[Influence of Tis108 on GA content and expression of key enzyme GeCYP714A1 involved in GA deactivation of Gastrodia elata].

To investigate the influence of the strigolactone inhibitor Tis108 on the growth of Gastrodia elata, this study treated G. elata tuber with Tis108 solution of 10 µmol·L~(-1) and measured the content of endogenous hormone gibberellin(GA) in the tuber. By using reverse transcription-polymerase chain reaction(RT-PCR) technology, the key enzyme GeCYP714A1 gene involved in GA deactivation was cloned. Bioinformatics analysis on the GeCYP714A1 gene was carried out by using ExPASy, SWISS-MODEL, MEGA, etc., and its expression levels in different parts of G. elata were determined. The results showed that after Tis108 treatment, GA content in G. elata tuber was significantly increased, and the transcription level of the GeCYP714A1 gene was significantly decreased. The full length of the coding region of the GeCYP714A1 gene is 1 173 bp, encoding 390 amino acids. The protein has a molecular weight of 44.85 kDa, a theoretical isoelectric point of 9.83, an instability index of 49.20, an aliphatic index of 89.03, and a grand average of hydropathicity of-0.235, classifying it as an unstable, basic, hydrophilic protein, and the GeCYP714A1 protein was localized in the mitochondria, lacking a signal peptide and a transmembrane structure. Phylogenetic tree analysis revealed that GeCYP714A1 was most closely related to the DcCYP714C2(PKU78454.1) protein from Dendrobium candidum, with a sequence identity of 67.25%. The qRT-PCR analysis of the expression patterns of the GeCYP714A1 gene indicated that GeCYP714A1 had the highest transcription level in G. elata tuber, followed by stem and inflorescence. The study represented that Tis108 inhibited the transcription level of GeCYP714A1 involved in GA deactivation in G. elata tuber, thereby increasing the accumulation of GA and affecting the growth of G. elata tuber. These results provided a basis for further studies of strigolactone regulation of GA signal and tuber development in G. elata.

Characteristics and functions of volatile organic compounds in the tripartite symbiotic system of Gastrodia elata-Armillaria gallica-Rahnella aceris HPDA25.

Tripartite interactions among plants, fungi, and bacteria are critical for maintaining plant growth and fitness, and volatile organic compounds (VOCs) play a significant role in these interactions. However, the functions of VOCs within the niche of mycoheterotrophic plants, which represent unique types of interactions, remain poorly understood. Gastrodia elata, a mycoheterotrophic orchid species, forms a symbiotic relationship with specific Armillaria species, serving as a model system to investigate this intriguing issue. Rahnella aceris HPDA25 is a plant growth-promoting bacteria isolated from G. elata, which has been found to facilitate the establishment of G. elata-Armillaria symbiosis. In this study, using the tripartite symbiotic system of G. elata-Armillaria gallica-R. aceris HPDA25, we investigate the role of VOCs in the interaction among mycoheterotrophic plants, fungi, and bacteria. Our results showed that 33 VOCs of HPDA25-inducible symbiotic G. elata elevated compared to non-symbiotic G. elata, indicating that VOCs indeed play a role in the symbiotic process. Among these, 21 VOCs were accessible, and six active VOCs showed complete growth inhibition activities against A. gallica, while R. aceris HPDA25 had no significant effect. In addition, three key genes of G. elata have been identified that may contribute to the increased concentration of six active VOCs. These results revealed for the first time the VOCs profile of G. elata and demonstrated its regulatory role in the tripartite symbiotic system involving G. elata, Armillaria, and bacteria.

Genome-wide identification of glycoside hydrolase family 1 members reveals GeBGL1 and GeBGL9 for degrading gastrodin in Gastrodia elata.

