Genome-wide identification of the histone modification gene family in Aquilaria sinensis and functional analysis of several HMs in response to MeJA and NaCl stress.

Histone modifications (HMs) play various roles in growth, development, and resistance to abiotic stress. However, HMs have been systematically identified in a few plants, and identification of HMs in medicinal plants is very rare. Aquilaria sinensis is a typical stress-induced medicinal plant, in which HMs remain unexplored. We conducted a comprehensive study to identify HMs and obtained 123 HMs. To conduct evolutionary analysis, we constructed phylogenetic trees and analyzed gene structures. To conduct functional analysis, we performed promoter, GO, and KEGG analyses and ortholog analyses against AtHMs. Based on the expression profiles of different tissues and different layers of Agar-Wit, some HMs of A. sinensis (AsHMs) were predicted to be involved in the formation of agarwood, and their response to MeJA and NaCl stress was tested by qRT-PCR analysis. By analyzing the enrichment of H3K4me3, H3K27me3, and H4K5ac in the promoter regions of two key sesquiterpene synthase genes, AsTPS13/18, we hypothesized that AsHMs play important roles in the synthesis of agarwood sesquiterpenes. We confirmed this hypothesis by conducting RNAi transgenic interference experiments. This study provided valuable information and important biological theories for studying epigenetic regulation in the formation of agarwood. It also provided a framework for conducting further studies on the biological functions of HMs.

Ice crystals and oxidative stress affect the viability of Areca catechu seeds following cryopreservation.

This study aimed to examine the effects of ice crystals and oxidative stress on seed viability in the context of cryopreservation, with the ultimate goal of identifying potential solutions to address the persistently low regeneration rate observed in recalcitrant medicinal plant seeds following cryopreservation. Using differential scanning calorimetry technology alongside seeds germination at different moisture levels after cryopreservation helped determine the best moisture content and freezing process for Areca catechu seeds. Seeds with 17-21 % moisture content and treated with PVS2 vitrification solution showed higher survival rates after cryopreservation. The oxidative markers of A. catechu seed embryos exhibited alterations due to vitrification freezing. However, there was no substantial association between seed viability and oxidative markers, such as reactive oxygen species (ROS), suggesting that oxidative damage mediated by ROS is not the primary factor contributing to the diminished viability of A. catechu seeds following cryopreservation. The inclusion of vitamin E, reduced glutathione (GSH), and 2-nitrobenzoic acid in the vitrification cryoprotectant achieved some improvement in seed viability of approximately 10 times, with GSH exhibiting the most pronounced optimizing effect.

Comparative transcriptome analysis reveals the differences in wound-induced agarwood formation between Chi-Nan and ordinary germplasm of Aquilaria sinensis.

Agarwood is a rare and valuable heartwood derived from Aquilaria sinensis in China. Compared with ordinary germplasm, Chi-Nan, a special germplasm of A. sinensis, has a better agarwood-producing capacity. However, the mechanisms underlying their different qualities remain poorly characterized. Here, a comparative transcriptome analysis of Chi-Nan and ordinary A. sinensis was carried out to investigate the wound responses of both germplasms. A total of 198.19 Gb of clean data were obtained with an average of 6.61 Gb of clean reads for each sample. By comparing with their control groups, more differentially expressed genes (DEGs) were observed in Chi-Nan germplasm. Kyoto Encyclopedia of Genes and Genomes (KEGG) and expression profile analysis suggested that Chi-Nan possesses a stronger ability to respond to wounding. Furthermore, the enrichment of biosynthetic pathways related to sesquiterpenes and 2-(2-phenylethyl) chromones (PECs) were more significant in Chi-Nan than in ordinary germplasm, and related genes showed significantly higher up-regulation in Chi-Nan after wounding. Sixteen candidate genes presumably involved in biosynthesis of agarwood components were identified and found to exhibit higher up-regulation in Chi-Nan than in ordinary germplasm in response to wounding. Overall, these results are helpful in explaining reasons for the higher agarwood-producing properties of Chi-Nan, and contribute to a further understanding of the mechanism of agarwood formation in A. sinensis.

