Anoectochilus roxburghii is a traditional Chinese herb with high medicinal value, with main bioactive constituents which are flavonoids. It commonly associates with mycorrhizal fungi for its growth and development. Moreover, mycorrhizal fungi can induce changes in the internal metabolism of host plants. However, its role in the flavonoid accumulation in A. roxburghii at different growth stages is not well studied. In this study, combined metabolome and transcriptome analyses were performed to investigate the metabolic and transcriptional profiling in mycorrhizal A. roxburghii (M) and non-mycorrhizal A. roxburghii (NM) growth for six months. An association analysis revealed that flavonoid biosynthetic pathway presented significant differences between the M and NM. Additionally, the structural genes related to flavonoid synthesis and different flavonoid metabolites in both groups over a period of six months were validated using quantitative real-time polymerase chain reaction (qRT-PCR) and high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS). The results showed that Ceratobasidium sp. AR2 could increase the accumulation of five flavonol-glycosides (i.e., narcissin, rutin, isorhamnetin-3-O-beta-d-glucoside, quercetin-7-O-glucoside, and kaempferol-3-O-glucoside), two flavonols (i.e., quercetin and isorhamnetin), and two flavones (i.e., nobiletin and tangeretin) to some degrees. The qRT-PCR showed that the flavonoid biosynthetic genes (PAL, 4CL, CHS, GT, and RT) were significantly differentially expressed between the M and NM. Overall, our findings indicate that AR2 induces flavonoid metabolism in A. roxburghii during different growth stages, especially in the third month. This shows great potential of Ceratobasidium sp. AR2 for the quality improvement of A. roxburghii.

Dendrobium officinale Kimura et Migo is a traditional and scarce medicinal orchid in China. Mycorrhizal fungi could supply nitrogen (N) to orchids for seed germination and seedling recruitment. However, the N transport mechanism between orchids and the fungus is poorly understand. Early studies found that the fungus MF23 (Mycena sp.) could promote the growth of D. officinale. To better dissect the molecular interactions involved in N transport between D. officinale and MF23, transcriptome and metabolome analyses were conducted on conventional and mycorrhizal cultivations of D. officinale. Moreover, validation tests were carried out in the greenhouse to measure net fluxes of NO3− and NH4+ of roots by a non-invasive micro-test technology (NMT), determine N assimilation enzyme activity by the ELISA, and analyze the expression level of differentially expressed genes (DEGs) of N transporters and DEGs involved in N metabolism by RT-qPCR. Combined transcriptome and metabolome analyses showed that MF23 may influence N metabolism in D. officinale. The expression of DoNAR2.1 (nitrate transporter-activating protein), DoAMT11 (ammonium transporter), DoATFs (amino acid transporters), DoOPTs (oligopeptide transporters), and DoGDHs (glutamate dehydrogenases) in symbiotic D. officinale was upregulated. NMT results showed a preference for NH4+ in D. officinale and indicated that MF23 could promote the uptake of NO3− andNH4+, especially for NH4+. ELISA results showed that MF23 could increase the activity of glutamine synthetase (GS) and glutamate dehydrogenase. This study suggested that MF23 increases the production of D. officinale by affecting N uptake and NH4+ assimilation capacity.

Gastrodia elata, a famous herbal medicine, has been received great attention on its treatments of headache, vertigo and epilepsy. Gastrodia nantoensis is a newly described species from central Taiwan with potential medicinal value. Gastrodia species are fully mycoheterotrophic orchids, and the courses of their seed development are more rapid as compared to the chlorophyllous orchids. A better understanding of their reproductive biology would provide insights into the propagation and conservation of the mycoheterotrophic orchid species. Based on the histological and histochemical investigations, we observed some notable features in ovule and embryo development. First, only the archesporial cell and/or megasporocyte are present within their ovaries at the time of anthesis. Second, their suspensors consist of a single cell and their mature embryos consist of a gradient of small to large cells. Nile red staining of a globular embryo reveals the presence of cuticular material in the surface wall of embryo proper and the lateral walls of suspensor cell, indicating that the basal wall of suspensor cell is the major route for nutrient supply from maternal tissues to embryo proper. Third, their seed coats are derived from a single integument, and lignin but not cuticular material is present in the outer most layer of seed coat and persists through seed maturation. The faster seed development of Gastrodia species is due to the speedy courses of ovule and embryo development. In the mature seeds, the presence of a differentiated apical zone in embryo proper suggests the easy-to-germinate character. This study provides basic knowledge for further molecular studies on embryo development and symbiotic germination of Gastrodia species.