KEY MESSAGE: We revealed the intrinsic transformation molecular mechanism of gastrodin by two ß-d-glucosidases (GeBGL1 and GeBGL9) during the processing of Gastrodia elata. Gastrodia elata is a plant resource with medicinal and edible functions, and its active ingredient is gastrodin. However, the intrinsic transformation molecular mechanism of gastrodin in G. elata has not been verified. We speculated that ß-d-glucosidase (BGL) may be the key enzymes hydrolyzing gastrodin. Here, we identified 11 GeBGL genes in the G. elata genome. These genes were unevenly distributed on seven chromosomes. These GeBGL proteins possessed motifs necessary for catalysis, namely, TF(I/M/L)N(T)E(Q)P and I(V/L)T(H/S)ENG(S). These GeBGLs were divided into five subgroups together with homologous genes from Arabidopsis thaliana, rice, and maize. Quantitative real-time PCR analysis showed GeBGL genes expression was tissue-specific. Gene cloning results showed two mutation sites in the GeBGL1 gene compared with the reference genome. And, the GeBGL4 gene has two indel fragments, which resulted in premature termination of translation and seemed to turn into a pseudogene. Furthermore, protein expression and enzyme activity results proved that GeBGL1 and GeBGL9 have the activity of hydrolyzing gastrodin into 4-hydroxybenzyl alcohol. This study revealed the function of ß-d-glucosidase in degrading active compounds during the G. elata processing for medicinal purposes. These results offer a theoretical foundation for elevating the standard and enhancing the quality of G. elata production.

Experimental study on the detection of Gastrodia elata by enzymatic recombinase amplification and immunochromatography.

OBJECTIVE: The objective of this research is to develop two methodologies, Enzymatic recombinase amplification (ERA) and Polymerase Chain Reaction (PCR) coupled with Lateral Flow Dipstick (LFD), for the swift authentication of Gastrodia elata. METHODOLOGY: Primers and nfo probes for the ERA of Gastrodia elata were developed based on the ITS2 genome sequences of Gastrodia elata and its counterfeits. Specific primers for the PCR analysis of Gastrodia elata were generated using the NCBI (National Center for Biotechnology Information) online platform. Through experimental validation, the optimal reaction system and conditions for both methodologies were established, and their efficacy was assessed. RESULTS: The methodologies developed herein are applicable for the targeted analysis of the medicinal species, Gastrodia elata. The sensitivity of the ERA-LFD detection method matched that of the conventional PCR-LFD approach, recorded at 1 ng µL-1. Consistency was observed in the results across three replicates of visualization test strips for both techniques. Upon evaluation, both the PCR-LFD and ERA-LFD methods demonstrated a total compliance rate of 100 %. CONCLUSION: The ERA-LFD and PCR-LFD methods facilitate reduced detection times and offer visual results. These techniques are particularly effective for on-site detection and quality control in the authentication of Gastrodia elata within traditional Chinese medicine markets and at the primary level of healthcare provision.

Diverse Mycena Fungi and Their Potential for Gastrodia elata Germination.

It remains to be determined whether there is a geographical distribution pattern and phylogenetic signals for the Mycena strains with seed germination of the orchid plant Gastrodia elata. This study analyzed the community composition and phylogenetics of 72 Mycena strains associated with G. elata varieties (G. elata. f. glauca and G. elata. f. viridis) using multiple gene fragments (ITS+nLSU+SSU). We found that (1) these diverse Mycena phylogenetically belong to the Basidiospore amyloid group. (2) There is a phylogenetic signal of Mycena for germination of G. elata. Those strains phylogenetically close to M. abramsii, M. polygramma, and an unclassified Mycena had significantly higher germination rates than those to M. citrinomarginata. (3) The Mycena distribution depends on geographic site and G. elata variety. Both unclassified Mycena group 1 and the M. abramsii group were dominant for the two varieties of G. elata; in contrast, the M. citrinomarginata group was dominant in G. elata f. glauca but absent in G. elata f. viridis. Our results indicate that the community composition of numerous Mycena resources in the Zhaotong area varies by geographical location and G. elata variety. Importantly, our results also indicate that Mycena's phylogenetic status is correlated with its germination rate.

