Effects of Combined Nitrogen-Phosphorus on Biomass Accumulation, Allocation, and Allometric Growth Relationships in Pinus yunnanensis Seedlings after Top Pruning.

Pinus yunnanensis (Franch), a species endemic to southwestern China, provides significant ecological and economic benefits. The quality of afforestation can be enhanced by promoting high-quality sprout growth. This study analyzed the effects of six fertilization treatments following top pruning: T1 (N: 0 g/plant-1; P: 0 g/plant-1), T2 (N: 0 g/plant-1; P: 2 g/plant-1), T3 (N: 0 g/plant-1; P: 4 g/plant-1), T4 (N: 0.6 g/plant-1; P: 0 g/plant-1), T5 (N: 0.6 g/plant-1; P: 2 g/plant-1), and T6 (N: 0.6 g/plant-1; P: 4 g/plant-1). The accumulation and allocation of aboveground biomass in roots, stems, and leaves of P. yunnanensis were measured, as well as changes in biomass per plant at 90 days (early stage), 180 days (middle stage), and 270 days (late stage) post-fertilization. At 90 days, root biomass accumulation in T3 was significantly higher, by 13.31%, compared to that in T1 (p < 0.05). The growth rates of stem and plant biomass followed the order T6 > T1 > T3 > T5 > T4 > T2. The biomass of sprouts and individual plants exhibited allometric growth under T1, T5, and T6 treatments. At 180 days, needle biomass allocation in T1 and T4 increased by 7.47% and 8.6%, respectively, compared to 90 days. Combined nitrogen-phosphorus fertilization significantly influenced aboveground biomass allocation, promoting growth more effectively than other treatments. By 270 days, the stem and individual biomass in T2 and T3 treatments showed significant differences (p < 0.01) compared to other treatments. Overall, root, stem, and sprouts were primarily influenced by phosphorus fertilization, while nitrogen fertilization notably promoted needle and leaf growth in later stages. The aboveground components were more affected by phosphorus fertilization. The combination of nitrogen and phosphorus fertilizers enhanced early-stage stem and sprouts, as well as late-stage root development.

Application of Bacillus tequilensis for the control of gray mold caused by Botrytis cinerea in blueberry and mechanisms of action: inducing phenylpropanoid pathway metabolism.

Background: Botrytis cinerea a blueberry gray mold, is one of the main diseases affecting postharvest storage, causing significant losses. Several studies have shown that Bacillus tequilensis can prevent the growth of plant pathogens by producing various antibacterial substances, and can induce plant resistance. However, research on the biological management of post-harvest gray mold in blueberries using B. tequilensis remains unclear. Methods: To better control the postharvest gray mold of blueberry, the effects of B. tequilensis KXF6501 fermentation solution (YY) and KXF6501 cell-free supernatant (SQ) on the induction of disease resistance in blueberry fruits were studied using biochemical and transcriptomic analyses. Results: We found that YY controlled the conidial germination and mycelial growth of B. cinerea in vitro, followed by SQ. After 3 d of culture, the lesion diameter and incidence of gray mold in blueberry fruits inoculated with YY and SQ were smaller than those in the control group. Therefore, gray mold in blueberries was effectively controlled during the prevention period, and the control effect of YY was better than that of SQ. Transcription spectrum analysis of blueberry peel tissue showed that the YY- and SQ-induced phenylpropane metabolic pathways had more differentially expressed genes (DEGs) than other biological pathways. In addition, biochemical analyses showed that YY treatment effectively enhanced the activity of enzymes related to the phenylpropane pathway (phenylalanine ammonialyase [PAL], cinnamate 4-hydroxylase [C4H], 4-coumarate CoA ligase [4CL], and polyphenol oxidase [PPO]) and stimulated the synthesis of lignin, total phenols, and flavonoids, followed by SQ. Compared with the control, the YY and SQ treatments reduced the weight loss rate and better maintained the appearance and nutritional quality of the blueberry fruits. Conclusion: Our findings suggest that B. tequilensis KXF6501 is potentially useful as a suitable bio-control agent in harvested blueberries.

