Identification of the entire set of transferred chloroplast DNA sequences in the mitochondrial genome of rice

Summary The entire set of transferred chloroplast DNA sequences in the mitochondrial genome of rice (Oryza sativa cv. Nipponbare) was identified using clone banks that cover the chloroplast and mitochondrial genomes. The mitochondrial fragments that were homologous to chloroplast DNA were mapped and sequenced. The nucleotide sequences around the termini of integrated chloroplast sequences in the rice mtDNA revealed no common sequences or structures that might enhance the transfer of DNA. Sixteen chloroplast sequences, ranging from 32 bases to 6.8 kb in length, were found to be dispersed throughout the rice mitochondrial genome. The total length of these sequences is equal to approximately 6% (22 kb) of the rice mitochondrial genome and to 19% of the chloroplast genome. The transfer of segments of chloroplast DNA seems to have occurred at different times, both before and after the divergence of rice and maize. The mitochondrial genome appears to have been rearranged after the transfer of chloroplast sequences as a result of recombination at these sequences. The rice mitochondrial DNA contains nine intact tRNA genes and three tRNA pseudogenes derived from the chloroplast genome.     

石刁柏雄性偏向核质体DNA的克隆与分析

该研究以雌雄异株植物石刁柏为材料,利用基因组消减杂交技术对石刁柏雌雄核基因组中的性别差异核质体DNA（nuclear plastid DNA,NUPTs）进行了分离和分析。结果表明：（1）通过构建消减杂交文库共获得了52个雄性偏向序列,序列长度分布在63~297 bp之间,其中有19个差异序列属于叶绿体来源序列（命名为Ao1~Ao19）,且这些序列与石刁柏叶绿体基因组的相似性均大于84%,Ao19与石刁柏叶绿体基因组相似性为100%。（2）利用基因组半定量PCR对19个NUPTs序列的性别差异分析表明,有4条序列为稳定的雄性偏向NUPTs序列,分别为Ao1、Ao3、Ao10和Ao18。（3）序列比对表明,转移到核基因组的NUPTs主要来源于叶绿体基因组的反向重复区（包含IRa和IRb区）,说明石刁柏叶绿体基因组重复区序列更容易向核基因组进行转移形成雄性偏向的NUPTs序列。     

Parallel rate heterogeneity in chloroplast and mitochondrial genomes of Brazil nut trees (Lecythidaceae) is consistent with lineage effects

We investigated whether relative rates of divergence were correlated between the mitochondrial and chloroplast genomes as expected under lineage effects or were genome specific as expected with locus-specific effects. Five mitochondrial noncoding regions (nad1B_C, nad4exon1_2, nad7exon2_3, nad7exon3_4, and rps14-cob) for 21 samples from Lecythidaceae were sequenced. Three chloroplast regions (rpl20-5'rps12, trnS-trnG, and psbA-trnH) were sequenced to expand the taxa in an existing data set. Absolute rates of nucleotide and insertion and deletion (indel) changes were 13 times faster in the chloroplast genome than in the mitochondrial genome. Similar indel length frequency distributions for both organelles suggested that common mechanisms were responsible for generating indels. Molecular clock tests applied to phylogenetic trees estimated from mitochondrial and chloroplast sequences revealed global rate heterogeneity of nucleotide substitution. Maximum likelihood and Tajima's 1D relative rate tests show that Lecythis zabucajo exhibited a rate acceleration for both the mitochondrial and chloroplast sequences. Whereas Eschweilera romeu-cardosoi showed a significant rate slowdown for chloroplast sequences, the mitochondrial sequences for 3 Eschweilera taxa showed evidence for a rate slowdown only when compared with L. zabucajo. Significant rate heterogeneity was also observed for indel changes in the mitochondrial genome but not for the chloroplast. The lack of mitochondrial nucleotide changes for some taxa as well as chloroplast indel homoplasy may have limited the power of relative rate tests to detect rate variation. Relative ratio tests consistently indicated rate proportionality among branch lengths between the mitochondrial and chloroplast phylogenetic trees. The relative ratio tests showed that taxa possessing rate heterogeneity had parallel relative divergence rates in both mitochondrial and chloroplast sequences as expected under lineage effects. A neutral replication-dependent model of rate heterogeneity for both nucleotide and indel changes provides a simple explanation for common patterns of rate heterogeneity across the 2 organelle genomes in Lecythidaceae. The lineage effects observed here were uncoupled from annual/perennial habit because all the species from this study are perennial.     

