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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.

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.

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.

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.

The Rieske Fe/S protein, a nuclear-encoded subunit of the cytochromeb 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 ΔpH-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 NH2-terminal membrane anchor, which lacks, however, the twin-arginine motif indicative of ΔpH/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.

Plastids of higher plants operate with at least two distinct DNA-dependent RNA polymerases, which are encoded in the organelle (PEP) and in the nucleus (NEP), respectively. Plastid run-on assays and Northern analyses were employed to analyse gene expression in tobacco mutant plastids lacking the PEP genes rpoA, rpoB or rpoC1. Hybridisation of run-on transcripts to restriction fragments representing the entire tobacco plastid chromosome, as well as to selected plastid gene-specific probes, shows that all parts of the plastid DNA are transcribed in rpo-deficient plastids. In comparison to wild-type chloroplasts, which are characterized by preferential transcription of photosynthesis-related genes in the light, mutant plastids exhibit a different transcription pattern with less pronounced differences in the hybridisation intensities between the individual genes. The analysis of steady-state transcript patterns and transcription rates of selected genes in both types of plastids demonstrates that differences in transcription rates are not necessarily paralleled by corresponding changes in transcript levels. The accumulation of large transcripts in the mutant plastids indicates that processing of primary transcripts may be impaired in the absence of PEP. These data suggest that, contrary to the prevailing view, much of the regulation of NEP-driven plastid gene expression in the rpo-deficient mutants is not based on differential promoter usage but is exerted at post-transcriptional levels.

The interaction between plastocyanin and the intact cytochrome bf complex, both from spinach, has been studied by stopped-flow kinetics with mutant plastocyanin to elucidate the site of electron transfer and the docking regions of the molecule. Mutation of Tyr-83 to Arg or Leu provides no evidence for a second electron transfer path via Tyr-83 of plastocyanin, which has been proposed to be the site of electron transfer from cytochrome f. The data found with mutations of acidic residues indicate that both conserved negative patches are essential for the binding of plastocyanin to the intact cytochrome bf complex. Replacing Ala-90 and Gly-10 at the flat hydrophobic surface of plastocyanin by larger residues slowed down and accelerated, respectively, the rate of electron transfer as compared with wild-type plastocyanin. These opposing effects reveal that the hydrophobic region around the electron transfer site at His-87 is divided up into two regions, of which only that with Ala-90 contributes to the attachment to the cytochrome bf complex. These binding sites of plastocyanin are substantially different from those interacting with photosystem I. It appears that each of the two binding regions of plastocyanin is split into halves, which are used in different combinations in the molecular recognition at the two membrane complexes.

We describe two related manganese-binding polypeptides with L-arginine metabolizing enzyme activity that can be detected as distinct components (designated PsbY-A1 and PsbY-A2, previously called L-AME) in membranes containing Photosystem II (PS II) from spinach. The polypeptides are bitopic and appear to exist in a heterodimeric form, but only in the chlorophyll a/b lineage of plants. Both proteins are encoded in the nucleus. In spinach and in Arabidopsis thaliana they are both derived from a single-copy gene (psbY) that is translated into a precursor polyprotein of approximately 20 kDa. The processing of the polyprotein is complex and includes at least four cleavage steps. Both polypeptides are exposed N-terminally to the lumenal and C-terminally to the stromal face of the thylakoid membrane.

The cDNAs encoding plantacyanin from spinach were isolated and characterized. In addition, four new cDNA sequences from Arabidopsis ESTs were identified that encode polypeptides resembling phytocyanins, plant-specific proteins constituting a distinct family of mononuclear blue copper proteins. One of them encodes plantacyanin from Arabidopsis, while three others, designated as uclacyanin 1, 2, and 3, encode protein precursors that are closely related to precursors of stellacyanins and a blue copper protein from pea pods. Comparative analyses with known phytocyanins allow further classification of these proteins into three distinct subfamilies designated as uclacyanins, stellacyanins, and plantacyanins. This specification is based on (1) their spectroscopic properties, (2) their glycosylation state, (3) the domain organization of their precursors, and (4) their copper-binding amino acids. The recombinant copper binding domain of Arabidopsis uclacyanin 1 was expressed, purified, and shown to bind a copper atom in a fashion known as "blue" or type 1. The mutant of cucumber stellacyanin in which the glutamine axial ligand was substituted by a methionine (Q99M) was purified and shown to possess spectroscopic properties similar to uclacyanin 1 rather than to plantacyanins. Its redox potential was determined by cyclic voltammetry to be +420 mV, a value that is significantly higher than that determined for the wild-type protein (+260 mV). The available structural data suggest that stellacyanins (and possibly other phytocyanins) might not be diffusible electron-transfer proteins participating in long-range electron-transfer processes. Conceivably, they are involved in redox reactions occurring during primary defense responses in plants and/or in lignin formation.

