Triterpenoid Saponin Biosynthesis Research Articles

Identification and Expression Analysis of Cytochrome P450 Genes Probably Involved in Triterpenoid Saponins Biosynthesis in Astragalus mongholicus.

Cytochromes P450 (P450s) are one of the largest enzymatic protein families and play critical roles in the synthesis and metabolism of plant secondary metabolites. Astragaloside IV (AS-IV) is one of the primary active components in Astragalus herbs, exhibiting diverse biological activities and pharmacological effects. However, P450s involved in the astragaloside biosynthesis have not been systematically analyzed in Astragalus mongholicus (A. mongholicus). In this study, we identified 209 P450 genes from the genome of A. mongholicus (AmP450s), which were classified into nine clans and 47 families and performed a systematic overview of their physical and chemical properties, phylogeny, gene structures and conserved motifs. Weighted gene co-expression network analysis (WGCNA) revealed that AmP450s are critical in the astragaloside biosynthesis pathway. The expression levels of these AmP450s were verified by quantitative real-time PCR (qRT-PCR) analysis in the root, stem and leaf, showing that most AmP450s are abundant in the root. Additionally, the correlation analysis between gene expressions and AS-IV content showed that twelve AmP450s, especially CYP71A28, CYP71D16 and CYP72A69, may have significant potential in the biosynthesis of astragaloside. This study systematically investigates the P450s of A. mongholicus and offers valuable insights into further exploring the functions of CYP450s in the astragaloside biosynthesis pathway.

Genome-Wide Identification of the CYP716 Gene Family in Platycodon grandiflorus (Jacq.) A. DC. and Its Role in the Regulation of Triterpenoid Saponin Biosynthesis.

The main type of saponins occurring in the root of Platycodon grandiflorus (Jacq.) A. DC. are oleanolic acid glycosides. The CYP716 gene family plays a major role in catalyzing the conversion of β-amyrin into oleanolic acid. However, studies on the CYP716 genes in P. grandiflorus are limited, and its evolutionary history remains poorly understood. In this study, 22 PgCYP716 genes were identified, distributed among seven subfamilies. Cis-acting elements of the PgCYP716 promoters were mainly involved in plant hormone regulation and responses to abiotic stresses. PgCYP716A264, PgCYP716A391, PgCYP716A291, and PgCYP716BWv3 genes were upregulated in the root and during saponin accumulation, as shown by RNA-seq analysis, suggesting that these four genes play an important role in saponin synthesis. The results of subcellular localization indicated that these four genes encoded membrane proteins. Furthermore, the catalytic activity of these four genes was proved in the yeast, which catalyzed the conversion of β-amyrin into oleanolic acid. We found that the content of β-amyrin, platycodin D, platycoside E, platycodin D3, and total saponins increased significantly when either of the four genes was over expressed in the transgenic hair root. In addition, the expression of PgSS, PgGPPS2, PgHMGS, and PgSE was also upregulated while these four genes were overexpressed. These data support that these four PgCYP716 enzymes oxidize β-amyrin to produce oleanolic acid, ultimately promoting saponin accumulation by activating the expression of upstream pathway genes. Our results enhanced the understanding of the functional variation among the PgCYP716 gene family involved in triterpenoid biosynthesis and provided a theoretical foundation for improving saponin content and enriching the saponin biosynthetic pathway in P. grandiflorus.

Identification and analysis of UGT genes associated with triterpenoid saponin in soapberry (Sapindus mukorossi Gaertn.)

