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

2021 ◽  
Vol 12 ◽  
Author(s):  
Lingye Su ◽  
Shufang Li ◽  
Hanhan Qiu ◽  
Hongfeng Wang ◽  
Congcong Wang ◽  
...  

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.

2021 ◽  
Author(s):  
Hanwen Yu ◽  
Mengli Liu ◽  
Minzhen Yin ◽  
Tingyu Shan ◽  
Huasheng Peng ◽  
...  

Abstract Background: Platycodon grandiflorus, a traditional Chinese medicine, contains considerable triterpene saponins with broad pharmacological activities. To date, information on the molecular mechanism of triterpenoid saponin biosynthesis in P. grandiflorus is limited. Here, single-molecule real-time (SMRT) and next-generation sequencing technologies were combined to comprehensively analyse the transcriptome and unveil triterpenoid saponin biosynthesis in P. grandiflorus.Results: We quantified four saponin monomers in P. grandiflorus, and found that the total content of the four saponins was the highest in the roots and the lowest in the stems and leaves. A total of 173,354 non-redundant transcripts generated from the PacBio platform were successfully annotated to seven functional databases, among which 1,765 transcripts were aligned to the "metabolism of terpenoids and polyketides" pathway in the KEGG database. Three full-length transcripts of β-amyrin synthase (β-AS), the key synthase of the β-amyrin, were identified. Furthermore, a total of 132,610 clean reads of BGISEQ sequences were utilised to explore key genes related to the triterpenoid saponin biosynthetic pathway in P. grandiflorus, and 96 differentially expressed genes (DEGs) involved were selected as candidates. Notably, 9 of the 96 DEGs showed the highest expression in the roots, which were considered key genes for synthesising triterpenoid saponins in P. grandiflorus. Furthermore, 3,469 genes encoding transcription factors (TFs) were identified and classified into 57 TF families, including MYB, bHLH, mTERF, and AP2-EREBP. The expression levels of genes were verified by quantitative real-time PCR.Conclusions: Our reliable transcriptome data provide valuable information on the related biosynthesis pathway and may provide new insights into the molecular mechanisms of triterpenoid saponin biosynthesis in P. grandiflorus.


2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Xinglong Su ◽  
Yingying Liu ◽  
Lu Han ◽  
Zhaojian Wang ◽  
Mengyang Cao ◽  
...  

AbstractPlatycodin 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.


Biologia ◽  
2014 ◽  
Vol 69 (7) ◽  
Author(s):  
Elnaz Nourozi ◽  
Bahman Hosseini ◽  
Abbas Hassani

AbstractHairy root culture system is a valuable tool to study the characteristics of gene expression, gene function, root biology, biochemical properties and biosynthesis pathways of secondary metabolites. In the present study, hairy roots were established in Anise hyssop (Agastache foeniculum) via Agrobacterium rhizogenes. Three strains of Agrobacterium rhizogenes (A4, A7 and 9435), were used for induction of hairy roots in four various explants (hypocotyl, cotyledon, one-month-old leaf and five-month-old leaf) of Anise hyssop. The highest frequency of transformation was achieved using A4 strain in one-month-old leaves (51.1%). The transgenic states of hairy root lines were confirmed by PCR (Polymerase chain reaction) method. High performance liquid chromatography analysis revealed that the production of rosmarinic acid (RA) in transformed roots of A. foeniculum was almost 4-fold higher than that of the non-transformed roots. In a separate experiment, hairy roots obtained from one-month-old leaves inoculated with A4 strain, were grown in liquid medium and the effects of different concentrations of salicylic acid (0.0, 0.01, 0.1 and 1 mM) and chitosan (0, 50, 100 and 150 mg L−1) (as elicitor) and sucrose (20, 30, 40 and 50 g L−1) on the growth of hairy roots were evaluated. The results showed that, 30 g L−1 sucrose and 100 mg L−1 chitosan increased the biomass of hairy root cultures and application of salicylic acid reduced the growth of hairy roots compared with control roots.