First report of the complete mitochondrial genome of 3 beetles (Coleoptera: Scarabaeidae) harming Gastrodia elata (Asparagales: Orchidaceae).

Gastrodia elata Blume, a valuable traditional Chinese medicine with significant clinical and nutritional importance, is a fungal heterotrophic orchid. We present the first report of the mitochondrial genome structure and characteristics of 3 Scarabaeidae pests affecting G. elata: Sophrops peronosporus Gu & Zhang, Anomala rufiventris Kollar & Redtenbacher, and Callistethus plagiicollis Fairmaire. Each mitogenome contained 13 protein-coding genes (PCGs), 22 transfer RNAs (tRNAs), 2 ribosomal RNAs (rRNAs), and a control region, with no gene rearrangements observed. All 21 tRNAs, except trnS1 that lacks a dihydrouridine, had a stable cloverleaf secondary structure. Maximum likelihood and Bayesian inference analyses based on the 13 PCGs produced 2 topologically similar phylogenetic trees, both of with high nodal support. Larvae of these Scarabaeidae pests cause substantial damage by gnawing on the tubers and roots of G. elata, leading to reduced yield and compromised quality. These findings contribute to phylogenetic studies of Scarabaeidae, expand knowledge of G. elata pests, and offer valuable reference materials for their identification and control.

Neuroprotection and Mechanism of Gas-miR36-5p from Gastrodia elata in an Alzheimer's Disease Model by Regulating Glycogen Synthase Kinase-3ß.

Alzheimer's disease (AD) is currently the most common neurodegenerative disease. Glycogen synthase kinase 3ß (GSK-3ß) is a pivotal factor in AD pathogenesis. Recent research has demonstrated that plant miRNAs exert cross-kingdom regulation on the target genes in animals. Gastrodia elata (G. elata) is a valuable traditional Chinese medicine that has significant pharmacological activity against diseases of the central nervous system (CNS). Our previous studies have indicated that G. elata-specific miRNA plays a cross-kingdom regulatory role for the NF-κB signaling pathway in mice. In this study, further bioinformatics analysis suggested that Gas-miR36-5p targets GSK-3ß. Through western blot, RT-qPCR, and assessments of T-AOC, SOD, and MDA levels, Gas-miR36-5p demonstrated its neuroprotective effects in an AD cell model. Furthermore, Gas-miR36-5p was detected in the murine brain tissues. The results of the Morris water maze test and western blot analysis provided positive evidence for reversing the learning deficits and hyperphosphorylation of Tau in AD mice, elucidating significant neuroprotective effects in an AD model following G. elata RNA administration. Our research emphasizes Gas-miR36-5p as a novel G. elata-specific miRNA with neuroprotective properties in Alzheimer's disease by targeting GSK-3ß. Consequently, our findings provide valuable insights into the cross-kingdom regulatory mechanisms underlying G. elata-specific miRNA, presenting a novel perspective for the treatment of Alzheimer's disease.

Ancient Horizontal Gene Transfers from Plastome to Mitogenome of a Nonphotosynthetic Orchid, Gastrodia pubilabiata (Epidendroideae, Orchidaceae).

