Dual pam 100

We used a Dual-PAM 100 measuring system (Heinz Walz, Effeltrich, German) to measure PSI and PSII parameters under atmospheric CO₂ condition. After illumination at 1455 μmol photons m⁻² s⁻¹ for 5 min to activate photosynthetic electron sinks, leaves were exposed to FL alternating between low light (59 μmol photons m⁻² s⁻¹, 2 min) and high light (1455 μmol photons m⁻² s⁻¹, 1 min). PSI parameters were calculated as follows: Y(I) = (P_m’ − P)/P_m; Y(ND) = P/P_m; Y(NA) = (P_m − P_m′)/P_m. Y(I), the quantum yield of PSI photochemistry; Y(ND), the quantum yield of PSI non-photochemical energy dissipation due to donor side limitation; Y(NA), the quantum yield of PSI non-photochemical energy dissipation due to acceptor side limitation. PSII parameters were calculated as follows: Y(II) = (F_m′ − F_s)/F_m′; NPQ = (F_m − F_m′)/F_m′; Y(NO) = F_s/F_m. Y(II), the effective quantum yield of PSII photochemistry; NPQ, non-photochemical quenching in PSII; Y(NO), the quantum yield of non-regulatory energy dissipation in PSII. The relative photosynthetic electron transport rate through PSI and PSII were calculated as: rETRI = PPFD × Y(I) × 0.84 × 0.5; rETRII = PPFD × Y(II) × 0.84 × 0.5. rETRI minus rETRII is assumed to be the rate of CEF.

A Dual-PAM 100 equipped with a P515/535 emitter-detector module (Heinz Walz) was used measure the electrochromic shift (ECS) signals. After light adaptation at 1455 μmol photons m⁻² s⁻¹ for 5 min, leaves were illuminated at 59 μmol photons m⁻² s⁻¹ for 2 min. Afterwards, light intensity was changed to 1455 μmol photons m⁻² s⁻¹, and ECS dark interval relaxation kinetics (DIRK_ECS) were recorded after this light transition for 10 s or 60 s. The proton gradient (ΔpH) component of proton motive force were calculated using DIRK_ECS [50 (link),51 (link)]. The chloroplast ATP synthase activity (g_H⁺) was estimated as the inverse of the decay time constant of the first-order ECS relaxation [52 (link)].

Cells were suspended with liquid TP medium at 20 μg chlorophyhll·mL⁻¹ before measurement. Chlorophyll fluorescence measurement was performed with a DUAL-PAM-100 (Walz, Effeltrich, Germany). For the measurement of light-induced absorbance changes at 705 nm, the cell suspension was mixed with the same volume of TP medium containing 20% (w/v) Ficol, and DCMU (1 μM) and DBMIB (1 μM) were added just before measurement. The samples were placed for 30 s in the darkness in a sample cuvette holder and illuminated with an orange actinic light at an intensity of 82 μmol photons m⁻² s⁻¹ for 5 s, followed by darkness for 5 s. The absorbance changes were recorded with a JTS-10 (BioLogic, Seyssinet-Pariset, France) using the following sequence program: 3(10msD)5s2(10ms)10msI{200µs,35,5s,J200µsD15µsI}20µsJ200µsD{2ms,35,5s,D}50msD.

A Dual-PAM 100 (Heinz Walz, Effeltrich, Germany) was used to measure the redox kinetics of P700 upon dark-to-light transition. After the mature leaves were adapted to dark conditions for at least 1 h, they were suddenly exposed to actinic light (1455 μmol photons m⁻² s⁻¹) and the redox changes in P700 were measured over 16 s. To measure P700 under anaerobic conditions, the detached leaves were induced in nitrogen for at least 1 h.

The chlorophyll fluorescence of PSII and the redox state of P700, an indicator of PSI activity, were measured in vivo concomitantly at room temperature using a Dual-PAM-100 fluorometer (Walz) connected to a PC with WinControl software. After dark adaptation of the leaves for 20 min, experiments were carried out by using the automated induction and recovery curve routine in the Dual-PAM software, with repetitive application of saturation pulses for the assessment of fluorescence and P700 parameters from which the quantum yields of PSI and PSII were calculated by the software. The fluorescence parameters were calculated as follows: Fv/Fm = (Fm-Fo)/Fm, Y (II) = (Fm'-F)/Fm' (Genty et al. 1989 (link)), Y (NO) = 1/ (NPQ + 1 + qL (Fm/Fo-1) (Kramer et al. 2004 (link)), Y (NPQ) = 1-Y (II) -1/(NPQ + 1 + qL (Fm/Fo-1) (Kornyeyev and Hendrickson 2007 (link); Kramer et al. 2004 (link)), Y (I) = 1- Y (ND) -Y (NA), Y (ND) = 1-P700 red, Y (NA) = (Pm-Pm') /Pm.

Chlorophyll fluorescence was measured using a Dual Pam-100 (Heinz Walz). Samples were placed into a cuvette under constant stirring at room temperature and were dark-adapted for 5–10 min before measurements. Light curves were recorded by increasing stepwise (3 min per step) the light intensity from 15 to 715 µmol photons m⁻² s⁻¹. Saturating flashes (10,000 µmol photons m⁻² s⁻¹, 200 ms duration) were applied to determine PSII yield, 1-qP, and ETRs [57 (link), 58 ].