A Dioxygenase Catalyzes Steroid 16α-Hydroxylation in ......2017/07/28 · 10 Article Title: A...

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Short title: 16α-Hydroxylase in Glycoalkaloid Biosynthesis (45 characters and spaces 1

<50) 2

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Corresponding author details: 4

Correspondence should be addressed to Masaharu Mizutani; 5

Address: Graduate School of Agricultural Science, Kobe University, Rokkoudai 1-1, Nada-ku, 6

Kobe, Hyogo 657-8501, Japan. 7

TEL, +81-78-803-5885; Fax, +81-78-803-5885; E-mail, [email protected] 8

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Article Title: A Dioxygenase Catalyzes Steroid 16α-Hydroxylation in Steroidal 10

Glycoalkaloid Biosynthesis 11

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Masaru Nakayasua, Naoyuki Umemotob,c, Kiyoshi Ohyamad, Yoshinori Fujimotod, Hyoung 14

Jae Leea, Bunta Watanabee, Toshiya Muranakaf, Kazuki Saitob,g, Yukihiro Sugimotoa, and 15

Masaharu Mizutania* 16

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aGraduate School of Agricultural Science, Kobe University, Rokkoudai 1-1, Nada-ku, Kobe, 18

Hyogo 657-8501, Japan. 19

Tel: +81-78-803-5885; email: [email protected] 20

bRIKEN Center for Sustainable Resource Science, Suehiro-cho 1-7-22, Tsurumi-ku, 21

Yokohama, Kanagawa 230-0045, Japan. 22

cCentral Laboratories for Key Technologies, Kirin Co., Ltd. Fukuura 1-13-5, Kanazawa-ku, 23

Plant Physiology Preview. Published on July 28, 2017, as DOI:10.1104/pp.17.00501

Copyright 2017 by the American Society of Plant Biologists

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Yokohama, Kanagawa 236-0004, Japan. 24

dDepartment of Chemistry and Materials Science, Tokyo Institute of Technology, Ookayama 25

2-12-1, Meguro-ku, Tokyo 152-8551, Japan. 26

eInstitute for Chemical Research, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan. 27

fDepartment of Biotechnology, Graduate School of Engineering, Osaka University, 28

Yamadaoka 2-1, Suita, Osaka 565-0871, Japan. 29

gGraduate School of Pharmaceutical Sciences, Chiba University, Inohana 1-8-1, Chuo-ku, 30

Chiba 260-8675, Japan. 31

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One sentence summary; 33

The 2-oxoglutarate-dependent dioxygenase 16DOX catalyzes steroid 16α-hydroxylation 34

in the steroidal glycoalkaloid (SGA) pathway and is a suitable target for controlling 35

toxic SGA levels in potato. (197 characters and spaces <200 characters) 36

37

Footnotes; 38

List of author contributions 39

M.N., N.U., K.O., K.S., and M.M. designed the research; M.M., Y.F., T.M., K.S., and Y.S. 40

supervised the experiments; M.N., N.U., K.O., H.L., and B.W. performed the research; and 41

M.N., N.U., and M.M. wrote the article. 42

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Funding information 44

This work was supported by the Program for Promotion of Basic and Applied Researches for 45

Innovations in Bio-oriented Industry (BRAIN), Japan, by the Scientific Technique Research 46



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Promotion Program for Agriculture, Forestry, Fisheries and Food Industry, Japan, and by the 47

Cross-ministerial Strategic Innovation Promotion Program (SIP), Japan. 48

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*Address correspondence to [email protected] 50

The author responsible for distribution of materials integral to the findings presented in this 51

article in accordance with the policy described in the Instructions for Authors 52

(www.plantphysiol.org) is: Masaharu Mizutani ([email protected]). 53

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Abstract 56

Steroidal glycoalkaloids (SGAs) are toxic specialized metabolites that are found in 57

Solanaceae. Potato (Solanum tuberosum) contains the SGAs α-solanine and α-chaconine, 58

while tomato (S. lycopersicum) contains α-tomatine, all of which are biosynthesized from 59

cholesterol. However, although two cytochrome P450 monooxygenases that catalyze the 22- 60

and 26-hydroxylation of cholesterol have been identified, the 16-hydroxylase remains 61

unknown. Feeding with deuterium-labeled cholesterol indicated that the 16α- and 62