The topology of the Rieske protein of the cytochrome b6/f complex in thylakoids from spinach chloroplasts was examined by protease protection experiments as well as polypeptide extraction assays using solutions of chaotropic salts or alkaline pH. While neither thermolysin nor trypsin cleave any of the Rieske protein when added to the stromal side of the thylakoid membrane, proteinase K is capable of removing approximately four residues from its NH2-terminus. The protein is resistant to membrane extraction by 0.1 M Na2CO3 or 2 M NaBr but is quantitatively released by 0.1 M NaOH. Treatment of thylakoids with 2 M NaSCN leads to extraction of variable amounts of the protein, depending on the presence or absence of sucrose in the medium which apparently stabilizes the cytochrome complex. From these results we conclude that the Rieske protein is an integral component of the cytochrome complex which spans the thylakoid membrane with a single hydrophobic segment and is anchored predominantly by electrostatic interactions.

Expression of chimeric uidA gene fusions (for bacterial beta-glucuronidase) with 5'-flanking sequences of the spinach AtpC and PetE genes (encoding the subunit gamma of the chloroplast ATP synthase and plastocyanin, respectively) requires sequences for the 5'-untranslated leaders. The sequence for the PetE leader does not exhibit significant similarities to those of other leader sequences. Closer inspection of PetE uncovered that the crucial region is located in the vicinity of the transcription start site (+5/+15, TTGTCATTTCT). In contrast, 3' deletions of sequences for the AtpC leader revealed that the region in the vicinity of the translation initiation codon is essential for uidA gene expression (+103/+176). This segment contains a CT-rich sequence (TTCTCTCTCCT), which is found identically or in a slightly modified form in sequences for 85 plant leaders deposited in the EMBL data bank. Site-directed mutagenesis of the CT-rich sequence resulted in a three-fold reduction of the transcription of the transgene. It is concluded (1) that different elements in the sequences for the spinach PetE and AtpC leaders control the expression of the uidA gene, (2) that these elements operate transcriptionally rather than post-transcriptionally and (3) that a CT-rich sequence represents a crucial cis element for the transcription of the AtpC::uidA gene fusion.

The cDNA encoding the 182 amino acid long precursor stellacyanin from Cucumis sativus was isolated and characterized. The protein precursor consists of four sequence domains: I, a 23 amino acid hydrophobic N-terminal signal peptide with features characteristic of secretory proteins; II, a 109 amino acid copper-binding domain; III, a 26 amino acid hydroxyproline- and serine-rich peptide characteristic of motifs found in the extension family, extracellular structural glycoproteins found in plant cell walls; and IV, a 22 amino acid hydrophobic extension. Maturation of the protein involves posttranslational processing of domains I and IV. The copper-binding domain (domain II), which shares high sequence identity with other stellacyanins, has been expressed without its carbohydrate attachment sites, refolded from the Escherichia coli inclusion bodies, purified, and characterized by electronic absorption, EPR, ESEEM, and RR spectroscopy. Its spectroscopic properties are nearly identical to those of stellacyanin from the Japanese lacquer tree Rhus vernicifera, the most extensively studied and best characterized stellacyanin, indicating that this domain folds correctly, even in the absence of its carbohydrate moiety. The presence of a hydroxyproline- and serine-rich domain III suggests that stellacyanin may have a function other than that of a diffusible electron transfer protein, conceivably participating in redox reactions localized at the plant cell wall, which are known to occur in response to wounding or infection of the plant.

Nuclear-encoded genes for proteins of the photosynthetic machinery represent a particular subset of genes. Their expression is cooperatively stimulated by discrete factors including the developmental stage of plastids and light. We have analyzed in transgenic tobacco the plastid- and light-dependent expression of a series of 5' promoter deletions of various nuclear genes from spinach, of fusions of defined promoter segments with the 90-bp 35S RNA CaMV minimal promoter, as well as with mutations in sequences with homologies to characterized cis-elements, to address the question of whether the plastid signal and light operate via the same or different cis-acting elements. In none of the 160 different transgenic lines (representing 32 promoter constructs from seven genes) analyzed, could significant differences be identified in the responses to the two regulatory pathways. The data are compatible with the idea that both signals control the expression of nuclear genes for plastid proteins via the same cis-acting elements.

The D1 reaction center protein of the Photosystem II complex in green plants is synthesized with a short carboxyl-terminal extension. Proteolytic cleavage and removal of this extension peptide in the thylakoid lumen are necessary for the assembly of a manganese cluster that is essential for the oxygen evolution activity of Photosystem II. We have isolated cDNAs encoding CtpA, the carboxyl-terminal processing protease for the D1 protein, from two higher plants, spinach and barley. In each of these organisms, CtpA is encoded by a single copy nuclear gene, and its steady-state mRNA levels are light-regulated. The CtpA protein is detectable in etiolated material, and its level increases approximately 5-fold upon illumination. Moreover, the CtpA gene is expressed in shoot tissues and not in roots. In its precursor form, the CtpA protein harbors a bipartite transit sequence characteristic for thylakoid lumenal proteins. Cell fractionation studies demonstrated that CtpA is associated with thylakoid membranes and is resistant to treatments with thermolysin, consistent with its localization in the lumen of thylakoids. Comparisons of the sequence of the higher plant CtpA enzyme with those of other related carboxyl-terminal processing proteases suggest that these proteins constitute a new family of proteases.

Approximately 4,200 nucleotides of the 16S/23S rDNA spacer and the 5' region flanking the rrn operon of the plastid chromosomes representing the five basic, phylogenetically related Euoenothera plastomes were sequenced and compared. The sequences that harbor the putative replication origins are almost identical except for a 785-bp intercistronic segment between the genes for the 16S rRNA and trnI. Differences are mainly caused by insertions/deletions and duplications; the predicted potential for formation of quite extensive secondary structure differs among the plastomes. Unexpected intraplastome variation has also been noted. Furthermore, the sequence-based and published genetically deduced plastome pedigrees differ significantly.

