Plastid Genome Of Higher Plants Research Articles

Comparative Chloroplast Genomics of Gossypium Species: Insights Into Repeat Sequence Variations and Phylogeny.

Cotton is one of the most economically important fiber crop plants worldwide. The genus Gossypium contains a single allotetraploid group (AD) and eight diploid genome groups (A–G and K). However, the evolution of repeat sequences in the chloroplast genomes and the phylogenetic relationships of Gossypium species are unclear. Thus, we determined the variations in the repeat sequences and the evolutionary relationships of 40 cotton chloroplast genomes, which represented the most diverse in the genus, including five newly sequenced diploid species, i.e., G. nandewarense (C1-n), G. armourianum (D2-1), G. lobatum (D7), G. trilobum (D8), and G. schwendimanii (D11), and an important semi-wild race of upland cotton, G. hirsutum race latifolium (AD1). The genome structure, gene order, and GC content of cotton species were similar to those of other higher plant plastid genomes. In total, 2860 long sequence repeats (>10 bp in length) were identified, where the F-genome species had the largest number of repeats (G. longicalyx F1: 108) and E-genome species had the lowest (G. stocksii E1: 53). Large-scale repeat sequences possibly enrich the genetic information and maintain genome stability in cotton species. We also identified 10 divergence hotspot regions, i.e., rpl33-rps18, psbZ-trnG (GCC), rps4-trnT (UGU), trnL (UAG)-rpl32, trnE (UUC)-trnT (GGU), atpE, ndhI, rps2, ycf1, and ndhF, which could be useful molecular genetic markers for future population genetics and phylogenetic studies. Site-specific selection analysis showed that some of the coding sites of 10 chloroplast genes (atpB, atpE, rps2, rps3, petB, petD, ccsA, cemA, ycf1, and rbcL) were under protein sequence evolution. Phylogenetic analysis based on the whole plastomes suggested that the Gossypium species grouped into six previously identified genetic clades. Interestingly, all 13 D-genome species clustered into a strong monophyletic clade. Unexpectedly, the cotton species with C, G, and K-genomes were admixed and nested in a large clade, which could have been due to their recent radiation, incomplete lineage sorting, and introgression hybridization among different cotton lineages. In conclusion, the results of this study provide new insights into the evolution of repeat sequences in chloroplast genomes and interspecific relationships in the genus Gossypium.

Structure of Plastid Genomes of Photosynthetic Eukaryotes.

This review presents current views on the plastid genomes of higher plants and summarizes data on the size, structural organization, gene content, and other features of plastid DNAs. Special emphasis is placed on the properties of organization of land plant plastid genomes (nucleoids) that distinguish them from bacterial genomes. The prospects of genetic engineering of chloroplast genomes are discussed.

Non-functional plastid ndh gene fragments are present in the nuclear genome of Norway spruce (Picea abies L. Karsch): insights from in silico analysis of nuclear and organellar genomes.

Many genes have been lost from the prokaryote plastidial genome during the early events of endosymbiosis in eukaryotes. Some of them were definitively lost, but others were relocated and functionally integrated to the host nuclear genomes through serial events of gene transfer during plant evolution. In gymnosperms, plastid genome sequencing has revealed the loss of ndh genes from several species of Gnetales and Pinaceae, including Norway spruce (Picea abies). This study aims to trace the ndh genes in the nuclear and organellar Norway spruce genomes. The plastid genomes of higher plants contain 11 ndh genes which are homologues of mitochondrial genes encoding subunits of the proton-pumping NADH-dehydrogenase (nicotinamide adenine dinucleotide dehydrogenase) or complex I (electron transport chain). Ndh genes encode 11 NDH polypeptides forming the Ndh complex (analogous to complex I) which seems to be primarily involved in chloro-respiration processes. We considered ndh genes from the plastidial genome of four gymnosperms (Cryptomeria japonica, Cycas revoluta, Ginkgo biloba, Podocarpus totara) and a single angiosperm species (Arabidopsis thaliana) to trace putative homologs in the nuclear and organellar Norway spruce genomes using tBLASTn to assess the evolutionary fate of ndh genes in Norway spruce and to address their genomic location(s), structure, integrity and functionality. The results obtained from tBLASTn were subsequently analyzed by performing homology search for finding ndh specific conserved domains using conserved domain search. We report the presence of non-functional plastid ndh gene fragments, excepting ndhE and ndhG genes, in the nuclear genome of Norway spruce. Regulatory transcriptional elements like promoters, TATA boxes and enhancers were detected in the upstream regions of some ndh fragments. We also found transposable elements in the flanking regions of few ndh fragments suggesting nuclear rearrangements in those regions. These evidences support the hypothesis that, at least in Picea, ndh translocations from the plastid to the nuclear genome have occurred, and that there might have been a functional machinery at some time during evolution to accommodate them within a nuclear-encoded environment, or attempts to form it.

