Pam 2100

According to the method of Zhang (2019a) , the following fluorescence parameters were determined. The sixth fully developed leaf was measured from 9:00 to 11:00 using a portable modulated chlorophyll fluorescence instrument (PAM-2100, WALZ, Germany). The initial fluorescence (F0) was determined after 20 min of dark acclimation, and the maximum fluorescence (Fm) was determined by irradiation saturation pulse (2,800.0 μmol·m⁻²·s⁻¹). The steady-state fluorescence (Ft) under light adaptation was measured by switching on the endogenous photochemical light (600.0 μmol·m⁻²·s⁻¹) for 5 min, and the maximum fluorescence (Fm′) under light adaptation was measured by switching on the saturation pulse (2,800.0 μmol·m⁻²·s⁻¹) at 20-s intervals. Fluorescence parameters and leaf light energy distribution were determined according to the following formulas.
We determined the light energy distribution of the leaf, including the dissipation part of antenna [D = 1 − (Fm′ − F0′)/Fm′], the photochemical reaction part [P = qP × (Fm′ − F0′)/Fm′], the dissipation part of the reaction center [E = (1 − qP) × (Fm′ − F0′)/Fm′], and the excitation energy distribution imbalance between photosystem (PS)II and PSI: β/α − 1 = (Fm′ − F0)/(Fm′ − Fs)− 1.

After heat stress for 12 d, three cucumber plants were collected and washed with ddH₂O. Then, the remaining water was sucked up, and the FW was detected using an electronic balance (Huazhi, Putian, China). The plant was enclosed in an envelope and placed in an oven (DHG-9030A, Shanghaiyiheng, China) for 30 min, which was set at 105°C. Then, the plant was dried at 75°C for 2 d to measure the DW. For measurement of the Chl fluorescence parameters, cucumber plants at 12 d of heat stress were placed in the dark for 30 min and were detected using a portable fluorimeter (PAM-2100, Walz, Effeltrich, Germany) [51 (link)].

The water potential (Ψ_w, MPa) was measured with a Schölander pressure pump (model PMS-1000, PMS Instruments, Corvallis, OR, United States). Stomatal conductance (g_s, mmol H₂O m⁻²s⁻¹), the sub-stomatal concentration of CO₂ (C_i), photosynthetic rate (A, μmol CO₂ m⁻²s⁻¹), transpiration (E, mmol H₂O m⁻²s⁻¹), water use efficiency (WUE, μmol CO₂ mmol⁻¹H₂O) and leaf temperature through infrared Thermometry (Tleaf, °C), were determined with a CIRAS-3 portable photosynthesis system (PP Systems, Amesbury MA). The measurements were recorded under saturating light conditions (1,500 μmol quanta m⁻² s⁻¹), with a temperature of 25°C, and ambient CO₂ concentration of 400 mol⁻¹ CO₂ and a relative humidity of ~55%. Chlorophyll fluorescence indices (i.e., Fv/Fm and Quantum yield) were measured with a portable pulse-amplitude modulated chlorophyll fluorometer (PAM-2100, Heinz Walz, Effeltrich, Germany). These measurements of the photosystem II efficiency were performed once the plants were adapted to darkness for 30 min, on the same leaves where stomatal conductance and photosynthesis were determined. All measures were performed on the third youngest full-developed leaf of each plant, analyzing a total of five plants per variety.

The photosynthetic parameters of leaf surface were measured by portable photosynthetic instrument LI-6400 (LI-Cor Inc., Lincoln, NE, USA). The light intensity was 1000 µmol/m²/s, and the flow rate was 500 mL/s when the parameters were measured. In addition, the temperature was 25–28 °C. We selected the third leaf for experiment, and three biological replicates were collected for each treatment.
Chlorophyll fluorescence was measured by the portable fluorescence instrument German WALZ PAM2100 after 0, 24, 48, and 72 h of treatment. The time of dark processing was 20–30 min. Three biological replicates were collected.
The chlorophyll content was measured by select the leaf of latest expanded for the experiment. We collected leaves of 0.1 g and cut them into filaments. We then put the filaments of the leaf into 25 mL acetone solution (80%) and soaked the sample for 24 h in the dark. OD was measured by spectrophotometer by colorimetric determination at 663 nm and 645 nm. The chlorophyll content was calculated as follows:

Ca and Cb, respectively, are concentrations of chlorophyll a and chlorophyll b. D663 and D645 are absorbance of 663 nm and 645 nm, respectively. W is fresh weight of sample, and V is volume of extract.

The net CO₂ assimilation rate (P_n), stomatal conductance (G_s), and intercellular CO₂ concentration (C_i) were measured with a portable open-flow gas exchange system (LI-6400, LI-COR Inc., United States), between 9:00 and 11:00 h, on fully expanded leaves that were at similar stages of development. During this time, relative air humidity, CO₂ concentration, and photon flux density were maintained at 60–70%, 380 μmol mol^-1 and 800 μmol m^-2 s^-1, respectively. Intrinsic water use efficiency (WUE_intr) was calculated by dividing the instantaneous values of P_n by G_s. The maximum quantum efficiency of photosystem II (F_v/F_m) of the leaves was measured with a portable pulse amplitude modulated fluorometer (PAM-2100, Walz, Effeltrich, Germany), where the leaves were dark-adapted with clips for 20 min. After this time, minimal fluorescence (F_o) was measured under a weak pulse of modulating light over 0.8 s, and maximal fluorescence (F_m) was induced by a saturating pulse of light (8,000 mmol m^-2 s^-1) applied over 0.8 s. F_v/F_m was calculated, where F_v was the difference between F_m and F_o.