The genomic organization of two satellite DNA sequences, pHvMWG2314 and pHvMWG2315, of barley (Hordeum vulgare, 2n=14, HH) was studied by comparative in situ hybridization (ISH) and PCR analysis. Both sequences are members of different RsaI families. The sequence pHvMWG2314 is a new satellite element with a monomer unit of 73 bp which is moderately amplified in different grasses and occurs in interstitial clusters on D-genome chromosomes of hexaploid wheat (Triticum aestivum, 2n=42, AABBDD). The 331-bp monomer pHvMWG2315 belongs to a tandemly amplified repetitive sequence family that is present in the Poaceae and preferentially amplified in Aegilops squarrosa (2n=14, DD), H. vulgare and Agropyron elongatum. (2n=14, EE). The first described representative of this family was pAs 1 from Ae. squarrosa. Different sequences of one satellite DNA family were amplified from Ae. squarrosa, A. elongatum and H. vulgare using PCR. Characteristic differences between members of the D and H genome occurred in a variable region which is flanked by two conserved segments. The heterogeneity within this element was exploited for the cytogenetic analysis of Triticeae genomes and chromosomes. Comparative ISH with pHvMWG2315 identified individual wheat and barley chromosomes under low (75%) and high (85%) hybridization stringency in homologous and heterologous systems. We propose the designation Tas330 for the Triticeae amplified sequence (Tas) satellite family with a 330 bp average monomer length.

A simple and efficient method for the preparation of a high number of plant metaphase spreads from a broad range of plant species suitable for light microscopy and high-resolution electron microscopy is described.

We report on microdissection, cloning and sequence, and Southern and fluorescence in situ hybridization (FISH) analysis of one moderately and one highly amplified repetitive DNA element, pHvMWG2314 and pHvMWG2315, respectively, isolated from barley (Hordeum vulgare L.) chromosome arm 3HL. The pHvMWG2315 sequence hybridizes to all 14 telomeric or subtelomeric regions of the barley chromosomes as determined by FISH. The 50 different hybridization sites that include intercalary signals allow the discrimination of all 14 chromosome arms and the construction of a karyotype of barley. The tandemly repeated subtelomeric element of 331 bp exists in all Triticeae species tested (H. vulgare, Agropyron elongatum, Secale cereale, Triticum tauschii, T. turgidum, and T. aestivum). It is AT rich (66%), exhibits 84% sequence homology to subfragments of the D genome "specific" 1-kb element pAsl of T. tauschii and 75% homology to the interspersed genome-specific DNA sequence pHcKB6 from H. chilense. The repetitive sequence pHvMWG2314 is moderately amplified in barley and highly amplified in hexaploid wheat. The in situ experiments revealed no distinct signals on barley chromosomes, indicating a dispersed character for the sequence. The significance of the results for the identification of chromosomes and chromosome aberrations in FISH experiments are discussed.Key words: karyotype, fluorescence in situ hybridization, FISH, DNA sequencing.

The nuclear-encoded proteins of the oxygen-evolving complex (OEC) of photosystem II are bound on the lumenal side of the thylakoid membrane and stabilize the manganese ion cluster forming the photosystem II electron donor side. The OEC proteins are released from their binding site(s) following light-induced degradation of reaction center II (RCII)-D1 protein in Chlamydomonas reinhardtii. The kinetics of OEC proteins release correlates with that of RCII-D1 protein degradation. Only a limited amount of RCII-D2 protein is degraded during the process, and no loss of the core proteins CP43 and CP47 is detected. The release of the OEC proteins is prevented when the photoinactivated RCII-D1 protein degradation is retarded by addition of 3-(3,5-dichlorophenyl)-1,1-dimethylurea or by a high PQH2/PQ ratio prevailing in membranes of the plastocyanin-deficient mutant Ac208. The released proteins are not degraded but persist in the thylakoid lumen for up to 8 h and reassociate with photosystem II when new D1 protein is synthesized in cells exposed to low light, thus allowing recovery of photosystem II function. Reassociation also occurs following D1 protein synthesis in darkness when RCII activity is only partially recovered. These results indicate that (i) the D1 protein participates in the formation of the lumenal OEC proteins binding site(s) and (ii) the photoinactivation of RCII-D1 protein does not alter the conformation of the donor side of photosystem II required for the binding of the OEC proteins.

A Sec-type system is responsible for the translocation of a subset of proteins across the thylakoid membrane in higher plant chloroplasts. Previous studies have suggested that the thylakoidal ΔpH plays a minor role in this translocation mechanism, but we show here that it can be essential for the translocation process, depending on the identity of the passenger protein and the concentration of ATP. Studies using chimeric proteins show that, whereas the presequence dictates the translocation pathway, the ΔpH requirement is dictated exclusively by the passenger protein; some passenger proteins are virtually ΔpH-independent whereas others are absolutely dependent. ΔpH requirement is not related to charge characteristics of the passenger proteins, ruling out an electrophoretic effect. Analysis of the 33-kDa photosystem II protein reveals an inverse relationship between ΔpH requirement and ATP concentration; import into isolated thylakoids is inhibited 14-fold by nigericin at moderate ATP concentrations, and totally inhibited when the ATP concentration is reduced to 2 μM. The results indicate that the roles of the ΔpH and ATP overlap and suggest that the ΔpH may be obligatory when the passenger protein is abnormally difficult to translocate, possibly due to the folding of the polypeptide chain. We compare the energetics of this system with those of prokaryotic systems from which the chloroplast system is believed to have evolved.