Bioactivity-Guided Isolation of Secondary Metabolites with Antioxidant and Antimicrobial Activities from Camellia fascicularis.

Camellia fascicularis has important ornamental, medicinal, and food values, which also have tremendous potential for exploiting bioactivities. We performed the bioactivity-guided (antioxidant and antimicrobial) screening of eight fractions obtained from the ethyl acetate phase of C. fascicularis. The antioxidant activity was measured by DPPH, ABTS, and FRAP, and the antibacterial activity was measured by the minimum inhibitory concentration (MIC) of Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus. The results of bioactivity-guided isolation indicated that the major antioxidant compounds in the ethanolic extracts of C. fascicularis may be present in fractions (Fr.) (A-G, obtained after silica gel column chromatography). Fr. (D-I, obtained after silica gel column chromatography) is a fraction of C. fascicularis with antimicrobial activity. The structures of compounds were determined by spectral analysis and nuclear magnetic resonance (NMR) combined with the available literature on secondary metabolites of C. fascicularis leaves. In this study, 17 compounds were identified, including four phenolics (1, 3-4, and 14), a phenylpropane (2), five terpenoids (5-7, 12, and 15), four flavonoids and flavonoid glycosides (8-10 and 16), and two lignins (13 and 17). Compounds 4-7, 13-15, and 17 were isolated from the genus Camellia for first time. The remaining compounds were also isolated from C. fascicularis for first time. The evaluation of antioxidant and antimicrobial activities revealed that compounds 1, 3, 9, 11, and 17 exhibited higher antioxidant activity than the positive control drug (ascorbic acid), and compounds 4, 8, 10, and 13 showed similar activity to ascorbic acid. The other compounds had weaker or no significant antioxidant activities. The MIC of antibacterial activity for compounds 4, 7, and 11-13 against P. aeruginosa was comparable to that of the positive control drug tetracycline at 125 µg/mL, and other secondary metabolites inhibited E. coli and S. aureus at 250-500 µg/mL. This is also the first report of antioxidant and antimicrobial activities of compounds 5-7, 13-15, and 17. The results of the study enriched the variety of secondary metabolites of C. fascicularis and laid the foundation for further research on the pharmacological efficacy and biological activity of this plant.

Comparative physiological and transcriptomic analyses provide induction resistance mechanisms of Bacillus tequilensis against Colletotrichum fructicola in Camellia oleifera.

Bacillus tequilensis DZY 6715 was isolated from healthy leaves in Camellia oleifera, and the strain DZY 6715 significantly inhibited anthracnose disease resulting from Colletotrichum fructicola in C. oleifera, besides, its associated mechanism of disease resistance was explored. B. tequilensis DZY 6715 treatment controlled mycelial growth of C. fructicola in C. oleifera, and significantly decreased C. oleifera anthracnose incidence and disease index compared with the control group. B. tequilensis DZY 6715 has strong biofilm forming ability, and also secretes extracellular ß-1, 3-glucanase and chitinase, which could cause cell membranes damage and increased cellular compound leakage. C.oleifera treated with DZY 6715 also effectively enhanced enzyme activities and stimulated the synthesis the substances related to phenylpropane metabolism and reactive oxygen metabolism. Moreover, transcript profiling analysis revealed more differentially expressed genes related to phenylpropanoid pathway metabolism and antioxidant system inducing by DZY 6715 compared with the control in C. oleifera. Thus, it can be concluded that B. tequilensis DZY 6715 is a suitable bio-control agent to control anthracnose disease in C. oleifera.

Secondary Metabolites with Antioxidant and Antimicrobial Activities from Camellia fascicularis.