Gastrodia pubilabiata is a nonphotosynthetic and mycoheterotrophic orchid belonging to subfamily Epidendroideae. Compared to other typical angiosperm species, the plastome of G. pubilabiata is dramatically reduced in size to only 30,698 base pairs (bp). This reduction has led to the loss of most photosynthesis-related genes and some housekeeping genes in the plastome, which now only contains 19 protein coding genes, three tRNAs, and three rRNAs. In contrast, the typical orchid species contains 79 protein coding genes, 30 tRNAs, and four rRNAs. This study decoded the entire mitogenome of G. pubilabiata, which consisted of 44 contigs with a total length of 867,349 bp. Its mitogenome contained 38 protein coding genes, nine tRNAs, and three rRNAs. The gene content of G. pubilabiata mitogenome is similar to the typical plant mitogenomes even though the mitogenome size is twice as large as the typical ones. To determine possible gene transfer events between the plastome and the mitogenome individual BLASTN searches were conducted, using all available orchid plastome sequences and flowering plant mitogenome sequences. Plastid rRNA fragments were found at a high frequency in the mitogenome. Seven plastid protein coding gene fragments (ndhC, ndhJ, ndhK, psaA, psbF, rpoB, and rps4) were also identified in the mitogenome of G. pubilabiata. Phylogenetic trees using these seven plastid protein coding gene fragments suggested that horizontal gene transfer (HGT) from plastome to mitogenome occurred before losses of photosynthesis related genes, leading to the lineage of G. pubilabiata. Compared to species phylogeny of the lineage of orchid, it was estimated that HGT might have occurred approximately 30 million years ago.

[Cloning and gene function of dicarboxylate-tricarboxylate carrier protein in Gastrodia elata].

The gene GeDTC encoding the dicarboxylate-tricarboxylate carrier protein in Gastrodia elata was cloned by specific primers which were designed based on the transcriptome data of G. elata. Bioinformatics analysis on GeDTC gene was carried out by using ExPASY, ClustalW, MEGA, etc. Positive transgenic plants and potato minituber were obtained by virtue of the potato genetic transformation system. Agronomic characters, such as size, weight, organic acid content, and starch content, of potato minituber were tested and analyzed and GeDTC gene function was preliminarily investigated. The results showed that the open reading frame of GeDTC gene was 981 bp in length and 326 amino acid residues were encoded, with a relative molecular weight of 35.01 kDa. It was predicted that the theoretical isoelectric point of GeDTC protein was 9.83, the instability coefficient was 27.88, and the average index of hydrophilicity was 0.104, which was indicative of a stable hydrophilic protein. GeDTC protein had a transmembrane structure and no signal peptide and was located in the inner membrane of mitochondria. The phylogenetic tree showed that GeDTC was highly homologous with DTC proteins of other plant species, among which GeDTC had the highest homology with DcDTC(XP＿020675804.1) in Dendrobium candidum, reaching 85.89%. GeDTC overexpression vector pCambia1300-35Spro-GeDTC was constructed by double digests, and transgenic potato plants were obtained by Agrobacterium-mediated gene transformation. Compared with the wild-type plants, transgenic potato minituber harvested by transplanting had smaller size, lighter weight, lower organic acid content, and no significant difference in starch content. It is preliminarily induced that GeDTC is the efflux channel of tricarboxylate and related to the tuber development, which lays a foundation for further elucidating the molecular mechanism of G. elata tuber development.

Whole genome sequencing and analysis of Armillaria gallica Jzi34 symbiotic with Gastrodia elata.

BACKGROUND: Armillaria species are plant pathogens, but a few Armillaria species can establish a symbiotic relationship with Gastrodia elata, a rootless and leafless orchid, that is used as a Chinese herbal medicine. Armillaria is a nutrient source for the growth of G. elata. However, there are few reports on the molecular mechanism of symbiosis between Armillaria species and G. elata. The genome sequencing and analysis of Armillaria symbiotic with G. elata would provide genomic information for further studying the molecular mechanism of symbiosis. RESULTS: The de novo genome assembly was performed with the PacBio Sequel platform and Illumina NovaSeq PE150 for the A. gallica Jzi34 strain, which was symbiotic with G. elata. Its genome assembly contained ~ 79.9 Mbp and consisted of 60 contigs with an N50 of 2,535,910 bp. There were only 4.1% repetitive sequences in the genome assembly. Functional annotation analysis revealed a total of 16,280 protein coding genes. Compared with the other five genomes of Armillaria, the carbohydrate enzyme gene family of the genome was significantly contracted, while it had the largest set of glycosyl transferase (GT) genes. It also had an expansion of auxiliary activity enzymes AA3-2 gene subfamily and cytochrome P450 genes. The synteny analysis result of P450 genes reveals that the evolutionary relationship of P450 proteins between A. gallica Jzi34 and other four Armillaria was complex. CONCLUSIONS: These characteristics may be beneficial for establishing a symbiotic relationship with G. elata. These results explore the characteristics of A. gallica Jzi34 from a genomic perspective and provide an important genomic resource for further detailed study of Armillaria. This will help to further study the symbiotic mechanism between A. gallica and G. elata.

