Plastid Intron Research Articles

On the importance of sequence alignment inspections in plastid phylogenomics - an example from revisiting the relationships of the water-lilies.

The water-lily clade represents the second earliest-diverging branch of angiosperms. Most of its species belong to Nymphaeaceae, of which the "core Nymphaeaceae"-comprising the genera Euryale, Nymphaea and Victoria-is the most diverse clade. Despite previous molecular phylogenetic studies on the core Nymphaeaceae, various aspects of their evolutionary relationships have remained unresolved. The length-variable introns and intergenic spacers are known to contain most of the sequence variability within the water-lily plastomes. Despite the challenges with multiple sequence alignment, any new molecular phylogenetic investigation on the core Nymphaeaceae should focus on these noncoding plastome regions. For example, a new plastid phylogenomic study on the core Nymphaeaceae should generate DNA sequence alignments of all plastid introns and intergenic spacers based on the principle of conserved sequence motifs. In this investigation, we revisit the phylogenetic history of the core Nymphaeaceae by employing such an approach. Specifically, we use a plastid phylogenomic analysis strategy in which all coding and noncoding partitions are separated and then undergo software-driven DNA sequence alignment, followed by a motif-based alignment inspection and adjustment. This approach allows us to increase the reliability of the character base compared to the default practice of aligning complete plastomes through software algorithms alone. Our approach produces significantly different phylogenetic tree reconstructions for several of the plastome regions under study. The results of these reconstructions underscore that Nymphaea is paraphyletic in its current circumscription, that each of the five subgenera of Nymphaea is monophyletic, and that the subgenus Nymphaea is sister to all other subgenera of Nymphaea. Our results also clarify many evolutionary relationships within the Nymphaea subgenera Brachyceras, Hydrocallis and Nymphaea. In closing, we discuss whether the phylogenetic reconstructions obtained through our motif-based alignment adjustments are in line with morphological evidence on water-lily evolution.

Maize requires Embryo defective27 for embryogenesis and seedling development.

The essential role of plastid translation in embryogenesis has been established in many plants, but a retrograde signal triggered by defective plastid translation machinery that may leads to embryogenesis arrest remains unknown. In this study, we characterized an embryo defective27 (emb27) mutant in maize (Zea mays), and cloning indicates that Emb27 encodes the plastid ribosomal protein S13. The null mutant emb27-1 conditions an emb phenotype with arrested embryogenesis; however, the leaky mutant emb27-2 exhibits normal embryogenesis but an albino seedling-lethal phenotype. The emb27-1/emb27-2 trans-heterozygotes display varying phenotypes from emb to normal seeds but albino seedlings. Analysis of the Emb27 transcription levels in these mutants revealed that the Emb27 expression level in the embryo corresponds with the phenotypic expression of the emb27 mutants. In the W22 genetic background, an Emb27 transcription level higher than 6% of the wild-type level renders normal embryogenesis, whereas lower than that arrests embryogenesis. Mutation of Emb27 reduces the level of plastid 16S rRNA and the accumulation of the plastid-encoded proteins. As a secondary effect, splicing of several plastid introns was impaired in emb27-1 and 2 other plastid translation-defective mutants, emb15 and emb16, suggesting that plastome-encoded factors are required for the splicing of these introns, such as Maturase K (MatK). Our results indicate that EMB27 is essential for plastid protein translation, embryogenesis, and seedling development in maize and reveal an expression threshold of Emb27 for maize embryogenesis.

Plastid phylogenomics and molecular evolution of Thismiaceae (Dioscoreales).

Species in Thismiaceae can no longer photosynthesize, and instead obtain carbon from soil fungi. Here we infer Thismiaceae phylogeny using plastid genome data, and also characterize the molecular evolution of this genome. 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 ω, the ratio of non-synonymous to synonymous changes. 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 likely were 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 likely retains a secondary role in heme biosynthesis, despite a probable loss of functionality in protein translation. Several cis-spliced group IIA introns are retained, despite the loss of the plastid intron maturase, matK. 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 provides a model system for uncovering the unique and divergent ways in which plastid genomes evolve in heterotrophic plants. This article is protected by copyright. All rights reserved.

The nuclear-encoded plastid ribosomal protein L18s are essential for plant development.