Camellia fascicularis has important ornamental, medicinal, and food value. It also has tremendous potential for exploiting bioactivities. However, the bioactivities of secondary metabolites in C. fascicularis have not been reported. The structures of compounds were determined by spectral analysis and nuclear magnetic resonance (NMR) combined with the available literature on secondary metabolites of C. fascicularis leaves. In this study, 15 compounds were identified, including 5 flavonoids (1-5), a galactosylglycerol derivative (6), a terpenoid (7), 4 lignans (8-11), and 4 phenolic acids (12-15). Compounds 6-7 and 9-12 were isolated from the genus Camellia for the first time. The remaining compounds were also isolated from C. fascicularis for the first time. Evaluation of antioxidant and antimicrobial activities revealed that compounds 5 and 8-11 exhibited stronger antioxidant activity than the positive drug ascorbic acid, while compounds 7, 13, and 15 showed similar activity to ascorbic acid. The minimum inhibitory concentration (MIC) of antibacterial activity for compounds 5, 7, 9, 11, and 13 against Pseudomonas aeruginosa was comparable to that of the positive control drug tetracycline at a concentration of 62.50 µg/mL; other secondary metabolites inhibited Escherichia coli and Staphylococcus aureus at concentrations ranging from 125-250 µg/mL.

Physiological Characteristics and Transcriptomic Responses of Pinus yunnanensis Lateral Branching to Different Shading Environments.

Pinus yunnanensis is an important component of China's economic development and forest ecosystems. The growth of P. yunnanensis seedlings experienced a slow growth phase, which led to a long seedling cultivation period. However, asexual reproduction can ensure the stable inheritance of the superior traits of the mother tree and also shorten the breeding cycle. The quantity and quality of branching significantly impact the cutting reproduction of P. yunnanensis, and a shaded environment affects lateral branching growth, development, and photosynthesis. Nonetheless, the physiological characteristics and the level of the transcriptome that underlie the growth of lateral branches of P. yunnanensis under shade conditions are still unclear. In our experiment, we subjected annual P. yunnanensis seedlings to varying shade intensities (0%, 25%, 50%, 75%) and studied the effects of shading on growth, physiological and biochemical changes, and gene expression in branching. Results from this study show that shading reduces biomass production by inhibiting the branching ability of P. yunnanensis seedlings. Due to the regulatory and protective roles of osmotically active substances against environmental stress, the contents of soluble sugars, soluble proteins, photosynthetic pigments, and enzyme activities exhibit varying responses to different shading treatments. Under shading treatment, the contents of phytohormones were altered. Additionally, genes associated with phytohormone signaling and photosynthetic pathways exhibited differential expression. This study established a theoretical foundation for shading regulation of P. yunnanensis lateral branch growth and provides scientific evidence for the management of cutting orchards.

The Response and Evaluation of Morphology, Physiology, and Biochemistry Traits in Triploid Passiflora edulis Sims 'Mantianxing' to Drought Stress.

As one of the most influential environmental factors, drought stress greatly impacts the development and production of plants. Triploid-induced Passiflora edulis Sims 'Mantianxing' is an important new cultivar for multi-resistance variety selective breeding, which is one of the P. edulis breeding essential targets. However, the performance of triploid 'Mantianxing' under drought stress is unknown. In order to study the drought resistance of triploid 'Mantianxing', our study compared drought-related indicators in diploids and triploids under natural drought experiments, including morphological, physiological, and biochemical characteristics. Results showed that triploid P. edulis 'Mantianxing' showed variable responses to drought treatment. Compared with diploids, triploids showed higher photosynthesis and chlorophyll fluorescence, osmotic adjustment substances, and antioxidant enzyme activity under drought stress and faster chlorophyll biosynthesis and growth recovery after rewatering. Generally speaking, these results indicate that the drought resistance of triploid P. edulis is superior to diploid. This study provides scientific information for breeding stress tolerance variety of P. edulis 'Mantianxing' new cultivar.

In vitro regeneration of triploid from mature endosperm culture of Passiflora edulis "Mantianxing".

