Author Correction: The Apostasia genome and the evolution of orchids.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Proteomic analysis identified proteins that are differentially expressed in the flavonoid and carotenoid biosynthetic pathways of Camellia Nitidissima flowers.

BACKGROUND: Camellia nitidissima Chi is a popular ornamental plant because of its golden flowers, which contain flavonoids and carotenoids. To understand the regulatory mechanism of golden color formation, the metabolites of C. nitidissima petals at five different developmental stages were detected, a proteome map of petals was first constructed via tandem mass tag (TMT) analysis, and the accuracy of the sequencing data was validated via parallel reaction monitoring (PRM). RESULTS: Nineteen color components were detected, and most of these components were carotenoids that gradually accumulated, while some metabolites were flavonoids that were gradually depleted. A total of 97,647 spectra were obtained, and 6,789 quantifiable proteins were identified. Then, 1,319 differentially expressed proteins (DEPs) were found, 55 of which belong to the flavonoid and carotenoid pathways, as revealed by pairwise comparisons of protein expression levels across the five developmental stages. Notably, most DEPs involved in the synthesis of flavonoids, such as phenylalanine ammonium lyase and 4-coumarate-CoA ligase, were downregulated during petal development, whereas DEPs involved in carotenoid synthesis, such as phytoene synthase, 1-deoxy-D-xylulose-5-phosphate synthase, and ß-cyclase, tended to be upregulated. Furthermore, protein‒protein interaction (PPI) network analysis revealed that these 55 DEPs formed two distinct PPI networks closely tied to the flavonoid and carotenoid synthesis pathways. Phytoene synthase and chalcone synthase exhibited extensive interactions with numerous other proteins and displayed high connectivity within the PPI networks, suggesting their pivotal biological functions in flavonoid and carotenoid biosynthesis. CONCLUSION: Proteomic data on the flavonoid and carotenoid biosynthesis pathways were obtained, and the regulatory roles of the DEPs were analyzed, which provided a theoretical basis for further understanding the golden color formation mechanism of C. nitidissima.

The complete mitochondrial genome of Castanopsis carlesii and Castanea henryi reveals the rearrangement and size differences of mitochondrial DNA molecules.

BACKGROUND: Castanopsis carlesii is a dominant tree species in subtropical evergreen broad-leaved forests and holds significant ecological value. It serves as an excellent timber tree species and raw material for cultivating edible fungi. Henry Chinquapin (Castanea henryi) wood is known for its hardness and resistance to water and moisture, making it an exceptional timber species. Additionally, its fruit has a sweet and fruity taste, making it a valuable food source. However, the mitogenomes of these species have not been previously reported. To gain a better understanding of them, this study successfully assembled high-quality mitogenomes of C. carlesii and Ca. henryi for the first time. RESULTS: Our research reveals that the mitochondrial DNA (mtDNA) of C. carlesii exhibits a unique multi-branched conformation, while Ca. henryi primarily exists in the form of two independent molecules that can be further divided into three independent molecules through one pair of long repetitive sequences. The size of the mitogenomes of C. carlesii and Ca. henryi are 592,702 bp and 379,929 bp respectively, which are currently the largest and smallest Fagaceae mitogenomes recorded thus far. The primary factor influencing mitogenome size is dispersed repeats. Comparison with published mitogenomes from closely related species highlights differences in size, gene loss patterns, codon usage preferences, repetitive sequences, as well as mitochondrial plastid DNA segments (MTPTs). CONCLUSIONS: Our study enhances the understanding of mitogenome structure and evolution in Fagaceae, laying a crucial foundation for future research on cell respiration, disease resistance, and other traits in this family.

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.

Comparative Phylogenomic Study of Malaxidinae (Orchidaceae) Sheds Light on Plastome Evolution and Gene Divergence.

