Complete plastid genome of Iris orchioides and comparative analysis with 19 Iris plastomes.

Iris is a cosmopolitan genus comprising approximately 280 species distributed throughout the Northern Hemisphere. Although Iris is the most diverse group in the Iridaceae, the number of taxa is debatable owing to various taxonomic issues. Plastid genomes have been widely used for phylogenetic research in plants; however, only limited number of plastid DNA markers are available for phylogenetic study of the Iris. To understand the genomic features of plastids within the genus, including its structural and genetic variation, we newly sequenced and analyzed the complete plastid genome of I. orchioides and compared it with those of 19 other Iris taxa. Potential plastid markers for phylogenetic research were identified by computing the sequence divergence and phylogenetic informativeness. We then tested the utility of the markers with the phylogenies inferred from the markers and whole-plastome data. The average size of the plastid genome was 152,926 bp, and the overall genomic content and organization were nearly identical among the 20 Iris taxa, except for minor variations in the inverted repeats. We identified 10 highly informative regions (matK, ndhF, rpoC2, ycf1, ycf2, rps15-ycf, rpoB-trnC, petA-psbJ, ndhG-ndhI and psbK-trnQ) and inferred a phylogeny from each region individually, as well as from their concatenated data. Remarkably, the phylogeny reconstructed from the concatenated data comprising three selected regions (rpoC2, ycf1 and ycf2) exhibited the highest congruence with the phylogeny derived from the entire plastome dataset. The result suggests that this subset of data could serve as a viable alternative to the complete plastome data, especially for molecular diagnoses among closely related Iris taxa, and at a lower cost.

Complete organelle genomes of Korean fir, Abies koreana and phylogenomics of the gymnosperm genus Abies using nuclear and cytoplasmic DNA sequence data.

Abies koreana E.H.Wilson is an endangered evergreen coniferous tree that is native to high altitudes in South Korea and susceptible to the effects of climate change. Hybridization and reticulate evolution have been reported in the genus; therefore, multigene datasets from nuclear and cytoplasmic genomes are needed to better understand its evolutionary history. Using the Illumina NovaSeq 6000 and Oxford Nanopore Technologies (ONT) PromethION platforms, we generated complete mitochondrial (1,174,803 bp) and plastid (121,341 bp) genomes from A. koreana. The mitochondrial genome is highly dynamic, transitioning from cis- to trans-splicing and breaking conserved gene clusters. In the plastome, the ONT reads revealed two structural conformations of A. koreana. The short inverted repeats (1186 bp) of the A. koreana plastome are associated with different structural types. Transcriptomic sequencing revealed 1356 sites of C-to-U RNA editing in the 41 mitochondrial genes. Using A. koreana as a reference, we additionally produced nuclear and organelle genomic sequences from eight Abies species and generated multiple datasets for maximum likelihood and network analyses. Three sections (Balsamea, Momi, and Pseudopicea) were well grouped in the nuclear phylogeny, but the phylogenomic relationships showed conflicting signals in the mitochondrial and plastid genomes, indicating a complicated evolutionary history that may have included introgressive hybridization. The obtained data illustrate that phylogenomic analyses based on sequences from differently inherited organelle genomes have resulted in conflicting trees. Organelle capture, organelle genome recombination, and incomplete lineage sorting in an ancestral heteroplasmic individual can contribute to phylogenomic discordance. We provide strong support for the relationships within Abies and new insights into the phylogenomic complexity of this genus.

Photoprotective mechanisms in Elysia species hosting Acetabularia chloroplasts shed light on host-donor compatibility in photosynthetic sea slugs.

Sacoglossa sea slugs have garnered attention due to their ability to retain intracellular functional chloroplasts from algae, while degrading other algal cell components. While protective mechanisms that limit oxidative damage under excessive light are well documented in plants and algae, the photoprotective strategies employed by these photosynthetic sea slugs remain unresolved. Species within the genus Elysia are known to retain chloroplasts from various algal sources, but the extent to which the metabolic processes from the donor algae can be sustained by the sea slugs is unclear. By comparing responses to high-light conditions through kinetic analyses, molecular techniques, and biochemical assays, this study shows significant differences between two photosynthetic Elysia species with chloroplasts derived from the green alga Acetabularia acetabulum. Notably, Elysia timida displayed remarkable tolerance to high-light stress and sophisticated photoprotective mechanisms such as an active xanthophyll cycle, efficient D1 protein recycling, accumulation of heat-shock proteins and α-tocopherol. In contrast, Elysia crispata exhibited absence or limitations in these photoprotective strategies. Our findings emphasize the intricate relationship between the host animal and the stolen chloroplasts, highlighting different capacities to protect the photosynthetic organelle from oxidative damage.

