Saponin Biosynthesis Research Articles

Characterization of UDP-glucose dehydrogenase isoforms in the medicinal legume Glycyrrhiza uralensis.

Uridine 5'-diphosphate (UDP)-glucose dehydrogenase (UGD) produces UDP-glucuronic acid from UDP-glucose as a precursor of plant cell wall polysaccharides. UDP-glucuronic acid is also a sugar donor for the glycosylation of various plant specialized metabolites. Nevertheless, the roles of UGDs in plant specialized metabolism remain poorly understood. Glycyrrhiza species (licorice), which are medicinal legumes, biosynthesize triterpenoid saponins, soyasaponins and glycyrrhizin, commonly glucuronosylated at the C-3 position of the triterpenoid scaffold. Often, several different UGD isoforms are present in plants. To gain insight into potential functional differences among UGD isoforms in triterpenoid saponin biosynthesis in relation to cell wall component biosynthesis, we identified and characterized Glycyrrhiza uralensis UGDs (GuUGDs), which were discovered to comprise five isoforms, four of which (GuUGD1-4) showed UGD activity in vitro. GuUGD1-4 had different biochemical properties, including their affinity for UDP-glucose, catalytic constant, and sensitivity to feedback inhibitors. GuUGD2 had the highest catalytic constant and highest gene expression level among the GuUGDs, suggesting that it is the major isoform contributing to the transition from UDP-glucose to UDP-glucuronic acid in planta. To evaluate the contribution of GuUGD isoforms to saponin biosynthesis, we compared the expression patterns of GuUGDs with those of saponin biosynthetic genes in methyl jasmonate (MeJA)-treated cultured stolons. GuUGD1-4 showed delayed responses to MeJA compared to those of saponin biosynthetic genes, suggesting that MeJA-responsive expression of GuUGDs compensates for the decreased UDP-glucuronic acid pool due to consumption during saponin biosynthesis.

Modification of ginsenoside saponin composition via the CRISPR/Cas9-mediated knockout of protopanaxadiol 6-hydroxylase gene in Panax ginseng

BackgroundThe roots of Panax ginseng contain two types of tetracyclic triterpenoid saponins, namely, protopanaxadiol (PPD)-type saponins and protopanaxatiol (PPT)-type saponins. In P. ginseng, the protopanaxadiol 6-hydroxylase (PPT synthase) enzyme catalyses protopanaxatriol (PPT) production from protopanaxadiol (PPD). In this study, we constructed homozygous mutant lines of ginseng by CRISPR/Cas9-mediated mutagenesis of the PPT synthase gene and obtained the mutant ginseng root lines having complete depletion of the PPT-type ginsenosides. MethodsTwo sgRNAs (single guide RNAs) were designed for target mutations in the exon sequences of the two PPT synthase genes (both PPTa and PPTg sequences) with the CRISPR/Cas9 system. Transgenic ginseng roots were generated through Agrobacterium-mediated transformation. The mutant lines were screened by ginsenoside analysis and DNA sequencing. ResultGinsenoside analysis revealed the complete depletion of PPT-type ginsenosides in three putative mutant lines (Cr4, Cr7, and Cr14). The reduction of PPT-type ginsenosides in mutant lines led to increased accumulation of PPD-type ginsenosides. The gene editing in the selected mutant lines was confirmed by targeted deep sequencing. ConclusionWe have established the genome editing protocol by CRISPR/Cas9 system in P. ginseng and demonstrated the mutated roots producing only PPD-type ginsenosides by depleting PPT-type ginsenosides. Because the pharmacological activity of PPD-group ginsenosides is significantly different from that of PPT-group ginsenosides, the new type of ginseng mutant producing only PPD-group ginsenosides may have new pharmacological characteristics compared to wild-type ginseng. This is the first report to generate target-induced mutations for the modification of saponin biosynthesis in Panax species using CRISPR–Cas9 system.

Cloning and Functional Characterization of a Squalene Synthase from Paris polyphylla var. yunnanensis.

