Comparative analysis of codon usage patterns in 16 chloroplast genomes of suborder Halimedineae.

The Halimedineae are marine green macroalgae that play crucial roles as primary producers in various habitats, including coral reefs, rocky shores, embayments, lagoons, and seagrass beds. Several tropical species have calcified thalli, which contribute significantly to the formation of coral reefs. In this study, we investigated the codon usage patterns and the main factors influencing codon usage bias in 16 chloroplast genomes of the suborder Halimedineae. Nucleotide composition analysis revealed that the codons of these species were enriched in A/U bases and preferred to end in A/U bases, and the distribution of GC content followed a trend of GC1 > GC2 > GC3. 30 optimal codons encoding 17 amino acids were identified, and most of the optimal codons and all of the over-expressed codons preferentially ended with A/U. The neutrality plot, effective number of codons (ENc) plot, and parity rule 2 (PR2) plot analysis indicated that natural selection played a major role in shaping codon usage bias of the most Halimedineae species. The genetic relationships based on their RSCU values and chloroplast protein-coding genes showed the closely related species have similar codon usage patterns. This study describes, for the first time, the codon usage patterns and characterization of Halimedineae chloroplast genomes, and provides new insights into the evolution of this suborder.

The complete chloroplast genome and phylogenetic analysis of Garcinia esculenta Y. H. Li. 1981 (Clusiaceae).

Garcinia esculenta Y. H. Li. 1981 (Clusiaceae) is an endemic tree species in China, primarily found in western and northwestern Yunnan Province. In this research, the complete chloroplast genome of G. esculenta was sequenced using the Illumina NovaSeq6000 platform. The result showed that the length of the complete chloroplast genome was 155,853 bp, which was composed of a large single-copy region (LSC) of 84,534 bp, a small single-copy region (SSC) of 17,175 bp, and a pair of inverted repeat (IR) regions of 27,072 bp. The overall GC content was 36.1%. The complete chloroplast genome encompassed 128 genes, comprising 83 protein-coding genes, 37 tRNA genes, and 8 rRNA genes. Phylogenetic analysis of the complete chloroplast genome sequences of 22 species revealed that G. esculenta is most closely related to G. oblongifolia.

The complete sequence of chloroplast genome of Baeckea frutescens Linaeus 1753 (Myrtoideae), a traditional folk medicinal plant.

Baeckea frutescens Linaeus 1753, as a traditional folk medicine in South East Asia, possesses sesquiterpenes, phloroglucinols, chromones, and essential oil, and is utilized for traditional Chinese medicinal purposes. The genetic diversity of the plant must be better understood, considering its significance. The complete chloroplast (cp) genome of B. frutescens was sequenced and assembled by using Illumina paired-end data, marking a significant advancement toward comprehending its genetic composition. The complete cp genome is 158,939 bp in length and contains 128 genes, consisting of 83 protein-coding genes, 8 ribosomal RNA genes, and 37 transfer RNA genes. Phylogenetic analyses indicated that B. frutescens and other the 13 were clustered to the family of Myrtaceae. These findings are crucial for the conservation and utilization of this important plant species. Additionally, they underscore the potential for future research on the evolution and preservation of B. frutescens, which could be advantageous in pharmaceutical applications.

The complete chloroplast genome of Calyptothecium philippinense Broth. (Pterobryaceae, Hypnales).

The genus Calyptothecium, currently comprising ca. 30 species worldwide, is the largest genus within the family Pterobryaceae. However, a comprehensive taxonomic revision of this genus is lacking. Calyptothecium philippinense Broth. 1899, a moss species widely found in the tropical regions of Asia, is characterized by the unique rugose leaves and large auriculate leaf bases. In this study, we sequenced the complete chloroplast genome (CPG) of C. philippinense using the Illumina NovaSeq 6000 platform. The length of the CPG of C. philippinense was determined to be 124,513 bp, with an AT content of 74%. The CPG of C. philippinense exhibited a standard quadripartite structure, consisting of one small single-copy (SSC) region (18,541 bp), one large single-copy region (LSC) (87,222 bp), and two inverted repeat (IR) regions (9375 bp each). A total of 126 genes from the CPG of C. philippinense were annotated, including 82 protein-coding genes, eight ribosomal RNA genes, and 36 transfer RNA genes. Phylogenetic analysis based on the CPGs of 25 bryophyte taxa revealed that the three Pterobryaceae species C. philippinense, Calyptothecium hookeri (Mitt.) Broth. and Pterobryopsis orientalis (Müll. Hal.) M. Fleisch. formed a robust clade. The findings could facilitate more accurate classification and help elucidate evolutionary relationships within Calyptothecium.

