On the translocation of proteins across the chloroplast envelope

Most of the chloroplast proteins are coded for in the nucleus and are synthesized in the cytosol from where they are subsequently transported into the different chloroplast compartments. The structural properties of the N-terminal extensions (transit peptides) of these nuclear-coded precursor proteins are discussed as well as the energy requirements for their translocation and the involvement of receptor proteins and that of other (ATP-dependent) factors.     

The activity of Triton X-100 soluble chlorophyllase in liposomes

Chlorophyllase (chlorophyll-chlorophyllidohydrolase, EC 3.1.1.14) was isolated and purified from Phaseolus vulgaris L. chloroplasts and etioplasts dissolved in 1% Triton X-100 and 10% glycerol. A 100 and 40-fold purification, respectively, was achieved. Enzyme preparations from both sources had similar affinities for chlorophyll a when assayed in a Triton X-100 medium. When electrophoresed in sodium dodecyl sulphate polyacrylamide gels the major band in both preparations migrated as a peptide of 30,000 daltons. Chlorophyll containing liposomes were also used as a substrate for chlorophyllase. The rate of hydrolysis did not follow Michaelis-Menten kinetics. When chlorophyllide a or methyl chlorophyllide a was incorporated in the liposomes, then in the presence of phytol dissolved in methanol, methylchlorophyllide a and chlorophyll a were shown to be synthesized. Apparently the purified enzyme in the presence of lipids, is endowed with both synthetic and hydrolytic activity.Abbreviations DEAE diethylaminoethyl - MeOH methanol - SDS sodium dodecyl sulphate     

Light-dependent particle movement in the outer envelope of chloroplasts fromCodium australicum (Silva)

Summary Chloroplasts from light treatedCodium australicum (Silva) show two regions of differing particle density in the outer envelope EF face, both in fixed and unfixed material. The region with high particle density is always oriented toward the cell wall. Dark treated chloroplasts show no such division. Particle size histograms indicate that this division is chiefly the result of a shift of existing large particles toward the cell wall in the light. Some addition of new material may also occur.     

A unique protein-kinase activity is responsible for the phosphorylation of the 26- and 14-kDa proteins but not of the 67-kDa protein in the chloroplast envelope membranes of spinach

Protein-phosphorylation activity has been reported in chloroplast envelope membranes of several species. In spinach (Spinacia oleracea L.), we found three major phosphoproteins after incubation in vitro of envelope membranes in the presence of [-³²P]ATP. A 67-kDa phosphoprotein was associated with both inner and outer envelope membranes whereas 26- and 14-kDa proteins were observed in the inner membrane. Although the phosphorylation of the 67-kDa protein is likely to take place via its phosphoglucomutase activity (Salvucci et al., 1990, Plant Physiol. 93, 105–109), the mechanism by which ³²P is incorporated into the 26- and 14-kDa proteins remains to be elucidated. To this aim, we have compared the conditions under which phosphorylation occurs in these three proteins. The effects of Mg²⁺, Ca²⁺, pH, ATP and H7 [1-(5-isoquinolinesulfonyl)-2-methylpiperazine], a specific inhibitor of protein-kinase C, as well as pulse-chase experiments with cold ATP, showed that the phosphorylation mechanism was identical for the 26- and 14-kDa proteins but quite different for the 67-kDa one. The protein kinase involved in the phosphorylation of the 26- and 14-kDa proteins was Ca²⁺-dependent, which was not the case of the 67-kDa protein. In addition, the use of a Triton X-114 phase-separation treatment indicated that both the 26- and 14-kDa proteins exhibited strong hydrophobic properties, in contrast to the hydrophilic character of the 67-kDa phosphoprotein. As indicated by analyses of phosphoamino acids, the three proteins were exclusively phosphorylated on serine residues. Furthermore, a treatment of envelopes by phospholipase C prior to the phosphorylation process inhibited ³²P incorporation into the three phospho-proteins to different extents (61%, 50% and 29% inhibition for the 67-, 14- and 26-kDa proteins, respectively). These results show that phosphatidylcholine and — or phosphatidylglycerol but not phosphatidylinositol were involved in this phosphorylation process.Abbreviations EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - H7 1-(5-isoquinolinesulfonyl)-2-methylpiperazine - M_r relative molecular mass - PAGE polyacrylamide gel electrophoresis - Rubisco ribulose-1,5-bisphosphate carboxylase-oxygenase - SDS sodium dodecyl sulfate The authors are grateful to Mrs. Delphine Herrmann and Mr. Daniel Leemann for their skillful technical assistance. This study was supported by the Swiss National Science Foundation (Grant No. 31.26386.89). This work is part of a doctoral program which is carried out by L.B. in the Laboratoire de Physiologie végétale, Université de Neuchâtel.     