Next generation synthetic vectors for transformation of the plastid genome of higher plants

Plastid transformation vectors are E. coli plasmids carrying a plastid marker gene for selection, adjacent cloning sites and flanking plastid DNA to target insertions in the plastid genome by homologous recombination. We report here on a family of next generation plastid vectors carrying synthetic DNA vector arms targeting insertions in the rbcL-accD intergenic region of the tobacco (Nicotiana tabacum) plastid genome. The pSS22 plasmid carries only synthetic vector arms from which the undesirable restriction sites have been removed by point mutations. The pSS24 vector carries a c-Myc tagged spectinomycin resistance (aadA) marker gene whereas in vector pSS30 aadA is flanked with loxP sequences for post-transformation marker excision. The synthetic vectors will enable direct manipulation of passenger genes in the transformation vector targeting insertions in the rbcL-accD intergenic region that contains many commonly used restriction sites.

Insensitivity of chloroplast gene expression to DNA methylation

Presence and possible functions of DNA methylation in plastid genomes of higher plants have been highly controversial. While a number of studies presented evidence for the occurrence of both cytosine and adenine methylation in plastid genomes and proposed a role of cytosine methylation in the transcriptional regulation of plastid genes, several recent studies suggested that at least cytosine methylation may be absent from higher plant plastid genomes. To test if either adenine or cytosine methylation can play a regulatory role in plastid gene expression, we have introduced cyanobacterial genes for adenine and cytosine DNA methyltransferases (methylases) into the tobacco plastid genome by chloroplast transformation. Using DNA cleavage with methylation-sensitive and methylation-dependent restriction endonucleases, we show that the plastid genomes in the transplastomic plants are efficiently methylated. All transplastomic lines are phenotypically indistinguishable from wild-type plants and, moreover, show no alterations in plastid gene expression. Our data indicate that the expression of plastid genes is not sensitive to DNA methylation and, hence, suggest that DNA methylation is unlikely to be involved in the transcriptional regulation of plastid gene expression.

A Guide to Choosing Vectors for Transformation of the Plastid Genome of Higher Plants

Plastid transformation, originally developed in tobacco (Nicotiana tabacum), has recently been extended to a number of crop species enabling in vivo probing of plastid function and biotechnological applications. In this article we report new plastid vectors that enable insertion of transgenes in the inverted repeat region of the plastome between the trnV and 3'rps12 or trnI and trnA genes. Efficient recovery of transplastomic clones is ensured by selection for spectinomycin (aadA) or kanamycin (neo) resistance genes. Expression of marker genes can be verified using commercial antibodies that detect the accumulation of neomycin phosphotranseferase II, the neo gene product, or the C-terminal c-myc tag of aminoglycoside-3''-adenylytransferase, encoded by the aadA gene. Aminoglycoside-3''-adenylytransferase, the spectinomycin inactivating enzyme, is translationally fused with green fluorescent protein in two vectors so that transplastomic clones can be selected by spectinomycin resistance and visually identified by fluorescence in ultraviolet light. The marker genes in the new vectors are flanked by target sites for Cre or Int, the P1 and phiC31 phage site-specific recombinases. When uniform transformation of all plastid genomes is obtained, the marker genes can be excised by Cre or Int expressed from a nuclear gene. Choice of expression signals for the gene of interest, complications caused by the presence of plastid DNA sequences recognized by Cre, and loss of transgenes by homologous recombination via duplicated sequences are also discussed to facilitate a rational choice from among the existing vectors and to aid with new target-specific vector designs.

