Title: CYP716A179 functions as a triterpene C-28 oxidase in tissue-cultured stolons of Glycyrrhiza uralensis

Journal: Plant Cell Reports

Authors:

Keita Tamura, Hikaru Seki, Hideyuki Suzuki, Mareshige Kojoma, Kazuki Saito, Toshiya Muranaka

Corresponding author:

Toshiya Muranaka

Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan

E-mail:

Fax: +81-6-6879-7426

Tel: +81-6-6879-7423

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Supplementary Fig. S1 Mass spectra of peaks identified in Fig. 2a–c and those of authentic standards. (a) β-amyrin, (b) erythrodiol, (c) oleanolic aldehyde, (d) oleanolic acid, (e) α-amyrin, (f) uvaol, (g) ursolic aldehyde, (h) ursolic acid, (i) 22α-hydroxy-α-amyrin, (j) lupeol, (k) betulin, (l) betulinic aldehyde, and (m) betulinic acid.


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Supplementary Fig. S1 (continued)


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l

Supplementary Fig. S1 (continued)


m

Supplementary Fig. S1 (continued)


Supplementary Fig. S2 GC-MS analysis of triterpenoid aglycones from tissue-cultured stolons and intact roots of G. uralensis. β-amyrin (1), oleanolic acid (4), betulinic acid (13), soyasapogenol B (14), and glycyrrhetinic acid (15) were identified in tissue-cultured stolons, whereas lupeol (10), soyasapogenol B (14), and glycyrrhetinic acid (15) were identified in intact roots. Echinocystic acid was used as an internal standard (IS)

Supplementary Fig. S3 Sequence alignment of CYP716A179 and CYP716A12 proteins. Multiple alignments were generated using GENETYX-MAC version 18.0.3 software

Supplementary Fig. S4 Phylogenetic tree of CYP716 proteins related to triterpenoid biosynthesis. The numbers above the branches indicate bootstrap values for 1,000 replicates. GuCYP72A154 was used as the outgroup. P450s that predominantly oxidize the C-28 position of β-amyrin are indicated in red. Aa, Artemisia annua; At, Arabidopsis thaliana; Bf, Bupleurum falcatum; Bv, Barbarea vulgaris; Cr, Catharanthus roseus; Gu, Glycyrrhiza uralensis; Ml, Maesa lanceolata; Mt, Medicago truncatula; Pg, Panax ginseng; Vv, Vitis vinifera


Supplementary Table S1 Primers used for polymerase chain reaction (PCR). The underlined sequences were added to facilitate unidirectional cloning of the product into pENTR/D-TOPO (Thermo Fisher Scientific)

Primer No. / Sequence (5’ to 3’)
1
2 / CACCATGGAGCATTTCTACATGTCC
TCAGGTATCGTGAGGATAAAGG


Supplementary Table S2 Primers used for quantitative real-time PCR analysis

Target gene / Forward primer (5’ to 3’) / Reverse primer (5’ to 3’)
β-tubulin / TCTTCGCAAACTGGCAGTGA / CGAGATGTGAGTGGGGCAAA
bAS / GGTGGTTTATCAGCGTGGGA / TGCTCAACTACAATGTCCGCA
LUS / GAAAGCAATACCCACAGCACAG / GCAAATTCCCCAACACCCATAC
CAS / CCGGCTGAGACTTTTGGTGA / TCTCGACGATGCCCAGGATA
CYP88D6 / GACGCCTCATTACTTCCCCAA / TTCAAGAGCTCAACGGGGTG
CYP72A154 / ATGGCGACCCTTACAAGCTC / AGATTCGTGGTGCAGCATCA
CYP716A179 / AGCGGTTCAAGTGGGAGATG / ATCGGGAGGTCATTTGCAGG
CYP93E3 / GCCGCCGATTACTTCTTCATTC / CGCGGATATTGACAAAGTGCTC

Supplementary Table S3 Summary of the RNA-seq analysis of tissue-cultured stolons of G. uralensis

Total read counts / 28,925,182
Number of contigs / 71,673
N50 of contigs (bp) / 1,639
Average length of contigs (bp) / 1,001
Minimum length of contigs (bp) / 224
Maximum length of contigs (bp) / 13,598

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