A High-Resolution Gene Map of the Chloroplast Genome of the Red Alga Porphyra purpurea

Extensive DNA sequencing of the chloroplast genome of the red alga Porphyra purpurea has resulted in the detection of more than 125 genes. Fifty-eight (approximately 46%) of these genes are not found on the chloroplast genomes of land plants. These include genes encoding 17 photosynthetic proteins, three tRNAs, and nine ribosomal proteins. In addition, nine genes encoding proteins related to biosynthetic functions, six genes encoding proteins involved in gene expression, and at least five genes encoding miscellaneous proteins are among those not known to be located on land plant chloroplast genomes. The increased coding capacity of the P. purpurea chloroplast genome, along with other characteristics such as the absence of introns and the conservation of ancestral operons, demonstrate the primitive nature of the P. purpurea chloroplast genome. In addition, evidence for a monophyletic origin of chloroplasts is suggested by the identification of two groups of genes that are clustered in chloroplast genomes but not in cyanobacteria.     

Absence of geographical structure of chloroplast DNA variation in sallow, Salix caprea L

In the present study, we have used PCR-RFLP markers to investigate the chloroplast DNA variation in 24 European populations of Salix caprea L. A subset of these populations has also been analysed with chloroplast microsatellites. The main feature of both markers is the absence of a clear geographic structure (G(ST(PCR-RFLP))=0.090, G(ST(microsatellites))=-0.017) and high levels of variation within populations. This lack of phylogeographic structure in S. caprea is suggested to be the consequence of the joint action of several factors: (i) presence of intermediate latitude refugia with large population sizes during the last glacial maximum, (ii) a high speed of recolonisation and dispersal ability, (iii) a high mutation rate and (iv) extensive hybridisation with other willow species. In addition to the S. caprea samples, a limited number of individuals from several other Salix species were also analysed with PCR-RFLP: S. cinerea, S. aurita, S. purpurea, S. atrocinerea, S. appendiculata, S. elaeagnos, S. fragilis and S. alba. Many of the haplotypes found in Salix caprea were also detected in S. cinerea, S. aurita, S. purpurea, S. atrocinerea and/ or S. appendiculata but not in S. alba, S. elaeagnos or S. fragilis. Our data suggest that hybridisation and gene flow have occurred within these two groups but not between them.     

The gene for the large subunit of ribulose-1,5-bisphosphate carboxylase in Euglena gracilis chloroplast DNA: location, polarity, cloning, and evidence for an intervening sequence

The gene for the large subunit (LS) of ribulose-1,5,-bisphosphate carboxylase of Euglena gracilis Z chloroplast DNA has been mapped by heterologous hybridization with DNA restriction fragments containing internal sequences from the Zea mays and Chlamydomonas reinhardii LS genes. The Euglena LS gene which has the same polarity as the Euglena rRNA genes has been located with respect to Pst I, Pvu I, and HindIII sites within the Eco RI fragment Eco A. The region of Euglena chloroplast DNA complementary to an 887 bp internal fragment from the Chlamydomonas chloroplast LS gene is interrupted by a 0.5-1.1 kbp non-complementary sequence. This is the first chloroplast protein gene located on the Euglena genome, and the first evidence for an intervening sequence within any chloroplast protein gene.     

Activation of the heterotrimeric G protein α-subunit GPA1 suppresses the ftsh-mediated inhibition of chloroplast development in Arabidopsis

Heterotrimeric G protein knock-out mutants have no phenotypic defect in chloroplast development, and the connection between the G protein signaling pathway and chloroplast development has only been inferred from pharmaceutical evidence. Thus, whether G protein signaling plays a role in chloroplast development remains an open question. Here, we present genetic evidence, using the leaf-variegated mutant thylakoid formation 1 ( thf1 ), indicating that inactivation or activation of the endogenous G protein α-subunit (GPA1) affects chloroplast development, as does the ectopic expression of the constitutively active Gα-subunit (cGPA1). Molecular biological and genetic analyses showed that FtsH complexes, which are composed of type-A (FtsH1/FtsH5) and type-B (FtsH2/FtsH8) subunits, are required for cGPA1-promoted chloroplast development in thf1 . Furthermore, the ectopic expression of cGPA1 rescues the leaf variegation of ftsh2 . Consistent with this finding, microarray analysis shows that ectopic expression of cGPA1 partially corrects mis-regulated gene expression in thf1 . This overlooked function of G proteins provides new insight into our understanding of the integrative signaling network, which dynamically regulates chloroplast development and function in response to both intracellular and extracellular signals.     