BackgroundSoapberry (Sapindus mukorossi) is an economically important multifunctional tree species. Triterpenoid saponins have many functions in soapberry. However, the types of uridine diphosphate (UDP) glucosyltransferases (UGTs) involved in the synthesis of triterpenoid saponins in soapberry have not been clarified.ResultsIn this study, 42 SmUGTs were identified in soapberry, which were unevenly distributed on 12 chromosomes and had sequence lengths of 450 bp to 1638 bp, with an average of 1388 bp. The number of amino acids in SmUGTs was 149 to 545, with an average of 462. Most SmUGTs were acidic and hydrophilic unstable proteins, and their secondary structures were mainly α-helices and random coils. All had conserved UDPGT and PSPG-box domains. Phylogenetic analysis divided them into four subclasses, which glycosylated different carbon atoms. Prediction of cis-acting elements suggested roles of SmUGTs in plant development and responses to environmental stresses. The expression patterns of SmUGTs differed according to the developmental stage of fruits, as determined by transcriptomics and RT-qPCR. Co-expression network analysis of SmUGTs and related genes/transcription factors in the triterpenoid saponin synthesis pathway was also performed. The results indicated potential roles for many transcription factors, such as SmERFs, SmGATAs and SmMYBs. A correlation analysis showed that 42 SmUGTs were crucial in saponin synthesis in soapberry.ConclusionsOur findings suggest optimal targets for manipulating glycosylation in soapberry triterpenoid saponin biosynthesis; they also provide a theoretical foundation for further evaluation of the functions of SmUGTs and analyses of their biosynthetic mechanisms.

Plant triterpenoid saponins function as susceptibility factors to promote the pathogenicity of Botrytis cinerea

The gray mold fungus Botrytis cinerea is a necrotrophic pathogen that causes diseases in hundreds of plant species, including high-value crops. Its polyxenous nature and pathogenic success are due to its ability to perceive host signals in its favor. In this study, we found that laticifer cells of Euphorbia lathyris are a source of susceptibility factors required by B. cinerea to cause disease. Consequently, poor-in-latex (pil) mutants, which lack laticifer cells, show full resistance to this pathogen, whereas lot-of-latex mutants, which produce more laticifer cells, are hypersusceptible. These S factors are triterpenoid saponins, which are widely distributed natural products of vast structural diversity. The downregulation of laticifer-specific oxydosqualene cyclase genes, which encode the first committed step enzymes for triterpene and, therefore, saponin biosynthesis, conferred disease resistance to B. cinerea. Likewise, the Medicago truncatula lha-1 mutant, compromised in triterpenoid saponin biosynthesis, showed enhanced resistance. Interestingly, the application of different purified triterpenoid saponins pharmacologically complemented the disease-resistant phenotype of pil and hla-1 mutants and enhanced disease susceptibility in different plant species. We found that triterpenoid saponins function as plant cues that signal transcriptional reprogramming in B. cinerea, leading to a change in its growth habit and infection strategy, culminating in the abundant formation of infection cushions, the multicellular appressoria apparatus dedicated to plant penetration and biomass destruction in B. cinerea. Taken together, these results provide an explanation for how plant triterpenoid saponins function as disease susceptibility factors to promote B. cinerea pathogenicity.

The genomes of seven economic Caesalpinioideae trees provide insights into polyploidization history and secondary metabolite biosynthesis

The Caesalpinioideae subfamily contains many well-known trees that are important for economic sustainability and human health, but a lack of genomic resources has hindered their breeding and utilization. Here, we present chromosome-level reference genomes for the two food and industrial trees Gleditsia sinensis (921 Mb) and Biancaea sappan (872 Mb), the three shade and ornamental trees Albizia julibrissin (705 Mb), Delonix regia (580 Mb), and Acacia confusa (566 Mb), and the two pioneer and hedgerow trees Leucaena leucocephala (1338 Mb) and Mimosa bimucronata (641 Mb). Phylogenetic inference shows that the mimosoid clade has a much higher evolutionary rate than the other clades of Caesalpinioideae. Macrosynteny comparison suggests that the fusion and breakage of an unstable chromosome are responsible for the difference in basic chromosome number (13 or 14) for Caesalpinioideae. After an ancient whole-genome duplication (WGD) shared by all Caesalpinioideae species (CWGD, ∼72.0 million years ago [MYA]), there were two recent successive WGD events, LWGD-1 (16.2–19.5 MYA) and LWGD-2 (7.1–9.5 MYA), in L. leucocephala. Thereafter, ∼40% gene loss and genome-size contraction have occurred during the diploidization process in L. leucocephala. To investigate secondary metabolites, we identified all gene copies involved in mimosine metabolism in these species and found that the abundance of mimosine biosynthesis genes in L. leucocephala largely explains its high mimosine production. We also identified the set of all potential genes involved in triterpenoid saponin biosynthesis in G. sinensis, which is more complete than that based on previous transcriptome-derived unigenes. Our results and genomic resources will facilitate biological studies of Caesalpinioideae and promote the utilization of valuable secondary metabolites.