The complete chloroplast genome of Camellia huulungensis Rosmann et Ninh, a golden Camellia species endemic to Vietnam.

Camellia huulungensis Rosmann & Ninh 1997, belonging to the sect. Chrysantha, holds important ornamental value and medicinal value. In this study, the complete chloroplast genome sequence of C. huulungensis was assembled using high-throughput sequencing technology. The entire length of chloroplast genome is 156,546 bp and contains a small single-copy region (18,257 bp), a large single-copy region (86,219 bp), and a pair of inverted repeat regions (26,035 bp). A total of 133 genes were annotated, including 88 protein-coding genes, 37 tRNA genes, and 8 rRNA genes. The overall GC content is 37.33%. The phylogenetic analysis showed that C. huulungensis is sister to C. aurea. The results can provide genetic data for further phylogenetic studies of Camellia.

The complete chloroplast genome of Fuchsia standishii J. Harrison, 1840 (Onagraceae) from Yunnan, China.

Fuchsia standishii J. Harrison, 1840, a perennial shrub, is renowned for its vividly colored and uniquely shaped blooms, which have an extended flowering season. Commonly cultivated as an ornamental potted plant, it is utilized in traditional Chinese medicine. In this study, we successfully sequenced and assembled the complete chloroplast genome of F. standishii using high-throughput Illumina sequencing technology. The assembled chloroplast genome displays a typical quadripartite structure, with a total length of 156,391 bp. It consists of a pair of inverted repeat regions (IRs), each measuring 25,069 bp, separated by a large single-copy region (LSC) of 87,754 bp and a small single-copy region (SSC) of 18,499 bp. The overall GC content of the genome is 37.60%. The genome includes a total of 129 genes, comprising 84 protein-coding genes, 37 tRNA genes, and eight rRNA genes. Phylogenetic analysis of 17 complete chloroplast genomes revealed that F. standishii forms a monophyletic group with the entire Circaea. This study provides a molecular foundation for future phylogenetic research on Fuchsia.

Plastid LPAT1 is an integral inner envelope membrane protein with the acyltransferase domain located in the stroma.

KEY MESSAGE: The N-terminal transmembrane domain of LPAT1 crosses the inner membrane placing the N terminus in the intermembrane space and the C-terminal enzymatic domain in the stroma. Galactolipids mono- and di-galactosyl diacylglycerol are the major and vital lipids of photosynthetic membranes. They are synthesized by five enzymes hosted at different sub-chloroplast locations. However, localization and topology of the second-acting enzyme, lysophosphatidic acid acyltransferase 1 (LPAT1), which acylates the sn-2 position of glycerol-3-phosphate (G3P) to produce phosphatidic acid (PA), remain unclear. It is not known whether LPAT1 is located at the outer or the inner envelope membrane and whether its enzymatic domain faces the cytosol, the intermembrane space, or the stroma. Even the size of mature LPAT1 in chloroplasts is not known. More information is essential for understanding the pathways of metabolite flow and for future engineering endeavors to modify glycerolipid biosynthesis. We used LPAT1 preproteins translated in vitro for import assays to determine the precise size of the mature protein and found that the LPAT1 transit peptide is at least 85 residues in length, substantially longer than previously predicted. A construct comprising LPAT1 fused to the Venus fluorescent protein and driven by the LPAT1 promoter was used to complement an Arabidopsis lpat1 knockout mutant. To determine the sub-chloroplast location and topology of LPAT1, we performed protease treatment and alkaline extraction using chloroplasts containing in vitro-imported LPAT1 and chloroplasts isolated from LPAT1-Venus-complemented transgenic plants. We show that LPAT1 traverses the inner membrane via an N-terminal transmembrane domain, with its N terminus protruding into the intermembrane space and the C-terminal enzymatic domain residing in the stroma, hence displaying a different membrane topology from its bacterial homolog, PlsC.