Plastid ribosomal proteins (PRPs) are necessary components for plastid ribosome biogenesis, playing essential roles in plastid development. The ribosomal protein L18 involved in the assemble of 5S rRNA and 23S rRNA, is vital for E. coli viability, but the functions of its homologs in plant plastid remain elusive. Here, we characterized the functions of the plant plastid ribosomal protein L18s (PRPL18s) in Arabidopsis and rice. AtPRPL18 was ubiquitously expressed in most of the plant tissues, but with higher expression levels in seedling shoots, leaves, and flowers. AtPRPL18 was localized in chloroplast. Genetic and cytological analyses revealed that a loss of function of AtPRPL18 resulted in embryo development arrest at globular stage. However, overexpression of AtPRPL18 did not show any visible phenotypical changes in Arabidopsis. The rice OsPRPL18 was localized in chloroplast. In contrast to AtPRPL18, knockout of OsPRPL18 did not affect embryo development, but led to an albino lethal phenotype at the seedling stage. Cytological analyses showed that chloroplast development was impaired in the osprpl18-1 mutant. Moreover, a loss-function of OsPRPL18 led to defects in plastid ribosome biogenesis and a serious reduction in the efficiency of plastid intron splicing. In all, these results suggested that PRPL18s play critical roles in plastid ribosome biogenesis, plastid intron splicing, and chloroplast development, and are essential for plant survival.

The pentatricopeptide repeat protein EMB1270 interacts with CFM2 to splice specific group II introns in Arabidopsis chloroplasts.

Chloroplast biogenesis requires the coordinated expression of chloroplast and nuclear genes. Here, we show that EMB1270, a plastid-localized pentatricopeptide repeat (PPR) protein, is required for chloroplast biogenesis in Arabidopsis thaliana. Knockout of EMB1270 led to embryo arrest, whereas a mild knockdown mutant of EMB1270 displayed a virescent phenotype. Almost no photosynthetic proteins accumulated in the albino emb1270 knockout mutant. By contrast, in the emb1270 knockdown mutant, the levels of ClpP1 and photosystem I (PSI) subunits were significantly reduced, whereas the levels of photosystem II (PSII) subunits were normal. Furthermore, the splicing efficiencies of the clpP1.2, ycf3.1, ndhA, and ndhB plastid introns were dramatically reduced in both emb1270 mutants. RNA immunoprecipitation revealed that EMB1270 associated with these introns in vivo. In an RNA electrophoretic mobility shift assay (REMSA), a truncated EMB1270 protein containing the 11 N-terminal PPR motifs bound to the predicted sequences of the clpP1.2, ycf3.1, and ndhA introns. In addition, EMB1270 specifically interacted with CRM Family Member 2 (CFM2). Given that CFM2 is known to be required for splicing the same plastid RNAs, our results suggest that EMB1270 associates with CFM2 to facilitate the splicing of specific group II introns in Arabidopsis.

Comprehensive genomic analyses with 115 plastomes from algae to seed plants: structure, gene contents, GC contents, and introns.

Chloroplasts are a common character in plants. The chloroplasts in each plant lineage have shaped their own genomes, plastomes, by structural changes and transferring many genes to nuclear genomes during plant evolution. Some plastid genes have introns that are mostly group II introns. This study aimed to get genomic and evolutionary insights on the plastomes from green algae to flowering plants. Plastomes of 115 species from green algae, bryophytes, pteridophytes (spore bearing vascular plants), gymnosperms, and angiosperms were mined from NCBI organelle genome database. Plastome structure, gene contents and GC contents were analyzed by the in-house developed Phyton code. Intronic features including presence/absence, length, intron phases were analyzed by manually in the annotated information in NCBI. The canonical quadripartite structures were retained in most plastomes except of a few plastomes that had lost an invert repeat (IR). Expansion or reduction or deletion of IRs resulted in the length variation of the plastomes. The number of protein coding genes ranged from 40 to 92 with an average 79.43 ± 5.84 per plastome and gene losses were apparent in specific lineages. The number of trn genes ranged from 13 to 33 with an average 21.19 ± 2.42 per plastome. Ribosomal RNA genes, rrn, were located in the IRs so that they were present in a duplicate except of the species that had lost one of the IR. GC contents were variable from 24.9 to 51.0% with an average 38.21 ± 3.27%, indicating bias to high AT contents. Plastid introns were present in 18 protein coding genes, six trn genes, and one rrn gene. Intron losses occurred among the orthologous genes in different plant lineages. The plastid introns were long compared with the nuclear introns, which might be related with the spliceosome nuclear introns and self-splicing group II plastid introns. The trnK-UUU intron contained the maturase encoding matK gene except in the chlorophyte algae and monilophyte ferns in which the trnK-UUU was lost, but matK retained. There were many annotation artefacts in the intron positions in the NCBI database. In the analysis of intron phases, phase 0 introns were more frequent than those of phase 2 and 3 introns. Phase polymorphism was observed in the introns of clpP which was derived from nucleotide insertion. Plastid trn introns were long compared to the archaeal or eukaryotic nuclear tRNA introns. Of the six plastid trn introns, one was at the D loop and other five were at the anticodon loop. The insertion sites were conserved among the trn genes in archaea, eukaryotic nuclear and plastid tRNA genes. Current study refurbrished the previous findings of structural variations, gene contents, and GC contents of the chloroplast genomes from green algae to flowering plants. The study also included some noble findings and discussions on the plastome introns including their length variations and phase variation. We also presented and corrected some false annotations on the introns in protein coding and tRNA genes in the genome database, which might be confirmed by the chloroplast transcriptome analysis in the future.

