Pam chlorophyll fluorometer

Chlorophyll fluorescence was measured with a Pulse-Amplitude-Modulation (PAM) Chlorophyll Fluorometer (Heinz-Walz GmbH, Effeltrich, Germany) as previously described [23 (link)]. Briefly, plants were dark-adapted for 30 min to measure the maximum quantum yield of photosystem II (PSII). Fv/Fm and electron transport rate (ETR) was recorded during a saturating photon pulse (4000 μmol m⁻² s⁻¹) using a whole leaf.

Chlorophyll fluorescence and the redox state of P700 were measured with PAM chlorophyll fluorometer (Walz, Effeltrich, Germany) with an emitter-detector (ED-101 US) for chlorophyll fluorescence and another (ED-P700DW-E) for P700 absorbance changes monitored by the absorbance at 810–830 nm. The fluorometer setup was as described as by the previous studies (Schreiber et al., 1986 (link)) and (Klughammer and Schreiber, 1998 ). The dark level chlorophyll fluorescence (F_o) was measured with a weak, modulated red light (650 nm, 0.09 μmol photons m^-2 s^-1). Maximum chlorophyll fluorescence (F_m) was measured after a 0.8 s pulse of saturated white light. Maximum quantum efficiency of PS II was determined by F_v/F_m. ΦPSII, the photochemical efficiency of PSII, was calculated as (F′_m - F)/F′_m; qP and qN were calculated as (F′_m - F)/(F′_m - F′_o) and 1 - (F′_m - F′_o)/(F_m - F_o), respectively, after steady-state photosynthesis was reached (15 min of light induction together with saturating pulses of 0.8 s every 30 s), and F_m here was determined before stress. The halftime of the oxidation of P700 was determined after reaching a steady state level of P700⁺ by illumination with FR (>705 nm, 5.2 μmol m^-2 s^-1) and that of re-reduction of the P700⁺ was determined after a 6 s illumination with FR.

Shoot and root length was determined by scale. Samples were oven dried at 70 °C for 24 h and then weighed.
Photosynthetic pigment contents were analyzed using the acetone extract method^{32 (link)}. Acetone (80%) was used to extract fresh leaf tissues, and absorbance read at 480 nm, 645 nm, 663 nm with a spectrophotometer (Beckman 640D, USA).
Leaf gas exchange measurements viz. net photosynthetic rate (Pn), carbon dioxide assimilation rate (A), stomatal conductance (gs) and transpiration rate (E) were measured on fully expanded horizontal leaves in full and bright sunlight between 10:00 h and 12:00 h using IRGA (LCA-4 model Analytical development Company, Hoddesdon England).
Chlorophyll fluorescence parameters were recorded with a junior PAM chlorophyll fluorometer (H. Walz, Effeltrich, Germany) on fully expanded horizontal tomato leaves^{33 (link)}.

Photosynthetic oxygen evolution was determined in vivo using a Clark-type oxygen electrode (Hansatech Instruments RS232, Norfolk, UK). Two milliliters of treated or untreated cultures were transferred to the measurement chamber. Oxygen formation was measured for 10 min at room temperature under illumination at 50 µE. PSII activity was analyzed in vivo with a WATER- pulse amplitude modulation (PAM) chlorophyll fluorometer (Walz GmbH, Effeltrich, Germany). All samples were dark-adapted for 5 min before measurement. Measurement of the PSII quantum yield (F_ν/F_m) was performed at room temperature with WinControl Data Acquisition software.

The redox change of PQ was monitored by Chl fluorescence, using a PAM Chlorophyll Fluorometer (Walz, Effeltrich, Germany) with an emitter–detector–cuvetter assembly (ED-101 US). Details for fluorometer setup were described previously (Schreiber et al., 1994 , 1995 (link)). The cell suspension was pipetted in a cuvetter with a thermostat. Cells were exposed to the actinic light (AL, 3 Wm^-2) for 30 s. Then AL was turned off and the transient increase kinetics in chlorophyll fluorescence was detected. Each sample was adapted in the dark for 2 min prior to measurement. As for treatment with the cyclic photophosphorylation cofactor, 100 μM of phenazine methosulphate (PMS) was added to the sample before measurement; and with the Calvin cycle inhibitor, iodoacetamide (IA) was pre-incubated with the sample at low concentration (40 μM) and high concentration (1 mM). As for dark starvation, cells of Synechocystis PCC 6803 were pre-treated in dark for 24–32 h before measurement. Every experiment was repeated three times at least.