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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 the 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 that was remarkably similar between the species studied. Nucleoids per organelle varied from a 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-fold 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 2600-3300 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-stranded 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., (© 2019 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

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.

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.

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., (© 2011 Blackwell Publishing Ltd.)

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 (8 microm 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.

Photosystem II (PSII) of oxygen-evolving cyanobacteria, algae, and land plants mediates electron transfer from the Mn(4)Ca 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 DeltapsbTc 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 DeltapsbTc. 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 DeltapsbTc, 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.

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 largely lacking. 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.

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 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 163 365 bp (plastome IV) to 165 728 bp (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 subsection-specific 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 ancestor.

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 DeltatrnN or DeltatrnC 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 DeltatrnC and DeltatrnN tissue exhibit different aberrations of the internal membrane systems and, more importantly, heteroplastomic DeltatrnN plants are variegated. Accumulation of tRNA-N and plastid-encoded proteins is reduced in white sectors of DeltatrnN 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.

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.

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 Delta psbM 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 Delta psbM, 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 Delta psbM plants because of accumulation of reduced plastoquinone in the dark and a limited photosystem II-mediated electron transport in the light. Charge recombination in Delta psbM measured by thermoluminescence oscillations significantly differs from the 2/6 patterns in the wild type. A simulation program of thermoluminescence oscillations indicates a higher Q(B)/Q(-)(B) ratio in dark-adapted mutant thylakoids relative to the wild type. The interaction of the Q(A)/Q(B) sites estimated by shifts in the maximal thermoluminescence emission temperature of the Q band, induced by binding of different herbicides to the Q(B) 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 (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 redox-regulated 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.

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.

We describe the identification of a novel chloroplast protein, designated TCP34 (tetratricopeptide-containing chloroplast protein of 34 kDa) due to the presence of three tandemly arranged tetratricopeptide repeat (TPR) arrays. The presence of the genes encoding this protein only in the genomes of higher plants but not in photosynthetic cyanobacterial prokaryotes suggests that TCP34 evolved after the separation of the higher plant lineage. The in vitro translated precursor could be imported into intact spinach chloroplasts and the processed products showed stable association with thylakoid membranes. Using a specific polyclonal antiserum raised against TCP34, three protein variants were detected. Two forms, T(1) and T(2), were associated with the thylakoid membranes and one, S(1), was found released in the stroma. TCP34 protein was not present in etioplasts and appeared only in developing chloroplasts. The ratio of membrane-bound and soluble forms was maximal at the onset of photosynthesis. The high molecular mass thylakoid TCP34 variant was found in association with a transcriptionally active protein/DNA complex (TAC) from chloroplasts and recombinant TCP34 showed specific binding to Spinacia oleracea chloroplast DNA. Two TCP34 forms, T(1) and S(1), were found to be phosphorylated. An as yet unidentified phosphorelay signal may modulate its capability for plastid DNA binding through the phosphorylation state of the putative response regulator-like domain. Based on the structural properties and biochemical analyses, we discuss the putative regulatory function of TCP34 in plastid gene expression.

The subgenomes of the plant cell, the nuclear genome, the plastome, and the chondriome are known to interact through various types of coevolving macromolecules. The combination of the organellar genome from one species with the nuclear genome of another species often leads to plants with deleterious phenotypes, demonstrating that plant subgenomes coevolve. The molecular mechanisms behind this nuclear-organellar incompatibility have been elusive, even though the phenomenon is widespread and has been known for >70 years. Here, we show by direct and reverse genetic approaches that the albino phenotype of a flowering plant with the nuclear genome of Atropa belladonna (deadly nightshade) and the plastome of Nicotiana tabacum (tobacco) develops as a result of a defect in RNA editing of a tobacco-specific editing site in the plastid ATPase alpha-subunit transcript. A plastome-wide analysis of RNA editing in these cytoplasmic hybrids and in plants with a tobacco nucleus and nightshade chloroplasts revealed additional defects in the editing of species-specific editing sites, suggesting that differences in RNA editing patterns in general contribute to the pigment deficiencies observed in interspecific nuclear-plastidial incompatibilities.