Evidence for the nuclear location of the gene for chloroplast elongation factor G.

The chloroplast protein synthesis factor responsible for the translocation step of polypeptide synthesis on chloroplast ribosomes (chloroplast elongation factor G [EF-G]) has been detected in whole cell extracts and in isolated chloroplasts from Euglena gracilis. This factor can be detected by its ability to catalyze translocation on 70 S prokaryotic ribosomes such as those from E. coli. Chloroplast EF-G is present in low levels when Euglena is grown in the dark and can be induced more than 20-fold when the organism is grown in the light. The induction of this factor by light is inhibited by cycloheximide, a specific inhibitor of protein synthesis on cytoplasmic ribosomes. However, inhibitors of chloroplast protein synthesis such as streptomycin or spectinomycin have no effect on the induction of this factor by light. Furthermore, chloroplast EF-G can be partially induced by light in an aplastidic mutant (strain W₃BUL) which has neither significant plastid structure nor detectable chloroplast DNA. These data strongly suggest that the genetic information for chloroplast EF-G resides in the nuclear genome, and that this protein is synthesized on cytoplasmic ribosomes prior to compartmentalization within the chloroplasts.     

Identification of a soybean chloroplast DNA replication origin-binding protein

Replication of chloroplast DNA (ctDNA) in several plants and in Chlamydomonas reinhardii has been shown to occur by a double displacement loop (D-loop) mechanism and potentially also by a rolling circle mechanism. D-loop replication origins have been mapped in several species. Minimal replication origin sequences used as probes identified two potential binding proteins by southwestern blot analysis. A 28 kDa (apparent molecular weight by SDS-PAGE analysis) soybean protein has been isolated by origin sequence-specific DNA affinity chromatography from total chloroplast proteins. Mass spectrometry analysis identified this protein as the product of the soybean C6SY33 gene (accession number ACU14156), which is annotated as encoding a putative uncharacterized protein with a molecular weight of 25,897 Da, very near the observed molecular weight of the purified protein based on gel electrophoresis. Western blot analysis using an antibody against a homologous Arabidopsis protein indicates that this soybean protein is localized specifically in chloroplasts. The soybean protein shares some homology within a single-stranded DNA binding (SSB) domain of E. coli SSB and an Arabidopsis thaliana mitochondrial-localized SSB of about 21 kDa (mtSSB). However, the soybean protein induces a specific electrophoretic mobility shift only when incubated with a double-stranded fragment containing the previously mapped ctDNA replication oriA region. This protein has no electrophoretic mobility shift activity when incubated with single-stranded DNA. In contrast, the Arabidopsis mtSSB causes a mobility shift only with single-stranded DNA but not with the oriA fragment or with control dsDNA of unrelated sequence. These results suggest that the 26 kDa soybean protein is a specific origin binding protein that may be involved in initiation of ctDNA replication.     

Chloroplast ribosomal protein L32 is encoded in the chloroplast genome

The 50 S subunit of chloroplast ribosomes was prepared from tobacco leaves. The proteins were fractionated and the N-terminal amino acid sequence of a 14 kDa protein was determined. This sequence matches the N-terminal sequence deduced from ORF55 located between ndhF and trnL on the small single-copy region of tobacco chloroplast DNA. The deduced protein shows homology to E. coli and B. stearothermophilus L32 proteins, and it has been named as CL32 and ORF55 as rpl32. The tobacco chloroplast genome therefore contains 21 different ribosomal protein genes.     

Plant mitochondrial F0F1 ATP synthase. Identification of the individual subunits and properties of the purified spinach leaf mitochondrial ATP synthase.

