Dendrobium Catenatum Research Articles

Effect of cultivation mode on bacterial and fungal communities of Dendrobium catenatum

BackgroundThe orchid growth and development often associate with microbes. However, the interaction between plant performance and microbial communities within and surrounding plants is less understood. Dendrobium catenatum, which used to be an endangered orchid species, has become a billion dollar industry in China. Simulated natural cultivation modes, such as living tree epiphytic (LT) and cliff epiphytic (CE) cultivations, improve the production or quality of D. catenatum and contribute to the development of D. catenatum industry. In a previous study, morphological characteristics, anatomical structure, and main bioactive components (polysaccharides and ethanol-soluble extractives) of D. catenatum grown under LT and CE significantly differed from a facility cultivation mode, pot (PO) cultivation, were observed. Whether cultivation mode affects bacterial and fungal communities of D. catenatum, thereby affecting the chemical quality of this plant, need to be explored.ResultsBoth three plant organs (leaf, stem, and root) and cultivating substrates obtained under three cultivation modes: living tree epiphytic (LT), cliff epiphytic (CE), and pot (PO) cultivation were examined by adopting high-throughput sequencing methods. Subsequently, bacterial and fungal correlations with D. catenatum main chemical components, stem polysaccharides and ethanol-soluble extractives and leaf phenols and flavonoids, were elucidated. The results showed that microbial communities of the plants and substrates are both influenced by the cultivation mode. However, the plants and their cultivating substrates exhibited different patterns of bacterial and fungal composition, with clearly distinguished dominant bacterial groups, but shared dominance among fungal groups. Bacteria and fungi differed in abundance, diversity, and community structure, depending on the cultivation environment and plant organ. Both bacterial and fungal communities were affected by cultivation mode and plant organ. In both plants and substrates, PO bacterial and fungal community structure differed significantly from those of LT and CE modes. Bacterial and fungal community structure differed significantly between roots and the other two plant organs examined (stems and leaves). Several bacteria and fungi were positively correlated with main chemical components in D. catenatum.ConclusionsThe findings indicate that microbial communities of the plants and substrates were both influenced by the cultivation mode and plant organ, and some of them were positively correlated with main chemical components in D. catenatum. The research would enhance our understanding of interactions between Dendrobium and the microbial environment, and to provide a theoretical basis for the development of improved D. catenatum cultivation methods.

Genome-wide identification and characterization of active ingredients related β-Glucosidases in Dendrobium catenatum

BackgroundDendrobium catenatum/D. officinale (here after D. catenatum), a well-known economically important traditional medicinal herb, produces a variety of bioactive metabolites including polysaccharides, alkaloids, and flavonoids with excellent pharmacological and clinical values. Although many genes associated with the biosynthesis of medicinal components have been cloned and characterized, the biosynthetic pathway, especially the downstream and regulatory pathway of major medicinal components in the herb, is far from clear. β-glucosidases (BGLUs) comprise a diverse group of enzymes that widely exist in plants and play essential functions in cell wall modification, defense response, phytohormone signaling, secondary metabolism, herbivore resistance, and scent release by hydrolyzing β-D-glycosidic bond from a carbohydrate moiety. The recent release of the chromosome-level reference genome of D. catenatum enables the characterization of gene families. Although the genome-wide analysis of the BGLU gene family has been successfully conducted in various plants, no systematic analysis is available for the D. catenatum. We previously isolated DcBGLU2 in the BGLU family as a key regulator for polysaccharide biosynthesis in D. catenatum. Yet, the exact number of DcBGLUs in the D. catenatum genome and their possible roles in bioactive compound production deserve more attention.ResultsTo investigate the role of BGLUs in active metabolites production, 22 BGLUs (DcBGLU1-22) of the glycoside hydrolase family 1 (GH1) were identified from D. catenatum genome. Protein prediction showed that most of the DcBGLUs were acidic and phylogenetic analysis classified the family into four distinct clusters. The sequence alignments revealed several conserved motifs among the DcBGLU proteins and analyses of the putative signal peptides and N-glycosylation site revealed that the majority of DcBGLU members dually targeted to the vacuole and/or chloroplast. Organ-specific expression profiles and specific responses to MeJA and MF23 were also determined. Furthermore, four DcBGLUs were selected to test their involvement in metabolism regulation. Overexpression of DcBGLU2, 6, 8, and 13 significantly increased contents of flavonoid, reducing-polysaccharide, alkaloid and soluble-polysaccharide, respectively.ConclusionThe genome-wide systematic analysis identified candidate DcBGLU genes with possible roles in medicinal metabolites production and laid a theoretical foundation for further functional characterization and molecular breeding of D. catenatum.