Structure of the multi-subunit chloroplast RNA polymerase.

Chloroplasts contain a dedicated genome that encodes subunits of the photosynthesis machinery. Transcription of photosynthesis genes is predominantly carried out by a plastid-encoded RNA polymerase (PEP), a nearly 1 MDa complex composed of core subunits with homology to eubacterial RNA polymerases (RNAPs) and at least 12 additional chloroplast-specific PEP-associated proteins (PAPs). However, the architecture of this complex and the functions of the PAPs remain unknown. Here, we report the cryo-EM structure of a 19-subunit PEP complex from Sinapis alba (white mustard). The structure reveals that the PEP core resembles prokaryotic and nuclear RNAPs but contains chloroplast-specific features that mediate interactions with the PAPs. The PAPs are unrelated to known transcription factors and arrange around the core in a unique fashion. Their structures suggest potential functions during transcription in the chemical environment of chloroplasts. These results reveal structural insights into chloroplast transcription and provide a framework for understanding photosynthesis gene expression.

Diversification of Plastid Structure and Function in Land Plants.

Plastids represent a largely diverse group of organelles in plant and algal cells that have several common features but also a broad spectrum of morphological, ultrastructural, biochemical, and physiological differences. Plastids and their structural and metabolic diversity significantly contribute to the functionality and developmental flexibility of the plant body throughout its lifetime. In addition to the multiple roles of given plastid types, this diversity is accomplished in some cases by interconversions between different plastids as a consequence of developmental and environmental signals that regulate plastid differentiation and specialization. In addition to basic plastid structural features, the most important plastid types, the newly characterized peculiar plastids, and future perspectives in plastid biology are also provided in this chapter.

Complex Endosymbioses I: From Primary to Complex Plastids, Serial Endosymbiotic Events.

A considerable part of the diversity of eukaryotic phototrophs consists of algae with plastids that evolved from endosymbioses between two eukaryotes. These complex plastids are characterized by a high number of envelope membranes (more than two) and some of them contain a residual nucleus of the endosymbiotic alga called a nucleomorph. Complex plastid-bearing algae are thus chimeric cell assemblies, eukaryotic symbionts living in a eukaryotic host. In contrast, the primary plastids of the Archaeplastida (plants, green algae, red algae, and glaucophytes) possibly evolved from a single endosymbiosis with a cyanobacterium and are surrounded by two membranes. Complex plastids have been acquired several times by unrelated groups of eukaryotic heterotrophic hosts, suggesting that complex plastids are somewhat easier to obtain than primary plastids. Evidence suggests that complex plastids arose twice independently in the green lineage (euglenophytes and chlorarachniophytes) through secondary endosymbiosis, and four times in the red lineage, first through secondary endosymbiosis in cryptophytes, then by higher-order events in stramenopiles, alveolates, and haptophytes. Engulfment of primary and complex plastid-containing algae by eukaryotic hosts (secondary, tertiary, and higher-order endosymbioses) is also responsible for numerous plastid replacements in dinoflagellates. Plastid endosymbiosis is accompanied by massive gene transfer from the endosymbiont to the host nucleus and cell adaptation of both endosymbiotic partners, which is related to the trophic switch to phototrophy and loss of autonomy of the endosymbiont. Such a process is essential for the metabolic integration and division control of the endosymbiont in the host. Although photosynthesis is the main advantage of acquiring plastids, loss of photosynthesis often occurs in algae with complex plastids. This chapter summarizes the essential knowledge of the acquisition, evolution, and function of complex plastids.

Plastid Transient and Stable Interactions with Other Cell Compartments.

Plastids are organelles delineated by two envelopes playing important roles in different cellular processes such as energy production or lipid biosynthesis. To regulate their biogenesis and their function, plastids have to communicate with other cellular compartments. This communication can be mediated by metabolites, signaling molecules, and by the establishment of direct contacts between the plastid envelope and other organelles such as the endoplasmic reticulum, mitochondria, peroxisomes, plasma membrane, and the nucleus. These interactions are highly dynamic and respond to different biotic and abiotic stresses. However, the mechanisms involved in the formation of plastid-organelle contact sites and their functions are still far from being understood. In this chapter, we summarize our current knowledge about plastid contact sites and their role in the regulation of plastid biogenesis and function.

