The complete plastid genome sequence of the enigmatic moss, Takakia lepidozioides (Takakiopsida, Bryophyta): evolutionary perspectives on the largest collection of genes in mosses and the intensive RNA editing.

KEY MESSAGE: Complete chloroplast genome sequence of a moss, Takakia lepidozioides (Takakiopsida) is reported. The largest collection of genes in mosses and the intensive RNA editing were discussed from evolutionary perspectives. We assembled the entire plastid genome sequence of Takakia lepidozioides (Takakiopsida), emerging from the first phylogenetic split among extant mosses. The genome sequences were assembled into a circular molecule 149,016 bp in length, with a quadripartite structure comprising a large and a small single-copy region separated by inverted repeats. It contained 88 genes coding for proteins, 32 for tRNA, four for rRNA, two open reading frames, and at least one pseudogene (tufA). This is the largest number of genes of all sequenced plastid genomes in mosses and Takakia is the only moss that retains the seven coding genes ccsA, cysA, cysT, petN rpoA, rps16 and trnPGGG. Parsimonious interpretation of gene loss suggests that the last common ancestor of bryophytes had all seven genes and that mosses lost at least three of them during their diversification. Analyses of the plastid transcriptome identified the extraordinary frequency of RNA editing with more than 1100 sites. We indicated a close correlation between the monoplastidy of vegetative tissue and the intensive RNA editing sites in the plastid genome in land plant lineages. Here, we proposed a hypothesis that the small population size of plastids in each vegetative cell of some early diverging land plants, including Takakia, might cause the frequent fixation of mutations in plastid genome through the intracellular genetic drift and that deleterious mutations might be continuously compensated by RNA editing during or following transcription.

HBD1 protein with a tandem repeat of two HMG-box domains is a DNA clip to organize chloroplast nucleoids in Chlamydomonas reinhardtii.

Compaction of bulky DNA is a universal issue for all DNA-based life forms. Chloroplast nucleoids (chloroplast DNA-protein complexes) are critical for chloroplast DNA maintenance and transcription, thereby supporting photosynthesis, but their detailed structure remains enigmatic. Our proteomic analysis of chloroplast nucleoids of the green alga Chlamydomonas reinhardtii identified a protein (HBD1) with a tandem repeat of two DNA-binding high mobility group box (HMG-box) domains, which is structurally similar to major mitochondrial nucleoid proteins transcription factor A, mitochondrial (TFAM), and ARS binding factor 2 protein (Abf2p). Disruption of the HBD1 gene by CRISPR-Cas9-mediated genome editing resulted in the scattering of chloroplast nucleoids. This phenotype was complemented when intact HBD1 was reintroduced, whereas a truncated HBD1 with a single HMG-box domain failed to complement the phenotype. Furthermore, ectopic expression of HBD1 in the mitochondria of yeast Δabf2 mutant successfully complemented the defects, suggesting functional similarity between HBD1 and Abf2p. Furthermore, in vitro assays of HBD1, including the electrophoretic mobility shift assay and DNA origami/atomic force microscopy, showed that HBD1 is capable of introducing U-turns and cross-strand bridges, indicating that proteins with two HMG-box domains would function as DNA clips to compact DNA in both chloroplast and mitochondrial nucleoids.

An Overview on Predicting Protein Subchloroplast Localization by using Machine Learning Methods.

The chloroplast is a type of subcellular organelle of green plants and eukaryotic algae, which plays an important role in the photosynthesis process. Since the function of a protein correlates with its location, knowing its subchloroplast localization is helpful for elucidating its functions. However, due to a large number of chloroplast proteins, it is costly and time-consuming to design biological experiments to recognize subchloroplast localizations of these proteins. To address this problem, during the past ten years, twelve computational prediction methods have been developed to predict protein subchloroplast localization. This review summarizes the research progress in this area. We hope the review could provide important guide for further computational study on protein subchloroplast localization.

Genes functioned in kleptoplastids of Dinophysis are derived from haptophytes rather than from cryptophytes.

Toxic dinoflagellates belonging to the genus Dinophysis acquire plastids indirectly from cryptophytes through the consumption of the ciliate Mesodinium rubrum. Dinophysis acuminata harbours three genes encoding plastid-related proteins, which are thought to have originated from fucoxanthin dinoflagellates, haptophytes and cryptophytes via lateral gene transfer (LGT). Here, we investigate the origin of these plastid proteins via RNA sequencing of species related to D. fortii. We identified 58 gene products involved in porphyrin, chlorophyll, isoprenoid and carotenoid biosyntheses as well as in photosynthesis. Phylogenetic analysis revealed that the genes associated with chlorophyll and carotenoid biosyntheses and photosynthesis originated from fucoxanthin dinoflagellates, haptophytes, chlorarachniophytes, cyanobacteria and cryptophytes. Furthermore, nine genes were laterally transferred from fucoxanthin dinoflagellates, whose plastids were derived from haptophytes. Notably, transcription levels of different plastid protein isoforms varied significantly. Based on these findings, we put forth a novel hypothesis regarding the evolution of Dinophysis plastids that ancestral Dinophysis species acquired plastids from haptophytes or fucoxanthin dinoflagellates, whereas LGT from cryptophytes occurred more recently. Therefore, the evolutionary convergence of genes following LGT may be unlikely in most cases.

