Photosynthesis and Ion Transport Mechanisms in Rice

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)].