The HD-ZIP II gene PaHAT14 increases cuticle deposition by down-regulating ERF gene PaERF105 in Phalaenopsis.

To analyze the gene involved in orchid floral development, a HD-Zip II gene PaHAT14, which specifically and highly expressed in perianth during early flower development was identified from Phalaenopsis. Transgenic Arabidopsis plants expressing 35S::PaHAT14 and 35S::PaHAT14+SRDX (fused with the repressor motif SRDX) exhibited similar altered phenotypes, including small leaves, early flowering, and bending petals with increased cuticle production. This suggests that PaHAT14 acts as a repressor. In contrast, transgenic Arabidopsis plants expressing 35S::PaHAT14+VP16 (fused with the activation domain VP16) exhibited curled leaves, late flowering, and folded petals with decreased cuticle production within hardly opened flowers. Additionally, the expression of the ERF gene DEWAX2, which negatively regulates cuticular wax biosynthesis, was down-regulated in 35S::PaHAT14 and 35S::PaHAT14+SRDX transgenic Arabidopsis, while it was up-regulated in 35S::PaHAT14+VP16 transgenic Arabidopsis. Furthermore, transient overexpression of PaHAT14 in Phalaenopsis petal/sepal increased cuticle deposition due to the down-regulation of PaERF105, a Phalaenopsis DEWAX2 orthologue. On the other hand, transient overexpression of PaERF105 decreased cuticle deposition, whereas cuticle deposition increased and the rate of epidermal water loss was reduced in PaERF105 VIGS Phalaenopsis flowers. Moreover, ectopic expression of PaERF105 not only produced phenotypes similar to those in 35S::PaHAT14+VP16 Arabidopsis but also compensated for the altered phenotypes observed in 35S::PaHAT14 and 35S::PaHAT14+SRDX Arabidopsis. These results suggest that PaHAT14 promotes cuticle deposition by negatively regulating downstream gene PaERF105 in orchid flowers.

Comprehensive polyphenolic profiling, antioxidant, antimicrobial, and cytotoxicity studies of Rhynchostylis retusa (L.) Blume: a medicinal orchid.

Rhynchostylis retusa (L.) Blume, commonly known as the Foxtail orchid, has garnered worldwide attention for its diverse medicinal properties. In this study, root extract and its fractions were evaluated for total polyphenols, flavonoids, targeted polyphenols, and antioxidant potential. The antimicrobial activity was assessed against Gram-positive and Gram-negative bacterial strains while cytotoxicity was assessed using the A549 and HCT-116 cell lines. The investigations showed that chloroform and ethyl acetate are the most effective solvents for fractionation of polyphenols from the parent extract. These fractions also exhibited strong antioxidant and cytotoxic potentials. The chloroform fraction showed maximum cell death of 87.35 and 92.36% in A549 and HCT- 116 cell lines respectively. All samples showed growth inhibition against bacterial strains except the n-hexane fraction, whereas the n-butanol fraction showed comparable antimicrobial activity with the tetracycline standard. The possible health benefits and thereby, application of R. retusa were thus revealed in this investigation.

Orchid Mycorrhizal Association of Cultivated Dendrobium Hybrid and Their Role in Seed Germination and Seedling Growth.

Orchids are crucial for the horticulture industry. Mycorrhizal fungi benefit crops by improving nutrition, plant growth, and disease resistance. However, the mycorrhizal association of horticultural hybrid orchids is poorly understood. To address this, we investigated mycorrhizal colonization in the entire root system and assessed the mycorrhizal community using a Dendrobium cultivar, D. Stardust 'Firebird', obtained from three nurseries. Additionally, we isolated and tested mycorrhizal fungi in symbiotic culture to assess their role in the seed germination and growth of Dendrobium species. All plants were colonized by mycorrhizal fungi, with a higher colonization rate in mature than in juvenile plants. Molecular identification of mycorrhizal fungi by Sanger and high-throughput sequencing revealed that the cultivar was associated with a phylogenetically diverse group of fungi, including mycorrhizal fungi from Tulasnellaceae, and several wood-decaying fungi. The Tulasnellaceae isolates significantly enhanced the seed germination of three Dendrobium species and increased the survival rate and growth of asymbiotic seedlings of D. moniliforme. This study is the first comprehensive examination of mycorrhizal associations in horticultural orchid hybrids, providing valuable insights for commercial production.