Genome-Wide Analysis of the WOX Transcription Factor Genes in Dendrobium catenatum Lindl.

The WUSCHEL-related homeobox (WOX) proteins are a class of transcription factors exclusive to plants. They can promote cell division or inhibit stem cell differentiation to regulate plant growth and development. However, the WOX transcription factor genes in the monocotyledon Dendrobium catenatum Lindl. remain relatively uncharacterized. Specifically, the effects of phytohormones on their expression levels are unclear. In this study, we identified and analyzed 10 candidate DcaWOX transcription factor genes in D. catenatum. The DcaWOX family was divided into the modern/WUS, intermediate, and ancient clades. The subcellular localization analysis detected DcaWOX-GFP fusion proteins in the tobacco epidermal leaf cell nucleus. In DcaWOX, members of the WUS clade with the WUS-box motif can significantly activate the expression of TPL in vivo, while members of the intermediate and ancient clades cannot. The expression of the DcaWOX genes varied among the examined tissues. Moreover, the DcaWOX expression patterns were differentially affected by the phytohormone treatments, with differences detected even between homologs of the same gene. Furthermore, the gene expression patterns were consistent with the predicted cis-acting elements in the promoters. The above results suggest that DcaWOX may have an important role in its growth and development and resistance to stress. The results of this comprehensive investigation of the DcaWOX gene family provide the basis for future studies on the roles of WOX genes in D. catenatum.

DhMYB22 and DhMYB60 regulate pigment intensity and floral organ shape in Dendrobium hybrid.

Flower pigment and shape are determined by the coordinated expression of a set of structural genes during flower development. R2R3-MYB transcription factors are known regulators of structural gene expression. The current study focused on two members of this large family of transcription factors that were predicted to have roles in pigment biosynthesis and organ shape development in orchids. Phylogenetic analysis was used to identify candidate Dendrobium catenatum R2R3-MYB (DcaMYB) sequences associated with pigment and cell shape development. Gene silencing of candidate DhMYBs in Dendrobium hybrid by direct application of dsRNA to developing flowers was followed by observation of gene expression level and flower phenotypes. Silencing of the structural gene chalcone synthase was used as a comparative control. Ten candidate flower-associated DcaMYBs were identified. Flowers treated with dsRNA of DhMYB22 and DhMYB60 sequences were less pigmented and had relatively low expression of anthocyanin biosynthetic genes (F3'H and DFR), lower total anthocyanin concentration and markedly lower levels of cyanidin-3-glucoside and cyanidin-3-rutinoside. Petals of DhMYB22-treated flowers and sepals of DhMYB60-treated flowers showed the greatest colour difference relative to the same organs in untreated flowers. DhMYB22-treated flowers had relatively narrow and constricted lips, while DhMYB60-treated flowers had narrow and constricted sepals. No significant difference in shape was observed for DhCHS-treated or untreated flowers. Our results demonstrate that DhMYB22 and DhMYB60 regulate pigment intensity and floral organ shape in Dendrobium. This is a first report of MYB regulation of floral organ shape in orchids.

Genome-wide analysis of basic helix-loop-helix genes in Dendrobium catenatum and functional characterization of DcMYC2 in jasmonate-mediated immunity to Sclerotium delphinii.