Malaxidinae is one of the most confusing groups in the Orchidaceae classification. Previous phylogenetic analyses have revealed that the relationships between the taxa in Malaxidinae have not yet been reliably established, using only a few plastome regions and nuclear ribosomal internal transcribed spacer (nrITS). In the present study, the complete plastomes of Oberonia integerrima and Crepidium purpureum were assembled using high-throughput sequencing. Combined with publicly available complete plastome data, this resulted in a dataset of 19 plastomes, including 17 species of Malaxidinae. The plastome features and phylogenetic relationships were compared and analyzed. The results showed the following: (1) Malaxidinae species plastomes possess the quadripartite structure of typical angiosperms, with sizes ranging from 142,996 to 158,787 bp and encoding from 125 to 133 genes. The ndh genes were lost or pseudogenized to varying degrees in six species. An unusual inversion was detected in the large single-copy region (LSC) of Oberonioides microtatantha. (2) Eight regions, including ycf1, matK, rps16, rpl32, ccsA-ndhD, clpP-psbB, trnFGAA-ndhJ, and trnSGCU-trnGUCC, were identified as mutational hotspots. (3) Based on complete plastomes, 68 protein-coding genes, and 51 intergenic regions, respectively, our phylogenetic analyses revealed the genus-level relationships in this subtribe with strong support. The Liparis was supported as non-monophyletic.

The Complete Mitogenome of Apostasia fujianica Y.Li & S.Lan and Comparative Analysis of Mitogenomes across Orchidaceae.

Apostasia fujianica belongs to the genus Apostasia and is part of the basal lineage in the phylogenetic tree of the Orchidaceae. Currently, there are only ten reported complete mitochondrial genomes in orchids, which greatly hinders the understanding of mitochondrial evolution in Orchidaceae. Therefore, we assembled and annotated the mitochondrial genome of A. fujianica, which has a length of 573,612 bp and a GC content of 44.5%. We annotated a total of 44 genes, including 30 protein-coding genes, 12 tRNA genes, and two rRNA genes. We also performed relative synonymous codon usage (RSCU) analysis, repeat sequence analysis, intergenomic transfer (IGT) analysis, and Ka/Ks analysis for A. fujianica and conducted RNA editing site analysis on the mitochondrial genomes of eight orchid species. We found that most protein-coding genes are under purifying selection, but nad6 is under positive selection, with a Ka/Ks value of 1.35. During the IGT event in A. fujianica's mitogenome, the trnN-GUU, trnD-GUC, trnW-CCA, trnP-UGG, and psaJ genes were identified as having transferred from the plastid to the mitochondrion. Compared to other monocots, the family Orchidaceae appears to have lost the rpl10, rpl14, sdh3, and sdh4 genes. Additionally, to further elucidate the evolutionary relationships among monocots, we constructed a phylogenetic tree based on the complete mitogenomes of monocots. Our study results provide valuable data on the mitogenome of A. fujianica and lay the groundwork for future research on genetic variation, evolutionary relationships, and breeding of Orchidaceae.

Genome-Wide Identification and Drought Stress Response Pattern of the NF-Y Gene Family in Cymbidium sinense.

Cymbidium sinense, a type of orchid plant, is more drought-resistant and ornamental than other terrestrial orchids. Research has shown that many members of the NUCLEAR FACTOR Y (NF-Y) transcription factor family are responsive to plant growth, development, and abiotic stress. However, the mechanism of the NF-Y gene family's response to abiotic stress in orchids has not yet been reported. In this study, phylogenetic analysis allowed for 27 CsNF-Y genes to be identified (5 CsNF-YAs, 9 CsNF-YBs, and 13 CsNF-YC subunits), and the CsNF-Ys were homologous to those in Arabidopsis and Oryza. Protein structure analysis revealed that different subfamilies contained different motifs, but all of them contained Motif 2. Secondary and tertiary protein structure analysis indicated that the CsNF-YB and CsNF-YC subfamilies had a high content of alpha helix structures. Cis-element analysis showed that elements related to drought stress were mainly concentrated in the CsNF-YB and CsNF-YC subfamilies, with CsNF-YB3 and CsNF-YC12 having the highest content. The results of a transcriptome analysis showed that there was a trend of downregulation of almost all CsNF-Ys in leaves under drought stress, while in roots, most members of the CsNF-YB subfamily showed a trend of upregulation. Additionally, seven genes were selected for real-time reverse transcription quantitative PCR (qRT-PCR) experiments. The results were generally consistent with those of the transcriptome analysis. The regulatory roles of CsNF-YB 1, 2, and 4 were particularly evident in the roots. The findings of our study may make a great contribution to the understanding of the role of CsNF-Ys in stress-related metabolic processes.