The regeneration of shoots from endosperm tissue is a highly effective method to obtain triploid plants. In this study, we elucidated the establishment of an in vitro regeneration system from endosperm culture for the production of Passiflora edulis "Mantianxing." The highest callus induction rate (83.33%) was obtained on the media supplemented with 1.0 mg/L TDZ. Meanwhile, the MS medium containing 1.0 mg/L 6-BA and 0.4 mg/L IBA gave the optimum 75% shoot bud induction. Chromosome analysis revealed that the chromosomal count of P. edulis "Mantianxing" regenerated from endosperm tissues was 27 (2n = 3x = 27), which indicated that shoots regenerated from endosperm tissues were triploids. Triploid P. edulis had more drought resistance than diploid plants. Our study provided a method for breeding of passion fruit by means of a stable and reproducible regeneration system from endosperm culture, leading to the generation of triploid plants.

Effects of salicylic acid-grafted bamboo hemicellulose on gray mold control in blueberry fruit: The phenylpropanoid pathway and peel microbial community composition.

Bamboo hemicellulose (HC) is a natural plant polysaccharide with good biocompatibility and biodegradability. But its poor antibacterial activity limits its application in fruits preservation. In this study, based on the good inducer of salicylic acid (SA) for plant diseases resistance, a novel antibacterial coating material was synthesized by grafting SA onto HC. The study aimed to investigate the synergistic effect of HC-g-SA on antibacterial ability, induces diseases resistance and microbial community composition of postharvest fruit. The graft copolymer treatment significantly reduced the incidence of gray mold caused by Botrytis cinerea in blueberries during storage (P < 0.05), and significantly stimulated the activity of key enzymes, including phenylalanine ammonia-lyase, chalcone isomerase, laccase, and polyphenol oxidase, leading to an increase in fungicidal compounds such as flavonoids, lignin, and total phenolics produced by the phenylpropanoid pathway in blueberries (P < 0.05). Moreover, the HC-g-SA coating altered bacterial and fungal community composition such that the abundance of postharvest fruit-peel pathogens was significantly reduced. After 8 days storage, the blueberry fruits treated by HC-g-SA had a weight loss rate of 12.42 ± 0.85 %. Therefore, the HC-g-SA graft copolymer had a positive impact on the control of gray mold in blueberry fruit during postharvest storage.

Evaluation of the in vitro antioxidant and antitumor activity of extracts from Camellia fascicularis leaves.

Camellia fascicularis is a unique plant rich in bioactive components. However, the isolation of the active substances in C. fascicularis leaves via sequential extraction with solvents of different polarity and the determination of their antioxidant and antitumor activities have not been reported. In this study, the total methanol extract of C. fascicularis leaves was sequentially extracted with different polar solvents, and the corresponding petroleum ether extract (PEE), ethyl acetate extract (EAE), and water extract (WE) were analyzed for their contents in active substances such as flavonoids, polyphenols, polysaccharides, and saponins. The antioxidant ability of the polar extracts was investigated by determining their reducing power and the radical scavenging rate on 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and hydroxyl radicals, and CCK-8 and Annexin-FITC/propidium iodide staining assays were conducted to investigate their inhibitory effects on HCCLM6 and HGC27 tumor cells. The results showed that PEE had a high saponin content of 197.35 ± 16.21 mg OAE/g, while EAE and WE exhibited a relatively higher polysaccharide content of 254.37 ± 1.99 and 373.27 ± 8.67 mg GE/g, respectively. The EAE demonstrated the greatest reducing power and the strongest clearing abilities on ABTS and DPPH radicals with respective EC50 values of 343.45 ± 20.12 and 14.07 ± 0.06 µg/ml. Moreover, the antitumor ability of the different polar extracts was dose-dependent, with WE showing the most potent inhibitory ability against HCCLM6 and HGC27 cells.

Imaging Effector Memory T-Cells Predicts Response to PD1-Chemotherapy Combinations in Colon Cancer.