HMGR and CHS gene cloning, characterizations and tissue-specific expressions in Polygala tenuifolia Willd.

Triterpenoid saponins and flavonoids have several pharmacological activities against P. tenuifolia. The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) and chalcone synthase (CHS) are the rate-limiting enzymes of triterpenoid saponin and flavonoid biosynthesis, respectively. In this study, HMGR and CHS genes were cloned from P. tenuifolia, and their bioinformatics analyses and tissue-specific expression were investigated. The results showed that the HMGR and CHS genes were successfully cloned, separately named the PtHMGR gene (NCBI accession: MK424118) and PtCHS gene (NCBI accession: MK424117). The PtHMGR gene is 2323 bp long, has an open reading frame (ORF) of 1782 bp, and encods 593 amino acids. The PtCHS gene is 1633 bp long with an ORF of 1170 bp, encoding 389 amino acids. PtHMGR and PtCHS were both hydrophobic, not signal peptides or secreted proteins, containing 10 conserved motifs. PtHMGR and PtCHS separately showed high homology with HMGR and CHS proteins from other species, and their secondary structures mainly included α-helix and random curl. The tertiary structure of PtHMGR was highly similarity to that the template 7ULI in RCSB PDB with 92.0% coverage rate. The HMG-CoA-binding domain of PtHMGR is located at 173-572 amino acid residues, including five bound sites. The tertiary structure of PtCHS showed high consistency with the template 1I86 in RCSB PDB with 100% coverage rate, contained malonyl CoA and 4-coumaroyl-CoA linkers. The expression of PtHMGR and PtCHS is tissue-specific. PtHMGR transcripts were mainly accumulated in roots, followed by leaves, and least in stems, and were significantly positively correlated with the contents of total saponin and tenuifolin. PtCHS was highly expressed in the stems, followed by the leaves, with low expression in the roots. PtCHS transcripts showed a significant positive correlation with total flavonoids content, however, they were significantly negatively correlated with the content of polygalaxanthone III (a type of flavonoids). This study provided insight for further revealing the roles of PtHMGR and PtCHS.

RNA sequencing analysis reveals PgbHLH28 as the key regulator in response to methyl jasmonate-induced saponin accumulation in Platycodon grandiflorus

Abstract Platycodon grandiflorus (Jacq.) A. DC, known for its saponin content, can potentially prevent and treat cerebrovascular diseases and COVID-19. Triterpenoid saponin biosynthesis in plants is enhanced by methyl jasmonate (MeJA) application. However, the underlying molecular mechanisms of MeJA-induced saponin biosynthesis remain unknown in P. grandiflorus. In the current study, exogenous application of 100 μmol/l MeJA was identified to be optimal for promoting saponin accumulation. RNA sequencing analysis demonstrated the PgbHLH28 gene as a key regulatory factor responding to MeJA during saponin accumulation. Overexpression of PgbHLH28 in P. grandiflorus increased saponin content, while silencing of PgbHLH28 significantly inhibited saponin synthesis, suggesting that PgbHLH28 acts as a positive regulator of saponin biosynthesis. Yeast one-hybrid and dual luciferase assays demonstrated that PgbHLH28 directly bound to the promoters of PgHMGR2 and PgDXS2 to activate gene expression. PgHMGR2 and PgDXS2 transformation promoted saponin accumulation, while silencing of these genes inhibited saponin biosynthesis. This study determined that MeJA promoted saponin accumulation in P. grandiflorus by inducing PgbHLH28 gene expression and activating downstream genes (PgHMGR2 and PgDXS2) involved in saponin biosynthesis. In conclusion, a complex regulatory network governing saponin biosynthesis following MeJA treatment was elucidated, offering a theoretical foundation for enhancing saponin content and biosynthesis efficacy in P. grandiflorus.