Characterization of the complete chloroplast genome sequence of Schisandra propinqua subsp. sinensis (Oliv.) R.M.K.Saunders 1997 (Schisandraceae).

Schisandra propinqua subsp. sinensis (Oliv.) R.M.K. Saunders 1997, a woody vine plant esteemed for its medicinal properties, has garnered attention in botanical research. In this study, we elucidated the complete chloroplast genome sequence of S. propinqua. The genome spans 145,562 bp and comprises a large single-copy (LSC) region of 94,164 bp, a small single-copy region of 18,294 bp (SSC), and a pair of inverted repeats (IR) of 16,552 bp. Notably, S. propinqua exhibits an overall GC content of 36.2%. Annotation revealed a total of 116 genes, encompassing 81 protein-coding genes, 27 transfer RNA (tRNA) genes, and 8 ribosomal RNA (rRNA) genes. Phylogenetic analysis unveiled a close relationship among Schisandraceae, indicating the evolutionary proximity. This comprehensive genomic dataset provides valuable insights into the genetic makeup and evolutionary dynamics within the Schisandra genus.

Genomic and biochemical analysis of lipid biosynthesis in Cyanophora paradoxa: Limited role of chloroplast in fatty acid synthesis.

Archaeplastida, a group of photosynthetic organisms with primary plastids, consists of green algae (plus plants), red algae, and glaucophytes. In contrast to green and red algae, information on lipids and lipid biosynthesis still needs to be included in the glaucophytes. The chloroplast is the site of photosynthesis and fatty acid synthesis in all photosynthetic organisms known to date. However, the genomic data of the glaucophyte Cyanophora paradoxa suggested the lack of acetyl CoA carboxylase and most components of fatty acid synthase in the chloroplast. Instead, multifunctional fatty acid synthase and acetyl CoA carboxylase are likely to reside in the cytosol. To examine this hypothesis, we measured fatty acid synthesis in isolated chloroplasts and whole cells using stable isotope labeling. The chloroplasts had very low activity of fatty acid synthesis, if any. Most processes of fatty acid synthesis, including elongation and desaturation, must be performed within the cytosol, and the fatty acids imported into the chloroplasts are assembled into the chloroplast lipids by the enzymes common to other algae and plants. Cyanophora paradoxa is a rare organism in which fatty acid synthesis and photosynthesis are not tightly linked. This could question the common origin of these two biosynthetic processes in Archaeplastida.

Complete chloroplast genome sequence of Artemisia littoricola (Asteraceae) from Dokdo Island Korea: genome structure, phylogenetic analysis, and biogeography study.

The Asteraceae family, particularly the Artemisia genus, presents taxonomic challenges due to limited morphological characteristics and frequent natural hybridization. Molecular tools, such as chloroplast genome analysis, offer solutions for accurate species identification. In this study, we sequenced and annotated the chloroplast genome of Artemisia littoricola sourced from Dokdo Island, employing comparative analyses across six diverse Artemisia species. Our findings reveal conserved genome structures with variations in repeat sequences and junction boundaries. Notably, the chloroplast genome of A. littoricola spans 150,985 bp, consistent with other Artemisia species, and comprises 131 genes, including 86 protein-coding, 37 tRNA, and 8 rRNA genes. Among these genes, 16 possess a single intron, while clpP and ycf3 exhibit two introns each. Furthermore, 18 genes display duplicated copies within the IR regions. Moreover, the genome possesses 42 Simple Sequence Repeats (SSRs), predominantly abundant in A/T content and located within intergenic spacer regions. The analysis of codon usage revealed that the codons for leucine were the most frequent, with a preference for ending with A/U. While the chloroplast genome exhibited conservation overall, non-coding regions showed lower conservation compared to coding regions, with the Inverted Repeat (IR) region displaying higher conservation than single-copy regions. Phylogenetic analyses position A. littoricola within subgenus Dracunculus, indicating a close relationship with A. scoparia and A. desertorum. Additionally, biogeographic reconstructions suggest ancestral origins in East Asia, emphasizing Mongolia, China (North East and North Central and South Central China), and Korea. This study underscores the importance of chloroplast genomics in understanding Artemisia diversity and evolution, offering valuable insights into taxonomy, evolutionary patterns, and biogeographic history. These findings not only enhance our understanding of Artemisia's intricate biology but also contribute to conservation efforts and facilitate the development of molecular markers for further research and applications in medicine and agriculture.

