Peng Di

Ginsenosides, the principal bioactive triterpenoid saponins of Panax ginseng C.A. Meyer, accumulate abundantly in leaves. They form a large family of secondary metabolites comprising more than 300 structurally distinct compounds, yet how leaf senescence reshapes their accumulation remains unclear. We profiled 376 saponin metabolite features across four sampling stages spanning the natural senescence progression (T1–T3) and a frost-terminated stage (T4) by ultra-performance liquid chromatography–high-resolution mass spectrometry (UPLC-HRMS), modeled temporal effects across the natural senescence gradient (T1–T3) with generalized estimating equations (GEE), analyzed the frost-terminated stage T4 as a distinct endpoint, verified senescence histologically, and analyzed stage T1–T3 transcriptomes with replicate-based statistical testing. Leaf senescence globally reshaped the saponin metabolome, with 326 of the 376 saponin features (86.7%) showing significant temporal effects across the natural senescence gradient (T1–T3). The four classical aglycone classes—protopanaxadiol (PPD), protopanaxatriol (PPT), ocotillol (OT) and oleanolic acid (OA)—followed divergent accumulation programs across the natural senescence progression (T1–T3): sustained accumulation (PPT, OT and PPD) versus a U-shaped curve (OA). At the frost-terminated stage, T4, the classes diverged sharply—PPD-type abundance fell back to the T1 level (abundance-weighted class-level T4/T3 = 0.63), whereas the other classes rose further—a frost-associated remodeling pattern rather than a continuation of the senescence program. The frost-associated PPD decline was glycosylation-dependent—low-glycosylation compounds fell far more sharply than highly glycosylated ones (median of per-compound ratios T4/T3 0.51 vs. 1.04; one-sided permutation p = 1.2 × 10⁻⁴)—and coincided with T3 induction of β-glucosidase genes (exploratory and hypothesis-level, based on two qualified T3 replicates, and requiring validation with larger replicate numbers) and histologically verified vacuolar collapse, consistent with a deglycosylation-associated catabolic mechanism. Senescence signaling was consistent with differential hormone responses (salicylic acid activation, jasmonate and ethylene attenuation), broad suppression of Ca²⁺-signaling components and WRKY/NAC-centered transcriptional control, and joint analysis suggested a dichotomous saponin–gene correlation pattern. Collectively, these results establish a temporal atlas of ginsenoside accumulation during ginseng leaf senescence, delineate the class-specific coupling of saponin secondary metabolism to the senescence program, and offer a new perspective on ginsenosides as metabolites embedded in the physiological activities of the ginseng life cycle.

Sweet wormwood (Artemisia annua) produces and deposits artemisinin in its glandular trichomes but the molecular mechanisms of trichome development remain poorly understood. Here, we conduct single-nucleus RNA sequencing of 10-day-old A. annua seedlings, revealing the specific RNA expression of the leaf trichome cells. The reconstruction of the developmental trajectory of trichome cells not only identifies novel expressed genes, but also elucidates trichome development involved in photosynthesis, auxin biosynthesis, and cutin biosynthesis. By integrating the developmental trajectory of trichome cells with histochemical assays, we identified several genes not previously reported to be involved in trichome development, such as TOE3, MYB1, WRKY10, and ZNF. Moreover, we identified a gene in subcluster 0 encoding a previously unknown WD40 repeat nuclear protein, WDR1, potentially involved in trichome development. We showed that WDR1 not only interacts with the transcription factor SPL9 and enhances its ability to activate HD1 expression, but also directly binds to the transcription factor HD1 itself, thereby forming a regulatory feedback loop that modulates trichome development in A. annua. This study reveals a previously uncharacterized gene that regulates trichome development in A. annua based on single-cell transcriptome analysis. The results presented here offer unprecedented insight into a new pathway for enhancing trichome density and artemisinin production.

Panax quinquefolius L. is a perennial medicinal herb particularly vulnerable to recurring cold stress throughout its life cycle. The GRAS family constitutes a class of plant-specific transcription factors with pivotal functions in growth, development, and environmental adaptation. Nevertheless, a comprehensive characterization of the GRAS transcription factor family in P. quinquefolius remains lacking. A total of 123 PqGRAS members were identified and classified into 13 subfamilies. Transcriptomic profiling implicated that the PAT1 and DELLA subfamilies likely play pivotal roles in integrating multiple environmental and hormonal signals to coordinate complex stress-adaptive responses in P. quinquefolius . Notably, PqGRAS086 (PAT1 subfamily) was significantly upregulated under both cold and methyl jasmonate (MeJA) treatments. Within the 2,000 bp upstream promoter of PqGRAS086 , MeJA-responsive regulatory elements were enriched, whereas low-temperature-responsive elements were entirely absent. Phylogenetic analysis further revealed that PqGRAS086 clustered with VaPAT1 , PtrPAT1 , and ZjCIGR1 , which have been demonstrated to play a positive regulatory role in enhancing plant cold tolerance. RT-qPCR analysis further demonstrated that exogenous MeJA enhanced PqGRAS086 expression during cold exposure, suggesting indirect involvement of PqGRAS086 in cold stress through the JA signaling pathway. This study provides the first genome-wide characterization of GRAS genes in P. quinquefolius and identifies PqGRAS086 as a candidate target for future functional studies and molecular breeding.

Phenolic acids are the major bioactive compounds in Perilla frutescens (L.) Britt; however, the regulatory roles of NAC transcription factors (TFs) in their biosynthesis remain unclear. Here, we performed a genome-wide identification and characterization of the NAC family in P. frutescens and explored their involvement in phenolic acid production. A total of 108 PfNAC genes were identified and classified into 17 subfamilies. Expression and promoter analyses suggested potential roles in secondary metabolism. PfNAC29 is located in the plasma membrane and necleus, while PfNAC40 and PfNAC80 are located in the nucleus.Yeast one-hybrid and dual-luciferase assays demonstrated that these TFs bind to the CATGTG motif in the PfC4H promoter and activate its transcription. Overexpression in transgenic hairy roots significantly increased rosmarinic acid, caffeic acid, and ferulic acid accumulation, accompanied by upregulation of key biosynthetic genes. These results indicate that PfNAC29, PfNAC40, and PfNAC80 act as positive regulators of phenolic acid biosynthesis and provide promising targets for metabolic engineering in medicinal plants.