Recent mobility of plastid encoded group II introns and twintrons in five strains of the unicellular red alga Porphyridium.

Group II introns are closely linked to eukaryote evolution because nuclear spliceosomal introns and the small RNAs associated with the spliceosome are thought to trace their ancient origins to these mobile elements. Therefore, elucidating how group II introns move, and how they lose mobility can potentially shed light on fundamental aspects of eukaryote biology. To this end, we studied five strains of the unicellular red alga Porphyridium purpureum that surprisingly contain 42 group II introns in their plastid genomes. We focused on a subset of these introns that encode mobility-conferring intron-encoded proteins (IEPs) and found them to be distributed among the strains in a lineage-specific manner. The reverse transcriptase and maturase domains were present in all lineages but the DNA endonuclease domain was deleted in vertically inherited introns, demonstrating a key step in the loss of mobility. P. purpureum plastid intron RNAs had a classic group IIB secondary structure despite variability in the DIII and DVI domains. We report for the first time the presence of twintrons (introns-within-introns, derived from the same mobile element) in Rhodophyta. The P. purpureum IEPs and their mobile introns provide a valuable model for the study of mobile retroelements in eukaryotes and offer promise for biotechnological applications.

Evolution of Red Algal Plastid Genomes: Ancient Architectures, Introns, Horizontal Gene Transfer, and Taxonomic Utility of Plastid Markers

Red algae have the most gene-rich plastid genomes known, but despite their evolutionary importance these genomes remain poorly sampled. Here we characterize three complete and one partial plastid genome from a diverse range of florideophytes. By unifying annotations across all available red algal plastid genomes we show they all share a highly compact and slowly-evolving architecture and uniquely rich gene complements. Both chromosome structure and gene content have changed very little during red algal diversification, and suggest that plastid-to nucleus gene transfers have been rare. Despite their ancient character, however, the red algal plastids also contain several unprecedented features, including a group II intron in a tRNA-Met gene that encodes the first example of red algal plastid intron maturase – a feature uniquely shared among florideophytes. We also identify a rare case of a horizontally-acquired proteobacterial operon, and propose this operon may have been recruited for plastid function and potentially replaced a nucleus-encoded plastid-targeted paralogue. Plastid genome phylogenies yield a fully resolved tree and suggest that plastid DNA is a useful tool for resolving red algal relationships. Lastly, we estimate the evolutionary rates among more than 200 plastid genes, and assess their usefulness for species and subspecies taxonomy by comparison to well-established barcoding markers such as cox1 and rbcL. Overall, these data demonstrates that red algal plastid genomes are easily obtainable using high-throughput sequencing of total genomic DNA, interesting from evolutionary perspectives, and promising in resolving red algal relationships at evolutionarily-deep and species/subspecies levels.

Phylogenetics of early branching eudicots: Comparing phylogenetic signal across plastid introns, spacers, and genes

Abstract Recent phylogenetic analyses revealed a grade with Ranunculales, Sabiales, Proteales, Trochodendrales, and Buxales as first branching eudicots, with the respective positions of Proteales and Sabiales still lacking statistical confidence. As previous analyses of conserved plastid genes remain inconclusive, we aimed to use and evaluate a representative set of plastid introns (group I: trnL; group II: petD, rpl16, trnK) and intergenic spacers (trnL‐F, petB‐petD, atpB‐rbcL, rps3‐rpl16) in comparison to the rapidly evolving matK and slowly evolving atpB and rbcL genes. Overall patterns of microstructural mutations converged across genomic regions, underscoring the existence of a general mutational pattern throughout the plastid genome. Phylogenetic signal differed strongly between functionally and structurally different genomic regions and was highest in matK, followed by spacers, then group II and group I introns. The more conserved atpB and rbcL coding regions showed distinctly lower phylogenetic information content. Parsimony, maximum likelihood, and Bayesian phylogenetic analyses based on the combined dataset of non‐coding and rapidly evolving regions (&gt;14 000 aligned characters) converged to a backbone topology of eudicots with Ranunculales branching first, a Proteales–Sabiales clade second, followed by Trochodendrales and Buxales. Gunnerales generally appeared as sister to all remaining core eudicots with maximum support. Our results show that a small number of intron and spacer sequences allow similar insights into phylogenetic relationships of eudicots compared to datasets of many combined genes. The non‐coding proportion of the plastid genome thus can be considered an important information source for plastid phylogenomics.