An effector protein of Fusarium graminearum targets chloroplasts and suppresses cyclic photosynthetic electron flow.

Chloroplasts are important photosynthetic organelles that regulate plant immunity, growth, and development. However, the role of fungal secretory proteins in linking the photosystem to the plant immune system remains largely unknown. Our systematic characterization of 17 chloroplast-targeting secreted proteins of Fusarium graminearum indicated that Fg03600 is an important virulence factor. Fg03600 translocation into plant cells and accumulation in chloroplasts depended on its chloroplast transit peptide. Fg03600 interacted with the wheat (Triticum aestivum L.) proton gradient regulation 5-like protein 1 (TaPGRL1), a part of the cyclic photosynthetic electron transport chain, and promoted TaPGRL1 homo-dimerization. Interestingly, TaPGRL1 also interacted with ferredoxin (TaFd), a chloroplast ferredoxin protein that transfers cyclic electrons to TaPGRL1. TaFd competed with Fg03600 for binding to the same region of TaPGRL1. Fg03600 expression in plants decreased cyclic electron flow (CEF) but increased the production of chloroplast-derived reactive oxygen species (ROS). Stably silenced TaPGRL1 impaired resistance to Fusarium head blight (FHB) and disrupted CEF. Overall, Fg03600 acts as a chloroplast-targeting effector to suppress plant CEF and increase photosynthesis-derived ROS for FHB development at the necrotrophic stage by promoting homo-dimeric TaPGRL1 or competing with TaFd for TaPGRL1 binding.

Diatom pyrenoids are encased in a protein shell that enables efficient CO2 fixation.

Pyrenoids are subcompartments of algal chloroplasts that increase the efficiency of Rubisco-driven CO2 fixation. Diatoms fix up to 20% of global CO2, but their pyrenoids remain poorly characterized. Here, we used in vivo photo-crosslinking to identify pyrenoid shell (PyShell) proteins, which we localized to the pyrenoid periphery of model pennate and centric diatoms, Phaeodactylum tricornutum and Thalassiosira pseudonana. In situ cryo-electron tomography revealed that pyrenoids of both diatom species are encased in a lattice-like protein sheath. Single-particle cryo-EM yielded a 2.4-Å-resolution structure of an in vitro TpPyShell1 lattice, which showed how protein subunits interlock. T. pseudonana TpPyShell1/2 knockout mutants had no PyShell sheath, altered pyrenoid morphology, and a high-CO2 requiring phenotype, with reduced photosynthetic efficiency and impaired growth under standard atmospheric conditions. The structure and function of the diatom PyShell provide a molecular view of how CO2 is assimilated in the ocean, a critical ecosystem undergoing rapid change.

Comparative study on chloroplast genome of Tamarix species.

Tamaricaceae comprises about 120 species and has a long evolutionary history, Tamarix Linn accounts for approximately 75% of the total species in this family. It is the most widely distributed and diverse genus in the family. They have important ecological significance for transforming deserts and improving climate conditions. However, Tamarix is the most poorly classified genera among flowering plants owing to its large variability and high susceptibility to interspecific hybridization. In this study, the complete chloroplast genomes of three Tamarix species and one draft chloroplast genome were obtained in this study. Combined with eight chloroplast genomes deposited in GenBank, complete chloroplast sequences of 12 Tamarix species were used for further analysis. There are 176 non-SSR-related indels and 681 non-indel-related SSRs in the 12 Tamarix chloroplast genomes. The mononucleotide SSRs are the most prevalent among all types of SSRs. The mVISTA results indicate high sequence similarities across the chloroplast genome, suggesting that the chloroplast genomes are highly conserved, except for sample Tamarix androssowii (ENC850343). The IR regions and the coding regions are more conserved than the single-copy and noncoding regions. The trnF-ndhJ, ndhC-trnM-CAU, ycf1, and trnL-UAG-ndhF regions are the most variable and have higher variability than those of the universal DNA markers. Finally, the first phylogenetic tree of Tamaricaceae was constructed which confirmed the monophyly of Tamarix in Tamaricaceae. The first phylogenetic tree of Tamarix was based on the complete chloroplast genome to date, the changes in branch length and support rate can potentially help us clarify the phylogenetic relationships of Tamarix. All the obtained genetic resources will facilitate future studies in population genetics, species identification, and conservation biology of Tamarix.