The Sec-1 locus (ω-secalin) of rye (Secale cereale L.) was mapped in the satellite of the short arm of chromosome 1R using fluorescence in situ hybridization and a genomic probe called pSec2B. Sec-1 is located in the middle of the satellite at the junction of the proximal euchromatic and the distal heterochromatic regions. Double hybridization experiments using rDNA and pSec2B showed that the NOR spans over the secondary constriction of the short arm of chromosome 1R and that there is a clearly visible gap between the NOR and Sec-1. Heterologous hybridization of pSec2B to barley visualized the B-hordein locus on chromosome 1H.Key words: fluorescence in situ hybridization, physical mapping, genetic mapping, secalin, rye, B-hordein, rDNA.

Thylakoid membranes of chloroplasts in higher plants harbor different pathways for the translocation of proteins. One of these routes is related to the prokaryotic Sec pathway, which mediates the secretion of particular proteins into the periplasmic space and involves the SecA protein as an essential component. We have isolated a full size cDNA of 3739 nucleotides encoding the SecA homologue from spinach. It contains an open reading frame of 1036 codons corresponding to a polypeptide with a calculated mass of 117 kDa. The deduced amino acid sequence shows between 43 and 49% identity to SecA proteins from bacteria and lower algae and 62% identity to SecA of the cyanobacterium Synechococcus sp. PCC7942. Compared with the Escherichia coli protein, spinach SecA carries an amino-terminal extension of approximately 80 residues. In organello experiments performed with the protein made in vitro by transcription of the cDNA and cell-free translation of the resulting RNA showed that this extension comprises a transit peptide that mediates the import of the protein into the chloroplast. The processed product of approximately 107 kDa accumulates predominantly in the stroma and to a lower extent associates with the thylakoid membrane. Comparably to E. coli, in which SecA activity can be inhibited by sodium azide, thylakoid translocation of a subset of lumenal proteins is sensitive to sodium azide in pea but not in spinach chloroplasts, suggesting that the latter contain an azide-resistant SecA variant.

Subunit 3 of photosystem I (PSI-3), the product of the nuclear psaF gene, is the docking protein for plastocyanin during photosynthetic electron transport in thylakoid membranes and is synthesized in the cytosol with a transit peptide that resembles structurally the bipartite targeting signals of hydrophilic, lumenal components such as plastocyanin. In organello import experiments performed with the authentic PSI-3 precursor and chimeric polypeptides consisting of residue-correct fusions of transit peptides and mature proteins derived from different plastid proteins demonstrate that the PSI-3 transit peptide is indeed capable of translocating proteins into the thylakoid lumen and that, conversely, mature PSI-3 depends on a bipartite transit peptide for its thylakoid transfer. Of the three recently described translocation/integration pathways for nucleus-encoded proteins carrying bipartite transit peptides that are distinct in their physiological requirements and strictly protein-specific, PSI-3, like plastocyanin and the 33-kDa protein of the oxygen-evolving complex, is translocated by a pathway that involves stromal factors but no proton gradient across the membrane. It is not affected by saturating amounts of the precursor for the 23-kDa protein of the oxygen-evolving complex that follows the latter route. Thylakoid translocation of PSI-3 is, however, impaired in the presence of sodium azide, which indicates that a homolog to the bacterial SecA protein might be involved in this process suggesting, thus, a prokaryote-like translocation pathway. The azide-sensitive factor appears to interact predominantly with the transit peptide of a precursor protein, since chimeras consisting of a presequence from an azide-resistant precursor and a mature part of an azide-sensitive polypeptide are still translocated in the presence of the inhibitor.

An improved in situ hybridization procedure is presented, based on synchronization of root meristems of barley and wheat, enzymatic digestion, a protoplast drop technique, and the use of the fluorescent dye Cy3. The combination of these approaches resulted in a significant increase of well-spread metaphases suitable for in situ hybridization as compared to squash preparations, and to a significantly enhanced number and intensity of hybridization signals as demonstrated for a B-hordein-specific low-copy probe of barley. In the case of Cy3 all metaphases displayed a signal, more than 60% of them on both chromatids of each gene-bearing chromosome.