Unlocking the Genetic Identity of Endangered Paphiopedilum Orchids: A DNA Barcoding Approach.

Orchids of the genus Paphiopedilum, also called slippers, are among the most valued representatives of the Orchidaceae family due to their aesthetic qualities. Due to overexploitation, deforestation, and illegal trade in these plants, especially in the vegetative phase, Paphiopedilum requires special protection. This genus is listed in Appendix I of the Convention on International Trade in Endangered Species of Wild Fauna and Flora. Their precise identification is of great importance for the preservation of genetic resources and biodiversity of the orchid family (Orchidaceae). Therefore, the main objective of the study was to investigate the usefulness of the DNA barcoding technique for the identification of endangered orchids of the genus Paphiopedilum and to determine the effectiveness of five loci: matK, rbcL, ITS2, atpF-atpH and trnH-psbA as potential molecular markers for species of this genus. Among single locus barcodes, matK was the most effective at identifying species (64%). Furthermore, matK, ITS2, matK + rbcL, and matK + trnH-psbA barcodes can be successfully used as a complementary tool to identify Paphiopedilum orchids while supporting morphological data provided by taxonomists.

Temporal turnover of Ceratobasidiaceae orchid mycorrhizal fungal communities with ontogenetic and phenological development in Prasophyllum (Orchidaceae).

BACKGROUND AND AIMS: Plant-fungus symbioses may experience temporal turnover during the host's ontogenetic or phenological development, which can influence the host plant's ecological requirements. This study investigates temporal turnover of Ceratobasidiaceae orchid mycorrhizal fungal (OMF) communities in Prasophyllum (Orchidaceae), asking if OMF communities are subject to temporal change due to orchid phenology or ontogeny. METHODS: Roots of adult Prasophyllum frenchii, P. lindleyanum and P. sp. aff. validum from Australia were sampled between autumn and spring. Seed was sown in situ as 'baits' to explore the mycorrhizal associations of germinating protocorms, which were compared to OMF in roots of co-occurring adult plants. Culture dependent and independent sequencing methods were used to amplify the internal transcribed spacer and mitochondrial large subunit loci, with sequences assigned to Operational Taxonomic Units (OTUs) in phylogenetic analyses. Germination trials were used to determine if fungal OTUs were mycorrhizal. KEY RESULTS: A persistent core of OMF associated with Prasophyllum, with Ceratobasidiaceae OMF dominant in all three species. Phenological turnover occurred in P. lindleyanum and P. sp. aff. validum, but not in P. frenchii, which displayed specificity to a single OTU. Ontogenetic turnover occurred in all species. However, phenological and ontogenetic turnover was typically driven by the presence or absence of infrequently detected OTUs in populations that otherwise displayed specificity to one or two dominant OTUs. Ex situ germination trials showed 13 of 14 tested OTUs supported seed germination in their host orchid, including eight OTUs that were not found in protocorms in situ. CONCLUSIONS: An understanding of OMF turnover can have practical importance for the conservation of threatened orchids and their mycorrhizal partners. However, frameworks for classifying OMF turnover should focus on OTUs important to the life cycle of the host plant, which we suggest are likely to be those that are frequently detected or functionally significant.

Osmophore Structure and Labellum Micromorphology in Ophrys speculum (Orchidaceae): New Interpretations of Floral Features and Implications for a Specific Sexually Deceptive Pollination Interaction.

Pollination by sexual deception specifically attracts male insects, through the floral scent and particular morphological features of the flower that serve as visual and tactile stimuli. The unique bond between the Ophrys speculum orchid and the male Dasyscolia ciliata wasp primarily stems from a few distinctive semiochemicals that mimic the female wasp's sex pheromone, although the floral scent comprises a variety of compounds. An osmophore producing highly volatile compounds has been documented in four close relatives of O. speculum and is now being also investigated in this species. Given the existing debates regarding the structure of the labellum and stigmatic cavity in O. speculum, this study details their micromorphology. Additionally, comparisons of O. speculum flowers and female D. ciliata wasps under stereomicroscopy and scanning electron microscopy are conducted to seek new evidence of visual and tactile mimicry. The findings confirm that (i) an osmophore is present at the apical margin of the labellum in O. speculum flowers; (ii) the labellum features a distinct basal field homologous to those found in other Ophrys species; and (iii) the basal labellum region closely mimics the female wasp's thorax and wings. The implications of these novel floral features are discussed within an evolutionary context.

