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...

Interfering RNA was used to suppress the expression of the genes At1g06680 and At2g30790 in Arabidopsis thaliana, which encode the PsbP-1 and PsbP-2 proteins, respectively, of photosystem II (PS II In higher plants, algae, and cyanobacteria, at least six intrinsic proteins appear to be required for oxygen evolution by PS II 2 (1-3). These are CP47, CP43, the D1 and D2 proteins, and the ␣ and ␤ subunits of cytochrome b 559 . Insertional inactivation or deletion of the genes for these components results in the disassembly of the PS II complex and the complete loss of oxygen evolution activity (for review, see Ref. 4). Additionally, a number of low molecular mass, intrinsic membrane protein components are associated with PS II (5-7), although the functions of many of these proteins remain obscure. Although PS II complexes containing only these intrinsic components can evolve oxygen in vitro, they do so at low rates (ϳ25-40% of control), are extremely susceptible to photoinactivation, and require high, nonphysiological levels of calcium and chloride for maximal activity (1, 3).)In higher plants and green algae, three extrinsic proteins, with apparent molecular masses of 33, 24, and 17 kDa, are required for high rates of oxygen evolution at physiological inorganic cofactor concentrations. The 33-kDa component, the PsbO protein, has been termed the manganese-stabilizing protein due to its stabilization of the manganese cluster during exposure to low chloride concentrations or to exogenous reductants. In vitro, the 24-and 17-kDa proteins (termed the PsbP and PsbQ proteins, respectively) appear to modulate the calcium and chloride requirements for efficient oxygen evolution. The precise roles of these proteins in oxygen evolution and PS II assembly/stability in vivo, however, remain unclear. These three extrinsic components interact with intrinsic membrane proteins and possibly with each other to yield fully functional oxygen-evolving complexes.The mature PsbP protein is highly conserved (8) in higher plants. In Arabidopsis, there are two putative genes, At1g06680 and At2g30790, which encode PsbP-1 and PsbP-2, respectively. It should be noted that initially only PsbP-1 was observed in Arabidopsis (9, 10) using two-dimensional IEF-SDS-PAGE. Recently, however, PsbP-2 has been detected during two-dimensional difference gel electrophoresis (11). In the cyanobacterium Synechocystis 6803, mutants in which the homologue of the psbP gene had been deleted exhibited reduced photoautotrophic growth as well as decreased water oxidation activity under CaCl 2 -limiting conditions (7, 12), whereas in Chlamydomonas, a mutant which did not accumulate PsbP was deficient in photoactivation (13).RNAi is a post-transcriptional gene-silencing process in which double-stranded RNA induces the degradation of homologous mRNA sequences (14). RNAi has been successfully applied as a powerful gene-silencing tool in a variety of organisms, including Caenorhabditis elegans and Drosophila melanogaster, and in mouse oocytes. It has also become a pop-* T...

In higher plants and cyanobacteria, at least six intrinsic proteins appear to be required for oxygen evolution by Photosystem II (PS II) 2 (1-3). These are CP47, CP43, the D1 and D2 proteins, 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); however, 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 (ϳ25-40% 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 molecular masses of 33, 24, and 17 kDa, are required for high rates of oxygen evolution at physiological inorganic cofactor concentrations (for review, see Ref. 7). The 33-kDa component, PsbO protein, is required for stabilization of the manganese cluster during exposure to low chloride concentrations or to exogenous reductants. The 24-and the 17-kDa proteins (termed the PsbP and PsbQ proteins, respectively) appear to modulate the calcium and chloride requirements for efficient oxygen evolution. These three extrinsic components interact with intrinsic membrane proteins and possibly with each other to yield fully functional oxygen-evolving complexes. It is unclear, however, whether the PsbP and PsbQ proteins act in concert in modulating the cofactor requirement or whether each has individual, discrete functions within the photosystem. Miyao and Murata (8) demonstrate that the PsbQ protein enhances oxygen evolution at chloride concentrations of Ͻ3 mM. Additionally, they show that the PsbQ protein, in concert with the PsbP component, slow the inactivation of oxygen evolution during chloride depletion and the activation of oxygen evolution during chloride reconstitution. As these authors point out, however, these experiments were performed under non-physiological conditions. It had been demonstrated earlier that the thylakoid membrane is highly permeable to chloride (9) and that the stromal chloride concentration is 30 -60 mM (10

a b s t r a c tInterfering RNA was used to suppress the expression of the genes At1g06680 and At2g30790 in Arabidopsis thaliana, which encode the PsbP-1 and PsbP-2 proteins, respectively, of Photosystem II. A phenotypic series of transgenic plants was recovered that expressed intermediate and low amounts of PsbP. Earlier we had documented significant alterations in a variety of Photosystem II parameters in these plant lines [Yi, X., Liu, H., Hargett, S. R., Frankel, L. K., Bricker, T. M. (2007). The PsbP protein is required for photosystem II complex assembly/stability and photoautotrophy in Arabidopsis thaliana. J. Biol. Chem. 34, 24833-24841]. In this communication, we document extensive defects in the thylakoid membrane architecture of these plants. Interestingly, strong interfering RNA suppression of the genes encoding the PsbQ protein (At4g21280 and At4g05180) was found to have no effect on the architecture of thylakoid membranes.

Interfering RNA was used to suppress simultaneously the expression of the four genes which encode the PsbO and PsbP proteins of Photosystem II in Arabidopsis (PsbO: At5g66570, At3g50820 and PsbP: At1g06680, At2g30790). A phenotypic series of transgenic plants was obtained that expressed variable amounts of the PsbO proteins and undetectable amounts of the PsbP proteins. Immunological studies indicated that the loss of PsbP expression was correlated with the loss of expression of the PsbQ, D2, and CP47 proteins, while the loss of PsbO expression was correlated with the loss of expression of the D1 and CP43 proteins. Q(A)(-) reoxidation kinetics in the absence of DCMU indicated that the slowing of electron transfer from Q(A)(-) to Q(B) was correlated with the loss of the PsbP protein. Q(A)(-) reoxidation kinetics in the presence of DCMU indicated that charge recombination between Q(A)(-) and donor side components of the photosystem was retarded in all of the mutants. Decreasing amounts of the PsbO protein in the absence of the PsbP component also led to a progressive loss of variable fluorescence yield (F(V)/F(M)). During fluorescence induction, the loss of PsbP was correlated with a more rapid O to J transition and a loss of the J to I transition. These results indicate that the losses of the PsbO and PsbP proteins differentially affect separate protein components and different PS II functions and can do so, apparently, in the same plant.