Often, patients fail to respond to immune checkpoint inhibitor (ICI) treatment despite favourable biomarker status. Numerous chemotherapeutic agents have been shown to promote tumour immunogenicity when used in conjunction with ICIs; however, little is known about whether such combination therapies lead to a lasting immune response. Given the potential toxicity of ICI-chemotherapy combinations, identification of biomarkers that accurately predict how individuals respond to specific treatment combinations and whether these responses will be long lasting is of paramount importance. In this study, we explored [18F]AlF-NOTA-KCNA3P, a peptide radiopharmaceutical that targets the Kv1.3 potassium channel overexpressed on T-effector memory (TEM) cells as a PET imaging biomarker for lasting immunological memory response. The first-line colon cancer chemotherapies oxaliplatin and 5-fluorouracil were assessed in a syngeneic colon cancer model, either as monotherapies or in combination with PD1, comparing radiopharmaceutical uptake to memory-associated immune cells in the tumour. [18F]AlF-NOTA-KCNA3P reliably separated tumours with immunological memory responses from non-responding tumours and could be used to measure Kv1.3-expressing TEM cells responsible for durable immunological memory response to combination therapy in vivo.

Extraction, characterization, antioxidant and anti-tumor activities of polysaccharides from Camellia fascicularis leaves.

The ultrasonic-assisted extraction of polysaccharides from Camellia fascicularis (PCF) was optimized using response surface methodology. After separation and purification with DEAE-52 cellulose and Sephadex G-200 glucan gel columns, the purified polysaccharide components of PCFa-1 and PCFc-1 were analyzed for their structural characterization, antioxidant and anti-tumor activities in vitro. The results indicated that liquid to material ratio of 42 mL/g, ultrasonic time of 53 min, ultrasonic temperature of 73 °C, and ultrasonic power of 215 W were the optimum extraction conditions for PCF with maximum yields (4.05 %). PCFa-1 and PCFc-1 contained 5.88 % and 9.58 % uronic acid content, with 7.53 and 108.91 kDa of average molecular weights, respectively. The PCFa-1 was mainly constituted of galactose, arabinose, and glucose, while PCFc-1 was primarily composed of arabinose, glucose, galacturonic acid, and rhamnose. Fourier transform infrared spectra revealed that PCFa-1 and PCFc-1 contained typical polysaccharide bands. Scanning electron microscopy showed that the surface of PCFa-1 and PCFc-1 were irregular and clumpy structures. Nuclear magnetic resonance showed that PCFa-1 and PCFc-1 were mainly α-glycosidic bond conformation. Furthermore, the PCFc-1 showed better antioxidant capacities than PCFa-1 against hydroxyl, DPPH, and ABTS radicals and exhibited more potent toxicity on A549 and HepG2 cells. These research results suggested that PCF, especially PCFc-1, possesses great potential as natural antioxidants and anti-tumor drugs.

Anti-Inflammatory Effects of Camellia fascicularis Polyphenols via Attenuation of NF-κB and MAPK Pathways in LPS-Induced THP-1 Macrophages.