Identification and Analysis of PEPC Gene Family Reveals Functional Diversification in Orchidaceae and the Regulation of Bacterial-Type PEPC.

Phosphoenolpyruvate carboxylase (PEPC) gene family plays a crucial role in both plant growth and response to abiotic stress. Approximately half of the Orchidaceae species are estimated to perform CAM pathway, and the availability of sequenced orchid genomes makes them ideal subjects for investigating the PEPC gene family in CAM plants. In this study, a total of 33 PEPC genes were identified across 15 orchids. Specifically, one PEPC gene was found in Cymbidium goeringii and Platanthera guangdongensis; two in Apostasia shenzhenica, Dendrobium chrysotoxum, D. huoshanense, Gastrodia elata, G. menghaiensis, Phalaenopsis aphrodite, Ph. equestris, and Pl. zijinensis; three in C. ensifolium, C. sinense, D. catenatum, D. nobile, and Vanilla planifolia. These PEPC genes were categorized into four subgroups, namely PEPC-i, PEPC-ii, and PEPC-iii (PTPC), and PEPC-iv (BTPC), supported by the comprehensive analyses of their physicochemical properties, motif, and gene structures. Remarkably, PEPC-iv contained a heretofore unreported orchid PEPC gene, identified as VpPEPC4. Differences in the number of PEPC homolog genes among these species were attributed to segmental duplication, whole-genome duplication (WGD), or gene loss events. Cis-elements identified in promoter regions were predominantly associated with light responsiveness, and circadian-related elements were observed in each PEPC-i and PEPC-ii gene. The expression levels of recruited BTPC, VpPEPC4, exhibited a lower expression level than other VpPEPCs in the tested tissues. The expression analyses and RT-qPCR results revealed diverse expression patterns in orchid PEPC genes. Duplicated genes exhibited distinct expression patterns, suggesting functional divergence. This study offered a comprehensive analysis to unveil the evolution and function of PEPC genes in Orchidaceae.

Genome-Wide Identification and Expression Analysis of the GRAS Gene Family and Their Responses to Heat Stress in Cymbidium goeringii.

The GRAS gene family, responsible for encoding transcription factors, serves pivotal functions in plant development, growth, and responses to stress. The exploration of the GRAS gene family within the Orchidaceae has been comparatively limited, despite its identification and functional description in various plant species. This study aimed to conduct a thorough examination of the GRAS gene family in Cymbidum goeringii, focusing on its physicochemical attributes, phylogenetic associations, gene structure, cis-acting elements, and expression profiles under heat stress. The results show that a total of 54 CgGRASs were pinpointed from the genome repository and categorized into ten subfamilies via phylogenetic associations. Assessment of gene sequence and structure disclosed the prevalent existence of the VHIID domain in most CgGRASs, with around 57.41% (31/54) CgGRASs lacking introns. The Ka/Ks ratios of all CgGRASs were below one, indicating purifying selection across all CgGRASs. Examination of cis-acting elements unveiled the presence of numerous elements linked to light response, plant hormone signaling, and stress responsiveness. Furthermore, CgGRAS5 contained the highest quantity of cis-acting elements linked to stress response. Experimental results from RT-qPCR demonstrated notable variations in the expression levels of eight CgGRASs after heat stress conditions, particularly within the LAS, HAM, and SCL4/7 subfamilies. In conclusion, this study revealed the expression pattern of CgGRASs under heat stress, providing reference for further exploration into the roles of CgGRAS transcription factors in stress adaptation.