D-optimal Design Optimization of Solvent Mixture for Flavonoid Extraction from Phalaenopsis Leaves with Antioxidant Activity.

Aims: This study aimed to optimize the extraction of flavonoids and antioxidants from Phalaenopsis leaves by using solvent mixtures. Method: The total flavonoid content (TFC) and antioxidant activity were evaluated using the colorimetric method and ferric-reducing antioxidant power (FRAP), respectively. Maceration extracts from fresh leaves were used for the analysis. The study used the Design Expert 13.0 program to optimize the solvents (water, acetone, and methanol) and their combined ratio. Result: The results showed that 100% acetone was the best solvent for both responses, with a desirability value of 0.884, TFC of 0.434 mg QE/g fresh weight (FW) and FRAP of 713.53 µmol TE/g FW. Screening of the most potent Phalaenopsis genotypes for obtaining the most active leaf extract showed that P. amboinensis and P. pantherina were the best genotypes for TFC (0.786-0.797 mg QE/g FW) and FRAP activity (862.25-891.48 µmol TE/g FW). Conclusion: This study demonstrates an easy and useful way to obtain flavonoids and antioxidants from Phalaenopsis materials that can be used in the flower-based industry to make new functional ingredients.

Perspective and challenges of mycorrhizal symbiosis in orchid medicinal plants.

The family Orchidaceae is of the most diverse taxon in the plant kingdom, and most of its members are highly valuable herbal medicines. Orchids have a unique mycorrhizal symbiotic relationship with specific fungi for carbohydrate and nutrient supplies in their whole lifecycle. The large-scale cultivation of the medicinal plant Gastodia elata is a successful example of using mycorrhizal symbiotic technology. In this review, we adopted G. elata and Dendrobium officinale as examples to describe the characteristics of orchid mycorrhiza and mycorrhizal benefits for host plants' growth and health (e.g. biotic and abiotic stress and secondary metabolite accumulation). The challenges in applying mycorrhizal technology to the cultivation of orchid medicinal plants in the future were also discussed. This review aims to serve as a theoretical guide for the cultivation of mycorrhizal technology in medicinal orchid plants.

MIR396-GRF/GIF enhances in planta shoot regeneration of Dendrobium catenatum.

Recent studies on co-transformation of the growth regulator, TaGRF4-GIF1 chimera (Growth Regulating Factor 4-GRF Interacting Factor 1), in cultivated wheat varieties (Triticum aestivum), showed improved regeneration efficiency, marking a significant breakthrough. Here, a simple and reproducible protocol using the GRF4-GIF1 chimera was established and tested in the medicinal orchid Dendrobium catenatum, a monocot orchid species. TaGRF4-GIF1 from T. aestivum and DcGRF4-GIF1 from D. catenatum were reconstructed, with the chimeras significantly enhancing the regeneration efficiency of D. catenatum through in planta transformation. Further, mutating the microRNA396 (miR396) target sites in TaGRF4 and DcGRF4 improved regeneration efficiency. The target mimicry version of miR396 (MIM396) not only boosted shoot regeneration but also enhanced plant growth. Our methods revealed a powerful tool for the enhanced regeneration and genetic transformation of D. catenatum.

Development and Application of a Duplex RT-RPA Assay for the Simultaneous Detection of Cymbidium mosaic virus and Odontoglossum ringspot virus.

Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV) are among the world's most serious and widespread orchid viruses; they often infect orchids, causing devastating losses to the orchid industry. Therefore, it is critical to establish a method that can rapidly and accurately detect viruses in the field using simple instruments, which will largely reduce the further spread of viruses and improve the quality of the orchid industry and is suitable for mass promotion and application at grassroots agrotechnical service points. In this investigation, we established a rapid amplification method for virus detection at 39 °C for 35 min to detect the presence of CymMV and ORSV simultaneously, sensitively, and specifically in orchids. Primers for the capsid protein (CP)-encoding genes of both viruses were designed and screened, and the reaction conditions were optimized. The experimental amplification process was completed in just 35 min at 39 °C. There were no instances of nonspecific amplification observed when nine other viruses were present. The RPA approach had detection limits of 104 and 103 copies for pMD19T-CymMV and pMD19T-ORSV, respectively. Moreover, the duplex RT-RPA investigation confirmed sensitivity and accuracy via a comparison of detection results from 20 field samples with those of a gene chip. This study presents a precise and reliable detection method for CymMV and ORSV using RT-RPA. The results demonstrate the potential of this method for rapid virus detection. It is evident that this method could have practical applications in virus detection processes.

Identification and Characterization of the AREB/ABF Gene Family in Three Orchid Species and Functional Analysis of DcaABI5 in Arabidopsis.

AREB/ABF (ABA response element binding) proteins in plants are essential for stress responses, while our understanding of AREB/ABFs from orchid species, important traditional medicinal and ornamental plants, is limited. Here, twelve AREB/ABF genes were identified within three orchids' complete genomes and classified into three groups through phylogenetic analysis, which was further supported with a combined analysis of their conserved motifs and gene structures. The cis-element analysis revealed that hormone response elements as well as light and stress response elements were widely rich in the AREB/ABFs. A prediction analysis of the orchid ABRE/ABF-mediated regulatory network was further constructed through cis-regulatory element (CRE) analysis of their promoter regions. And it revealed that several dominant transcriptional factor (TF) gene families were abundant as potential regulators of these orchid AREB/ABFs. Expression profile analysis using public transcriptomic data suggested that most AREB/ABF genes have distinct tissue-specific expression patterns in orchid plants. Additionally, DcaABI5 as a homolog of ABA INSENSITIVE 5 (ABI5) from Arabidopsis was selected for further analysis. The results showed that transgenic Arabidopsis overexpressing DcaABI5 could rescue the ABA-insensitive phenotype in the mutant abi5. Collectively, these findings will provide valuable information on AREB/ABF genes in orchids.

The road less taken: Dihydroflavonol 4-reductase inactivation and delphinidin anthocyanin loss underpins a natural intraspecific flower colour variation.

Visual cues are of critical importance for the attraction of animal pollinators, however, little is known about the molecular mechanisms underpinning intraspecific floral colour variation. Here, we combined comparative spectral analysis, targeted metabolite profiling, multi-tissue transcriptomics, differential gene expression, sequence analysis and functional analysis to investigate a bee-pollinated orchid species, Glossodia major with common purple- and infrequent white-flowered morphs. We found uncommon and previously unreported delphinidin-based anthocyanins responsible for the conspicuous and pollinator-perceivable colour of the purple morph and three genetic changes underpinning the loss of colour in the white morph - (1) a loss-of-function (LOF; frameshift) mutation affecting dihydroflavonol 4-reductase (DFR1) coding sequence due to a unique 4-bp insertion, (2) specific downregulation of functional DFR1 expression and (3) the unexpected discovery of chimeric Gypsy transposable element (TE)-gene (DFR) transcripts with potential consequences to the genomic stability and post-transcriptional or epigenetic regulation of DFR. This is one of few known cases where regulatory changes and LOF mutation in an anthocyanin structural gene, rather than transcription factors, are important. Furthermore, if TEs prove to be a frequent source of mutation, the interplay between environmental stress-induced TE evolution and pollinator-mediated selection for adaptive colour variation may be an overlooked mechanism maintaining floral colour polymorphism in nature.

Cymensifin A: a promising pharmaceutical candidate to defeat lung cancer via cellular reactive oxygen species-mediated apoptosis.

