4.5. Chlorophyll Fluorescence Measurements and Analysis of Absorbance Changes

Fluorescence images of intact pea plants were recorded by IMAGING-PAM (MAXI version; Walz, Germany) equipped with an IMAG-MAX/L LED-array illumination unit (blue) and a CCD camera (IMAG-K7, 640 × 480 pixels). The minimum (F₀) and maximum (F_m) fluorescence were determined in 30-min dark-adapted plants and were used to calculate the maximum quantum yield of photosystem II (PSII) as F_v/F_m = (F_m − F₀)/F_m. During plant adaptation to the specific light level (180 µmol m⁻² s⁻¹), saturating pulses (over 6000 µmol photons m⁻² s⁻¹ PPFD) with the duration of 0.8 s were applied in order to determine the steady-state fluorescence (F′) and the maximum fluorescence (F_m′) in the light. The quantum efficiency of PSII photochemistry (Φ_PSII) was calculated using the formula (F_m′ − F′)/F_m′ [21]. The redox state of PSII (q_L), which is a measure of the fraction of open PSII reaction centers was calculated using the formula q_L = (F_q′/F_v′)/(F₀′/F′), where F_q′ is the difference in fluorescence between F_m′ and F′ and F_v′ is variable fluorescence from the light-adapted leaf [20]. The minimal fluorescence in the light (F₀′) was estimated using the equation F₀′ = F₀/((F_v/F_m) + F₀/F_m′) [66] and the maximum PSII efficiency in light-adapted leaves was determined as F_v′/F_m′. The non-photochemical quenching parameter (NPQ) reflecting downregulation of PSII as a protective mechanism against excess light was defined according to the equation NPQ = (F_m − F_m′)/F_m′ [67].

The electrochromic shift (ECS) was determined by MultispeQ V2.0 (PhotosynQ Inc., East Lansing, MI, USA) linked to the PhotosynQ platform (www.photosynq.org) [68]. The amplitude of the first-order decay kinetics of the ECS trace in the first 300 ms (ECSt) was used to assess the proton motive force (pmf) [69,70]. The thylakoid conductivity to protons (g_H+) was determined by the ECS dark interval relaxation kinetics at 520 nm [47].