4.5. Chlorophyll Fluorescence Analysis

Chlorophyll fluorescence was measured in dark-adapted A. thaliana young and mature leaves using an imaging-PAM fluorometer (Walz, Effeltrich, Germany), as described previously [91]. Light curves were used for the calculation of various fluorescence parameters at photosynthetic photon flux density (PPFD) of 0, 6, 46, 136, 226, 336, 436, 636, 736, 894, 1011, 1211 and 1386 μmol photons m⁻² s⁻¹. The chlorophyll fluorescence parameters measured were the minimum chlorophyll a fluorescence in the dark (Fo), the maximum chlorophyll a fluorescence in the dark (F_m), the maximum chlorophyll a fluorescence in the light (F_m′) and the steady-state photosynthesis in the light (F_s). The minimum chlorophyll a fluorescence in the light was calculated by the Imaging Win V2.41a software (Heinz Walz GmbH, Effeltrich, Germany) as Fo′ = Fo/(F_v/F_m + Fo/F_m′) [122]. The maximum efficiency of PSII photochemistry (F_v/F_m, where F_v = F_m − Fo) [123], the effective quantum yield of PSII photochemistry (Φ_PSII = [F_m′ − F_s]/F_m′) [124,125], the quantum yield of regulated non-photochemical energy loss in PSII (Φ_NPQ = F_s/F_m′ − F_s/F_m) [126] and the quantum yield of non-regulated energy dissipated in PSII (Φ_NO = F_s/F_m) [126] were calculated. We also measured the relative PSII electron transport rate (ETR = Φ_PSII × PAR × c × abs, where PAR is the photosynthetically active radiation, c is 0.5 and abs is the total light absorption of the leaf taken as 0.84), the proportion of closed PSII reaction centers, referred to as excitation pressure and calculated as 1 − q_p [98], where q_p = [F_m′ − F_s]/[F_m′ − Fo′], the non-photochemical quenching, that reflects heat dissipation of excitation energy (NPQ = [F_m − F_m′]/F_m′ [127], and the excess excitation energy (EXC = F_v′/F_m′ × (1 − q_p) [60].

A linear regression analysis was also performed [14].