Efficient and Stable Transformation of Lactuca sativa L. cv. Cisco (lettuce) Plastids

Transgenic plastids offer unique advantages in plant biotechnology, including high-level foreign protein expression. However, broad application of plastid genome engineering in biotechnology has been largely hampered by the lack of plastid transformation systems for major crops. Here we describe the development of a plastid transformation system for lettuce, Lactuca sativa L. cv. Cisco. The transforming DNA carries a spectinomycin-resistance gene (aadA) under the control of lettuce chloroplast regulatory expression elements, flanked by two adjacent lettuce plastid genome sequences allowing its targeted insertion between the rbcL and accD genes. On average, we obtained 1 transplastomic lettuce plant per bombardment. We show that lettuce leaf chloroplasts can express transgene-encoded GFP to ~36% of the total soluble protein. All transplastomic T0 plants were fertile and the T1 progeny uniformly showed stability of the transgene in the chloroplast genome. This system will open up new possibilities for the efficient production of edible vaccines, pharmaceuticals, and antibodies in plants.     

Reappraising plastid markers of the red algae for phylogenetic community ecology in the genomic era

Selection of appropriate genetic markers to quantify phylogenetic diversity is crucial for community ecology studies. Yet, systematic evaluation of marker genes for this purpose is scarcely done. Recently, the combined effort of phycologists has produced a rich plastid genome resource with taxonomic representation spanning all of the major lineages of the red algae (Rhodophyta). In this proof‐of‐concept study, we leveraged this resource by developing and applying a phylogenomic strategy to seek candidate plastid markers suitable for phylogenetic community analysis. We ranked the core genes of 107 published plastid genomes based on various sequence‐derived properties and their tree distance to plastid genome phylogenies. The resulting ranking revealed that the most widely used marker, rbcL, is not necessarily the optimal marker, while other promising markers might have been overlooked. We designed and tested PCR primers for several candidate marker genes, and successfully amplified one of them, rpoC1, in a taxonomically broad set of red algal specimens. We suggest that our general marker identification methodology and the rpoC1 primers will be useful to the phycological community for investigating the biodiversity and community ecology of the red algae.     

Plastid phylogenomics and molecular evolution of Thismiaceae (Dioscoreales)

Premise

Species in Thismiaceae can no longer photosynthesize and instead obtain carbon from soil fungi. Here we infer Thismiaceae phylogeny using plastid genome data and characterize the molecular evolution of this genome.

Methods

We assembled five Thismiaceae plastid genomes from genome skimming data, adding to previously published data for phylogenomic inference. We investigated plastid-genome structural changes, considering locally colinear blocks (LCBs). We also characterized possible shifts in selection pressure in retained genes by considering changes in the ratio of nonsynonymous to synonymous changes (ω).

Results

Thismiaceae experienced two major pulses of gene loss around the early diversification of the family, with subsequent scattered gene losses across descendent lineages. In addition to massive size reduction, Thismiaceae plastid genomes experienced occasional inversions, and there were likely two independent losses of the plastid inverted repeat (IR) region. Retained plastid genes remain under generally strong purifying selection (ω << 1), with significant and sporadic weakening or strengthening in several instances. The bifunctional trnE-UUC gene of Thismia huangii may retain a secondary role in heme biosynthesis, despite a probable loss of functionality in protein translation. Several cis-spliced group IIA introns have been retained, despite the loss of the plastid intron maturase, matK.

Conclusions

We infer that most gene losses in Thismiaceae occurred early and rapidly, following the initial loss of photosynthesis in its stem lineage. As a species-rich, fully mycoheterotrophic lineage, Thismiaceae provide a model system for uncovering the unique and divergent ways in which plastid genomes evolve in heterotrophic plants.     

Plastid genome of <Emphasis Type="Italic">Seseli montanum</Emphasis>: Complete sequence and comparison with plastomes of other members of the Apiaceae family

This work reports the complete plastid (pt) DNA sequence of Seseli montanum L. of the Apiaceae family, determined using next-generation sequencing technology. The complete genome sequence has been deposited in GenBank with accession No. KM035851. The S. montanum plastome is 147,823 bp in length. The plastid genome has a typical structure for angiosperms and contains a large single-copy region (LSC) of 92,620 bp and a small single-copy region (SSC) of 17,481 bp separated by a pair of 18,861 bp inverted repeats (IRa and IRb). The composition, gene order, and AT-content in the S. montanum plastome are similar to that of a typical flowering plant pt DNA. One hundred fourteen unique genes have been identified, including 30 tRNA genes, four rRNA genes, and 80 protein genes. Of 18 intron-containing genes found, 16 genes have one intron, and two genes (ycf3, clpP) have two introns. Comparative analysis of Apiaceae plastomes reveals in the S. montanum plastome a LSC/IRb junction shift, so that the part of the ycf2 (4980 bp) gene is located in the LSC, but the other part of ycf2 (1301 bp) is within the inverted repeat. Thus, structural rearrangements in the plastid genome of S. montanum result in an enlargement of the LSC region by means of capture of a large part of ycf2, in contrast to eight Apiaceae plastomes where the complete ycf2 gene sequence is located in the inverted repeat.     