To investigate the nuclear-controlled mechanisms of [4Fe-4S] cluster assembly in chloroplasts, we selected Arabidopsis thaliana mutants with a decreased content of photosystem I (PSI) containing three [4Fe-4S] clusters. One identified gene, ACCUMULATION OF PHOTOSYSTEM ONE1 (APO1), belongs to a previously unknown gene family with four defined groups (APO1 to APO4) only found in nuclear genomes of vascular plants. All homologs contain two related motifs of approximately 100 amino acid residues that could potentially provide ligands for [4Fe-4S] clusters. APO1 is essentially required for photoautotrophic growth, and levels of PSI core subunits are below the limit of detection in the apo1 mutant. Unlike other Arabidopsis PSI mutants, apo1 fails to accumulate significant amounts of the outer antenna subunits of PSI and PSII and to form grana stacks. In particular, APO1 is essentially required for stable accumulation of other plastid-encoded and nuclear-encoded [4Fe-4S] cluster complexes within the chloroplast, whereas [2Fe-2S] cluster-containing complexes appear to be unaffected. In vivo labeling experiments and analyses of polysome association suggest that translational elongation of the PSI transcripts psaA and psaB is specifically arrested in the mutant. Taken together, our findings suggest that APO1 is involved in the stable assembly of several [4Fe-4S] cluster-containing complexes of chloroplasts and interferes with translational events probably in association with plastid nucleoids.

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.

The psbEFLJ operon of tobacco plastids encodes four bitopic low molecular mass transmembrane components of photosystem II. Here, we report the effect of inactivation of psbL on the directional forward electron flow of photosystem II as compared to that of the wild type and the psbJ deletion mutant, which is impaired in PSII electron flow to plastoquinone [Regel et al. (2001) J. Biol. Chem. 276, 41473-41478]. Exposure of Delta psbL plants to a saturating light pulse gives rise to the maximal fluorescence emission, Fm(L), which is followed within 4-6 s by a broader hitherto not observed second fluorescence peak in darkness, Fm(D). Conditions either facilitating oxidation or avoiding reduction of the plastoquinone pool do not affect the Fm(L) level of Delta psbL plants but prevent the appearance of Fm(D). The level of Fm(D) is proportional to the intensity and duration of the light pulse allowing reduction of the plastoquinone pool in dark-adapted leaves prior to the activation of PSI and oxidation of plastoquinol. Lowering the temperature decreases the Fm(D) level in the Delta psbL mutant, whereas it increases considerably the lifetime of Q(A)*- in the Delta psbJ mutant. The thermoluminescence signal generated by Q(A)*-/S(2) charge recombination is not affected; on the other hand, charge recombination of Q(B)*-/S(2,3) could not be detected in Delta psbL plants. PSII is highly sensitive to photoinhibition in Delta psbL. We conclude that PsbL prevents reduction of PSII by back electron flow from plastoquinol protecting PSII from photoinactivation, whereas PsbJ regulates forward electron flow from Q(A)*- to the plastoquinone pool. Therefore, both proteins contribute substantially to ensure unidirectional forward electron flow from PSII to the plastoquinone pool.

Scanning electron microscopy (SEM) proves to be an appropriate technique for imaging chromatin organization in meiosis I and II of rye (Secale cereale) down to a resolution of a few nanometers. It could be shown for the first time that organization of basic structural elements (coiled and parallel fibers, chromomeres) changes dramatically during the progression to metaphase I and II. Controlled loosening with proteinase K (after fixation with glutaraldehyde) provides an enhanced insight into chromosome architecture even of highly condensed stages of meiosis. By selective staining with platinum blue, DNA content and distribution can be visualized within compact chromosomes as well as in a complex arrangement of fibers. Chromatin interconnecting threads, which are typically observed in prophase I between homologous and non-homologous chromosomes, stain clearly for DNA. In zygotene transversion of chromatid strands to their homologous counterparts becomes evident. In pachytene segments of synapsed and non-synapsed homologs alternate. At synapsed regions pairing is so intimate that homologous chromosomes form one filament of structural entity. Chiasmata are characterized by chromatid strands which traverse from one homolog to its counterpart. Bivalents are characteristically fused at their telomeric regions. In metaphase I and II there is no structural evidence for primary and secondary constrictions., (Copyright 2004 S. Karger AG, Basel)