Paris polyphylla Smith var. yunnanensis (Franch.) Hand. - Mazz. is a precious traditional Chinese medicine, and steroidal saponins are its major bioactive constituents possessing extensive biological activities. Squalene synthase (SQS) catalyzes the first dedicated step converting two molecular of farnesyl diphosphate (FDP) into squalene, a key intermediate in the biosynthetic pathway of steroidal saponins. In this study, a squalene synthase gene (PpSQS1) was cloned and functionally characterized from P. polyphylla var. yunnanensis, representing the first identified SQS from the genus Paris. The open reading frame of PpSQS1 is 1239 bp, which encodes a protein of 412 amino acids showing high similarity to those of other plant SQSs. Expression of PpSQS1 in Escherichia coli resulted in production of soluble recombinant proteins. Gas chromatography-mass spectrometry analysis showed that the purified recombinant PpSQS1 protein could produce squalene using FDP as a substrate in the in vitro enzymatic assay. qRT-PCR analysis indicated that PpSQS1 was highly expressed in rhizomes, consistent with the dominant accumulation of steroidal saponins there, suggesting that PpSQS1 is likely involved in the biosynthesis of steroidal saponins in the plant. The findings lay a foundation for further investigation on the biosynthesis and regulation of steroidal saponins, and also provide an alternative gene for manipulation of steroid production using synthetic biology.

Full-length transcriptome sequences by a combination of sequencing platforms applied to isoflavonoid and triterpenoid saponin biosynthesis of Astragalus mongholicus Bunge

BackgroundAstragalus mongholicus Bunge is an important medicinal plant used in traditional Chinese medicine. It is rich in isoflavonoids and triterpenoid saponins. Although these active constituents of A. mongholicus have been discovered for a long time, the genetic basis of isoflavonoid and triterpenoid saponin biosynthesis in this plant is virtually unknown because of the lack of a reference genome. Here, we used a combination of next-generation sequencing (NGS) and single-molecule real-time (SMRT) sequencing to identify genes involved in the biosynthetic pathway of secondary metabolites in A. mongholicus.ResultsIn this study, NGS, SMRT sequencing, and targeted compound analysis were combined to investigate the association between isoflavonoid and triterpenoid saponin content, and specific gene expression in the root, stem, and leaves of A. mongholicus. Overall, 643,812 CCS reads were generated, yielding 121,107 non-redundant transcript isoforms with an N50 value of 2124 bp. Based on these highly accurate transcripts, 104,756 (86.50%) transcripts were successfully annotated by any of the seven databases (NR, NT, Swissprot, KEGG, KOG, Pfam and GO). Levels of four isoflavonoids and four astragalosides (triterpenoid saponins) were determined. Forty-four differentially expressed genes (DEGs) involved in isoflavonoid biosynthesis and 44 DEGs from 16 gene families that encode enzymes involved in triterpenoid saponin biosynthesis were identified. Transcription factors (TFs) associated with isoflavonoid and triterpenoid saponin biosynthesis, including 72 MYBs, 53 bHLHs, 64 AP2-EREBPs, and 11 bZIPs, were also identified. The above transcripts showed different expression trends in different plant organs.ConclusionsThis study provides important genetic information on the A. mongholicus genes that are essential for isoflavonoid and triterpenoid saponin biosynthesis, and provides a basis for developing the medicinal value of this plant.

Triterpenoid saponin biosynthesis genes and their expression patterns during the development of sea cucumber Apostichopus japonicus

Triterpenoid saponin biosynthesis genes and their expression patterns during the development of sea cucumber Apostichopus japonicus

Chitosan elicitation of saponin accumulation in Psammosilene tunicoides hairy roots by modulating antioxidant activity, nitric oxide production and differential gene expression

