Illuminating the biosynthesis pathway genes involved in bioactive specific monoterpene glycosides in Paeonia veitchii Lynch by a combination of sequencing platforms.

BACKGROUND: Paeonia veitchii Lynch, a well-known herb from the Qinghai-Tibet Plateau south of the Himalayas, can synthesize specific monoterpene glycosides (PMGs) with multiple pharmacological activities, and its rhizome has become an indispensable ingredient in many clinical drugs. However, little is known about the molecular background of P. veitchii, especially the genes involved in the biosynthetic pathway of PMGs. RESULTS: A corrective full-length transcriptome with 30,827 unigenes was generated by combining next-generation sequencing (NGS) and single-molecule real-time sequencing (SMRT) of six tissues (leaf, stem, petal, ovary, phloem and xylem). The enzymes terpene synthase (TPS), cytochrome P450 (CYP), UDP-glycosyltransferase (UGT), and BAHD acyltransferase, which participate in the biosynthesis of PMGs, were systematically characterized, and their functions related to PMG biosynthesis were analysed. With further insight into TPSs, CYPs, UGTs and BAHDs involved in PMG biosynthesis, the weighted gene coexpression network analysis (WGCNA) method was used to identify the relationships between these genes and PMGs. Finally, 8 TPSs, 22 CYPs, 7 UGTs, and 2 BAHD genes were obtained, and these putative genes were very likely to be involved in the biosynthesis of PMGs. In addition, the expression patterns of the putative genes and the accumulation of PMGs in tissues suggested that all tissues are capable of biosynthesizing PMGs and that aerial plant parts could also be used to extract PMGs. CONCLUSION: We generated a large-scale transcriptome database across the major tissues in P. veitchii, providing valuable support for further research investigating P. veitchii and understanding the genetic information of plants from the Qinghai-Tibet Plateau. TPSs, CYPs, UGTs and BAHDs further contribute to a better understanding of the biology and complexity of PMGs in P. veitchii. Our study will help reveal the mechanisms underlying the biosynthesis pathway of these specific monoterpene glycosides and aid in the comprehensive utilization of this multifunctional plant.

[Selection and validation of reference genes for quantitative real-time PCR analysis in Paeonia veitchii].

Paeonia veitchii and P. lactiflora are both original plants of the famous Chinese medicinal drug Paeoniae Radix Rubra in the Chinese Pharmacopoeia. They have important medicinal value and great potential in the flower market. The selection of stable and reliable reference genes is a necessary prerequisite for molecular research on P. veitchii. In this study, two reference genes, Actin and GAPDH, were selected as candidate genes from the transcriptome data of P. veitchii. The expression levels of the two candidate genes in different tissues(phloem, xylem, stem, leaf, petiole, and ovary) and different growth stages(bud stage, flowering stage, and dormant stage) of P. veitchii were detected using real-time fluorescence quantitative technology(qRT-PCR). Then, the stability of the expression of the two reference genes was comprehensively analyzed using geNorm, NormFinder, BestKeeper, ΔCT, and RefFinder. The results showed that the expression patterns of Actin and GAPDH were stable in different tissues and growth stages of P. veitchii. Furthermore, the expression levels of eight genes(Pv-TPS01, Pv-TPS02, Pv-CYP01, Pv-CYP02, Pv-CYP03, Pv-BAHD01, Pv-UGT01, and Pv-UGT02) in different tissues were further detected based on the transcriptome data of P. veitchii. The results showed that when Actin and GAPDH were used as reference genes, the expression trends of the eight genes in different tissues of P. veitchii were consistent, validating the reliability of Actin and GAPDH as reference genes for P. veitchii. In conclusion, this study finds that Actin and GAPDH can be used as reference genes for studying gene expression levels in different tissues and growth stages of P. veitchii.

A new cyclic peptide from Selaginella tamariscina.

A new cyclic peptide selapeptin B (1), together with one known nor-lignan glycoside moellenoside C (2), was isolated from Selaginella tamariscina. The structures of 1 and 2 were elucidated on the basis of chemical and spectral analysis, including 1D, 2D NMR analyses and HRESIMS. Two compounds were evaluated for cytotoxic activities against B16F10, MDA-MB-231, and MDA-MB-468 cell lines by MTT assay. Compound 1 showed the potent activity against B16F10 melanoma cell lines.

[A new cyclopeptide from Selaginella tamariscina].

One new cyclopeptide was isolated from the ethyl acetate fraction of the 75% EtOH extract of Selaginella tamariscina by various column chromatography methods(HP-20, polyamide and semi-preparative HPLC). Its structure was identified as selapeptin A(1) by extensive spectroscopic analysis(HR-ESI-MS, 1 D and 2 D NMR). Compound 1 was evaluated for cytotoxic activities by MTT assay. It showed potent cytotoxic activity against B16 F10 with the inhibition rate of 51.57%±4.34% at 40 µmol·L~(-1) while had no impacts on MDA-MB-231 and MDA-MB-468 at 100 µmol·L~(-1).