The protein assembly and stability of photosystem II (PSII) (sub)complexes were studied in mature leaves of four plastid mutants of tobacco (Nicotiana tabacum L), each having one of the psbEFLJ operon genes inactivated. In the absence of psbL, no PSII core dimers or PSII-light harvesting complex (LHCII) supercomplexes were formed, and the assembly of CP43 into PSII core monomers was extremely labile. The assembly of CP43 into PSII core monomers was found to be necessary for the assembly of PsbO on the lumenal side of PSII. The two other oxygen-evolving complex (OEC) proteins, PsbP and PsbQ, were completely lacking in Delta psbL. In the absence of psbJ, both intact PSII core monomers and PSII core dimers harboring the PsbO protein were formed, whereas the LHCII antenna remained detached from the PSII dimers, as demonstrated by 77 K fluorescence measurements and by the lack of PSII-LHCII supercomplexes. The Delta psbJ mutant was characterized by a deficiency of PsbQ and a complete lack of PsbP. Thus, both the PsbL and PsbJ subunits of PSII are essential for proper assembly of the OEC. The absence of psbE and psbF resulted in a complete absence of all central PSII core and OEC proteins. In contrast, very young, vigorously expanding leaves of all psbEFLJ operon mutants accumulated at least traces of D2, CP43 and the OEC proteins PsbO and PsbQ, implying developmental control of the expression of the PSII core and OEC proteins. Despite severe problems in PSII assembly, the thylakoid membrane complexes other than PSII were present and correctly assembled in all psbEFLJ operon mutants.

Structural investigation and morphometry of meiotic chromosomes by scanning electron microscopy (in comparison to light microscopy) of all stages of condensation of meiosis I + II show remarkable differences during chromosome condensation in mitosis and meiosis I of rye (Secale cereale) with respect to initiation, mode and degree of condensation. Mitotic chromosomes condense in a linear fashion, shorten in length and increase moderately in diameter. In contrast, in meiosis I, condensation of chromosomes in length and diameter is a sigmoidal process with a retardation in zygotene and pachytene and an acceleration from diplotene to diakinesis. The basic structural components of mitotic chromosomes of rye are "parallel fibers" and "chromomeres" which become highly compacted in metaphase. Although chromosome architecture in early prophase of meiosis seems similar to mitosis in principle, there is no equivalent stage during transition to metaphase I when chromosomes condense to a much higher degree and show a characteristic "smooth" surface. No indication was found for helical winding of chromosomes either in mitosis or in meiosis. Based on measurements, we propose a mechanism for chromosome dynamics in mitosis and meiosis, which involves three individual processes: (i) aggregation of chromatin subdomains into a chromosome filament, (ii) condensation in length, which involves a progressive increase in diameter and (iii) separation of chromatids., (Copyright 2003 S. Karger AG, Basel)

The genetic transformation of plastids of higher plants has developed into a powerful approach for both basic research and biotechnology. Due to the high copy number of the plastid genome per plastid and per cell, repeated cycles of shoot regeneration under conditions selective for the modified plastid chromosome are required to obtain transformants entirely lacking wild-type plastid genomes. The presence of promiscuous plastid DNA in nuclear and/or mitochondrial genomes that generally contaminate even gradient-purified plastid fractions reduces the applicability of the highly sensitive PCR approach to monitor the absence of residual wild-type plastid chromosomes in transformed lines. It is therefore difficult, or even impossible, to assess reliably the hetero- or homoplastomic state of plastid transformants in this manner. By analysing wild-type and transplastomic mutants of tobacco, we demonstrate that separation of plastid chromosomes isolated from gradient-purified plastid fractions by pulsed-field gel electrophoresis can overcome the problem of (co)amplification of interfering promiscuous plastid DNA. PCR analyses with primers specific for plastid, mitochondrial and nuclear genes reveal an impressive purity of such plastid DNA fractions at a detection limit of less than one wild-type plastid chromosome copy per ten transplastomic cells.