Isolation of High-Quality Plastids from the Diatom Phaeodactylum tricornutum.

Phaeodactylum tricornutum, a model pennate diatom, carries a secondary plastid surrounded by four membranes. Its biological function remains mysterious, supposed to combine features of the primary chloroplast and the endomembrane system. Isolation of high-quality plastid from the diatom enables a more conclusive understanding of the special structure and metabolic pathways in the plastid. Due to the direct continuity between the chloroplast endoplasmic reticulum membrane (cERM) and the outer nuclear envelope together with the integration of cERM into the cellular endoplasmic reticulum (ER) system, the plastid isolation is still challenging. In this study, highly purified P. tricornutum plastids with the four-layered membrane are obtained by Percoll density gradient centrifugation. The isolated plastids are unlikely to contain any residue of nuclear and coatomer compartments, and they might contain a relatively small contamination of mitochondrion and ER debris.

The Main Functions of Plastids.

Plastids are semi-autonomous organelles like mitochondria and derive from a cyanobacterial ancestor that was engulfed by a host cell. During evolution, they have recruited proteins originating from the nuclear genome, and only parts of their ancestral metabolic properties were conserved and optimized to limit functional redundancy with other cell compartments. Furthermore, large disparities in metabolic functions exist among various types of plastids, and the characterization of their various metabolic properties is far from being accomplished. In this review, we provide an overview of the main functions, known to be achieved by plastids or shared by plastids and other compartments of the cell. In short, plastids appear at the heart of all main plant functions.

Monitoring of Lipid Fluxes Between Host and Plastid-Bearing Apicomplexan Parasites.

Apicomplexan parasites are unicellular eukaryotes responsible for major human diseases such as malaria and toxoplasmosis, which cause massive social and economic burden. Toxoplasmosis, caused by Toxoplasma gondii, is a global chronic infectious disease affecting ~1/3 of the world population and is a major threat for any immunocompromised patient. To date, there is no efficient vaccine against these parasites and existing treatments are threatened by rapid emergence of parasite resistance. Throughout their life cycle, Apicomplexa require large amount of nutrients, especially lipids for propagation and survival. Understanding lipid acquisition is key to decipher host-parasite metabolic interactions. Parasite membrane biogenesis relies on a combination of (a) host lipid scavenging, (b) de novo lipid synthesis in the parasite, and (c) fluxes of lipids between host and parasite and within. We recently uncovered that parasite need to store the host-scavenged lipids to avoid their toxic accumulation and to mobilize them for division. How can parasites orchestrate the many lipids fluxes essential for survival? Here, we developed metabolomics approaches coupled to stable isotope labelling to track, monitor, and quantify fatty acid and lipids fluxes between the parasite, its human host cell, and its extracellular environment to unravel the complex lipid fluxes in any physiological environment the parasite could meet.

Determining the Subcellular Localization of Proteins in the Different Membranes of Diatom Secondary Plastid.

Diatoms such as Phaeodactylum tricornutum arose through a process termed secondary endosymbiosis, in which red alga-derived plastids are surrounded by a complicated membrane system. Subcellular marker proteins provide defined localizations on the compartmental and even sub-compartmental levels in the complex plastids of diatoms. Here we introduce how to use subcellular marker proteins and in vivo co-localization in the diatom P. tricornutum by presenting a step-by-step method allowing the determination of subcellular localization of proteins in different membranes of the secondary plastid. This chapter describes the materials required and the procedures of transformation and microscopic observation.

Analysis of a Super-Complex at Contact Sites Between Mitochondria and Plastids.

Plastids are organelles playing fundamental roles in different cellular processes such as energy metabolism or lipid biosynthesis. To fulfill their biogenesis and their function in the cell, plastids have to communicate with other cellular compartments. This communication can be mediated by the establishment of direct contact sites between plastids envelop and other organelles. These contacts are dynamic structures regulated in response to stress. For example, during phosphate (Pi) starvation, the number of contact sites between plastids and mitochondria significantly increases. In this situation, these contacts play an important role in the transfer of galactoglycerolipids from plastids to mitochondria. Recently, Pi starvation stress was used to identify key proteins involved in the traffic of galactoglycerolipids from plastids to mitochondria in Arabidopsis thaliana. A mitochondrial lipoprotein complex called MTL (Mitochondrial Transmembrane Lipoprotein) was identified. This complex contains mitochondrial proteins but also proteins located in the plastid envelope, suggesting its presence at the plastid-mitochondria junction. This chapter describes the protocol to isolate the MTL complex by clear-native polyacrylamide gel electrophoresis (CN-PAGE) from the mitochondrial fraction of Arabidopsis cell cultures and the methods to study different features of this complex.

