Chloroplast nucleoids are large, compact nucleoprotein structures containing multiple copies of the plastid genome. Studies on structural and quantitative changes of plastid DNA (ptDNA) during leaf development are scarce and have produced controversial data. We have systematically investigated nucleoid dynamics and ptDNA quantities in mesophyll of Arabidopsis , tobacco, sugar beet, and maize from the early post-meristematic stage until necrosis. DNA of individual nucleoids was quantified by DAPI-based supersensitive epifluorescence microscopy. Nucleoids occurred in scattered, stacked or ring-shaped arrangements and in recurring patterns during leaf development remarkably similar between the species studied. Nucleoids per organelle varied from few in meristematic plastids to >30 in mature chloroplasts (corresponding to about 20-750 nucleoids per cell). Nucleoid ploidies ranged from haploid to >20-fold even within individual organelles, with average values between 2.6- and 6.7-fold and little changes during leaf development. DNA quantities per organelle increased gradually from about a dozen plastome copies in tiny plastids of apex cells to 70-130 copies in chloroplasts of about 7 μm diameter in mature mesophyll tissue, and from about 80 plastome copies in meristematic cells to 2,600-3,300 copies in mature diploid mesophyll cells without conspicuous decline during leaf development. Pulsed-field electrophoresis, restriction of high-molecular weight DNA from chloroplasts and gerontoplasts, and CsCl equilibrium centrifugation of single- and double-stranded ptDNA revealed no noticeable fragmentation of the organelle DNA during leaf development, implying that plastid genomes in mesophyll tissues are remarkably stable until senescence. Significance Statement Plastid DNA is organized in nucleoids that are highly dynamic in organization, structure and amount during leaf development. The present investigation fully resolves now this dynamic and is a precise cytogenetic characterization of nucleoids DNA spanning the entire life cycle of the leaf.

The fate of plastid DNA (ptDNA) during leaf development has become a matter of contention. Reports on little change in ptDNA copy number per cell contrast with claims of complete or nearly complete DNA loss already in mature leaves. We employed high-resolution fluorescence microscopy, transmission electron microscopy, semithin sectioning of leaf tissue, and real-time quantitative PCR to study structural and quantitative aspects of ptDNA during leaf development in four higher plant species (Arabidopsis thaliana, sugar beet [Beta vulgaris], tobacco [Nicotiana tabacum], and maize [Zea mays]) for which controversial findings have been reported. Our data demonstrate the retention of substantial amounts of ptDNA in mesophyll cells until leaf necrosis. In ageing and senescent leaves of Arabidopsis, tobacco, and maize, ptDNA amounts remain largely unchanged and nucleoids visible, in spite of marked structural changes during chloroplast-to-gerontoplast transition. This excludes the possibility that ptDNA degradation triggers senescence. In senescent sugar beet leaves, reduction of ptDNA per cell to ∼30% was observed reflecting primarily a decrease in plastid number per cell rather than a decline in DNA per organelle, as reported previously. Our findings are at variance with reports claiming loss of ptDNA at or after leaf maturation.

The chloroplast-encoded low molecular weight protein PsbN is annotated as a photosystem II (PSII) subunit. To elucidate the localization and function of PsbN, encoded on the opposite strand to the psbB gene cluster, we raised antibodies and inserted a resistance cassette into PsbN in both directions. Both homoplastomic tobacco (Nicotiana tabacum) mutants psbN-F and psbN-R show essentially the same PSII deficiencies. The mutants are extremely light sensitive and failed to recover from photoinhibition. Although synthesis of PSII proteins was not altered significantly, both mutants accumulated only ∼25% of PSII proteins compared with the wild type. Assembly of PSII precomplexes occurred at normal rates, but heterodimeric PSII reaction centers (RCs) and higher order PSII assemblies were not formed efficiently in the mutants. The psbN-R mutant was complemented by allotopic expression of the PsbN gene fused to the sequence of a chloroplast transit peptide in the nuclear genome. PsbN represents a bitopic trans-membrane peptide localized in stroma lamellae with its highly conserved C terminus exposed to the stroma. Significant amounts of PsbN were already present in dark-grown seedling. Our data prove that PsbN is not a constituent subunit of PSII but is required for repair from photoinhibition and efficient assembly of the PSII RC.

Understanding the molecular basis of how new species arise is a central question and prime challenge in evolutionary biology and includes understanding how genomes diversify. Eukaryotic cells possess an integrated compartmentalized genetic system of endosymbiotic ancestry. The cellular subgenomes in nucleus, mitochondria and plastids communicate in a complex way and co-evolve. The application of hybrid and cybrid technologies, most notably those involving interspecific exchanges of plastid and nuclear genomes, has uncovered a multitude of species-specific nucleo-organelle interactions. Such interactions can result in plastome-genome incompatibilities, which can phenotypically often be recognized as hybrid bleaching, hybrid variegation or disturbance of the sexual phase. The plastid genome, because of its relatively low number of genes, can serve as a valuable tool to investigate the origin of these incompatibilities. In this article, we review progress on understanding how plastome-genome co-evolution contributes to speciation. We genetically classify incompatible phenotypes into four categories. We also summarize genetic, physiological and environmental influence and other possible selection forces acting on plastid-nuclear co-evolution and compare taxa providing molecular access to the underlying loci. It appears that plastome-genome incompatibility can establish hybridization barriers, comparable to the Dobzhansky-Muller model of speciation processes. Evidence suggests that the plastid-mediated hybridization barriers associated with hybrid bleaching primarily arise through modification of components in regulatory networks, rather than of complex, multisubunit structures themselves that are frequent targets.

Salient features of the first meiotic division are independent segregation of chromosomes and homologous recombination (HR). In non-sexually reproducing, homozygous species studied to date HR is absent. In this study, we constructed the first linkage maps of homozygous, bivalent-forming Oenothera species and provide evidence that HR was exclusively confined to the chromosome ends of all linkage groups in our population. Co-segregation of complementary DNA-based markers with the major group of AFLP markers indicates that HR has only a minor role in generating genetic diversity of this taxon despite its efficient adaptation capability. Uneven chromosome condensation during meiosis in Oenothera may account for restriction of HR. The use of plants with ancient chromosomal arm arrangement demonstrates that limitation of HR occurred before and independent from species hybridizations and reciprocal translocations of chromosome arms—a phenomenon, which is widespread in the genus. We propose that consecutive loss of HR favored the evolution of reciprocal translocations, beneficial superlinkage groups and ultimately permanent translocation heterozygosity.