Elicitation is one of the most effective strategies for enhancing plant bioactive compounds, such as triterpenoid saponins. Chitosan gained worldwide attention for biostimulant activity, but little is known about its roles in the elicitation of triterpenoid saponin in medicinal plants. Here, we explored the regulatory network of chitosan on saponin accumulation in hairy root cultures of Psammosilene tunicoides, a valuable medicinal herb known for its pain-relieving properties endemic to China. Compared with control, the highest total saponin accumulation exhibited a 4.55-fold enhancement in hairy roots elicited by 200 mg L−1 chitosan for nine days. High-performance liquid chromatography (HPLC) revealed the yields of quillaic acid, gypsogenin and gypsogenin-3-O-β-D-glucuronopyranoside were significantly increased after chitosan treatments. Moreover, exogenous chitosan application dramatically triggered the reactive oxygen species (ROS) scavenging enzyme activities and nitric oxide (NO) content in hairy roots. Comparative transcriptome analysis from chitosan-treated (1 and 9 d) or control groups revealed that differentially expressed genes (DEGs) were greatly enriched in plant-pathogen interaction and metabolic processes. The transcriptions of candidate DEGs involved in chitosan-elicited saponin metabolism were increased, especially genes encoding antioxidant enzymes (SOD, POD and GR), stress-responsive transcription factors (WRKYs and NACs) and terpenoid biosynthetic enzymes (DXS, GPPS and SE). Taken together, these results indicate that chitosan elicitor promotes triterpenoid saponin biosynthesis by enhancing antioxidant activities, NO production and differential gene expression in P. tunicoides hairy roots.

A candidate gene identified in converting platycoside E to platycodin D from Platycodon grandiflorus by transcriptome and main metabolites analysis

Platycodin D and platycoside E are two triterpenoid saponins in Platycodon grandiflorus, differing only by two glycosyl groups structurally. Studies have shown β-Glucosidase from bacteria can convert platycoside E to platycodin D, indicating the potential existence of similar enzymes in P. grandiflorus. An L9(34) orthogonal experiment was performed to establish a protocol for calli induction as follows: the optimal explant is stems with nodes and the optimum medium formula is MS + NAA 1.0 mg/L + 6-BA 0.5 mg/L to obtain callus for experimental use. The platycodin D, platycoside E and total polysaccharides content between callus and plant organs varied wildly. Platycodin D and total polysaccharide content of calli was found higher than that of leaves. While, platycoside E and total polysaccharide content of calli was found lower than that of leaves. Associating platycodin D and platycoside E content with the expression level of genes involved in triterpenoid saponin biosynthesis between calli and leaves, three contigs were screened as putative sequences of β-Glucosidase gene converting platycoside E to platycodin D. Besides, we inferred that some transcription factors can regulate the expression of key enzymes involved in triterpernoid saponins and polysaccharides biosynthesis pathway of P. grandiflorus. Totally, a candidate gene encoding enzyme involved in converting platycoside E to platycodin D, and putative genes involved in polysaccharide synthesis in P. grandiflorus had been identified. This study will help uncover the molecular mechanism of triterpenoid saponins biosynthesis in P. grandiflorus.

Activity and function studies of the promoter cis-acting elements of the key enzymes in saponins biosynthesis of DS from Panax notoginseng.

Panax notoginseng is a traditional Chinese medicine for the treatment of blood diseases, in which saponins were the main active components. Dammarenediol synthase (DS) is a key enzyme in the saponin synthesis pathway of P. notoginseng. The promoter is an important region to regulate gene expression, and the study of the promoter sequence provides important evidence for revealing the mechanism of gene expression regulation. However, there was still little research on the promoter function of P. notoginseng. In this study, the 1382bp promoter upstream of DS from P. notoginseng was cloned and sequenced. The promoter sequence was analyzed by online databases. The plant expression vector fused with the β-glucuronidase gene was constructed and transferred into Agrobacterium tumefaciens. Then tobacco was injected, and its response to exogenous hormones (gibberellin and abscisic acid) was studied by transient expression to verify its unique action elements. The results showed that the tobacco leaves transferred with DS promoter had significantly increased GUS protease activity after spraying GA and ABA, indicating that both DS promoter can specifically and significantly respond to exogenous GA and ABA signal. These findings will help us to better understand the regulatory mechanisms of the upstream region of the DS gene and provide a basis for future research on the interaction of cis-acting elements of promoters with related transcription factors.