First Record of Comparative Plastid Genome Analysis and Phylogenetic Relationships among Corylopsis Siebold & Zucc. (Hamamelidaceae).

Corylopsis Siebold & Zucc. (Hamamelidaceae) is widely used as a horticultural plant and comprises approximately 25 species in East Asia. Molecular research is essential to distinguish Corylopsis species, which are morphologically similar. Molecular research has been conducted using a small number of genes but not in Corylopsis. Plastid genomes of Corylopsis species (Corylopsis gotoana, Corylopsis pauciflora, and Corylopsis sinensis) were sequenced using next-generation sequencing techniques. Repeats and nucleotide diversity that could be used as DNA markers were also investigated. A phylogenetic investigation was carried out using 79 protein-coding genes to infer the evolutionary relationships within the genus Corylopsis. By including new plastomes, the overall plastid genome structure of Corylopsis was similar. Simple sequence repeats of 73-106 SSRs were identified in the protein-coding genes of the plastid genomes, and 33-40 long repeat sequences were identified in the plastomes. The Pi value of the rpl33_rps18 region, an intergenic spacer, was the highest. Phylogenetic analysis demonstrated that Corylopsis is a monophyletic group and Loropetalum is closely related to Corylopsis. C. pauciflora, C. gotoana, and C. spicata formed a clade distributed in Japan, whereas C. sinensis, C. glandulifera, and C. velutina formed a clade that was distributed in China.

Cryo-EM structures of the plant plastid-encoded RNA polymerase.

Chloroplasts are green plastids in the cytoplasm of eukaryotic algae and plants responsible for photosynthesis. The plastid-encoded RNA polymerase (PEP) plays an essential role during chloroplast biogenesis from proplastids and functions as the predominant RNA polymerase in mature chloroplasts. The PEP-centered transcription apparatus comprises a bacterial-origin PEP core and more than a dozen eukaryotic-origin PEP-associated proteins (PAPs) encoded in the nucleus. Here, we determined the cryo-EM structures of Nicotiana tabacum (tobacco) PEP-PAP apoenzyme and PEP-PAP transcription elongation complexes at near-atomic resolutions. Our data show the PEP core adopts a typical fold as bacterial RNAP. Fifteen PAPs bind at the periphery of the PEP core, facilitate assembling the PEP-PAP supercomplex, protect the complex from oxidation damage, and likely couple gene transcription with RNA processing. Our results report the high-resolution architecture of the chloroplast transcription apparatus and provide the structural basis for the mechanistic and functional study of transcription regulation in chloroplasts.

Structure of the plant plastid-encoded RNA polymerase.

Chloroplast genes encoding photosynthesis-associated proteins are predominantly transcribed by the plastid-encoded RNA polymerase (PEP). PEP is a multi-subunit complex composed of plastid-encoded subunits similar to bacterial RNA polymerases (RNAPs) stably bound to a set of nuclear-encoded PEP-associated proteins (PAPs). PAPs are essential to PEP activity and chloroplast biogenesis, but their roles are poorly defined. Here, we present cryoelectron microscopy (cryo-EM) structures of native 21-subunit PEP and a PEP transcription elongation complex from white mustard (Sinapis alba). We identify that PAPs encase the core polymerase, forming extensive interactions that likely promote complex assembly and stability. During elongation, PAPs interact with DNA downstream of the transcription bubble and with the nascent mRNA. The models reveal details of the superoxide dismutase, lysine methyltransferase, thioredoxin, and amino acid ligase enzymes that are subunits of PEP. Collectively, these data provide a foundation for the mechanistic understanding of chloroplast transcription and its role in plant growth and adaptation.

An RNA polymerase that became a Swiss army knife.

RNA polymerases (RNAPs) control the first step of gene expression in all forms of life by transferring genetic information from DNA to RNA, a process known as transcription. In this issue of Cell, Webster et al. and Wu et al. report three-dimensional structures of RNAP complexes from chloroplasts.