Plastid genomes (plastomes) are part of the integrated compartmentalised genetic system of photoautotrophic eukaryotes. They are highly redundant and generally dispersed in several regions (nucleoids) within organelles. DNA quantities and number of DNA-containing regions per plastid vary and are developmentally regulated in a way not yet understood. Reliable quantitative data describing these patterns are scarce. We present a protocol to isolate fractions of pure plastids with varying average sizes from leaflets (≤1 mm) and leaves of different developmental stages continuously up to maturity (25 cm) from Beta vulgaris L. (sugar beet) to determine DNA amounts per organelle. The approach is based on plastid purification from homogenates of moderately fixed tissue by differential and isopycnic gradient centrifugations and on application of two different DNA specific colorimetric reactions after removing potentially interfering compounds. The sensitive fluorochrome DAPI (4′,6-diamidino-2-phenylindole) was used to estimate numbers and emission intensity of nucleoids per plastid. The amounts determined ranged from 0.15 to 4.9 × 10 −2 pg DNA for plastids of 1→8 μm average diameter, corresponding from approximately a dozen to 330 genome equivalents per organelle and on average four to seven copies per nucleoid. The ratio of plastid/nuclear DNA changed continuously during leaf development from as little as 0.4% to about 20% in fully developed leaves. On the other hand, mesophyll cells of mature leaves differing in ploidy (di-, tri- and tetraploid) appeared to maintain a relatively constant nuclear genome/plastome ratio, equivalent to about 1,700 copies per C -value.

The characterisation of transcript levels of chloroplast genes and their changes under different conditions is an initial step towards understanding chloroplast gene expression and the functional integration of the plastid chromosome into the entire integrated compartmentalised genome of the plant cell. Using RNA from cells of 12 different developmental stages and stress treatments, we have studied the transcript patterns of all 96 genes of the circular plastid chromosome of Euglena gracilis, Pringsheim strain Z, by a macroarray-based approach and Northern analysis of selected genes representing approximately half a dozen operons. The unicellular alga possesses complex, triple-envelope chloroplasts that were acquired by secondary endosymbiosis. (1) Transcripts were detected from all genes, although stationary concentrations varied substantially between individual loci. No obvious economy in the expression pattern with respect to transcription units and genes for complex structures was noted. (2) The chromosome appears to be constitutively expressed under all chosen conditions including stresses such as UV light, temperature, antiplastidial agents, herbicide and heavy metal exposure. (3) The euglenoid organelle transcriptome is qualitatively relatively insensitive to the environment, but exhibited marked overall quantitative changes. The more or less global changes demonstrate that primarily RNA turnover, translational, proteolytic and/or metabolic control regulate organelle gene expression in the alga.

The flowering plant genus Oenothera is uniquely suited for studying molecular mechanisms of speciation. It assembles an intriguing combination of genetic features, including permanent translocation heterozygosity, biparental transmission of plastids, and a general interfertility of well-defined species. This allows an exchange of plastids and nuclei between species often resulting in plastome– genome incompatibility. For evaluation of its molecular determinants we present the complete nucleotide sequences of the five basic, genetically distinguishable plastid chromosomes of subsection Oenothera (=Euoenothera) of the genus, which are associated in distinct combinations with six basic genomes. Sizes of the chromosomes range from 163365bp (plastome IV) to 165 728bp (plastome I), display between 96.3% and 98.6% sequence similarity and encode a total of 113 unique genes. Plastome diversification is caused by an abundance of nucleotide substitutions, small insertions, deletions and repetitions. The five plastomes deviate from the general ancestral design of plastid chromosomes of vascular plants by a subsectionspecific 56 kb inversion within the large single-copy segment. This inversion disrupted operon structures and predates the divergence of the subsection presumably 1 My ago. Phylogenetic relationships suggest plastomes I–III in one clade, while plastome IV appears to be closest to the common

A unique combination of genetic features and a rich stock of information make the flowering plant genus Oenothera an appealing model to explore the molecular basis of speciation processes including nucleus-organelle coevolution. From representative species, we have recently reported complete nucleotide sequences of the 5 basic and genetically distinguishable plastid chromosomes of subsection Oenothera (I-V). In nature, Oenothera plastid genomes are associated with 6 distinct, either homozygous or heterozygous, diploid nuclear genotypes of the 3 basic genomes A, B, or C. Artificially produced plastome-genome combinations that do not occur naturally often display interspecific plastome-genome incompatibility (PGI). In this study, we compare formal genetic data available from all 30 plastome-genome combinations with sequence differences between the plastomes to uncover potential determinants for interspecific PGI. Consistent with an active role in speciation, a remarkable number of genes have high Ka/Ks ratios. Different from the Solanacean cybrid model Atropa/tobacco, RNA editing seems not to be relevant for PGIs in Oenothera. However, predominantly sequence polymorphisms in intergenic segments are proposed as possible sources for PGI. A single locus, the bidirectional promoter region between psbB and clpP, is suggested to contribute to compartmental PGI in the interspecific AB hybrid containing plastome I (AB-I), consistent with its perturbed photosystem II activity.

The genus Oenothera has an outstanding scientific tradition. It has been a model for studying aspects of chromosome evolution and speciation, including the impact of plastid nuclear co-evolution. A large collection of strains analyzed during a century of experimental work and unique genetic possibilities allow the exchange of genetically definable plastids, individual or multiple chromosomes, and/or entire haploid genomes (Renner complexes) between species. However, molecular genetic approaches for the genus are largelylacking. In this study, we describe the development of efficient PCR-based marker systems for both the nuclear genome and the plastome. They allow distinguishing individual chromosomes, Renner complexes, plastomes, and subplastomes. We demonstrate their application by monitoring interspecific exchanges of genomes, chromosome pairs, and/or plastids during crossing programs, e.g., to produce plastome-genome incompatible hybrids. Using an appropriate partial permanent translocation heterozygous hybrid, linkage group 7 of the molecular map could be assigned to chromosome 9.8 of the classical Oenothera map. Finally, we provide the first direct molecular evidence that homologous recombination and free segregation of chromosomes in permanent. translocation heterozygous strains is suppressed.

Photosystem II (PSII) of oxygen-evolving cyanobacteria, algae, and land plants mediates electron transfer from the Mn4Ca cluster to the plastoquinone pool. It is a dimeric supramolecular complex comprising more than 30 subunits per monomer, of which 16 are bitopic or peripheral, low-molecular-weight components. Directed inactivation of the plastid gene encoding the low-molecular-weight peptide PsbTc in tobacco (Nicotiana tabacum) does not prevent photoautotrophic growth. Mutant plants appear normal green, and levels of PSII proteins are not affected. Yet, PSII-dependent electron transport, stability of PSII dimers, and assembly of PSII light-harvesting complexes (LHCII) are significantly impaired. PSII light sensitivity is moderately increased and recovery from photoinhibition is delayed, leading to faster D1 degradation in ΔpsbTc under high light. Thermoluminescence emission measurements revealed alterations of midpoint potentials of primary/secondary electron-accepting plastoquinone of PSII interaction. Only traces of CP43 and no D1/D2 proteins are phosphorylated, presumably due to structural changes of PSII in ΔpsbTc. In striking contrast to the wild type, LHCII in the mutant is phosphorylated in darkness, consistent with its association with PSI, indicating an increased pool of reduced plastoquinone in the dark. Finally, our data suggest that the secondary electron-accepting plastoquinone of PSII site, the properties of which are altered in ΔpsbTc, is required for oxidation of reduced plastoquinone in darkness in an oxygen-dependent manner. These data present novel aspects of plastoquinone redox regulation, chlororespiration, and redox control of LHCII phosphorylation.