Extensive Reorganization of the Plastid Genome of <Emphasis Type="Italic">Trifolium subterraneum</Emphasis> (Fabaceae) Is Associated with Numerous Repeated Sequences and Novel DNA Insertions

The plastid genome of Trifolium subterraneum is 144,763 bp, about 20 kb longer than those of closely related legumes, which also lost one copy of the large inverted repeat (IR). The genome has undergone extensive genomic reconfiguration, including the loss of six genes (accD, infA, rpl22, rps16, rps18, and ycf1) and two introns (clpP and rps12) and numerous gene order changes, attributable to 14–18 inversions. All endpoints of rearranged gene clusters are flanked by repeated sequences, tRNAs, or pseudogenes. One unusual feature of the Trifolium subterraneum genome is the large number of dispersed repeats, which comprise 19.5% (ca. 28 kb) of the genome (versus about 4% for other angiosperms) and account for part of the increase in genome size. Nine genes (psbT, rbcL, clpP, rps3, rpl23, atpB, psbN, trnI-cau, and ycf3) have also been duplicated either partially or completely. rpl23 is the most highly duplicated gene, with portions of this gene duplicated six times. Comparisons of the Trifolium plastid genome with the Plant Repeat Database and searches for flanking inverted repeats suggest that the high incidence of dispersed repeats and rearrangements is not likely the result of transposition. Trifolium has 19.5 kb of unique DNA distributed among 160 fragments ranging in size from 30 to 494 bp, greatly surpassing the other five sequenced legume plastid genomes in novel DNA content. At least some of this unique DNA may represent horizontal transfer from bacterial genomes. These unusual features provide direction for the development of more complex models of plastid genome evolution. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

An aberrant plastid ribosomal RNA gene cluster in the root parasite Conopholis americana

The plastid ribisomal RNA (rRNA) operon of the achlorophyllous root parasite Conopholis americana was completely sequenced. Full-length rRNA genes are retained in the gene cluster, but significant divergence has occurred in the 16S, 23S and 5S genes. Both the 16S–23S intergenic spacer and the 4.5S–5S intergenic spacer have suffered substantial deletions, including the two tRNA genes typically found in prokaryotic and plastid 16S–23S spacers.     

Abundance of plastid DNA insertions in nuclear genomes of rice and Arabidopsis

Pairwise comparison of whole plastid and draft nuclear genomic sequences of Arabidopsis thaliana and Oryza sativa L. ssp. indica shows that rice nuclear genomic sequences contain homologs of plastid DNA covering about 94 kb (83%) of plastid genome and including one or more full-length intact (without mutations resulting in premature stop codons) homologues of 26 known protein-coding (KPC) plastid genes. By contrast, only about 20 kb (16%) of chloroplast DNA, including a single intact plastid-derived KPC gene, is presented in the nucleus of A. thaliana. Sixteen rice plastid genes have at least one nuclear copy without any mutation or with only synonymous substitutions. Nuclear copies for other ten plastid genes contain both synonymous and non-synonymous substitutions. Multiple ESTs for 25 out of 26 KPC genes were also found, as well as putative promoters for some of them. The study of substitutions pattern shows that some of nuclear homologues of plastid genes may be functional and/or are under the pressure of the positive natural selection. The similar comparative analysis performed on rice chromosome 1 revealed 27 contigs containing plastid-derived sequences, totalling about 84 kb and covering two thirds of chloroplast DNA, with the intact nuclear copies of 26 different KPC genes. One of these contigs, AP003280, includes almost 57 kb (45%) of chloroplast genome with the intact copies of 22 KPC genes. At the same time, we observed that relative locations of homologues in plastid DNA and the nuclear genome are significantly different.     

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.     

