Terpene synthases (TPSs) are crucial for the diversification of terpenes, catalyzing the formation of a wide variety of terpenoid compounds. However, genome-wide systematic characterization of TPS genes in Cymbidium ensifolium has not been reported. Within the genomic database of C. ensifolium, we found 30 CeTPS genes for this investigation. CeTPS genes were irregularly distributed throughout the seven chromosomes and primarily expanded through tandem duplications. The CeTPS proteins were classified into three TPS subfamilies, including 17 TPS-b members, 8 TPS-a members, and 5 TPS-c members. Conserved motif analysis showed that most CeTPSs contained DDxxD and RRX₈W motifs. Cis-element analysis of CeTPS gene promoters indicated regulation primarily by plant hormones and stress. Transcriptome analysis revealed that CeTPS1 and CeTPS18 had high expression in C. ensifolium flowers. qRT-PCR results showed that CeTPS1 and CeTPS18 were predominantly expressed during the flowering stage. Furthermore, CeTPS1 and CeTPS18 proteins were localized in the chloroplasts. These results lay the theoretical groundwork for future research on the functions of CeTPSs in terpenoid biosynthesis.

In the Red List of Threatened Species, released by International Union for Conservation of Nature (IUCN), Paphiopedilum helenae has been classified as an endangered species. It exhibits exceptional decorative value and germplasm resource potential. To elucidate the ecological adaptation of this species and the characteristics of its rhizosphere microbiome, bacterial 16S rRNA and fungal ITS sequences of three wild populations of P. helenae were investigated using Illumina high-throughput sequencing technology and the microbial community structures and diversities were systematically compared. These three populations were spanned across distinct geographical locations in Longzhou County, Guangxi. The results showed that the bacterial community in the rhizosphere soil of P. helenae comprised 31 phyla, primarily including Actinobacteriota, Proteobacteria, Chloroflexi and Acidobacteriota. On the other hand, the fungal community consisted of 10 phyla, dominated by Ascomycota and Basidiomycota. There were significant differences in the diversity of rhizosphere microbes across different populations of P. helenae. The LG population had the highest bacterial richness (Chao index: 2912.71 ± 131.73; p < 0.05) and diversity (Shannon index: 6.40 ± 0.06; p < 0.01), while the MQ population had the lowest diversity (Shannon index: 3.47 ± 0.24; p < 0.01) of fungi. The degree of variation in fungal β-diversity was significantly higher than that of bacteria. Soil organic matter (SOM) and available nitrogen (AN) contents were the core factors shaping the microbial communities in the rhizosphere soil of P. helenae, which jointly explained 49.87% and 16.39% of variations in the bacterial and fungal communities. Furthermore, population-specific enrichment of functionally significant microorganisms was evident. Population MQ was enriched with plant growth-promoting and stress-resistant fungi, such as Geminibasidium, Trichoderma, etc. Population LG was enriched with oligotrophic bacteria (e.g., Patescibacteria), while population SL exhibited an overwhelming dominance of Ascomycota (93.25%) and enrichment of pathogenic fungal genus Nigrospora. This research revealed the variations in the functional adaptation strategy of P. helenae and the microbial communities in the rhizosphere soils across different geographical locations. This suggests that microbial community imbalance in rhizosphere soil may be one of the factors leading to the endangerment of this plant species. The study proposed a differentiated protection strategy for endangered plant species based on microbial resources. The results provide a theoretical basis for development of a “microorganism-assisted protection” strategy for ecological restoration and sustainable utilization of endangered orchid plants.

This study explores the metabolite diversity and potential medicinal value of three Paphiopedilum species—P. dianthum, P. micranthum, and P. barbigerum—using widely targeted metabolomics via HPLC-MS/MS in conjunction with in vitro antioxidant assays. A total of 2201 metabolites were detected across the three species, with flavonoids emerging as the dominant class (480 compounds, accounting for 21.8% of total metabolites). Comparative metabolomic analysis showed that flavonoid levels varied most prominently among the species. Notably, the metabolic profile of P. barbigerum (PB) diverged substantially from those of P. dianthum (PD) and P. micranthum (PM), which shared a higher degree of similarity with each other. Quantitative evaluation of antioxidant-associated metabolites revealed that PB exhibited the greatest enrichment in compounds with antioxidant potential, particularly flavonoids and phenolic acids, followed by PM and PD. These results were corroborated by antioxidant assays, in which PB demonstrated the highest free radical scavenging activity, with PM and PD displaying progressively lower effects. Differences in flavonoid content likely underpin these variations in antioxidant capacity. KEGG pathway enrichment analysis indicated that differentially expressed metabolites were primarily involved in flavonoid-associated biosynthetic routes, notably flavonoid biosynthesis (ko00941) and isoflavonoid biosynthesis (ko00943), with ko00941 being the most enriched. Within this pathway, PB showed eight significantly upregulated flavonoid metabolites, while PM and PD had seven and five, respectively. The observed differences may stem from species-specific expression of key biosynthetic enzymes such as flavonoid 3′-hydroxylase (F3′H) in PM and flavonoid 3′,5′-hydroxylase (F3′5′H) in PB, which influence both flavonoid composition and antioxidant potential.