GelFAP v2.0: an improved platform for Gene functional analysis in Gastrodia elata.

BACKGROUND: Gastrodia elata (tianma), a well-known medicinal orchid, is widely used to treat various kinds of diseases with its dried tuber. In recent years, new chromosome-level genomes of G.elata have been released in succession, which offer an enormous resource pool for understanding gene function. Previously we have constructed GelFAP for gene functional analysis of G.elata. As genomes are updated and transcriptome data is accumulated, collection data in GelFAP cannot meet the need of researchers. RESULTS: Based on new chromosome-level genome and transcriptome data, we constructed co-expression network of G. elata, and then we annotated genes by aligning with sequences from NR, TAIR, Uniprot and Swissprot database. GO (Gene Ontology) and KEGG (Kyoto Encylopaedia of Genes and Genomes) annotations were predicted by InterProScan and GhostKOALA software. Gene families were further predicted by iTAK (Plant Transcription factor and Protein kinase Identifier and Classifier), HMMER (hidden Markov models), InParanoid. Finally, we developed an improved platform for gene functional analysis in G. elata (GelFAP v2.0) by integrating new genome, transcriptome data and processed functional annotation. Several tools were also introduced to platform including BLAST (Basic Local Alignment Search Tool), GSEA (Gene Set Enrichment Analysis), Heatmap, JBrowse, Motif analysis and Sequence extraction. Based on this platform, we found that the flavonoid biosynthesis might be regulated by transcription factors (TFs) such as MYB, HB and NAC. We also took C4H and GAFP4 as examples to show the usage of our platform. CONCLUSION: An improved platform for gene functional analysis in G. elata (GelFAP v2.0, www.gzybioinformatics.cn/Gelv2 ) was constructed, which provides better genome data, more transcriptome resources and more analysis tools. The updated platform might be preferably benefit researchers to carry out gene functional research for their project.

Transcriptomic heterochrony and completely cleistogamous flower development in the mycoheterotrophic orchid Gastrodia.

Cleistogamy, in which plants can reproduce via self-fertilization within permanently closed flowers, has evolved in > 30 angiosperm lineages; however, consistent with Darwin's doubts about its existence, complete cleistogamy - the production of only cleistogamous flowers - has rarely been recognized. Thus far, the achlorophyllous orchid genus, Gastrodia, is the only known genus with several plausible completely cleistogamous species. Here, we analyzed the floral developmental transcriptomes of two recently evolved, completely cleistogamous Gastrodia species and their chasmogamous sister species to elucidate the possible changes involved in producing common cleistogamous traits. The ABBA-BABA test did not support introgression and protein sequence convergence as evolutionary mechanisms leading to cleistogamy, leaving convergence in gene expression as a plausible mechanism. Regarding transcriptomic differentiation, the two cleistogamous species had common modifications in the expression of developmental regulators, exhibiting a gene family-wide signature of convergent expression changes in MADS-box genes. Our transcriptomic pseudotime analysis revealed a prolonged juvenile state and eventual maturation, a heterochronic pattern consistent with partial neoteny, in cleistogamous flower development. These findings indicate that transcriptomic partial neoteny, arising from changes in the expression of conserved developmental regulators, might have contributed to the rapid and repeated evolution of cleistogamous flowers in Gastrodia.

