Pam 200

Plants at the V3 stage were transferred to 14 °C/12 °C (moderately low temperature) for four days, then to 8 °C/6 °C (severe cold stress) for four days and finally to 24 °C/22 °C for two days (regrowth). At the end of each period the plants were photographed.
The maximum quantum efficiency of PSII primary photochemistry (Fv/Fm) and PSII operating quantum efficiency (Φ_PSII) were measured with a fluorometer (PAM 200, H. Walz, Germany) at time points specified in the legend to Fig. 3. Separate measurements were done for plants transferred directly (without a cold-acclimation period) from 24 °C/22 °C to 8 °C/6 °C. For the measurement of Fv/Fm, the saturating one-second light flash intensity was about 3500 μmol quanta m^-2 s^-1. Before the measurements the plants were dark-adapted for 30 min at 24 °C. For the measurement of Φ_PSII, leaves were exposed to eight two-minute periods of light of 200 μmol quanta m^-2 s^-1 intensity, each period terminating with a saturating one-second light flash of ca. 3500 μmol quanta m^-2 s^-1. Independent experiments were repeated three times with three to five plants per line per experimental variant.

To assess the effect of freezing on the photosynthetic activity of white clover, analysis of photosystem II Fv/Fm was measured during the experiment. We measured photosynthetic activity at each of the five time points noted above from a single green leaflet on each whole plant. All plants were dark-acclimated followed by measuring the maximum photochemical yield of photosystem II (Fv/Fm) using a photosynthesis yield analyzer JUNIOR-PAM (Walz Teaching-PAM fluorometer (PAM-200), Heinz Walz GmbH, Effeltrich, Germany) and WinControl-3 software. Three independent measurements were averaged for each plant per time point.

Maximum quantum efficiency of PSII primary photochemistry (Fv/Fm) and PSII operating quantum efficiency (Φ_PSII) were measured with a fluorometer (PAM 200, H. Walz, Germany) in control plants and in plants subjected to low temperature (8/6 °C day/night) for 14, 38 or 110 h (i.e., 4 h after beginning of the light phase of photoperiod). For the measurement of Fv/Fm, the saturating one-second light flash intensity was about 3,500 μmol quanta m⁻² s⁻¹. Before measurements of Fv/Fm, the plants were dark-adapted for 30–60 min at 24 °C. For the measurement of Φ_PSII at 24 °C, leaves were exposed to eight 2-min periods of light of 200 μmol quanta m⁻² s⁻¹ intensity, each period terminated with a saturating 1-s light flash of ca. 3,500 μmol quanta m⁻² s⁻¹ intensity. Experiments were repeated three times using three plants per line per experimental variant. Data were subjected to two-way ANOVA with the use of PROC GLM procedure (SAS 9.1.3; SAS Institute, Cary, NC). Effects of line, variant and their interaction were considered as fixed effects while Fv/Fm and ΦPSII were considered as responding variables. Analyzes were performed at 0.05 significance level.