Zymographic Method for Distinguishing Different Classes of Superoxide Dismutases in Plants.

In plants, especially in chloroplasts, superoxide radical is generated when an electron is transferred to dimolecular O2 due to decreased activity of Photosystem I. The superoxide (O2-) radical accumulation is more rampant in plants exposed to abiotic stresses due to oxidation of photosystem components. Excessive superoxide radical accumulation will lead to oxidative damage to the cellular macromolecules. The ubiquitous superoxide dismutases (SODs) represent critical enzymatic antioxidant system present in cells, which can catalyze the disproportion of superoxide (O2-) radical rapidly into hydrogen peroxide (H2O2) and molecular oxygen. Depending on the metal cofactor present, the plant SODs are classified into Cu/ZnSOD, MnSOD, and FeSOD. The activity of SODs can be quantified zymographically. Additionally, using this method, different classes of SODs can be distinguished by using H2O2, KCN, and NaN3.

Proteomics Provides Insight into the Interaction between Mulberry and Silkworm.

Mulberry leaves have been selected as a food source for the silkworm (Bombyx mori) for over 5000 years. However, the interaction mechanisms of mulberry-silkworm remain largely unknown. We explore the interaction between mulberry and silkworm at the protein level. Total proteins were extracted from mulberry leaves and silkworm feces on day 5 of the fifth larval instar and analyzed on shotgun liquid chromatography-tandem mass spectrometry, respectively. In total, 2076 and 210 foliar proteins were identified from mulberry leaves and silkworm feces, respectively. These proteins were classified into four categories according to their subcellular location: chloroplast proteins, mitochondrial proteins, secretory-pathway proteins, and proteins of other locations. Chloroplast proteins accounted for 68.3% in mulberry leaves but only 23.2% in the feces. In contrast, secretory-pathway proteins had low abundance in mulberry leaves (7.3%) but were greatly enriched to the largest component in the feces (60.1%). Most of the foliar secretory-pathway proteins in the feces were found to be resistant to silkworm feeding by becoming involved in primary metabolite, proteinase inhibition, cell-wall remodeling, redox regulation, and pathogen-resistant processes. On the contrary, only six defensive proteins were identified in the fecal chloroplast proteins including two key proteins responsible for synthesizing jasmonic acid, although chloroplast proteins were the second largest component in the feces. Collectively, the comparative proteomics analyses indicate that mulberry leaves not only provide amino acids to the silkworm but also display defense against silkworm feeding, although the silkworm grows very well by feeding on mulberry leaves, which provides new insights into the interactions between host-plant and insect herbivores.

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.

Genome-wide identification, evolution and expression analysis of mTERF gene family in maize.

Plant mitochondrial transcription termination factor (mTERF) genes comprise a large family with important roles in regulating organelle gene expression. In this study, a comprehensive database search yielded 31 potential mTERF genes in maize (Zea mays L.) and most of them were targeted to mitochondria or chloroplasts. Maize mTERF were divided into nine main groups based on phylogenetic analysis, and group IX represented the mitochondria and species-specific clade that diverged from other groups. Tandem and segmental duplication both contributed to the expansion of the mTERF gene family in the maize genome. Comprehensive expression analysis of these genes, using microarray data and RNA-seq data, revealed that these genes exhibit a variety of expression patterns. Environmental stimulus experiments revealed differential up or down-regulation expression of maize mTERF genes in seedlings exposed to light/dark, salts and plant hormones, respectively, suggesting various important roles of maize mTERF genes in light acclimation and stress-related responses. These results will be useful for elucidating the roles of mTERF genes in the growth, development and stress response of maize.

Evolution of chloroplast J proteins.

Hsp70 chaperones are involved in multiple biological processes and are recruited to specific processes by designated J domain-containing cochaperones, or J proteins. To understand the evolution and functions of chloroplast Hsp70s and J proteins, we identified the Arabidopsis chloroplast J protein constituency using a combination of genomic and proteomic database searches and individual protein import assays. We show that Arabidopsis chloroplasts have at least 19 J proteins, the highest number of confirmed J proteins for any organelle. These 19 J proteins are classified into 11 clades, for which cyanobacteria and glaucophytes only have homologs for one clade, green algae have an additional three clades, and all the other 7 clades are specific to land plants. Each clade also possesses a clade-specific novel motif that is likely used to interact with different client proteins. Gene expression analyses indicate that most land plant-specific J proteins show highly variable expression in different tissues and are down regulated by low temperatures. These results show that duplication of chloroplast Hsp70 in land plants is accompanied by more than doubling of the number of its J protein cochaperones through adding new J proteins with novel motifs, not through duplications within existing families. These new J proteins likely recruit chloroplast Hsp70 to perform tissue specific functions related to biosynthesis rather than to stress resistance.