The genus Oenothera shows an intriguing extent of permanent translocation heterozygosity. Reciprocal translocations of chromosome arms in species or populations result in various kinds of chromosome multivalents in diakinesis. Early meiotic events conditioning such chromosome behaviour are poorly understood. We found a surprising uniformity of the leptotene-diplotene period, regardless of the chromosome configuration at diakinesis (ring of 14, 7 bivalents, mixture of bivalents and multivalents). It appears that the earliest chromosome interactions at Oenothera meiosis are untypical, since they involve pericentromeric regions. During early leptotene, proximal chromosome parts cluster and form a highly polarized Rabl configuration. Telomeres associated in pairs were seen at zygotene. The high degree of polarization of meiotic nuclei continues for an exceptionally long period, i.e., during zygotene-pachytene into the diplotene contraction stage. The Rabl-polarized meiotic architecture and clustering of pericentromeres suggest a high complexity of karyotypes, not only in structural heterozygotes but also in bivalent-forming homozygous species.

Higher plant chloroplast genomes code for a conserved set of 30 tRNAs. This set is believed to be sufficient to support translation, although import of cytosolic tRNA has been proposed to provide additional tRNA species to the chloroplast. Previous knock-outs of tRNA genes, or the pronounced reduction of the level of selected tRNAs, has not led to severe phenotypes. We deleted the two tRNA genes trnN-GUU and trnC-GCA independently from the plastid chromosome of tobacco. No homoplastomic tissue of either Delta trnN or Delta trnC plants could be isolated. Both mutants exhibit occasional loss of leaf sectors, and mutant plastid chromosomes are rapidly lost upon relief of selective pressure. This suggests that the knock-out of both trn genes is lethal, and that both tRNA species are required for cell survival. Surprisingly, the impact on chloroplast and cell development differs pronouncedly between the two mutants. Heteroplastomic Delta trnC and Delta trnN tissue exhibit different aberrations of the internal membrane systems and, more importantly, heteroplastomic Delta trnN plants are variegated. Accumulation of tRNA-N and plastid-encoded proteins is reduced in white sectors of Delta trnN plants, and differentiation of palisade cells is abolished. Our data demonstrate that plastid tRNAs are essential, i.e. not complemented by cytosolic tRNA, and have a differential impact on chloroplast and plant cell development.