Photosystem (PSII) is a supramolecular polypeptide complex found in oxygenic photosynthetic membranes, which is capable of extracting electrons from water for the reduction of plastoquinone. An intriguing feature of this assembly is the fact that it includes more than a dozen low-mass polypeptides of generally unknown function. Using a transplastomic approach, we have individually disrupted the genes of the psbEFLJoperon in Nicotiana tabacum, which encode four such polypeptides, without impairing expression of downstream loci of the operon. All four mutants exhibited distinct phenotypes; none of them was capable of photoautotrophic growth. All mutants bleached rapidly in the light. Disruption of psbEand psbF, which code for the alpha and beta apoproteins of cytochrome b(559), abolished PSII activity, as expected; Delta psbL and Delta psbJ plants displayed residual PSII activity in young leaves. Controlled partial solubilisation of thylakoid membranes uncovered surprisingly severe impairment of PSII structure, with subunit and assembly patterns varying depending on the mutant considered. In the Delta psbL mutant PSII was assembled primarily in a monomeric form, the homodimeric form was preponderant in Delta psbJ, and, unlike the case in Delta psbZ, the thylakoids of both mutants released some PSII supercomplexes. On the other hand, Photosystem I (PSI), the cytochrome b(6)f complex, ATP synthase, LHCII, and CP24/CP26/CP29 antennae were present in near wild-type levels. The data are discussed in terms of their implications for structural, biogenetic and functional aspects of PSII.

The plant cell operates with an integrated, compartmentalized genome consisting of nucleus/cytosol, plastids and mitochondria that, in its entirety, is regulated in time, quantitatively, in multicellular organisms and also in space. This genome, as do genomes of eukaryotes in general, originated in endosymbiotic events, with at least three cells, and was shaped phylogenetically by a massive and highly complex restructuring and intermixing of the genetic potentials of the symbiotic partners and by lateral gene transfer. This was accompanied by fundamental changes in expression signals in the entire system at almost all regulatory levels. The gross genome rearrangements contrast with a highly specific compartmental interplay, which becomes apparent in interspecific nuclear-plastid cybrids or hybrids. Organelle exchanges, even between closely related species, can greatly disturb the intracellular genetic balance ("hybrid bleaching"), which is indicative of compartmental coevolution and is of relevance for speciation processes. The photosynthetic machinery of plastids, which is embedded in that genetic machinery, is an appealing model to probe into genomic and organismic evolution and to develop functional molecular genomics. We have studied the reciprocal Atropa belladonna-Nicotiana tabacum cybrids, which differ markedly in their phenotypes, and found that transcriptional and post-transcriptional processes can contribute to genome/plastome incompatibility. Allopolyploidy can influence this phenomenon by providing an increased, cryptic RNA editing potential and the capacity to maintain the integrity of organelles of different taxonomic origins.

Redox-controlled, reversible phosphorylation of the thylakoid light harvesting complex II (LHCII) regulates its association with photosystems (PS) I or II and thus, energy distribution between the two photosystems (state transition). Illumination of solubilized LHCII enhances exposure of the phosphorylation site at its N-terminal domain to protein kinase(s) and tryptic cleavage in vitro [Zer et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 8277-8282]. Here we report that short illumination (5-10 min, 15-30 micromol m(-2) s(-1)) enhances the accessibility of LHCII phosphorylation site to kinase(s) activity also in isolated thylakoids. However, prolonged illumination or higher light intensities (30 min, 80-800 micromol m(-2) s(-1)) prevent phosphorylation of LHCII in the isolated membranes as well as in vivo, although redox-dependent protein kinase activity persists in the illuminated thylakoids toward exogenous solubilized LHCII. This phenomenon, ascribed to light-induced inaccessibility of the phosphorylation site to the protein kinase(s), affects in a similar way the accessibility of thylakoid LHCII N-terminal domain to tryptic cleavage. The illumination effect is not redox related, decreases linearly with temperature from 25 to 5 degrees C and may be ascribed to light-induced conformational changes in the complex causing lateral aggregation of dephosphorylated LHCII bound to and/or dissociated from PSII. The later state occurs under conditions allowing turnover of the phospho-LHCII phosphate. The light-induced inaccessibility of LHCII to the membrane-bound protein kinase reverses readily in darkness only if induced under LHCII-phosphate turnover conditions. Thus, phosphorylation prevents irreversible light-induced conformational changes in LHCII allowing lateral migration of the complex and the related state transition process.