Complete sequence and analysis of plastid genomes of two economically important red algae: Pyropia haitanensis and Pyropia yezoensis.

BACKGROUND: Pyropia haitanensis and P. yezoensis are two economically important marine crops that are also considered to be research models to study the physiological ecology of intertidal seaweed communities, evolutionary biology of plastids, and the origins of sexual reproduction. This plastid genome information will facilitate study of breeding, population genetics and phylogenetics. PRINCIPAL FINDINGS: We have fully sequenced using next-generation sequencing the circular plastid genomes of P. hatanensis (195,597 bp) and P. yezoensis (191,975 bp), the largest of all the plastid genomes of the red lineage sequenced to date. Organization and gene contents of the two plastids were similar, with 211-213 protein-coding genes (including 29-31 unknown-function ORFs), 37 tRNA genes, and 6 ribosomal RNA genes, suggesting a largest coding capacity in the red lineage. In each genome, 14 protein genes overlapped and no interrupted genes were found, indicating a high degree of genomic condensation. Pyropia maintain an ancient gene content and conserved gene clusters in their plastid genomes, containing nearly complete repertoires of the plastid genes known in photosynthetic eukaryotes. Similarity analysis based on the whole plastid genome sequences showed the distance between P. haitanensis and P. yezoensis (0.146) was much smaller than that of Porphyra purpurea and P. haitanensis (0.250), and P. yezoensis (0.251); this supports re-grouping the two species in a resurrected genus Pyropia while maintaining P. purpurea in genus Porphyra. Phylogenetic analysis supports a sister relationship between Bangiophyceae and Florideophyceae, though precise phylogenetic relationships between multicellular red alage and chromists were not fully resolved. CONCLUSIONS: These results indicate that Pyropia have compact plastid genomes. Large coding capacity and long intergenic regions contribute to the size of the largest plastid genomes reported for the red lineage. Possessing the largest coding capacity and ancient gene content yet found reveal that Pyropia are more primitive multicellular red algae.

Plastid genome evolution across the genus Cuscuta (Convolvulaceae): two clades within subgenus Grammica exhibit extensive gene loss.

The genus Cuscuta (Convolvulaceae, the morning glory family) is one of the most intensely studied lineages of parasitic plants. Whole plastome sequencing of four Cuscuta species has demonstrated changes to both plastid gene content and structure. The presence of photosynthetic genes under purifying selection indicates that Cuscuta is cryptically photosynthetic. However, the tempo and mode of plastid genome evolution across the diversity of this group (~200 species) remain largely unknown. A comparative investigation of plastid genome content, grounded within a phylogenetic framework, was conducted using a slot-blot Southern hybridization approach. Cuscuta was extensively sampled (~56% of species), including groups previously suggested to possess more altered plastomes compared with other members of this genus. A total of 56 probes derived from all categories of protein-coding genes, typically found within the plastomes of flowering plants, were used. The results indicate that two clades within subgenus Grammica (clades 'O' and 'K') exhibit substantially more plastid gene loss relative to other members of Cuscuta. All surveyed members of the 'O' clade show extensive losses of plastid genes from every category of genes typically found in the plastome, including otherwise highly conserved small and large ribosomal subunits. The extent of plastid gene losses within this clade is similar in magnitude to that observed previously in some non-asterid holoparasites, in which the very presence of a plastome has been questioned. The 'K' clade also exhibits considerable loss of plastid genes. Unlike in the 'O' clade, in which all species seem to be affected, the losses in clade 'K' progress phylogenetically, following a pattern consistent with the Evolutionary Transition Series hypothesis. This clade presents an ideal opportunity to study the reduction of the plastome of parasites 'in action'. The widespread plastid gene loss in these two clades is hypothesized to be a consequence of the complete loss of photosynthesis. Additionally, taxa that would be the best candidates for entire plastome sequencing are identified in order to investigate further the loss of photosynthesis and reduction of the plastome within Cuscuta.

Phylogenetic viewpoints on regulation of light harvesting and electron transport in eukaryotic photosynthetic organisms.

The comparative study of photosynthetic regulation in the thylakoid membrane of different phylogenetic groups can yield valuable insights into mechanisms, genetic requirements and redundancy of regulatory processes. This review offers a brief summary on the current understanding of light harvesting and photosynthetic electron transport regulation in different photosynthetic eukaryotes, with a special focus on the comparison between higher plants and unicellular algae of secondary endosymbiotic origin. The foundations of thylakoid structure, light harvesting, reversible protein phosphorylation and PSI-mediated cyclic electron transport are traced not only from green algae to vascular plants but also at the branching point between the "green" and the "red" lineage of photosynthetic organisms. This approach was particularly valuable in revealing processes that (1) are highly conserved between phylogenetic groups, (2) serve a common physiological role but nevertheless originate in divergent genetic backgrounds or (3) are missing in one phylogenetic branch despite their unequivocal importance in another, necessitating a search for alternative regulatory mechanisms and interactions.