Dendrobium catenatum, belonging to the Orchidaceae, is a precious Chinese herbal medicine. Sclerotium delphinii (P1) is a broad-spectrum fungal disease, which causes widespread loss in the near-wild cultivation of D. catenatum. Thus, resistance breeding of D. catenatum has become the key to solve this problem. The basic helix–loop–helix (bHLH) gene family is closely related to plant resistance to external stresses, but the related research in D. catenatum is not deep enough yet. Phylogenetic analysis showed that 108 DcbHLH genes could be divided into 23 subgroups. Promoter cis-acting elements revealed that DcbHLHs contain a large number of stress-related cis-acting elements. Transcriptome analysis of MeJA and P1 treatment manifested that exogenous MeJA can change the expression pattern of most bHLH genes, especially the IIIe subgroup, including inhibiting the expression of DcbHLH026 (MYC2a) and promoting the expression of DcbHLH027 (MYC2b). Subcellular localization indicated that they were located in the nucleus. Furthermore, exogenous MeJA treatment significantly delayed disease time and reduced lesion size after infection with P1. DcMYC2b-overexpression Arabidopsis lines showed significantly smaller lesions after being infected with P1 than the wild type, indicating that DcMYC2b functions as an important positive regulator in D. catenatum defense against P1. Our findings shed more insights into the critical role of the DcbHLH family in plants and the resistance breeding of D. catenatum.

Developmental Characteristics and Auxin Response of Epiphytic Root in Dendrobium catenatum.

Dendrobium catenatum, a traditional precious Chinese herbal medicine, belongs to epiphytic orchids. Its special life mode leads to the specialization of roots, but there is a lack of systematic research. The aerial root in D. catenatum displays diverse unique biological characteristics, and it initially originates from the opposite pole of the shoot meristem within the protocorm. The root development of D. catenatum is not only regulated by internal cues but also adjusts accordingly with the change in growth environments. D. catenatum root is highly tolerant to auxin, which may be closely related to its epiphytic life. Exogenous auxin treatment has dual effects on D. catenatum roots: relatively low concentration promotes root elongation, which is related to the induced expression of cell wall synthesis genes; excessive concentration inhibits the differentiation of velamen and exodermis and promotes the overproliferation of cortical cells, which is related to the significant upregulation of WOX11-WOX5 regeneration pathway genes and cell division regulatory genes. Overexpression of D. catenatum WOX12 (DcWOX12) in Arabidopsis inhibits cell and organ differentiation, but induces cell dedifferentiation and callus production. Therefore, DcWOX12 not only retains the characteristics of ancestors as stem cell regulators, but also obtains stronger cell fate transformation ability than homologous genes of other species. These findings suggest that the aerial root of D. catenatum evolves special structure and developmental characteristics to adapt to epiphytic life, providing insight into ideal root structure breeding of simulated natural cultivation in D. catenatum and a novel target gene for improving the efficiency of monocot plant transformation.

Polyketide synthases (PKSs) of secondary metabolism: in silico identification and characterization in orchids

Type III polyketide synthases (PKSs) catalyse the formation of an array of polyketides with diverse structures that play an important role in secondary metabolism in plants. This group of enzymes is encoded by a multigene family, the Type III polyketide synthase (PKS) gene family. Vast reserves of secondary metabolites in orchids make these plants suitable candidates for research in the area. In this study, genome-wide searches lead to the identification of five PeqPKS, eight DcaPKS and six AshPKS genes in Phalaenopsis equestris, Dendrobium catenatum and Apostasia shenzhenica, respectively. All the members showed the presence of two characteristic conserved domains (Chal_sti_synt_N and Chal_sti_synt_C) and were generally localised in the cytoplasm. The phylogenetic analysis led to the classification of these proteins into two groups: CHS (chalcone synthase (CHS) and non-CHS. A single protein in P. equestris and two proteins each in D. catenatum and A. shenzhenica clustered within the CHS clade. The majority of the genes exhibited similar structural patterns with a single intron. Expression profiling revealed the tissue-specific expression of these genes with high expression in reproductive tissues for most genes. A number of stress-responsive cis-regulatory elements were predicted, noteworthy amongst these are, ABRE and CGTCA that are chiefly responsible for responding to abscisic acid and methyl jasmonate, respectively. Our study provides a reference framework for future studies involving functional elucidation of PKS genes and biotechnological production of polyketides. Communicated by Ramaswamy H. Sarma

Metabolic Profiling of Terpene Diversity and the Response of Prenylsynthase-Terpene Synthase Genes during Biotic and Abiotic Stresses in Dendrobium catenatum.