A Review on Saponin Biosynthesis and its Transcriptomic Resources in Medicinal Plants

Saponins are naturally occurring glycosides which are produced by various plant species with diverse biological properties. The surface-active properties of saponins differentiates them from other glycosides. They are classified mainly into steroid and triterpenoid glycosides based on the structure of hydrophobic aglycone unit. The synthesis of saponins and the genes involved in its biosynthesis can be identified by transcriptome profiling. Using high-throughput sequencing technologies, the steroid or triterpenoid saponin biosynthesis related genes have been discovered which can help boosting the molecular research in this biosynthetic pathway. This review mainly illustrates the importance of saponins and the transcriptome resources of important medicinal plants for identifying genes related to its biosynthesis. Furthermore, the significance of unexplored plants which are rich sources of saponins in future studies are also discussed in the current review.

β-Amyrin Synthase1 Controls the Accumulation of the Major Saponins Present in Pea (Pisum sativum).

The use of pulses as ingredients for the production of food products rich in plant proteins is increasing. However, protein fractions prepared from pea or other pulses contain significant amounts of saponins, glycosylated triterpenes that can impart an undesirable bitter taste when used as an ingredient in foodstuffs. In this article, we describe the identification and characterization of a gene involved in saponin biosynthesis during pea seed development, by screening mutants obtained from two Pisum sativum TILLING (Targeting Induced Local Lesions INGenomes) populations in two different genetic backgrounds. The mutations studied are located in a gene designated PsBAS1 (β-amyrin synthase1), which is highly expressed in maturing pea seeds and which encodes a protein previously shown to correspond to an active β-amyrin synthase. The first allele is a nonsense mutation, while the second mutation is located in a splice site and gives rise to a mis-spliced transcript encoding a truncated, nonfunctional protein. The homozygous mutant seeds accumulated virtually nosaponin without affecting the seed nutritional or physiological quality. Interestingly, BAS1 appears to control saponin accumulation in all other tissues of the plant examined. These lines represent a first step in the development of pea varieties lacking bitterness off-flavors in their seeds. Our work also shows that TILLING populations in different genetic backgrounds represent valuable genetic resources for both crop improvement and functional genomics.

Full-Length Transcriptome Analyses of Genes Involved in Triterpenoid Saponin Biosynthesis of Psammosilene tunicoides Hairy Root Cultures With Exogenous Salicylic Acid.

Triterpenoid saponins constitute a diverse class of bioactive compounds in medicinal plants. Salicylic acid (SA) is an efficient elicitor for secondary metabolite production, but a transcriptome-wide regulatory network of SA-promoted triterpenoid saponin biosynthesis remains little understood. In the current study, we described the establishment of the hairy root culture system for Psammosilene tunicoides, a triterpenoid saponin-producing medicinal herb in China, using genetic transformation by Agrobacterium rhizogenes. Compared to controls, we found that total saponin content was dramatically increased (up to 2.49-fold) by the addition of 5 mg/L SA in hairy roots for 1 day. A combination of single-molecule real-time (SMRT) and next-generation sequencing (Illumina RNA-seq) was generated to analyze the full-length transcriptome data for P. tunicoides, as well as the transcript profiles in treated (8 and 24 h) and non-treated (0 h) groups with 5 mg/L SA in hairy roots. A total of 430,117 circular consensus sequence (CCS) reads, 16,375 unigenes and 4,678 long non-coding RNAs (lncRNAs) were obtained. The average length of unigenes (2,776 bp) was much higher in full-length transcriptome than that derived from single RNA-seq (1,457 bp). The differentially expressed genes (DEGs) were mainly enriched in the metabolic process. SA up-regulated the unigenes encoding SA-binding proteins and antioxidant enzymes in comparison with controls. Additionally, we identified 89 full-length transcripts encoding enzymes putatively involved in saponin biosynthesis. The candidate transcription factors (WRKY, NAC) and structural genes (AACT, DXS, SE, CYP72A) might be the key regulators in SA-elicited saponin accumulation. Their expression was further validated by quantitative real-time PCR (qRT-PCR). These findings preliminarily elucidate the regulatory mechanisms of SA on triterpenoid saponin biosynthesis in the transcriptomic level, laying a foundation for SA-elicited saponin augmentation in P. tunicoides.

Appropriate Reference Genes for RT-qPCR Normalization in Various Organs of Anemone flaccida Fr. Schmidt at Different Growing Stages.

