A Seed-Specific Regulator of Triterpene Saponin Biosynthesis in <i>Medicago truncatula</i>

Ribeiro, Bianca; Lacchini, Elia; Bicalho, Keylla Utherdyany; Mertens, Jan; Arendt, Philipp; Bossche, Robin Vanden; Calegário, Gabriela; Gryffroy, Lore; Ceulemans, Evi; Buitink, Julia; Goossens, Alain; Pollier, Jacob

doi:10.1105/tpc.19.00609

Cited by 37 publications

(52 citation statements)

References 83 publications

(147 reference statements)

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“…bHLH TFs have emerged as crucial components in the gene regulatory networks controlling many biological processes in plants, including light and phytohormone signaling (Kazan & Manners, 2013; Lee et al., 2006; Leivar et al., 2008; Yin et al., 2005), stress responses (Abe et al., 1997), shoot branching (Yang et al., 2012), and tissue and organ development (Kanaoka et al., 2008; Morohashi et al., 2007; Rajani & Sundaresan, 2001; Sorensen et al., 2003; Szécsi et al., 2006). In addition, bHLH TFs regulate the biosynthesis of plant specialized metabolites, such as nicotine in tobacco (Shoji & Hashimoto, 2011; Zhang et al., 2012), glucosinolates in Arabidopsis (Schweizer et al., 2013), cucurbitacin in cucumber (Shang et al., 2014), phytoalexins in rice (Yamamura et al., 2015), artemisinin in Artemisia annua (Shen et al., 2016), paclitaxel in Taxus cuspidata (Lenka et al., 2015), saponins in Medicago truncatula (Mertens, Pollier, et al., 2016; Ribeiro et al., 2020), amygdalin in almond (Sanchez‐Perez et al., 2019), and anthocyanins in many plant species (Patra et al., 2013; Xu et al., 2015). The bHLH TFs belonging to subgroup IIId, IIIe, and IIIf are well characterized for their roles in plant specialized metabolite biosynthesis.…”

Section: Introductionmentioning

confidence: 99%

BHLH IRIDOID SYNTHESIS 3 is a member of a bHLH gene cluster regulating terpenoid indole alkaloid biosynthesis in Catharanthus roseus

et al. 2021

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Basic helix‐loop‐helix (bHLH) transcription factors (TFs) are key regulators of plant specialized metabolites, including terpenoid indole alkaloids (TIAs) in Catharanthus roseus. Two previously characterized subgroup‐IVa bHLH TFs, BIS1 (bHLH Iridoid Synthesis 1) and BIS2 regulate iridoid biosynthesis in the TIA pathway. We reanalyzed the recently updated C. roseus genome sequence and discovered that BIS1 and BIS2 are clustered on the same genomic scaffold with a previously uncharacterized bHLH gene, designated as BIS3. Only a few bHLH gene clusters have been studied to date. Comparative analysis of 49 genome sequences from different plant lineages revealed the presence of analogous bHLH clusters in core angiosperms, including the medicinal plants Calotropis gigantea (giant milkweed) and Gelsemium sempervirens (yellow jessamine), but not in the analyzed basal angiosperm and lower plants. Similar to the iridoid pathway genes, BIS3 is highly expressed in roots and induced by methyl jasmonate. BIS3 activates the promoters of iridoid branch genes, geraniol synthase (GES), geraniol 10‐hydroxylase (G10H), 8‐hydroxygeraniol oxidoreductase (8HGO), iridoid synthase (IS), 7‐deoxyloganetic acid glucosyl transferase (7‐DLGT), and 7‐deoxyloganic acid hydroxylase (7DLH), but not iridoid oxidase (IO). Transactivation of the promoters was abolished when BIS3 is converted to a dominant repressor by fusing with the ERF‐associated amphiphilic repression (EAR) sequence. In addition, BIS3 acts synergistically with BIS1 and BIS2 to activate the G10H promoter in tobacco cells. Mutation of the known bHLH TF binding motif, G‐box (CACGTG) in the G10H promoter significantly reduced but did not abolish the transactivation by BIS3. Promoter deletion analysis of G10H suggests that the sequences adjacent to the G‐box are also involved in the regulation by BIS3. Overexpression of BIS3 in C. roseus flower petals significantly upregulated the expression of iridoid biosynthetic genes and increased loganic acid accumulation. BIS2 expression was significantly induced by BIS3 although BIS3 did not directly activate the BIS2 promoter. Our results advance our understanding of the regulation of plant specialized metabolites by bHLH TF clusters.