Cryopreservation of Medicinal Plant Seeds: Strategies for Genetic Diversity Conservation and Sustainability.

The depletion of medicinal plant resources leads to the irreversible loss of their genetic diversity. The preservation of medicinal plant germplasm using cryobanks is crucial for maintaining the sustainability of these resources. This study examined the efficacy of cryopreservation on 164 medicinal plant seeds, identified general principles for preserving medicinal plant seeds at ultra-low temperatures, and established a cryobank for dry-sensitive medicinal plant seeds. Over 90% of orthodox seeds were unaffected by freezing, with optimal conditions being a 5-10% moisture content and direct freezing. Intermediate seeds were best frozen with a 7-15% moisture content, and those with a lower initial moisture content were best suited to direct freezing. While recalcitrant seeds' freezing was most influenced by moisture content, there was no specific range. Direct freezing is appropriate for recalcitrant seeds possessing a hard seed coat and a firm seed kernel, whereas seeds with a brittle or soft seed coat are better suited for vitrification or stepwise freezing methods. There was no significant correlation between alterations in physiological and biochemical indicators and microscopic structures of seeds before and following liquid nitrogen freezing, as well as their storage characteristics. The findings of this research offer evidence in favor of the extended conservation of plant seeds and the extensive utilization of ultra-low temperature technology and provides an example of protecting the genetic diversity of plant resources.

Successful development of molecular diagnostic technology combining mini-barcoding and high-resolution melting for traditional Chinese medicine agarwood species based on single-nucleotide polymorphism in the chloroplast genome.

Agarwood is a valuable traditional medicine and fragrance. The production process is a typical injury-induced defense response. Currently, there are approximately 22 known species in the genus Aquilaria Lam., all of which can produce agarwood, whereas there are only two legal species of traditional Chinese medicinal agarwood, Aquilaria sinensis (Lour.) Spreng. and Aquilaria agallocha (Lour.) Roxb. The Taiwan herbal Pharmacopoeia of China stipulates that the medicinal agarwood species are A. sinensis and its relatives in the same genus. Moreover, there are five species of agarwood available for clinical medicinal use in Japan, including A. agallocha and A. sinensis, which are often confused with each other or used in a mixed way in the trade process. Therefore, accurate identification of traditional Chinese medicinal agarwood species is important to ensure the authenticity of traditional medicines and to guide the safety of clinical medication. In this study, 59 specific single-nucleotide polymorphism loci were screened and obtained from the chloroplast genomes of 12 species of the genus Aquilaria Lam. We established an identification method for traditional Chinese medicinal agarwood using mini-barcoding combined with high-resolution melting (HRM) and designed and validated 10 pairs of primers from the psbM-trnD, psbA, rps16, petN, ndhE-psaC, rps4, atpE, ycf1, rps15-trnN, and matK regions. The amplification products were all less than 200 bp, with a high success rate of amplification. The method was applied to successfully identify traditional Chinese medicinal agarwood species from commercial agarwood samples. Overall, the sensitivity of this method was sufficient to detect 1% of adulterants in medicinal agarwood products, proving that mini-barcoding HRM is a powerful and flexible tool. This method can be used as a fast and effective high-throughput method for authenticity testing of traditional Chinese medicinal agarwood and its raw materials containing agarwood-containing proprietary Chinese medicines and is recommended for industrial applications.

2-(2-Phenylethyl)chromones increase in Aquilaria sinensis with the formation of agarwood.