Spinach leaf mitochondrial F0F1 ATPase has been purified and is shown to consist of twelve polypeptides. Five of the polypeptides constitute the F1 part of the enzyme. The remaining polypeptides, with molecular masses of 28 kDa, 23 kDa, 18.5 kDa, 15 kDa, 10.5 kDa, 9.5 kDa and 8.5 kDa, belong to the F0 part of the enzyme. This is the first report concerning identification of the subunits of the plant mitochondrial F0. The identification of the components is achieved on the basis of the N-terminal amino acid sequence analysis and Western blot technique using monospecific antibodies against proteins characterized in other sources. The 28-kDa protein crossreacts with antibodies against the subunit of bovine heart ATPase with N-terminal Pro-Val-Pro- which corresponds to subunit F0b of Escherichia coli F0F1. Sequence analysis of the N-terminal 32 amino acids of the 23-kDa protein reveals that this protein is similar to mammalian oligomycin-sensitivity-conferring protein and corresponds to the F1 delta subunit of the chloroplast and E. coli ATPases. The 18.5-kDa protein crossreacts with antibodies against subunit 6 of the beef heart F0 and its N-terminal sequence of 14 amino acids shows a high degree of sequence similarity to the conserved regions at N-terminus of the ATPase subunits 6 from different sources. ATPase subunit 6 corresponds to subunit F0a of the E. coli enzyme. The 15-kDa protein and the 10.5-kDa protein crossreact with antibodies against F6 and the endogenous ATPase inhibitor protein of beef heart F0F1-ATPase, respectively. The 9.5-kDa protein is an N,N'-dicyclohexylcarbodiimide-binding protein corresponding to subunit F0c of the E. coli enzyme. The 8.5-kDa protein is of unknown identity. The isolated spinach mitochondrial F0F1 ATPase catalyzes oligomycin-sensitive ATPase activity of 3.5 mumol.mg-1.min-1. The enzyme catalyzes also hydrolysis of GTP (7.5 mumol.mg-1.min-1) and ITP (4.4 mumol.mg-1.min-1). Hydrolysis of ATP was stimulated fivefold in the presence of amphiphilic detergents, however the hydrolysis of other nucleotides could not be stimulated by these agents. These results show that the plant mitochondrial F0F1 ATPase complex differs in composition from the other mitochondrial, chloroplast and bacterial ATPases. The enzyme is, however, more closely related to the yeast mitochondrial ATPase and to the animal mitochondrial ATPase than to the chloroplast enzyme. The plant mitochondrial enzyme, however, exhibits catalytic properties which are characteristic for the chloroplast enzyme.     

Is E37, a major polypeptide of the inner membrane from plastid envelope,an S-adenosyl methionine-dependent methyltransferase?

Using antibodies raised against E37, one of the major polypeptides of the inner membrane from the chloroplast envelope, it has been demonstrated that a single immunologically related polypeptide was present in total protein extracts from various higher plants (monocots and dicots), in photosynthetic and non-photosynthetic tissues from young spinach plantlets, as well as in the cytoplasmic membrane from the cyanobacteria Synechococcus . This ubiquitous distribution of E37 strongly suggests that this protein plays an envelope-specific function common to all types of plastids. Comparison of tobacco and spinach E37 amino acid sequences deduced from the corresponding cDNA demonstrates that consensus motifs for S-adenosyl methionine-dependent methyltransferases are located in both sequences. This hypothesis was confirmed using a biochemical approach. It was demonstrated that E37, together with two minor spinach chloroplast envelope polypeptides of 32 and 39 kDa, can be specifically photolabeled with [³H]-S-adenosyl methionine upon UV-irradiation. Identification of E37 as a photolabeled polypeptide was established by immunoprecipitation. Furthermore, photolabeling of the three envelope polypeptides was specifically inhibited by very low concentration of S-adenosyl homocysteine, thus providing evidence for the presence within these proteins of S-adenosyl methionine- and S-adenosyl homocysteine-binding sites that were closely associated. Taken as a whole these results strongly suggest that E37 is an ubiquitous plastid envelope protein that probably has an S-adenosyl methionine-dependent methyltransferase activity. The 32 and 39 kDa envelope polypeptides probably have a similar methyltransferase activity.     