Background: The upgrade of natural products for cancer treatment is essential since current anticancer drugs still pose severe side effects. Cymensifin A (Cym A) isolated from an orchid Cymbidium ensifolium has shown its potential to induce the death of several cancer cells; however, its underlying molecular mechanisms are hitherto unknown. Methods: Here, we conducted a set of in vitro preliminary tests to assess the cytotoxic effects of Cym A on non-small-cell lung cancer (NSCLC) cells (A549, H23, H292, and H460). A flow cytometry system and Western blot analyses were employed to unveil molecular mechanisms underlying cancer cell apoptosis caused by Cym A. Results: Cym A at 25-50 µM caused the death of all NSCLC cells tested, and its cytotoxicity was comparable to cisplatin, a currently used anticancer drug. The compound induced apoptosis of all NSCLC cells in a dose-dependent manner (5-50 µM), proven by flow cytometry, but H460 cells showed more resistance compared to other cells tested. Cym A-treated H460 cells demonstrated increased reactive oxygen species (ROS) and downregulated antioxidants (catalase, superoxide dismutase, and thioredoxin). The compound also upregulated the tumor suppressor P53 and the pro-apoptotic protein BAX but downregulated pro-survival proteins (BCL-2 and MCL-1) and deactivated survival signals (AKT and ERK) in H460 cells. Cym A was proven to trigger cellular ROS formation, but P53 and BAX were 2-fold more activated by Cym A compared to those treated with hydrogen peroxide. Our findings also supported that Cym A exerted its roles in the downregulation of nuclear factor erythroid 2-related factor 2 (a regulator of cellular antioxidant activity) and the increased levels of cleaved poly (ADP-ribose) polymerase (PARP) and cleaved caspase 3/7 during apoptosis. Conclusion: We propose that Cym A induces lung cancer cell death via ROS-mediated apoptosis, while the modulation of cellular ROS/antioxidant activity, the upregulation of P53 and BAX, the downregulation or deactivation of BCL-2, MCL-1, AKT, and ERK, and the increased cleavage of PARP and caspase 3/7, were the elucidated underlying molecular mechanisms of this phytochemical. The compound can be a promising candidate for future anticancer drug development.

We know very little about pollination in the Platanthera Rich (Orchidaceae: Orchidoideae).

The Platanthera Rich. (Orchidoideae) comprise a speciose genus of orchids primarily in the northern hemisphere, with up to 200 known species worldwide. Individual species are known to self-pollinate, but many rely on insect pollinators with characteristics such as floral color, timing of floral odor emissions, nectar rewards, and spur length associated with particular pollination syndromes. As with many orchids, some orchid-pollinator associations are likely highly co-evolved, but we also know that some Platanthera spp. are the result of hybridization events, which implies a lack of pollinator fidelity in some cases. Some Platanthera spp. occur in large numbers which, coupled with the numerous Platanthera-pollinator systems, make them accessible as study species and useful for co-evolutionary studies. Due to the likely effects of climate change and ongoing development on Platanthera spp. habitats, these orchids and their associated pollinators should be a focus of conservation attention and management. However, while there is a fairly substantial literature coverage of Platanthera-pollinator occurrence and interactions, there are still wide gaps in our understanding of the species involved in these systems. In this systematic review, we outline what is current knowledge and provide guidance on further research that will increase our understanding of orchid-insect co-evolutionary relationships. Our review covers 157 orchid species and about 233 pollinator species interacting with 30 Platanthera spp. We provide analyses on aspects of these interactions such as flower morphology, known insect partners of Platanthera species, insect-Platanthera specificity, pollination visitor timing (diurnal vs. nocturnal), floral rewards, and insect behavior affecting pollination outcomes (e.g., pollinia placement). A substantial number of Platanthera spp. and at least a few of their known pollinators are of official (IUCN) conservation concern - and many of their pollinators remain unassessed or even currently unknown - which adds to the urgency of further research on these co-evolved relationships.

Co-occurring orchid species associated with different low-abundance mycorrhizal fungi from the soil in a high-diversity conservation area in Denmark.