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Section: Introductionmentioning

confidence: 99%

BHLH IRIDOID SYNTHESIS 3 is a member of a bHLH gene cluster regulating terpenoid indole alkaloid biosynthesis in Catharanthus roseus

et al. 2021

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“…TRITERPENE SAPONIN BIOSYNTHESIS ACTIVATING REGULATOR1 (MtTSAR1) upregulates the soyasaponin pathway in M. truncatula [20]. MtTSARs 2 and 3 are factors that activate hemolytic saponin accumulation, with differences in tissue speci city [20,21]. We recently identi ed GubHLH3 as a positive regulator of soyasaponin biosynthesis in G. uralensis [22], and this protein is closely related to MtTSAR2 but not MtTSAR1.…”

Section: Introductionmentioning

confidence: 99%

“…Interestingly, the functions of MtTSARs and CrBIS1 were shown to be interchangeable through heterologous expression of MtTSARs in C. roseus and CrBIS1 in M. truncatula [26]. In addition, production of both saponins and MIAs were commonly regulated by MeJA [5,21,24,27].…”

Section: Introductionmentioning

confidence: 99%

“…Numerous studies have reported genome-wide identi cation and classi cation of bHLH factors in plants [18,19,28,29,30]. Although the genomes of Arabidopsis thaliana and Oryza sativa possess four and six subclade IVa members, respectively [19], more than 30 subclade IVa bHLH genes were found in the genomes of Glycine max and M. truncatula [21,28]. This nding suggests that Fabaceae plants may have acquired a large number of subclade IVa members during the evolution of saponin biosynthesis.…”

Section: Introductionmentioning

confidence: 99%

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Insights into the Diversification of Subclade IVa bHLH Transcription Factors in Fabaceae

Suzuki¹,

Seki²,

Muranaka³

2020

Preprint

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Background: Fabaceae 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.Results: In 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.Conclusions: This 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.

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“…There are 127 CYP families from plants are identified and they are categorized into 11 clans of which include single or multiple sub-clades on a phylogenetic tree (Nelson and Werck, 2011). So far, many CYP families are reported to participate in pentacyclic triterpenoid structural modifications, including CYP51, CYP71, CYP72, CYP87, CYP88, CYP93, and CYP716 (Ghosh, 2017;Miettinen et al, 2017b;Ribeiro et al, 2020).…”

Section: Introductionmentioning

confidence: 99%

The genome analysis ofTripterygium wilfordiireveals TwCYP712K1 andTwCYP712K2responsible for oxidation of friedelin in celastrol biosynthesis pathway

Pei

Yan

Kong

et al. 2020

Preprint

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ABSTRACTTripterygium wilfordii is a Traditional Chinese Medicine (TCM) from family Celastraceae and celastrol is one of the strongest active ingredients belonging to friedelane-type pentacyclic triterpenoid, which has a large clinical application value of anti-tumor, immunosuppression, and obesity treatment. The first committed biosynthesis step of celastrol is the cyclization of 2, 3-oxidosqualene to friedelin, catalyzed by the oxidosqualene cyclase, while the rest of this pathway is still unclear. In this study, we reported a reference genome assembly of T. wilfordii with high-quality annotation by using a hybrid sequencing strategy (Nanopore, Bionano, Illumina HiSeq, and Pacbio), which obtained a 340.12 Mb total size and contig N50 reached 3.09 Mb. In addition, we successfully anchored 91.02% sequences into 23 pseudochromosomes using Hi-C technology and the super-scaffold N50 reached 13.03 Mb. Based on integration genome, transcriptom and metabolite analyses, as well as in vivo and in vitro enzyme assays, two CYP450 genes, TwCYP712K1 and TwCYP712K2 have been proven for C-29 position oxidation of friedelin to produce polpunonic acid, which clarifies the second biosynthesis step of celastrol. Syntenic analysis revealed that TwCYP712K1 and TwCYP712K2 derived from the common ancestor. These results have provided insight into illustrating pathways for both celastrol and other bioactive compounds found in this plant.

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A Seed-Specific Regulator of Triterpene Saponin Biosynthesis in Medicago truncatula

Abstract: Short title: Seed-specific saponin biosynthesis in Medicago One-sentence summary: The discovery of a seed-specific regulator of hemolytic saponin biosynthesis in Medicago truncatula led to the identification of the missing P450 of the hemolytic saponin biosynthesis branch.

Cited by 37 publications

References 83 publications

BHLH IRIDOID SYNTHESIS 3 is a member of a bHLH gene cluster regulating terpenoid indole alkaloid biosynthesis in Catharanthus roseus

BHLH IRIDOID SYNTHESIS 3 is a member of a bHLH gene cluster regulating terpenoid indole alkaloid biosynthesis in Catharanthus roseus

Insights into the Diversification of Subclade IVa bHLH Transcription Factors in Fabaceae

The genome analysis ofTripterygium wilfordiireveals TwCYP712K1 andTwCYP712K2responsible for oxidation of friedelin in celastrol biosynthesis pathway

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