[Chemical constituents of phenylethanoid glycosides from Forsythiae Fructus and their antitumor activities].

Four phenylethanoid glycosides were isolated from the 75% EtOH extract of Forsythiae Fructus by various column chromatography methods(MCI, silica gel, ODS and semi-preparative HPLC). Their structures were identified as forsythenside M(1), forsythenside K(2), forsythoside I(3) and forsythoside A(4) by physicochemical properties and extensive spectroscopic analysis(UV, 1 D and 2 D NMR, HR-ESI-MS). Among them, compound 1 was one new phenylethanoid glycoside. The in vitro cytotoxic activities of these compounds against MCF-7, A-375, SGC-7901 and B16 F10 were evaluated. The results showed that compounds 1-4 had cytotoxic activities against MCF-7, A-375, SGC-7901 and B16 F10 at 40 µmol·L~(-1).

Characterizing glycosyltransferases by a combination of sequencing platforms applied to the leaf tissues of Stevia rebaudiana.

BACKGROUND: Stevia rebaudiana (Bertoni) is considered one of the most valuable plants because of the steviol glycosides (SGs) that can be extracted from its leaves. Glycosyltransferases (GTs), which can transfer sugar moieties from activated sugar donors onto saccharide and nonsaccharide acceptors, are widely distributed in the genome of S. rebaudiana and play important roles in the synthesis of steviol glycosides. RESULTS: Six stevia genotypes with significantly different concentrations of SGs were obtained by induction through various mutagenic methods, and the contents of seven glycosides (stevioboside, Reb B, ST, Reb A, Reb F, Reb D and Reb M) in their leaves were considerably different. Then, NGS and single-molecule real-time (SMRT) sequencing were combined to analyse leaf tissue from these six different genotypes to generate a full-length transcriptome of S. rebaudiana. Two phylogenetic trees of glycosyltransferases (SrUGTs) were constructed by the neighbour-joining method and successfully predicted the functions of SrUGTs involved in SG biosynthesis. With further insight into glycosyltransferases (SrUGTs) involved in SG biosynthesis, the weighted gene co-expression network analysis (WGCNA) method was used to characterize the relationships between SrUGTs and SGs, and forty-four potential SrUGTs were finally obtained, including SrUGT85C2, SrUGT74G1, SrUGT76G1 and SrUGT91D2, which have already been reported to be involved in the glucosylation of steviol glycosides, illustrating the reliability of our results. CONCLUSION: Combined with the results obtained by previous studies and those of this work, we systematically characterized glycosyltransferases in S. rebaudiana and forty-four candidate SrUGTs involved in the glycosylation of steviol glucosides were obtained. Moreover, the full-length transcriptome obtained in this study will provide valuable support for further research investigating S. rebaudiana.

Metabolome and transcriptome associated analysis of sesquiterpenoid metabolism in Nardostachys jatamansi.

Background: Nardostachys jatamansi, an extremely endangered valuable plant of the alpine Himalayas, can synthesize specific sesquiterpenoids with multiple effective therapies and is widely exploited for the preparation of drugs, cosmetics and even religious functions (e.g., well-known spikenard). However, how accumulation trend of the sesquiterpenoids in tissues and the molecular mechanisms underlying the production of the active ingredients are not well understood. Methods: The single-molecule real-time (SMRT) and RNA-seq transcriptome sequencing were combined to analyse the roots, rhizomes, leaves, flowers and anthocaulus of N. jatamansi. The phytochemical analysis was performed by gas chromatography‒mass spectrometry (GC‒MS) and ultrahigh-performance liquid chromatography (UPLC). Results: A high-quality full-length reference transcriptome with 26,503 unigenes was generated for the first time. For volatile components, a total of sixty-five compounds were successfully identified, including fifty sesquiterpenoids. Their accumulation levels in five tissues were significantly varied, and most of the sesquiterpenoids were mainly enriched in roots and rhizomes. In addition, five aromatic compounds were only detected in flowers, which may help the plant attract insects for pollination. For nonvolatile ingredients, nardosinone-type sesquiterpenoids (nardosinone, kanshone C, and isonardosinone) were detected almost exclusively in roots and rhizomes. The candidate genes associated with sesquiterpenoid biosynthesis were identified by transcriptome analysis. Consistently, it was found that most biosynthesis genes were abundantly expressed in the roots and rhizomes according to the functional enrichment and expression patterns results. There was a positive correlation between the expression profile of genes related to the biosynthesis and the accumulation level of sesquiterpenoids in tissues. Gene family function analysis identified 28 NjTPSs and 43 NjCYPs that may be involved in the biosynthesis of the corresponding sesquiterpenoids. Furthermore, gene family functional analysis and gene coexpression network analysis revealed 28 NjTPSs and 43 NjCYPs associated with nardosinone-type sesquiterpenoid biosynthesis. Conclusion: Our research results reveal the framework of sesquiterpenoids accumulation and biosynthesis in plant tissues and provide valuable support for further studies to elucidate the molecular mechanisms of sesquiterpenoid regulation and accumulation in N. jatamansi and will also contribute to the comprehensive utilization of this alpine plant.