Species relationships in the Hordeum murinum aggregate viewed from chloroplast DNA restriction fragment patterns

Summary Three annual widespread species of Hordeum were investigated by the fragment pattern method on their chloroplast (cp) DNA. The species were H. glaucum, H. leporinum and H. murinum; H. vulgare was surveyed for comparison. Twelve restriction enzymes were used, nine recognizing 6 bp, one 5 bp and two 4 bp, thus, randomly surveyed, a total of 2,113 bp or 1.6% of the cp genome. Differences in patterns were found in three enzymes, HindIII, CfoI and MspI. CfoI characterizes H. glaucum from the other two species. HindIII and MspI revealed polymorphisms within species. These results confirm previous numerical taxonomic relationships among these three closely related species. Furthermore, cpDNA polymorphism in Hordeum is discussed in view of earlier reports on cpDNA polymorphism in H. vulgare. The taxonomic implications of cpDNA polymorphism are discussed after reviewing several articles using the fragment pattern method on cpDNA. The importance of using material from several populations representative of a species is stressed.     

Localization and function of SulP,a nuclear-encoded chloroplast sulfate permease in<Emphasis Type="Italic"> Chlamydomonas reinhardtii</Emphasis>

Recent work [H.-C. Chen et al. (2003) Planta 218:98-106] reported on the genomic, proteomic, phylogenetic and evolutionary aspects of a putative nuclear gene ( SulP) encoding a chloroplast sulfate permease in the model green alga Chlamydomonas reinhardtii. In this article, evidence is provided for the envelope localization of the SulP protein and its function in the uptake and assimilation of sulfate by the chloroplast. Localization of the SulP protein in the chloroplast envelope was concluded upon isolation of C. reinhardtii chloroplasts, followed by fractionation into envelope and thylakoid membranes and Western blotting of these fractions with specific polyclonal antibodies raised against the recombinant SulP protein. The function of the SulP protein was probed in antisense transformants of C. reinhardtii having lower expression levels of the SulP gene. Results showed that cellular sulfate uptake capacity was lowered as a consequence of attenuated SulP gene expression in the cell, directly affecting rates of de novo protein biosynthesis in the chloroplast. The antisense transformants exhibited phenotypes of sulfate-deprived cells, displaying slow rates of light-saturated oxygen evolution, low levels of Rubisco in the chloroplast and low steady-state levels of the photosystem-II D1 reaction-center protein. The role of the chloroplast sulfate transport in the uptake and assimilation of sulfate in C. reinhardtii is discussed along with its impact on the repair of photosystem-II from a frequently occurring photo-oxidative damage and potential use for the elucidation of the H(2)-evolution-related metabolism in this green alga.     

Proteomics of chloroplast envelope membranes

Proteomics is a very powerful approach to link the information contained in sequenced genomes, like Arabidopsis, to the functional knowledge provided by studies of plant cell compartments, such as chloroplast envelope membranes. This review summarizes the present state of proteomic analyses of highly purified spinach and Arabidopsis envelope membranes. Methods targeted towards the hydrophobic core of the envelope allow identifying new proteins, and especially new transport systems. Common features were identified among the known and newly identified putative envelope inner membrane transporters and were used to mine the complete Arabidopsis genome to establish a virtual plastid envelope integral protein database. Arabidopsis envelope membrane proteins were extracted using different methods, that is, chloroform/methanol extraction, alkaline or saline treatments, in order to retrieve as many proteins as possible, from the most to the less hydrophobic ones. Mass spectrometry analyses lead to the identification of more than 100 proteins. More than 50% of the identified proteins have functions known or very likely to be associated with the chloroplast envelope. These proteins are (a) involved in ion and metabolite transport, (b) components of the protein import machinery and (c) involved in chloroplast lipid metabolism. Some soluble proteins, like proteases, proteins involved in carbon metabolism or in responses to oxidative stress, were associated with envelope membranes. Almost one third of the newly identified proteins have no known function. The present stage of the work demonstrates that a combination of different proteomics approaches together with bioinformatics and the use of different biological models indeed provide a better understanding of chloroplast envelope biochemical machinery at the molecular level.     