Phage-Type RNA Polymerase RPOTmp Transcribes the rrn Operon from the PC Promoter at Early Developmental Stages in Arabidopsis

The plastid genome of higher plants is transcribed by two different types of RNA polymerases named nucleus encoded RNA polymerase (NEP) and plastid encoded RNA polymerase. Plastid encoded RNA polymerase is a multimeric enzyme comparable to eubacterial RNA polymerases. NEP enzymes represent a small family of monomeric phage-type RNA polymerases. Dicotyledonous plants harbor three different phage-type enzymes, named RPOTm, RPOTp, and RPOTmp. RPOTm is exclusively targeted to mitochondria, RPOTp is exclusively targeted to plastids, and RPOTmp is targeted to plastids as well as to mitochondria. In this article, we have made use of RPOTp and RPOTmp T-DNA insertion mutants to answer the question of whether both plastid-located phage-type RNA polymerases have overlapping or specific functions in plastid transcription. To this aim, we have analyzed accD and rpoB messenger RNAs (mRNA; transcribed from type I NEP promoters), clpP mRNA (transcribed from the -59 type II NEP promoter), and the 16S rRNA (transcribed from the exceptional PC NEP promoter) by primer extension. Results suggest that RPOTp represents the principal RNA polymerase for transcribing NEP-controlled mRNA genes during early plant development, while RPOTmp transcribes specifically the rrn operon from the PC promoter during seed imbibition.

Tobacco plastid ribosomal protein S18 is essential for cell survival

Plastid genomes contain a conserved set of genes most of which are involved in either photosynthesis or gene expression. Among the ribosomal protein genes present in higher plant plastid genomes, rps18 is special in that it is absent from the plastid genomes of several non-green unicellular organisms, including Euglena longa and Toxoplasma gondii. Here we have tested whether the ribosomal protein S18 is required for translation by deleting the rps18 gene from the tobacco plastid genome. We report that, while deletion of the rps18 gene was readily obtained, no homoplasmic Δrps18 plants or leaf sectors could be isolated. Instead, segregation into homoplasmy led to severe defects in leaf development suggesting that the knockout of rps18 is lethal and the S18 protein is required for cell survival. Our data demonstrate that S18 is indispensable for plastid ribosome function in tobacco and support an essential role for plastid translation in plant development. Moreover, we demonstrate the occurrence of flip-flop recombination on short inverted repeat sequences which generates different isoforms of the transformed plastid genome that differ in the orientation a 70 kb segment in the large single-copy region. However, infrequent occurrence of flip-flop recombination and random segregation of plastid genomes result in the predominant presence of only one of the isoforms in many tissue samples. Implications for the interpretation of chloroplast transformation experiments and vector design are discussed.

Elimination of deleterious mutations in plastid genomes by gene conversion

Asexual reproduction is believed to be detrimental, mainly because of the accumulation of deleterious mutations over time, a hypothesis known as Muller's ratchet. In seed plants, most asexually reproducing genetic systems are polyploid, with apomictic species (plants forming seeds without fertilization) as well as plastids and mitochondria providing prominent examples. Whether or not polyploidy helps asexual genetic systems to escape Muller's ratchet is unknown. Gene conversion, particularly when slightly biased, represents a potential mechanism that could allow asexual genetic systems to reduce their mutation load in a genome copy number-dependent manner. However, direct experimental evidence for the operation of gene conversion between genome molecules to correct mutations is largely lacking. Here we describe an experimental system based on transgenic tobacco chloroplasts that allows us to analyze gene conversion events in higher plant plastid genomes. We provide evidence for gene conversion acting as a highly efficient mechanism by which the polyploid plastid genetic system can correct deleterious mutations and make one good genome out of two bad ones. Our finding that gene conversion can be biased may provide a molecular link between asexual reproduction, high genome copy numbers and low mutation rates.