The translocation of plastocyanin across the thylakoid membrane in Pisum sativum has been studied in reconstitution assays and using chimeric constructs. The reconstitution assays demonstrate that plastocyanin translocation is absolutely dependent on the presence of a stromal factor(s) and nucleotide triphosphates (NTPs), whereas neither element is required for the translocation of the 23 or 16 kDa proteins of the oxygen-evolving complex. Previous studies had revealed that the transthylakoidal delta pH is essential for translocation of the 23 and 16 kDa proteins but unnecessary for plastocyanin translocation. The basis for these mechanistic differences has been tested by analysing the translocation of a chimeric construct consisting of the presequence of the 23 kDa protein linked to the mature plastocyanin sequence. This construct is efficiently imported into thylakoids in the absence of stromal extracts or NTPs and translocation across the thylakoid membrane within intact chloroplasts is totally inhibited by the uncoupler nigericin: the translocation requirements are thus identical to those of the pre-23 kDa protein and diametrically opposite to those of pre-plastocyanin. Transport across the thylakoid membrane of a second fusion protein, consisting of the presequence of the 16 kDa protein linked to mature plastocyanin, is also dependent on a delta pH. The data suggest that two distinct systems are involved in the translocation of proteins across the thylakoid membrane, with each system recognizing specific signals within the presequences of a subset of lumenal protein precursors.

We have constructed an EcoRI-based YAC (yeast artificial chromosome) library from barley (Hordeum vulgare L. cv. Franka) using the vector pYAC4. The library consists of approximately 18,000 recombinant YACs with insert sizes ranging between 100 and 1000 kb (average of 160 kb) corresponding to 50% of the barley genome. Size fractionation after ligation resulted in an increased average insert size (av. 370 kb) but also in a substantial decrease in cloning efficiency. Less than 1% of the colonies showed homology to a plastome-specific probe; approximately 50% of the colonies displayed a signal with a dispersed, highly repetitive barley-specific probe. Using a primer combination deduced from the sequence of a member of the small Hor1 gene family coding for the C-hordein storage proteins, the library was screened by polymerase chain reaction and subsequently by the colony hybridization technique. A single YAC, designated Y66C11, with a 120 kb insert was isolated. This DNA fragment represents a coherent stretch from the terminal part of the Hor1 gene region as judged from the correspondence of the restriction patterns between Y66C11 DNA and barley DNA after hybridization with the Hor1-specific probe. Restriction with the isoschizomeric enzymes HpaII/MspI suggests a high degree of methylation of the Hor1 region in mesophyll cells but not in YAC-derived (yeast) DNA.

Plastocyanin and the 16-, 23-, and 33-kDa polypeptides of the oxygen-evolving complex associated with photosystem II are hydrophilic, nuclear encoded components of the photosynthetic machinery that are all located in the lumen of thylakoid membranes. All four proteins are therefore imported into chloroplasts and, in addition, translocated across the thylakoid membrane. They share functionally equivalent, bipartite transit peptides, which are removed in two steps during or after import into the organelle and translocation across the thylakoid membrane, respectively. The transit peptides lack any homology at the sequence level but possess remarkably similar predicted secondary structures. We have studied the targeting potential of the authentic precursor molecules and all possible chimeric combinations generated by a specific, commonly applicable cassette system, which facilitates codon-correct reciprocal exchanges of transit peptides and mature parts. An unexpected specificity of import and sorting processes was found. All constructs can be imported into the organelle, though with greatly differing efficiency. On the other hand, the lumen-targeting parts are essential but not in all cases sufficient for correct intraorganellar routing. This implies that translocation across the thylakoid membrane appears not to depend merely on simple interactions of charged or hydrophobic regions between protein and membrane but requires an additional quality of information that includes the functional co-evolution of a transit peptide with its mature protein. Signaling and sorting appear to be essential at almost every step of the entire process for proper traffic regulation since distinct steps can be impaired (rate-limiting or arrested) in the individual combinations: the transfer across the envelope membranes (e.g. 16/PC) and the interaction with (e.g. 16/33) or the translocation across the thylakoid membrane (e.g. 33/23).

The B-hordein gene family consists of at least 13 genes that appear to be clustered in two regions on the barley chromosome 1H (5). Using fluorescence in situ hybridization techniques, we have localized the B-hordein locus (Hor2) to the distal end of the short arm of chromosome 1H (5), corresponding to the genetically determined position, 91% distal on the short arm. This result is based on appropriate barley lines (i.e. the two cultivars Igri and Betzes, the telotrisomic and ditelotetrasomic lines 1HS and the translocation line T21), on two experimental approaches and on a signal frequency of between 20% and 60% on metaphase chromosomes.

We have isolated the genes encoding 11 photosystem I (PSI) subunits from Synechococcus sp., from which this reaction center has been crystallized. The recombinant DNAs, including psaA, psaB, psaC, psaD, psaE, psaF, psal, psaJ, psaK and psaL , were obtained by heterologous hybridization with probes from appropriate cDNAs or genes from spinach and Synechocystis sp. PCC 6803, or with synthetic oligodeoxyribonucleotides. Genes psaA/psaB, psaF/psaJ and psaL/psal are each closely linked. The open reading frames predict polypeptides of 83 kDa (subunits la and Ib, encoded by genes psaA and psaB , respectively), 15.4 kDa (II, psaD ), 17.7 kDa (III, psaF ), 8.4 kDa (IV, psaE ), 8.8 kDa (VII, psaC ), 4.6 kDa (VIII, psal ), 4.8 kDa (IX, psaJ ), 8.5 kDa (X, psaK ) and 15.5 kDa (XI, psaL ). A novel subunit (XII, psaM ) was also identified. Subunits II, III, IV and VII seem to be peripheral, while the others seem to be intrinsic components of the reaction center. These data imply a striking similarity of cyanobacterial and eukaryotic PSI. All subunits studied are encoded by single-copy genes which seem to be transcribed into monocistronic ( psaC, psaD, psaC, psaK ) or dicistronic ( psaA/psaB, psaF/psaJ, psaL/psal ) RNA species. Subunit III is translated as a 17.7-kDa precursor, including a transit peptide of 23 amino acid residues. This is consistent with its location in the thylakoid lumen.