Cytonuclear Interactions and Subgenome Dominance Shape the Evolution of Organelle-Targeted Genes in the Brassica Triangle of U.

The interaction and coevolution between nuclear and cytoplasmic genomes are one of the fundamental hallmarks of eukaryotic genome evolution and, 2 billion yr later, are still major contributors to the formation of new species. Although many studies have investigated the role of cytonuclear interactions following allopolyploidization, the relative magnitude of the effect of subgenome dominance versus cytonuclear interaction on genome evolution remains unclear. The Brassica triangle of U features 3 diploid species that together have formed 3 separate allotetraploid species on similar evolutionary timescales, providing an ideal system for understanding the contribution of the cytoplasmic donor to hybrid polyploid. Here, we investigated the evolutionary pattern of organelle-targeted genes in Brassica carinata (BBCC) and 2 varieties of Brassica juncea (AABB) at the whole-genome level, with particular focus on cytonuclear enzyme complexes. We found partial evidence that plastid-targeted genes experience selection to match plastid genomes, but no obvious corresponding signal in mitochondria-targeted genes from these 2 separately formed allopolyploids. Interestingly, selection acting on plastid genomes always reduced the retention rate of plastid-targeted genes encoded by the B subgenome, regardless of whether the Brassica nigra (BB) subgenome was contributed by the paternal or maternal progenitor. More broadly, this study illustrates the distinct selective pressures experienced by plastid- and mitochondria-targeted genes, despite a shared pattern of inheritance and natural history. Our study also highlights an important role for subgenome dominance in allopolyploid genome evolution, even in genes whose function depends on separately inherited molecules.

Organelle Genomes of Epipogium roseum Provide Insight into the Evolution of Mycoheterotrophic Orchids.

Epipogium roseum, commonly known as one of the ghost orchids due to its rarity and almost transparent color, is a non-photosynthetic and fully mycoheterotrophic plant. Given its special nutritional strategies and evolutionary significance, the mitogenome was first characterized, and three plastomes sampled from Asia were assembled. The plastomes were found to be the smallest among Orchidaceae, with lengths ranging from 18,339 to 19,047 bp, and exhibited high sequence variety. For the mitogenome, a total of 414,552 bp in length, comprising 26 circular chromosomes, were identified. A total of 54 genes, including 38 protein-coding genes, 13 tRNA genes, and 3 rRNA genes, were annotated. Multiple repeat sequences spanning a length of 203,423 bp (45.47%) were discovered. Intriguingly, six plastid regions via intracellular gene transfer and four plastid regions via horizontal gene transfer to the mitogenome were observed. The phylogenomics, incorporating 90 plastomes and 56 mitogenomes, consistently revealed the sister relationship of Epipogium and Gastrodia, with a bootstrap percentage of 100%. These findings shed light on the organelle evolution of Orchidaceae and non-photosynthetic plants.

Modified Crosstalk between Phytohormones in Arabidopsis Mutants for PEP-Associated Proteins.

Plastid-encoded RNA polymerase (PEP) forms a multisubunit complex in operating chloroplasts, where PEP subunits and a sigma factor are tightly associated with 12 additional nuclear-encoded proteins. Mutants with disrupted genes encoding PEP-associated proteins (PAPs) provide unique tools for deciphering mutual relationships among phytohormones. A block of chloroplast biogenesis in Arabidopsis pap mutants specifying highly altered metabolism in white tissues induced dramatic fluctuations in the content of major phytohormones and their metabolic genes, whereas hormone signaling circuits mostly remained functional. Reprogramming of the expression of biosynthetic and metabolic genes contributed to a greatly increased content of salicylic acid (SA) and a concomitant decrease in 1-aminocyclopropane-1-carboxylic acid (ACC) and oxophytodienoic acid (OPDA), precursors of ethylene and jasmonic acid, respectively, in parallel to reduced levels of abscisic acid (ABA). The lack of differences in the free levels of indole-3-acetic acid (IAA) between the pap mutants and wild-type plants was accompanied by fluctuations in the contents of IAA precursors and conjugated forms as well as multilayered changes in the expression of IAA metabolic genes. Along with cytokinin (CK) overproduction, all of these compensatory changes aim to balance plant growth and defense systems to ensure viability under highly modulated conditions.