The PsbP Protein Is Required for Photosystem II Complex Assembly/Stability and Photoautotrophy in Arabidopsis thaliana

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). A phenotypic series of transgenic plants was recovered that expressed intermediate and low amounts of PsbP. Chlorophyll fluorescence induction and QA– decay kinetics analyses were performed. Decreasing amounts of expressed PsbP protein led to the progressive loss of variable fluorescence and a marked decrease in the fluorescence quantum yield (FV/FM). This was primarily due to the loss of the J to I transition. Analysis of the fast fluorescence rise kinetics indicated no significant change in the number of PS IIβ centers present in the mutants. Analysis of QA– decay kinetics in the absence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea indicated a defect in electron transfer from QA– to QB, whereas experiments performed in the presence of this herbicide indicated that charge recombination between QA– and the oxygen-evolving complex was seriously retarded in the plants that expressed low amounts of the PsbP protein. These results demonstrate that the amount of functional PS II reaction centers is compromised in the plants that exhibited intermediate and low amounts of the PsbP protein. Plants that lacked detectable PsbP were unable to survive in the absence of sucrose, indicating that the PsbP protein is required for photoautotrophy. Immunological analysis of the PS II protein complement indicated that significant losses of the CP47 and D2 proteins, and intermediate losses of the CP43 and D1 proteins, occurred in the absence of the PsbP protein. This demonstrates that the extrinsic protein PsbP is required for PS II core assembly/stability. 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). A phenotypic series of transgenic plants was recovered that expressed intermediate and low amounts of PsbP. Chlorophyll fluorescence induction and QA– decay kinetics analyses were performed. Decreasing amounts of expressed PsbP protein led to the progressive loss of variable fluorescence and a marked decrease in the fluorescence quantum yield (FV/FM). This was primarily due to the loss of the J to I transition. Analysis of the fast fluorescence rise kinetics indicated no significant change in the number of PS IIβ centers present in the mutants. Analysis of QA– decay kinetics in the absence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea indicated a defect in electron transfer from QA– to QB, whereas experiments performed in the presence of this herbicide indicated that charge recombination between QA– and the oxygen-evolving complex was seriously retarded in the plants that expressed low amounts of the PsbP protein. These results demonstrate that the amount of functional PS II reaction centers is compromised in the plants that exhibited intermediate and low amounts of the PsbP protein. Plants that lacked detectable PsbP were unable to survive in the absence of sucrose, indicating that the PsbP protein is required for photoautotrophy. Immunological analysis of the PS II protein complement indicated that significant losses of the CP47 and D2 proteins, and intermediate losses of the CP43 and D1 proteins, occurred in the absence of the PsbP protein. This demonstrates that the extrinsic protein PsbP is required for PS II core assembly/stability. In higher plants, algae, and cyanobacteria, at least six intrinsic proteins appear to be required for oxygen evolution by PS II 2The abbreviations used are: PS I and IIphotosystems I and IIDCMU3-(3,4-dichlorophenyl)-1,1-dimethylureaLiDSlithium dodecyl sulfateTricineN-tris[hydroxymethyl]methyl glycine (1Murata N. Miyao M. Omata T. Matsunami H. Kuwabara T. Biochim. Biophys. Acta. 1984; 765: 363-369Crossref Scopus (181) Google Scholar, 2Burnap R.L. Sherman L.A. Biochemistry. 1991; 30: 440-446Crossref PubMed Scopus (180) Google Scholar, 3Bricker T.M. Biochemistry. 1992; 31: 4623-4628Crossref PubMed Scopus (121) Google Scholar). These are CP47, CP43, the D1 and D2 proteins, and the α and β subunits of cytochrome b559. 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. 4Nelson N. Yocum C.F. Annu. Rev. Plant Biol. 2006; 57: 521-565Crossref PubMed Scopus (736) Google Scholar). Additionally, a number of low molecular mass, intrinsic membrane protein components are associated with PS II (5Bricker T.M. Ghanotakis D.F. Ort D.R. Yocum C.F. Oxygenic Photosynthesis: The Light Reactions. Kluwer Academic Publishers, Dordrecht, Netherlands1996: 113-136Google Scholar, 6Ikeuchi M. Koike H. Inoue Y. FEBS Lett. 1989; 242: 263-269Crossref PubMed Scopus (86) Google Scholar, 7Thornton L.E. Roose J.L. Pakrasi H.B. Ikeuchi M. Wydrzynski T.J. Satoh K. Photosystem II: The Light-Driven Water: Plastoquinone Oxidoreductase. Springer, Dordrecht, Netherlands2005: 121-138Google Scholar), 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 (∼25–40% of control), are extremely susceptible to photoinactivation, and require high, nonphysiological levels of calcium and chloride for maximal activity (1Murata N. Miyao M. Omata T. Matsunami H. Kuwabara T. Biochim. Biophys. Acta. 1984; 765: 363-369Crossref Scopus (181) Google Scholar, 3Bricker T.M. Biochemistry. 1992; 31: 4623-4628Crossref PubMed Scopus (121) Google Scholar). photosystems I and II 3-(3,4-dichlorophenyl)-1,1-dimethylurea lithium dodecyl sulfate N-tris[hydroxymethyl]methyl glycine 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 (8Bricker T.M. Burnap R.L. Wydrzynski T.J. Satoh K. Photosystem II: The Light-Driven Water: Plastoquinone Oxidoreductase. Springer, Dordrecht, Netherlands2005: 95-120Google Scholar) 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 (9Peltier J.