Obtained from Aquilaria Lam. and Gyrinops Gaertn., agarwood is a prestigious perfume and medicinal material in the world. Its primary chemical constituents and indicators of agarwood's development are 2-(2-phenylethyl)chromones (PECs). However, how PECs affect its quality, accumulation, and transformation pattern is still unclear. The present study investigated this issue by monitoring resin filling in agarwood generated by the whole-tree agarwood-inducing technique over a span of a year, observing the ethanol extract concentration at different sampling times, and statistically examining PECs in agarwood from each sampling period. In agarwood, the resin accumulated over time, except during the 4th-6th month due to the creation of a barrier layer. The relative content of total PECs demonstrated an overall increase throughout the year but a decrease from the 4th month to the 6th month, and the relative content of 19 PECs that persisted throughout the year was positively correlated with the content of ethanol extracts. In addition, the process of chromone accumulation was accompanied by the production and transformation of different types of chromones, with flindersia type 2-(2-phenylethyl)chromones, epoxy-2-(2-phenylethyl)chromones, and diepoxy-2-(2-phenylethyl)chromones being the major chromone components; in addition, the content of 5,6,7,8-tetrahydro-2-(2-phenylethyl)chromones kept increasing after 6 months of agarwood formation. Three main trends were identified from 58 analogs of PECs, each with notable variation. The first type had the highest content at the beginning of resin formation. The second type had the highest content at 6 months and then started to decrease, and the third type had a slowly increasing content. As a whole, this study systematically investigated the accumulation of PECs during injury-induced agarwood production in A. sinensis, which is of scientific significance in resolving the transformation of PECs and revealing the secret of agarwood formation.

The LOB domain protein, a novel transcription factor with multiple functions: A review.

The LATERAL ORGAN BOUNDARIES DOMAIN (LBD) protein, named for its LATERAL ORGAN BOUNDARIES (LOB) domain, is a member of a class of specific transcription factors commonly found in plants and is absent from all other groups of organisms. LBD TFs have been systematically identified in about 35 plant species and are involved in regulating various aspects of plant growth and development. However, research on the signaling network and regulatory functions of LBD TFs is insufficient, and only a few members have been studied. Moreover, a comprehensive review of these existing studies is lacking. In this review, the structure, regulatory mechanism and function of LBD TFs in recent years were reviewed in order to better understand the role of LBD TFs in plant growth and development, and to provide a new perspective for the follow-up study of LBD TFs.

Comprehensive Analysis Reveals the Difference in Volatile Oil between Bupleurum marginatum var. stenophyllum (Wolff) Shan et Y. Li and the Other Four Medicinal Bupleurum Species.

Volatile oil serves as a traditional antipyretic component of Bupleuri Radix. Bupleurum marginatum var. stenophyllum (Wolff) Shan et Y. Li belongs to the genus Bupleurum and is distinguished for its high level of saikosaponins and volatile oils; nonetheless, prevailing evidence remains inconclusive regarding its viability as an alternative resource of other official species. This study aims to systematically compare the volatile oil components of both dried and fresh roots of B. marginatum var. stenophyllum and the four legally available Bupleurum species across their chemical, molecular, bionics, and anatomical structures. A total of 962 compounds were determined via GC-MS from the dried roots; B. marginatum var. stenophyllum showed the greatest differences from other species in terms of hydrocarbons, esters, and ketones, which was consistent with the results of fresh roots and the e-nose analysis. A large number of DEGs were identified from the key enzyme family of the monoterpene synthesis pathway in B. marginatum var. stenophyllum via transcriptome analysis. The microscopic observation results, using different staining methods, further showed the distinctive high proportion of phloem in B. marginatum var. stenophyllum, the structure which produces volatile oils. Together, these pieces of evidence hold substantial significance in guiding the judicious development and utilization of Bupleurum genus resources.

Single-cell transcriptome profiling reveals the spatiotemporal distribution of triterpenoid saponin biosynthesis and transposable element activity in Gynostemma pentaphyllum shoot apexes and leaves.