Genetic polymorphism in Gymnodinium galatheanum chloroplast DNA sequences and development of a molecular detection assay

Nuclear and chloroplast-encoded small subunit ribosomal DNA sequences were obtained from several strains of the toxic dinoflagellate Gymnodinium galatheanum. Phylogenetic analyses and comparison of sequences indicate that the chloroplast sequences show a higher degree of sequence divergence than the nuclear homologue. The chloroplast sequences were chosen as targets for the development of a 5'--3' exonuclease assay for detection of the organism. The assay has a very high degree of specificity and has been used to screen environmental water samples from a fish farm where the presence of this dinoflagellate species has previously been associated with fish kills. Various hypotheses for the derived nature of the chloroplast sequences are discussed, as well as what is known about the toxicity of the species.     

Accumulation of chloroplast DNA sequences on the Y chromosome of Silene latifolia

Silene latifolia is a model dioecious plant with heteromorphic sex chromosomes. The Y chromosome is the largest in this species. Theoretical models propose an accumulation of repetitive DNA sequences in non-recombining parts of the Y chromosome. In this study, we isolated a BAC7H5 clone preferentially hybridizing to the Y chromosome of S. latifolia. Sequence analysis revealed that this BAC7H5 contains part of the chloroplast genome, indicating that these chloroplast sequences have accumulated on the Y chromosome and also may contribute to its large size. We constructed Y chromosome- and X chromosome-specific libraries and screened them to find Y- and/or X-linked copies of chloroplast sequences. Sequence analysis revealed higher divergence of a non-genic region of the chloroplast sequences located on the Y chromosome while genic regions tested showed only very low (max 0.9%) divergence from their chloroplast homologues.     

Many parallel losses of infA from chloroplast DNA during angiosperm evolution with multiple independent transfers to the nucleus

We used DNA sequencing and gel blot surveys to assess the integrity of the chloroplast gene infA, which codes for translation initiation factor 1, in >300 diverse angiosperms. Whereas most angiosperms appear to contain an intact chloroplast infA gene, the gene has repeatedly become defunct in approximately 24 separate lineages of angiosperms, including almost all rosid species. In four species in which chloroplast infA is defunct, transferred and expressed copies of the gene were found in the nucleus, complete with putative chloroplast transit peptide sequences. The transit peptide sequences of the nuclear infA genes from soybean and Arabidopsis were shown to be functional by their ability to target green fluorescent protein to chloroplasts in vivo. Phylogenetic analysis of infA sequences and assessment of transit peptide homology indicate that the four nuclear infA genes are probably derived from four independent gene transfers from chloroplast to nuclear DNA during angiosperm evolution. Considering this and the many separate losses of infA from chloroplast DNA, the gene has probably been transferred many more times, making infA by far the most mobile chloroplast gene known in plants.     

Prokaryotic features of a nucleus-encoded enzyme. cDNA sequences for chloroplast and cytosolic glyceraldehyde-3-phosphate dehydrogenases from mustard (Sinapis alba)

Two cDNA clones, encoding cytosolic and chloroplast glyceraldehyde-3-phosphate dehydrogenases (GAPDH) from mustard (Sinapis alba), have been identified and sequenced. Comparison of the deduced amino acid sequences with one another and with the GAPDH sequences from animals, yeast and bacteria demonstrates that nucleus-encoded subunit A of chloroplast GAPDH is distinct from its cytosolic counterpart and the other eukaryotic sequences and relatively similar to the GAPDHs of thermophilic bacteria. These results are compatible with the hypothesis that the nuclear gene for subunit A of chloroplast GAPDH is of prokaryotic origin. They are in puzzling contrast with a previous publication demonstrating that Escherichia coli GAPDH is relatively similar to the eukaryotic enzymes [Eur. J. Biochem. 150, 61-66 (1985)].     