Invasion potential and host shifts of Australian and African ectomycorrhizal fungi in mixed eucalypt plantations

• Transportation of forestry materials results in unintended co-introduction of nonnative species that may cause enormous ecological or economic damage. While the invasion ecology of plants and animals is relatively well-known, that of microorganisms, except aboveground pathogens, remains poorly understood. • This work addresses host shifts and invasion potential of root symbiotic ectomycorrhizal fungi that were co-introduced with Australian eucalypts and planted in clear-cut miombo woodlands in Zambia, south-central Africa. • By use of rDNA and plastid intron sequence analysis for identification and phylogenetic techniques for inferring fungal origin, we demonstrated that host shifts were uncommon in the Australian fungi, but frequent in the African fungi, especially in mixed plantations where roots of different trees intermingle. • There was evidence for naturalization, but not for invasion by Australian ectomycorrhizal fungi. Nevertheless, the fungi introduced may pose an invasion risk along with further adaptation to local soil environment and host trees. Inoculation of eucalypts with native edible fungi may ameliorate the potential invasion risks of introduced fungi and provide an alternative source of nutrition.

From trace evidence to bioinformatics: Putting bryophytes into molecular biology education

Students benefit most from their science education when they participate fully in the process of science in the context of real-world problems. We describe a student-directed open-inquiry lab experience that has no predetermined outcomes and requires students to engage in all components of scientific inquiry from posing a question through evaluating and reporting results. Over 5 weeks, students learn how bryophytes are used in forensics and become proficient in important molecular biology lab skills including DNA isolation, polymerase chain reaction, gel electrophoresis, capillary electrophoresis, and genotyping. For this portion of the experience, there is no specialized equipment necessary outside of gel electrophoresis supplies and a thermocycler. In an optional extension of the experience, students sequence a plastid intron and use introductory bioinformatics skills to identify species related to their forensics case. Students who participated in the lab experience performed well on content-based assessment, and student attitudes toward the experience were positive and indicative of engaged learning. The lab experience is easily modified for higher or lower level courses and can be used in secondary education.

Parallel Loss of Plastid Introns and Their Maturase in the Genus Cuscuta

Plastid genome content and arrangement are highly conserved across most land plants and their closest relatives, streptophyte algae, with nearly all plastid introns having invaded the genome in their common ancestor at least 450 million years ago. One such intron, within the transfer RNA trnK-UUU, contains a large open reading frame that encodes a presumed intron maturase, matK. This gene is missing from the plastid genomes of two species in the parasitic plant genus Cuscuta but is found in all other published land plant and streptophyte algal plastid genomes, including that of the nonphotosynthetic angiosperm Epifagus virginiana and two other species of Cuscuta. By examining matK and plastid intron distribution in Cuscuta, we add support to the hypothesis that its normal role is in splicing seven of the eight group IIA introns in the genome. We also analyze matK nucleotide sequences from Cuscuta species and relatives that retain matK to test whether changes in selective pressure in the maturase are associated with intron deletion. Stepwise loss of most group IIA introns from the plastid genome results in substantial change in selective pressure within the hypothetical RNA-binding domain of matK in both Cuscuta and Epifagus, either through evolution from a generalist to a specialist intron splicer or due to loss of a particular intron responsible for most of the constraint on the binding region. The possibility of intron-specific specialization in the X-domain is implicated by evidence of positive selection on the lineage leading to C. nitida in association with the loss of six of seven introns putatively spliced by matK. Moreover, transfer RNA gene deletion facilitated by parasitism combined with an unusually high rate of intron loss from remaining functional plastid genes created a unique circumstance on the lineage leading to Cuscuta subgenus Grammica that allowed elimination of matK in the most species-rich lineage of Cuscuta.

