Decoding the Root Lignification Mechanism of Angelica sinensis through Genome-wide Methylation Analysis.

Angelica sinensis is a traditional Chinese herbal medicine with significant economic and medicinal value. However, early bolting and flowering can occur during the second year of the vegetative growth period, rendering the roots unviable for medicinal use and resulting in substantial economic losses. Consequently, the growing interest in studying the molecular mechanisms underlying early bolting or increased root lignification in A. sinensis. Here, we conducted whole-genome bisulfite sequencing and observed an increase in whole-genome DNA methylation levels on chromosomes after bolting in A. sinensis. Comparative analysis revealed methylation patterns in the upstream, gene body, and downstream regions in the context of CG, CHG, and CHH, suggesting a possible association between CHH-type methylation of promoters and phenylpropanoid biosynthesis. Furthermore, joint analysis of transcriptomic and methylomics data revealed a positive correlation between DNA methylation and gene expression. We identified the hyperDMR gene in the CHH background within the promoter region; this gene is also a key gene (AsCOMT1), exhibiting dual catalytic activity and facilitating the synthesis of both ferulic acid and lignin. Enzyme kinetic analysis demonstrated that AsCOMT1 preferentially catalyzes the synthesis of lignin monomer precursors. These findings highlight the important regulatory role of DNA methylation in bolting and the synthesis of secondary metabolites in A. sinensis, providing valuable insights into the underlying molecular mechanisms. Therefore, as DNA methylation plays an important regulatory role in A. sinensis bolting and secondary metabolite synthesis, it has potential significance in the analysis of the underlying molecular mechanism.

Elucidation of AsANS controlling pigment biosynthesis in Angelica sinensis through hormonal and transcriptomic analysis.

Angelica sinensis, a traditional Chinese medicinal plant, has been primarily reported due to its nutritional value. Pigmentation in this plant is an important appearance trait that directly affects its commercial value. To understand the mechanism controlling purpleness in A. sinensis, hormonal and transcriptomic analyses were performed in three different tissues (leave, root and stem), using two cultivars with contrasting colors. The two-dimensional data set provides dynamic hormonal and gene expression networks underpinning purpleness in A. sinensis. We found abscisic acid as a crucial hormone modulating anthocyanin biosynthesis in A. sinensis. We further identified and validated 7 key genes involved in the anthocyanin biosynthesis pathway and found a specific module containing ANS as a hub gene in WGCNA. Overexpression of a candidate pigment regulatory gene, AsANS (AS08G02092), in transgenic calli of A. sinensis resulted in increased anthocyanin production and caused purpleness. Together, these analyses provide an important understanding of the molecular networks underlying A. sinensis anthocyanin production and its correlation with plant hormones, which can provide an important source for breeding.

Optimization of saponin extraction from the leaves of Panax notoginseng and Panax quinquefolium and evaluation of their antioxidant, antihypertensive, hypoglycemic and anti-inflammatory activities.

Panax notoginseng and Panax quinquefolium are important economic plants that utilize dried roots for medicinal and food dual purposes; there is still insufficient research of their stems and leaves, which also contain triterpenoid saponins. The extraction process was developed with a total saponin content of 12.30 ± 0.34% and 12.19 ± 0.64% for P. notoginseng leaves (PNL) and P. quinquefolium leaves (PQL) extracts, respectively. PNL and PQL saponin extracts showed good antioxidant, antihypertensive, hypoglycemic, and anti-inflammatory properties in vitro and RAW264.7 cells. A total of 699 metabolites were identified in PNL and PQL saponin extracts, with the majority being triterpenoid saponins, flavonoids and amino acids. Fourteen ginsenosides, 18 flavonoids or alkaloids, and 16 amino acids were enriched in both saponin extracts. Overall, the utilization of saponins from medicinal plants PNL and PQL has been developed to facilitate systematic research in the functional food and natural product industries.

A Systematic Review and Developmental Perspective on Origin of CMS Genes in Crops.