Characterization of a protein of the plastid inner envelope having homology to animal inorganic phosphate,chloride and organic-anion transporters

A protein from Arabidopsis thaliana (L.) Heynh. showing homology to animal proteins of the NaPi-1 family, involved in the transport of inorganic phosphate, chloride, glutamate and sialic acid, has been characterized. This protein, named ANTR2 (for anion transporters) was shown by chloroplast subfractionation to be localized to the plastid inner envelope in both A. thaliana and Spinacia oleracea (L.). Immunolocalization revealed that ANTR2 was expressed in the leaf mesophyll cells as well as in the developing embryo at the upturned-U stage. Five additional homologues of ANTR2 are found in the Arabidopsis genome, of which one was shown by green fluorescent protein (GFP) fusion to be also located in the chloroplast. All ANTR proteins share homology to the animal NaPi-1 family, as well as to other organic-anion transporters that are members of the Anion:Cation Symporter (ACS) family, and share the main features of transporters from this family, including the presence of 12 putative transmembrane domains and of a 7-amino acid motif in the fourth putative transmembrane domain. ANTR2 thus represent a novel protein of the plastid inner envelope that is likely to be involved in anion transport.Abbreviations ACS Anion:Cation Symporter - GFP green fluorescent protein - Pi inorganic phosphate     

Selectable marker recycling in the chloroplast

The bacterial geneaadA is an important and widely used selectable marker for manipulation of the chloroplast genome through biolistic transformation. Because no other such marker is available, two strategies for recycling of theaadA cassette have been developed. One utilizes homologous recombination between two direct repeats flanking theaadA cassette to allow its loss under non-selective growth conditions. A second strategy is to perform co-transformation with a plasmid containing a modified, non-essential chloroplast gene and another plasmid in which theaadA cassette disrupts a chloroplast gene known to be essential for survival. Under selective growth conditions the first mutation can be transferred to all chloroplast DNA copies whereas theaadA insertion remains heteroplasmic. Loss of the selectable marker can be achieved subsequently by growing the cells on non-selective media. In both cases it is possible to reuse theaadA cassette for the stepwise disruption or mutagenesis of any gene in the same strain.     

Phytogeny of Brassica and allied genera based on variation in chloroplast and mitochondrial DNA patterns: molecular and taxonomic classifications are incongruous

Summary Chloroplast DNA (cpDNA) variability of 60 taxa of the genus Brassica and allied genera comprising 50 species was studied. RFLPs for seven enzymes were generated and F values were estimated from five frequently cutting enzymes. Phenetic clusterings indicated a clear division of Brassica coenospecies into two distinct lineages referred to as the Brassica and Sinapis lineages. Two unexplored genera, Diplotaxis and Erucastrum, also exhibited two lineages in addition to the genera Brassica and Sinapis. This finding is inconsistent with the existing taxonomic classification based on morphology. Mitochondrial DNA (mtDNA) variability studied from EcoRI RFLP patterns, by hybridizing total DNA with four cosmid clones containing non-overlapping mtDNA fragments, did not show any congruence with cpDNA variation patterns. However, at the cytodeme level, the patterns of genetic divergence suggested by the cpDNA data could be correlated with mtDNA variation. In the Brassica lineage, Diplotaxis viminea was identified as the female parent of the allotetraploid D. muralis. The chloroplast DNAs of Erucastrum strigosum and Er. abyssinicum were found to be very closely related. In the Sinapis lineage, Brassica maurorum was found to be the diploid progenitor of autotetraploid B. cossoneana. B. amplexicaulis showed a very different cpDNA pattern from other members of the subtribe. Brassica adpressa was closest to Erucastrum laevigatum and could be the diploid progenitor of autotetraploid Er. laevigatum. Based on the close similarity of the cpDNA pattern of Diplotaxis siifolia with that of D. assurgens, we have proposed the retention of this species in the genus Diplotaxis. The taxonomic positions of some other species have also been discussed.     