Chloroplast elongation factor Tu: Evidence that it is the product of a chloroplast gene in Euglena

The chloroplast protein synthesizing factor responsible for the binding of aminoacyl-tRNA to ribosomes (EF-Tu_chl) has been identified in extracts of Euglena gracilis. This factor is present in low levels when Euglena is grown in the dark and can be induced more than 10-fold when the organism is exposed to light. The induction of the chloroplast EF-Tu by light is inhibited by streptomycin, an inhibitor of protein synthesis on chloroplast ribosomes, indicating that protein synthesis within the chloroplast itself is required for the induction of this factor. The induction of the chloroplast EF-Tu by light is also inhibited by cycloheximide, a specific inhibitor of protein synthesis on cytoplasmic ribosomes. The effect of cycloheximide probably results from the inhibition of chloroplast ribosome synthesis which requires the synthesis of many proteins by the cytoplasmic translational system. Chloroplast EF-Tu cannot be induced by light in an aplastidic mutant (strain W₃BUL) of Euglena which has neither significant plastid structure nor detectable chloroplast DNA. These data strongly suggest that the genetic information for chloroplast EF-Tu resides in the chloroplast genome and that this protein is synthesized within the organelle itself.     

Development of a luciferase reporter gene, luxCt, for Chlamydomonas reinhardtii chloroplast

Luciferase reporter genes have been successfully used in a variety of organisms to examine gene expression in living cells, but are yet to be successfully developed for use in chloroplast. Green fluorescent protein (gfp) has been used as a reporter of chloroplast gene expression, but because of high auto-fluorescence, very high levels of GFP accumulation are required for visualization in vivo. We have developed a luciferase reporter for chloroplast by synthesizing the two-subunit bacterial luciferase (lux)AB, as a single fusion protein in Chlamydomonas reinhardtii chloroplast codon bias. We expressed a chloroplast luciferase gene, luxCt, in C. reinhardtii chloroplasts under the control of the ATPase alpha subunit (atpA) or psbA promoter and 5' untranslated regions (UTRs) and the rubisco large subunit (rbcL) 3' UTR. We show that luxCt is a sensitive reporter of chloroplast gene expression, and that luciferase activity can be measured in vivo using a charge coupled device (CCD) camera or in vitro using a luminometer. We further demonstrate that luxCt protein accumulation, as measured by Western blot analysis, is proportional to luminescence, as determined both in vivo and in vitro, and that luxCt is capable of reporting changes in chloroplast gene expression during a dark to light shift. These data demonstrate the utility of the luxCt gene as a versatile and sensitive reporter of chloroplast gene expression in living cells.     

D-lysergic acid activation and cell-free synthesis of D-lysergyl peptides in enzyme fractions from the ergot fungus Claviceps purpurea

The D-lysergic acid activating enzyme from the ergot fungus Claviceps purpurea was purified to near homogeneity. It has a native Mr of about 245,000 and in its denatured form is a single polypeptide chain of Mr 62,000. The enzyme catalyzes the ATP-pyrophosphate exchange reaction dependent on D-lysergic acid and, though much less, that dependent on dihydrolysergic acid. Western blot analysis of SDS electropherograms of crude protein extracts from C. purpurea using monospecific antibodies directed against the D-lysergic acid activating enzyme revealed the immunostaining of one particular band which was identical with that of the D-lysergic acid activating enzyme. No significant immunoreactive band with higher molecular weight was seen, which precludes the possibility that the enzyme had arisen from the proteolysis of a high molecular weight ergot peptide synthetase. An ammonium sulfate fractionated enzyme fraction was prepared from C. purpurea strain C1 that catalyzed the incorporation of D-lysergic acid into two peptides which besides D-lysergic acid contained alanine, phenylalanine, and proline. Dihydrolysergic acid was efficiently incorporated into the corresponding dihydrolysergic acid containing analogues of the two compounds. Radiochemical analysis and degradation studies suggest that the two D-lysergic acid containing peptides most probably are N-[N-(D-lysergyl)-L-alanlyl]-L-phenylalanyl-L-proline lactam and N-[N-(D-lysergyl)-L-analyl]-L-phenylalanyl-D-proline lactam, respectively. N-[N-(D-Lysergyl)-L-alanyl]-L-phenylalanyl-L-proline lactam is considered to be the immediate precursor of ergotamine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Determination of the site of synthesis of some Euglena cytoplasmic and chloroplast ribosomal proteins.