Identification of the Key Residues of the Uridine Diphosphate Glycosyltransferase 91D2 and its Effect on the Accumulation of Steviol Glycosides in Stevia rebaudiana.

Stevia (Stevia rebaudiana Bertoni) possesses substantial value for its unique sweet compounds-steviol glycosides (SGs). In the metabolic glycosylation grid of SGs, SrUGT91D2 has been shown to catalyze formation of 1,2-ß-d-glucoside linkages at the C13- and C19-positions and play a crucial role in the synthesis of SGs, including the formation of stevioside (ST), rebaudioside E (RE), and rebaudioside D (RD). However, the key residues of the SrUGT91D2 enzyme and how SrUGT91D2 affects the accumulation of SGs in S. rebaudiana remain unclear. In the present study, cloning and functional analysis of full-length SrUGT91D2 gene sequences were performed in 10 different S. rebaudiana genotypes with divergent SG compositions. After sequence analysis, it was found that most of the sequences of this gene (more than 50%) in each genotype were consistent with the UGT91D2e_No.5 allele, which has been reported to exert catalytic activity on 1,2-ß-d-glucoside. Moreover, six variants (UGT91D2e_No.5, SrUGT91D2-11-14, SrUGT91D2-110, SrUGT91D2-023, SrUGT91D2-N01, and SrUGT91D2-N04) of this gene were obtained, and their activities were identified. Although there were some differences among these variants, the only type of mutation was partial base substitution at a very low level. In addition, the expression analysis of SrUGT91D2 in each genotype showed that the expression level of the gene was significantly different among genotypes, and a significant positive correlation was found between the content of RD (which was closely influenced by SrUGT91D2) and the expression level of SrUGT91D2 in each genotype (correlation coefficient = 0.91). Thus, it was indicated that SrUGT91D2 was relatively conserved in S. rebaudiana, and the differential effect of SrUGT91D2 on the accumulation of related SGs mainly derived from its expression level. Furthermore, based on homologous modeling and molecular docking analysis, T84, T144, A194, S284, E285, V286, G365, E369, R404, and G409 were predicted to be key residues in the glucosylation of SGs by SrUGT91D2. After site-mutation and enzyme assays, it was confirmed that T84, T144, R404, A194, and G409 are the key residues in the SrUGT91D2 protein, especially T144 and G409. This work provided valuable information for understanding the structure-activity relationship of the SrUGT91D2 protein and the molecular mechanism of SG accumulation in stevia.

Mutations in the uridine diphosphate glucosyltransferase 76G1 gene result in different contents of the major steviol glycosides in Stevia rebaudiana.

In the metabolic glycosylation grid of steviol glycosides, UGT76G1 was shown to catalyze at least eight different glucosylation steps, including the formation of rebaudioside B (Reb B) and rebaudioside A (Reb A) (Olsson et al., 2016). In this study, the accumulation of steviolbioside, Reb B, stevioside (ST) and Reb A in more than 140 samples of stevia leaves collected from different regions in China were analyzed by high-performance liquid chromatography (HPLC), and five genotypes, 'N01-N05', with significantly different levels of the abovementioned glycosides were discovered. Mutations in the UGT76G1 gene cloned from cDNAs from these five genotypes were identified, and the functions of the recombinant UGT76G1 variants were ascertained by adding steviolbioside and ST substrates. In addition, homology modeling and molecular docking were used to elucidate the functional differences between variants and UGT76G1. Comparing the sequences of the five variants 'N01-N05' with UGT76G1 (AY345974.1) revealed that base substitutions were not observed in 'N01'. By contrast, 'N02' exhibited 9 single nucleotide polymorphisms (SNPs) and 9 associated amino acid substitutions or insertions with notable variations in the protein structure; however, an enzyme assay showed similar functionalities between the variant and UGT76G1. In 'N03', 49 SNPs and 29 associated amino acid substitutions or insertions were identified and shown to induce significant variations in the protein structure, especially in the binding pocket, resulting in the lack of functionality of this variant in the enzyme assay. These results were in agreement with the docking profiles. Moreover, a nonsense mutation of p.1090T > G in 'N04' and an insertion of a 68 base fragment in 'N05' were found, and both produced a premature protein without any catalytic activity. Therefore, UGT76G1, which is vital to the content of main steviol glycosides, should be a key gene marker for the molecular breeding of Stevia rebaudiana. Our investigations also revealed the location and orientation of active groups of the receptors and donors in the UGT76G1 enzyme, which play key roles in determining whether the enzyme has any enzymatic activity.