Gynostemma pentaphyllum (Thunb.) Makino is an important producer of dammarene-type triterpenoid saponins. These saponins (gypenosides) exhibit diverse pharmacological benefits such as anticancer, antidiabetic, and immunomodulatory effects, and have major potential in the pharmaceutical and health care industries. Here, we employed single-cell RNA sequencing (scRNA-seq) to profile the transcriptomes of more than 50,000 cells derived from G. pentaphyllum shoot apexes and leaves. Following cell clustering and annotation, we identified five major cell types in shoot apexes and four in leaves. Each cell type displayed substantial transcriptomic heterogeneity both within and between tissues. Examining gene expression patterns across various cell types revealed that gypenoside biosynthesis predominantly occurred in mesophyll cells, with heightened activity observed in shoot apexes compared to leaves. Furthermore, we explored the impact of transposable elements (TEs) on G. pentaphyllum transcriptomic landscapes. Our findings the highlighted the unbalanced expression of certain TE families across different cell types in shoot apexes and leaves, marking the first investigation of TE expression at the single-cell level in plants. Additionally, we observed dynamic expression of genes involved in gypenoside biosynthesis and specific TE families during epidermal and vascular cell development. The involvement of TE expression in regulating cell differentiation and gypenoside biosynthesis warrant further exploration. Overall, this study not only provides new insights into the spatiotemporal organization of gypenoside biosynthesis and TE activity in G. pentaphyllum shoot apexes and leaves but also offers valuable cellular and genetic resources for a deeper understanding of developmental and physiological processes at single-cell resolution in this species.

Utilizing metabolomics and network analysis to explore the effects of artificial production methods on the chemical composition and activity of agarwood.

Introduction: Agarwood is a traditional aromatic southern medicine. It has a long history of being used in traditional Chinese aromatherapy to treat insomnia, anxiety and depression. Due to the scarcity of wild resources, people have planted trees successfully and begun to explore various agarwood-inducing techniques. This study comparative analysis of volatile metabolites in agarwood produced by various inducing techniques and its potential sleep-promoting, anti-anxiety and anti-depressant network pharmacological activities. Methods: A total of 23 batches of two types of agarwood were collected, one of which was produced by artificial techniques, including 6 batches of TongTi (TT) agarwood produced by "Agar-Wit" and 6 batches of HuoLao (HL) agarwood produced by "burning, chisel and drilling", while the other was collected from the wild, including 6 batches of BanTou (BT) agarwood with trunks broken due to natural or man-made factors and 5 batches of ChongLou (CL) agarwood with trunks damaged by moth worms. The study employed metabolomics combined with network analysis to compare the differences in volatile metabolites of agarwood produced by four commonly used inducing techniques, and explored their potential roles and possible action targets in promoting sleep, reducing anxiety, and alleviating depression. Results: A total of 147 volatile metabolites were detected in agarwood samples, mainly including small aromatic hydrocarbons, sesquiterpenes and 2-(2-phenylethyl) chromone and their pyrolysis products. The results showed composition of metabolites was minimally influenced by the agarwood induction method. However, their concentrations exhibited significant variations, with 17 metabolites showing major differences. The two most distinct metabolites were 6-methoxy-2-(2-phenylethyl) chromone and 6,7-dimethoxy-2-(2-phenylethyl) chromone. Among the volatile metabolites, 142 showed promising potential in treating insomnia, anxiety, and depression, implicating various biological and signaling pathways, predominantly ALB and TNF targets. The top three active metabolites identified were 2-(2-phenylethyl) chromone, 1,5-diphenylpent-1-en-3-one, and 6-methoxy-2-[2-(4'-methoxyphenyl) ethyl] chromone, with their relative content in the four types of agarwood being TT>HL>CL>BT. Conclusion: The differences in the content of 2-(2-phenylethyl) chromones suggest that they may be responsible for the varying therapeutic activities observed in different types of agarwood aromatherapy. This study offers theoretical support for the selection of agarwood in aromatherapy practices.

Screening and validation of reference genes in Dracaena cochinchinensis using quantitative real-time PCR.

