P515 535 accessory module

In order to estimate the magnitude of the pmf, we measured the light‐induced absorbance difference between 550 and 515 nm referred to as the electrochromic shift (ECS). We used the Dual‐PAM 100 spectrometer and its P515/535 accessory module (Walz; Schreiber and Klughammer 2008), with a measuring light at a 2000 Hz pulse frequency. Plants were dark‐adapted for 30 min, after which detached leaves were illuminated sequentially with 22, 53, and 127, as well as 39, 216, and 826 μmol of photons m⁻² s⁻¹ of AL (620 nm). Each light intensity step was applied for 3 min, after which the AL was switched off for 1 min to monitor the post‐illumination response of the ECS signal. Partitioning of the total pmf to ΔpH and ΔΨ was determined from the post‐illumination response of the ECS signal as described by Schreiber and Klughammer (2008). All measured values were normalized with the maximal ECS change (ECS_ST) induced by a saturating single‐turnover flash of 20 μs and 14 000 μmol photons m⁻² s⁻¹ at the beginning of each measurement from a dark‐adapted leaf. Measurements from three to eight individual leaves were made from each line.

To measure the magnitude and kinetics of pmf formation and to determine the conductivity of the ATP synthase to protons, changes in the ECS (P515) signal were recorded with a Dual‐PAM‐100 spectrometer and its P515/535 accessory module (Walz, Effeltrich, Germany) (Schreiber and Klughammer, 2008; Klughammer et al., 2013). A measuring light at a 2000‐Hz pulse frequency was used in all ECS measurements as described in Nikkanen et al. (2018). Dark intervals of 250 msec were applied at time‐points specified in Figure S5 to determine the magnitude of light‐induced pmf (ECS_T), and to calculate the conductivity parameters of the ATP synthase (g_H+) as well as the proton flux parameter (v_H+), as described previously (Cruz et al., 2001; Cruz et al., 2005; Nikkanen et al., 2018).