Dendrobium catenatum is a widely cultivated Chinese orchid herb rich in abundant secondary metabolites, such as terpenes. However, terpene distribution and characterization of terpene biosynthesis-related genes remain unknown in D. catenatum. In this study, metabolic profiling was performed to analyze terpene distribution in the root, stem, leaf, and flower of D. catenatum. A total of 74 terpene compounds were identified and classified. Clustering analysis revealed that terpene compounds exhibited a tissue-specific accumulation, including monoterpenes in the flowers, sesquiterpenes in the stems, and triterpenes in the roots. Transcriptome analysis revealed that the ‘terpenoid backbone biosynthesis’ pathway was only significantly enriched in root vs. flower. The expression of terpene biosynthesis-related genes was spatiotemporal in the flowers. Prenylsynthase-terpene synthases (PS-TPSs) are the largest and core enzymes for generating terpene diversity. By systematic sequence analysis of six species, 318 PS-TPSs were classified into 10 groups and 51 DcaPS-TPSs were found in eight of them. Eighteen DcaPS-TPSs were regulated by circadian rhythm under drought stress. Most of the DcaPS-TPSs were influenced by cold stress and fungi infection. The cis-element of the majority of the DcaPS-TPS promoters was related to abiotic stress and plant development. Methyl jasmonate levels were significantly associated with DcaTPSs expression and terpene biosynthesis. These results provide insight into further functional investigation of DcaPS-TPSs and the regulation of terpene biosynthesis in Dendrobium.

Phylogenetic analysis and expression profiling of Nuclear Factor-Y gene family in Dendrobium catenatum Lindl. (Orchidaceae)

Nuclear factor-Y (NF-Y) are transcription factors that play vital role in various developmental processes such as flowering, embryogenesis, root formation, nodulation and stress tolerance. In the present study, 27 NF-Y genes were identified in Dendrobium catenatum which were grouped into three sub-families: DcNF-YA (5), DcNF-YB (10) and DcNF-YC (12), on the basis of presence of specific conserved domains. The phylogenetic relationship between DcNF-Y protein sequences with their orthologs in Vanilla planifolia and Arabidopsis thaliana confirmed the classification of these proteins into the specified three groups. Physico-chemical characterization reported a wide range in the protein length (111 to 433 amino acids), with DcNF-YAs being longer than the others. Strong interaction between most of the DcNF-Y proteins suggests towards the formation of complexes which might be playing a role in different developmental processes. Gene architectural studies predicted the presence of multiple (3–6) introns in DcNF-YA in contrast with the intron-less status of majority of DcNF-YB and DcNF-YC genes. The abundance of stress related cis-regulatory elements in the promoter regions of DcNF-Y genes indicate the potential of these genes in stress tolerance. Additionally, variable expression of these genes in diverse tissues point towards their widespread role in growth and development. This work can act as a harbinger for functional characterization of NF-Y genes with a view for genetic improvement in Dendrobium catenatum Lindl.

Water-soluble non-starch polysaccharides of wild-simulated Dendrobium catenatum Lindley plantings on rocks and bark of pear trees.

The total water-soluble polysaccharide (TP) of Dendrobium catenatum is composed of starch and active non-starch polysaccharides (NSPs) with glucomannan as the main structural type. Although the TP content has been used as a quality assessment indicator for many years, the NSPs content in samples from different environments and growth seasons have not been reported. In this study, we found that NSPs had stronger antioxidant activity than TP. The NSPs content was higher in wild-simulated environments including rocks and trees compared to plantings grown in greenhouse. The culture mode and growth period affected the ratio of NSPs and starch. Facility cultivation provided optimal growth conditions but produced more starch, whereas wild-simulated cultivation resulted in a higher ratio of NSPs, particularly in September. Therefore, cultivation by lithophytation and epiphytation may be preferable to facility plantings, which is expected to be enormously useful for the current production and quality control of D. catenatum.

Identification and analysis of sucrose synthase gene family associated with polysaccharide biosynthesis in Dendrobium catenatum by transcriptomic analysis.