PnNAC2 promotes the biosynthesis of Panax notoginseng saponins and induces early flowering.

PnNAC2 positively regulates saponin biosynthesis by binding the promoters of key biosynthetic genes, including PnSS, PnSE, and PnDS. PnNAC2 accelerates flowering through directly associating with the promoters of FT genes. NAC transcription factors play an important regulatory role in both terpenoid biosynthesis and flowering. Saponins with multiple pharmacological activities are recognized as the major active components of Panax notoginseng. The P. notoginseng flower is crucial for growth and used for medicinal and food purposes. However, the precise function of the P. notoginseng NAC transcription factor in the regulation of saponin biosynthesis and flowering remains largely unknown. Here, we conducted a comprehensive characterization of a specific NAC transcription factor, designated as PnNAC2, from P. notoginseng. PnNAC2 was identified as a nuclear-localized protein with transcription activator activity. The expression profile of PnNAC2 across various tissues mirrored the accumulation pattern of total saponins. Knockdown experiments of PnNAC2 in P. notoginseng calli revealed a significant reduction in saponin content and the expression level of pivotal saponin biosynthetic genes, including PnSS, PnSE, and PnDS. Subsequently, Y1H assays, dual-LUC assays, and electrophoretic mobility shift assays (EMSAs) demonstrated that PnNAC2 exhibits binding affinity to the promoters of PnSS, PnSE and PnDS, thereby activating their transcription. Additionally, an overexpression assay of PnNAC2 in Arabidopsis thaliana witnessed the acceleration of flowering and the induction of the FLOWERING LOCUS T (FT) gene expression. Furthermore, PnNAC2 demonstrated the ability to bind to the promoters of AtFT and PnFT genes, further activating their transcription. In summary, these results revealed that PnNAC2 acts as a multifunctional regulator, intricately involved in the modulation of triterpenoid saponin biosynthesis and flowering processes.

PgDDS Changes the Plant Growth of Transgenic Aralia elata and Improves the Production of Re and Rg3 in Its Leaves.

Aralia elata (Miq.) Seem is a medicinal plant that shares a common pathway for the biosynthesis of triterpenoid saponins with Panax ginseng. Here, we transferred the dammarenediol-II synthase gene from P. ginseng (PgDDS; GenBank: AB122080.1) to A. elata. The growth of 2-year-old transgenic plants (L27; 9.63 cm) was significantly decreased compared with wild-type plants (WT; 74.97 cm), and the leaflet shapes and sizes of the transgenic plants differed from those of the WT plants. Based on a terpene metabolome analysis of leaf extracts from WT, L13, and L27 plants, a new structural skeleton for ursane-type triterpenoid saponins was identified. Six upregulated differentially accumulated metabolites (DAMs) were detected, and the average levels of Rg3 and Re in the leaves of the L27 plants were 42.64 and 386.81 μg/g, respectively, increased significantly compared with the WT plants (15.48 and 316.96 μg/g, respectively). Thus, the expression of PgDDS in A. elata improved its medicinal value.

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

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 β-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.