Photosystem II, the oxygen-evolving complex of photosynthetic organisms, includes an intriguingly large number of low molecular weight polypeptides, including PsbM. Here we describe the first knock-out of psbM using a transplastomic, reverse genetics approach in a higher plant. Homoplastomic ΔpsbM plants exhibit photoautotrophic growth. Biochemical, biophysical, and immunological analyses demonstrate that PsbM is not required for biogenesis of higher order photosystem II complexes. However, photosystem II is highly light-sensitive, and its activity is significantly decreased in ΔpsbM, whereas kinetics of plastid protein synthesis, reassembly of photosystem II, and recovery of its activity are comparable with the wild type. Unlike wild type, phosphorylation of the reaction center proteins D1 and D2 is severely reduced, whereas the redox-controlled phosphorylation of photosystem II light-harvesting complex is reversely regulated in ΔpsbM plants because of accumulation of reduced plastoquinone in the dark and a limited photosystem II-mediated electron transport in the light. Charge recombination in ΔpsbM measured by thermoluminescence oscillations significantly differs from the 2/6 patterns in the wild type. A simulation program of thermoluminescence oscillations indicates a higher QB/Q –B ratio in dark-adapted mutant thylakoids relative to the wild type. The interaction of the QA/QB sites estimated by shifts in the maximal thermoluminescence emission temperature of the Q band, induced by binding of different herbicides to the QB site, is changed indicating alteration of the activation energy for back electron flow. We conclude that PsbM is primarily involved in the interaction of the redox components important for the electron flow within, outward, and backward to photosystem II. Photosystem II, the oxygen-evolving complex of photosynthetic organisms, includes an intriguingly large number of low molecular weight polypeptides, including PsbM. Here we describe the first knock-out of psbM using a transplastomic, reverse genetics approach in a higher plant. Homoplastomic ΔpsbM plants exhibit photoautotrophic growth. Biochemical, biophysical, and immunological analyses demonstrate that PsbM is not required for biogenesis of higher order photosystem II complexes. However, photosystem II is highly light-sensitive, and its activity is significantly decreased in ΔpsbM, whereas kinetics of plastid protein synthesis, reassembly of photosystem II, and recovery of its activity are comparable with the wild type. Unlike wild type, phosphorylation of the reaction center proteins D1 and D2 is severely reduced, whereas the redox-controlled phosphorylation of photosystem II light-harvesting complex is reversely regulated in ΔpsbM plants because of accumulation of reduced plastoquinone in the dark and a limited photosystem II-mediated electron transport in the light. Charge recombination in ΔpsbM measured by thermoluminescence oscillations significantly differs from the 2/6 patterns in the wild type. A simulation program of thermoluminescence oscillations indicates a higher QB/Q –B ratio in dark-adapted mutant thylakoids relative to the wild type. The interaction of the QA/QB sites estimated by shifts in the maximal thermoluminescence emission temperature of the Q band, induced by binding of different herbicides to the QB site, is changed indicating alteration of the activation energy for back electron flow. We conclude that PsbM is primarily involved in the interaction of the redox components important for the electron flow within, outward, and backward to photosystem II. Deletion of PsbM in tobacco alters the QB site properties and the electron flow within photosystem II.Journal of Biological ChemistryVol. 293Issue 29PreviewVOLUME 282 (2007) PAGES 9758–9767 Full-Text PDF Open Access Photosystem II (PSII), 5The abbreviations used are: PSI and PSII, photosystem I and II, respectively; BN, blue native; CP, chlorophyll-protein; DCMU, 3(3,4-dichlorophenyl)-1,1-dimethylurea; LHC, light-harvesting complex of photosystem II; LMWs, low molecular weight subunits; PQ, plastoquinone; PQH2, plastoquinol; TL, thermoluminescence; WT, wild type; bis-Tris, 2-[bis(2-hydroxyethyl) amino]-2-(hydroxymethyl)propane-1,3-diol; NPQ, nonphotochemical quenching; qP, photochemical quenching; E, einstein. a supramolecular pigment-protein complex of photosynthetic organisms, utilizes absorbed light energy to oxidize water, releasing dioxygen and electrons that serve as the major source of reducing power in photosynthetic activity. The mechanisms of this process have been studied extensively. Based on biochemical (reviewed in Ref. 1Nelson N. Yocum C.F. Annu. Rev. Plant Biol. 2006; 57: 521-565Crossref PubMed Scopus (728) Google Scholar), biophysical (2Vrettos J.S. Brudvig G.W. Philos. Trans. R. Soc. Lond. B Biol. Sci. 2002; 357: 1395-1404Crossref PubMed Scopus (49) Google Scholar, 3Clausen J. Debus R.J. Junge W. Biochim. Biophys. Acta. 2004; 1655: 184-194Crossref PubMed Scopus (64) Google Scholar), and structural analysis, including electron microscopy (4Hankamer B. Morris E. Nield J. Gerle C. Barber J. J. Struct. Biol. 2001; 135: 262-269Crossref PubMed Scopus (76) Google Scholar, 5Dekker J.P. Boekema E.J. Biochim. Biophys. Acta. 2005; 1706: 12-39Crossref PubMed Scopus (676) Google Scholar) and x-ray diffraction (6Kamiya N. Shen J.R. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 98-103Crossref PubMed Scopus (994) Google Scholar, 7Biesiadka J. Loll B. Kern J. Irrgang K.D. Zouni A. Phys. Chem. Chem. 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Chem. 2006; 281: 14981-14990Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar), as well as on the components involved in the biogenesis of PSII (12Meurer J. Plücken H. Kowallik K.V. Westhoff P. EMBO J. 1998; 17: 5286-5297Crossref PubMed Scopus (178) Google Scholar, 13Ossenbühl F. Inaba-Sulpice M. Meurer J. Soll J. Eichacker L.A. Plant Cell. 2004; 18: 2236-2246Crossref Scopus (50) Google Scholar, 14Peng L. Ma J. Chi W. Guo J. Zhu S. Lu Q. Lu C. Zhang L. Plant Cell. 2006; 18: 955-969Crossref PubMed Scopus (184) Google Scholar). Most if not all genes encoding PSII subunits have been identified in cyanobacteria and higher plants, and their function, expression, and regulation have been studied extensively (15Rochaix J.D. Plant Physiol. 2001; 125: 142-144Crossref PubMed Scopus (46) Google Scholar, 16Shi L.-X. Schröder W.P. Biochim. Biophys. Acta. 2004; 1608: 75-96Crossref PubMed Scopus (131) Google Scholar, 17Thornton L.E. Roose J.L. Pakrasi H.B. Ikeuchi M. Wydrzynski T. Satoh K. Photosystem II: The Water/Plastoquinone Oxidoreductase in Photosynthesis. Kluwer Academic Publishers Group, Dordrecht, The Netherlands2005: 121-138Google Scholar). PSII is the most intricate assembly of thylakoid membrane systems consisting of more than 30 subunits. It assembles as a dimer together with the minor light-harvesting antenna CP24, CP26, and CP29 for each monomer and is further surrounded by trimeric LHCII protein complexes (5Dekker J.P. Boekema E.J. Biochim. Biophys. Acta. 2005; 1706: 12-39Crossref PubMed Scopus (676) Google Scholar). Based on the similarity of subunit sequences, composition, and activity of PSII, it is generally accepted that the structure of the PSII core in eukaryotes is basically similar to that of cyanobacteria, for which x-ray diffraction structures between 3.8 and 3.0 Å resolution have been obtained (8Ferreira K.N. Iverson T.M. Maghlaoui K. Barber J. Iwata S. Science. 2004; 303: 1831-1837Crossref PubMed Scopus (2854) Google Scholar, 9Loll B. Kern J. Saenger W. Zouni A. Biesiadka J. Nature. 2005; 438: 1040-1044Crossref PubMed Scopus (1611) Google Scholar). However, despite sustained attempts to obtain a higher structural resolution for the PSII of higher plants (10Liu Z. Yan H. Wang K. Kuang T. Zhang J. Gui L. An X. Chang W. Nature. 2004; 428: 287-292Crossref PubMed Scopus (1380) Google Scholar), the heterogeneity of the photochemical center of PSII caused by light-induced changes has so far prevented the formation of crystals allowing a higher resolution of the complex. One of the most intriguing features of the PSII core is the presence of 16 bitopic, intrinsic, or peripheral low molecular weight proteins. Knowledge about their roles in the overall photosynthetic process is still fragmentary. In eukaryotic PSII, 11 of them are encoded by plastid chromosomes, notably PsbE, -F, -H, -I, -J, -K, -L, -M, -N, -Tc, and -Z (reviewed in Refs. 5Dekker J.P. Boekema E.J. Biochim. Biophys. Acta. 2005; 1706: 12-39Crossref PubMed Scopus (676) Google Scholar and 16Shi L.-X. Schröder W.P. Biochim. Biophys. Acta. 2004; 1608: 75-96Crossref PubMed Scopus (131) Google Scholar, 17Thornton L.E. Roose J.L. Pakrasi H.B. Ikeuchi M. Wydrzynski T. Satoh K. Photosystem II: The Water/Plastoquinone Oxidoreductase in Photosynthesis. Kluwer Academic Publishers Group, Dordrecht, The Netherlands2005: 121-138Google Scholar, 18Nelson N. Ben-Shem A. Nat. Rev. Mol. Cell Biol. 2004; 12: 971-982Crossref Scopus (421) Google Scholar, 19Minagawa J. Takahashi Y. Photosynth. Res. 2004; 82: 241-263Crossref PubMed Google Scholar). The fact that most low molecular weight subunits (LMWs) of PSII have been highly conserved throughout the evolution implies that they perform essential functions, as indeed this has been established for PsbI, PsbT, the α and β subunits of the two-chain cytochrome b559, PsbE and PsbF, respectively, as well as for PsbL and PsbJ, which fulfill crucial structural and functional roles (20Whitmarsh J. Pakrasi H.B. Ort D.R. Yocum C.F. Oxygenic Photosynthesis: The Light Reactions. Kluwer Academic Publishers Group, Dordrecht, The Netherlands1996: 249-264Google Scholar, 21Stewart D.H. Brudvig G.W. Biochim. Biophys. Acta. 1998; 1367: 63-87Crossref PubMed Scopus (216) Google Scholar, 22Bondarava N. De Pascalis L. Al-Babili S. Goussias C. Golecki J.R. Beyer P. Bock R. Krieger-Liszkay A. J. Biol. Chem. 2003; 278: 13554-13560Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 23Regel R.E. Ivleva N.B. Zer H. Meurer J. Shestakov S.V. Herrmann R.G. Pakarasi H.B. Ohad I. J. Biol. 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However, only limited information is available on the function and exact position of the other LMWs within the PSII complex. Moreover, studies on distinct LMWs from various organisms have indicated different roles (reviewed in Refs. 16Shi L.-X. Schröder W.P. Biochim. Biophys. Acta. 2004; 1608: 75-96Crossref PubMed Scopus (131) Google Scholar, 17Thornton L.E. Roose J.L. Pakrasi H.B. Ikeuchi M. Wydrzynski T. Satoh K. Photosystem II: The Water/Plastoquinone Oxidoreductase in Photosynthesis. Kluwer Academic Publishers Group, Dordrecht, The Netherlands2005: 121-138Google Scholar) or control of the same function, albeit to a different degree, as is the case for PsbJ in Synechocystis sp. PCC 6803 and tobacco (23Regel R.E. Ivleva N.B. Zer H. Meurer J. Shestakov S.V. Herrmann R.G. Pakarasi H.B. Ohad I. J. Biol. Chem. 2001; 276: 41473-41478Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Apparently, the management of PSII electron flow in terms of energy dissipation and avoiding generation of oxygen radicals has to cope with different requirements in various organisms. Such differences may depend on the eco-physiological conditions under which different organisms thrive and the divergence of the outer antenna. It is therefore conceivable that the properties of the LMWs of PSII vary in different organisms. The study of all chloroplast-encoded LMWs in one organism offers a better chance to understand the roles for each one of them in the same assembly. Therefore, we have inactivated plastid genes encoding LMWs of PSII using a transplastomic approach in tobacco, and we reported on the roles of six of them, including PsbE, PsbF, PsbL, PsbJ, PsbI, and PsbZ (23Regel R.E. Ivleva N.B. Zer H. Meurer J. Shestakov S.V. Herrmann R.G. Pakarasi H.B. Ohad I. J. Biol. Chem. 2001; 276: 41473-41478Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 24Ohad I. Dal Bosco C. Herrmann R.G. Meurer J. Biochemistry. 2004; 43: 2297-2308Crossref PubMed Scopus (44) Google Scholar, 25Schwenkert S. Umate P. Dal Bosco C. Volz S. Mlèochová L. Zoryan M. Eichacker L.A. Ohad I. Herrmann R.G. Meurer J. J. Biol. Chem. 2006; 281: 34227-34238Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 26Swiatek M. Kuras R. Sokolenko A. Higgs D. Olive J. Cinque G. Müller B. Eichacker L.A. Stern D.B. Bassi R. Herrmann R.G. Wollmann F.A. Plant Cell. 2001; 13: 1347-1367Crossref PubMed Google Scholar, 27Ohnishi N. Kashino Y. Satoh K. Ozawa S.I. Takahashi Y. J. Biol. Chem. 2007; 28210.1074/jbc.M606763200Google Scholar, 28Swiatek M. Regel R.E. Meurer J. Wanner G. Pakarasi H.B. Ohad I. Herrmann R.G. Mol. Genet. Genomics. 2003; 268: 699-710Crossref PubMed Scopus (59) Google Scholar). The PsbM polypeptide has been detected in PSII complexes isolated from Chlamydomonas reinhardtii (29de Vitry C. Diner B.A. Popo J.L. J. Biol. Chem. 1991; 266: 16614-16621Abstract Full Text PDF PubMed Google Scholar), Synechocystis sp. PCC 6803 (30Ikeuchi M. Inoue Y. Vermaas W. Mathis P. Photosynthesis: From Light to Biosphere. Kluwer Academic Publishers Group, Dordrecht, The Netherlands1995: 297-300Google Scholar), and Synechococcus vulcanus (31Ikeuchi M. Koike H. Inoue Y. FEBS Lett. 1989; 253: 178-182Crossref PubMed Scopus (45) Google Scholar), and its presence in Synechocystis sp. PCC 6803 (32Kashino Y. Lauber W.M. Carroll J.A. Wang Q. Whitmarsh J. Satoh K. Pakarasi H.B. Biochemistry. 2002; 41: 8004-8012Crossref PubMed Scopus (279) Google Scholar) and pea (33Gomez S.M. Nishio J.N. Faull K.F. Whitelegge J.P. Mol. Cell. Proteomics. 2002; 1: 46-59Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar) has been confirmed recently using proteomics approaches. However, mutation studies of PsbM have not yet been reported from any organism. Moreover, the exact position of PsbM within the PSII assembly as well as its function remains to be established (8Ferreira K.N. Iverson T.M. Maghlaoui K. Barber J. Iwata S. Science. 