Targeted disruption of chloroplast genes in Chlamydomonas reinhardtii.

Summary We have developed an efficient procedure for the disruption of Chlamydomonas chloroplast genes. Wild-type C. reinhardtii cells were bombarded with microprojectiles coated with a mixture of two plasmids, one encoding selectable, antibiotic-resistance mutations in the 16S ribosomal RNA gene and the other containing either the atpB or rbcL photosynthetic gene inactivated by an insertion of 0.48 kb of yeast DNA in the coding sequence. Antibiotic-resistant transformants were selected under conditions permissive for growth of nonphotosynthetic mutants. Approximately half of these transformants were initially heteroplasmic for copies of the disrupted atpB or rbcL genes integrated into the recipient chloroplast genome but still retained photosynthetic competence. A small fraction of the transformants (1.1% for atpB; 4.3% for rbcL) were nonphotosynthetic and homoplasmic for the disrupted gene at the time they were isolated. Single cell cloning of the initially heteroplasmic transformants also yielded nonphotosynthetic segregants that were homoplasmic for the disrupted gene. Polypeptide products of the disrupted atpB and rbcL genes could not be detected using immunoblotting techniques. We believe that any nonessential Chlamydomonas chloroplast gene, such as those involved in photosynthesis, should be amenable to gene disruption by cotransformation. The method should prove useful for the introduction of site-specific mutations into chloroplast genes and flanking regulatory sequences with a view to elucidating their function.     

Mechanistic analysis of wheat chlorophyllase

Chlorophyllase catalyzes the initial step in the degradation of chlorophyll and plays a key role in leaf senescence and fruit ripening. Here, we report the cloning of chlorophyllase from Triticum aestivum (wheat) and provide a detailed mechanistic analysis of the enzyme. Purification of recombinant chlorophyllase from an Escherichia coli expression system indicates that the enzyme functions as a dimeric protein. Wheat chlorophyllase hydrolyzed the phytol moiety from chlorophyll (k(cat) = 566 min(-1); K(m) = 63 microM) and was active over a broad temperature range (10-75 degrees C). In addition, the enzyme displays carboxylesterase activity toward p-nitrophenyl (PNP)-butyrate, PNP-decanoate, and PNP-palmitate. The pH-dependence of the reaction showed the involvement of an active site residue with a pK(a) of approximately 6.5 for both k(cat) and k(cat)/K(m) with chlorophyll, PNP-butyrate, and PNP-decanoate. Using these substrates, solvent kinetic isotope effects ranging from 1.5 to 1.9 and from 1.4 to 1.9 on k(cat) and k(cat)/K(m), respectively, were observed. Proton inventory experiments suggest the transfer of a single proton in the rate-limiting step. Our analysis of wheat chlorophyllase indicates that the enzyme uses a charge-relay mechanism similar to other carboxylesterases for catalysis. Understanding the activity and mechanism of chlorophyllase provides insight on the biological and chemical control of senescence in plants and lays the groundwork for biotechnological improvement of this enzyme.     

The coding site of chloroplast ferredoxin

Ferredoxins were isolated and purified from leaves of different species of Nicotiana and Petunia and from spinach leaves. Their spectral properties, degree of homogeneity, and molecular weights were determined. The preparations were further analyzed by polyacrylamide gel electrophoresis of tryptic hydrolysates. This allowed us to distinguish between not only ferredoxins of Nicotiana, Petunia, and spinach, but even ferredoxins of various Nicotiana species. We used the differences in tryptic peptide compositions as phenotypic markers to study the mode of inheritance of chloroplast ferredoxin to see whether the coding site is in the chloroplast or in the nucleus. Analysis of the tryptic peptide composition of ferredoxin from different interspecific hybrids of Nicotiana showed that the characteristics of both parental ferredoxins were present. The results indicate that the primary structure of at least the male ferredoxin is coded for in the nucleus. In some of the hybrids the relative contribution of the male parent appeared to be low, suggesting that the female genome (presumably that part located in the plastome) exerted a dominating influence.     