Cloning and gene function of dicarboxylate-tricarboxylate carrier protein in Gastrodia elata / 中国中药杂志

The gene GeDTC encoding the dicarboxylate-tricarboxylate carrier protein in Gastrodia elata was cloned by specific primers which were designed based on the transcriptome data of G. elata. Bioinformatics analysis on GeDTC gene was carried out by using ExPASY, ClustalW, MEGA, etc. Positive transgenic plants and potato minituber were obtained by virtue of the potato genetic transformation system. Agronomic characters, such as size, weight, organic acid content, and starch content, of potato minituber were tested and analyzed and GeDTC gene function was preliminarily investigated. The results showed that the open reading frame of GeDTC gene was 981 bp in length and 326 amino acid residues were encoded, with a relative molecular weight of 35.01 kDa. It was predicted that the theoretical isoelectric point of GeDTC protein was 9.83, the instability coefficient was 27.88, and the average index of hydrophilicity was 0.104, which was indicative of a stable hydrophilic protein. GeDTC protein had a transmembrane structure and no signal peptide and was located in the inner membrane of mitochondria. The phylogenetic tree showed that GeDTC was highly homologous with DTC proteins of other plant species, among which GeDTC had the highest homology with DcDTC(XP＿020675804.1) in Dendrobium candidum, reaching 85.89%. GeDTC overexpression vector pCambia1300-35Spro-GeDTC was constructed by double digests, and transgenic potato plants were obtained by Agrobacterium-mediated gene transformation. Compared with the wild-type plants, transgenic potato minituber harvested by transplanting had smaller size, lighter weight, lower organic acid content, and no significant difference in starch content. It is preliminarily induced that GeDTC is the efflux channel of tricarboxylate and related to the tuber development, which lays a foundation for further elucidating the molecular mechanism of G. elata tuber development.

[Construction of protoplast genetic transformation system for Mycena--symbiont of Gastrodia elata].

Mycena, a symbiont of Gastrodia elata, promotes seed germination of G. elata and plays a crucial role in the sexual reproduction of G. elata. However, the lack of genetic transformation system of Mycena blocks the research on the interaction mechanism of the two. In order to establish the protoplast transformation system of Mycena, this study analyzed the protoplast enzymatic hydrolysis system, screened the resistance markers and regeneration medium, and explored the transient transformation. After hydrolysis of Mycena hyphae with complexes enzymes for 8 h and centrifugation at 4 000 r·min~(-1), high-concentration and quality protoplast was obtained. The optimum regeneration medium for Mycena was RMV, and the optimum resistance marker was 50 mg·mL~(-1) hygromycin. The pLH-HygB-HuSHXG-GFP-HdSHXG was transformed into the protoplast of Mycena which then expressed GFP. The established protoplast transformation system of Mycena laid a foundation for analyzing the functional genes of Mycena and the molecular mechanism of the symbiosis of Mycena and G. elata.

The Gastrodia menghaiensis (Orchidaceae) genome provides new insights of orchid mycorrhizal interactions.

BACKGROUND: To illustrate the molecular mechanism of mycoheterotrophic interactions between orchids and fungi, we assembled chromosome-level reference genome of Gastrodia menghaiensis (Orchidaceae) and analyzed the genomes of two species of Gastrodia. RESULTS: Our analyses indicated that the genomes of Gastrodia are globally diminished in comparison to autotrophic orchids, even compared to Cuscuta (a plant parasite). Genes involved in arbuscular mycorrhizae colonization were found in genomes of Gastrodia, and many of the genes involved biological interaction between Gatrodia and symbiotic microbionts are more numerous than in photosynthetic orchids. The highly expressed genes for fatty acid and ammonium root transporters suggest that fungi receive material from orchids, although most raw materials flow from the fungi. Many nuclear genes (e.g. biosynthesis of aromatic amino acid L-tryptophan) supporting plastid functions are expanded compared to photosynthetic orchids, an indication of the importance of plastids even in totally mycoheterotrophic species. CONCLUSION: Gastrodia menghaiensis has the smallest proteome thus far among angiosperms. Many of the genes involved biological interaction between Gatrodia and symbiotic microbionts are more numerous than in photosynthetic orchids.