PCR and sequencing from a single pollen grain

In order to eliminate the laborious step of DNA extraction preceding all studies within the field of plant molecular biology we attempted to do PCR amplifications directly on pollen grains. Successful PCR amplification was obtained in reactions including a single pollen grain from Hordeum vulgare or Secale strictum. Both the plastid gene encoding ribulose-1,5-biphosphate carboxylase/oxygenase (rbcL) and the nuclear-encoded internal transcribed spacer regions (ITS) and the 5.8S rDNA region were amplified and sequenced to verify PCR amplification.     

Phylogeny of Plastids Based on Cladistic Analysis of Gene Loss Inferred from Complete Plastid Genome Sequences

Based on the recent hypothesis on the origin of eukaryotic phototrophs, red algae, green plants, and glaucophytes constitute the primary photosynthetic eukaryotes (whose plastids may have originated directly from a cyanobacterium-like prokaryote via primary endosymbiosis), whereas the plastids of other lineages of eukaryotic phototrophs appear to be the result of secondary or tertiary endosymbiotic events (involving a phototrophic eukaryote and a host cell). Although phylogenetic analyses using multiple plastid genes from a wide range of eukaryotic lineages have been carried out, some of the major phylogenetic relationships of plastids remain ambiguous or conflict between different phylogenetic methods used for nucleotide or amino acid substitutions. Therefore, an alternative methodology to infer the plastid phylogeny is needed. Here, we carried out a cladistic analysis of the loss of plastid genes after primary endosymbiosis using complete plastid genome sequences from a wide range of eukaryotic phototrophs. Since it is extremely unlikely that plastid genes are regained during plastid evolution, we used the irreversible Camin-Sokal model for our cladistic analysis of the loss of plastid genes. The cladistic analysis of the 274 plastid protein-coding genes resolved the 20 operational taxonomic units representing a wide range of eukaryotic lineages (including three secondary plastid-containing groups) into two large monophyletic groups with high bootstrap values: one corresponded to the red lineage and the other consisted of a large clade composed of the green lineage (green plants and Euglena) and the basal glaucophyte plastid. Although the sister relationship between the green lineage and the Glaucophyta was not resolved in recent phylogenetic studies using amino acid substitutions from multiple plastid genes, it is consistent with the rbcL gene phylogeny and with a recent phylogenetic study using multiple nuclear genes. In addition, our analysis robustly resolved the conflicting/ambiguous phylogenetic positions of secondary plastids in previous phylogenetic studies: the Euglena plastid was sister to the chlorophycean (Chlamydomonas) lineage, and the secondary plastids from the diatom (Odontiella) and cryptophyte (Guillardia) were monophyletic within the red lineage.     

Complete sequence and analysis of the plastid genome of the unicellular red alga Cyanidioschyzon merolae.

The complete nucleotide sequence of the plastid genome of the unicellular primitive red alga Cyanidioschyzon merolae 10D (Cyanidiophyceae) was determined. The genome is a circular DNA composed of 149,987 bp with no inverted repeats. The G + C content of this plastid genome is 37.6%. The C. merolae plastid genome contains 243 genes, which are distributed on both strands and consist of 36 RNA genes (3 rRNAs, 31 tRNAs, tmRNA, and a ribonuclease P RNA component) and 207 protein genes, including unidentified open reading frames. The striking feature of this genome is the high degree of gene compaction; it has very short intergenic distances (approximately 40% of the protein genes were overlapped) and no genes have introns. This genome encodes several genes that are rarely found in other plastid genomes. A gene encoding a subunit of sulfate transporter (cysW) is the first to be identified in a plastid genome. The cysT and cysW genes are located in the C. merolae plastid genome in series, and they probably function together with other nuclear-encoded components of the sulfate transport system. Our phylogenetic results suggest that the Cyanidiophyceae, including C. merolae, are a basal clade within the red lineage plastids.     

A regional approach to plant DNA barcoding provides high species resolution of sedges (Carex and Kobresia, Cyperaceae) in the Canadian Arctic Archipelago