PURPOSE: Plant polyphenols possess beneficial functions against various diseases. This study aimed to identify phenolic ingredients in Camellia fascicularis (C. fascicularis) and investigate its possible underlying anti-inflammatory mechanism in lipopolysaccharide (LPS)-induced human monocytes (THP-1) macrophages. METHODS: C. fascicularis polyphenols (CFP) were characterized by ultra-performance liquid chromatography (UPLC) combined with quadrupole-time-of-flight mass/mass spectrometry (Q-TOF-MS/MS). The THP-1 cells were differentiated into macrophages under the stimulation of phorbol 12-myristate 13-acetate (PMA) and then treated with LPS to build a cellular inflammation model. The cell viability was detected by CCK-8 assay. The levels of reactive oxygen species (ROS) were assessed by flow cytometry. The secretion and expression of inflammatory cytokines were tested by enzyme-linked immunosorbent assay (ELISA) and real-time polymerase chain reaction (RT-PCR). In addition, the nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways were analyzed by Western blotting. RESULTS: Twelve phenolic constituents including (-)-epicatechin, casuariin, agastachoside, etc. in CFP were identified. The CCK-8 assay showed that CFP exhibited no significant cytotoxicity between 100 and 300 µg/mL. After treated with CFP, the release of ROS was significantly suppressed. CFP inhibited inflammation in macrophages by attenuating the polarization of LPS-induced THP-1 macrophages, down-regulating the expression of the pro-inflammatory cytokines IL-6, IL-1ß and TNF-α, and up-regulating the expression of the anti-inflammatory cytokine IL-10. Western blotting experiments manifested that CFP could markedly inhibit the phosphorylation of p65, ERK and JNK, thereby suppressing the activation of NF-κB and MAPK signaling pathways. CONCLUSION: These findings indicated that CFP exerted anti-inflammatory activity by inhibiting the activation NF-κB and MAPK pathways which may induce the secretion of pro-inflammatory cytokines. This study offers a reference for C. fascicularis as the source of developing natural, safe anti-inflammatory agents in the future.

Enhanced spreading, migration and osteodifferentiation of HBMSCs on macroporous CS-Ta - A biocompatible macroporous coating for hard tissue repair.

Macroporous tantalum (Ta) coating was produced on titanium alloy implant for bone repair by cold spray (CS) technology, which is a promising technology for oxygen sensitive materials. The surface characteristics as well as in vitro cytocompatibility were systematically evaluated. The results showed that a rough and macroporous CS-Ta coating was formed on the Ti6Al4V (TC4) alloy surfaces. The surface roughness showed a significant enhancement from 17.06 µm (CS-Ta-S), 27.48 µm (CS-Ta-M) to 39.21 µm (CS-Ta-L) with the increase of the average pore diameter of CS-Ta coatings from 138.25 µm, 198.25 µm to 355.56 µm. In vitro results showed that macroporous CS-Ta structure with tantalum pentoxide (Ta2O5) was more favorable to induce human bone marrow derived mesenchymal stem cells (HBMSCs) spreading, migration and osteodifferentiation than TC4. Compared with the micro-scaled structure outside the macropores, the surface micro-nano structure inside the macropores was more favorable to promote osteodifferentiation with enhanced alkaline phosphatase (ALP) activity and extracellular matrix (ECM) mineralization. In particular, CS-Ta-L with the largest pore size showed significantly enhanced integrin-α5 expression, cell migration, ALP activity, ECM mineralization as well as osteogenic-related genes including ALP, osteopontin (OPN) and osteocalcin (OCN) expression. Our results indicated that macroporous Ta coatings by CS, especially CS-Ta-L, may be promising for hard tissue repairs.

Identification of two UDP-glycosyltransferases involved in the main oleanane-type ginsenosides in Panax japonicus var. major.

MAIN CONCLUSION: Two UDP-glycosyltransferases from Panax japonicus var. major were identified, and the biosynthetic pathways of three oleanane-type ginsenosides (chikusetsusaponin IVa, ginsenoside Ro, zingibroside R1) were elucidated. Chikusetsusaponin IVa and ginsenoside Ro are primary active components formed by stepwise glycosylation of oleanolic acid in five medicinal plants of the genus Panax. However, the key UDP-glycosyltransferases (UGTs) in the biosynthetic pathway of chikusetsusaponin IVa and ginsenoside Ro are still unclear. In this study, two UGTs (PjmUGT1 and PjmUGT2) from Panax japonicus var. major involved in the biosynthesis of chikusetsusaponin IVa and ginsenoside Ro were identified based on bioinformatics analysis, heterologous expression and enzyme assays. The results show that PjmUGT1 can transfer a glucose moiety to the C-28 carboxyl groups of oleanolic acid 3-O-ß-D-glucuronide and zingibroside R1 to form chikusetsusaponin IVa and ginsenoside Ro, respectively. Meanwhile, PjmUGT2 can transfer a glucose moiety to oleanolic acid 3-O-ß-D-glucuronide and chikusetsusaponin IVa to form zingibroside R1 and ginsenoside Ro. This work uncovered the biosynthetic mechanism of chikusetsusaponin IVa and ginsenoside Ro, providing the rational production of valuable saponins through synthetic biology strategy.