2004; 303: 1831-1837Crossref PubMed Scopus (2854) Google Scholar, 17Thornton L.E. Roose J.L. Pakrasi H.B. Ikeuchi M. Wydrzynski T. Satoh K. Photosystem II: The Water/Plastoquinone Oxidoreductase in Photosynthesis. Kluwer Academic Publishers Group, Dordrecht, The Netherlands2005: 121-138Google Scholar). The analyses of the first PsbM knock-out presented here demonstrate that the biogenesis of PSII is not significantly altered in the absence of PsbM. However, properties of the QB site and its interaction with QA and charge recombination within PSII are specifically impaired in ΔpsbM resulting in a shift of thermoluminescence (TL) B band oscillations, a decreased rate of oxygen evolution and forward electron flow, and thus an increased light-induced photoinactivation as well as dephosphorylation of LHCII. Because of a high plastoquinol (PQH2) content in dark-adapted mutant plants, levels of LHCII phosphorylation are significantly elevated as compared with the wild type. Phosphorylation of the reaction center proteins D1/D2 is faintly detectable presumably because of conformational changes induced by loss of PsbM proteins. Clone Construction to Inactivate the psbM Gene in Tobacco Chloroplasts—The recombinant plasmid B20 (tobacco plastome clone bank) (34Sugiura M. Shinozaki K. Zaita N. Kusuda M. Kumano M. Plant Sci. 1986; 44: 211-216Crossref Scopus (183) Google Scholar) containing a 17,235-bp insertion (nucleotide positions 26,191–43,426 in the plastid chromosome, accession number Z00044) in the vector pBR322 was digested with BamHI, and the resulting 2,096-bp fragment containing psbM was subcloned into the singular BamHI restriction site of pBluescript II KS– (Stratagene Inc., La Jolla, CA). Inactivation of the psbM gene (nucleotide position 30,861 (N) to 30,757 (C) bp, accession number Z00044) was achieved by insertion of the aadA cassette, including a terminator signal (35Koop H.U. Steinmüller K. Wagner H. Rossler C. Eibl C. Sacher L. Planta. 1996; 199: 193-201Crossref PubMed Scopus (137) Google Scholar) at a unique BsgI restriction site in the N-terminal part of the gene (nucleotide position 30,844). Transformation of Nicotiana tabacum cv. Petit Havanna, the selection procedure, and in vitro propagation of transformants were carried out essentially as described (25Schwenkert S. Umate P. Dal Bosco C. Volz S. Mlèochová L. Zoryan M. Eichacker L.A. Ohad I. Herrmann R.G. Meurer J. J. Biol. Chem. 2006; 281: 34227-34238Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Isolation of plastid chromosomes by orthogonal pulse-field gel electrophoresis was carried out as described (36Swiatek M. Greiner S. Kemp S. Drescher A. Koop H.U. Herrmann R.G. Maier R.M. Curr. Genet. 2003; 43: 45-53Crossref PubMed Scopus (18) Google Scholar). Tobacco lines carrying the aadA cassette in a neutral insertion site and referred to as RV plants were used as wild type (WT) control plants (25Schwenkert S. Umate P. Dal Bosco C. Volz S. Mlèochová L. Zoryan M. Eichacker L.A. Ohad I. Herrmann R.G. Meurer J. J. Biol. Chem. 2006; 281: 34227-34238Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Preparation and Handling of Thylakoid Membranes—Thylakoid membranes isolated from 4-week-old plants grown under greenhouse conditions were chosen for immunoblot analysis, phosphorylation experiments, and analysis of their composition by the blue native gel (BN) method as described (25Schwenkert S. Umate P. Dal Bosco C. Volz S. Mlèochová L. Zoryan M. Eichacker L.A. Ohad I. Herrmann R.G. Meurer J. J. Biol. Chem. 2006; 281: 34227-34238Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). For separation of photosynthetic chlorophyll-protein complexes by sucrose gradient centrifugation, 4-week-old in vitro grown (10–20 μEm–2 s–1 light intensity; 12-h photoperiod) plants were used. Isolated thylakoid membranes (final chlorophyll concentration 1 mg/ml) were partially lysed during an incubation of 45 min on ice in darkness in bis-Tris/n-dodecyl-β-d-maltoside buffer (final concentrations of 20 mm bis-Tris/HCl, pH 6.5, 5 mm NaCl, 5 mm MgCl2, and 1.0% n-dodecyl-β-d-maltoside). After centrifugation at 18,000 × g and 4 °C for 12 min, the solubilized fraction was layered onto a linear (0.1–1.0 m) sucrose gradient in bis-Tris/n-dodecyl-β-d-maltoside buffer. The gradient was run at 4 °C for 18 h in a Beckman SW40Ti rotor at 200,000 × g. Blue native-PAGE (BN-PAGE) was performed as described earlier with modifications (25Schwenkert S. Umate P. Dal Bosco C. Volz S. Mlèochová L. Zoryan M. Eichacker L.A. Ohad I. Herrmann R.G. Meurer J. J. Biol. Chem. 2006; 281: 34227-34238Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). The appearing spots were sequenced by mass spectrometry and assigned accordingly (37Granvogl B. Reisinger V. Eichacker L.A. Proteomics. 2006; 6: 3681-3695Crossref PubMed Scopus (53) Google Scholar). For TL measurements, thylakoids were prepared by grinding a few leaves in a buffer containing 20 mm Tris-HCl, pH 7.4, 5 mm MgCl2, 20 mm NaCl, and 100 mm sorbitol. The material homogenized at 0 °C was filtered through nylon micromeshes and used immediately for measurements. Chlorophyll a Fluorescence Induction Kinetics—Chlorophyll a fluorescence induction kinetics was measured using a pulse amplitude-modulated fluorimeter (PAM-101, Waltz, Effeltrich, Germany) (38Schreiber U. Bilger W. Hormannn H. Neubauer C. Raghavendra A.S. Photosynthesis: A Comprehensive Treatise. Cambridge University Press, Cambridge, UK1998: 320-336Google Scholar). Prior to measurements, leaves were dark-adapted for 5 min. The potential maximum quantum yield of PSII was measured as (Fm – Fo)/Fm = Fv/Fm. Red actinic light (650 nm, 20 and 250 μEm–2 s–1) was used for measurements of fluorescence quenching. Photochemical (qP) and nonphotochemical (NPQ) quenching were determined by repetitive saturation pulses. The quenching coefficients, NPQ and qP, were calculated as (Fm – Fm′)/Fm′ and (Fm′– F)/(Fm ′– Fo), respectively (38Schreiber U. Bilger W. Hormannn H. Neubauer C. Raghavendra A.S. Photosynthesis: A Comprehensive Treatise. Cambridge University Press, Cambridge, UK1998: 320-336Google Scholar). State Transition and Thylakoid Protein Phosphorylation— State transition in intact leaves was calculated using the PAM-101 fluorimeter as (Fm′– Fm″)/Fm′ (39Lunde C. Jensen P.E. Haldrup A. Knoetzel J. Scheller H.V. Nature. 2000; 30: 613-615Crossref Scopus (287) Google Scholar). Protein phosphorylation was carried out using isolated thylakoids as described (25Schwenkert S. Umate P. Dal Bosco C. Volz S. Mlèochová L. Zoryan M. Eichacker L.A. Ohad I. Herrmann R.G. Meurer J. J. Biol. Chem. 2006; 281: 34227-34238Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 40Zer H. Vink M. Shochat S. Herrmann R.G. Andersson B. Ohad I. Biochemistry. 2003; 42: 728-738Crossref PubMed Scopus (43) Google Scholar). All buffers used during thylakoid preparation contained 10 mm NaF. Detection of the phosphorylation level of thylakoid membrane proteins was carried out by immunoblotting using anti-phosphothreonine antibodies (New England Biolabs) as described earlier (25Schwenkert S. Umate P. Dal Bosco C. Volz S. Mlèochová L. Zoryan M. Eichacker L.A. Ohad I. Herrmann R.G. Meurer J. J. Biol. Chem. 2006; 281: 34227-34238Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Low Temperature Fluorescence Measurements—Thylakoid suspensions (20 μg of chlorophyll/ml) of WT and mutant leaves were frozen by immersing a liquid nitrogen-cooled glass rod (4 mm diameter) into the thylakoid suspension and rapidly returning it to the Dewar vessel of the sample holder of the fluorimeter filled with liquid nitrogen (Fluoromax-3, Horiba Jobin-Yvon, France). Fluorescence emission spectra were recorded using 430 nm excitation and 1.5 nm slits for both excitation and emission monochromators. Photosystem I (PSI) Redox State—The redox state of PSI was measured on leaves using the PSI attachment of PAM101 (Walz, Effeltrich, Germany). The oxidation status of PSI at the light intensities indicated was expressed as the ratio ΔA/ΔAmax (41Klughammer C. Schreiber U. Planta. 1994; 192: 261-268Crossref Google Scholar). Thermoluminescence Measurements—TL of thylakoid suspensions was measured using a home-built apparatus as described (42Zer H. Prasil O. Ohad I. J. Biol. Chem. 1994; 269: 17670-17676Abstract Full Text PDF PubMed Google Scholar). Samples (400 μl) of 40 μg of chlorophyll/ml were placed on the TL stage, dark-adapted at 20 °C for 3 min, and rapidly frozen to –20 °C by a stream of liquid nitrogen. The sample was then excited by saturating flashes delivered by a xenon arc discharge lamp (0.05 microfarad capacitor, charged at 1000 V, 3 μs at 70% light emission; EG and G), then heated at a rate of 0.6 °C s–1, and photons were counted. For measurements of the intensity of the B band emission oscillations (Q –B/S2, S3 charge recombination) as a function of the numbers of single turnover excitations, a train of flashes (1–6 flashes, about 300-ms interval between flashes) was given at 0 °C followed by rapid freezing. For detecting the TL signal resulting from QA–/S2 recombination (Q band), the herbicides DCMU or ioxynil (Serva, Heidelberg, Germany), both binding to the QB site and thus preventing QA– oxidation, were added at concentrations as indicated before dark adaptation. Concentrations of ioxynil higher than 20 μm were avoided because of the high fluorescence quenching induced by this herbicide. Glycerol (25% v/v) was added to the samples to avoid distortion of the linear heating rate during the ice-melting stage. The kinetics of the QB– decay in darkness was measured following excitation of the dark-adapted sample by a single flash at 20 °C, followed by further incubation in darkness for the indicated periods, and followed by rapid freezing to –20 °C and starting the heating of the sample and photon-counting process. A computer-based simulation program allowing the prediction of the S-states ratio and the occupancy ratio QB/QB– was employed (supplemental S1). The program simulates predicted oscillation profiles and checks for the correlation between simulated and measured values. The free parameters in the simulation are as follows: 1) the S-states and QB/QB– occupancy levels in the dark adapted state, which can vary between 0 and 1 in 0.1 steps; 2) the misses, i.e. the fraction of reaction centers that is not excited by a flash, which may vary between 0 and 0.5; and 3) the double hits, i.e. the fraction of reaction centers that are excited twice by a single flash, which may vary between 0 and 0.5. The value ∑1–6|intensitymeasured – intensitysimulated|, where 1–6 represents the number of flashes, was used to estimate the correlation between measured and simulated results. The program used is given in the supplemental S1. Minimization of the cost function was performed by a simulated annealing algorithm (43Corana A. Marchesi M. Martini C. Ridella S. ACM Transactions on Mathematical Software. 1987; 13: 262-280Crossref Scopus (1259) Google Scholar). Oxygen Evolution Measurements—Photosynthetic electron flow was determined using thylakoids isolated as described for TL measurements. The PSII specific electron acceptor p-benzoquinone was used under saturating light conditions using a Clark-type oxygen electrode for measuring oxygen evolution. Measurements of Photoinhibition and Recovery Process—The sensitivity of PSII to oxidative stress has been determined with leaf disks (10 mm diameter, 5 disks per sample) of WT and ΔpsbM plants exposed to 500 μEm–2 s–1 heterochromatic light. The photoinactivation of PSII was measured as changes in the Fv/Fm parameter as a function of exposure time. To estimate the contribution of the PSII recovery process during treatment with high light, leaf disks were infiltrated with a solution of d-threo-chloramphenicol (200 μgml–1) in darkness for 30 min prior to the exposure to high light. As a control, leaf disks were incubated in water. To assess the inhibition kinetics and the capacity to recover PSII activity, leaf disks were exposed to 1,500 μEm–2 s–1 until an Fv/Fm of 0.17 was reached in both WT and ΔpsbM. The recovery was followed in low light (3 μE m–2 s–1) for up to 6 h measuring the Fv/Fm level every 1 h. Inactivation of psbM—Nine independent transformants with an identical phenotype were initially obtained, and three lines were used for further studies. Their homoplastomic status was confirmed by sequencing of the insertion site and by PCR analysis using isolated plastid chromosomes as template (Fig. 1A). Northern analysis with a strand-specific probe containing the coding region of the psbM gene demonstrated that not even traces of psbM transcripts were detectable confirming the homoplastomic state of the mutant (Fig. 1B). Levels and Compositions of Thylakoid Membrane Complexes in ΔpsbM Resemble Those of the WT—Inactivation of psbMin tobacco caused a quite distinct phenotype. Different from several other LMWs mutants of the chloroplast, such as ΔpsbE, ΔpsbF, ΔpsbL, and ΔpsbJ, ΔpsbM plants are capable of photoautotrophic growth on soil. However, mutant leaves appeared bleached if the light intensity exceeded ∼200 μEm–2 s–1, thus indicating increased light sensitivity of the photosynthetic apparatus. To elucidate the function of PsbM, homoplastomic ΔpsbM mutants in tobacco were analyzed by biochemical and biophysical approaches. The relative amounts and sizes of pigment-containing thylakoid membrane complexes in sucrose gradients of ΔpsbM did not differ significantly from those of the WT (Fig. 2A). Only PSII-LHCII supercomplexes were faintly diminished, and consequently trimeric LHCII antennae complexes showed a slight increase in ΔpsbM. Moreover, a significant increase in the LHCII-CP24-CP29 complex was observed in the mutant compared with WT. These re