Chloroplast genome sequences from total DNA for plant identification

Chloroplast DNA sequence data are a versatile tool for plant identification or barcoding and establishing genetic relationships among plant species. Different chloroplast loci have been utilized for use at close and distant evolutionary distances in plants, and no single locus has been identified that can distinguish between all plant species. Advances in DNA sequencing technology are providing new cost‐effective options for genome comparisons on a much larger scale. Universal PCR amplification of chloroplast sequences or isolation of pure chloroplast fractions, however, are non‐trivial. We now propose the analysis of chloroplast genome sequences from massively parallel sequencing (MPS) of total DNA as a simple and cost‐effective option for plant barcoding, and analysis of plant relationships to guide gene discovery for biotechnology. We present chloroplast genome sequences of five grass species derived from MPS of total DNA. These data accurately established the phylogenetic relationships between the species, correcting an apparent error in the published rice sequence. The chloroplast genome may be the elusive single‐locus DNA barcode for plants.     

Chloroplast DNA Sequence Homologies among Vascular Plants

The extent of sequence conservation in the chloroplast genome of higher plants has been investigated. Supercoiled chloroplast DNA, prepared from pea seedlings, was labeled in vitro and used as a probe in reassociation experiments with a high concentration of total DNAs extracted from several angiosperms, gymnosperms, and lower vascular plants. In each case the probe reassociation was accelerated, demonstrating that some chloroplast sequences have been highly conserved throughout the evolution of vascular plants. Only among the flowering plants were distinct levels of cross-reaction with the pea chloroplast probe evident; broad bean and barley exhibited the highest and lowest levels, respectively. With the hydroxylapatite assay these levels decreased with a decrease in probe fragment length (from 1,860 to 735 bases), indicating that many conserved sequences in the chloroplast genome are separated by divergent sequences on a rather fine scale. Despite differences observed in levels of homology with the hydroxylapatite assay, S1 nuclease analysis of heteroduplexes showed that outside of the pea family the extent of sequence relatedness between the probe and various heterologous DNAs is approximately the same: 30%. In our interpretation, the fundamental changes in the chloroplast genome during angiosperm evolution involved the rearrangement of this 30% with respect to the more rapidly changing sequences of the genome. These rearrangements may have been more extensive in dicotyledons than in monocotyledons. We have estimated the amount of conserved and divergent DNA interspersed between one another.     

Three distinct ribonucleoproteins from tobacco chloroplasts: each contains a unique amino terminal acidic domain and two ribonucleoprotein consensus motifs.

Chloroplasts contain their own genetic system. Eighteen different split genes have been found among approximately 130 chloroplast genes from higher plants. However, little is known about the chloroplast splicing system. Mammalian heterogeneous nuclear ribonucleoproteins (hnRNP proteins) have been shown to be involved in splicing. We applied a purification procedure developed for HeLa cell hnRNP proteins, which uses a single-stranded DNA (ssDNA) affinity column, directly to the tobacco chloroplast lysate to isolate their chloroplast counterparts. Four proteins (mol. wt approximately 30 kd) bound strongly to the column. The amino-terminal sequences of three of them were determined and their cDNA clones were isolated from a tobacco leaf cDNA library. Sequence analysis of these clones revealed that all three proteins contain two ribonucleoprotein consensus sequences (RNP-CS), confirming their ribonucleoprotein (RNP) nature. The presence of putative transit peptides in their predicted protein sequences, and an in vitro import experiment confirmed they are located in the chloroplast. This is the first report of organellar proteins containing RNP-CS. In addition, these three chloroplast proteins have a very acidic amino-terminal domain, a novel feature among RNP proteins identified so far. They are expressed both in leaves and roots; their mRNA levels showed different light modulation in mature leaves. The three proteins might be involved in splicing and/or processing of chloroplast RNAs.     

Wheat phosphoglycerate kinase: evidence for recombination between the genes for the chloroplastic and cytosolic enzymes.

We have isolated and sequenced cDNA clones containing the entire coding region of both the chloroplast and cytosolic versions of phosphoglycerate kinase from wheat. Comparison of these sequences reveals a higher than expected level of similarity between the nucleic acids and encoded proteins. Analysis of this data in relation to that for phosphoglycerate kinase sequences of mammals, prokaryotes and yeasts suggests that the wheat genes have recombined. This has resulted in the chloroplast and cytosolic kinases being more similar to each other than would be expected if the chloroplast enzyme had evolved directly from that of a prokaryotic progenitor.     