Comparative transcriptome and metabolome analyses provide new insights into the molecular mechanisms underlying taproot thickening in Panax notoginseng.

BACKGROUND: Taproot thickening is a complex biological process that is dependent on the coordinated expression of genes controlled by both environmental and developmental factors. Panax notoginseng is an important Chinese medicinal herb that is characterized by an enlarged taproot as the main organ of saponin accumulation. However, the molecular mechanisms of taproot enlargement are poorly understood. RESULTS: A total of 29,957 differentially expressed genes (DEGs) were identified during the thickening process in the taproots of P. notoginseng. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment revealed that DEGs associated with "plant hormone signal transduction," "starch and sucrose metabolism," and "phenylpropanoid biosynthesis" were predominantly enriched. Further analysis identified some critical genes (e.g., RNase-like major storage protein, DA1-related protein, and Starch branching enzyme I) and metabolites (e.g., sucrose, glucose, fructose, malate, and arginine) that potentially control taproot thickening. Several aspects including hormone crosstalk, transcriptional regulation, homeostatic regulation between sugar and starch, and cell wall metabolism, were identified as important for the thickening process in the taproot of P. notoginseng. CONCLUSION: The results provide a molecular regulatory network of taproot thickening in P. notoginseng and facilitate the further characterization of the genes responsible for taproot formation in root medicinal plants or crops.

Transcriptome analysis of Panax zingiberensis identifies genes encoding oleanolic acid glucuronosyltransferase involved in the biosynthesis of oleanane-type ginsenosides.

MAIN CONCLUSION: Oleanolic acid glucuronosyltransferase (OAGT) genes synthesizing the direct precursor of oleanane-type ginsenosides were discovered. The four recombinant proteins of OAGT were able to transfer glucuronic acid at C-3 of oleanolic acid that yields oleanolic acid 3-O-ß-glucuronide. Ginsenosides are the primary active components in the genus Panax, and great efforts have been made to elucidate the mechanisms underlying dammarane-type ginsenoside biosynthesis. However, there is limited information on oleanane-type ginsenosides. Here, high-performance liquid chromatography analysis demonstrated that oleanane-type ginsenosides (particularly ginsenoside Ro and chikusetsusaponin IV and IVa) are the abundant ginsenosides in Panax zingiberensis, an extremely endangered Panax species in southwest China. These ginsenosides are derived from oleanolic acid 3-O-ß-glucuronide, which may be formed from oleanolic acid catalyzed by an unknown oleanolic acid glucuronosyltransferase (OAGT). Transcriptomic analysis of leaves, stems, main roots, and fibrous roots of P. zingiberensis was performed, and a total of 46,098 unigenes were obtained, including all the identified homologous genes involved in ginsenoside biosynthesis. The most upstream genes were highly expressed in the leaves, and the UDP-glucosyltransferase genes were highly expressed in the roots. This finding indicated that the precursors of ginsenosides are mainly synthesized in the leaves and transported to different parts for the formation of particular ginsenosides. For the first time, enzyme activity assay characterized four genes (three from P. zingiberensis and one from P. japonicus var. major, another Panax species with oleanane-type ginsenosides) encoding OAGT, which particularly transfer glucuronic acid at C-3 of oleanolic acid to form oleanolic acid 3-O-ß-glucuronide. Taken together, our study provides valuable genetic information for P. zingiberensis and the genes responsible for synthesizing the direct precursor of oleanane-type ginsenosides.