The cytochrome b(6)f (Cyt b(6)f) complex in flowering plants contains nine conserved subunits, of which three, PetG, PetL, and PetN, are bitopic plastid-encoded low-molecular-weight proteins of largely unknown function. Homoplastomic knockout lines of the three genes have been generated in tobacco (Nicotiana tabacum 'Petit Havana') to analyze and compare their roles in assembly and stability of the complex. Deletion of petG or petN caused a bleached phenotype and loss of photosynthetic electron transport and photoautotrophy. Levels of all subunits that constitute the Cyt b(6)f complex were faintly detectable, indicating that both proteins are essential for the stability of the membrane complex. In contrast, DeltapetL plants accumulate about 50% of other Cyt b(6)f subunits, appear green, and grow photoautotrophically. However, DeltapetL plants show increased light sensitivity as compared to wild type. Assembly studies revealed that PetL is primarily required for proper conformation of the Rieske protein, leading to stability and formation of dimeric Cyt b(6)f complexes. Unlike wild type, phosphorylation levels of the outer antenna of photosystem II (PSII) are significantly decreased under state II conditions, although the plastoquinone pool is largely reduced in DeltapetL, as revealed by measurements of PSI and PSII redox states. This confirms the sensory role of the Cyt b(6)f complex in activation of the corresponding kinase. The reduced light-harvesting complex II phosphorylation did not affect state transition and association of light-harvesting complex II to PSI under state II conditions. Ferredoxin-dependent plastoquinone reduction, which functions in cyclic electron transport around PSI in vivo, was not impaired in DeltapetL.