BackgroundDendrobium catenatum is a valuable traditional medicinal herb with high commercial value. D. catenatum stems contain abundant polysaccharides which are one of the main bioactive components. However, although some genes related to the synthesis of the polysaccharides have been reported, more key genes need to be further elucidated.ResultsIn this study, the contents of polysaccharides and mannose in D. catenatum stems at four developmental stages were compared, and the stems’ transcriptomes were analyzed to explore the synthesis mechanism of the polysaccharides. Many genes involved in starch and sucrose metabolisms were identified by KEGG pathway analysis. Further analysis found that sucrose synthase (SUS; EC 2.4.1.13) gene maybe participated in the polysaccharide synthesis. Hence, we further investigated the genomic characteristics and evolution relationships of the SUS family in plants. The result suggested that the SUS gene of D. catenatum (DcSUS) had undergone the expansion characterized by tandem duplication which might be related to the enrichment of the polysaccharides in D. catenatum stems. Moreover, expression analyses of the DcSUS displayed significant divergent patterns in different tissues and could be divided into two main groups in the stems with four developmental stages.ConclusionIn general, our results revealed that DcSUS is likely involved in the metabolic process of the stem polysaccharides, providing crucial clues for exploiting the key genes associated with the polysaccharide synthesis.

Genome-Wide Analysis of LBD Transcription Factor Genes in Dendrobiumcatenatum.

The LATERAL ORGAN BOUNDARIES DOMAIN (LBD) gene family comprises plant-specific transcription factors that control cell proliferation and differentiation during growth and development in many plant species. However, to date, no studies of the LBD gene family in Dendrobium catenatum have been reported. In this study, a genome-wide analysis of LBD genes was performed in D. catenatum and 24 LBD genes were identified. The genes were classified into two classes (I and II) based on phylogenetic relationships and motif structure. Subcellular localization analysis for DcaLBD6 and DcaLBD18 from class I and DcaLBD37 and DcaLBD41 from class II revealed that the proteins were localized in the nucleus. Transient expression analysis of DcaLBD6, DcaLBD18, DcaLBD37, and DcaLBD41 indicated that class I and class II members have opposite roles in regulating VASCULAR-RELATED NAC-DOMAIN 7 (VND7) expression. DcaLBD genes showed diverse expression patterns in response to different phytohormone treatments. Heat maps revealed diverse patterns of DcaLBD gene expression in different organs. These results lay the foundation for further detailed studies of the LBD gene family in D. catenatum.

Weighted gene co-expression network analysis revealed the synthesis of aromatic compounds in Dendrobium catenatum

Aromatic compounds in Dendrobium catenatum could affect brewing quality. Methyl jasmonate (MeJA) treatment enhances the content of aromatic compounds in D. catenatum, improving the wine quality in brewing industry and medicinal value. In this study, two D. catenatum genotypes, A6 and B1, were used to investigate the effect of MeJA treatment on aromatic compounds. A total of 52 aromatic metabolites were identified using metabolome technique. Based on RNA-seq, weighted gene co-expression network analysis (WGCNA) of 16, 841 highly expressed transcripts uncovered black, cyan and turquoise modules that are significantly associated with the variation of aromatic compounds. These results reveal the changes of aromatic metabolites during MeJA treatment and provide insight into the biosynthesis mechanism of aromatic compounds in D. catenatum, which could be conducive to the synthesis of active bioactive compounds and their use as industrial by-products.

Identification and Expression Analysis of WRKY Gene Family in Response to Abiotic Stress in Dendrobium catenatum.