Genome-Based Identification of the Dof Gene Family in Three Cymbidium Species and Their Responses to Heat Stress in Cymbidium goeringii.

As an important genus in Orchidaceae, Cymbidium has rich ecological diversity and significant economic value. DNA binding with one zinc finger (Dof) proteins are pivotal plant-specific transcription factors that play crucial roles in the growth, development, and stress response of plants. Although the Dof genes have been identified and functionally analyzed in numerous plants, exploration in Orchidaceae remains limited. We conducted a thorough analysis of the Dof gene family in Cymbidium goeringii, C. ensifolium, and C. sinensis. In total, 91 Dof genes (27 CgDofs, 34 CeDofs, 30 CsDofs) were identified, and Dof genes were divided into five groups (I-V) based on phylogenetic analysis. All Dof proteins have motif 1 and motif 2 conserved domains and over half of the genes contained introns. Chromosomal localization and collinearity analysis of Dof genes revealed their evolutionary relationships and potential gene duplication events. Analysis of cis-elements in CgDofs, CeDofs, and CsDofs promoters showed that light-responsive cis-elements were the most common, followed by hormone-responsive elements, plant growth-related elements, and abiotic stress response elements. Dof proteins in three Cymbidium species primarily exhibit a random coil structure, while homology modeling exhibited significant similarity. In addition, RT-qPCR analysis showed that the expression levels of nine CgDofs changed greatly under heat stress. CgDof03, CgDof22, CgDof27, CgDof08, and CgDof23 showed varying degrees of upregulation. Most upregulated genes under heat stress belong to group I, indicating that the Dof genes in group I have great potential for high-temperature resistance. In conclusion, our study systematically demonstrated the molecular characteristics of Dof genes in different Cymbidium species, preliminarily revealed the patterns of heat stress, and provided a reference for further exploration of stress breeding in orchids.

The Complete Chloroplast Genomes of Bulbophyllum (Orchidaceae) Species: Insight into Genome Structure Divergence and Phylogenetic Analysis.

Bulbophyllum is one of the largest genera and presents some of the most intricate taxonomic problems in the family Orchidaceae, including species of ornamental and medical importance. The lack of knowledge regarding the characterization of Bulbophyllum chloroplast (cp) genomes has imposed current limitations on our study. Here, we report the complete cp genomes of seven Bulbophyllum species, including B. ambrosia, B. crassipes, B. farreri, B. hamatum, B. shanicum, B. triste, and B. violaceolabellum, and compared with related taxa to provide a better understanding of their genomic information on taxonomy and phylogeny. A total of 28 Bulbophyllum cp genomes exhibit typical quadripartite structures with lengths ranging from 145,092 bp to 165,812 bp and a GC content of 36.60% to 38.04%. Each genome contained 125-132 genes, encompassing 74-86 protein-coding genes, 38 tRNA genes, and eight rRNA genes. The genome arrangements, gene contents, and length were similar, with differences observed in ndh gene composition. It is worth noting that there were exogenous fragment insertions in the IR regions of B. crassipes. A total of 18-49 long repeats and 38-80 simple sequence repeats (SSRs) were detected and the single nucleotide (A/T) was dominant in Bulbophyllum cp genomes, with an obvious A/T preference. An analysis of relative synonymous codon usage (RSCU) revealed that leucine (Leu) was the most frequently used codon, while cysteine (Cys) was the least used. Six highly variable regions (rpl32-trnLUAG > trnTUGU-trnLUAA > trnFGAA-ndhJ > rps15-ycf1 > rbcL-accD > psbI-trnSGCU) and five coding sequences (ycf1 > rps12 > matK > psbK > rps15) were identified as potential DNA markers based on nucleotide diversity. Additionally, 31,641 molecular diagnostic characters (MDCs) were identified in complete cp genomes. A phylogenetic analysis based on the complete cp genome sequences and 68 protein-coding genes strongly supported that 28 Bulbophyllum species can be divided into four branches, sects. Brachyantha, Cirrhopetalum, and Leopardinae, defined by morphology, were non-monophyly. Our results enriched the genetic resources of Bulbophyllum, providing valuable information to illustrate the complicated taxonomy, phylogeny, and evolution process of the genus.