-B. Emanuelsson O. Kalume D.E. Ytterberg J. Friso Plant PubMed Scopus Google Scholar, M. T. J. Biol. PubMed Scopus Google Scholar) however, PsbP-2 has been during M. T. L.A. Plant J. 2006; PubMed Scopus Google Scholar). In the in which the of the been exhibited activity L.E. Roose J.L. Pakrasi H.B. Ikeuchi M. Wydrzynski T.J. Satoh K. Photosystem II: The Light-Driven Water: Plastoquinone Oxidoreductase. Springer, Dordrecht, Netherlands2005: 121-138Google Scholar, Biochemistry. PubMed Scopus Google Scholar), whereas in a which PsbP was in J. Biol. PubMed Scopus Google Scholar). is a in which RNA the of PubMed Scopus Google Scholar). has been a in a of and and in It has a for the physiological functions of genes in plants Rev. PubMed Scopus Google Scholar). to PS II membrane proteins, has been used to the of the PsbO and PsbQ proteins in Arabidopsis T.M. J. Biol. 2006; PubMed Scopus Google Scholar, M. T.M. J. Biol. PubMed Scopus Google Scholar) the PsbP and PsbQ proteins in K. J. Plant PubMed Scopus Google Scholar). The performed in indicated that of the PsbQ proteins led to no of the PsbP proteins led to of quantum yield of PS loss of and manganese cluster that in the of the intrinsic PS II core proteins and the extrinsic PsbO protein in plants was to that in K. J. Plant PubMed Scopus Google Scholar). performed in Arabidopsis that the of PsbQ led to the assembly/stability of PS II in low in the loss of T.M. J. Biol. 2006; PubMed Scopus Google Scholar). In the of PsbP expression in from performed in K. J. Plant PubMed Scopus Google Scholar) in that in Arabidopsis, the PsbP protein to be required for and in its significant in the amounts of the PS II core proteins D2 and CP47 fluorescence yield and to the that PsbP is required for PS II assembly/stability and in RNA and Plant J. PubMed Scopus Google Scholar) was used to RNA The to of the was to suppress the expression of PsbP-1 and This be to The for were and for the and and for the of was performed a in in containing reaction of of of of and of Arabidopsis in were a at for by at for at for and at for and a at for The was in and the of the and by the expression was from with and for of the by the M. M. J. PubMed Scopus Google Scholar). Arabidopsis plants were by the Plant J. PubMed Google Scholar). were with and for with and for by three with were containing sucrose, and and for at in the and the of occurred at in and the plants were containing with sucrose, and at a of for plants were from which was presence of the in the was by the of with to the and the of the of the plants that exhibited the exhibited the which was in the plants plants were for the presence of the PsbP protein by was in a and to a in the presence of of the a protein was by the of and the were for at least The were at for a and and were used by exposure to and the mature PsbP was to with the PsbP protein from Arabidopsis and was used in these that were used to the of the Photosystem II complex were the from many induction was with a of the in Ref. M. J. Scopus Google Scholar). and were by The exhibited a for low and only from for of the fluorescence from and plants were and for of the In the fluorescence induction experiments were in a series between and the of In the fluorescence induction the kinetics of the fluorescence rise a by were for with in the presence of were at a of with The of PS and PS IIβ centers were M. J. Scopus Google Scholar). In the fluorescence decay the kinetics of the electron transfer between QA– and were in the absence of the recombination of QA– with PS II components were in the presence of these experiments were between a were the Biochemistry. PubMed Scopus Google Scholar). In this three decay components and a were In the the were in in for to the fluorescence were the and by Immunological of a analysis of the protein complement of the were from and that expressed low levels of PsbP. These from the were and were in a with a The was two of and the were by at for The were in a amount of and the was by the of Plant PubMed Google Scholar). was performed a with of and with and were of the a was and the was to the the were with a at and of from and from plants with the were containing and with each of and Although containing these a were recovered from plants with the however, from the plants were containing and sucrose, many were recovered The presence of the in plants was by of the of the plants that exhibited exhibited the indicating the presence of the of the transgenic plants for the presence of the PsbP proteins, analysis with a that the PsbP proteins was performed. The results from a are in In plants, two were observed that the protein The of the is at this although a of the PsbP-1 protein. It should be noted that this is the PsbP-2 protein, this at a apparent molecular M. T. L.A. Plant J. 2006; PubMed Scopus Google Scholar). was at the PsbP-2 in the plants, which in this plants with the a or complete loss of the PsbP-1 In plants were for the presence of the PsbP protein. These results indicated that of the plants expression levels to that of for the PsbP-1 protein, exhibited intermediate of and of the transgenic plants exhibited complete loss of the PsbP-1 protein. These results are with the results in other other In of of the protein M. M. J. PubMed Scopus Google Scholar), the of a phenotypic series of in the Plants of PsbP containing that were and were to the that lacked PsbP-1 was green the or plants that detectable amounts of PsbP-1 plants and the that lacked detectable levels of PsbP-1 and whereas the other plants, which expressed detectable amounts of These results that PsbP is required for in in by of the a fluorescence rise observed in Arabidopsis and in the plants. 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Springer, Dordrecht, Netherlands2005: 95-120Google Scholar), these for of the of the higher that loss of PsbO in higher plants primarily to the loss of CP43 M. T.M. J. Biol. PubMed Scopus Google Scholar) the loss of PsbP primarily to the loss of CP47, roles for these two extrinsic proteins in the assembly/stability of these intrinsic membrane results that the of has a in the of a of PsbP in amounts of PsbP led to a loss of the progressive loss of variable fluorescence and a marked decrease in the fluorescence quantum yield functional PS II reaction and a significant loss of PS II reaction with D2 and CP47 that the PsbP protein is required for PS II core complex assembly/stability and in