Chromosome-level genome assembly of the fully mycoheterotrophic orchid Gastrodia elata.

Gastrodia elata, an obligate mycoheterotrophic orchid, requires complete carbon and mineral nutrient supplementation from mycorrhizal fungi during its entire life cycle. Although full mycoheterotrophy occurs most often in the Orchidaceae family, no chromosome-level reference genome from this group has been assembled to date. Here, we report a high-quality chromosome-level genome assembly of G. elata, using Illumina and PacBio sequencing methods with Hi-C technique. The assembled genome size was found to be 1045 Mb, with an N50 of 50.6 Mb and 488 scaffolds. A total of 935 complete (64.9%) matches to the 1440 embryophyte Benchmarking Universal Single-Copy Orthologs were identified in this genome assembly. Hi-C scaffolding of the assembled genome resulted in 18 pseudochromosomes, 1008 Mb in size and containing 96.5% of the scaffolds. A total of 18,844 protein-coding sequences (CDSs) were predicted in the G. elata genome, of which 15,619 CDSs (82.89%) were functionally annotated. In addition, 74.92% of the assembled genome was found to be composed of transposable elements. Phylogenetic analysis indicated a significant contraction of genes involved in various biosynthetic processes and cellular components and an expansion of genes for novel metabolic processes and mycorrhizal association. This result suggests an evolutionary adaptation of G. elata to a mycoheterotrophic lifestyle. In summary, the genomic resources generated in this study will provide a valuable reference genome for investigating the molecular mechanisms of G. elata biological functions. Furthermore, the complete G. elata genome will greatly improve our understanding of the genetics of Orchidaceae and its mycoheterotrophic evolution.

Construction of protoplast genetic transformation system for Mycena--symbiont of Gastrodia elata / 中国中药杂志

Mycena, a symbiont of Gastrodia elata, promotes seed germination of G. elata and plays a crucial role in the sexual reproduction of G. elata. However, the lack of genetic transformation system of Mycena blocks the research on the interaction mechanism of the two. In order to establish the protoplast transformation system of Mycena, this study analyzed the protoplast enzymatic hydrolysis system, screened the resistance markers and regeneration medium, and explored the transient transformation. After hydrolysis of Mycena hyphae with complexes enzymes for 8 h and centrifugation at 4 000 r·min~(-1), high-concentration and quality protoplast was obtained. The optimum regeneration medium for Mycena was RMV, and the optimum resistance marker was 50 mg·mL~(-1) hygromycin. The pLH-HygB-HuSHXG-GFP-HdSHXG was transformed into the protoplast of Mycena which then expressed GFP. The established protoplast transformation system of Mycena laid a foundation for analyzing the functional genes of Mycena and the molecular mechanism of the symbiosis of Mycena and G. elata.

A chromosome-scale Gastrodia elata genome and large-scale comparative genomic analysis indicate convergent evolution by gene loss in mycoheterotrophic and parasitic plants.