The targeting of cytosolically synthesized proteins into the thylakoid lumen is mediated by an aminoterminal pre-sequence consisting of an "envelope transit" and a "thylakoid transfer" signal in tandem. We have investigated the structural characteristics of several thylakoid transfer signals by determining the intermediate sites at which the stromal processing peptidase cleaves to remove the transit sequences. Using this approach we have found that the thylakoid transfer signals of Silene pratensis plastocyanin, 23-kDa oxygen-evolving complex protein from wheat, and 33-kDa oxygen-evolving complex protein from wheat, are 25, 39, and 48 residues in length, respectively. All of the transfer signals contain hydrophobic core sequences and a "-3,-1" motif reminiscent of those found in signal sequences, but the amino-terminal regions of the transfer signals of the 23- and 33-kDa proteins are both longer and more highly charged. The net charge of each amino-terminal region of the transfer sequences is +1, including the amino-terminal amino group. In each case, the stromal processing peptidase cleaves immediately after a positively charged residue, but otherwise the cleavage sites exhibit no common elements of either primary or secondary structure.

We have isolated recombinant lambda gt11 phages which carry cDNA clones for the major light-harvesting chlorophyll a/b -binding proteins of photosystem I (LHCPI) and II (LHCPII), subunit II of photosystem I, a chlorophyll a/b -binding protein of photosystem II (CP24), the Rieske iron—sulphur protein of the cytochrome b 6 / f complex, and the 33, 23 and 16 kDa proteins of the water-oxidizing complex of photosystem II from Nicotiana tabacum . The nucleotide sequences of cDNA clones encoding the precursors for LHCPI and the FeS protein are presented. If tobacco or tomato seedlings, or seedlings of a phytochrome-deficient aurea mutant of tomato which lacks more than 95% of the phytochrome of the isogenic wild type, are kept in blue light, the transcript level of each of these genes is higher than in seedlings grown in red light suggesting the involvement of a blue—UVA-light photoreceptor. In the case of LHCPI, a 1 min blue-light pulse applied to red-light-grown seedlings is sufficient to increase the transcript levels to those present in blue-light-grown seedlings, whereas almost no increase is observed for transcripts encoding the FeS and 33 kDa proteins. If dark-grown tomato seedlings receive a single far-red-light pulse, significant stimulation is detected for LHCPI transcripts, whereas transcripts encoding the FeS and 33 kDa proteins are not stimulated. It is concluded that the lower light requirement for the increase in the LHCPI transcript level is not specific for one of the light-dependent signal transduction chains.

Transit peptides are N-terminal extensions that serve to route nuclearencoded proteins into chloroplasts. We have collected amino acid sequences for approximately 260 transit peptides, and have constructed a database that presently runs under FileMaker Plus on Macintosh personal computers. In addition, the collection is available as an ASCII-file, either in a tab-delimited format suitable for import into other database programs, or in a format with line-identifiers similar to the widely used Swiss Prot database. We hope that the plant molecular biology community will help us ensure that the collection is kept up to date by submitting new sequences directly to us.

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.

A technique is described for using standard squash preparations of mitotic and meiotic chromosomes for both light microscopy and subsequent high-resolution scanning electron microscopy for investigation of the same specimen. Depending on the microscope and conditions of preparation, a resolution of a few nanometers is routinely possible. Tilting of the specimen provides a three-dimensional insight into chromosomal structures. Combination of material-dependent signals of backscattered electrons with the secondary electron image allows an unambiguous localization of surface markers.

The promotors of the single-copy genes for subunit delta of the chloroplast ATP synthase (atpD) and plastocyanin (PC) from spinach have been sequenced, dissected and analysed in transgenic F0 and F1 tobacco plants using the bacterial GUS gene as a reporter for promotor activity. The transcription of these genes is photo-controlled. The results have been compared with those obtained for the spinach rbcS-1 gene, one of the light-regulated genes encoding the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase, and for the cauliflower mosaic virus (CaMV) 35S RNA promotor. We find that the 5' upstream regions of about 1200 nucleotides contain all the sequences required for light regulation, organ-, tissue- and development-specific expression, and that they are structurally diverse. Their cis-acting elements are functionally defined. The proximal regions of the spinach promotors contain potential TATA, CAAT and T-cyt boxes at appropriate positions, but only sequence elements with low similarity to published light-responsive elements. Positive light-stimulated regions, regions with constitutive, light-independent enhancing effects and with 'silencer'-like activity in complete darkness are found in proximal and far upstream promotor segments. Highest activity of these promotors is correlated with the presence of chloroplasts but is not confined to photosynthetic tissue. Surprisingly, expression is observed in the phloem regions of transgenic leaves, leaf and floral stems, in the vascular area of anthers and in pollen. No histochemical staining has been detected in roots. The distal region of atpD located between -1137 and -590 contains elements for expression in the outer phloem, the region from -590 to -185 for activity in the inner phloem of floral stems. Similar tissue-specific patterns are observed with a fusion between the caufliflower mosaic virus 35S RNA promotor and the GUS gene.