Light-harvesting complex II (LHCII) prepared from isolated thylakoids of either broken or intact chloroplasts by three independent methods, exhibits proteolytic activity against LHCII. This activity is readily detectable upon incubation of these preparations at 37 degrees C (without addition of any chemicals or prior pre-treatment), and can be monitored either by the LHCII immunostain reduction on Western blots or by the Coomassie blue stain reduction in substrate-containing "activity gels". Upon SDS-sucrose density gradient ultracentrifugation of SDS-solubilized thylakoids, a method which succeeds in the separation of the pigment-protein complexes in their trimeric and monomeric forms, the protease activity copurifies with the LHCII trimer, its monomer exhibiting no activity. This LHCII trimer, apart from being "self-digested", also degrades the Photosystem II (PSII) core proteins (D1, D2) when added to an isolated PSII core protein preparation containing the D1/D2 heterodimer. Under our experimental conditions, 50% of LHCII or the D1, D2 proteins are degraded by the LHCII-protease complex within 30 min at 37 degrees C and specific degradation products are observed. The protease is light-inducible during chloroplast biogenesis, stable in low concentrations of SDS, activated by Mg(2+), and inhibited by Zn(2+), Cd(2+), EDTA and p-hydroxy-mercury benzoate (pOHMB), suggesting that it may belong to the cysteine family of proteases. Upon electrophoresis of the LHCII trimer on substrate-containing "activity gels" or normal Laemmli gels, the protease is released from the complex and runs in the upper part of the gel, above the LHCII trimer. A polypeptide of 140 kDa that exhibits proteolytic activity against LHCII, D1 and D2 has been identified as the protease. We believe that this membrane-bound protease is closely associated to the LHCII complex in vivo, as an LHCII-protease complex, its function being the regulation of the PSII unit assembly and/or adaptation.

The nuclear and plastid genomes of the plant cell form a coevolving unit which in interspecific combinations can lead to genetic incompatibility of compartments even between closely related taxa. This phenomenon has been observed for instance in Atropa-Nicotiana cybrids. We have sequenced the plastid chromosome of Atropa belladonna (deadly nightshade), a circular DNA molecule of 156,688 bp, and compared it with the corresponding published sequence of its relative Nicotiana tabacum (tobacco) to understand how divergence at the level of this genome can contribute to nuclear-plastid incompatibilities and to speciation. It appears that (1) regulatory elements, i.e., promoters as well as translational and replicational signal elements, are well conserved between the two species; (2) genes--including introns--are even more highly conserved, with differences residing predominantly in regions of low functional importance; and (3) RNA editotypes differ between the two species, which makes this process an intriguing candidate for causing rapid reproductive isolation of populations.

A set of 62 genes that encode the entire peptidase complement of Synechocystis sp. PCC 6803 has been identified in the genome database of that cyanobacterium. Sequence comparisons with the Arabidopsis genome uncovered the presumably homologous chloroplast components inherited from their cyanobacterial ancestor. A systematic gene disruption approach was chosen to individually inactivate, by customary transformation strategies, the majority of the cyanobacterial genes encoding peptidase subunits that are related to chloroplast enzymes. This allowed classification of the peptidases that are required for cell viability or are involved in specific stress responses. The comparative analysis between Synechocystis and Arabidopsis chloroplast peptidases showed that: (1) homologous enzymes that arose by gene duplications in cyanobacteria are functionally diverse and frequently do not complement each other, (2) the chloroplast appears to house a number of distinct peptidase polypeptide chains of cyanobacterial origin (49) which is comparable with a cyanobacterial cell (62) and (3) the peptidase complement in plastids results from a combination of the loss of some cyanobacterial peptidases and the gain or diversification of subclasses of peptidases. This reorganization in the pattern of proteolytic enzymes may reflect distinct environmental and physiological changes between prokaryotic and organellar systems.

Transcription of plastid chromosomes in vascular plants is accomplished by at least two RNA polymerases of different phylogenetic origin: the ancestral (endosymbiotic) cyanobacterial-type RNA polymerase (PEP), of which the core is encoded in the organelle chromosome, and an additional phage-type RNA polymerase (NEP) of nuclear origin. Disruption of PEP genes in tobacco leads to off-white phenotypes. A macroarray-based approach of transcription rates and of transcript patterns of the entire plastid chromosome from leaves of wild-type as well as from transplastomic tobacco lacking PEP shows that the plastid chromosome is completely transcribed in both wild-type and PEP-deficient plastids, though into polymerase-specific profiles. Different probe types, run-on transcripts, 5' or 3' labelled RNAs, as well as cDNAs, have been used to evaluate the array approach. The findings combined with Northern and Western analyses of a selected number of loci demonstrate further that frequently no correlation exists between transcription rates, transcript levels, transcript patterns, and amounts of corresponding polypeptides. Run-on transcription as well as stationary RNA concentrations may increase, decrease or remain similar between the two experimental materials, independent of the nature of the encoded gene product or of the multisubunit assembly (thylakoid membrane or ribosome). Our findings show (i) that the absence of photosynthesis-related, plastome-encoded polypeptides in PEP-deficient plants is not directly caused by a lack of transcription by PEP, and demonstrate (ii) that the functional integration of PEP and NEP into the genetic system of the plant cell during evolution is substantially more complex than presently supposed.