Integrated metabolome and transcriptome analysis reveals the regulatory mechanism of low nitrogen-driven biosynthesis of saponins and flavonoids in Panax notoginseng

BackgroundNitrogen (N) is an important macronutrient involved in the biosynthesis of primary and secondary metabolites in plants. However, the metabolic regulatory mechanism of low-N-induced triterpenoid saponin and flavonoid accumulation in rhizomatous medicinal Panax notoginseng (Burk.) F. H. Chen remains unclear. MethodsTo explore the potential regulatory mechanism and metabolic basis controlling the response of P. notoginseng to N deficiency, the transcriptome and metabolome were analysed in the roots. ResultsThe N content was significantly reduced in roots of N0-treated P. notoginseng (0 kg·N·667 m−2). The C/N ratio was enhanced in the N-deficient P. notoginseng. N deficiency promotes the accumulation of amino acids (L-proline, L-leucine, L-isoleucine, L-norleucine, L-arginine, and L-citrulline) and sugar (arabinose, xylose, glucose, fructose, and mannose), thus providing precursor metabolites for the biosynthesis of flavonoids and triterpenoid saponins. Downregulation of key structural genes (PAL, PAL3, ACC1, CHS2, PPO, CHI3, F3H, DFR, and FGT), in particular with the key genes of F3H, involved in the flavonoid biosynthesis pathway possibly induced the decrease in flavonoid content with increased N supply. Notoginsenoside R1, ginsenoside Re, Rg1, Rd, F1, R1 + Rg1 + Rb1 and total triterpenoid saponins were enhanced in the N0 groups than in the N15 (15 kg·N·667 m−2) plants. Higher phosphoenolpyruvate (an intermediate of glycolyticwith pathway metabolism) and serine (an intermediate of photorespiration) levels induced by N deficiency possibly promote saponin biosynthesis through mevalonic acid (MVA) and methylerythritol (MEP) pathways. Genes (MVD2, HMGS, HMGR1, HMGR2, DXR, and HMGR1) encoding the primary enzymes HMGS, HMGR, DXR, and MVD in the MVA and MEP pathways were significantly upregulated in the N0-treated P. notoginseng. The saponin biosynthesis genes DDS, DDS, CYP716A52, CYP716A47, UGT74AE2, and FPS were upregulated in the N-deficient plants. Upregulation of genes involved in saponin biosynthesis promotes the accumulation of triterpenoid saponins in the N0-grown P. notoginseng. ConclusionsN deficiency enhances primary metabolisms, such as amino acids and sugar accumulation, laying the foundation for the synthesis of flavonoids and triterpenoid saponins in P. notoginseng. F3H, DDS, FPS, HMGR, HMGS and UGT74AE2 can be considered as candidates for functional characterisation of the N-regulated accumulation of triterpenoid saponins and flavonoids in future.

Integrated metabolome and transcriptome analysis identifies candidate genes involved in triterpenoid saponin biosynthesis in leaves of Centella asiatica (L.) Urban.

Centella asiatica (L.) Urban is a well-known medicinal plant which has multiple pharmacological properties. Notably, the leaves of C. asiatica contain large amounts of triterpenoid saponins. However, there have only been a few studies systematically elucidating the metabolic dynamics and transcriptional differences regarding triterpenoid saponin biosynthesis during the leaf development stages of C. asiatica. Here, we performed a comprehensive analysis of the metabolome and transcriptome to reveal the dynamic patterns of triterpenoid saponin accumulation and identified the key candidate genes associated with their biosynthesis in C. asiatica leaves. In this study, we found that the key precursors in the synthesis of terpenoids, including DMAPP, IPP and β-amyrin, as well as 22 triterpenes and eight triterpenoid saponins were considered as differentially accumulated metabolites. The concentrations of DMAPP, IPP and β-amyrin showed significant increases during the entire stage of leaf development. The levels of 12 triterpenes decreased only during the later stages of leaf development, but five triterpenoid saponins rapidly accumulated at the early stages, and later decreased to a constant level. Furthermore, 48 genes involved in the MVA, MEP and 2, 3-oxidosqualene biosynthetic pathways were selected following gene annotation. Then, 17 CYP450s and 26 UGTs, which are respectively responsible for backbone modifications, were used for phylogenetic-tree construction and time-specific expression analysis. From these data, by integrating metabolomics and transcriptomics analyses, we identified CaHDR1 and CaIDI2 as the candidate genes associated with DMAPP and IPP synthesis, respectively, and CaβAS1 as the one regulating β-amyrin synthesis. Two genes from the CYP716 family were confirmed as CaCYP716A83 and CaCYP716C11. We also selected two UGT73 families as candidate genes, associated with glycosylation of the terpenoid backbone at C-3 in C. asiatica. These findings will pave the way for further research on the molecular mechanisms associated with triterpenoid saponin biosynthesis in C. asiatica.