Cytoplasmic male sterility (CMS) arises from the incompatibility between the nucleus and cytoplasm as typical representatives of the chimeric structures in the mitochondrial genome (mitogenome), which has been extensively applied for hybrid seed production in various crops. The frequent occurrence of chimeric mitochondrial genes leading to CMS is consistent with the mitochondrial DNA (mtDNA) evolution. The sequence conservation resulting from faithfully maternal inheritance and the chimeric structure caused by frequent sequence recombination have been defined as two major features of the mitogenome. However, when and how these chimeric mitochondrial genes appear in the context of the highly conserved reproduction of mitochondria is an enigma. This review, therefore, presents the critical view of the research on CMS in plants to elucidate the mechanisms of this phenomenon. Generally, distant hybridization is the main mechanism to generate an original CMS source in natural populations and in breeding. Mitochondria and mitogenomes show pleomorphic and dynamic changes at key stages of the life cycle. The promitochondria in dry seeds develop into fully functioning mitochondria during seed imbibition, followed by massive mitochondria or mitogenome fusion and fission in the germination stage along with changes in the mtDNA structure and quantity. The mitogenome stability is controlled by nuclear loci, such as the nuclear gene Msh1. Its suppression leads to the rearrangement of mtDNA and the production of heritable CMS genes. An abundant recombination of mtDNA is also often found in distant hybrids and somatic/cybrid hybrids. Since mtDNA recombination is ubiquitous in distant hybridization, we put forward a hypothesis that the original CMS genes originated from mtDNA recombination during the germination of the hybrid seeds produced from distant hybridizations to solve the nucleo-cytoplasmic incompatibility resulting from the allogenic nuclear genome during seed germination.

Genome-wide profiling of WRKY genes involved in flavonoid biosynthesis in Erigeron breviscapus.

The transcription factors of WRKY genes play essential roles in plant growth, stress responses, and metabolite biosynthesis. Erigeron breviscapus, a traditional Chinese herb, is abundant in flavonoids and has been used for centuries to treat cardiovascular and cerebrovascular diseases. However, the WRKY transcription factors that regulate flavonoid biosynthesis in E. breviscapus remain unknown. In this study, a total of 75 EbWRKY transcription factors were predicted through comprehensive genome-wide characterization of E. breviscapus and the chromosomal localization of each EbWRKY gene was investigated. RNA sequencing revealed transient responses of 74 predicted EbWRKY genes to exogenous abscisic acid (ABA), salicylic acid (SA), and gibberellin 3 (GA3) after 4 h of treatment. In contrast, the expression of key structural genes involved in flavonoid biosynthesis increased after 4 h in GA3 treatment. However, the content of flavonoid metabolites in leaves significantly increased at 12 h. The qRT-PCR results showed that the expression patterns of EbWRKY11, EbWRKY30, EbWRKY31, EbWRKY36, and EbWRKY44 transcription factors exhibited a high degree of similarity to the 11 structural genes involved in flavonoid biosynthesis. Protein-DNA interactions were performed between the key genes involved in scutellarin biosynthesis and candidate WRKYs. The result showed that F7GAT interacts with EbWRKY11, EbWRKY36, and EbWRKY44, while EbF6H has a self-activation function. This study provides comprehensive information on the regulatory control network of flavonoid accumulation mechanisms, offering valuable insights for breeding E. breviscapus varieties with enhanced scutellarin content.

Chemical composition and antioxidant activity of Polygonatum kingianum processed by the traditional method of "Nine Cycles of Steaming and Sun-Drying".

Polygonatum kingianum Coll. et (Hemsl) is a famous Chinese traditional food and medicine analogous plant. The rhizome of P. kingianum showed a decrease in levels of alkaloids, amino acids and derivatives, terpenoids, and an increase in organic acid and saccharides when it was processed by the traditional method of "Nine Cycles of Steaming and Sun-Drying". The relative content of 341 metabolites were increased (fold change, FC > 2; variable importance in projection, VIP > 1 and P-value, P < 0.05); while 456 metabolites were decreased (FC < 0.5, VIP > 1, and P < 0.05). The changes in chemical components result in a decrease in numb taste and an increase in sweetness. The increased antioxidant activity was observed in the processed samples. Together, this work has advanced the mechanism of reducing numb taste and enhancing antioxidant activity in the resource plants, such as P. kingianum, processed by the traditional method.