Anemone flaccida Fr. Schmidt is a traditional medicinal herb in southwestern China and has multiple pharmacological effects on bruise injuries and rheumatoid arthritis (RA). A new drug with a good curative effect on RA has recently been developed from the extract of A. flaccida rhizomes, of which the main medicinal ingredients are triterpenoid saponins. Due to excessive exploitation, the wild population has been scarce and endangered in a few of its natural habitats and research on the cultivation of the plant commenced. Studies on the gene expressions related to the biosynthesis of triterpenoid saponins are not only helpful for understanding the effects of environmental factors on the medicinal ingredient accumulations but also necessary for monitoring the herb quality of the cultivated plants. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) as a sensitive and powerful technique has been widely used to detect gene expression across tissues in plants at different stages; however, its accuracy and reliability depend largely on the reference gene selection. In this study, the expressions of 10 candidate reference genes were evaluated in various organs of the wild and cultivated plants at different stages, using the algorithms of geNorm, NormFinder and BestKeeper, respectively. The purpose of this study was to identify the suitable reference genes for RT-qPCR detection in A. flaccida. The results showed that two reference genes were sufficient for RT-qPCR data normalization in A. flaccida. PUBQ and ETIF1a can be used as suitable reference genes in most organs at various stages because of their expression stabilitywhereas the PUBQ and EF1Α genes were desirable in the rhizomes of the plant at the vegetative stage.

Insights into the diversification of subclade IVa bHLH transcription factors in Fabaceae

BackgroundFabaceae plants appear to contain larger numbers of subclade IVa basic-helix-loop-helix (bHLH) transcription factors than other plant families, and some members of this subclade have been identified as saponin biosynthesis regulators. We aimed to systematically elucidate the diversification of this subclade and obtain insights into the evolutionary history of saponin biosynthesis regulation in Fabaceae.ResultsIn this study, we collected sequences of subclade IVa bHLH proteins from 40 species, including fabids and other plants, and found greater numbers of subclade IVa bHLHs in Fabaceae. We confirmed conservation of the bHLH domain, C-terminal ACT-like domain, and exon-intron organisation among almost all subclade IVa members in model legumes, supporting the results of our classification. Phylogenetic tree-based classification of subclade IVa revealed the presence of three different groups. Interestingly, most Fabaceae subclade IVa bHLHs fell into group 1, which contained all legume saponin biosynthesis regulators identified to date. These observations support the co-occurrence and Fabaceae-specific diversification of saponin biosynthesis regulators. Comparing the expression of orthologous genes in Glycine max, Medicago truncatula, and Lotus japonicus, orthologues of MtTSAR1 (the first identified soyasaponin biosynthesis regulatory transcription factor) were not expressed in the same tissues, suggesting that group 1 members have gained different expression patterns and contributions to saponin biosynthesis during their duplication and divergence. On the other hand, groups 2 and 3 possessed fewer members, and their phylogenetic relationships and expression patterns were highly conserved, indicating that their activities may be conserved across Fabaceae.ConclusionsThis study suggests subdivision and diversification of subclade IVa bHLHs in Fabaceae plants. The results will be useful for candidate selection of unidentified saponin biosynthesis regulators. Furthermore, the functions of groups 2 and 3 members are interesting targets for clarifying the evolution of subclade IVa bHLH transcription factors in Fabaceae.

Response of bitter and sweet Chenopodium quinoa varieties to cucumber mosaic virus: Transcriptome and small RNASeq perspective.