Mycoheterotrophic and parasitic plants are heterotrophic and parasitize on fungi and plants, respectively, to obtain nutrients. Large-scale comparative genomics analysis has not been conducted in mycoheterotrophic or parasitic plants or between these two groups of parasites. We assembled a chromosome-level genome of the fully mycoheterotrophic plant Gastrodia elata (Orchidaceae) and performed comparative genomic analyses on the genomes of G. elata and four orchids (initial mycoheterotrophs), three parasitic plants (Cuscuta australis, Striga asiatica, and Sapria himalayana), and 36 autotrophs from various angiosperm lineages. It was found that while in the hemiparasite S. asiatica and initial mycoheterotrophic orchids, approximately 4-5% of the conserved orthogroups were lost, the fully heterotrophic G. elata and C. australis both lost approximately 10% of the conserved orthogroups, indicating that increased heterotrophy is positively associated with gene loss. Importantly, many genes that are essential for autotrophs, including those involved in photosynthesis, the circadian clock, flowering time regulation, immunity, nutrient uptake, and root and leaf development, were convergently lost in both G. elata and C. australis. The high-quality genome of G. elata will facilitate future studies on the physiology, ecology, and evolution of mycoheterotrophic plants, and our findings highlight the critical role of gene loss in the evolution of plants with heterotrophic lifestyles.

Transcriptome Reveals Roles of Lignin-Modifying Enzymes and Abscisic Acid in the Symbiosis of Mycena and Gastrodia elata.

Gastrodia elata is a well-known medicinal and heterotrophic orchid. Its germination, limited by the impermeability of seed coat lignin and inhibition by abscisic acid (ABA), is triggered by symbiosis with fungi such as Mycena spp. However, the molecular mechanisms of lignin degradation by Mycena and ABA biosynthesis and signaling in G. elata remain unclear. In order to gain insights into these two processes, this study analyzed the transcriptomes of these organisms during their dynamic symbiosis. Among the 25 lignin-modifying enzyme genes in Mycena, two ligninolytic class II peroxidases and two laccases were significantly upregulated, most likely enabling Mycena hyphae to break through the lignin seed coats of G. elata. Genes related to reduced virulence and loss of pathogenicity in Mycena accounted for more than half of annotated genes, presumably contributing to symbiosis. After coculture, upregulated genes outnumbered downregulated genes in G. elata seeds, suggesting slightly increased biological activity, while Mycena hyphae had fewer upregulated than downregulated genes, indicating decreased biological activity. ABA biosynthesis in G. elata was reduced by the downregulated expression of 9-cis-epoxycarotenoid dioxygenase (NCED-2), and ABA signaling was blocked by the downregulated expression of a receptor protein (PYL12-like). This is the first report to describe the role of NCED-2 and PYL12-like in breaking G. elata seed dormancy by reducing the synthesis and blocking the signaling of the germination inhibitor ABA. This study provides a theoretical basis for screening germination fungi to identify effective symbionts and for reducing ABA inhibition of G. elata seed germination.

Comparative transcriptome analysis of tubers, stems, and flowers of Gastrodia elata Blume reveals potential genes involved in the biosynthesis of phenolics.

Orchidaceae, well known for its fascinating flowers, is one of the largest and most diverse families of flowering plants. There are many kinds of plants in this family; these are distributed practically globally and have high ornamental and medicinal values. Gastrodia elata Blume, a traditional Chinese medicinal herb, is a rootless and leafless achlorophyllous orchid. Phenolic compounds are considered to be the major bioactive constituents in G. elata, with antioxidant, antiangiogenic, neuroprotective, antidepressant, anxiolytic, and sedative activities. In this study, we determined the contents of six main phenolic components in tubers, stems and flowers from G. elata. Meanwhile, the transcriptomes of the tuber, stem and flower tissues of G. elata were obtained using the BGISEQ-500 platform. A total of 58.29 Gb of data and 113,067 unigenes were obtained, of which 74,820 unigenes were functionally annotated against seven public databases. Differentially expressed genes between tuber, stem and flower tissues were identified. A total of 76 DEGs encoding eight key enzymes were identified as candidate genes involved in the biosynthesis of phenolics in G. elata. For further validation, the expression levels of unigenes were measured using quantitative real-time PCR. Our results greatly enrich the transcriptomic data of G. elata and provide valuable information for the identification of candidate genes involved in the biosynthesis of secondary metabolites.