Imaging pam chlorophyll fluorescence imaging system

The chlorophyll fluorescence of leaves was measured in vivo using an IMAGING-PAM chlorophyll fluorescence imaging system (WALZ, Effeltrich, Germany) on three representative plants, avoiding the leaf veins as much as possible. The measurements were repeated three times, and the fluorescence parameters were analyzed.
The chlorophyll content of the leaves was determined by a 80% acetone extraction. About 0.1 g of the second pair of old leaves of the plant was taken and weighed in a 10-ml centrifuge tube after removing the veins, 5 ml of 80% acetone was added, and the leaves were soaked for 1 week under dark conditions until they turned white, and the chlorophyll content was determined by spectrophotometry (Wellburn, 1994 (link)). Three replicates of each index were determined.

The experimental methods refer to previous descriptions [75 (link)]. The IMAGING-PAM chlorophyll fluorescence imaging system (Heinz Walz, Germany) was selected for the determination of chlorophyll fluorescence parameters, and the leaves were dark-adapted for 30 min before testing. Fo (minimal fluorescence) was induced by measured light, and Fm (maximum fluorescence) was produced by excitation with saturated pulsed light. When the fluorescence was reduced to Fo, fluorescence kinetics were induced using photochemical light (1600 μmol·m⁻²·s⁻¹), and saturating pulses were turned on at 20 s intervals to determine the maximum fluorescence yield, Fm, when all the PS II reaction centers were off, and the actual fluorescence intensity, F, at any given time. The final parameters measured include Fv/Fm (maximum efficiency of PSII photochemistry), Y(II) (actual efficiency of PSII photochemistry), and Fo (initial fluorescence) (Table 4).