Identification of basic helix-loop-helix transcription factors that activate betulinic acid biosynthesis by RNA-sequencing of hydroponically cultured<i>Lotus japonicus</i>

Suzuki, Hayato; Takahashi, Hirokazu; Fukushima, Ery Odette; Nakazono, Mikio; Muranaka, Toshiya; Seki, Hikaru

doi:10.1101/2022.11.16.516519

Cited by 5 publications

(4 citation statements)

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“…The triterpenoid profiles of LORE1 insertion mutant lines of L. japonicus accession Gifu B-129 ( Fukai et al., 2012 ; Urbański et al., 2012 ) were analyzed to investigate the effect of loss-of-function of either LjCPR1 or LjCPR2 genes. Based on a previous study, LUS and CYP716A s genes that are responsible for lupeol and betulinic acid production, respectively, were upregulated in secondary aerenchyma of hydroponic-cultured L. japonicus ( Suzuki et al., 2022 ) and soybean plants under flooded conditions ( Takahashi et al., 2022 ). Therefore, to investigate if plant culture condition affects LjCPRs’ involvement with specific CYPs, the Ljcpr1 and Ljcpr2-1 loss-of-function mutant lines were both cultured in soil and a hydroponic system.…”

Section: Resultsmentioning

confidence: 99%

Class I and II NADPH-cytochrome P450 reductases exhibit different roles in triterpenoid biosynthesis in Lotus japonicus

et al. 2023

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Cytochrome P450 monooxygenases (CYPs) are enzymes that play critical roles in the structural diversification of triterpenoids. To perform site-specific oxidations of the triterpene scaffold, CYPs require electrons transferred by NADPH-cytochrome P450 reductase (CPR), which is classified into two main classes, class I and class II, based on their structural difference. Lotus japonicus is a triterpenoids-producing model legume with one CPR class I gene (LjCPR1) and a minimum of two CPR class II genes (LjCPR2-1 and LjCPR2-2). CPR classes I and II from different plants have been reported to be involved in different metabolic pathways. By performing gene expression analyses of L. japonicus hairy root culture treated with methyl jasmonate (MeJA), this study revealed that LjCPR1, CYP716A51, and LUS were down-regulated which resulted in no change in betulinic acid and lupeol content. In contrast, LjCPR2s, bAS, CYP93E1, and CYP72A61 were significantly upregulated by MeJA treatment, followed by a significant increase of the precursors for soyasaponins, i.e. β-amyrin, 24-OH β-amyrin, and sophoradiol content. Triterpenoids profile analysis of LORE1 insertion and hairy root mutants showed that the loss of the Ljcpr2-1 gene significantly reduced soyasaponins precursors but not in Ljcpr1 mutants. However, Ljcpr1 and Ljcpr2-1 mutants showed a significant reduction in lupeol and oleanolic, ursolic, and betulinic acid contents. Furthermore, LjCPR1, but not LjCPR2, was crucial for seed development, supporting the previous notion that CPR class I might support plant basal metabolism. This study suggests that CPR classes I and II play different roles in L. japonicus triterpenoid biosynthesis.

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Section: Resultsmentioning

confidence: 99%

Class I and II NADPH-cytochrome P450 reductases exhibit different roles in triterpenoid biosynthesis in Lotus japonicus

et al. 2023

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“…Most of the identified markers are distant from target biosynthesis genes; thus, their functional interactions with them need to be verified, considering that long‐distance chromatin interactions between loci might occur and chromatin loops might connect regulatory elements to their target genes (Peng et al ., 2019 ). The analysis of the genomic regions surrounding the identified markers resulted in numerous TFs, some belonging to families known to be involved in the regulation of triterpenoid biosynthesis like bHLH, AP2‐like ethylene‐responsive and WRKY (Mertens et al ., 2016 ; Tamura et al ., 2018 ; da Silva Magedans et al ., 2021 ; Suzuki et al ., 2023 ). Further studies will assess their role in the regulation of olive triterpenoid biosynthesis.…”

Section: Discussionmentioning

confidence: 99%

OeBAS and CYP716C67 catalyze the biosynthesis of health‐beneficial triterpenoids in olive (Olea europaea) fruits

et al. 2023

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Summary The bioactive properties of olive (Olea europaea) fruits and olive oil are largely attributed to terpenoid compounds, including diverse triterpenoids such as oleanolic, maslinic and ursolic acids, erythrodiol, and uvaol. They have applications in the agri‐food, cosmetics, and pharmaceutical industries. Some key steps involved in the biosynthesis of these compounds are still unknown. Genome mining, biochemical analysis, and trait association studies have been used to identify major gene candidates controlling triterpenoid content of olive fruits. Here, we identify and functionally characterize an oxidosqualene cyclase (OeBAS) required for the production of the major triterpene scaffold β‐amyrin, the precursor of erythrodiol, oleanolic and maslinic acids, and a cytochrome P450 (CYP716C67) that mediates 2α oxidation of the oleanane‐ and ursane‐type triterpene scaffolds to produce maslinic and corosolic acids, respectively. To confirm the enzymatic functions of the entire pathway, we have reconstituted the olive biosynthetic pathway for oleanane‐ and ursane‐type triterpenoids in the heterologous host, Nicotiana benthamiana. Finally, we have identified genetic markers associated with oleanolic and maslinic acid fruit content on the chromosomes carrying the OeBAS and CYP716C67 genes. Our results shed light on the biosynthesis of olive triterpenoids and provide new gene targets for germplasm screening and breeding for high triterpenoid content.

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“…The triterpenoid profiles of LORE1 insertion mutant lines of L. japonicus accession Gifu B-129 (Fukai et al, 2012;Urbański et al, 2012) were analyzed to investigate the effect of loss-of-function of either LjCPR1 or LjCPR2 genes. Based on a previous study, LUS and CYP716As genes that are responsible for lupeol and betulinic acid production, respectively, were upregulated in secondary aerenchyma of hydroponic-cultured L. japonicus (Suzuki et al, 2022) and soybean plants under flooded conditions (Takahashi et al, 2022). Therefore, to investigate if plant culture condition affects LjCPRs' involvement with specific CYPs, the Ljcpr1 and Ljcpr2-1 loss-of-function mutant lines were both cultured in soil and a hydroponic system.…”

Section: Ljcpr1 and Ljcpr2-1 Loss-of-function Mutant Plantsmentioning

confidence: 99%

Advances in Discoveries of Plant Phytochemicals

2024

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Identification of basic helix-loop-helix transcription factors that activate betulinic acid biosynthesis by RNA-sequencing of hydroponically culturedLotus japonicus

Cited by 5 publications

References 79 publications

Class I and II NADPH-cytochrome P450 reductases exhibit different roles in triterpenoid biosynthesis in Lotus japonicus

Class I and II NADPH-cytochrome P450 reductases exhibit different roles in triterpenoid biosynthesis in Lotus japonicus

OeBAS and CYP716C67 catalyze the biosynthesis of health‐beneficial triterpenoids in olive (Olea europaea) fruits

Advances in Discoveries of Plant Phytochemicals

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