Comparative Analysis of Six Complete Plastomes of Tripterospermum spp.

The Tripterospermum, comprising 34 species, is a genus of Gentianaceae. Members of Tripterospermum are mostly perennial, entwined herbs with high medicinal value and rich in iridoids, xanthones, flavonoids, and triterpenes. However, our inadequate understanding of the differences in the plastid genome sequences of Tripterospermum species has severely hindered the study of their evolution and phylogeny. Therefore, we first analyzed the 86 Gentianae plastid genomes to explore the phylogenetic relationships within the Gentianae subfamily where Tripterospermum is located. Then, we analyzed six plastid genomes of Tripterospermum, including two newly sequenced plastid genomes and four previously published plastid genomes, to explore the plastid genomes' evolution and phylogenetic relationships in the genus Tripterospermum. The Tripterospermum plastomes have a quadripartite structure and are between 150,929 and 151,350 bp in size. The plastomes of Tripterospermum encoding 134 genes were detected, including 86 protein-coding genes (CDS), 37 transfer RNA (tRNA) genes, eight ribosomal RNA (rRNA) genes, and three pseudogenes (infA, rps19, and ycf1). The result of the comparison shows that the Tripterospermum plastomes are very conserved, with the total plastome GC content ranging from 37.70% to 37.79%. In repeat sequence analysis, the number of single nucleotide repeats (A/T) varies among the six Tripterospermum species, and the identified main long repeat types are forward and palindromic repeats. The degree of conservation is higher at the SC/IR boundary. The regions with the highest divergence in the CDS and the intergenic region (IGS) are psaI and rrn4.5-rrn5, respectively. The average pi of the CDS and the IGS are only 0.071% and 0.232%, respectively, indicating that the Tripterospermum plastomes are highly conserved. Phylogenetic analysis indicated that Gentianinae is divided into two clades, with Tripterospermum as a sister to Sinogeniana. Phylogenetic trees based on CDS and CDS + IGS combined matrices have strong support in Tripterospermum. These findings contribute to the elucidation of the plastid genome evolution of Tripterospermum and provide a foundation for further exploration and resource utilization within this genus.

Genome-Wide Identification and Expression Pattern Analysis of TIFY Family Genes Reveal Their Potential Roles in Phalaenopsis aphrodite Flower Opening.

The TIFY gene family (formerly known as the zinc finger proteins expressed in inflorescence meristem (ZIM) family) not only functions in plant defense responses but also are widely involved in regulating plant growth and development. However, the identification and functional analysis of TIFY proteins remain unexplored in Orchidaceae. Here, we identified 19 putative TIFY genes in the Phalaenopsis aphrodite genome. The phylogenetic tree classified them into four subfamilies: 14 members from JAZ, 3 members from ZML, and 1 each from PPD and TIFY. Sequence analysis revealed that all Phalaenopsis TIFY proteins contained a TIFY domain. Exon-intron analysis showed that the intron number and length of Phalaenopsis TIFY genes varied, whereas the same subfamily and subgroup genes had similar exon or intron numbers and distributions. The most abundant cis-elements in the promoter regions of the 19 TIFY genes were associated with light responsiveness, followed by MeJA and ABA, indicating their potential regulation by light and phytohormones. The 13 candidate TIFY genes screened from the transcriptome data exhibited two types of expression trends, suggesting their different roles in cell proliferation and cell expansion of floral organ growth during Phalaenopsis flower opening. Overall, this study serves as a background for investigating the underlying roles of TIFY genes in floral organ growth in Phalaenopsis.

A Comprehensive Analysis of Auxin Response Factor Gene Family in Melastoma dodecandrum Genome.