Dendrobium catenatum has become a rare and endangered medicinal plant due to habitat loss in China. As one of the most important and largest transcription factors, WRKY plays a critical role in response to abiotic stresses in plants. However, little is known regarding the functions of the WRKY family in D. catenatum. In this study, a total of 62 WRKY genes were identified from the D. catenatum genome. Phylogenetic analysis revealed that DcWRKY proteins could be divided into three groups, a division supported by the conserved motif compositions and intron/exon structures. DcWRKY gene expression and specific responses under drought, heat, cold and salt stresses were analyzed through RNA-seq data and RT-qPCR assay. The results showed that these genes had tissue-specificity and displayed different expression patterns in response to abiotic stresses. The expression levels of DcWRKY22, DcWRKY36 and DcWRKY45 were up-regulated by drought stress. Meanwhile, DcWRKY22 was highly induced by heat in roots, and DcWRKY45 was significantly induced by cold stress in leaves. Furthermore, DcWRKY27 in roots and DcWRKY58 in leaves were extremely induced under salt treatment. Finally, we found that all the five genes may function in ABA- and SA-dependent manners. This study identified candidate WRKY genes with possible roles in abiotic stress and these findings not only contribute to our understanding of WRKY family genes, but also provide valuable information for stress resistance development in D. catenatum.

Preparation, Structural Features and in vitro Immunostimulatory Activity of a Glucomannan From Fresh Dendrobium catenatum Stems.

Dendrobium catenatum polysaccharides (DCPs) have attracted attention due to their multiple physiological activities and health benefits. In this study, a novel water-soluble DCP was obtained from fresh D. catenatum stems through three-phase partitioning and ethanol precipitation at room temperature. Its structural characteristics, rheological property, and in vitro immunostimulatory activity were evaluated. Results demonstrated that DCP was a homogenous polysaccharide with a carbohydrate content of 92.75% and a weight-average molecular weight of 2.21 × 105 Da. This polysaccharide is an O-acetylated glucomannan comprised by glucose, mannose, and galacturonic acid in a molar ratio of 30.2:69.5:0.3 and mainly comprises (1→4)-β-D-mannopyranosyl (Manp), 2-O-acetyl-(1→4)-β-D-Manp, (1→6)-α-D-glucopyranosyl (Glcp), and (1→4)-α-D-Glcp residues. DCP exhibits an extended rigid chain in an aqueous solution and favorable steady shear fluid and dynamic viscoelastic behaviors. In vitro immunostimulating assays indicated that DCP activates RAW264.7 cells, thus markedly promoting macrophage proliferation and phagocytosis and increasing the levels of nitric oxide, interferon-γ, interleukin-6, and interleukin-1β. Moreover, the presence of O-acetyl group and high Mw in DCP might be responsible for its potent immunostimulatory activity in vitro. Therefore, our data suggested that DCP could be developed as a promising immunostimulant in functional food and pharmaceutical industries.

Cold Response Transcriptome Analysis of the Alternative Splicing Events Induced by the Cold Stress in D. catenatum.

Dendrobium catenatum Lindl is a valuable medicinal herb and gardening plant due to its ornamental value and special medical value. Low temperature is a major bottleneck restricting D. catenatum expansion towards the north, which influences the quality and yield of D. catenatum. In this study, we analysed the cold response of D. catenatum by RNA-Seq. A total of 4302 differentially expressed genes were detected under cold stress, which were mainly linked to protein kinase activity, membrane transport and the glycan biosynthesis and metabolism pathway. We also identified 4005 differential alternative events in 2319 genes significantly regulated by cold stress. Exon skipping and intron retention were the most common alternative splicing isoforms. Numerous genes were identified that differentially modulated under cold stress, including cold-induced transcription factors and splicing factors mediated by AS (alternative splicing). GO enrichment analysis found that differentially alternatively spliced genes without differential expression levels were related to RNA/mRNA processing and spliceosomes. DAS (differentially alternative splicing) genes with different expression levels were mainly enriched in protein kinase activity, plasma membrane and cellular response to stimulus. We further identified and cloned DcCBP20 in D. catenatum; we found that DcCBP20 promotes the generation of alternative splicing variants in cold-induced genes under cold stress via genetic experiments and RT–PCR. Taken together, our results identify the main cold-response pathways and alternative splicing events in D. catenatum responding to cold treatment and that DcCBP20 of D. catenatum get involved in regulating the AS and gene expression of cold-induced genes during this process. Our study will contribute to understanding the role of AS genes in regulating the cold stress response in D. catenatum.