2020 ◽  
Author(s):  
Minzhen Yin ◽  
Shanshan Chu ◽  
Tingyu Shan ◽  
Liangping Zha ◽  
Huasheng Peng

Abstract Background: Astragalus mongholicus Bunge is an important medicinal plant and has been used in traditional Chinese medicine for a long history, which is rich in isoflavonoids and triterpenoid saponins. Although these active constituents in A. mongholicus have been discovered for a long time, the molecular genetic basis of the isoflavonoid and triterpenoid saponin biosynthesis pathways is virtually unknown due to the lack of a reference genome. The combination of next-generation sequencing (NGS) and single-molecule real-time (SMRT) sequencing to analyze genes involved in the biosynthetic pathways of secondary metabolites in medicinal plants has been widely recognized.Results: In this study, NGS, SMRT sequencing, and targeted compounds were combined to investigate the association between isoflavonoids and triterpenoid saponins and gene expression in roots, stems and leaves of A. mongholicus. A total of four main isoflavonoids and four astragalosides (belong to triterpenoid saponins) were measured, and 44 differentially expressed genes (DEGs) of nine gene families, 44 DEGs of 16 gene families that encode for enzymes involved in isoflavonoid and triterpenoid saponin biosynthesis were identified, separately. Additionally, transcription factors (TFs) associated with isoflavonoid and triterpenoid saponin biosynthesis were analyzed, including 72 MYBs, 53 bHLHs, 64 AP2-EREBPs and 11 bZIPs. The above transcripts exhibit different expression trends in different organs.Conclusions: Our study provides important genetic information for the essential genes of isoflavonoid and triterpenoid saponin biosynthesis in A. mongholicus, and provides a basis for developing its medicinal value.


2020 ◽  
Vol 48 (2) ◽  
pp. 839-848
Author(s):  
Shuang ZHAO ◽  
Hong TANG

Valtrate is a pharmacologically active epoxyiridoid ester found in the roots and rhizomes of Valeriana jatamansi Jones. The plant produces only small amounts of this metabolite naturally, and so induction of hairy roots as well as elicitation can be useful to increase its commercial production. In this study, strain R1601 of Agrobacterium rhizogenes was used to induce hairy roots in V. jatamansi, and stable hairy root cultures of V. jatamansi were established successfully. The influence of three exogenous elicitors including methyl jasmonate (MJ), jasmonic acid (JA) and salicylic acid (SA) on valtrate production in the hairy root cultures of V. jatamansi was also investigated, and the 25-day-old hairy root cultures were treated with different concentrations of the elicitors at exposure time of 7 days. This present study showed that MJ (100 mg/L) highly promoted valtrate production at 7 days after elicitation, to a level of 3.63 times higher than that of non-elicited control. SA did not significantly increase the production of valtrate. This is the first-time study to assess the elicitation of hairy root cultures to promote valtrate biosynthesis in V. jatamansi and the resulting experiments demonstrated that MJ was indeed a potent inducer of valtrate biosynthesis.


BMC Genomics ◽  
2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Weifang Liao ◽  
Zhinan Mei ◽  
Lihong Miao ◽  
Pulin Liu ◽  
Ruijie Gao

Abstract Background Entada phaseoloides (L.) Merr. is an important traditional medicinal plant. The stem of Entada phaseoloides is popularly used as traditional medicine because of its significance in dispelling wind and dampness and remarkable anti-inflammatory activities. Triterpenoid saponins are the major bioactive compounds of Entada phaseoloides. However, genomic or transcriptomic technologies have not been used to study the triterpenoid saponin biosynthetic pathway in this plant. Results We performed comparative transcriptome analysis of the root, stem, and leaf tissues of Entada phaseoloides with three independent biological replicates and obtained a total of 53.26 Gb clean data and 116,910 unigenes, with an average N50 length of 1218 bp. Putative functions could be annotated to 42,191 unigenes (36.1%) based on BLASTx searches against the Non-redundant, Uniprot, KEGG, Pfam, GO, KEGG and COG databases. Most of the unigenes related to triterpenoid saponin backbone biosynthesis were specifically upregulated in the stem. A total of 26 cytochrome P450 and 17 uridine diphosphate glycosyltransferase candidate genes related to triterpenoid saponin biosynthesis were identified. The differential expressions of selected genes were further verified by qPT-PCR. Conclusions The dataset reported here will facilitate the research about the functional genomics of triterpenoid saponin biosynthesis and genetic engineering of Entada phaseoloides.


