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Plant Commun
Plant Commun
Plant Communications
2590-3462
Elsevier

S2590-3462(24)00202-5
10.1016/j.xplc.2024.100932
100932
Correspondence
Insights into dammarane-type triterpenoid saponin biosynthesis from the telomere-to-telomere genome of Gynostemma pentaphyllum
Yun Lingling 15
Zhang Chuyi 15
Liang Tongtong 1
Tian Yu 1
Ma Guoxu 1
Courdavault Vincent 2
Sun Sijie 14
Ma Baiping 3
Li Ziqin 1
Li Rucan 1
Cao Feng 1
Shen Xiaofeng 1
Wei Jianhe 1
Li Ying liying@implad.ac.cn
1∗
Guo Baolin blguo@implad.ac.cn
1∗∗
Sun Chao csun@implad.ac.cn
1∗∗∗
1 Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100193, China
2 Biomolécules et Biotechnologies Végétales, EA2106, Université de Tours, 37200 Tours, France
3 Beijing Institute of Radiation Medicine, Beijing 100850, China
∗ Corresponding author liying@implad.ac.cn
∗∗ Corresponding author blguo@implad.ac.cn
∗∗∗ Corresponding author csun@implad.ac.cn
4 Present address: National Genomics Data Center, Beijing Institute of Genomics, Chinese Academy of Sciences and China National Center for Bioinformation, Beijing 100101, China

5 These authors contributed equally to this article.

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https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Published: April 30, 2024
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pmcDear Editor,

Gynostemma pentaphyllum (Thunb.) Makino, a perennial climbing vine in the Cucurbitaceae family, has been widely used in traditional medicine for over 600 years (Blumert and Liu, 1999). It serves as a valuable natural source of over 200 dammarane-type saponins with notable bioactive properties, including anti-cancer, cardioprotective, hepatoprotective, neuroprotective, and anti-diabetic activities (Li et al., 2016; Nguyen et al., 2021). Interestingly, G. pentaphyllum has been found to contain ginsenosides and other structurally similar dammarane triterpenoids despite its distant phylogenetic relationship to Panax ginseng (Fan et al., 2017; Ahmed et al., 2023). In addition, G. pentaphyllum offers advantages over P. ginseng in terms of easier accessibility and significantly higher levels of dammarane-type saponins (Razmovski-Naumovski et al., 2005). Hence, G. pentaphyllum represents a promising resource for the development of dammarane-type saponin-derived pharmaceuticals.

Here, we successfully assembled a high-quality telomere-to-telomere (T2T) genome of G. pentaphyllum by integrating 30.54 Gb (∼51×) PacBio HiFi circular consensus sequencing reads, 103.71 Gb (∼173×) Oxford Nanopore Technologies (ONT) ultra-long reads, 65.63 Gb (∼109×) high-throughput chromosome conformation capture data, and Bionano optical data (Figure 1A and Supplemental Figure 1; Supplemental Table 1). The primary assembly was generated using HiFi reads, which were then corrected with Bionano optical maps and high-throughput chromosome conformation capture data, leaving behind 28 gaps (Supplemental Table 2). We next closed the gaps by manually assembling ONT ultra-long reads and polished the filled gaps with HiFi reads (Figure 1B). Finally, a high-quality T2T genome of G. pentaphyllum (Gp_T2Tv1.0) with a total length of 599.38 Mb was generated, and all 22 telomeres (CAATAAn) and 11 centromeres were identified on the 11 chromosomes (Supplemental Table 3). The accuracy and completeness of Gp_T2Tv1.0 were assessed by multiple methods. Analysis with Benchmarking Universal Single-Copy Orthologs estimated that the genome completeness was as high as 98.70%. The long terminal repeat assembly index (LAI) value for Gp_T2Tv1.0 was 14.89, which is comparable to LAI values reported for previously published T2T genomes of other species, such as 15.05 for cabbage (Zhang et al., 2023) and 12.92 for potato (Yang et al., 2023). The average base quality value of the 11 chromosomes was 36.57, highlighting the base-level accuracy and completeness of the assembly (Supplemental Table 4). The mapping rates of Illumina short reads, HiFi reads, and ONT ultra-long reads were >99%, and Bionano optical maps showed high consistency across all assembled chromosomes (Supplemental Table 5; Supplemental Figures 2 and 3). Approximately 70.48% of the genome sequences were repetitive, 65.80% of which were transposon elements (Supplemental Table 6). We predicted 26 003 protein-coding genes in the genome, with an average coding-sequence length of 4567 bp and an average of 5.38 exons per gene (Supplemental Table 7). The centromere regions consisted of 68% transposable elements and 32% tandem repeats and exhibited significant variations in length and structural composition among the 11 chromosomes (Supplemental Table 8). A total of 101 genes were annotated in these regions, enriching the genetic resources for this species.Figure 1 T2T genome of G. pentaphyllum and screening, characterization, and evolutionary analysis of DS in G. pentaphyllum.