Plant-fungal interactions are ubiquitous across ecosystems and contribute significantly to plant ecology and evolution. All orchids form obligate symbiotic relationships with specific fungi for germination and early growth, and the distribution of terrestrial orchid species has been linked to occurrence and abundance of specific orchid mycorrhizal fungi (OMF) in the soil. The availability of OMF can therefore be a habitat requirement that is relevant to consider when establishing management and conservation strategies for threatened orchid species, but knowledge on the spatial distribution of OMF in soil is limited. We here studied the mycorrhizal associations of three terrestrial orchid species (Anacamptis pyramidalis, Orchis purpurea and Platanthera chlorantha) found in a local orchid diversity hotspot in eastern Denmark, and investigated the abundance of the identified mycorrhizal fungi in the surrounding soil. We applied ITS metabarcoding to samples of orchid roots, rhizosphere soil and bulk soil collected at three localities, supplemented with standard barcoding of root samples with OMF specific primers, and detected 22 Operational Taxonomic Units (OTUs) putatively identified as OMF. The three orchid species displayed different patterns of OMF associations, supporting the theory that association with specific fungi constitutes part of an orchid's ecological niche allowing co-occurrence of many species in orchid-rich habitats. The identified mycorrhizal partners in the basidiomycete families Tulasnellaceae and Ceratobasidiaceae (Cantharallales) were detected in low abundance in rhizosphere soil, and appeared almost absent from bulk soil at the localities. This finding highlights our limited knowledge of the ecology and trophic mode of OMF outside orchid tissues, as well as challenges in the detection of specific OMF with standard methods. Potential implications for management and conservation strategies are discussed.

Antimicrobial potential, GCMS analysis and molecular docking studies of Coelogyne suaveolens extracts: Identification of bioactive compounds with mechanism of action.

Coelogyne suaveolens has been used as a traditional medicine for many years, and its potential as a natural source of antibacterial agents is of great interest. This investigation aimed to identify the bioactive compounds in the plant extract and assess their antibacterial properties. To achieve this, we identified the bioactive compounds using Gas chromatography mass spectrometry (GCMS) analysis on the extract's ethyl acetate fraction and used the disc diffusion method to determine the antibacterial effect. Additionally, molecular docking were performed to predict the binding affinities of selected phytochemicals against specific proteins in order to identify the root cause of bacterial inhibition. Our results revealed that the extract exhibited significant antibacterial activity against Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, and Klebsiella pneumoniae, which are common and problematic pathogens. Furthermore, molecular docking studies identified eight best-selected compounds, of which {androstan-17-one, oxime, (5.alpha.)-}, diethofencarb, tetraconazole, {3,6-dimethyl-2,3,3a,4,5,7a-hexahydrobenzofuran}, and geranyl acetate showed a significant binding affinity with best binding interaction with the target enzymes. This suggests that binding to these specific proteins might lead to the mechanism of action of the evaluated antibacterial action. In conclusion, the present study contributes to the growing body of knowledge on natural antimicrobial agents and could have significant implications for the development of new and effective antibacterial agents.

Transcriptome data reveals the conservation genetics of Cypripedium forrestii, a plant species with extremely small populations endemic to Yunnan, China.

The Cypripedium forrestii is an orchid species with extremely small populations (PSESP) in Yunnan, China. C. forrestii is range-restricted and less-studied than many orchid species, and it is exposed to various threats to its survival. We investigated its potential habitats and collected 52 samples from eight locations, as well as two outgroup species for reference. We developed genetic markers (SNPs) for C. forrestii based on transcriptome sequencing (RNA-seq) data, and analyzed the genetic diversity, population structure, gene flow and demographic history of C. forrestii in detail. C. forrestii is a taxonomically independent species to protect. We found that the genetic diversity of C. forrestii was very low (1.7e-4) compared with other endangered species. We identified three genetic clusters, and several populations with distinct genetic backgrounds. Most genetic diversity was found within sampling sites (87.87%) and genetic clusters (91.39%). Gene flow has been greatly limited over the most recent generations, probably due to geographical distance, historical climate change and habitat fragmentation. We also detected a severe bottleneck event brought about by the recent population constraints. These factors, together with its reproductive characteristics, contribute to the population fragmentation and low genetic diversity of C. forrestii. Based on our findings, we suggest an integrative conservation strategy to protect and recover the genetic diversity of C. forrestii and a further comprehensive study of its ecological traits in the future.

Complete genome sequence of new Microbacterium sp. strain ET2, isolated from roots of leafless orchid.

Whole-genome sequence of ET2 strain, isolated from the roots of leafless orchid, constitutes a single circular chromosome of 3,604,840 bp (69.44% G + C content). BLAST+-based average nucleotide identity (ANIb) and digital DNA-DNA hybridization values indicate that ET2 may be a novel Microbacterium species. Genes putatively involved in plant-microbial interactions were predicted.