The cytochrome b6f (Cyt b6f) complex in flowering plants contains nine conserved subunits, of which three, PetG, PetL, and PetN, are bitopic plastid-encoded low-molecular-weight proteins of largely unknown function. Homoplastomic knockout lines of the three genes have been generated in tobacco (Nicotiana tabacum 'Petit Havana') to analyze and compare their roles in assembly and stability of the complex. Deletion of pet Go rpetN caused a bleached phenotype and loss of photosynthetic electron transport and photoautotrophy. Levels of all subunits that constitute the Cyt b6f complex were faintly detectable, indicating that both proteins are essential for the stability of the membrane complex. In contrast, DpetL plants accumulate about 50% of other Cyt b6f subunits, appear green, and grow photoautotrophically. However, DpetL plants show increased light sensitivity as compared to wild type. Assembly studies revealed that PetL is primarily required for proper conformation of the Rieske protein, leading to stability and formation of dimeric Cyt b6f complexes. Unlike wild type, phosphorylation levels of the outer antenna of photosystem II (PSII) are significantly decreased under state II conditions, although the plastoquinone pool is largely reduced in DpetL, as revealed by measurements of PSI and PSII redox states. This confirms the sensory role of the Cyt b6f complex in activation of the corresponding kinase. The reduced light-harvesting complex II phosphorylation did not affect state transition and association of light-harvesting complex II to PSI under state II conditions. Ferredoxin-dependent

