Shanghai Chenshan Plant Science Research Center

Dracocephalum moldavica L., an annual herb valued for its medicinal and ornamental properties, produces flavonoid glycosides like apigenin 7-O-glucuronide, scutellarein-7-O-glucuronide, and vitexin, which offer cardiovascular benefits. However, the UDP-glycosyltransferases (UGTs) involved in their biosynthesis have not been fully characterized. In the present investigation, we identified five UGTs, which comprise two bifunctional flavonoid UDP-glucuronosyl/glucosyltransferase genes, DmUGT1 and DmUGT2; two flavonoid UDP-glucosyltransferase genes, DmUGT3 and DmUGT4; and one type I di-C-glycosyltransferase gene, DmCGT1. The UDP-glucuronosyl/glucosyltransferase DmUGT1 showed effective glycosylation activity and exhibited a wide substrate promiscuity, facilitating the synthesis of the principal flavonoid glycosides in D. moldavica, including bioactive compounds such as scutellarein-7-O-glucuronide. Homology modeling and site-directed mutagenesis of the bifunctional DmUGT1 indicated that the amino acids Ser127 and Tyr373 are critical determinants of sugar donor specificity. DmCGT1 could catalyze phloretin to form phloretin-3'-C-glycoside and phloretin-3',5'-di-C-glycoside. Additionally, we engineered Escherichia coli strains that utilized DmUGT1 and DmCGT1, complemented with plasmids designed to enhance the intracellular supply of UDP-glucuronic acid and UDP-glucose in E. coli. These engineered strains successfully enabled the in vivo production of scutellarein-7-O-glucuronide and phloretin-3',5'-di-C-glycoside, achieving yields of 195 and 196 mg/L, respectively. This study provides a systematic elucidation of the glycosylation mechanisms of flavonoids in D. moldavica and offers candidate genes and methodologies for the biosynthesis of bioactive glycoside compounds through synthetic biology approaches.

Background: Shancigu is a traditional Chinese medicine which is effective at clearing heat, detoxifying, dissipating masses, and resolving nodules. It consists of the dried pseudobulbs of orchids such as Cremastra appendiculata, or Pleione yunnanensis. To deeply understand the differences in the compositional and pharmacological active compounds in Shancigu, this study employed widely targeted metabolomics to analyze differential metabolites between two Shancigu species, C. appendiculata and P. yunnanensis. Methods: In this study, ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) was used to qualitatively, quantitatively, and differentially analyze the metabolites of C. appendiculata and P. yunnanensis. Results: Metabolite profiling identified 2890 compounds across 13 classes. Within these, 687 metabolites showed significant differential abundance (23.76% total), including 331 upregulated and 356 downregulated compounds. Pathway enrichment analysis revealed these differential metabolites primarily concentrated in stilbenoid biosynthesis (types I and II) and flavonoid aglycone biosynthesis. The most highly expressed metabolites in the Cremastra group were L-tyrosine, dopamine and 3,4-dihydroxybenzaldehyde-xylose-glucoside, while in the Pleione group, the most abundant metabolites were 3,5-dihydroxy-2’-methoxy-4-methylbibenzyl, Shancigusin F and aloifol I. C. appendiculata preferentially accumulates flavonoids and phenolic acids whereas P. yunnanensis favors terpenoid and nucleotide derivative production. Conclusions: This study identifies key differential metabolites in C. appendiculata and P. yunnanensis, providing basic data for the overall evaluation and breeding of Shancigu, laying a foundation for further quality control and precise medication of Shancigu.

This study established an efficient in vitro regeneration system using stem nodes from root collar suckers as explants. Subsequently, regenerated shoots were used to establish an in vitro medicinal production protocol that achieved ginkgolic acid production. The self-developed Ginkgo biloba medium (GBM), first reported in this study, was pivotal to system establishment. The plantlet propagation system showed that the bases of stem nodes dipped in GBM with 2 mg·L−1 6-benzyladenine (BA) and 0.2 mg·L−1 1-naphthaleneacetic acid (NAA) achieved near-complete axillary bud induction (99.56%). Adventitious shoot induction reached 82.22% (3.5 shoots/explant) using GBM with 0.2 mg·L−1 BA, 0.02 mg·L−1 kinetin (Kin) and 0.2 g·L−1 proline (Pro). Maximum adventitious shoot elongation (92.22%, average 3.35 cm) was observed on GBM containing 0.1 mg·L−1 zeatin (ZT) and 0.01 mg·L−1 BA. After 3-week preculture with 15 mg·L−1 phloroglucinol (PG), treatment with 0.6 mg·L−1 indole-3-butyric acid (IBA) and 0.2% activated carbon (AC) yielded 96.67% rooting (6.19 roots/explant) and 85% acclimatization survival. For medicinal resource production, bud cluster induction at 94.44% (20.89 buds/explant) on GBM with 1 mg·L−1 BA, 0.03 mg·L−1 Kin, and 0.2 g·L−1 Pro. Leaf organs in GBM with 0.3 mg·L−1 BA, 0.01 mg·L−1 Kin, 0.01 mg·L−1 IBA, 0.3 g·L−1 Pro, and 0.01 mg·L−1 glutamine (Gln) accumulated 20.64 g fresh weight and 41.910 mg·g−1 DW ginkgolic acids, representing a 4.93-fold increase over mother plants. This system enables large-scale Ginkgo biloba L. propagation and provides an in vitro strategy for producing medicinal compounds in endangered plants.