Identification of stress-related characteristics of the <i>WRKY</i> gene family: A case study of <i>Dendrobium catenatum</i>

As one of the largest families of transcription factors (TFs) in plants, the WRKY TF family plays a key role in regulating plant responses to various biotic and abiotic stresses. However, there is no confirmed method to quickly identify stress-responsive members from the <italic>WRKY</italic> gene family. In this study, all reported functional <italic>WRKY</italic> genes were first analyzed, and the amino acid patterns in response to stress were identified in group II-c (T-R/K-S/T-E/Q/D-V/I/L-E/D-I/V/H/N-L/M-D/E-D-G/E-F/Y-K/R-WRKYG-Q/K-K-A/T-VKN-S/N-P), group II-d (VPA-I/V-S-X-K-M/L/V/I-ADIP-P/A/V-D-D/E-Y/F-S-WRKYGQKPIKGSP-H/Y-PRGYYKCS-S/T-V/M-RGCPARKVER), and group II-e (PSD-S/A/L-WAWRKYGQKPIKGSPYPR-G/S-YYRCSSSKGC). <italic>WRKY</italic> genes in<italic> Dendrobium catenatum</italic> were used to validate the accuracy of these patterns. A total of 63 <italic>DcaWRKY</italic> genes were identified, their gene structures, conserved motifs, and gene expression patterns were analyzed, and a phylogenetic tree was constructed. Gene expression patterns were then analyzed under drought stress, and seven <italic>DcaWRKY</italic> genes (<italic>Dca002550</italic>, <italic>Dca002715</italic>, <italic>Dca005648</italic>, <italic>Dca007842</italic>, <italic>Dca010430</italic>, <italic>Dca016437</italic>, and <italic>Dca006787</italic>) were randomly selected to determine their expression levels and verify their expression patterns by quantitative real-time polymerase chain reaction analysis. The identified amino acid patterns were validated by drought-responsive <italic>WRKY</italic> genes in <italic>D. catenatum</italic>, confirming the accuracy of these amino acid patterns and providing valuable insights into further research of the <italic>WRKY</italic> family in <italic>D. catenatum</italic>.

The Differences and Genetic Relationships of 4 Dendrobium Species Based on High Throughput GBS-SNP Markers

The differences and genetic relationships of 4 Dendrobium species ( Dendrobium huoshanense, Dendrobium officinale, Dendrobium moniliforme, Dendrobium Fanjingshanense ) were analyzed based on&nbsp;high throughput&nbsp;SNP markers obtained by GBS, using&nbsp; Dendrobium catenatum &nbsp;as the reference genome, for providing reference for the study of these 4 Dendrobium species. The results showed that the number of clean reads per each sample in&nbsp; D.&nbsp; huoshanense ,&nbsp; D .&nbsp; officinale ,&nbsp; D .&nbsp; moniliforme ,&nbsp; D . &nbsp;Fanjingshanense &nbsp;were 1 300 724, 1 286 162, 1 380 009 and 1 170 337, respectively, and the number of SNPs per each sample were 1 507 746, 893 333, 1 364 605 and 1 227 006, respectively. The genetic structure based on Admixture software revealed that the samples of&nbsp; D . &nbsp;officinale were clustered into one branch, and the remaining Dendrobium samples were clustered into the other branch, when the best K value was 2; when k = 3, the samples of&nbsp; D .&nbsp; officinale &nbsp;were grouped into one cluster, some samples of&nbsp; D . &nbsp;huoshanense , all samples of&nbsp; D .&nbsp; moniliforme &nbsp;and&nbsp; D. fanjingshanense &nbsp;were grouped into the second cluster, and some samples of&nbsp; D .&nbsp; huoshanense &nbsp;were grouped into the third cluster; When k = 4, it was divided into four branches: the samples of &nbsp;D .&nbsp; officinale &nbsp;were grouped one branch, the samples of&nbsp; D .&nbsp; moniliforme&nbsp; and &nbsp;D .&nbsp; fanjingshanense &nbsp;were grouped one branch, the samples of&nbsp; D .&nbsp; huoshanense were divided into two branches, which was similar to the result of an unrooted neighbor-joining phylogenetic tree (NJ tree); Furthermore, principal coordinates analyses&nbsp;(PCoA)&nbsp;revealed that 91 Dendrobium samples were classed into three cluster. All samples of&nbsp; D. of ficinale &nbsp;and&nbsp; D. hu oshanense &nbsp;were grouped together, respectively, and the samples of&nbsp; D. monil iforme&nbsp; and &nbsp;D . fanjingshan ense &nbsp;were grouped together. According to the results of this study, the genetic relation of&nbsp; D. off icinale &nbsp;and the other 3 species of Dendrobium are relatively distant, and the genetic relationship of&nbsp; D. mo niliforme &nbsp;and&nbsp; D.&nbsp; fanjingshanense &nbsp;were relatively close, 72 samples of&nbsp; D .&nbsp; huoshanense &nbsp;in the study&nbsp;were divided into two branches, the genetic relationship in one of which was relatively close to&nbsp; D .&nbsp; moniliforme &nbsp;and&nbsp; D .&nbsp; fanjingshanense . The study verified that the genetic relationship between different Dendrobium species or different species of plants was investigated using SNPs detected by GBS.