Analysis of codon usage patterns in complete plastomes of four medicinal Polygonatum species (Asparagaceae).

Polygonati Rhizoma and Polygonati odorati Rhizoma, known as "Huangjing" and "Yuzhu" in China, are medicinal Polygonatum species resources with top-grade medical and edible properties. The chloroplast (cp) genome has been used to study species diversity, evolution, and breeding of species for applications in genetic engineering. Codon usage bias (CUB), a common and complex natural phenomenon, is essential for studies of codon optimization of exogenous genes, genetic engineering, and molecular evolution. However, the CUB of medicinal Polygonatum species chloroplast genomes has not been systematically studied. In our study, a detailed analysis of CUB was performed in the medicinal Polygonatum species chloroplast genomes. We investigated the codon bias of 204 plastid protein-coding genes (PCGs) in 4 medicinal Polygonatum species using CodonW and CUSP online software. Through the analysis of the codon bias index, we found that the medicinal Polygonatum species chloroplast genomes had weak codon usage bias. In addition, our results also showed a high preference for AT bases in medicinal Polygonatum species chloroplast genomes, and the preference to use AT-ending codons was observed in these species chloroplast genomes. The neutrality plot, ENC plot, PR2-Bias plot, and correspondence analysis showed that compared with mutation pressure, natural selection was the most important factor of CUB. Based on the comparative analysis of high-frequency codons and high expression codons, we also determined the 10-11 optimal codons of investigative medicinal Polygonatum species. Furthermore, the result of RSCU-based cluster analysis showed that the genetic relationship between different medicinal Polygonatum species could be well reflected. This study provided an essential understanding of CUB and evolution in the medicinal Polygonatum species chloroplast genomes.

Comparison of organelle genomes between endangered mangrove plant Dolichandrone spathacea to terrestrial relative provides insights into its origin and adaptative evolution.

Dolichandrone spathacea is a mangrove associate with high medicinal and ecological values. However, due to the dual-pressure of climate change and human activities, D. spathacea has become endangered in China. Moreover, misidentification between D. spathacea and its terrestrial relative D. cauda-felina poses further challenges to field protection and proper medicinal usage of D. spathacea. Thus, to address these problems, we sequenced and assembled mitochondrial (mt) and chloroplast (cp) genomes for both D. spathacea and D. cauda-felina. Comparative analysis revealed apparently different size and scaffold number between the two mt genomes, but a high similarity between the cp genomes. Eight regions with high sequence divergence were identified between the two cp genomes, which might be used for developing candidate DNA markers for distinguishing the two species. The splitting between D. spathacea and D. cauda-felina was inferred to occur at ~6.8 - 7.7 million years ago (Mya), which may be driven by the environment fluctuations in late Miocene. In the cp genome, 12 genes related to the expression of photosynthesis-associated proteins were detected with signatures of positive selection, which may contribute to the origin and evolutionary adaptation of Dolichandrone mangrove species. These new findings do not only enrich organelle genomic resources of Dolichandrone species, but also provide important genetic clues for improving the conservation and proper usage of endangered mangrove associate D. spathacea.

The complete chloroplast genome of Tabebuia rosea (Bignoniaceae).

Tabebuia rosea is a world-renowned woody plant with colorful flowers in full bloom. In addition to its high ornamental value, it also has ecological and medicinal value. In this study, the complete circular chloroplast genome of T. rosea was reconstructed and annotated using Illumina sequencing. The chloroplast genome was 158,919 bp in size with GC content of 38.21%, including a large single-copy region of 85,823 bp, a small single-copy region of 12,816 bp, and a pair of inverted repeats of 30,140 bp. It encoded 132 genes, including 87 protein-coding genes, 37 tRNA genes, and 8 rRNA genes. Based on current available chloroplast genome sequences, the phylogenetic analysis indicated that T. rosea was clustered with T. nodosa and H. chrysanthus. This study provided insights into the evolutionary relationships among different species of Bignoniaceae.

Whole chloroplast genome sequence and phylogenetic analysis of Calanthe discolor (Orchidaceae).