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 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.

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.

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 cytochrome b6 polypeptide present in cytochrome b6/| preparations from spinach thylakoids is phosphorylated concomitantly with the autophosphorylation of the 64 kDa polypeptide identified as the redox-controlled LHCII kinase. The N-terminal sequence of the 64 kDa kinase and sequence analysis of cytochrome b6 indicate the existence of putative phosphorylation sites in both proteins.

Summary The plastid encoded RNA polymerase subunit genes rpoA, B and C1 of tobacco were disrupted individually by PEG-mediated plastid transformation. The resulting off-white mutant phenotype is identical for inactivation of the different genes. The mutants pass through a normal ontogenetic cycle including flower formation and production of fertile seeds. Their plastids reveal a poorly developed internal membrane system consisting of large vesicles and, occasionally, flattened membranes, reminiscent of stacked thylakoids. The rpo–material is capable of synthesising pigments and lipids, similar in composition but at lower amounts than the wild-type. Western analysis demonstrates that plastids contain nuclear-coded stroma and thylakoid polypeptides including terminally processed lumenal components of the Sec but not of the ΔpH thylakoid translocation machineries. Components using the latter route accumulate as intermediates. In striking contrast, polypeptides involved in photosynthesis encoded by plastid genes could not be detected by Western analysis, although transcription of plastid genes, including the rrn operon, by the plastid RNA polymerase of nuclear origin is found as expected. Remarkably, ultrastructural, sedimentation and Northern analyses as well as pulse experiments suggest that rpo–plastids contain functional ribosomes. The detection of the plastid-encoded ribosomal protein Rpl2 is consistent with these results. The findings demonstrate that the consequences of rpo gene disruption, and implicitly the integration of the two plastid polymerase types into the entire cellular context, are considerably more complex than presently assumed.

The Clp proteases represent a large, ancient ATP-dependent protease family which in higher plants is known to be located in chloroplasts. The soluble, presumably multisubunit, enzyme of the organelle stroma is of dual genetic origin. It consists of a nuclear-encoded, regulatory subunit ClpC, which is an ATPase, and a plastid-encoded proteolytic subunit ClpP, which is a serine protease. An additional, nuclear-encoded proteolytic subunit resembling ClpP has been recently reported from tomato (Schaller and Ryan, 1995 plant gene Register 95–00). We demonstrate that in both tomato Lycopersicon esculentum Mill. and Arabidopsis thaliana, (L.) Heynh. the nuclear-encoded ClpP (nClpP) is made as a precursor molecule that can be imported into isolated intact chloroplasts of spinach (Spinacia oleracea L.) and processed in two or three steps, respectively, to the size of the authentic protein. Furthermore, both gel electrophoresis under non-denaturing conditions and size-exclusion chromatography verified that the three proteins can form distinct heteromeric supramolecular complexes of approximately 860, 1380 and 1700 kDa (probably also of 600 kDa) molecular mass. The size ranges of the former two are reminiscent of those of Clp complexes described from Escherichia coli. In addition, various complexes between 160 and 560 kDa are detectable with the individual components. Both the processing “intermediates” and the mature nClpP are found in assembled form.

We describe the identification of the first immunophilin associated with the photosynthetic membrane of chloroplasts. This complex 40 kDa immunophilin, designated TLP40 (thylakoid lumen PPIase), located in the lumen of the thylakoids, was found to play a dual role in photosynthesis involving both biogenesis and intraorganelle signalling. It originates in a single‐copy nuclear gene, is made as a precursor of 49.2 kDa with a bipartite lumenal targeting transit peptide, and is characterized by a structure including a cyclophilin‐like C‐terminal segment of 20 kDa, a predicted N‐terminal leucine zipper and a potential phosphatase‐binding domain. It can exist in different oligomeric conformations and attach to the inner membrane surface. It is confined predominantly to the non‐appressed thylakoid regions, the site of protein integration into the photosynthetic membrane. The isolated protein possesses peptidyl‐prolyl cis–trans isomerase protein folding activity characteristic of immunophilins, but is not inhibited by cyclosporin A. TLP40 also exerts an effect on dephosphorylation of several key proteins of photosystem II, probably as a constituent of a transmembrane signal transduction chain. This first evidence for a direct role of immunophilins in a photoautotrophic process suggests that light‐mediated protein phosphorylation in photosynthetic membranes and the role of the thylakoid lumen are substantially more complex than anticipated.

The molecular recognition between plastocyanin and photosystem I was studied. Photosystem I and plastocyanin can be cross-linked to an active electron transfer complex. Immunoblots and mass spectrometric analysis of proteolytic peptides indicate that the two negative patches conserved in plant plastocyanins are cross-linked with lysine residues of a domain near the N-terminus of the PsaF subunit of photosystem I. Conversion of these negative to uncharged patches of plastocyanin by site-directed mutation D42N/E43Q/D44N/E45Q and E59Q/E60Q/D61N respectively, reveals the first patch to be essential for the electrostatic interaction in the electron transfer complex with photosystem I and the second one to lower the redox potential. The domain in PsaF, not found in cyanobacteria, is predicted to fold into two amphipathic alpha-helices. The interacting N-terminal helix lines up six lysines on one side which may guide a fast one-dimensional diffusion of plastocyanin and provide the electrostatic attraction at the attachment site, in addition to the hydrophobic interaction in the area where the electron is transferred to P700 in the reaction center of photosystem I. This two-step interaction is likely to increase the electron transfer rate by more than two orders of magnitude in plants as compared with cyanobacteria. Our data resolve the controversy about the function of PsaF.