Dragon's blood, the red resin derived from the wounded Dracaena, is a precious traditional medicine used by different culture. Dracaena cochinchinensis is one of the main species of Dracaena, and is the endangered medicinal plants in China. The vulnerable status severely limits the medicinal value and wide application of dragon's blood. Therefore, it's essential to analyze the mechanisms that form dragon's blood in order to increase artificial production. To clarify the mechanisms forming dragon's blood, understanding gene expression in the flavonoid biosynthesis pathway is the foundation. However, reference genes of D. cochinchinensis haven't been analyzed. In this study, expression profiles of seven commonly used housekeeping genes (Actin, α-EF, UBC, ß-tubulin, 18S, GAPDH, His) were evaluated by using quantitative real-time PCR combined with the algorithms geNorm, NormFinder, BestKeeper, and RefFinder. On the basis of overall stability ranking, the best reference genes were the combinations ß-tubulin +UBC for wounded stems and α-EF +18S + Actin for different organs. Reliability of the recommended reference genes was validated by normalizing relative expression of two key enzyme genes PAL1 and CHI1 in the flavonoid biosynthesis pathway. The results provide a foundation to study gene expression in future research on D. cochinchinensis or other Dracaena.

Pharmacokinetics and molecular docking of the cardioprotective flavonoids in Dalbergia odorifera.

The purpose of this research was to investigate the cardioprotective effects and pharmacokinetics of Dalbergia odorifera flavonoids. The cardioprotective effects were detected by hematoxylin-eosin staining histopathological observations and the detection of myocardial enzymes by kits in serum, peroxidation and antioxidant levels and ATPase activities by kits in the homogenate supernatant, and antioxidant and apoptosis-related protein expression in heart tissue by immunohistochemistry. The pharmacokinetics parameters of the flavonoids in rat plasma were investigated by ultra-high-performance liquid chromatography coupled with tandem mass spectrometry. Molecular docking of the compounds absorbed by the blood with specific proteins was carried out. D. odorifera flavonoids significantly reduced the levels of creatinine kinase, alanine transaminase, nitric oxide, and Hydrogen peroxide, elevated the levels of glutathione, superoxide dismutase, and ATPase, significantly reduced the pathological degree of heart tissue and had obvious anti-myocardial ischemia efficacy. Nine out of the 17 flavonoids were detected in rat plasma. The peak concentration and the area under the plasma concentration-time curve values of 3'-O-methylviolanone and sativanone were significantly higher than those of other ingredients. The peak time of most flavonoids (except for Genistein and Pruneion) was lower than 2 h, while the half-life of elimination of the nine flavonoids ranged from 3.32 to 21.5 h. The molecular docking results showed that daidzein, dalbergin, formononetin, and genistein had the potential to bind to the target proteins. The results of the study provide an important basis for understanding the cardioprotective effects and clinical application of D. odorifera.

First Report of Sclerotinia Rot Caused by Sclerotinia sclerotiorum on Artemisia capillaris in China.

