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A chimeric gene encoding an anti‐sense RNA of the 10 kd protein of the water‐splitting apparatus of photosystem II of higher plants under the control of the CaMV 35S promoter was introduced into potato using Agrobacterium based vectors. The expression of the anti‐sense RNA led to a significant reduction of the amounts of the 10 kd protein and RNA in a number of transgenic plants. In three out of 36 plants tested, the level of the 10 kd protein was only up to 1‐3% compared with the wild‐type control. The drastic reduction of the 10 kd protein did not influence the accumulation of other photosystem II associated polypeptides at both the RNA and protein level. Furthermore no phenotypic differences were observed between potato plants expressing wild‐type and drastically reduced levels of the 10 kd protein with respect to growth rate, habitus or ultrastructure of the chloroplasts. Measurements of the relaxation of the flash‐induced enhancement in the fluorescence quantum yield as determined in intact leaves and the rates and characteristic oscillation pattern of O2 evolution as determined in isolated thylakoid samples however, show that the elimination of the 10 kd protein on the one hand retards reoxidation of QA‐ and on the other hand introduces a general disorder into the PSII complex.

The 690 nm absorption change reflecting the turnover of the system-II-reaction center chlorophyll, Chl-aII (often referred to as P 680), has been investigated under different experimental conditions in spinach chloroplasts. A comparison was made with oxygen evolution and with absorption changes of Chl-aI measured at 703 nm, both indicating the number of electrons produced by system II. It was found: 1. The dependency on actinic flash intensity of the initial amplitudes of the measured 690 nm absorption change, ∆A0 (Chl-an), markedly differs for normal and for Tris-washed chloroplasts, respectively. 2. The saturation curve of ∆A0 (Chl-an) in Tris-washed chloroplasts is similar to that for the total amplitude of the 703 nm absorption change, ∆A0 (Chl-aI), in normal chloroplasts, and can be described by an exponential function. On the other hand, ∆A0 (Chl-aII) in normal chloroplasts exhibits a more complex biphasic dependency and much higher flash intensities are required for saturation. 3. Under repetitive flash group excitation and in the presence of an A D R Y (= acceleration of the deactivation reactions of the water-splitting enzyme system Y)-reagent the initial amplitude of the 690 nm absorption change oscillates in the same characteristic pattern as the oxygen evolution. 4. The initial amplitude of the 690 nm absorption change, ∆A0(Chl-aII), in Tris-washed chloroplasts becomes significantly smaller (more than 50%) by the addition of system-II-electron donors (benzidine, p-phenylendiamine, tetraphenylboron), whereas the total amplitude of the 703 nm absorption change, ∆A0(Chl-aI) increases 3 -4-fold. In order to explain these results, the existance of a very fast reduction kinetics of Chl-aII+ is postulated, which is not detectable by our measuring equipment. The half time of this reaction is ≦ 1 μs. Reaction centers with the very fast “undetected” Chl-aII+-reduction are photochemically transformed into slower one by double hit processes with a comparatively low quantum yield. Furthermore, it is inferred, that the dark recovery kinetics of Chl-aII is dependent on the charge accumulation state of the watersplitting enzyme system Y. This phenomenon is shown to explain also the oscillation pattern of delayed fluorescence. On the basis of the present results two alternative reaction schemes for the functional organization of the electron transport on the donor side of system II are discussed.

The functional connection between redox component Y z identified as Tyr-161 of polypeptide D-1 (Debus et al. 1988) and P680(+) was analyzed by measurements of laser flash induced absorption changes at 830 nm in PS II membrane fragments from spinach. It was found that neither DCMU nor the ADRY agent 2-(3-chloro-4-trifluoromethyl) anilino-3,5-dinitrothiophene (ANT 2p) affects the rate of P680(+) reduction by Y z under conditions where the catalytic site of water oxidation stays in the redox state S1. In contrast to that, a drastic retardation is observed after mild trypsin treatment at pH=6.0. This effect which is stimualted by flash illumination can be largely reversed by Ca(2+). The above mentioned data lead to the following conclusions: (a) the segment of polypeptide D-1 containing Tyr-161 and coordination sites of P680 is not allosterically affected by structural changes due to DCMU binding at the QB-site which is also located in D-1. (b) ANT 2p as a strong protonophoric uncoupler and ADRY agent does not modify the reaction coordinate of P680(+) reduction by Y z , and (c) Ca(2+) could play a functional role for the electronic and vibrational coupling between the redox groups Y z and P680. The electron transport from Y z to P680(+) is discussed within the framework of a nonadiabatic process. Based on thermodynamic considerations the reorganization energy is estimated to be in the order of 0.5 V.

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