Saponins are secondary metabolites with antiviral properties. Low saponin (sweet) varieties of quinoa (Chenopodium quinoa) have been developed because seeds high in saponins taste bitter. The aim of this study was to elucidate the role of saponin in resistance of quinoa to Cucumber mosaic virus (CMV). Differential gene expression was studied in time-series study of CMV infection. High-throughput transcriptome sequence data were obtained from 36 samples (3 varieties × +/- CMV × 1 or 4 days after inoculation × 3 replicates). Translation, lipid, nitrogen, amino acid metabolism, and mono- and sesquiterpenoid biosynthesis genes were upregulated in CMV infections. In ‘Red Head’ (bitter), CMV-induced systemic symptoms were concurrent with downregulation of a key saponin biosynthesis gene, TSARL1, four days after inoculation. In local lesion responses (sweet and semi-sweet), TSARL1 levels remained up-regulated. Known microRNAs (miRNA) (81) from 11 miR families and 876 predicted novel miRNAs were identified. Differentially expressed miRNA and short interfering RNA clusters (24nt) induced by CMV infection are predicted to target genomic and intergenic regions enriched in repetitive elements. This is the first report of integrated RNASeq and sRNASeq data in quinoa-virus interactions and provides comprehensive understanding of involved genes, non-coding regions, and biological pathways in virus resistance.

Saponins: Extraction, bio-medicinal properties and way forward to anti-viral representatives

Medicinal or herbal plants are widely used for their many favourable properties and are generally safe without any side effects. Saponins are sugar conjugated natural compounds which possess a multitude of biological activities such as medicinal properties, antimicrobial activity, antiviral activity, etc. Saponin production is a part of the normal growth and development process in a lot of plants and plant extracts such as liquorice and ginseng which are exploited as potential drug sources. Herbal compounds have shown a great potential against a wide variety of infectious agents, including viruses such as the SARS-CoV; these are all-natural products and do not show any adverse side effects. This article reviews the various aspects of saponin biosynthesis and extraction, the need for their integration into more mainstream medicinal therapies and how they could be potentially useful in treating viral diseases such as COVID-19, HIV, HSV, rotavirus etc. The literature presents a close review on the saponin efficacy in targeting mentioned viral diseases that occupy a high mortality rate worldwide. This manuscript indicates the role of saponins as a source of dynamic plant based anti-viral remedies and their various methods for extraction from different sources.

Advances in understanding of the biosynthetic pathway and regulatory mechanism of triterpenoid saponins in plants

Triterpenoid saponins are a type of triterpenoid glycoside. Widely distributed in plants, triterpenoid saponins play a role in resisting pathogenic microorganisms and herbivores, but they also have a variety of pharmacological activities that have been widely adopted in medicine, common chemicals, food, and agriculture, to name a few. In recent years, with the development of science and technology and the deepening of research, the basic framework and regulatory mechanisms of triterpenoid saponin biosynthesis in plants have become better understood. Plant triterpenoid saponins have multiple types of complex structures and, although they share a common precursor synthesis pathway, their post-modification stages are highly specific and diverse. In this paper, the biosynthetic pathway, key enzyme genes, transcriptional regulation, and microbial cell engineering of triterpenoid saponins were reviewed, and the future development direction of triterpenoid saponins, which could provide theoretical reference for related research in the abovementioned fields, was prospected.

Effects of shading on triterpene saponin accumulation and related gene expression of Aralia elata (Miq.) Seem

Aralia elata (Miq.) Seem is widely used as a medicinal plant and functional food in China. In this study, A. elata plants were exposed to full sunlight (CK), 40% shading (LS), 60% shading (MS), and >80% shading (ES) condition to investigate the effects of shading treatments on growth, stress levels, antioxidant enzymes activity, araloside content and related gene expression. The greatest growth and leaf biomass were achieved in 40% shading, and leaf biomass per plant increased by 16.09% compared to the non-shading treatment. Furthermore, the lowest reactive oxide species (ROS) production and lipid peroxidation resulting from increasing antioxidant enzyme activity were also observed in LS treatment. Overall, shading percentage negatively regulated the expression of key enzymes (squalene synthase, SS; squalene epoxidase, SE and β-amyrin synthase, bAS) involved in the saponin biosynthesis, resulting in the greatest yields of total and four selected aralosides in A. elata leaves were achieved in sunlight group. However, the greatest yield of total saponin in the leaves was observed in the 40% shading group due to higher leaf biomass. The results suggest that optimizing the field growing conditions would be important for obtaining the greatest yield of bioactive components. Total saponin and selected aralosides also have a significant correlation with ROS production and antioxidant enzyme activity, these indicated the increased yield of these saponins may be part of a defense response. The study concludes that the production of saponin was the interaction of oxidative stress and photosynthesis.