In higher plants, a small nuclear gene family encodes mitochondrial as well as chloroplast RNA polymerases (RNAP) homologous to the bacteriophage T7-enzyme. The Arabidopsis genome contains three such RpoT genes, while in monocotyledonous plants only two copies have been found. Analysis of Nicotiana tabacum, a natural allotetraploid, identified six different RpoT sequences. The study of the progenitor species of tobacco, N. sylvestris and N. tomentosiformis, uncovered that the sequences represent two orthologous sets each of three RpoT genes (RpoT1, RpoT2 and RpoT3). Interestingly, while the organelles are inherited exclusively from the N. sylvestris maternal parent, all six RpoT genes are expressed in N. tabacum. GFP-fusions of Nicotiana RpoT1 revealed mitochondrial targeting properties. Constructs containing the amino-terminus of RpoT2 were imported into mitochondria as well as into plastids. Thus, the dual-targeting feature, first described for Arabidopsis RpoT;2, appears to be conserved among eudicotyledonous plants. Tobacco RpoT3 is targeted to chloroplasts and the RNA is differentially expressed in plants lacking the plastid-encoded RNAP. Remarkably, translation of RpoT3 mRNA has to be initiated at a CUG codon to generate a functional plastid transit peptide. Thus, besides AGAMOUS in Arabidopsis, Nicotiana RpoT3 provides a second example for a non-viral plant mRNA that is exclusively translated from a non-AUG codon.

Editing of plastid RNAs proceeds by C-to-U, in hornwort species also by extensive U-to-C, transitions, which predominantly lead to the restoration of codons for structurally and/or functionally important, conserved amino acid residues. So far, only one instance of editing outside coding regions has been reported - in the psbL/ psbF intergenic region of Ginkgo biloba. This site was proposed to have no functional importance. Here we present an evaluation of an editing site in the ndhI/ ndhG intergenic region in a related group of monocot plants. Efficient editing of this site, as well as the phylogenetic conservation of the resulting uridine residue, point at an important role for the sequence restored by editing. Two potential functions can be envisaged. (1) RNA secondary structure predictions suggest that the C-to-U conversion at this site can lead to a modified stem/loop structure of the ndhG 5' UTR, which could influence ndhG expression. (2) Alternatively, editing of the ndhI/ ndhG intergenic region may tag a so far unidentified small (12-codon) ORF, and lead to the restoration of a conserved phenylalanine codon. A screen with specific antibodies elicited against the putative peptide failed to detect such a peptide in chloroplast fractions. However, this failure may be attributable to its low and/or development-specific expression.

The Rieske Fe/S protein, a nuclear-encoded subunit of the cytochrome b(6)/f complex in chloroplasts, is retarded in the stromal space after import into the chloroplast and only slowly translocated further into the thylakoid membrane system. As shown by the sensitivity to nigericin and to specific competitor proteins, thylakoid transport takes place by the DeltapH-dependent TAT pathway. The Rieske protein is an untypical TAT substrate, however. It is only the second integral membrane protein shown to utilize this pathway, and it is the first authentic substrate without a cleavable signal peptide. Transport is instead mediated by the NH(2)-terminal membrane anchor, which lacks, however, the twin-arginine motif indicative of DeltapH/TAT-dependent transport signals. Furthermore, transport is affected by sodium azide as well as by competitor proteins for the Sec pathway in chloroplasts, demonstrating for the first time some cross-talk of the two pathways. This might take place in the stroma where the Rieske protein accumulates after import in several complexes of high molecular mass, among which the cpn60 complex is the most prominent. These untypical features suggest that the Rieske protein represents an intermediate or early state in the evolution of the thylakoidal protein transport pathways.