Integration of transcriptome, metabolome and high-throughput amplicon sequencing to compare the performance of wild and cultivated psammosilene tunicoides to reveal the beneficial plant-microbe interactions for domestication

Psammosilene tunicoides is an herbal medicine with analgesic, anti-inflammatory and immunoregulatory properties, and it is listed as an International Union for the Conservation of Nature (IUCN) endangered species. The plants have been domesticated in some areas of Yunnan Province, China, for commercial and medical use. However, the performance of domesticated plants and the negative effects of domestication remain unclear. Multi-omics analyses were performed to compare the performance between wild P. tunicoides (WP) and cultivated P. tunicoides (CP). Transcriptomic and metabolomic data were generated from the roots, while microbiome data were obtained from the rhizospheres. The transcriptomic data showed that there were different patterns of gene expression between the WP and CP. The genes HMGS, HMGR, MPD and SQS, which are involved in the biosynthesis of triterpenoid saponins, were significantly more highly expressed in the WP than in the CP. The metabolomic analysis also found that significantly more triterpenoids were produced in the WP compared with the CP. The microbiome data showed that most of the plant growth-promoting rhizobacteria (PGPR), such as Burkholderia-Caballeronia-Paraburkholderia and others, were enriched in the WP type, while some pathogenic microorganisms were enriched in the CP type. A weighted gene co-expression network analysis revealed that the accumulation of triterpenoid saponins positively correlated with the modules of genes that primarily participated in the plant hormone signal transduction pathway and the PGPR in modules. This indicated that the triterpenoid saponins may function as signaling molecules to recruit the functional microbes. The regulatory “plant-microbe” network of microorganisms and the genes associated with triterpenes was detected in P. tunicoides. This suggests that the management of these interactions should be considered as a sustainable tool to enhance the performance of domesticated plants.

Investigation of the response of Platycodongrandiflorus (Jacq.) A. DC to salt stress using combined transcriptomics and metabolomics

BackgroundPlatycodon grandiflorus (Jacq.) A. DC is a famous traditional Chinese medicine in China and an authentic medicine in Inner Mongolia. It has been traditionally used as an expectorant in cough and also has anti-inflammatory and other pharmacological effects. As a homologous plant of medicine and food, P. grandiflorus is widely planted in Northeast China. Soil salinity isa limiting factor for its cultivation. In this study, we comprehensively described the physiological characteristics of P. grandiflorus and combined transcriptomics and metabolomics to study the response of roots of P. grandiflorus to salt stress.ResultsOverall, 8,988 differentially expressed genes were activated and significantly altered the metabolic processes. In total, 428 differentially abundant metabolites were affected by salt stress. After moderate and severe salt stress, most of the differentially abundant metabolites were enriched in the L-phenylalanine metabolic pathway. Through the comprehensive analysis of the interaction between key genes and metabolites, the main pathways such as lignin compound biosynthesis and triterpene saponin biosynthesis were completed. The relative content of compounds related to lignin biosynthesis, such as caffeic acid, coniferin, and syringing, increased under salt stress, and the related genes such as PAL, C4H, and the key enzyme gene UGT72E2 were activated to adapt to the salt stress. Platycodon saponin is one of the major triterpene saponins in P. grandiflorus, and Platycodin D is its most abundant major bioactive component. Under severe salt stress, Platycodin D level increased by nearly 1.77-fold compared with the control group. Most of the genes involved insynthetic pathway of Platycodin D, such as HMGCR, GGPS, SE, and LUP, were upregulated under salt stress.ConclusionSalt stress led to a decrease in the biomass and affected the activities of antioxidant enzymes and contents of osmotic regulators in the plant. These results provided not only novel insights into the underlying mechanisms of response of P. grandiflorus to salt stress but also a foundation for future studies on the function of genes related to salt tolerance in the triterpenoid saponin biosynthesis pathway.