Artemisia capillaris (Asteraceae) is an annual herb found in ˃10 provinces in China. It is cultivated on ˃670 ha, with annual production around 2,500 tons. Its shoot is used in traditional Chinese medicine (Liu et al. 2021). From April to May 2023, Sclerotinia rot symptoms were seen at the Institute of Medicinal Plant Development (40.04°N, 116.28°E), Beijing, China. Disease incidence was up to 10% in the field through investigation of 300 plants. Initial symptoms were irregular tan-brown lesions (0.5 to 5.0 mm) that expended to circumferential necrosis on the roots and basal stem, aerial mycelia and sclerotia were developed on them. The leaves and stem tips were withered and droopy in severe cases. Twelve symptomatic primary roots of 12 plants from two sites were cut into 5 × 5 mm pieces, surface sterilized with 75% ethanol for 30 s and 5% NaClO for 60 s, rinsed with distilled water for three times, dried with sterile filter paper, put on potato dextrose agar (PDA), and incubated at 25°C in the dark for 2 days. Two Sclerotinia-like isolates were obtained using the hyphaltip method. White aerial mycelia were sparse and appressed for isolate YC1-3 and dense for isolate YC1-7. After incubated at 25°C in the dark for 15 days, 10 to 25 sclerotia were developed near the colony margin. Sclerotia of isolate YC1-3 were 1.0 to 3.9 × 1.2 to 4.5 (mean 1.8 × 2.2) mm (n = 60), ovoid or arc-shaped. Sclerotia of isolate YC1-7 were 1.5 to 3.4 × 2.7 to 9.2 (mean 2.3 × 4.3) mm (n = 60), ovoid, dumbbell shaped or curved. The isolates were identified as Sclerotinia sclerotiorum based on morphology (Maas 1998). To further identify the pathogens, molecular identification was performed with isolates YC1-3 and YC1-7. DNA of the two isolates were extracted by the cetyltrimethylammonium bromide (CTAB) method. Polymerase chain reaction was performed with primers ITS1/ITS4 for the internal transcribed spacer (ITS) region (Choi et al. 2020; White et al. 1990) and primers G3PDHfor/G3PDHrev for the glyceraldehyde 3-phosphate dehydrogenase (G3PDH) gene (Garfinkel. 2021). BLAST search analysis revealed that the ITS sequence (GenBank OR229758 and OR229762) was ≥99% similar to S. sclerotiorum (MN099281, MZ379265, KX781301, etc.), and the G3PDH sequence (OR778388 and OR761975) was too (MZ493894, JQ036048, OQ790148, etc.). Phylogenetic trees were computed with ITS and G3PDH sequences using the Maximum Likelihood in MEGA 11. Nine two-month-old seedlings of A. capillaris were used to test pathogenicity. The epidermis layer of each primary root was slightly wounded (2 × 2 mm, 1 mm deep) using a sterile dissecting blade. Three plants were inoculated with mycelial plugs (5 mm in diameter) of YC1-3 and YC1-7 that cultured on PDA for 7 days. Control plants were inoculated with sterile PDA plugs. All seedlings were then incubated at 25oC and 90% relative humidity. After isolate YC1-7 inoculation 3 days and isolate YC1-3 inoculation 5 days, inoculated roots had symptoms like those in the field, controls had no symptoms. S. sclerotiorum was consistently re-isolated from diseased roots, fulfilling Koch's postulates. Diseases caused by S. sclerotiorum have been reported threatens several important economical crops (Marin and Peres 2020; Guan et al. 2022). To our knowledge, this is the first report of S. sclerotiorum causes Sclerotinia rot on A. capillaris. To avoid of significant economic losses, it is urgent to establish an effective disease-management strategy.

Exploration of the B3 transcription factor superfamily in Aquilaria sinensis reveal their involvement in seed recalcitrance and agarwood formation.

The endangered tree species of the Aquilaria genus produce agarwood, a high value material produced only after wounding; however, conservation of Aquilaria seeds is difficult. The B3 transcription factor family has diverse important functions in plant development, especially in seed development, although their functions in other areas, such as stress responses, remain to be revealed. Here germination tests proved that the seeds of A. sinensis were recalcitrant seeds. To provide insights into the B3 superfamily, the members were identified and characterized by bioinformatic approaches and classified by phylogenetic analysis and domain structure. In total, 71 members were identified and classified into four subfamilies. Each subfamily not only had similar domains, but also had conserved motifs in their B3 domains. For the seed-related LAV subfamily, the B3 domain of AsLAV3 was identical to that of AsVALs but lacked a typical zf-CW domain such as VALs. AsLAV5 lacks a typical PHD-L domain present in Arabidopsis VALs. qRT-PCR expression analysis showed that the LEC2 ortholog AsLAV4 was not expressed in seeds. RAVs and REMs induced after wound treatment were also identified. These findings provide insights into the functions of B3 genes and seed recalcitrance of A. sinensis and indicate the role of B3 genes in wound response and agarwood formation.This is the first work to investigate the B3 family in A. sinensis and to provide insights of the molecular mechanism of seed recalcitrance.This will be a valuable guidance for studies of B3 genes in stress responses, secondary metabolite biosynthesis, and seed development.