Genome-wide identification and analysis of the PAL genes from the orchids Apostasia shenzhenica, Dendrobium catenatum and Phalaenopsis equestris

Phenylalanine ammonia-lyase (PAL) is a key gateway enzyme that connects the phenylpropanoid pathway to primary metabolism. The phenylpropanoid pathway plays a vital role in the growth and environmental adaptation of many plants leading to the production of valuable bioactive compounds with industrial and medical applications. In the present study, nine putative PAL genes from three orchids were identified; five in Apostasia shenzhenica and two each in Dendrobium catenatum and Phalaenopsis equestris. Eighteen motifs and four major conserved functional domains were identified as reported in PAL proteins of other species. All the nine PALs were stable based on their computed physicochemical properties and localized in the cytoplasm. The three-dimensional structures of PALs revealed a homo-tetrameric structure consisting of four identical subunits. A total of 21 cis-regulatory elements with known functions were identified from the promoter regions of all PALs which are responsible for various plant responses to light, stress and growth regulators like auxins, gibberellins and abscisic acid. Phylogenetic analysis showed that the studied PAL proteins clustered in two major clades (clade I and II), placing dicot and monocot PALs in two separate monophyletic clades. In silico gene expression of the identified PALs in different vegetative and reproductive tissues revealed the differential expressions based on tissue type and disclosed that the expression of PAL genes was upregulated in all the tissues examined with an exception of PePAL leaf samples where no expression was detected, however, the same being highly expressed in reproductive tissues (PePAL1-labellum; PePAL2-sepal). In case of AsPALs, the expression was found to be highest in reproductive tissues (AsPAL4-maximum in inflorescence). On the other hand, the expression of DcPALs was found to be highest in vegetative tissues (DcPAL2-maximum in root). Based on the medicinal importance of orchids and the significant role of PAL genes in synthesis of bioactive compounds, the functional characterization of PAL genes can be further exploited in genetic improvement of medicinal orchids. Communicated by Ramaswamy H. Sarma

Genome-Wide Analysis of the TCP Transcription Factor Genes in Dendrobium catenatum Lindl.

Teosinte branched1/cycloidea/proliferating cell factor (TCP) gene family members are plant-specific transcription factors that regulate plant growth and development by controlling cell proliferation and differentiation. However, there are no reported studies on the TCP gene family in Dendrobium catenatum Lindl. Here, a genome-wide analysis of TCP genes was performed in D. catenatum, and 25 TCP genes were identified. A phylogenetic analysis classified the family into two clades: Class I and Class II. Genes in the same clade share similar conserved motifs. The GFP signals of the DcaTCP-GFPs were detected in the nuclei of tobacco leaf epidermal cells. The activity of DcaTCP4, which contains the miR319a-binding sequence, was reduced when combined with miR319a. A transient activity assay revealed antagonistic functions of Class I and Class II of the TCP proteins in controlling leaf development through the jasmonate-signaling pathway. After different phytohormone treatments, the DcaTCP genes showed varied expression patterns. In particular, DcaTCP4 and DcaTCP9 showed opposite trends after 3 h treatment with jasmonate. This comprehensive analysis provides a foundation for further studies on the roles of TCP genes in D. catenatum.