New vectors and marker excision systems mark progress in engineering the plastid genome of higher plants

The transformation of the plastid genome, until recently restricted to tobacco, is now being extended to a rapidly growing list of crops. This perspective provides an overview of emerging trends of technology development in the field with a focus on vector design and marker excision systems. The new tools will facilitate engineering of the photosynthetic machinery and enable novel agricultural and industrial applications.

DNA microarray analysis of plastid gene expression in an Arabidopsis mutant deficient in a plastid transcription factor sigma, SIG2.

The plastid genome of higher plants contains more than one hundred genes for photosynthesis, gene expression, and other processes. Plastid transcription is done by two types of RNA polymerase, PEP and NEP. PEP is a eubacteria-type RNA polymerase that is essential for chloroplast development. In Arabidopsis thaliana, six sigma factors (SIG1-6) are encoded by the nuclear genome, and postulated to determine the transcription specificity of PEP. In this study, we constructed a DNA microarray for all of the plastid protein-coding genes, and analyzed the effects of the sig2 lesion on the global plastid gene expression. Of the 79 plastid protein genes, it was found that only the psaJ transcript was decreased in the mutant, whereas transcripts of 47 genes were rather increased. Since many of the up-regulated genes are under the control of NEP, it was suggested that the NEP activity was increased in the sig2-1 mutant.

Plastidic genome of higher plants and algae: structure and function

The data on the structure and gene content in completely sequenced plastid (predominantly chloroplast) genomes of higher plants and algae are reviewed. In higher plants, genome structure and gene content are highly conserved. Plastid genomes of algae are less conserved and contain several unique genes, which are not found in chloroplast DNAs of higher plants. Plastid genomes encode proteins involved in transcription and translation, as well as proteins of the photosynthetic apparatus and photosynthetic metabolism. The problems of origin and evolution of plastids are briefly discussed. The data published by the first half of 2002 are presented. Use was made of databases OGMP (http://megasun.bch.umontreal.ca/ogmp/projects/other/cp_list.html) and NCBI (www.ncbi.nlm.nih.gov).

Cytosine methylation of plastid genome in higher plants. Fact or artefact?

DNA methylation of chloroplast genome has been studied in a large variety of angiosperm species using restriction enzyme analysis of three genomic loci (totally encompassing about 10% of chloroplast genome) and bisulfite genomic sequencing of tobacco ribulose bisphosphate carboxylase/oxygenase (large subunit) gene ( rbcL). Except for CCWGG (W=A or T) sites that were partially refractory to the cleavage with methylation sensitive EcoRII in all loci, no cytosine methylation was found at the CCGG ( MspI/ HpaII) and several other restriction sites tested. However, EcoRII was unable to completely digest an unmethylated CCWGG site in the cloned rbcL gene on plasmid. Further a bisulfite genomic sequencing performed on EcoRII-restricted DNA failed to show any 5-methylcytosine either within or outside inspected EcoRII sites along the 3′ end of rbcL coding region. In conclusion our results do not support evidence for methylated cytosine residues in plant chloroplast genomes and we suggest that results obtained with EcoRII should be interpreted with great care especially when small differences in methylation levels are analysed.

Chloroplast genes are expressed during intracellular symbiotic association of Vaucheria litorea plastids with the sea slug Elysia chlorotica.

