Du800 ultraviolet spectrophotometer

During filling stage, the photosynthetic rates of the flag leaves were measured using a Li‐6400 portable photosynthesis system (Li‐Cor, Lincoln, NE, USA) under the following conditions: 400 μmol mol⁻¹ ambient CO₂; 1200 μmol m⁻²sec⁻¹ photosynthetic photon flux density and 500 μmol sec⁻¹ air flow rate. The chlorophyll content was determined by spectrophotometry (Ye et al., 2016). The middle parts of flag leaves were collected from wild‐type and mutant plants cut into segments of about 1 cm each. The leaf samples were immersed in 80% acetone for 24 h (26°C in dark) and measured by DU800 ultraviolet spectrophotometer (Beckman, Fullerton, CA, USA).

Chlorophyll was extracted from fresh fifth true leaves (0.1 g), using 80% (V/V) ethanol in acetone in darkness for 24 h. Thereafter, the absorbance of extract was measured at 470, 645, and 663 nm, using a DU800 Ultraviolet Spectrophotometer (Beckman Coulter, La Brea, CA, USA). The control was 80% (V/V) ethanol in acetone. The total chlorophyll, chlorophyll a, chlorophyll b, and carotenoid contents in the plants were calculated as described by Holm (1954 ). Three biological repeats were determined for each material, and three times the technical repeats were performed in each biological repeat.