Stellacyanins are blue (type I) copper glycoproteins that differ from other members of the cupredoxin family in their spectroscopic and electron transfer properties. Until now, stellacyanins have eluded structure determination. Here we report the three-dimensional crystal structure of the 109 amino acid, non-glycosylated copper binding domain of recombinant cucumber stellacyanin refined to 1.6 A resolution. The crystallographic R-value for all 18,488 reflections (sigma > 0) between 50-1.6 A is 0.195. The overall fold is organized in two beta-sheets, both with four beta-stands. Two alpha-helices are found in loop regions between beta-strands. The beta-sheets form a beta-sandwich similar to those found in other cupredoxins, but some features differ from proteins such as plastocyanin and azurin in that the beta-barrel is more flattened, there is an extra N-terminal alpha-helix, and the copper binding site is much more solvent accessible. The presence of a disulfide bond at the copper binding end of the protein confirms that cucumber stellacyanin has a phytocyanin-like fold. The ligands to copper are two histidines, one cysteine, and one glutamine, the latter replacing the methionine typically found in mononuclear blue copper proteins. The Cu-Gln bond is one of the shortest axial ligand bond distances observed to date in structurally characterized type I copper proteins. The characteristic spectroscopic properties and electron transfer reactivity of stellacyanin, which differ significantly from those of other well-characterized cupredoxins, can be explained by its more exposed copper site, its distinctive amino acid ligand composition, and its nearly tetrahedral ligand geometry. Surface features on the cucumber stellacyanin molecule that could be involved in interactions with putative redox partners are discussed.

Thylakoid membranes of chloroplasts are composed of approx. 75 polypeptide species. Nearly 60% originate in nuclear genes, the remainder in plastid genes. In order to localize representatives of the nuclear-encoded gene complement in a eukaryotic plant genome (sugar beet, Beta vulgaris L.), we have investigated the RFLP patterns of 21 cDNAs from spinach that code for thylakoid proteins or proteins peripherally associated with thylakoid membranes. Differences in gene dosage were noted between both related species. Polymorphism was found for 12 cDNA loci in a segregating sugar beet F2 population. These loci were mapped along with genomic RFLP, isozyme, and morphological markers, and shown to be distributed in six of the nine sugar beet linkage groups. The lack of positional clustering even of genes that encode components of the same supramolecular membrane assembly is commensurate with phylogenetically independent gene translocations from the plastid (endosymbiont), and raises the question of the functional integration of various translocated genes into common signal transduction chains.

Run-on assays with isolated nuclei demonstrate that the transcription rates of AtpC and AtpD (gene products: the CF1 subunits gamma and delta of the chloroplast ATP synthase) are comparable in spinach seedlings. However, chimeric GUS gene fusions with 5'-flanking regions of the AtpC gene direct an approximately 10-fold lower GUS level in transgenic tobacco compared with equivalent fragments from the AtpD gene. Both promoters contain sequences in the vicinity of the respective TATA boxes, which are sufficient to direct light-regulated, plastid-dependent and organ-specific expression of the GUS gene. In contrast, the upstream regions of both promoters differ the higher GUS level directed by the AtpD promoter is caused by enhancer-like elements located upstream of the region involved in the regulated expression, while nucleotides upstream of -73 in the AtpC promoter contribute relatively little to the promoter activity. 5'-Deletion analyses and site-directed mutagenesis studies indicated that the -73/-48 bp AtpC region contains cis-elements crucial for this regulated expression. If five nucleotides within this region (-59/-55) are exchanged, the GUS gene is constitutively expressed and the activity in etiolated seedlings, in seedlings with photobleached plastids and in roots increases to the level detectable in green cotyledons. It is concluded that signal transduction pathways from different regulators converge prior to gene regulation and that these five nucleotides are part of a cis-element which functions as a repressor in darkness, in tissues with impaired plastids and in roots.

The delta pH-driven and Sec-related thylakoidal protein translocases recognise distinct types of thylakoid transfer signal, yet all transfer signals resemble bacterial signal peptides in structural terms. Comparison of known transfer signals reveals a single concrete difference: signals for the delta pH-dependent system contain a common twin-arginine motif immediately before the hydrophobic region. We show that this motif is critical for the delta pH-driven translocation process; substitution of the arg-arg by gln-gln or even arg-lys totally blocks translocation across the thylakoid membrane, and replacement by lys-arg reduces the rate of translocation by > 100-fold. The targeting information in this type of signal thus differs fundamentally from that of bacterial signal peptides, where the required positive charge can be supplied by any basic amino acid. Insertion of a twin-arg motif into a Sec-dependent substrate does not alter the pathway followed but reduces translocation efficiency, suggesting that the motif may also repel the Sec-type system. Other information must help to specify the choice of translocation mechanism, but this information is unlikely to reside in the hydrophobic region because substitution by a hydrophobic section from an integral membrane protein does not affect the translocation pathway.