Previous research on barcoding sedges (Carex) suggested that basic searches within a global barcoding database would probably not resolve more than 60% of the world’s some 2000 species. In this study, we take an alternative approach and explore the performance of plant DNA barcoding in the Carex lineage from an explicitly regional perspective. We characterize the utility of a subset of the proposed protein-coding and noncoding plastid barcoding regions (matK, rpoB, rpoC1, rbcL, atpF-atpH, psbK-psbI) for distinguishing species of Carex and Kobresia in the Canadian Arctic Archipelago, a clearly defined eco-geographical region representing 1% of the Earth’s landmass. Our results show that matK resolves the greatest number of species of any single-locus (95%), and when combined in a two-locus barcode, it provides 100% species resolution in all but one combination (matK + atpFH) during unweighted pair-group method with arithmetic mean averages (UPGMA) analyses. Noncoding regions were equally or more variable than matK, but as single markers they resolve substantially fewer taxa than matK alone. When difficulties with sequencing and alignment due to microstructural variation in noncoding regions are also considered, our results support other studies in suggesting that protein-coding regions are more practical as barcoding markers. Plastid DNA barcodes are an effective identification tool for species of Carex and Kobresia in the Canadian Arctic Archipelago, a region where the number of co-existing closely related species is limited. We suggest that if a regional approach to plant DNA barcoding was applied on a global scale, it could provide a solution to the generally poor species resolution seen in previous barcoding studies.     

The First Complete Chloroplast Genome Sequences in Actinidiaceae: Genome Structure and Comparative Analysis

Actinidia chinensis is an important economic plant belonging to the basal lineage of the asterids. Availability of a complete Actinidia chloroplast genome sequence is crucial to understanding phylogenetic relationships among major lineages of angiosperms and facilitates kiwifruit genetic improvement. We report here the complete nucleotide sequences of the chloroplast genomes for Actinidia chinensis and A. chinensis var deliciosa obtained through de novo assembly of Illumina paired-end reads produced by total DNA sequencing. The total genome size ranges from 155,446 to 157,557 bp, with an inverted repeat (IR) of 24,013 to 24,391 bp, a large single copy region (LSC) of 87,984 to 88,337 bp and a small single copy region (SSC) of 20,332 to 20,336 bp. The genome encodes 113 different genes, including 79 unique protein-coding genes, 30 tRNA genes and 4 ribosomal RNA genes, with 16 duplicated in the inverted repeats, and a tRNA gene (trnfM-CAU) duplicated once in the LSC region. Comparisons of IR boundaries among four asterid species showed that IR/LSC borders were extended into the 5’ portion of the psbA gene and IR contraction occurred in Actinidia. The clap gene has been lost from the chloroplast genome in Actinidia, and may have been transferred to the nucleus during chloroplast evolution. Twenty-seven polymorphic simple sequence repeat (SSR) loci were identified in the Actinidia chloroplast genome. Maximum parsimony analyses of a 72-gene, 16 taxa angiosperm dataset strongly support the placement of Actinidiaceae in Ericales within the basal asterids.     

Polymorphism and gene arrangement among plastomes of ten Epilobium species

Summary Plastid DNAs of ten different Epilobium species from four continents have been analysed using the restriction endonucleases BamHI, BglI, BglII, EcoRI, PstI, PvuII and SalI. With respect to the position of cleavage sites of those enzymes, each species has a specific plastome. Fragment patterns of different species from the same continent show a higher degree of similarity than those from different continents. Physical maps of the circular plastid DNA molecule have been constructed for each of the ten species by localising the cleavage sites of the enzymes BglI, PvuII and SalI. As in most other higher plants, the plastid DNA of Epilobium is segmentally organized into two inverted repeats separated by a large and a small single copy region. In heterologous hybridization experiments using radioactively labelled gene probes, the positions of structural genes coding for the rRNAs and for seven polypeptides have been determined. In contrast to its closest relative, Oenothera, the gene arrangement of Epilobium plastomes has the same order as in spinach. This indicates that changes in gene arrangement may be genus-specific and not the result of one or several events affecting all members of a plant family.Abbreviations kbp kilobase pairs - ptDNA plastid DNA - rDNA ribosomal DNA - rRNA ribosomal RNA - SDS sodium dodecyl sulfate     

A divergent plastid genome inConopholis americana,an achlorophyllous parasitic plant

We have used heterologous probes to investigate the degree of sequence conservation in the plastid genome ofConopholis americana, a totally achlorophyllous angiosperm which exists as a root parasite on red oaks. AlthoughConopholis is completely nonphotosynthetic, it retains a plastid genome in which certain regions, including that which contains the ribosomal RNA genes, are highly conserved. Other regions, including those containing the genes for numerous photosynthesis proteins, are either absent or highly divergent. We also find that the 16S and 23S ribosomal genes of theConopholis plastid are transcribed and processed, implying a potentially functional genetic apparatus. These results are in agreement with findings reported recently for a related root parasite,Epifagus virginiana (de Pamphilis and Palmer, 1990). Furthermore, the plastid genome is maintained in high copy number in fruit tissue, whereas mature seeds have an approximately 10-fold lower copy number.     

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.