Huerteales sister to Brassicales plus Malvales, and newly circumscribed to include Dipentodon, Gerrardina, Huertea, Perrottetia, and Tapiscia

ABSTRACTSequence data from the matK gene, the trnK group II intron, the trnL group I intron and the trnL‐F spacer were analysed for a broad sampling of the rosids and other eudicots. For the first time all putative genera of Dipentodontaceae and Tapisciaceae (Dipentodon, Huertea, Perrottetia, Tapiscia), as well as the recently described Gerrardinaceae were included in a molecular phylogenetic dataset. All genera were found in a well supported Huerteales clade. Moreover, with the rapidly evolving and non‐coding plastid sequence data we were able to resolve the Huerteales clade to branch after Sapindales, and to be sister to a Brassicales‐Malvales clade. Increased resolution and support among the malvids underscore the potential of plastid introns and spacers as well as the matK gene as phylogenetic markers in rosids.

Recent horizontal intron transfer to a chloroplast genome

Evidence is presented for the recent, horizontal transfer of a self-splicing, homing group II intron from a cyanobacteria to the chloroplast genome of Euglena myxocylindracea. The psbA gene of E.myxocylindracea was found to contain a single 2566 nt group II intron with a gene in domain 4 for a 575 amino acid maturase. The predicted secondary structure and tertiary interactions of the group II intron, as well as the derived maturase primary sequence, most closely resemble the homing intron of the cyanobacterium Calothrix and the rnl introns of Porphyra purpurea mitochondria, while being only distantly related to all other Euglena plastid introns and maturases. All main functional domains of the intron-encoded proteins of known homing introns are conserved, including reverse transcriptase domains 1-7, the zinc finger domain and domain X. The close relationship with cyanobacterial introns was confirmed by phylogenetic analysis. Both the full-length psbA intron and a Delta-maturase variant self-splice in vitro in two independent assays. The psbA intron is the first example of a self-splicing chloroplast group II intron from any organism. These results support the conclusion that the psbA intron is the result of a recent horizontal transfer into the E.myxocylindracea chloroplast genome from a cyanobacterial donor and should prompt a reconsideration of horizontal transfer mechanisms to account for the origin of other chloroplast genetic elements.

Phylogénie moléculaire du genre Moraea (Iridaceae: Irideae): apports du séquençage d'une région d'ADN chloroplastique

DNA sequence analysis of the plastid intron rps 16 from each of 66 species in the African genus Moraea (Iridaceae: Irideae), including examples of all the major species clusters in the genus, brings new data for the phylogenetic tree. The results show that the current subgeneric classification poorly reflects the phylogenetic relationships of species within Moraea. This work confirms the results of a phylogenetic study using morphological characters. Moraea is paraphyletic if Barnardiella, Galaxia, Hexaglottis, Homeria and Gynandriris are recognized as separate genera.

What Is the Relationship among Hernandiaceae, Lauraceae, and Monimiaceae, and Why Is This Question So Difficult to Answer?

Molecular and morphological phylogenetic studies in the Laurales have found that Hernandiaceae, Lauraceae, and Monimiaceae sensu stricto form a monophyletic group. Because of the paucity of phylogenetically informative substitutions, however, relationships among families within this clade remain unclear. In general, molecular phylogenies may conflict because of a variety of factors, including substitution rate variation among sites and lineages, taxon sampling, outgroup choice, and base compositional biases. We analyzed a total of 2846 aligned nucleotides from a plastid intron, three spacers, and a portion of the nuclear 26S rRNA gene in a sample of Hernandiaceae, Lauraceae, and Monimiaceae; we used four outgroups with differing substitution rates. Despite obtaining single best topologies with maximum likelihood, minimum evolution, and parsimony approaches, family relationships remained as poorly supported as they were in the previous molecular studies. Exploration of the data indicates that varying substitution rates across lineages or sites, insufficient taxon sampling, fast‐evolving outgroups, or biased base composition are unlikely to explain the difficult reconstruction. Exclusion of some of the longest branched taxa (the hemiparasite Cassytha, selected Hernandiaceae, and two of the four outgroups) had no effect on topologies. To resolve relationships among the three families one could now complement existing five‐gene data sets by adding the basal genera of Lauraceae, Monimiaceae, and Hernandiaceae, which are newly sampled here, or, our preferred strategy, by sequencing low‐copy nuclear genes for the key genera to obtain different kinds of data.

Ins and outs of plastid genome evolution

Recent findings have established cracks in the straight-laced image of the plastid genome as a molecule whose sole function is photosynthesis and whose gene content is highly conserved. Genes for numerous non-photosynthetic functions have been identified. Algal plastid genomes contain many genes with no homologs in angiosperms, and the recent transfer of genes from the plastid to the nuclear genome has been described. Wholesale abandonment of genes encoding photosynthetic and gene-expression functions has occurred in the plastid genomes of a non-green plant and alga. The origins of plastid DNA, its use in phylogenetic studies, and the origins of plastid introns are also reviewed.