The dependence of the degrees of sigmoidicities of fluorescence induction curves in spinach chloroplasts on the duration of actinic pulses in pump-probe experiments

Utilizing a pump-probe double flash method, the shapes of the fluorescence induction curves in spinach chloroplasts (0°C), and the induction ratio R = F_M/F₀ (F₀ and F_M are the fluorescence yields when all PS II reaction centers are open, and closed, respectively) were studied as a function of: (1) the duration (t₁) of the pump flash, (2) the number of pump flashes, (3) absence or presence of DCMU, and (4) the time interval Δt between the pump and the probe flashes. In the pump-probe technique, an actinic pulse (P₁) of different fluences closes a fraction q of the PS II reaction centers. After a variable time interval Δt a second, weak pulse (P₂), is used to measure the variable fluorescence yield F_v. The shapes of the fluorescence curves (F_v vs. the fluence of the P₁ pulse) were analyzed in terms of the standard eqution F_v = (1 - p)q/(1 - pq), where p is a parameter describing the connectivity between different photosynthetic units (Joliot, A. and Joliot, P. (1964) C. R. Acad. Sci. 13, 4622-4625). The shapes of the F_v curves are found to depend only on the width (t₁) of the pump pulses. For t₁ ≤ 300 ps, the curves are exponential in shape with p = 0.0 and R < 2.5. However, for pulse durations t₁ in the millisecond range, and employing the same probe flash method for measuring the variable fluorescence, the F_v curves assume the same familiar sigmoidal shapes as in the case of conventional steady-state illumination. With t₁ in the microsecond range (t₁ ≈ 0.7-2 μs), the F_v curves are more nearly exponential than sigmoidal in shape with p values generally around ≈0.3 and R < 3. For t₁ >/ 50 μs, the fluorescence induction curves are sigmoidal in shape and, generally, p values of 0.55-0.60 are observed with R > 3. A sequential hit model (Valkunas, L., Geacintov, N.E., France, L. and Breton, J. (1991) Biophys. J. 59, 397-408), in which the PS II reaction centers evolve through different states characterized by differences in fluorescence yields, a process characterized by a dark-time t₁ or ≈2-50 μs between two successive hits, can account for these results. This model is consistent with the concept of interunit transfer of excitations. However, the shapes of the fluorescence induction curves cannot provide any information on the value of p within the context of this model.