Ribosomal protein synthesis during chloroplast development in Euglena gracilis has been studied by using inhibitors specific for either chloroplast or cytoplasmic protein syntheses. Fifty proteins of cytoplasmic and 39 of chloroplast ribosomes have been examined. Synthesis of all cytoplasmic ribosomal proteins is strongly inhibited by cycloheximide. Lincomycin (LIN) seems to have no effect on the synthesis of these proteins. In contrast, formation of 12 chloroplast ribosomal proteins is inhibited by cycloheximide (CHI), that of 9 by lincomycin, and that of 6 by both of these antibiotics; the technique used in this study did not permit definite determination of the sites of synthesis of the remaining proteins.     

OEP80, an essential protein paralogous to the chloroplast protein translocation channel Toc75, exists as a 70-kD protein in the Arabidopsis thaliana chloroplast outer envelope

Toc75 and OEP80 are paralogous proteins found in the Viridiplantae lineages, and appear to have evolved from a protein in the outer membrane of an ancient cyanobacterium. Toc75 is known to act as a protein translocation channel at the outer membrane of the chloroplast envelope, whereas the exact function of OEP80 is not understood. In Arabidopsis thaliana, each protein is encoded by a single gene, and both are essential for plant viability from embryonic stages onward. Sequence annotation and immunoblotting data with an antibody against its internal sequence (αOEP80(325-337)) indicated that the molecular weight of OEP80 is ca. 80?kD. Here we present multiple data to show that the size of A. thaliana OEP80 is smaller than previously estimated. First, we prepared the antibody against a recombinant protein consisting of annotated full-length A. thaliana OEP80 with an N-terminal hexahistidine tag (αOEP80(1-732)). This antibody recognized a 70-kD protein in the A. thaliana chloroplast membrane fraction which migrated faster than the His-tagged antigen and the protein recognized by the αOEP80(325-337) antibody on SDS-PAGE. Immunoprecipitation followed by LC-MS/MS analysis confirmed that the 70-kD protein was encoded by the OEP80 cDNA. Next, we performed a genetic complementation assay using embryo-lethal oep80-null plants and constructs encoding OEP80 and its variants. The results revealed that the nucleotide sequence encoding the 52 N-terminal amino acids was not required for functional expression of OEP80 and accumulation of the 70-kD protein. The data also indicated that an additional C-terminal T7 tag remained intact without disrupting the functionality of OEP80, and was not exposed to the cytoplasmic surface of the chloroplast envelope. Finally, OEP80-T7 and Toc75 showed distinct migration patterns on blue native-PAGE. This study provides molecular tools to investigate the function of OEP80, and also calls for caution in using an anti-peptide antibody.     

Comparative proteomics analyses of intraspecific differences in the response of Stipa purpurea to drought

Stipa purpurea is widely distributed along a large precipitation gradient on the Tibetan Plateau.This implies that S.purpurea from different populations may have different responses to drought stress.To explore this we compared the morphological and physiological changes of S.purpurea seedlings cultivated from seeds from Gar County and Nagqu County after 7 and 14 days of drought stress and subsequent rewatering.The results showed that S.purpurea plants from the more arid Gar area were more tolerant to drought stress than that from Nagqu.To investigate the potential mechanisms underlying this difference,we used i TRAQ quantitative proteomics technology to analyze protein dynamics in S.purpurea samples treated with 7 days of drought stress and subsequent re-watering.The results indicated that,during the process of drought and re-watering treatments,there were differentially expressed proteins in either or both S.purpurea populations.These differential proteins were divided into 24 functional categories that were mainly associated with stress response,the antioxidant system,photosynthesis,carbohydrate metabolism,and post-translational modifications.According to these results,we concluded that the molecular basis of stronger drought resistance likely lies in the specific up-regulation or higher expression of many proteins involved in stress response,the antioxidant system,post-translational modification and osmotic regulation in S.purpurea from Gar County compared with that from Nagqu.This study improves our understanding of the intraspecific differences in drought resistance within S.purpurea populations,which helps to understand the distribution of S.purpurea along the moisture gradient,as well as the effect of climate change on this species.     