Synergistic effect on biosynthesis of Panax notoginseng saponins by overexpressing a transcription factor PnbHLH and RNA interference of cycloartenol synthase gene

This study cloned the transcription factor gene PnbHLH which held an open reading frame of 966 bp encoding 321 amino acids. This study constructed the overexpression vector of transcription factor PnbHLH of Panax notoginseng. The combination of PnbHLH overexpression and RNAi of the key enzyme gene PnCAS involved in the phytosterol biosynthesis was achieved in P. notoginseng cells, thus exploring the biosynthetic regulation of P. notoginseng saponins(PNS) by the synergistic effect of PnbHLH overexpression and PnCAS RNAi. The results showed that the PnbHLH transcription factor interacted with the promoters of key enzyme genes PnDS, PnSS and PnSE in the biosynthetic pathway of PNS, and then regulated the expression levels of key enzyme genes and affected the biosynthesis of saponins indirectly. Further study indicated that the synergistic effect of PnbHLH overexpression and PnCAS RNAi was a more effective approach to regulate the biosynthesis of saponins. Compared with the wild type and PnCAS RNAi cells of P. notoginseng, the contents of total saponins and monomeric saponins(Rd, Rb_1, Re, Rg_1 and R_1) were increased to some extent in the cell lines of PnbHLH overexpression and PnCAS RNAi. This indicated that the two ways of forward regulation and reverse regulation of saponin biosynthesis showed superposition effect. This study explored a more rational and efficient regulation strategy of PNS biosynthesis based on the advantages of multi-point regulation of transcription factors as well as the down-regulation of by-product synthesis of saponins.

Saponin production from Quillaja genus species. An insight into its applications and biology

ABSTRACT: Quillaja genus (Quillajaceae family) is endemic to South America, where is represented by two species, Quillaja saponaria and Quillaja brasiliensis. One outstanding characteristic of these forest tree species is their production of saponins, a family of amphipathic glycosides, involved in the defensive response of plants against biotic and abiotic factors. Saponins are metabolites of economic importance due to their chemical and physical properties. Basic and applied research efforts performed during the last decades, mainly on Q. saponaria, have placed these compounds as an important raw material in industrial areas, such as food and beverage, cosmetics, vaccine production, biopesticides, among others. In this review, we summarize information on saponins from Quillaja species during the last years, analyzing current developments by application areas, as well as their chemical composition and properties. We also describe the general advances in revealing saponin biosynthesis pathways, related genes and Quillaja genomes, as well as the conservation status, domestication processes, and perspectives in the context of implementing genetic improvement programs.

Lowering Nitrogen and Increasing Potassium Application Level Can Improve the Yield and Quality of Panax notoginseng.

Excessive nitrogen (N) application and potassium (K) supplement deficiency is a common problem in Panax notoginseng cultivation. However, synergistic effects of lowering N and increasing K application on yield and quality of P. notoginseng have not been reported. Field experiments in two locations with different N and K combined application were conducted to study the effects on yield and quality. Then, the saponin accumulation mechanisms were explored by pot and hydroponic culture with 2- or 3-year-old seedlings. The investigation showed that 70% of P. notoginseng cultivation fields reached abundant levels of total nitrogen (TN) but had deficient levels of total potassium (TK), which may be detrimental to balance the N/K uptake of P. notoginseng. Moreover, the average biomass was 18.9 g, and P. notoginseng saponin (PNS) content was 6.95%; both were influenced by the N/K values of P. notoginseng. The field experiments indicated that compared to the conventional N and K application (N:K = 2:1), lowering N and increasing K application (N:K = 1:2) decreased root rot rate by 36.4–46.1% and increased survival rate, root biomass, and yield, as well as PNS content by 17.9–18.3, 5.7–32.9, 27.8–57.1, and 5–10%, respectively. The mechanism of lowering N and increasing K application on the PNS content improving was due to the decreasing of N/K value, which promoted photosynthesis, sugar accumulation, and the expression of saponin biosynthesis genes. Therefore, lowering N and increasing K application to the ratio of 1:2 would have great potential to improve the synergistic effect on yield and quality of P. notoginseng cultivation.