Promiscuous Oxidosqualene Cyclases from Neoalsomitra integrifoliola Catalyzing the Formation of Tetracyclic, Pentacyclic, and Heterocyclic Triterpenes.

Six oxidosqualene cyclases (NiOSC1-NiOSC6) from Neoalsomitra integrifoliola were characterized for the biosynthesis of diverse triterpene scaffolds, including tetracyclic and pentacyclic triterpenes from the 2,3-oxidosqualene (1) and oxacyclic triterpenes from the 2,3:22,23-dioxidosqualene (2). NiOSC1 showed high efficiency in the production of naturally rare (20R)-epimers of oxacyclic triterpenes. Mutagenesis results revealed that the NiOSC1-F731G mutant significantly increased the yields of (20R)-epimers compared to the wild type. Homology modeling and molecular docking elucidated the origin of the (20R)-configuration in the epoxide addition step.

Integrated metabolome and transcriptome analyses reveal the molecular mechanism underlying dynamic metabolic processes during taproot development of Panax notoginseng.

BACKGROUND: Panax notoginseng (Burk) F. H. Chen is one of the most famous Chinese traditional medicinal plants. The taproot is the main organ producing triterpenoid saponins, and its development is directly linked to the quality and yield of the harvested P. notoginseng. However, the mechanisms underlying the dynamic metabolic changes occurring during taproot development of P. notoginseng are unknown. RESULTS: We carried out metabolomic and transcriptomic analyses to investigate metabolites and gene expression during the development of P. notoginseng taproots. The differentially accumulated metabolites included amino acids and derivatives, nucleotides and derivatives, and lipids in 1-year-old taproots, flavonoids and terpenoids in 2- and 3-year-old taproots, and phenolic acids in 3-year-old taproots. The differentially expressed genes (DEGs) are related to phenylpropanoid biosynthesis, metabolic pathway and biosynthesis of secondary metabolites at all three developmental stages. Integrative analysis revealed that the phenylpropanoid biosynthesis pathway was involved in not only the development of but also metabolic changes in P. notoginseng taproots. Moreover, significant accumulation of triterpenoid saponins in 2- and 3-year-old taproots was highly correlated with the up-regulated expression of cytochrome P450s and uridine diphosphate-dependent glycosyltransferases genes. Additionally, a gene encoding RNase-like major storage protein was identified to play a dual role in the development of P. notoginseng taproots and their triterpenoid saponins synthesis. CONCLUSIONS: These results elucidate the molecular mechanism underlying the accumulation of and change relationship between primary and secondary metabolites in P. notoginseng taproots, and provide a basis for the quality control and genetic improvement of P. notoginseng.

Development of a stable genetic transformation system in Erigeron breviscapus: a case study with EbYUC2 in relation to leaf number and flowering time.

MAIN CONCLUSION: A stable genetic transformation system for Erigeron breviscapus was developed. We cloned the EbYUC2 gene and genetically transformed it into Arabidopsis thaliana and E. breviscapus. The leaf number, YUC2 gene expression, and the endogenous auxin content in transgenic plants were significantly increased. Erigeron breviscapus is a prescription drug for the clinical treatment of cardiovascular and cerebrovascular diseases. The rosette leaves have the highest content of the major active compound scutellarin and are an important component in the yield of E. breviscapus. However, little is known about the genes related to the leaf number and flowering time of E. breviscapus. In our previous study, we identified three candidate genes related to the leaf number and flowering of E. breviscapus by combining resequencing data and genome-wide association study (GWAS). However, their specific functions remain to be characterized. In this study, we cloned and transformed the previously identified full-length EbYUC2 gene into Arabidopsis thaliana, developed the first stable genetic transformation system for E. breviscapus, and obtained the transgenic plants overexpressing EbYUC2. Compared with wild-type plants, the transgenic plants showed a significant increase in the number of leaves, which was correlated with the increased expression of EbYUC2. Consistently, the endogenous auxin content, particularly indole-3-acetic acid, in transgenic plants was also significantly increased. These results suggest that EbYUC2 may control the leaf number by regulating auxin biosynthesis, thereby laying a foundation for revealing the molecular mechanism governing the leaf number and flowering time of E. breviscapus.

Biosynthetic pathway of prescription cucurbitacin IIa and high-level production of key triterpenoid intermediates in engineered yeast and tobacco.