Isolation and phenotypic characterization ofChlamydomonas reinhardtii mutants defective in chloroplast DNA segregation

Summary Each wild-typeChlamydomonas reinhardtii cell has one large chloroplast containing several nuclei (nucleoids). We used DNA insertional mutagenesis to isolate Chlamydomonas mutants which contain a single, large chloroplast (cp) nucleus and which we namedmoc (monokaryotic chloroplast). DAPI-fluorescence microscopy and microphotometry observations revealed thatmoc mutant cells only contain one cp-nucleus throughout the cell division cycle, and that unequal segregation of cpDNA occurred during cell division in themoc mutant. One cell with a large amount of cpDNA and another with a small amount of cpDNA were produced after the first cell division. Unequal segregation also occurred in the second cell division, producing one cell with a large amount (about 70 copies) of cpDNA and three other cells with a small amount (only 2–8 copies) of cpDNA. However, most individualmoc cells contained several dozen cpDNA copies 12 h after the completion of cell division, suggesting that cpDNA synthesis was activated immediately after chloroplast division. In contrast to the cpDNA, the mitochondrial (mt) DNA of themoc mutants was observed as tiny granules scattered throughout the entire cell. These segregated to each daughter cell equally during cell division. Electron-microscopic observation of the ultrastructure ofmoc mutants showed that a low-electron-density area, which was identified as the cp-nucleus by immunoelectron microscopy with anti-DNA antibody, existed near the pyrenoid. However, there were no other structural differences between the chloroplasts of wild-type cells andmoc mutants. The thylakoid membranes and pyrenoid were identical. Therefore, we propose that the novelmoc mutants are only defective in the dispersion and segregation of cpDNA. This strain should be useful to elucidate the mechanism for the segregation of cpDNA.Abbreviations DAPI 4,6-diamidino-2-phenylindole - VIMPCS video-intensified microscope photon-counting system     

Cytokinin treatment of embryos inhibits the synthesis of chloroplast proteins in Norway spruce

A pulse treatment of Norway spruce (Picea abies (L.) Karst) embryos with the cytokinin N⁶-benzyladenine induces the formation of adventitious buds from subepidermal cells in the hypocotyl and cotyledons. In addition the treatment also inhibits elongation growth, a key process during germination. In this report we demonstrate that these effects on development of the plant are associated with a suppression of the accumulation of several major chloroplast proteins during germination. These proteins include the large subunit of ribulose bisphosphate/carboxylase oxygenase, two subunits of the chloroplast ATPase, protochlorophyllide reductase and a 23000-M_r component of photosystem II. For two nuclear-encoded proteins, the small subunit of ribulose bisphosphate carboxylase/oxygenase and the light-harvesting chlorophyll a/b-binding protein, a corresponding suppression of the increase in the steady-state amounts of mRNA is recorded. The suppression of chloroplast protein synthesis is consistant with the previously documented delay in greening that results from cytokinin treatment, but the effect is opposite to that found in other plants, where cytokinins promote the synthesis of chloroplast proteins, and stimulate chloroplast biogenesis. We believe that this difference is explained by the cytokinin primarily suppressing organ development, and a strict dependance of chloroplast biogenesis on the developmental state of the organs.Abbreviations Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - CF1 coupling-factor 1 of chloroplast ATPase - LHCP light-harvesting chlorophyll a/b-binding protein - LSU large subunit of Rubisco - NADPH-protochlorophyllide oxidoreductase Pchlide reductase - SDS sodium dodecyl sulfate - SSU small subunit of Rubisco We thank K. Hutchison (Dept. of Biochemistry, University of Maine, Orono, Maine, USA) and P. Gustafsson (Dept. of Plant Physiology, University of Umeå, Sweden) for providing the Larix and Pinus clones, and M. Ryberg (Dept. of Plant Physiology, University of Göteborg, Sweden), R. Ölmüller (Botanisches Institut, Universität München, FRG) and W. Lockau (Institut für Botanik, Universität Regensburg, FRG), for the gift of antisera towards Pchlide reductase, RuBPCase and LHCP, and ATPase, respectively. Supported by the Swedish Council for Forestry and Agricultural Research and the Swedish Natural Sciences Research Council.     