Deciphering triterpenoid saponin biosynthesis by leveraging transcriptome response to methyl jasmonate elicitation in Saponaria vaccaria

Methyl jasmonate (MeJA) is a known elicitor of plant specialized metabolism, including triterpenoid saponins. Saponaria vaccaria is an annual herb used in traditional Chinese medicine, containing large quantities of oleanane-type triterpenoid saponins with anticancer properties and structural similarities to the vaccine adjuvant QS-21. Leveraging the MeJA-elicited saponin biosynthesis, we identify multiple enzymes catalyzing the oxidation and glycosylation of triterpenoids in S. vaccaria. This exploration is aided by Pacbio full-length transcriptome sequencing and gene expression analysis. A cellulose synthase-like enzyme can not only glucuronidate triterpenoid aglycones but also alter the product profile of a cytochrome P450 monooxygenase via preference for the aldehyde intermediate. Furthermore, the discovery of a UDP-glucose 4,6-dehydratase and a UDP-4-keto-6-deoxy-glucose reductase reveals the biosynthetic pathway for the rare nucleotide sugar UDP-d-fucose, a likely sugar donor for fucosylation of plant natural products. Our work enables the production and optimization of high-value saponins in microorganisms and plants through synthetic biology approaches.

Genome assembly of Polygala tenuifolia provides insights into its karyotype evolution and triterpenoid saponin biosynthesis.

Polygala tenuifolia is a perennial medicinal plant that has been widely used in traditional Chinese medicine for treating mental diseases. However, the lack of genomic resources limits the insight into its evolutionary and biological characterization. In the present work, we reported the P. tenuifolia genome, the first genome assembly of the Polygalaceae family. We sequenced and assembled this genome by a combination of Illumnina, PacBio HiFi, and Hi-C mapping. The assembly includes 19 pseudochromosomes covering ~92.68% of the assembled genome (~769.62Mb). There are 36 463 protein-coding genes annotated in this genome. Detailed comparative genome analysis revealed that P. tenuifolia experienced two rounds of whole genome duplication that occurred ~39-44 and ~18-20 million years ago, respectively. Accordingly, we systematically reconstructed ancestral chromosomes of P. tenuifolia and inferred its chromosome evolution trajectories from the common ancestor of core eudicots to the present species. Based on the transcriptomics data, enzyme genes and transcription factors involved in the synthesis of triterpenoid saponin in P. tenuifolia were identified. Further analysis demonstrated that whole-genome duplications and tandem duplications play critical roles in the expansion of P450 and UGT gene families, which contributed to the synthesis of triterpenoid saponins. The genome and transcriptome data will not only provide valuable resources for comparative and functional genomic researches on Polygalaceae, but also shed light on the synthesis of triterpenoid saponin.

The ricebean genome provides insight into Vigna genome evolution and facilitates genetic enhancement.

The ricebean genome provides insight into Vigna genome evolution and facilitates genetic enhancement.