Stereocontrol of C-N Axial and Spiro-Central Chirality via Rh(II)-Catalyzed Enantioselective N-H Bond Insertion of Indolinone-Spiroacetal.

A rhodium-catalyzed carbene N-H insertion protocol for simultaneously controlling the C-N axial and spiro-central chiralities is disclosed, resulting in the rapid assembly of enantiopure N-arylindolinone-spiroacetal derivatives in high yields with excellent enantioselectivities. This promising strategy features the chiral C-N axis, spiro-central chirality, functional group tolerance, and late-stage diversification. DFT calculations indicate that the N-H insertion is the axial-chirality-determining step and that the 1,5-H shift step is regiospecifically caused by the spirocycle.

Feeding-induced plant metabolite responses to a phoretic gall mite, its carrier psyllid and both, after detachment.

Phoresy is one of the most distinctive relationships between mites and insects, and the off-host interaction between phoretic mites and their carriers is the most critical factor sustaining the phoretic association. As phoretic associations commonly occur in temporary habitats, little is known about off-host interactions between phoronts and carriers. However, an off-host interaction has been reported, in which the plant-mediated competition between a phoretic gall mite, Aceria pallida, and its psyllid vector, Bactericera gobica, after detachment decreases leaf abscission caused by B. gobica and then directly facilitates their phoretic association. In this obligate phoresy, A. pallida seasonally attaches to B. gobica for overwinter survival and they share the same host plant, Lycium barbarum, during the growing season. It is unknown how the host plant responds to these two herbivores and what plant metabolites are involved in their interspecific interaction. Here, effects of A. pallida and B. gobica on the host plant's transcriptome and metabolome, and on enzymes involved in plant defence, at various infestation stages were studied by inoculating A. pallida and B. gobica either separately or simultaneously on leaves of L. barbarum. Our results showed that (a) A. pallida significantly promoted primary and secondary metabolite accumulation, (b) B. gobica markedly inhibited primary and secondary metabolite accumulation and had little influence on defence enzyme activity, and (c) under simultaneous A. pallida and B. gobica infestation, an intermediate response was predicted. These findings indicate that A. pallida and B. gobica have different effects on host plants, A. pallida inhibits B. gobica mainly by increasing the secondary metabolism of L. barbarum, whereas B. gobica inhibits A. pallida mainly by decreasing the primary metabolism of L. barbarum. In conjunction with our previous research, we speculate that this trade-off in host plant metabolite response between A. pallida and B. gobica after detachment promotes a stable phoretic association.

Identification and characterization of novel sesquiterpene synthases TPS9 and TPS12 from Aquilaria sinensis.

Sesquiterpenes are characteristic components and important quality criterions for agarwood. Although sesquiterpenes are well-known to be biosynthesized by sesquiterpene synthases (TPSs), to date, only a few TPS genes involved in agarwood formation have been reported. Here, two new TPS genes, namely, TPS9 and TPS12, were isolated from Aquilaria sinensis (Lour.) Gilg, and their functions were examined in Escherichia coli BL21(DE3), with farnesyl pyrophosphate (FPP) and geranyl pyrophosphate (GPP) as the substrate of the corresponding enzyme activities. They were both identified as a multiproduct enzymes. After incubation with FPP, TPS9 liberated ß-farnesene and cis-sesquisabinene hydrate as main products, with cedrol and another unidentified sesquiterpene as minor products. TPS12 catalyzes the formation of ß-farnesene, nerolidol, γ-eudesmol, and hinesol. After incubation with GPP, TPS9 generated citronellol and geraniol as main products, with seven minor products. TPS12 converted GPP into four monoterpenes, with citral as the main product, and three minor products. Both TPS9 and TPS12 showed much higher expression in the two major tissues emitting floral volatiles: flowers and agarwood. Further, RT-PCR analysis showed TPS9 and TPS12 are typical genes mainly expressed during later stages of stress response, which is better known than that of chromone derivatives. This study will advance our understanding of agarwood formation and provide a solid theoretical foundation for clarifying its mechanism in A. sinensis.

