Interfering RNA was used to suppress the expression of two genes that encode the manganese-stabilizing protein of photosystem II in Arabidopsis thaliana, MSP-1 (encoded by psbO-1, At5g66570), and MSP-2 (encoded by psbO-2, At3g50820). A phenotypic series of transgenic plants was recovered that expressed high, intermediate, and low amounts of these two manganese-stabilizing proteins. Chlorophyll fluorescence induction and decay analyses were performed. Decreasing amounts of expressed protein led to the progressive loss of variable fluorescence and a marked decrease in the fluorescence quantum yield (F v /F m ) in both the absence and the presence of dichloromethylurea. This result indicated that the amount of functional photosystem II reaction centers was compromised in the plants that exhibited intermediate and low amounts of the manganese-stabilizing proteins. An analysis of the decay of the variable fluorescence in the presence of dichlorophenyldimethylurea indicated that charge recombination between Q A ؊ and the S 2 state of the oxygen-evolving complex was seriously retarded in the plants that expressed low amounts of the manganesestabilizing proteins. This may have indicated a stabilization of the S 2 state in the absence of the extrinsic component. Immunological analysis of the photosystem II protein complement indicated that significant losses of the CP47, CP43, and D1 proteins occurred upon the loss of the manganese-stabilizing proteins. This indicated that these extrinsic proteins were required for photosystem II core assembly/stability. Additionally, although the quantity of the 24-kDa extrinsic protein was only modestly affected by the loss of the manganese-stabilizing proteins, the 17-kDa extrinsic protein dramatically decreased. The control proteins ribulose bisphosphate carboxylase and cytochrome f were not affected by the loss of the manganese-stabilizing proteins; the photosystem I PsaB protein, however, was significantly reduced in the low expressing transgenic plants. Finally, it was determined that the transgenic plants that expressed low amounts of the manganese-stabilizing proteins could not grow photoautotrophically.In higher plants and cyanobacteria, at least six intrinsic proteins appear to be required for oxygen evolution by photosystem II (PS II) 1 (1-3). These are CP 47, CP 43, the D1 and D2proteins, and the ␣ and ␤ subunits of cytochrome b 559 . Insertional inactivation or deletion of the genes for these components results in the complete loss of oxygen evolution activity. Additionally, a number of low molecular mass, intrinsic membrane protein components are associated with PS II (4 -6), although the functions of many of these proteins remain obscure. Although PS II complexes containing only these intrinsic components can evolve oxygen, they do so at low rates (about 25-40% of control), are extremely susceptible to photoinactivation, and require high, non-physiological levels of calcium and chloride for maximal activity (1, 3). In higher plants, three extrinsic proteins, with apparent...

The psbC gene encodes CP43, a component of Photosystem II (PSII) in higher plants, algae, and cyanobacteria. Previous work demonstrated that alteration of an arginine residue occurring at position 305 to serine produced a strain (R305S) with altered PSII activity (Knoepfle, N., Bricker, T. M., and Putnam-Evans, C. (1999) Biochemistry 38, 1582-1588. This strain grew at wild-type rates in complete BG-11 media (480 µM chloride) and evolved oxygen at rates that were 60-70% of the observed wildtype rates. The R305S strain assembled approximately 70-80% of the functional PSII centers contained in the control strain, and these PSII centers were very sensitive to photoinactivation at high light intensities. We recently observed that the R305S mutant exhibited a pronounced chloride effect. When this mutant was grown in media depleted of chloride (30 µM chloride), it exhibited a severely reduced photoautotrophic growth rate. The effect of chloride depletion on the growth rate of the mutant was reversed by the addition of 480 µM bromide to the chloride-depleted BG-11 media. Oxygen evolution rates for the mutant were further depressed to about 22% of that observed in control cells under chloride-limiting conditions. Addition of bromide restored these rates to those observed under chloride-sufficient conditions. The mutant exhibited a significantly lower relative quantum yield for oxygen evolution than did the control strain, and this was exacerbated under chloride-limiting conditions. Fluorescence yield measurements indicated that both the mutant and the control strains assembled fewer PSII reaction centers under chloride-limiting conditions. The reaction centers assembled by the mutant exhibited an enhanced sensitivity to photoinactivation under chloride-limiting conditions, with a t 1/2 of photoinactivation of 2.6 min under chloride-limiting conditions as compared to a t 1/2 of 4.7 min under normal growth conditions. The mutant also exhibited an enhanced stability of its S 2 state and increased number of centers in the S 1 state following dark incubation. These results indicate that the mutant R305S exhibits a defect in its ability to utilize chloride in support of efficient oxygen evolution in PSII. This is the first mutant of this type described in the CP43 protein.