Reductive titration of photosystem I and differential extinction coefficient of P700+ at 810-950 nm in leaves

We describe a method of reductive titration of photosystem I (PSI) density in leaves by generating a known amount of electrons (e-) in photosystem II (PSII) and measuring the resulting change in optical signal as these electrons arrive at pre-oxidized PSI. The method complements a recently published method of oxidative titration of PSI donor side e- carriers P700, plastocyanin (PC) and cytochrome f by illuminating a darkened leaf with far-red light (FRL) V. Oja, H. Eichelmann, R.B. Peterson, B. Rasulov, A. Laisk, Decyphering the 820 nm signal: redox state of donor side and quantum yield of photosystem I in leaves, Photosynth. Res. 78 (2003) 1-15], presenting a nondestructive way for the determination of PSI density in intact leaves. Experiments were carried out on leaves of birch (Betula pendula Roth) and several other species grown outdoors. Single-turnover flashes of different quantum dose were applied to leaves illuminated with FRL, and the FRL was shuttered off immediately after the flash. The number of e- generated in PSII by the flash was measured as four times O2 evolution following the flash. Reduction of the pre-oxidized P700 and PC was followed as a change in leaf transmittance using a dual-wavelength detector ED P700DW (810 minus 950 nm, H. Walz, Effeltrich, Germany). The ED P700DW signal was deconvoluted into P700+ and PC+ components using the abovementioned oxidative titration method. The P700+ component was related to the absolute number of e- that reduced the P700+ to calculate the extinction coefficient. The effective differential extinction coefficient of P700+ at 810-950 nm was 0.40+/-0.06 (S.D.)% of transmittance change per micromol P700+ m(-2) or 17.6+/-2.4 mM(-1) cm(-1). The result shows that the scattering medium of the leaf effectively increases the extinction coefficient by about two times and its variation (+/-14% S.D.) is mainly caused by light-scattering properties of the leaf.