Auxin Response Factors (ARFs) mediate auxin signaling and govern diverse biological processes. However, a comprehensive analysis of the ARF gene family and identification of their key regulatory functions have not been conducted in Melastoma dodecandrum, leading to a weak understanding of further use and development for this functional shrub. In this study, we successfully identified a total of 27 members of the ARF gene family in M. dodecandrum and classified them into Class I-III. Class II-III showed more significant gene duplication than Class I, especially for MedARF16s. According to the prediction of cis-regulatory elements, the AP2/ERF, BHLH, and bZIP transcription factor families may serve as regulatory factors controlling the transcriptional pre-initiation expression of MedARF. Analysis of miRNA editing sites reveals that miR160 may play a regulatory role in the post-transcriptional expression of MeARF. Expression profiles revealed that more than half of the MedARFs exhibited high expression levels in the stem compared to other organs. While there are some specific genes expressed only in flowers, it is noteworthy that MedARF16s, MedARF7A, and MedARF9B, which are highly expressed in stems, also demonstrate high expressions in other organs of M. dodecandrum. Further hormone treatment experiments revealed that these MedARFs were sensitive to auxin changes, with MedARF6C and MedARF7A showing significant and rapid changes in expression upon increasing exogenous auxin. In brief, our findings suggest a crucial role in regulating plant growth and development in M. dodecandrum by responding to changes in auxin. These results can provide a theoretical basis for future molecular breeding in Myrtaceae.

General Analysis of Heat Shock Factors in the Cymbidium ensifolium Genome Provided Insights into Their Evolution and Special Roles with Response to Temperature.

Heat shock factors (HSFs) are the key regulators of heat stress responses and play pivotal roles in tissue development and the temperature-induced regulation of secondary metabolites. In order to elucidate the roles of HSFs in Cymbidium ensifolium, we conducted a genome-wide identification of CeHSF genes and predicted their functions based on their structural features and splicing patterns. Our results revealed 22 HSF family members, with each gene containing more than one intron. According to phylogenetic analysis, 59.1% of HSFs were grouped into the A subfamily, while subfamily HSFC contained only two HSFs. And the HSF gene families were differentiated evolutionarily between plant species. Two tandem repeats were found on Chr02, and two segmental duplication pairs were observed on Chr12, Chr17, and Chr19; this provided evidence for whole-genome duplication (WGD) events in C. ensifolium. The core region of the promoter in most CeHSF genes contained cis-acting elements such as AP2/ERF and bHLH, which were associated with plant growth, development, and stress responses. Except for CeHSF11, 14, and 19, each of the remaining CeHSFs contained at least one miRNA binding site. This included binding sites for miR156, miR393, and miR319, which were responsive to temperature and other stresses. The HSF gene family exhibited significant tissue specificity in both vegetative and floral organs of C. ensifolium. CeHSF13 and CeHSF15 showed relatively significant expression in flowers compared to other genes. During flower development, CeHSF15 exhibited markedly elevated expression in the early stages of flower opening, implicating critical regulatory functions in organ development and floral scent-related regulations. During the poikilothermic treatment, CeHSF14 was upregulated over 200-fold after 6 h of heat treatment. CeHSF13 and CeHSF14 showed the highest expression at 6 h of low temperature, while the expression of CeHSF15 and CeHSF21 continuously decreased at a low temperature. The expression patterns of CeHSFs further confirmed their role in responding to temperature stress. Our study may help reveal the important roles of HSFs in plant development and metabolic regulation and show insight for the further molecular design breeding of C. ensifolium.

Comparative and phylogenetic analysis of Chiloschista (Orchidaceae) species and DNA barcoding investigation based on plastid genomes.