Evidence in favor of the symbiotic origin of chloroplasts: primary structure and evolution of tobacco glyceraldehyde-3-phosphate dehydrogenases

We report nucleotide sequences of cDNAs for the nuclear genes encoding chloroplast (GapA and GapB) and cytosolic (GapC) glyceraldehyde-3-phosphate dehydrogenases (GAPDH) from N. tabacum. Comparison of nucleotide sequences indicates that the GapA and GapB genes evolved following duplication of an ancestral gene about 450 million years ago. However, the divergence of GapA/B and GapC occurred much earlier in evolution than the divergence of GapC and GAPDH genes of animals and fungi, suggesting that chloroplast and cytosolic GAPDHs evolved from different lineages. Comparison of amino acid sequences shows that the chloroplast GAPDHs are related to GAPDHs found in thermophilic bacteria, while the cytosolic GAPDH is related to the GAPDH found in mesophilic prokaryotes. These results strongly support the symbiotic origin of chloroplasts.     

甘薯叶绿体表达载体构建及其融合基因在叶绿体中的瞬间表达

利用叶绿体基因组进化中高度保守的特点,根据烟草叶绿体基因组全序列设计引物,从甘薯(Ipomoea batatas)叶绿体基因组中克隆了2个相邻的功能基因rbcL(GenBank登录号为AY942199)和accD(GenBank登录号为AY942200),并以此作为定点整合外源基因的同源重组片段.以来自叶绿体基因组的强启动子Prrn和RpsbA-pro分别驱动选择标记基因aadA及phaC-gfp融合基因,构建成表达盒prrn-aadA-TpsbA-ter与RpsbA-pro-phaC-gfp-RpsbA-ter,然后将这2个表达盒串联在一起克隆进甘薯叶绿体同源片段中,获得甘薯叶绿体定点整合表达载体pSC-GFP.酶切分析证明,所构建的载体符合预期设计;采用该载体对甘薯叶片进行基因枪转化,结果显示,phaC-gfp融合基因可在叶绿体特异启动子和终止子的调控下在甘薯幼嫩叶片中瞬间表达,证明构建的载体pSC-GFP可用于甘薯叶绿体转化.     

Chloroplast transit peptides: structure, function and evolution

It is thought that two to three thousand different proteins are targeted to the chloroplast, and the 'transit peptides' that act as chloroplast targeting sequences are probably the largest class of targeting sequences in plants. At a primary structural level, transit peptide sequences are highly divergent in length, composition and organization. An emerging concept suggests that transit peptides contain multiple domains that provide either distinct or overlapping functions. These functions include direct interaction with envelope lipids, chloroplast receptors and the stromal processing peptidase. The genomic organization of transit peptides suggests that these domains might have originated from distinct exons, which were shuffled and streamlined throughout evolution to yield a modern, multifunctional transit peptide. Although still poorly characterized, this evolutionary process could yield transit peptides with different domain organizations. The plasticity of transit peptide design is consistent with the diverse biological functions of chloroplast proteins.     

Acyl carrier protein-derived sequence encoded by the chloroplast genome in the marine diatom Cylindrotheca sp. strain N1.

The chloroplast genome of chromophytic and rhodophytic algae differs from the plastid genome of plants and green algae in that it encodes the gene for the small subunit (rbcS) of ribulose 1,5-bisphosphate carboxylase/oxygenase. Hybridization studies indicated that there was a second region of chloroplast DNA from the marine diatom Cylindrotheca sp. strain N1 that strongly hybridized to a previously isolated Cylindrotheca fragment that contained the rbcS gene and flanking sequences. Subsequent determination of the oligonucleotide sequence of this second chloroplast DNA fragment, however, indicated that hybridization was due to identical sequences 3' to the previously cloned Cylindrotheca chloroplast rbcL rbcS genes. Sequences derived from the 5' end of the second chloroplast DNA fragment contained a short open reading frame of 80 amino acids which was found to be highly homologous to bacterial acyl carrier protein and nuclear-encoded acyl carrier protein from plants. Amino acid residues in the environment of Ser-36 of the Escherichia coli protein, which is bound to a 4'-phosphopantetheine moiety, are virtually identical in the Cylindrotheca deduced sequence and all other sources of this protein. Unlike plant acyl carrier-deduced amino acid sequences, there was no leader peptide sequence found for the presumptive Cylindrotheca protein, consistent with the location of this DNA fragment on the chloroplast genome of this organism. DNA encoding the putative acyl carrier protein gene and rbcS thus represent two genes that are chloroplast-encoded in the chromophytic marine diatom Cylindrotheca, a significant departure from the organization of such genes in plants.     