The photosystem II (PSII) complex of photosynthetic oxygen evolving membranes comprises a number of small proteins whose functions remain unknown. Here we report that the low molecular weight protein encoded by the psbJ gene is an intrinsic component of the PSII complex. Fluorescence kinetics, oxygen flash yield, and thermoluminescence measurements indicate that inactivation of the psbJ gene in Synechocystis 6803 cells and tobacco chloroplasts lowers PSII-mediated oxygen evolution activity and increases the lifetime of the reduced primary acceptor Q(A)(-) (more than a 100-fold in the tobacco DeltapsbJ mutant). The decay of the oxidized S(2,3) states of the oxygen-evolving complex is considerably accelerated, and the oscillations of the Q(B)(-)/S(2,3) recombination with the number of exciting flashes are damped. Thus, PSII can be assembled in the absence of PsbJ. However, the forward electron flow from Q(A)(-) to plastoquinone and back electron flow to the oxidized Mn cluster of the donor side are deregulated in the absence of PsbJ, thereby affecting the efficiency of PSII electron flow following the charge separation process.

A new component of the chloroplast proteolytic machinery from Arabidopsis thaliana was identified as a SppA-type protease. The sequence of the mature protein, deduced from a full-length cDNA, displays 22% identity to the serine-type protease IV (SppA) from Escherichia coli and 27% identity to Synechocystis SppA1 (sll1703) but lacks the putative transmembrane spanning segments predicted from the E. coli sequence. The N-terminal sequence exhibits typical features of a cleavable chloroplast stroma-targeting sequence. The chloroplast localization of SppA was confirmed by in organello import experiments using an in vitro expression system and by immunodetection with antigen-specific antisera. Subfractionation of intact chloroplasts demonstrated that SppA is associated exclusively with thylakoid membranes, predominantly stroma lamellae, and is a part of some high molecular mass complex of about 270 kDa that exhibits proteolytic activity. Treatments with chaotropic salts and proteases showed that SppA is largely exposed to the stroma but that it behaves as an intrinsic membrane protein that may have an unusual monotopic arrangement in the thylakoids. We demonstrate that SppA is a light-inducible protease and discuss its possible involvement in the light-dependent degradation of antenna and photosystem II complexes that both involve serine-type proteases.

RNA editing is unique among post-transcriptional processes in plastids, as it exhibits extraordinary phylogenetic dynamics leading to species-specific editing site patterns. The evolutionary loss of a site is considered to entail the loss of the corresponding nuclear-encoded site-specific factor, which prevents the editing of foreign, i.e. heterologous, sites. We investigated the editing of short 'spliced' and 'unspliced' ndhA gene fragments from spinach in Nicotiana tabacum (tobacco) in vivo using biolistic transformation. Surprisingly, it turned out that the spinach site is edited in the heterologous nuclear background. Furthermore, only exon-exon fusions were edited, whereas intron-containing messages remained unprocessed. A homologue of the spinach site was found to be present and edited in Nicotiana tomentosiformis, representing the paternal parent, but absent from Nicotiana sylvestris, representing the maternal parent of tobacco. Our data show that: (i) the cis-determinants for ndhA editing are split by an intron; (ii) the editing capacity cannot be deduced from editing sites; and (iii) allopolyploidization can increase the editing capacity, which implies that it can influence speciation processes in evolution.

We have characterized the biochemical nature and the function of PsbZ, the protein product of a ubiquitous open reading frame, which is known as ycf9 in Chlamydomonas and ORF 62 in tobacco, that is present in chloroplast and cyanobacterial genomes. After raising specific antibodies to PsbZ from Chlamydomonas and tobacco, we demonstrated that it is a bona fide photosystem II (PSII) subunit. PsbZ copurifies with PSII cores in Chlamydomonas as well as in tobacco. Accordingly, PSII mutants from Chlamydomonas and tobacco are deficient in PsbZ. Using psbZ-targeted gene inactivation in tobacco and Chlamydomonas, we show that this protein controls the interaction of PSII cores with the light-harvesting antenna; in particular, PSII-LHCII supercomplexes no longer could be isolated from PsbZ-deficient tobacco plants. The content of the minor chlorophyll binding protein CP26, and to a lesser extent that of CP29, also was altered substantially under most growth conditions in the tobacco mutant and in Chlamydomonas mutant cells grown under photoautotrophic conditions. These PsbZ-dependent changes in the supramolecular organization of the PSII cores with their peripheral antennas cause two distinct phenotypes in tobacco and are accompanied by considerable modifications in (1) the pattern of protein phosphorylation within PSII units, (2) the deepoxidation of xanthophylls, and (3) the kinetics and amplitude of nonphotochemical quenching. The role of PsbZ in excitation energy dissipation within PSII is discussed in light of its proximity to CP43, in agreement with the most recent structural data on PSII.

