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.

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.

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.

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.

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.

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.

Orchids acquire fungal carbon for seed germination: pathways and players.

To germinate in nature, orchid seeds strictly rely on seed germination-promoting orchid mycorrhizal fungi (sgOMFs) for provision of carbon nutrients. The underlying delivery pathway, however, remains elusive. We develop here a plausible model for sugar transport from sgOMFs to orchid embryonic cells to fuel germination. Orchids exploit sgOMFs to induce the formation of pelotons, elaborate intracellular hyphal coils in orchid embryos. The colonized orchid cells then obtain carbon nutrients by uptake from living hyphae and peloton lysis, primarily as glucose derived from fungal trehalose hydrolyzed by orchid-specific trehalases. The uptake of massive fungally derived glucose is likely to be mediated by two classes of membrane proteins, namely, sugars will eventually be exported transporters (SWEETs) and H+-hexose symporters. The proposed model serves as a launch pad for further research to better understand and improve orchid seed germination and conservation.

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.

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.

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.

Orchid seeds are not always short lived in a conventional seed bank!

BACKGROUND AND AIMS: Orchid seeds are reputed to be short lived in dry, cold storage conditions, potentially limiting the use of conventional seed banks for long-term ex situ conservation. This work explores whether Cattleya seeds are long lived or not during conventional storage (predried to ~12 % relative humidity, then stored at -18 °C). METHODS: We explored the possible interaction of factors influencing seed lifespan in eight species of the genus Cattleya using physiological (germination and vigour), biochemical (gas chromatography), biophysical (differential scanning calorimetry) and morphometric methods. Seeds were desiccated to ~3 % moisture content and stored at -18 °C for more than a decade, and seed quality was measured via three in vitro germination techniques. Tetrazolium staining was also used to monitor seed viability during storage. The morphometric and germination data were subjected to ANOVA and cluster analysis, and seed lifespan was subjected to probit analysis. KEY RESULTS: Seeds of all Cattleya species were found to be desiccation tolerant, with predicted storage lifespans (P50y) of ~30 years for six species and much longer for two species. Cluster analysis showed that the three species with the longest-lived seeds had smaller (9-11 %) airspaces around the embryo. The post-storage germination method impacted the quality assessment; seeds equilibrated at room temperature for 24 h or in 10 % sucrose solution had improved germination, particularly for the seeds with the smallest embryos. Chromatography revealed that the seeds of all eight species were rich in linoleic acid, and differential scanning calorimetry identified a peak that might be auxiliary to selecting long-lived seeds. CONCLUSIONS: These findings show that not all orchids produce seeds that are short lived, and our trait analyses might help to strengthen prediction of seed longevity in diverse orchid species.

The mycorrhizal symbiosis: research frontiers in genomics, ecology, and agricultural application.

Mycorrhizal symbioses between plants and fungi are vital for the soil structure, nutrient cycling, plant diversity, and ecosystem sustainability. More than 250 000 plant species are associated with mycorrhizal fungi. Recent advances in genomics and related approaches have revolutionized our understanding of the biology and ecology of mycorrhizal associations. The genomes of 250+ mycorrhizal fungi have been released and hundreds of genes that play pivotal roles in regulating symbiosis development and metabolism have been characterized. rDNA metabarcoding and metatranscriptomics provide novel insights into the ecological cues driving mycorrhizal communities and functions expressed by these associations, linking genes to ecological traits such as nutrient acquisition and soil organic matter decomposition. Here, we review genomic studies that have revealed genes involved in nutrient uptake and symbiosis development, and discuss adaptations that are fundamental to the evolution of mycorrhizal lifestyles. We also evaluated the ecosystem services provided by mycorrhizal networks and discuss how mycorrhizal symbioses hold promise for sustainable agriculture and forestry by enhancing nutrient acquisition and stress tolerance. Overall, unraveling the intricate dynamics of mycorrhizal symbioses is paramount for promoting ecological sustainability and addressing current pressing environmental concerns. This review ends with major frontiers for further research.

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.

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.

Micropropagation of Guarianthe skinneri (Bateman) Dressler & W. E. Higging in Temporary Immersion Bioreactors.

Guarianthe skinneri (Bateman) Dressler & W. E. Higgins is an orchid valued for its ornamental characteristics. However, it is an orchid classified as threatened with extinction due to the illegal extraction from its natural habitat. In addition, its propagation through seed germination is very low, as is the case with most members of the family Orchidaceae. Its asexual propagation through pseudobulb separation is slow and produces a few propagules. For this reason, in vitro propagation techniques are an alternative to increase the number of plants obtained and thus be able to recover this valuable plant genetic resource. Temporary immersion systems (TIS) offer the advantage of mass-propagating plants for different purposes. This chapter describes a large-scale micropropagation protocol for Guarianthe skinneri using temporary immersion bioreactors (TIB).

Micropropagation of Encyclia cordigera (Kunth) Dressler in Ebb-and-Flow Bioreactor.

The use of new technologies for micropropagation such as temporary immersion systems (TISs) is important, because it reduces costs by 40% lowering labor, agar and containers. TISs are containers designed for large-scale, semiautomatic production of plants in a liquid medium, which has been used in propagation of commercial orchids. This tool has high potential for application in micropropagation of medicinal and endangered orchids for conservation and commercial purposes. In this chapter, we describe a detailed protocol for propagation and development of Encyclia cordigera to be used in research projects for small-scale production. This protocol comprises all steps from explant preparation to the establishment orchids plantlets.

Orchid Micropropagation Using Temporary Immersion Systems: A Review.

Temporary immersion systems (TIS) have been used for orchid micropropagation. The main advantage of TIS use for micropropagation is that the explant is periodically immersed in nutrient media, and then, the nutrient solution is drained, which allows the explant tissue to stay in air. The current review resumes the application of TIS in orchid propagation. Fifty-three papers are discussed considering: explant, culture media, TIS bioreactor type, frequency and immersion time, and the TIS effects in acclimatization phase.