The orchid Calanthe discolor, which has high ornamental and medicinal value, is mainly distributed in Zhejiang, Jiangsu, and southeast Hubei Provinces of China, as well as in Japan and the southern Korean peninsula. In this study, the whole chloroplast genome sequence of C. discolor was first assembled using high-throughput Illumina paired-end technology, providing data to evaluate the evolution of this species. The C. discolor chloroplast genome was158,286 bp long, including a large single-copy region of 87,095 bp, a small single-copy region of 18,407 bp, and two copies of a repeat region (26,392-bp each). The overall G + C content was 41.2%. A total of 133 genes were predicted from the genome, including 87 protein-coding genes, eight ribosomal RNAs, 38 transfer RNAs. Phylogenetic analysis indicated a close relationship between C. discolor and C. bicolor.

Revisiting the early light-induced protein hypothesis in the sustained thermal dissipation mechanism in yew leaves.

Overwintering evergreen trees in boreal regions continuously convert absorbed light energy into heat through a process known as 'sustained thermal dissipation'. To better understand this mechanism, this study examined the alterations in the photosynthetic apparatus and transcriptomes of yew (Taxus cuspidata) leaves throughout the year, comparing sun-exposed and shaded leaves. The Y(II) parameter, conventionally used to estimate the quantum yield of photosystem II (PSII), suggests the occurrence of temperature-dependent thermal dissipation during winter. On the other hand, the levels of photosystem subunits, including the D1 subunit of the PSII reaction center, remain relatively stable year-round, suggesting that typical photoinhibition is unlikely to occur. Time-resolved chlorophyll fluorescence analysis revealed that heat dissipation at the PSII antenna is prominent in winter. Winter transcriptomes are notably characterized by a predominance of Elip transcripts encoding early light-induced protein (ELIP), which constitute 20% of the total transcripts, as deduced from RNA-seq analysis. Furthermore, ELIP protein concentration increases to nearly half that of the major light-harvesting complexes. The predicted structure of ELIP includes potential chlorophyll a and carotenoid binding sites. Considering a previous report showing ELIP's capacity for energy dissipation, these findings lead to a reevaluation of its significant role in sustained thermal dissipation.

The complete chloroplast genome of Buxus sinica var. parvifolia (Buxaceae) and its phylogenetic analysis.

Buxus sinica var. parvifolia is a shrub or small arbor of the Buxaceae family, rich in various medicinal alkaloids and of great horticultural value. In this study, we sequenced, assembled, and annotated the complete chloroplast genome of B. sinica var. parvifolia for the first time. The length of the chloroplast genome is 158,995 bp with 38.1% overall GC content. It includes a large single-copy (LSC) region of 88,140 bp, a small single-copy (SSC) region of 17,761 bp, and two inverted repeat regions of 26,547 bp. Additionally, 132 functional genes in the genome are identified, including 87 protein-coding genes, eight ribosomal RNA genes, and 37 transfer RNA genes. Phylogenetic analysis showed that B. sinica var. parvifolia is closely related to Buxus microphylla. The complete chloroplast genome sequence of B. sinica var. parvifolia and its phylogenetic analysis provides useful genomic information for the further study of B. sinica var. parvifolia and other Buxus species.

The complete chloroplast genomes and phylogenetic analysis of Exbucklandia longipetala and Exbucklandia populnea (Hamamelidaceae).

Exbucklandia longipetala and Exbucklandia populnea are two evergreen trees of the genus Exbucklandia in the family Hamamelidaceae. In this study, the complete chloroplast genomes of E. longipetala and E. populnea were sequenced, assembled, and annotated. The total lengths of the chloroplast genomes were 160,723 bp and 160,744 bp, respectively, and both had a GC content of 38.1%. The complete chloroplast genomes of these two species had typical quadripartite structures: LSC region (88,972 bp and 88,989 bp), SSC region (18,907 bp and 18,911 bp) and a pair of inverted repeats both of 26,422 bp. Both species contained 114 unique genes, including 80 protein-coding genes, 30 tRNA genes, and 4 rRNA genes. Phylogenetic analysis indicated that E. longipetala and E. populnea are sister species to each other. Our results provide useful genetic resources for further studies on the origin and evolution of Hamamelidaceae.