16β-hydrogen atoms of cholesterol were eliminated to form α-solanine and α-chaconine in 63

potato, while only the 16α-hydrogen atom was eliminated in α-tomatine biosynthesis, 64

suggesting that a single oxidation at C-16 takes place during tomato SGA biosynthesis while a 65

two-step oxidation occurs in potato. Here we show that a 2-oxoglutarate-dependent 66

dioxygenase (2OGD), designated as 16DOX, is involved in SGA biosynthesis. We found that 67

the transcript of potato 16DOX (St16DOX) was expressed at high levels in the tuber sprouts, 68

where large amounts of SGAs are accumulated. Biochemical analysis of the recombinant 69

St16DOX protein revealed that St16DOX catalyzes the 16α-hydroxylation of 70

hydroxycholesterols and that (22S)-22,26-dihydroxycholesterol was the best substrate among 71

the nine compounds tested. St16DOX-silenced potato plants contained significantly lower 72

levels of SGAs and a detailed metabolite analysis revealed that they accumulated the 73

glycosides of (22S)-22,26-dihydroxycholesterol. Analysis of the tomato 16DOX (Sl16DOX) 74

gene gave essentially the same results. These findings clearly indicate that 16DOX is a steroid 75

16α-hydroxylase that functions in the SGA biosynthetic pathway. Furthermore, 76

St16DOX-silencing did not affect potato tuber yield, indicating that 16DOX may be a suitable 77

target for controlling toxic SGA levels in potato. (249 words < 250) 78



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Introduction 81

Steroidal glycoalkaloids (SGAs) are typically found in plants in the family Solanaceae 82

(Harrison, 1990; Helmut, 1998; Petersen et al., 1993). In particular, potato (Solanum 83

tuberosum) is known to contain the SGAs α-solanine and α-chaconine, while tomato (S. 84

lycopersicum) contains α-tomatine, which are toxic to fungi, bacteria, insects, animals, and 85

humans (Friedman, 2002, 2006). The mechanisms of toxicity include the disruption of 86

membranes and the inhibition of acetylcholine esterase activity (Roddick, 1989). α-Solanine 87

and α-chaconine occur in most tissues of potato plants, but are present at particularly high 88

levels in floral and tuber sprout tissues (Kozukue and Mizuno, 1985, 1989; Ginzberg et al., 89

2009). Therefore, since these potato SGAs are toxic and cause a bitter taste, controlling their 90

content in potato tubers is an important focus of potato breeding (Friedman, 2006). Similarly, 91

α-tomatine exists in all tomato plant tissues, but is richly accumulated in the leaves and 92

immature fruit, with its content decreasing during fruit ripening (Friedman, 2002). 93

SGAs are composed of C27 steroids with an oligosaccharide at the hydroxy group at 94

the C-3 position (Friedman, 2002, 2006). In α-solanine and α-chaconine in potato, the 95

oligosaccharides solatriose and chacotriose are attached to the C-3 hydroxy group of 96

solanidine, which is a solanidane-type aglycone; and similarly, in α-tomatine in tomato, 97

lycotetraose is linked to the C-3 hydroxy group of tomatidine, which is spirosolane-type 98

aglycone. These SGAs are biosynthesized from cholesterol (Sawai et al., 2014), which is 99

subsequently modified via oxidation at the C-16, C-22, and C-26 positions, amination at the 100

C-26 position, and glycosylation at the C-3 hydroxy group (Friedman, 2002; Ginzberg et al., 101

2009; Petersen et al., 1993; Ohyama et al., 2013). Cytochrome P450 monooxygenases (CYPs) 102

are likely involved in the oxidative modification of cholestrol, while a transaminase catalyzes 103



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the amination at C-26, and UDP-dependent glycosyltransferases (UGTs) also function in the 104

glycosylation at the C-3 hydroxy group (Fig. 1). Some UGTs have now been identified as the 105



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enzymes that are involved in the glycosylation steps for SGA biosynthesis (Moehs CP et al., 106

1997; McCue KF et al., 2005, 2006, 2007; Itkin et al., 2011). Itkin et al. (2013) reported that 107

several genes that are involved in the SGA biosynthetic pathways exist as gene clusters on 108

chromosomes 7 and 12 in the genomes of tomato and potato. In addition, sterol side chain 109

reductase 2 (SSR2) has been identified as a gene that is committed to cholesterol biosynthesis 110

and involved in SGA production (Sawai et al., 2014). However, the genes that are involved in 111

the later steps of the SGA biosynthetic pathways remain to be completely elucidated. 112