The marine slug Elysia chlorotica (Gould) forms an intracellular symbiosis with photosynthetically active chloroplasts from the chromophytic alga Vaucheria litorea (C. Agardh). This symbiotic association was characterized over a period of 8 months during which E. chlorotica was deprived of V. litorea but provided with light and CO2. The fine structure of the symbiotic chloroplasts remained intact in E. chlorotica even after 8 months of starvation as revealed by electron microscopy. Southern blot analysis of total DNA from E. chlorotica indicated that algal genes, i.e., rbcL, rbcS, psaB, psbA, and 16S rRNA are present in the animal. These genes are typically localized to the plastid genome in higher plants and algae except rbcS, which is nuclear-encoded in higher plants and green (chlorophyll a/b) algae. Our analysis suggests, however, that similar to the few other chromophytes (chlorophyll a/c) examined, rbcS is chloroplast encoded in V. litorea. Levels of psbA transcripts remained constant in E. chlorotica starved for 2 and 3 months and then gradually declined over the next 5 months corresponding with senescence of the animal in culture and in nature. The RNA synthesis inhibitor 6-methylpurine reduced the accumulation of psbA transcripts confirming active transcription. In contrast to psbA, levels of 16S rRNA transcripts remained constant throughout the starvation period. The levels of the photosystem II proteins, D1 and CP43, were high at 2 and 4 months of starvation and remained constant at a lower steady-state level after 6 months. In contrast, D2 protein levels, although high at 2 and 4 months, were very low at all other periods of starvation. At 8 months, de novo synthesis of several thylakoid membrane-enriched proteins, including D1, still occurred. To our knowledge, these results represent the first molecular evidence for active transcription and translation of algal chloroplast genes in an animal host and are discussed in relation to the endosymbiotic theory of eukaryote origins.

Deletion of rpoB reveals a second distinct transcription system in plastids of higher plants.

The plastid genome in higher plants encodes subunits of an Escherichia coli-like RNA polymerase which initiates transcription of plastid genes from sequences resembling E.coli sigma70-type promoters. By deleting the gene for the essential beta subunit of the tobacco E.coli-like RNA polymerase, we have established the existence of a second plastid transcription system which does not utilize E.coli-like promoters. In contrast to the E.coli-like RNA polymerase, the novel transcription machinery preferentially transcribes genetic system genes rather than photosynthetic genes. Although the mutant plants are photosynthetically defective, transcription by this polymerase is sufficient for plastid maintenance and plant development.

Extrachromosomal elements in tobacco plastids.

The plastid genome of higher plants is a circular double-stranded DNA molecule which is present in multiple identical copies. We report here an 868-bp plastid DNA minicircle, NICE1, that formed in tobacco (Nicotiana tabacum) plastids during transformation, as an unexpected product of homologous recombination. Such extrachromosomal elements are normally absent in plastids of higher plants. We have constructed shuttle plasmids containing NICE1 sequences which are maintained extrachromosomally when reintroduced into plastids by particle bombardment. Furthermore, recombination between homologous sequences in the shuttle plasmids and the main plastid genome occurs. Recombination products were characterized after recovery of the shuttle plasmids in Escherichia coli and of recombinant plastid genomes in the progeny of transformed plants. Our findings indicate that shuttle plasmids can be used to engineer plastid genes without concomitant integration of foreign DNA and to recover plastid mutations in E. coli.

Functional loss of all ndh genes in an otherwise relatively unaltered plastid genome of the holoparasitic flowering plant Cuscuta reflexa

We have cloned and sequenced an area of about 9.0 kb of the plastid DNA (ptDNA) from the holoparasitic flowering plant Cuscuta reflexa to investigate the evolutionary response of plastid genes to a reduced selective pressure. The region contains genes for the 16S rRNA, a subunit of a plastid NAD(P)H dehydrogenase (ndhB), three transfer RNAs (trnA, trnI, trnV) as well as the gene coding for the ribosomal protein S7 (rps7). While the other genes are strongly conserved in C. reflexa, the ndhB gene is a pseudogene due to many frameshift mutations. In addition we used heterologous gene probes to identify the other ndh genes encoded by the plastid genome in higher plants. No hybridization signals could be obtained, suggesting that these genes are either lost or strongly altered in the ptDNA of C. reflexa. Together with evidence of deleted genes in the ptDNA of C. reflexa, the plastid genome can be grouped into four classes reflecting a different evolutionary rate in each case. The phylogenetic position of Cuscuta and the significance of ndh genes in the plastid genome of higher plants are discussed.

Organization and sequences of genes for the subunits of ATP synthase in the thermophilic cyanobacterium Synechococcus 6716.