Cucurbitacin IIa is a triterpenoid isolated exclusively from Hemsleya plants and a non-steroidal anti-inflammatory drug that functions as the main ingredient of prescription Hemslecin capsules and tablets in China. Synthetic biology provides new strategies for production of such valuable cucurbitacins at a large scale; however, the biosynthetic pathway of cucurbitacin IIa has been unknown, and the heterologous production of cucurbitacins in galactose medium has been expensive and low yielding. In this study, we characterized the functions of genes encoding two squalene epoxidases (HcSE1-2), six oxidosqualene cyclases (HcOSC1-6), two CYP450s (HcCYP87D20 and HcCYP81Q59), and an acyltransferase (HcAT1) in cucurbitacin IIa biosynthesis by heterologous expression in Saccharomyces cerevisiae and Nicotiana benthamiana. We achieved high-level production of the key cucurbitacin precursor 11-carbonyl-20ß-hydroxy-Cuol from glucose in yeast via modular engineering of the mevalonate pathway and optimization of P450 expression levels. The resulting yields of 46.41 mg/l 11-carbonyl-20ß-hydroxy-Cuol and 126.47 mg/l total cucurbitacin triterpenoids in shake flasks are the highest yields yet reported from engineered microbes. Subsequently, production of 11-carbonyl-20ß-hydroxy-Cuol by transient gene expression in tobacco resulted in yields of 1.28 mg/g dry weight in leaves. This work reveals the key genes involved in biosynthesis of prescription cucurbitacin IIa and demonstrates that engineered yeast cultivated with glucose can produce high yields of key triterpenoid intermediates. We describe a low-cost and highly efficient platform for rapid screening of candidate genes and high-yield production of pharmacological triterpenoids.

Low-concentration exogenous 3-indoleacetic acid improves fruit-setting rate of Marsdenia tenacissima by inhibiting the expression of embryo abortion-related genes.

Marsdenia tenacissima is a medicinal plant characterized by many flowers, few fruits, and a low fruit-setting rate. Exogenous auxins can improve the fruit-setting rate of plants; however, their impacts on M. tenacissima and regulatory mechanisms remain unclear. In this study, we conducted a field experiment to determine the fruit-setting rate, seed-setting rate, fruit size, and changes in transcriptional expression of related genes by spraying 10 and 50 mg·L-1 of 3-indoleacetic acid (IAA). The control plants were sprayed with distilled water. Our results indicated that the fruit-setting rate was 0.15 when treated with 10 mg·L-1 of IAA, which was 2.76-fold higher than that of the control. Compared with that of the control, the number of differentially expressed genes (DEGs) regulated by 10 mg·L-1 of IAA was 28.6-fold higher than that regulated by 50 mg·L-1 of IAA. These DEGs were closely related to hormone metabolism and fruit development. By transcriptome analysis, spraying 10 mg·L-1 of IAA increased the expressions of STP6, MYB17, and LAX3 and reduced those of CXE18, ILR1-like 3, and SAUR50; this possibly affected the ovule, embryo, and fruit development, thereby elevating the fruit-setting rate of M. tenacissima. Our results indicated that low IAA concentration increased the fruit-setting rate of M. tenacissima, providing theoretical and practical support for promoting the seed yield of M. tenacissima.

Functional characterization of genes related to triterpene and flavonoid biosynthesis in Cyclocarya paliurus.

