Li 6400xt

Five fully expanded new leaves were selected from each pot (the third leaf from the top of the plant) to determine net photosynthetic rate, stomatal conductance, intercellular CO₂ concentration, and transpiration rate, using a LI6400XT portable photosynthesis apparatus (LI-6400XT, Li-Cor, Inc., Lincoln, NE, USA). Because the photosynthesis rates are highest at midday, which can cause the leaf stomata to close and affect the test results, and in the afternoon, when the light intensity decreases, the measurements were performed between 9:00 and 11:00 am (full sunlight).

The photosynthetic rate of maize leaves (the first leaf above the ear of the maize plant in each experimental treatment) was measured by a Li-6400XT portable photosynthesis system (Li-6400XT, Li-Cor, Inc., Lincoln, NE, United States) at the silking stage and the grain-filling stage (75 and 100 days after planting, DAP), respectively. The measurements were performed during the morning of a sunny day (9:00–11:00). The leaf chamber light intensity of the head light source was set to 1600 μmol m^–2 s^–1 photosynthetic photon flux density (PPFD).

Light response curves of Photosynthesis were measured using a LI-6400XT (Li-Cor, United States). The parameters were measured on uppermost and fully expanded leaves from 09:00 to 11:30 am on a clear day. The temperature and CO₂ concentration of leaf chamber were maintained at 25°C and 380 μmol mol^-1, respectively. PAR was increased from 0 to 1500 μmol photons m^-2 s^-1 (0, 20, 50, 80, 100, 200, 400, 600, 800, 1000, 1200, 1500 μmol m^-2 s^-1, 36 min). And then, after linear fitting, light compensation point (LCP), LSP and light-saturated net photosynthetic rate (A_max), apparent quantum efficiency (AQE) and dark respiration rate (R_d) were estimated by the method of Ye (2007) (link).
Diurnal variation of leaf gas exchange was measured on a clear sunny day. Photosynthesis was measured with a LI-6400XT (Li-Cor, United States) equipped with 2 cm × 3 cm clear chamber. P_n and C_i were recorded at intervals of 2 h from 08:30 am to 16:30 pm. The measured leaves were kept at their natural angle of posture exposing to direct irradiance outside leaf chamber. The temperature and CO₂ concentration of leaf chamber were kept at natural environment.

The chlorophyll index of the upper surface of one fully developed apical leaf was measured, using a Dualex optical sensor (Force-A, Paris, France), which determines a chlorophyll index as follows:
Chlorophyll Index $=\frac{lighttransmittedinfar-red-lighttransmittedinred}{lighttransmittedinred}$
The net photosynthesis rate (A), the stomatal conductance (g_s) and intercellular CO₂ concentration (C_i) of each leaf chosen for the chlorophyll concentration measurement were also measured 5–8 h after the beginning of the lighting period, using a gas exchange analyser LI-6400 XT (LI-COR, Lincoln, NE, United States), equipped with a transparent leaf chamber. Inside the flow cell, the CO₂ concentration was 400 μmol mol⁻¹, the temperature was 20°C, the air flow was 500 μmol s⁻¹ and relative humidity was 65%.
The intrinsic water use efficiency (WUEi) and the instantaneous carboxylation efficiency (A/C_i) were calculated by dividing the net photosynthesis rate by the stomatal conductance and intercellular CO₂ concentration, respectively.
The measurements were carried out on eight plants per cultivar and light condition, taken from 20 plants in the centre of the shelf, at the ‘VFB stage of the order 2 axes carried by the order 1 long axis in apical position’. The experiment was carried out in duplicate.

To understand the functional stay-greenness of the mutants, four individual flag leaves were labeled at heading stage to measure the photosynthetic activity of SG mutants and WT. The net photosynthetic rate was measured using portable photosynthesis system (Li-6400XT, LI-COR Biosciences, Lincoln, NE, USA) at 10:30 am–12:30 pm, under 1200 μmol photon m⁻²·s⁻¹ light intensity, post-anthesis till physiological maturity in seven days interval. During the entire time course of the observations, the atmospheric CO₂ concentration, air temperature, and relative air humidity were 390–400 μmol mol⁻¹, 35–38 °C, and 46–52%, respectively.

Photosynthetic parameters were estimated in the seventh leaf of all the Arabidopsis lines using an LI-6400XT portable photosynthesis system (LICOR, Lincoln, NE, USA) ten days after treatment [27 (link)]. P_n, C_i, g_s, transpiration rate, Fv’/Fm’, ΦPSII, and ETR were estimated at 300 mmol⁻¹ of flow of air m⁻² $·$ s⁻¹, 1000 mol $\mathrm{photons}\mathrm{of}\mathrm{PAR}$ m⁻² $·$ s⁻¹, 23–24 °C, and 400 mol $·$ mol⁻¹ of CO₂.