Modifications in steroid and triterpenoid metabolism in Calendula officinalis plants and hairy root culture in response to chitosan treatment

BackgroundChitosan, a deacetylated derivative of chitin, is one of the most preferred biopolymers for use as biostimulants and biofertilizers in organic agriculture and as elicitors to enhance the productivity of plant in vitro cultures. Valued as a non-toxic, biodegradable, and environment-friendly agent, it is widely applied to improve plant growth and yield, the content of bioactive specialized metabolites, and resistance to stress conditions and pathogens. However, the influence of chitosan on the growth-defense trade-off, particularly the interplay between steroid and triterpenoid metabolism, has not been extensively investigated.ResultsIn this study, Calendula officinalis pot plants and hairy root cultures exposed to chitosan treatment displayed reduced biomass and altered steroid and triterpenoid metabolism. Biosynthesis and accumulation of free forms of sterols (particularly stigmasterol) were inhibited, while the content of sterol esters increased remarkably. The content of some triterpenoids (mainly free triterpenoid acids) was slightly enhanced; however, the biosynthesis of triterpenoid saponins was negatively affected.ConclusionsThese results indicate that in certain plants, chitosan treatment might not positively influence the growth and metabolite production. Therefore, to avoid unexpected effects, initial studies of the conditions of chitosan treatment are recommended, including the dose and the number of chitosan applications, the type of treatment (e.g., foliar or soil), and the vegetative stage of the treated plants.

Heterologous synthesis of ginsenoside F2 in Saccharomyces cerevisiae by pathway and UDP-glycosyltransferase engineering

Ginsenoside F2 (F2) is a rare tetracyclic triterpenoid saponin with a variety of bioactivities, such as anti-cancer, anti-inflammatory, antioxidant etc. In this study, we constructed the biosynthetic pathway of F2 in Saccharomyces cerevisiae by expressing UDP-glycosyltransferase 1 (UGT1) and glycosyltransferase yojK1 (GTK1) in a high protopanaxadiol (PPD) production strain WLT-MVA5. Subsequently, we fused the two glycosyltransferases and modified the endogenous pathways of S. cerevisiae, increasing the titer of F2 to 56.31 mg/L. Then, a combined semi-rational design and directed evolution method was applied to engineer GTK1 and a mutant GTK1F81W/F178S was finally obtained which further increased the F2 titer to 86.80 mg/L. Finally, the best performance strain was applied to scale up in the 5 L bioreactor and F2 titer reached 375.38 mg/L. This research realizes the high-production of F2 in S. cerevisiae and provides a reference method for the biosynthesis of other triterpenoid saponins.

Melatonin improves saponin biosynthesis and primary root growth in Psammosilene tunicoides hairy roots through multiple hormonal signaling and transcriptional pathways

Elicitation represents one of the most efficient techniques for improving metabolite biosynthesis and biomass accumulation in plant cell cultures. Melatonin (MLT) has been extensively studied for its multiregulatory functions, but its elicitation roles in medicinal plants remain largely unknown. Herein, we investigated the effects of MLT on triterpenoid saponin biosynthesis and root growth in hairy root cultures of Psammosilene tunicoides, an endangered Chinese medicinal plant famous for its pain-relieving capabilities. Exogenous MLT significantly boosted saponin accumulation, promoted primary root thickness and elongation, and increased biomass production compared with the control in P. tunicoides hairy roots, particularly when 10 mg L− 1 MLT was applied for two days. Furthermore, the reactive oxygen species (ROS), nitric oxide (NO), and Ca2+ contents, as well as antioxidant enzyme activities, were initially triggered after MLT treatments. Additionally, MLT raised the diverse phytohormone profiles that were related to plant secondary metabolism (salicylic acid, jasmonic acid, abscisic acid) and growth (indoleacetic acid (IAA) conjugates, cytokinins, and strigolactone) but decreased free IAA levels in hairy roots. Analysis of comparative transcriptome data revealed that multiple differentially expressed genes (DEGs) might be the key regulators in MLT-elicited networks, especially genes that were associated with signal transduction (POD, NR, and CDPK), transcription factor regulation (WRKYs, NACs, and AR2/ERFs), terpenoid metabolism (HMGS, DXS, GPPS, and SE) and root growth (COBL7 and Egase). These findings demonstrated that MLT elicitors could simultaneously improve saponin biosynthesis and primary root growth in P. tunicoides hairy roots, mainly by enhancing the ROS-mediated signaling cascade and coregulating the associated phytohormonal and transcriptional pathways.