MAIN CONCLUSION: The oxidosqualene cyclases (OSCs) generating triterpenoid skeletons in Cyclocarya paliurus were identified for the first time, and two uridine diphosphate (UDP)-glycosyltransferases (UGTs) catalyzing the glycosylation of flavonoids were characterized. Cyclocarya paliurus, a native rare dicotyledonous plant in China, contains an abundance of triterpenoid saponins and flavonoid glycosides that exhibit valuable pharmaceutical effects in preventing hypertension, hyperlipidemia, and diabetes. However, the molecular mechanism explaining the biosynthesis of triterpenoid saponin and flavonoid glycoside in C. paliurus remains unclear. In this study, the triterpene content in different tissues and the expression pattern of genes encoding the key enzymes associated with triterpenoid saponin and flavonoid glycoside biosynthesis were studied using transcriptome and metabolome analysis. The eight upstream oxidosqualene cyclases (OSCs) involved in triterpenoid saponin biosynthesis were functionally characterized, among them CpalOSC6 catalyzed 2,3;22,23-dioxidosqualene to form 3-epicabraleadiol; CpalOSC8 cyclized 2,3-oxidosqualene to generate dammarenediol-II; CpalOSC2 and CpalOSC3 produced ß-amyrin and CpalOSC4 produced cycloartenol, while CpalOSC2-CpalOSC5, CpalOSC7, and CpalOSC8 all produced lanosterol. However, no catalytic product was detected for CpalOSC1. Moreover, two downstream flavonoid uridine diphosphate (UDP)-glycosyltransferases (UGTs) (CpalUGT015 and CpalUGT100) that catalyze the last step of flavonoid glycoside biosynthesis were functionally elucidated. These results uncovered the key genes involved in the biosynthesis of triterpenoid saponins and flavonoid glycosides in C. paliurus that could be applied to produce flavonoid glycosides and key triterpenoid saponins in the future via a synthetic strategy.

Simple and biodegradable mesoporous silica nanocarriers for enhancing antitumor therapy through photochemical synergism.

The biosafety and degradability of nanocarriers have always been an important factor restricting their entry into the clinic. In this work, a new nano-system was prepared by coating the photothermal effect of dopamine-doped mesoporous silica nanoparticles with carboxymethyl chitin through electrostatic interaction, and is further anchored with folic acid on the surface for targeted delivery of anti-cancer the drug doxorubicin (DOX). The nano-system (DOX@PDA/MSN-CMCS-FA) is simply modified CMCS after being loaded with DOX and has good dispersibility, and the drug loading is 10.6%. In vitro release studies have shown that the release rate of PDA/MSN-CMCS-FA is 40% in pH 5.5. Effective degradation is debris in 14 d acidic environments. Due to the anti-infrared photothermal effects of PDA doping and DOX chemotherapy, the semi-lethal concentration (IC50) of nanoparticles (NPS) was 14.95 µg/mL, which can inhibit tumor cell growth by photochemical synergistic treatment, and have certain degradation performance.

Multilayered regulation of secondary metabolism in medicinal plants.

Medicinal plants represent a huge reservoir of secondary metabolites (SMs), substances with significant pharmaceutical and industrial potential. However, obtaining secondary metabolites remains a challenge due to their low-yield accumulation in medicinal plants; moreover, these secondary metabolites are produced through tightly coordinated pathways involving many spatiotemporally and environmentally regulated steps. The first regulatory layer involves a complex network of transcription factors; a second, more recently discovered layer of complexity in the regulation of SMs is epigenetic modification, such as DNA methylation, histone modification and small RNA-based mechanisms, which can jointly or separately influence secondary metabolites by regulating gene expression. Here, we summarize the findings in the fields of genetic and epigenetic regulation with a special emphasis on SMs in medicinal plants, providing a new perspective on the multiple layers of regulation of gene expression.

Combined Metabolome and Transcriptome Analysis of Creamy Yellow and Purple Colored Panax notoginseng Roots.

Panax notoginseng (Burk.) F.H. Chen is a species of the Araliaceae family that inhabits southwestern China, Burma, and Nepal. It is cultivated on a commercial scale in Yunnan province, China, owing to its significance in traditional Chinese medicine. Panax notoginseng roots are usually yellow-white (HS); however, purple roots (ZS) have also been reported. The majority of P. notoginseng research has concentrated on the identification and production of natural chemicals in HS; however, there is little to no information about the composition of ZS. Using UPLC-MS/MS, we investigated the global metabolome profile of both ZS- and HS-type roots and discovered 834 metabolites from 11 chemical groups. There were 123 differentially accumulated metabolites (DAM) in the HS and ZS roots, which were classified as lipids and lipid-like molecules, polyketides, organoheterocyclic chemicals, and organooxygen compounds. We investigated the associated compounds in the DAMs because of the importance of anthocyanins in color and saponins and ginsenosides in health benefits. In general, we discovered that pigment compounds such as petunidin 3-glucoside, delphinidin 3-glucoside, and peonidin-3-O-beta-galactoside were more abundant in ZS. The saponin (eight compounds) and ginsenoside (26 compounds) content of the two varieties of roots differed as well. Transcriptome sequencing revealed that flavonoid and anthocyanin production genes were more abundant in ZS than in HS. Similarly, we found differences in gene expression in genes involved in terpenoid production and related pathways. Overall, these findings suggest that the purple roots of P. notoginseng contain varying amounts of ginsenosides and anthocyanins compared to roots with a creamy yellow color.

