 °C. All of the measurements were conducted at 9:00–11:00 a.m. The soluble sugar was determined using the anthrone-H₂SO₄ colorimetry method [56 (link)], and soluble protein in fresh leaves was determined by the method of Bradford [57 (link)]. The activity of V-H⁺-ATPase was evaluated by determining the release of phosphate (Pi) and expressed in μmol Pi·mg⁻¹ protein·h⁻¹. Membrane proteins were extracted from fresh samples, and 10 μg of microsomal membranes was incubated for 40 min at 28 °C. Afterward, 40 mM of citric acid was added to impede the reaction. Meanwhile, 10 μg of bovine serum albumin was used as a reference. The V-H⁺-ATPase activity was calculated, as described by Zhang et al. [22 (link)].

For leaf gas exchange and chlorophyll fluorescence analysis, plants were placed in a growth chamber at 25°C and held for 1 h at a photon flux density of 600 μmol⋅m^-2⋅s^-1 before measurement. Leaf gas exchange and chlorophyll fluorescence parameters were measured simultaneously using a portable photosynthesis system (LI-6400XT, LI-COR, USA) equipped with a leaf chamber fluorimeter (LI6400-40, LI-COR, USA) under both photorespiratory (21% O₂) and non-photorespiratory (2% O₂) conditions (Zhou et al., 2004 (link)). The air temperature, CO₂ concentration and photosynthetic photon flux intensity (PPFD) were set at 25°C, 350 μmol⋅mol^-1, 600 μmol⋅m^-2⋅s^-1. The A-Ci curves under PPFD 300 and 600 μmol⋅m^-2⋅s^-1 were measured between 0 and 1200 μmol⋅mol^-1 CO₂ at 21% O₂ and 25°C (Brooks and Farquhar, 1985 (link)).