(A) Distribution of genomic features in the Gp_T2Tv1.0 genome. Displayed tracks show chromosomes (A), tandem repeat density (B), GC content (C), CG methylation density (D), gene density (E), TE density (F), and LTR density (G). The inner lines present links between synteny-selected paralogs. All distributions are drawn in a window size of 1 Mb.

(B) Schematic of gap filling in the centromeric region of chromosome 2. Red and blue lines on the HiFi and ONT reads represent regions with >10 and ≤10 mismatched bases, respectively. Bionano optical map alignments confirm the accuracy of gap filling.

(C) Comparative transcriptome analysis of dammarane-type triterpenoid saponin biosynthesis in G. pentaphyllum. The enzymes highlighted in red are involved in the pathway: IDI , isopentenyl diphosphate isomerase; GPPS, geranylgeranyl pyrophosphate synthase; FPS, farnesyl pyrophosphate synthase; SS, squalene synthase; SE, squalene epoxidase; DS, dammarenediol-II synthase; CYP450, cytochrome P450; UGT, UDP-glycosyltransferases. The expression value of each identified candidate gene is colored in transcripts per million in six tissues: root (AR), tendril (AT), mature leaf (ML), young leaf (YL), mature stem (MS), and young stem (YS). Low to high expression is indicated by the change in color from green to red.

(D) Schematic diagram of the molecular docking of dammarenediol-II with the predicted GpDS.

(E) Overlay of gas chromatography–mass spectrometry traces, showing representative compound peaks for the target products of wild GpDS and its mutants.

(F) Maximum-likelihood phylogenetic analysis of the OSC family. The paralogous A and B branches are denoted by distinct color series; i.e., blue and purple, respectively. Characterized OSCs with different functions are represented by different tip colors. The red star marks the DSs of P. ginseng and G. pentaphyllum. The color of the outer circle denotes the plant lineage to which the species containing the OSC gene belongs.

The cyclization of 2,3-oxidosqualene catalyzed by oxidosqualene cyclases (OSCs) is the first committed branch point in triterpenoid biosynthesis (Haralampidis et al., 2002; Li et al., 2023). A total of 11 OSCs were annotated in our T2T G. pentaphyllum genome, and their functions and classifications were predicted on the basis of a phylogenetic tree (Supplemental Figure 4; Supplemental Table 9). We examined the expression patterns of 11 OSCs and farnesyl pyrophosphate synthase, squalene synthase, and squalene epoxidase genes in the upstream pathways, revealing that GpOSC1 demonstrated a strong co-expression relationship with these upstream genes (Figure 1C). We cloned the full-length open reading frame of GpOSC1 and characterized its function through a yeast expression assay and transient gene expression system in Nicotiana benthamiana, enabling comprehensive verification both in vivo and in vitro. The results of gas chromatography–mass spectrometry analysis demonstrated that GpOSC1 could catalyze the formation of dammarenediol-II from 2,3-oxidosqualene through cyclization (Supplemental Figures 5–8). To gain deeper insights into the catalytic mechanism of GpOSC1 (referred to as GpDS), we predicted its 3D structure using AlphaFold2 and performed molecular docking with dammarenediol-II, revealing that 25 amino acid residues at the active center are within 5 Å of the dammarenediol-II molecule (Figure 1D and Supplemental Figures 9 and 10). We constructed Y259H, W418A, D485N, C564A, S412F, H479N, and C486A mutants of GpDS based on the results of docking and conserved motifs (Figure 1D; Supplemental Table 10; Supplemental Figures 11 and 12). The single-site mutants D485N, S412F, and W418A exhibited complete loss of activity, and the C486A, C564A, H479N, and Y259H mutants had significantly reduced activity (Figure 1E). Our investigation indicated that several residues at the active site of GpDS play a crucial role in its enzymatic activity, potentially by interacting with the substrate and shaping the overall conformation. These findings are consistent with previous reports on dammarenediol-II synthase (DS) in P. ginseng (PgDS; ID: ACZ71036.1) (Liu et al., 2017; Chen et al., 2021), suggesting that GpDS shares a similar catalytic mechanism with PgDS. Overall, we speculated that the acidic residue D485 initiates 2,3-oxidosqualene cyclization by acting as a proton donor, while C486 and C564 increase the acidity through hydrogen bonds with D485. The Y259 residue in the VWCYFR motif is critical for stabilizing the intermediate cation and facilitating dammarenediol-II formation.