Tissue Engineering of JAK Inhibitor-Loaded Hierarchically Biomimetic Nanostructural Scaffold Targeting Cellular Senescence for Aged Bone Defect Repair and Bone Remolding.

Cell senescence or apoptosis contributes to self-failure and functional loss in specialized cells, leading to incapacity of the body to repair specific damages. Senescent bone marrow mesenchymal stem cells (BMSCs) lose their proliferative abilities and secrete senescence-associated secretory phenotype (SASP), hindering their participation in bone defect repair. Hence, the effective suppression of cell senescence is crucial to restore the self-repair capacity of body to treat bone defects. Since the Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway is associated with SASP secretion, herein, a new strategy is proposed to inhibit this pathway to suppress SASP secretion and enhance osteoblast activity based on a novel hierarchically biomimetic nanostructural electrospun scaffold with JAK inhibitors (JAKi, Ruxolitinib) loaded. As validated by in vitro and in vivo experiments, the JAKi loaded scaffold reduces SASP expression effectively and alleviates senescent cell burden, creating a pro-regeneration microenvironment that enhances osteoblast function and mineralization activity as well as rejuvenating the bone repair capacity. These findings offer insights into the regulatory role of cellular senescence in bone aging and provide a new and effective strategy to treat age-related bone defects by delivery of JAKi to locally aging bone defect sites.

EbbHLH80 Enhances Salt Responses by Up-Regulating Flavonoid Accumulation and Modulating ROS Levels.

bHLH transcription factors are involved in multiple aspects of plant biology, such as the response to abiotic stress. Erigeron breviscapus is a composite plant, and its rich flavonoids have strong preventive and therapeutic effects on cardio cerebral vascular disease. EbbHLH80, a gene from E. breviscapus that positively regulates flavonoid synthesis, was previously characterized. However, it is unclear whether EbbHLH80 increases flavonoid accumulation, which affects salt tolerance. The function of EbbHLH80 in transgenic tobacco seeds was identified by phylogenetic analysis and metabolome-transcriptome analysis. We investigated the role of EbbHLH80 in salt stress response. Our results showed that the expression of EbbHLH80 increased following salt treatment. Integrating the metabolome and transcriptome analysis of EbbHLH80-OE and Yunyan 87 (WT) seeds, we identified several genes and metabolites related to flavonoid biosynthesis and salt stress. Moreover, EbbHLH80-OE plants displayed higher salt tolerance than wild-type plants during seed germination and seedling growth. After salt treatment, transgenic tobacco had significantly lower levels of reactive oxygen species (ROS) than WT, with enhanced levels of antioxidant enzyme expression. Altogether, our results demonstrated that EbbHLH80 might be a positive regulator, promoting salt tolerance by modulating ROS scavenging and increasing stress-responsive genes.

Production of the antidepressant orcinol glucoside in Yarrowia lipolytica with yields over 6,400-fold higher than plant extraction.

Orcinol glucoside (OG), mainly found in the rhizome of the traditional Chinese herb Curculigo orchioides Gaertn, is noted for its antidepressant effects. In this study, an efficient screening pipeline was established for identifying the highly active orcinol synthase (ORS) and UDP-dependent glycosyltransferase (UGT) involved in the biosynthesis of OG by combining transcriptome analysis, structure-based virtual screening, and in vitro enzyme activity assays. By enhancing the downstream pathway, metabolic engineering and fermentation optimization, the OG production in Yarrowia lipolytica was improved 100-fold, resulting in a final yield of 43.46 g/L (0.84 g/g DCW), which is almost 6,400-fold higher than the extraction yield from C. orchioides roots. This study provides a reference for rapid identification of functional genes and high-yield production of natural products.