2021 ◽  
Vol 43 (3) ◽  
Author(s):  
Abdulwadood S. M. Alsoufi ◽  
Klaudia Staśkiewicz ◽  
Michał Markowski

AbstractHairy root cultures are an efficient tool for the biotechnological production of plant metabolites and a convenient experimental model for analyzing the effect of various compounds on plant metabolism. In contrast to many other types of in vitro plant cultures, hairy roots do not require an external supply of phytohormones to the medium. Consequently, plant growth regulators such as auxins and cytokinins are rarely used as elicitors in hairy root in vitro cultures; however, they can strongly influence plant defense responses. The aim of this study was to investigate the influence of two auxins: natural indole-3-acetic acid (IAA) and synthetic 1-naphthaleneacetic acid (NAA), as well as two cytokinins: natural kinetin and synthetic 6-benzylaminopurine (BAP) at a concentration of 0.75 mg/L on the metabolism of sterols and triterpenoids in Calendula officinalis hairy roots. Auxins prevented the accumulation of triterpenoid saponins (oleanolic acid glycosides), while cytokinin BAP increased their accumulation by 17% and their release into the culture medium by a factor of 10. Other cytokinins and kinetins increased the sterol levels by 17%, the level of stigmasterol by 15%, and the level of isofucosterol by 7 times.


2020 ◽  
Author(s):  
Weifang Liao ◽  
Lihong Miao ◽  
Pulin Liu ◽  
Ruijie Gao ◽  
Zhinan Mei

Abstract Background Entada phaseoloides (L.) Merr. is an important traditional medicinal plant. The stem of Entada phaseoloides is popularly used as traditional medicine because of its significance in dispelling wind and dampness and remarkable anti-inflammatory activities. Triterpenoid saponins are the major bioactive compounds of Entada phaseoloides. However, genomic or transcriptomic technologies have not been used to study the triterpenoid saponin biosynthetic pathway in this plant.Results We performed comparative transcriptome analysis of the root, stem, and leaf tissues of Entada phaseoloides with three independent biological replicates and obtained a total of 53.26 Gb clean data and 116,910 unigenes, with an average N50 length of 1218 bp. Putative functions could be annotated to 42,191 unigenes (36.1%) based on BLASTx searches against the Non-redundant, Uniprot, KEGG, Pfam, GO, KEGG and COG databases. Most of the unigenes related to triterpenoid saponin backbone biosynthesis were specifically upregulated in the stem. A total of 26 cytochrome P450 and 17 uridine diphosphate glycosyltransferase candidate genes related to triterpenoid saponin biosynthesis were identified. The differential expressions of selected genes were further verified by qPT-PCR.Conclusions The dataset reported here will facilitate the research about the functional genomics of triterpenoid saponin biosynthesis and genetic engineering of Entada phaseoloides .


2010 ◽  
Vol 5 (7) ◽  
pp. 1934578X1000500 ◽  
Author(s):  
Chang Ling Zhao ◽  
Xiu Ming Cui ◽  
Yan Ping Chen ◽  
Quan Liang

Triterpenoid saponins are one of the key active components of many medicinal plants. The biosynthetic pathway of triterpenoid saponins in higher plants and a lot of experimental results both indicated that the key enzymes involved in triterpenoid saponin synthesis are squalene synthase (SS), squalene epoxidase (SE), lupeol synthase (LS), dammarenediol synthase (DS), β-amyrin synthase (β-AS), cytochrome P450-dependent monooxygenase (PDMO), and glycosyltransferase (GT). The activities and coding genes of the key enzymes could be induced by a range of factors in various plant species. However, the effects of the factors on the content and composition of the triterpenoid saponins in specific plants are not certainly coincident, and different factors appear to induce the gene expressions of the key enzymes by different signal pathways and at different levels. This paper could provide a reference for strengthening the triterpenoid saponin-synthesizing capability of specific medicinal plants at enzyme and/or gene expression levels in order to improve the plants’ commercial values.


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