BACKGROUND: Chiloschista (Orchidaceae, Aeridinae) is an epiphytic leafless orchid that is mainly distributed in tropical or subtropical forest canopies. This rare and threatened orchid lacks molecular resources for phylogenetic and barcoding analysis. Therefore, we sequenced and assembled seven complete plastomes of Chiloschista to analyse the plastome characteristics and phylogenetic relationships and conduct a barcoding investigation. RESULTS: We are the first to publish seven Chiloschista plastomes, which possessed the typical quadripartite structure and ranged from 143,233 bp to 145,463 bp in size. The plastomes all contained 120 genes, consisting of 74 protein-coding genes, 38 tRNA genes and eight rRNA genes. The ndh genes were pseudogenes or lost in the genus, and the genes petG and psbF were under positive selection. The seven Chiloschista plastomes displayed stable plastome structures with no large inversions or rearrangements. A total of 14 small inversions (SIs) were identified in the seven Chiloschista plastomes but were all similar within the genus. Six noncoding mutational hotspots (trnNGUU-rpl32 > rpoB-trnCGCA > psbK-psbI > psaC-rps15 > trnEUUC-trnTGGU > accD-psaI) and five coding sequences (ycf1 > rps15 > matK > psbK > ccsA) were selected as potential barcodes based on nucleotide diversity and species discrimination analysis, which suggested that the potential barcode ycf1 was most suitable for species discrimination. A total of 47-56 SSRs and 11-14 long repeats (> 20 bp) were identified in Chiloschista plastomes, and they were mostly located in the large single copy intergenic region. Phylogenetic analysis indicated that Chiloschista was monophyletic. It was clustered with Phalaenopsis and formed the basic clade of the subtribe Aeridinae with a moderate support value. The results also showed that seven Chiloschista species were divided into three major clades with full support. CONCLUSION: This study was the first to analyse the plastome characteristics of the genus Chiloschista in Orchidaceae, and the results showed that Chiloschista plastomes have conserved plastome structures. Based on the plastome hotspots of nucleotide diversity, several genes and noncoding regions are suitable for phylogenetic and population studies. Chiloschista may provide an ideal system to investigate the dynamics of plastome evolution and DNA barcoding investigation for orchid studies.

Molecular mechanism of different flower color formation of Cymbidium ensifolium.

Cymbidium ensifolium is one of the national orchids in China, which has high ornamental value with changeable flower colors. To understand the formation mechanism of different flower colors of C. ensifolium, this research conducted transcriptome and metabolome analyses on four different colored sepals of C. ensifolium. Metabolome analysis detected 204 flavonoid metabolites, including 17 polyphenols, 27 anthocyanins, 75 flavones, 34 flavonols, 25 flavonoids, 18 flavanones, and 8 isoflavones. Among them, purple-red and red sepals contain a lot of anthocyanins, including cyanidin, pelargonin, and paeoniflorin, while yellow-green and white sepals have less anthocyanins detected, and their metabolites are mainly flavonols, flavanones and flavonoids. Transcriptome sequencing analysis showed that the expression levels of the anthocyanin biosynthetic enzyme genes in red and purple-red sepals were significantly higher than those in white and yellow-green sepals of C. ensifolium. The experimental results showed that CeF3'H2, CeDFR, CeANS, CeF3H and CeUFGT1 may be the key genes involved in anthocyanin production in C. ensifolium sepals, and CeMYB104 has been proved to play an important role in the flower color formation of C. ensifolium. The results of transformation showed that the CeMYB104 is involved in the synthesis of anthocyanins and can form a purple-red color in the white perianth of Phalaenopsis. These findings provide a theoretical reference to understand the formation mechanism of flower color in C. ensifolium.

Insights into cryptochrome modulation of ABA signaling to mediate dormancy regulation in Marchantia polymorpha.