A ribosomal protein is encoded in the chloroplast DNA in a lower plant but in the nucleus in angiosperms. Isolation of the spinach L21 protein and cDNA clone with transit and an unusual repeat sequence

The distribution of chloroplast ribosomal protein genes between the organelle DNA and the nuclear DNA is highly conserved in land plants, but a notable exception is rpl21. This gene has been found in the completely sequenced chloroplast genome of a lower plant but not in that of two higher plants. We describe the purification and characterization of the spinach chloroplast ribosomal protein L21 and the isolation and nucleotide sequence of a cDNA clone that encodes its cytoplasmic precursor. The mature protein, identified by NH2-terminal sequencing, has 201 residues (Mr 22,766) and is thus substantially larger than either its Escherichia coli (103 residues) or the lower plant homologue (116 residues). The extra length is in peptide extensions at both amino and carboxyl termini. The COOH-terminal extension is unusual in that it comprises seven Ala-Glu repeats, a feature not found in any other ribosomal proteins described so far. The cDNA clone also encodes a 55-residue long transit peptide (with a high proportion of the polar residues, threonine and serine), to target the L21 protein into chloroplasts. The identification of rpl21 as a nuclear gene in a higher plant (spinach) and chloroplast gene in a lower plant (liverwort) suggests an organelle-to-nucleus gene relocation during the evolution of the former.     

叶绿体蛋白质组学研究进展

蛋白质组分析是鉴定蛋白质种类和功能的有力工具之一。叶绿体作为光合作用的重要细胞器,叶绿体蛋白质组学成为了研究的热点,涉及的领域包括叶绿体的总蛋白质组学、亚细胞蛋白质组学、差异蛋白质组学和蛋白质的功能等。现主要介绍蛋白质组学的常用技术以及叶绿体蛋白质组学的最新研究进展。     

Presence in the stroma of chloroplasts of a large pool of a ribosomal protein not structurally related to any Escherichia coli ribosomal protein

Summary A search was made for the presence of a pool of free ribosomal proteins in the stroma of the spinach chloroplast. The results showed that a relatively large amount of one protein, CS-S5, is present in the stroma. Immunoprecipitation experiments showed that this protein is encoded by the nuclear genome. Clones were isolated from a cDNA library constructed in the expression vector lambda gtll, using specific antibodies raised against the CS-S5 protein. A full-length cDNA was sequenced which contains an open reading frame (ORF) for the precursor of the CS-S5 protein, as shown by immunoprecipitation. This precursor contains a putative transit peptide of 66 amino acids and the mature product has no significant homology with any of the Escherichia coli ribosomal proteins, in contrast to the other ribosomal protein gene products so far identified in spinach chloroplasts.     

Zeolin is a recombinant storage protein with different solubility and stability properties according to its localization in the endoplasmic reticulum or in the chloroplast

Several strategies have been exploited to maximize heterologous protein accumulation in the plant cell. Recently, it has been shown that a portion of a maize prolamin storage protein, gamma-zein, can be used for the high accumulation of a recombinant protein in novel endoplasmic reticulum (ER)-derived protein bodies of vegetative tissues. In this study, we investigate whether this protein can be expressed in the chloroplast. Our long-term purpose is to use zeolin to produce value-added proteins by fusing these polypeptides with its gamma-zein portion and targeting the recombinant proteins to the ER or to the chloroplast. We show here that zeolin accumulates in the chloroplast to lower levels than in the ER and its stability is compromised by chloroplast proteolytic activity. Co-localization of zeolin and the ER chaperone BiP in the chloroplast does not have a beneficial effect on zeolin accumulation. In this organelle, zeolin is not stored in protein bodies, nor do zeolin polypeptides seem to be linked by inter-chain disulfide bonds, which are usually formed by the six cysteine of the gamma-zein portion, indicating abnormal folding of the recombinant protein. Therefore, it is concluded that to accumulate zeolin in the chloroplast it is necessary to facilitate inter-chain disulfide bond formation.