The sequences of the genes for the nine subunits of ATP synthase in the thermophilic cyanobacterium Synechococcus 6716 have been determined. The genes were identified by comparison of the encoded proteins with sequences of ATP synthase subunits in other species, and confirmed for subunits alpha, beta, delta and epsilon, by determining their N-terminal sequences. They are arranged at three separate loci. Six of them are in one cluster in the order a: c: b': b: delta: alpha, and those for the beta and epsilon subunits form a second and separate cluster. The gene for the gamma-subunit is at a third site. As in other bacteria, the gene for subunit a is immediately preceded by a gene coding for a small hydrophobic protein of unknown function, known as uncI in Escherichia coli. The gene orders in Synechococcus 6716 are related to the orders of ATP synthase genes in the plastid genomes of higher plants, and particularly of a red alga and a diatom. The sequences of the subunits are similar to those of chloroplast ATP synthase, the alpha, beta and c subunits being particularly well conserved. Differences in the primary structures of the Synechococcus 6716 and chloroplast gamma subunits probably underlie different mechanisms of activation of ATP synthase. The nucleotide sequences that are presented also contain 12 other open reading frames. One of them encodes a protein sequence related to the E. coli DNA repair enzyme, photolyase, and another codes for a protein that contains internal repeats related to sequences in the myosin heavy chain.

Studies on the expression of NDH-H, a subunit of the NAD(P)H-plastoquinone-oxidoreductase of higher-plant chloroplasts

The plastid genomes of higher plants contain eleven reading frames (ndhA-K) that are homologous to genes encoding subunits of the mitochondrial NADH-ubiquinone-oxidoreductase (complex I). The carboxyterminal end of the NDH-H subunit from rice (Oryza sativa L.) was expressed as a fusion protein in Escherichia coli and antibodies against the fusion protein were generated in rabbits. The antibody was used to study the expression of NDH-H, and the following results were obtained: (i) NDH-H is expressed in mono- and dicotyledonous plants, (ii) NDH-H is localized on the stroma lamellae of the thylakoid membrane and (iii) NDH-H is expressed in etioplasts. Together with the finding that two other ndh genes (ndhI and ndhK) are expressed in plastids, these results point to the existence of an NAD(P)H-plastoquinone-oxidoreductase on the thylakoid membrane. The possible function of the enzyme in plastids is discussed and it is suggested that it works in balancing the ATP/ADP and the NADPH/NADP ratios during changing external (i.e. light) or internal (i.e. ATP and NADPH demands of biosynthetic pathways of the plastid) conditions.

Chloroplast rps15 and the rpoB/C1/C2 gene cluster are strongly transcribed in ribosome-deficient plastids: evidence for a functioning non-chloroplast-encoded RNA polymerase.

Transcription of plastid genes and transcript accumulation were investigated in white leaves of the albostrians mutant of barley (Hordeum vulgare) and in heat-bleached leaves of rye (Secale cereale) as well as in normal green leaves of both species. Cells of white leaves of the mutant and cells of heat-bleached leaves bear undifferentiated plastids lacking ribosomes and, consequently, plastid translation products, among them the subunits of a putative chloroplast RNA polymerase encoded by the plastid genes rpoA, B, C1 and C2. The following results were obtained. (i) Plastid genes are transcribed despite the lack of chloroplast gene-encoded RNA polymerase subunits. The plastid origin of these transcripts was proven. This finding provides evidence for the existence of a plastid RNA polymerase encoded entirely by nuclear genes. (ii) Transcripts of the rpo genes and of rps15, but not of genes involved in photosynthesis and related processes (psbA, rbcL, atpI-H), were abundantly accumulated in ribosome-deficient plastids. In contrast, chloroplasts accumulated transcripts of photosynthetic, but not of the rpo genes. (iii) Differences in transcript accumulation between chloroplasts and ribosome-deficient plastids are due to different relative transcription rates and different transcript stability. (iv) The observed differences in transcription are not caused by an altered pattern of methylation of plastid DNA. Thus, the prokaryotic plastid genome of higher plants is transcribed by two RNA polymerases. The observed differences in transcription between chloroplasts and undifferentiated plastids might reflect different functions of the two enzymes.