Widespread occurrence of small inversions in the chloroplast genomes of land plants

Large inversions are well characterized in the chloroplast genomes of land plants. In contrast, reports of small inversions are rare and involve limited plant groups. In this study, we report the widespread occurrence of small inversions ranging from 5 to 50 bp in fully and partially sequenced chloroplast genomes of both monocots and dicots. We found that small inversions were much more common than large inversions. The small inversions were scattered over the chloroplast genome including the IR, SSC, and LSC regions. Several small inversions were uncovered in chloroplast genomes even though they shared the same overall gene order. The majority of these small inversions were located within 100 bp downstream of the 3' ends of genes. All had inverted repeat sequences, ranging from 11 to 24 bp, at their ends. Such small inversions form stem-loop hairpin structures that usually have the function of stabilizing the corresponding mRNA molecules. Intra-molecular recombination between the inverted sequences in the stem-forming regions are responsible for generating flip-flop orientations of the loops. The presence of two different orientations of the stem-loop in the trnL-F noncoding region of a single species of Jasminum elegans suggests that a short inversion can be generated within a short period of time. Small inversions of non-coding sequences may influence sequence alignment and character interpretation in phylogeny reconstructions, as shown in nine species of Jasminum. Many small inversions may have been generated by parallel or back mutation events during chloroplast genome evolution. Our data indicate that caution is needed when using chloroplast non-coding sequences for phylogenetic analysis.     

Nucleotide sequences of chloroplast 5S ribosomal ribonucleic acid in flowering plants.

Evidence for the sequence of duckweed (Lemna minor) chloroplast 5S rRNA was derived from the analysis of partial and complete enzymic digests of the 32P-labelled molecule. The possible sequence of the chloroplast 5S rRNA from three other flowering plants was deduced by complete digestion with T1 ribonuclease and comparison of the sequences of the oligonucleotide products with homologous sequences in the duckweed 5S rRNA. This analysis indicates that the chloroplast 5S rNA species differ appreciably from their cytosol counterparts but bear a strong resemblance to one another and to the 5S rRNA species of prokaryotes. Structural features apparently common to all 5S rRNA molecules are also discussed.     

Variable copy number DNA sequences in rice

Summary We have cloned two types of variable copy number DNA sequences from the rice embryo genome. One of these sequences, which was cloned in pRB301, was amplified about 50-fold during callus formation and diminished in copy number to the embryonic level during regeneration. The other clone, named pRB401, showed the reciprocal pattern. The copy numbers of both sequences were changed even in the early developmental stage and eliminated from nuclear DNA along with growth of the plant. Sequencing analysis of the pRB301 insert revealed some open reading frames and direct repeat structures, but corresponding sequences were not identified in the EMBL and LASL DNA databases. Sequencing of the nuclear genomic fragment cloned in pRB401 revealed the presence of the 3rps12-rps7 region of rice chloroplast DNA. Our observations suggest that during callus formation (dedifferentiation), regeneration and the growth process the copy numbers of some DNA sequences are variable and that nuclear integrated chloroplast DNA acts as a variable copy number sequence in the rice genome. Based on data showing a common sequence in mitochondria and chloroplast DNA of maize (Stern and Lonsdale 1982) and that the rps12 gene of tobacco chloroplast DNA is a divided gene (Torazawa et al. 1986), it is suggested that the sequence on the inverted repeat structure of chloroplast DNA may have the character of a movable genetic element.