The outer envelope protein OEP24 from pea chloroplasts can functionally replace the mitochondrial VDAC in yeast

The chloroplastic outer envelope protein OEP24 from pea forms a high-conductance low specificity solute channel as shown by in vitro studies. In order to establish its function also in an in vivo-like system, the gene encoding OEP24 was transformed into a yeast strain which lacks the general mitochondria solute channel porin, also known as voltage-dependent anion channel (VDAC). Transformation of the yeast VDAC(-) strain with the OEP24 gene resulted in the recovery of a phenotype indistinguishable from the wild-type. The OEP24 polypeptide is targeted to the mitochondrial outer membrane in this heterologous system. We conclude that OEP24 forms a solute channel in pea chloroplasts in planta.     

Absence of mitochondrial and chloroplast DNA recombinations in Brassica napus plants regenerated from protoplasts,protoplast fusions and anther culture

Summary Over 400 Brassica napus plants regenerated from individual protoplasts, from protoplast fusions and from anther culture were analysed for chloroplast and mitochondrial genome rearrangements by restriction fragment length polymorphisms. None were detected, attesting to the fidelity of the tissue culture procedures employed. In the majority of protoplast fusion products, the cytoplasmic organelles had completely sorted out at the callus stage but three regenerated plants possessed mixed parental populations of mitochondrial genomes and one regenerant contained mixed chloroplast genomes. In all four examples, the cytoplasmic genome sorted out in planta in favor of one parental type which was faithfully maternally transmitted to progeny.     

Phytoene synthase and phytoene dehydrogenase associated with envelope membranes from spinach chloroplasts

Envelope membranes of spinach chloroplasts contain appreciable activities of the carotenogenic enzymes phytoene synthase (formation of phytoene by condensation of two molecules geranylgeranyl pyrophosphate) and phytoene dehydrogenase (formation of lycopene from phytoene), plus a phosphatase activity. These results were obtained by coincubation experiments using isolated envelope membranes and either a phytoene-forming in vitro system (from [1-¹⁴C]isopentenyl pyrophosphate) or [¹⁴C]geranylgeranyl pyrophosphate or a geranylgeranyl-pyrophosphate-forming in vitro system (from [1-¹⁴C]isopentenyl pyrophosphate). Within thylakoids carotenogenic enzymes could not be detected. It is concluded that the chloroplast envelope is at least a principal site of the membrane-bound steps of carotenoid biosynthesis in chloroplasts.Abbreviastions Chlorophyll a_GC Chlorophyll a, esterified with geranylgeraniol - GGPP geranylgeranyl pyrophosphate - HPLC high pressure liquid chromatography - IPP isopentenyl pyrophosphate     

The phosphate translocator of the chloroplast envelope. Isolation of the carrier protein and reconstitution of transport

This report describes the solubilization and purification of the phosphate translocator of spinach chloroplasts and the reconstitution of its activity by incorporation into liposomes. (1) Prior to the isolation, the carrier is specifically labelled by treatment with 2,4,6-trinitrobenzenesulfonic acid and NaB[³H]H₄. (2) After preextraction of purified envelope membranes with Brij 58 for removing other loosely bound membrane proteins, the phosphate translocator is extracted with Triton X-100. After passing the resulting extract over a DEAE-Sepharose column followed by sucrose density gradient ultracentrifugation, the translocator protein is purified to apparent homogeneity. The 5–6-fold purification thus obtained concurs with earlier findings that the phosphate translocator protein represents 15–20% of the envelope membrane protein. This highly purified protein is suitable for studies of the hydrodynamic parameters of the translocator. (3) Since the exposure to detergents affects the activity of the translocator protein, alternatively, a rapid batch procedure for the purification of the translocator protein employing hydroxyapatite is used, yielding within 15 min the phosphate translocator protein of about 70% purity. (4) After incorporation of this protein fraction into liposomes, a specific transport of phosphate into these liposomes is observed, which van be terminated by inhibitor stop with pyridoxal 5′-phosphate. This uptake is only observed when the liposomes have been preloaded with phosphate or 3-phosphoglycerate, but not with 2-phosphoglycerate. Thus, like in intact chloroplasts, also the reconstituted transport facilitates an obligatory and specific counter exchange of anions. The apparent K_m for the transport of phosphate by this reconstituted system is about 0.8 mM, which is comparable to the corresponding value in intact chloroplasts. The calculated turnover of 150–300 min⁻¹ (20°C) accounts for 3–6% of the original activity.