Plant physiological assay

Osmotic potential in the flag leaf of ‘Hom Nil’ rice was measured, according to Lanfermeijer et al. (1991). In brief, one hundred milligrams of fresh tissue were chopped into small pieces, transferred to 1.5 mL micro tube, and then crushed using a glass rod. The 20 μL of the extracted solution was dropped directly onto a filter paper in an osmometer chamber (5520 Vapro^®, Wescor, Utah, USA) and subsequently the data were collected. Then, the osmolarity (mmol kg⁻¹) was converted to osmotic potential (MPa) using conversion factor of osmotic potential measurement.

Chlorophyll a (Chl_a), chlorophyll b (Chl_b), total chlorophyll (TC) concentration in the flag leaf tissues were analyzed according to the method of Shabala et al. (1998), whereas total carotenoid (C_x+c) concentration was assayed following Lichtenthaler (1987) method. One hundred milligrams of leaf tissue was homogenized in glass vials using 10 mL of 99.5% acetone and blended using a homogenizer. The glass vials were sealed with Parafilm^® (Sigma-Aldrich, USA) to prevent evaporation, and then stored at 4°C for 48 h. Chl_a and Chl_b concentrations were measured at 662 nm and 644 nm, whereas C_x+c concentration was measured at 470 nm using UV–VIS spectrophotometer against acetone (99.5%) as a blank.

Chlorophyll fluorescence emission was measured from the adaxial surface of flag leaf using a fluorescence monitoring system (model FMS 2; Hansatech Instruments Ltd., Norfolk, UK) in the pulse amplitude modulation mode (Loggini et al. 1999). A leaf kept in dark for 30 min was initially exposed to the modulated measuring beam of far-red light (LED source) with typical peak at wavelength 735 nm. Initial fluorescence (F₀) and maximum (F_m) fluorescence yields were measured under weakly modulated red light (< 85 μmol m⁻² s⁻¹) with 1.6 s pulses of saturating light (> 1500 μmol m⁻² s⁻¹ PPFD) and calculated using FMS software for Windows^®. The variable fluorescence yield (F_v) was calculated using the equation: F_v = F_m–F₀. The ratio of variable to maximum fluorescence (F_v/F_m) was calculated as the maximum quantum yield of PSII photochemistry. The photon yield of PSII (Φ_PSII) in the light was calculated as: Φ_PSII = (F_m′ − F)/F_m′ after 45 s of illumination, when steady state was achieved (Maxwell and Johnson 2000).

Net photosynthetic rate (P_n; μmol m⁻² s⁻¹), transpiration rate (E; mmol H₂O m⁻² s⁻¹) and stomatal conductance (g_s; mmol m⁻² s⁻¹) were measured using a Portable Photosynthesis System with an Infra-red Gas Analyzer (Model LI 6400, LI-COR^® Inc., Lincoln, Nebraska, USA). All parameters were measured continuously by monitoring the content of the air entering and existing in the IRGA headspace chamber, according to Cha-um et al. (2007).