To investigate the origin and evolutionary trajectory of DS in flowering plants, we proposed a model for OSC evolution through a combination of phylogenetic and synteny analyses (Figure 1F and Supplemental Figures 13–15). A comprehensive phylogenetic analysis of 428 OSC sequences from 115 plant species across the angiosperm lineage (Supplemental Table 9) revealed that the basal angiosperm Amborella trichopoda contains a solitary OSC with homology to cycloartenol synthases identified in ferns and gymnosperms, suggesting that all OSCs in angiosperms evolved from an ancestral cycloartenol synthase-like protein. The remaining OSCs were initially divided into two primary clades, designated clade A and clade B, with each clade encompassing OSCs derived from species representing major taxonomic groups across the angiosperms, suggesting that most angiosperms share a common OSC gene duplication event that occurred after the divergence of A. trichopoda. Synteny analysis revealed the presence of tandem-duplicated OSC genes within syntenic blocks of both Nymphaea colorata and Aristolochia fimbriata, suggesting that the OSC duplication occurred prior to the speciation of N. colorata (Supplemental Figure 15). Following the duplication event, the paralogous OSC genes A and B showed a tripartite branching pattern in core eudicots, with subclades A1, A2, and A3 evolving from clade A and subclades B1, B2, and B3 from clade B. In grape, the synonymous substitution values (Ks) of paralogous OSC genes within each subclade (i.e., between A1, A2, and A3 and between B1, B2, and B3) ranged from 1.14 to 1.59, which is more generally similar to the Ks peak corresponding to the whole-genome triplication (γ-WGT) event (Supplemental Figure 16). This suggests that the tripartite branching pattern of A/B may have originated from the whole-genome triplication event of core eudicots. In addition, phylogenetic analysis of the OSCs demonstrated that the B2 subclade has undergone pronounced neofunctionalization, with GpDS from the Cucurbitaceae family nested within the β-amyrin synthase genes in this clade. By contrast, the PgDS from the Araliaceae family is clustered with the multifunctional OSCs in the B3 subclade. Therefore, we speculate that DSs evolved independently in G. pentaphyllum and P. ginseng.

In summary, we generated a high-quality T2T genome assembly of G. pentaphyllum and functionally characterized a DS from 11 putative OSC genes identified in the genome. Our findings demonstrate a similar catalytic mechanism between the GpDS characterized here and the well-studied PgDS from P. ginseng. Phylogenetic and synteny analyses further elucidated the independent evolutionary trajectories of the GpDS and PgDS genes. Overall, this study offers valuable resources for molecular-assisted breeding and the production of high-value dammarane-type triterpenoid saponins in cell factories.

Accession numbers

The assembled genomes in this study have been deposited in the National Center for Biotechnology Information (NCBI) and China National Genomics Data Center with accession numbers PRJNA1030181 and PRJCA025561, respectively. Source data are provided in this paper.

Funding

This work was supported by the 10.13039/501100005150 Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences (grant 2021-I2M-1-032 ). The funders had no role in the design of the study; the collection, analyses, or interpretation of data; the writing of the manuscript; or the decision to publish the results.

Author contributions

Conceptualization, C.S. and B.G.; investigation, L.Y., T.L., G.M., Z.L., R.L., and F.C.; formal analysis, C.Z., Y.L., Y.T., and S.S.; writing – original draft, L.Y., C.Z., and Y.L.; writing – review & editing, C.S., B.G., B.M., J.W., X.S., and V.C.

Supplemental information

Document S1. Supplemental methods and Supplemental Figures 1–16

Data S1. Supplemental Tables 1–10

Document S2. Article plus supplemental information

Acknowledgments

No conflict of interest is declared.

Published by the Plant Communications Shanghai Editorial Office in association with Cell Press, an imprint of Elsevier Inc., on behalf of CSPB and CEMPS, CAS.

Supplemental information can be found online at https://doi.org/10.1016/j.xplc.2024.100932.
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