Supporting Information

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The spectrophotometric technique developed by D. Béal and P.J. uses similar electronic and computational devices than those described in ref. 1. Detecting flashes (10-ms square pulses) are provided by LEDs peaking at 710, 870, and 530 nm, respectively. Monochromatic flashes at 705 and 520 nm are obtained using interference filters (half-bandwidth of 10 nm). The detecting beam is split in two parts by a glass sheet. The transmitted light (≈90% of the incident light) is directed on the leaf (measuring beam, 6 × 3 mm). Light transmitted through the leaf is measured by a PIN Hamamatsu S3204 photodiode that collects most of scattered light. The reflected light (≈10% of incident light) is used as reference beam that is measured by another photodiode. Measuring and reference signals are integrated with time constant of 10 m s.

When using detecting flashes at 705 nm, the photodiode is protected from the fluorescence emission at l < 690 nm by a BG 39 Schott filter. For detecting flashes at 870 nm, two Wratten filters, and a BG39 Schott filter protect the photodiode from actinic light and fluorescence. At 870 nm, the scattered light within the leaf is weakly absorbed, which leads to a larger optical path than at 705 nm. In addition, a piece of white paper is introduced between the Wratten filters and the photodiode. A part of the detecting light is scattered back toward the leaf, further increasing the optical path.

Under far-red actinic excitation, the measuring photodiode cannot be protected from the actinic illumination. To avoid saturation of the measuring channel, the actinic light is switched off for 150-m s dark periods that are distributed during the course of the actinic illumination. Detecting flashes are fired 130 m s after the beginning of each dark period. Approximately 60 detecting flashes are fired to sample the absorption changes induced by a 20-s far-red illumination. We have checked that no significant reduction of P₇₀₀ occurs during the 150-m s dark periods. The total duration of the dark periods (60 × 150 m s) is negligible as compared to the time of actinic illumination (≈20 s).

A weak variable fluorescence signal induced by the actinic far-red illumination is superimposed to the absorption changes associated with P₇₀₀ and PC oxidation when detected at 705 nm. The fluorescence induction kinetics is measured by firing detecting flashes at 520 nm (in place of 705 nm) during the 150-m s dark periods. In the conditions of Fig. 2, the amplitude of the fluorescence increase is F_V/F₀ = ≈0.25 and ≈0.15 in dark-adapted and preilluminated leaf, respectively. The amplitude of the fluorescence increase detected at 520 nm has been compared to that detected at 705 nm as follows: a dark-adapted leaf is submitted to 2 s of saturating green illumination that induces the reduction of Q_A and PQ pool. During the following dark period, P₇₀₀ and PC are fully reduced in <1 s, whereas fluorescence decays for 20 s. It implies that the signal detected at 705 nm beyond 1 s is exclusively associated with fluorescence emission, which allows a calibration of the signal detected at 705 nm with respect to that detected at 520 nm. The amplitude of the fluorescence change measured at 705 nm is ≈3% of that associated with P₇₀₀ oxidation. In addition, a small absorption increase of unknown origin (less than ≈2% of the full P₇₀₀ signal) is observed during the first hundreds of milliseconds of illumination. This absorption increase that is shorter than the lag phase in P₇₀₀ oxidation is subtracted from P₇₀₀ kinetics. The comparison of Fig. 2 A (705 nm) and B (P₇₀₀) shows that omission of these corrections introduces no major changes in the kinetics. Nevertheless, they are required for accurate determination of the equilibrium constant between P₇₀₀ and PC (Fig. 5).

The PSI photochemical rate constant (k_{i PSI}) under far-red illumination has been measured as follows: the membrane potential increase induced by a 400-m s far-red light pulse is measured at 520 nm. In these conditions, the membrane potential increase is proportional to the number of photoinduced charge separations at the level of PSI and PSII, with no significant contribution of the cyt bf turnover that occurs in a longer time range. The membrane potential increased induced by the 400-m s far-red pulse is normalized to the membrane potential increase induced by a 10-ns saturating laser flash, as described in ref. 1. The computed value of K_{i PSI} corresponds to the average value of the photochemical rate constants for the different PSI that differs by the size of their antenna (2). On the basis of PSI and PSII action spectrum (3) and of the actinic light spectrum, we estimate that ≈90% of the absorbed photons sensitize PSI and ≈10% or less sensitized PS II antenna.

1. Joliot, P., Beal, D. & Joliot, A. (2004) Biochim. Biophys. Acta 1656, 166-176.

2. Danielsson, R., Albertsson, P. A., Mamedov, F. & Styring, S. (2004) Biochim. Biophys. Acta 1608, 53-61.

3. Joliot, P., Joliot, A. & Kok, B. (1968) Biochim. Biophys. Acta 153, 635-652.