Coevolution of cellular genetic compartments is a fundamental aspect in eukaryotic genome evolution that becomes apparent in serious developmental disturbances after interspecific organelle exchanges. The genus Oenothera represents a unique, at present the only available, resource to study the role of the compartmentalized plant genome in diversification of populations and speciation processes. An integrated approach involving cDNA cloning, EST sequencing, and bioinformatic data mining was chosen using Oenothera elata with the genetic constitution nuclear genome AA with plastome type I. The Gene Ontology system grouped 1621 unique gene products into 17 different functional categories. Application of arrays generated from a selected fraction of ESTs revealed significantly differing expression profiles among closely related Oenothera species possessing the potential to generate fertile and incompatible plastid/nuclear hybrids (hybrid bleaching). Furthermore, the EST library provides a valuable source of PCR-based polymorphic molecular markers that are instrumental for genotyping and molecular mapping approaches.

Phylloquinone is a compound present in all photosynthetic plants serving as cofactor for Photosystem I-mediated electron transport. Newly identified seedling-lethal Arabidopsis thaliana mutants impaired in the biosynthesis of phylloquinone possess reduced Photosystem I activity. The affected gene, called PHYLLO, consists of a fusion of four previously individual eubacterial genes, menF, menD, menC, and menH, required for the biosynthesis of phylloquinone in photosynthetic cyanobacteria and the respiratory menaquinone in eubacteria. The fact that homologous men genes reside as polycistronic units in eubacterial chromosomes and in plastomes of red algae strongly suggests that PHYLLO derived from a plastid operon during endosymbiosis. The principle architecture of the fused PHYLLO locus is conserved in the nuclear genomes of plants, green algae, and the diatom alga Thalassiosira pseudonana. The latter arose from secondary endosymbiosis of a red algae and a eukaryotic host indicating selective driving forces for maintenance and/or independent generation of the composite gene cluster within the nuclear genomes. Besides, individual menF genes, encoding active isochorismate synthases (ICS), have been established followed by splitting of the essential 3' region of the menF module of PHYLLO only in genomes of higher plants. This resulted in inactivation of the ICS activity encoded by PHYLLO and enabled a metabolic branch from the phylloquinone biosynthetic route to independently regulate the synthesis of salicylic acid required for plant defense. Therefore, gene fusion, duplication, and fission events adapted a eubacterial multienzymatic system to the metabolic requirements of plants.

Photosystem II ( PSII) core complexes consist of CP47, CP43, D1, D2 proteins and of several low molecular weight integral membrane polypeptides, such as the chloroplast-encoded PsbE, PsbF, and PsbI proteins. To elucidate the function of PsbI in the photosynthetic process as well as in the biogenesis of PSII in higher plants, we generated homoplastomic knock-out plants by replacing most of the tobacco psbI gene with a spectinomycin resistance cartridge. Mutant plants are photoautotrophically viable under green house conditions but sensitive to high light irradiation. Antenna proteins of PSII accumulate to normal amounts, but levels of the PSII core complex are reduced by 50%. Bioenergetic and fluorescence studies uncovered that PsbI is required for the stability but not for the assembly of dimeric PSII and supercomplexes consisting of PSII and the outer antenna (PSII-LHCII). Thermoluminescence emission bands indicate that the presence of PsbI is required for assembly of a fully functional Q(A) binding site. We show that phosphorylation of the reaction center proteins D1 and D2 is light and redoxregulated in the wild type, but phosphorylation is abolished in the mutant, presumably due to structural alterations of PSII when PsbI is deficient. Unlike wild type, phosphorylation of LHCII is strongly increased in the dark due to accumulation of reduced plastoquinone, whereas even upon state II light phosphorylation is decreased in Delta psbI. These data attest that phosphorylation of D1/D2, CP43, and LHCII is regulated differently.

Reversible phosphorylation of chl a/b protein complex II (LHCII), the mobile light-harvesting antenna, regulates its association and energy transfer/dissipation to photosystem (PS) II or I (state transition). Excitation of LHCII induces conformational changes affecting the exposure of the phosphorylation site at the N-terminal domain to protein kinase(s) [Zer, H., et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 8277-8282; Zer, H., et al. (2003) Biochemistry 42, 728-738]. Thus, it was of interest to examine whether the pigment composition of LHCII affects the light-induced modulation of LHCII phosphorylation and state transition. To this end, we have used thylakoids of wild-type Chlamydomonas reinhardtii and xanthophyll deficient mutants npq1, lor1, npq2, npq1 lor1, and npq2 lor1. Phosphorylated protein bands P11, P13, and P17 are considered components of the mobile C. reinhardtii LHCII complex. The protein composition of these bands has been analyzed by mass spectrometry using Qtof-2 with a nanospray attachment. P11 and P13 contain C. reinhardtii light-harvesting chlorophyll a/b binding protein LhcII type I. P17 contains C. reinhardtii LhcII types III and IV. Illumination of isolated thylakoids inhibits the redox-controlled phosphorylation of polypeptide bands P13 and P17 and to a lower extent that of P11. The light-induced inhibition of LHCII phosphorylation and the state transition process are not influenced by extensive differences in the xanthophyll composition of the mutants. Thus, LHCII can be visualized as possessing two functionally distinct, independent domains: (i) the pigment binding transmembrane domain regulating the extent of energy transfer/dissipation and (ii) the surface-exposed phosphorylation site regulating the association of LHCII with PSII or PSI.