A homozygous insertion mutant with the inactivated clpP2 gene, which encodes the proteolytic subunit of ATP-dependent peptidase, was obtained in the unicellular cyanobacterium Synechocystis sp. PCC 6803. The mutant cannot grow under photoautotrophic conditions, but cells grown under heterotrophic conditions in a glucose-containing medium have active photosystems I and II (PS I and PS II). The loss of capacity for photoautotrophic growth is determined by a high sensitivity of mutant cells to the inactivating effect of light. Their incubation under light with an intensity above 10 microE m-2 s-1 inhibits cell growth in culture and causes degradation of photosynthetic pigments. It is proposed that the ClpP2 peptidase is involved in the protection of Synechocystis 6803 cells from photoinhibition.

Landsberg erecta x Columbia F8 recombinant inbred lines of Arabidopsis thaliana, arrayed BAC clones covering most of the genome, and databank sequence information were used to map the positions of 69 genes in the genome of A. thaliana. These genes encode all known constituents of the photosynthetic thylakoid membrane, some regulatory factors involved in its biogenesis, and the RNA polymerases of nuclear origin that operate in chloroplasts and mitochondria. Designations of novel genes are proposed. The data of these three approaches are generally consistent, although ambiguities have been noted for some genome segments and with gene duplications. For thylakoid multi-subunit structures, no positional clustering of genes has been found, not even for genes encoding different subunits of the same membrane complex. The genes of the lhc superfamily encoding antenna apoproteins and their relatives are a particularly intriguing example. The lack of positional clustering is consistent with phylogenetically independent gene translocations from the plastid (endosymbiont) to the nucleus. This raises the basic question of how independently translocated genes which acquired different promoter sequences and transit peptides were functionally integrated into common signal transduction chains.

The chloroplast chromosome of spinach (Spinacia oleracea) is a double-stranded circular DNA molecule of 150,725 nucleotide pairs. A comparison of this chromosome with those of the three other autotrophic dicotyledons for which complete DNA sequences of plastid chromosomes are available confirms a conserved overall structure. Three classes of open reading frames were distinguished: (1) genes of known function which include 108 unique loci, (2) three hypothetical chloroplast reading frames (ycfs) that are highly conserved interspecifically, and (3) species-specific or rapidly diverging 'open reading frames'. A detailed transcript study of one of the latter (ycf15) shows that these loci may be transcribed, but do not constitute protein-coding genes.

A new approach for comparative cytogenetic banding analysis of plant chromosomes has been established. The comparative GISH (cGISH) technique is universally applicable to various complex genomes of Monocotyledonae (Triticum aestivum, Agropyron elongatum, Secale cereale, Hordeum vulgare, Allium cepa, Muscari armenaticum and Lilium longiflorum) and Dicotyledonae (Vicia faba, Beta vulgaris, Arabidopsis thaliana). Labelled total genomic DNA of A. thaliana generates signals at conserved chromosome regions. The nucleolus organizing regions (NORs) containing the majority of tandemly repeated rDNA sequences, N-band regions containing satellite DNA, conserved homologous sequences at telomeres and additional chromosome-characteristic markers were detected in heterologous FISH experiments. Multicolour FISH analysis with repetitive DNA probes simultaneously revealed the chromosome assignment of 56 cGISH signals in rye and 61 cGISH signals in barley. Further advantages of this technique are: (1) the fast and straightforward preparation of the probe; (2) the generation of signals with high intensity and reproducibility even without signal amplification; and (3) no requirement of species-specific sequences suitable for molecular karyotype analysis. Hybridization can be performed without competitive DNA. Signal detection without significant background is possible under low stringency conditions. The universal application of this fast and simple one-step fluorescence banding technique for plant cytogenetic and plant genome evolution is discussed.