Agarwood extract mitigates alcoholic fatty liver in C57 mice via anti­oxidation and anti­inflammation.

Alcoholic fatty liver disease (AFLD) is a disease with a high incidence rate among individuals who drink alcohol. Our previous study found that agarwood alcohol extracts (AAEs) have a protective effect against drug­induced liver damage via anti­inflammatory and antioxidant mechanisms. Therefore, we hypothesized that agarwood may have a protective effect against AFLD. The present study assessed the potential protective effects and the underlying mechanism of action of AAEs for the treatment of an AFL in vivo model. The AFLD mouse model was established by continuous high fat diet and alcohol gavage in C57 mice. After treatment with AAEs, blood was collected, liver and adipose tissues were removed and liver and adipose indexes were analyzed. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglyceride (TG) and cholesterol (CHO) in serum were detected. The liver tissue was assessed using pathological sections. Biochemical methods were used to detect the levels of oxidative stress in the supernatant of liver tissue homogenate. The levels of pro­inflammatory cytokines in the serum were detected by ELISA. The protein expression levels of nuclear erythroid 2­related factor 2 (Nrf2) and nuclear factor kappa­B (NF­κB) in liver tissues were detected using western blotting. AAE treatment decreased the liver and adipose indexes, reduced the levels of AST, ALT, TG and CHO, improved the liver pathological characteristics and enhanced antioxidant and anti­inflammatory activities. In addition, AAEs increased the protein expression level of Nrf2 and decreased the protein expression level of NF­κB compared with AFL mice. AAE­treated animals exhibited reduced metabolic enzyme and blood lipid levels, demonstrated improved liver function and relieved the pathological damage of AFLD induced by consuming a high fat and alcohol diet. AAEs have potential protective effects in AFLD via antioxidant and anti­inflammatory mechanisms.

Biosynthesis and Pharmacological Activities of Flavonoids, Triterpene Saponins and Polysaccharides Derived from Astragalus membranaceus.

Astragalus membranaceus (A. membranaceus), a well-known traditional herbal medicine, has been widely used in ailments for more than 2000 years. The main bioactive compounds including flavonoids, triterpene saponins and polysaccharides obtained from A. membranaceus have shown a wide range of biological activities and pharmacological effects. These bioactive compounds have a significant role in protecting the liver, immunomodulation, anticancer, antidiabetic, antiviral, antiinflammatory, antioxidant and anti-cardiovascular activities. The flavonoids are initially synthesized through the phenylpropanoid pathway, followed by catalysis with corresponding enzymes, while the triterpenoid saponins, especially astragalosides, are synthesized through the universal upstream pathways of mevalonate (MVA) and methylerythritol phosphate (MEP), and the downstream pathway of triterpenoid skeleton formation and modification. Moreover, the Astragalus polysaccharide (APS) possesses multiple pharmacological activities. In this review, we comprehensively discussed the biosynthesis pathway of flavonoids and triterpenoid saponins, and the structural features of polysaccharides in A. membranaceus. We further systematically summarized the pharmacological effects of bioactive ingredients in A. membranaceus, which laid the foundation for the development of clinical candidate agents. Finally, we proposed potential strategies of heterologous biosynthesis to improve the industrialized production and sustainable supply of natural products with pharmacological activities from A. membranaceus, thereby providing an important guide for their future development trend.