Some fig species introduced outside of their native range have become invasive when colonized by their obligate pollinating wasps, but how these pollinators migrated and adapted to novel environments are less studied. Here, we focus on Eupristina verticillata, the obligate pollinating wasp of an invasive fig tree species (Ficus microcarpa), to uncover its demography and the molecular basis for adaptations to novel environments. We find that only one of the three cryptic species colonized in the sampling locations outside of its native range. This dominant cryptic species migrated simultaneously from the native range to the Americas and to the Mediterranean c. 130 years ago. Moreover, selective sweep analyses reveal several positively selected genes associated with adaptations to the nonnative range. Genome-wide association detect a nonsynonymous substitution in a dopamine N-acetyltransferase gene significantly linked with brood size. Our study outlines the route to colonization and genetic adaptations of an invasive mutualism.

Abstract Background Angraecum sesquipedale, commonly known as Darwin’s orchid, is a distinctive ornamental species uniquely susceptible to biotic and abiotic stresses, primarily owing to its confinement to specialized habitats. WRKY transcription factors play pivotal roles in plant stress responses, growth and development regulation, and secondary metabolism. However, research on this gene family in A. sesquipedale remains unreported. Results In this study, 70 WRKY genes (AsWRKYs) were identified in A. sesquipedale through genome-wide analysis. Phylogenetic analysis, integrating WRKY genes from four model plants (Arabidopsis thaliana, Solanum lycopersicum, Oryza sativa, and Glycine max), classified these 70 AsWRKYs into three major groups, with Group Ⅱ further subdivided into five subgroups. Genes within the same group exhibited high conservation in gene structure and motif composition. Tissue expression profiling revealed that several AsWRKYs (AsWRKY21 and AsWRKY49) exhibit root-preferential expression, suggesting their potential involvement in regulating root growth and development in A. sesquipedale. The expression patterns under cold stress showed that 7 genes in roots and 4 genes in leaves exhibited early rapid responses, while 15 genes in leaves and 4 genes in roots exhibited long-term sustained response features. Integrating expression patterns with phylogenetic relationships, key candidate genes potentially implicated in cold stress regulation, including AsWRKY17, AsWRKY23, AsWRKY30, AsWRKY56, and AsWRKY58, were identified. Conclusions This study identified 70 WRKY genes in A. sesquipedale, elucidated their classification, evolutionary characteristics, and expression patterns, and unveiled the potential mechanisms of AsWRKYs in cold stress responses. The findings establish a foundation for understanding the evolutionary adaptability of AsWRKYs and the cold regulatory network in A. sesquipedale, and lay a foundation for molecular breeding and genetic improvement of stress resistance in orchids.

Triadica sebifera, an economically and medicinally valuable tree species native to China, was investigated for its in vitro regeneration potential using leaf explants from nodal cuttings of young stems and sprouts. This study evaluated the effects of basal media, plant growth regulators (PGRs), explant sources, and incision methods on adventitious shoot induction, supplemented by histological analysis. The highest shoot regeneration frequency (98.89%) and maximum shoot number (72) were achieved via direct organogenesis using sucker-derived nodal cuttings cultured on MS medium with 2 mg/L 6- benzyladenine (6-BA), 0.3 mg/L kinetin (KT), and 0.2 mg/L α-naphthaleneacetic acid (NAA). Under identical conditions, branch-derived explants showed lower regeneration (84.44%, 64 shoots). Transverse midvein incision proved most effective, with sucker-derived leaves exhibiting superior regeneration. Shoots elongated completely (100%) on Murashige and Skoog (MS) medium containing 0.3 mg/L 6-BA, 0.03 mg/L NAA, and activated charcoal. Rooting was optimal on MS medium with 0.3 mg/L indole-3-butyric acid (IBA), yielding a 98% acclimatization survival rate. Histological analysis revealed de novo meristem formation from parenchyma cells, confirming direct organogenesis without callus intermediation, further validating the enhanced regenerative capacity of sprout-derived explants. This efficient in vitro regeneration system provides a foundation for large-scale propagation and germplasm conservation of T. sebifera, while offering insights for woody plant regeneration studies.