The acquisition of dormancy capabilities has enabled plants to survive in adverse terrestrial environmental conditions. Dormancy accumulation and release is coupled with light signaling, which is well studied in Arabidopsis, but it is unclear in the distant nonvascular relative. We study the characteristics and function on dormancy regulation of a blue light receptor cryptochrome in Marchantia polymorpha (MpCRY). Here, we identified MpCRY via bioinformatics and mutant complement analysis. The biochemical characteristics were assessed by multiple protein-binding assays. The function of MpCRY in gemma dormancy was clarified by overexpression and mutation of MpCRY, and its mechanism was analyzed via RNA sequencing and quantitative PCR analyses associated with hormone treatment. We found that the unique MpCRY protein in M. polymorpha undergoes both blue light-promoted interaction with itself (self-interaction) and blue light-dependent phosphorylation. MpCRY has the specific characteristics of blue light-induced nuclear localization and degradation. We further demonstrated that MpCRY transcriptionally represses abscisic acid (ABA) signaling-related gene expression to suppress gemma dormancy, which is dependent on blue light signaling. Our findings indicate that MpCRY possesses specific biochemical and molecular characteristics, and modulates ABA signaling under blue light conditions to regulate gemma dormancy in M. polymorpha.

R2R3-MYB genes coordinate conical cell development and cuticular wax biosynthesis in Phalaenopsis aphrodite.

Petals of the monocot Phalaenopsis aphrodite (Orchidaceae) possess conical epidermal cells on their adaxial surfaces, and a large amount of cuticular wax is deposited on them to serve as a primary barrier against biotic and abiotic stresses. It has been widely reported that subgroup 9A members of the R2R3-MYB gene family, MIXTA and MIXTA-like in eudicots, act to regulate the differentiation of conical epidermal cells. However, the molecular pathways underlying conical epidermal cell development and cuticular wax biosynthesis in monocot petals remain unclear. Here, we characterized two subgroup 9A R2R3-MYB genes, PaMYB9A1 and PaMYB9A2 (PaMYB9A1/2), from P. aphrodite through the transient overexpression of their coding sequences and corresponding chimeric repressors in developing petals. We showed that PaMYB9A1/2 function to coordinate conical epidermal cell development and cuticular wax biosynthesis. In addition, we identified putative targets of PaMYB9A1/2 through comparative transcriptome analyses, revealing that PaMYB9A1/2 acts to regulate the expression of cell wall-associated and wax biosynthetic genes. Furthermore, a chemical composition analysis of cuticular wax showed that even-chain n-alkanes and odd-chain primary alcohols are the main chemical constituents of cuticular wax deposited on petals, which is inconsistent with the well-known biosynthetic pathways of cuticular wax, implying a distinct biosynthetic pathway occurring in P. aphrodite flowers. These results reveal that the function of subgroup 9A R2R3-MYB family genes in regulating the differentiation of epidermal cells is largely conserved in monocots and dicots. Furthermore, both PaMYB9A1/2 have evolved additional functions controlling the biosynthesis of cuticular wax.

Origin and diversification of a Himalayan orchid genus Pleione.

Pleione is an orchid endemically distributed in high mountain areas across the Hengduan Mountains (HDM), Himalayas, Southeast Asia and South of China. The unique flower shapes, rich colors and immense medicinal importance of Pleione are valuable ornamental and economic resources. However, the phylogenetic relationships and evolutionary history of the genus have not yet been comprehensively resolved. Here, the evolutionary history of Pleione was investigated using single-copy gene single nucleotide polymorphisms and chloroplast genome datasets. The data revealed that Pleione could be divided into five clades. Discordance in topology between the two phylogenetic trees and network and D-statistic analyses indicated the occurrence of reticulate evolution in the genus. The evolution could be attributed to introgression and incomplete lineage sorting. Ancestral area reconstruction suggested that Pleione was originated from the HDM. Uplifting of the HDM drove rapid diversification by creating conditions favoring rapid speciation. This coincided with two periods of consolidation of the Asian monsoon climate, which caused the first rapid diversification of Pleione from 8.87 to 7.83 Mya, and a second rapid diversification started at around 4.05 Mya to Pleistocene. The interaction between Pleione and climate changes, especially the monsoons, led to the current distribution pattern and shaped the dormancy characteristic of the different clades. In addition to revealing the evolutionary relationship of Pleione with orogeny and climate changes, the findings of this study provide insights into the speciation and diversification mechanisms of plants in the East Asian flora.