CYPs are heme-thiolate membrane proteins that are generally bound to the 113

cytoplasmic surface of the endoplasmic reticulum and are known to be involved in the 114

oxidation steps during the metabolism of various plant natural products. A number of CYPs 115

have been reported to catalyze the oxidation of triterpene skeletons, such as oleanane-, lupine-, 116

ursane- and dammarane-types (Moses et al., 2014; Seki et al., 2015). In addition, several CYP 117

genes have been identified as being responsible for the oxidation of the steroid backbone in 118

the biosynthesis and catabolism of brassinosteroids, which are a plant steroid hormones 119

(Ohnishi et al., 2009). Therefore, we previously investigated SGA biosynthesis in potato and 120

tomato by selecting several CYP genes that show high expression in SGA-rich organs as 121

candidate oxidases of cholesterol using the public expressed sequence tag (EST) databases of 122

potato and tomato. This showed that two CYPs (PGA1/CYP72A208 and PGA2/CYP72A188) 123

are involved in the hydroxylation of cholesterol at the C-26 and C-22 positions (Umemoto et 124

al., 2016). In addition, another CYP (PGA3/GAME4/CYP88B1) was also identified as being 125

involved in SGA biosynthesis, but its catalytic function remains unknown (Umemoto and 126

Sasaki, 2013; Itkin et al., 2013). We were unable to find a C-16 hydroxylase among the 127

candidate CYPs, however. 128



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The 2-oxoglutarate dependent dioxygenase (2OGD) superfamily is the second 129

largest enzyme family, following the CYP superfamily, in the plant genome (Kawai et al., 130

2014). 2OGDs are non-heme iron-containing proteins that localize in the cytosol as soluble 131

proteins. Many 2OGDs play crucial roles in the oxidation steps in the biosynthesis of diverse 132

specialized metabolites such as flavonoids and phytohormones (Kawai et al., 2014). However, 133

unlike CYPs, no 2OGDs have been identified as being involved in the biosynthesis of 134

triterpenoids and steroids to date. Here, we characterized the 2OGD gene named 16DOX and 135

found that it is involved in SGA biosynthesis. The 16DOX gene was co-expressed with the 136

previously identified SGA biosynthetic genes in potato and tomato, and the 16DOX protein 137

was found to catalyze the hydroxylation of cholesterol at the C-16α position. Furthermore, 138

16DOX-silencing in transgenic potato plants led to significantly reduced endogenous SGA 139

levels and suppression of potato tuber sprouting, which are similar to the phenotypes 140

observed in PGA1- and PGA2-silenced plants (Umemoto et al., 2016). Thus, 16DOX is the 141

first 2OGD to be identified as functioning as a steroid 16α-hydroxylase involved in SGA 142

biosynthesis. 143

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RESULTS 147

SGA analysis of potato shoots and tomato seedlings fed with stable isotope-labeled 148

compounds 149

To examine the fates of hydrogen atoms at the C-16 position during SGA biosynthesis 150

in potato shoots and tomato seedlings, we conducted feeding experiments using three kinds of 151

stable isotope-labeled compounds: [15,15,16α,17α-2H4]cholesterol, 152

[15,15,16β,17α-2H4]cholesterol, and [15,15,17α-2H3]cholesterol. The accumulated SGAs in 153

the harvested shoots and seedlings were extracted and analyzed by liquid 154

chromatography-mass spectrometry (LC-MS) (Fig. 2). In the case of potato shoots, labeled 155

α-solanine and α-chaconine were detected by monitoring the m/z 871 and 855 ions [M+H]+, 156

respectively (Fig. 2A-F), while non-labeled α-solanine and α-chaconine were detected by 157

monitoring the m/z 868 and 852 ions [M+H]+, respectively, in shoots that were fed with the 158

three labeled cholesterols. These results indicate that the 15- and 17-hydrogens of cholesterol 159

were retained but both hydrogens at the 16α and 16β positions were eliminated in the process 160

of potato SGA biosynthesis. In the case of tomato seedlings , labeled α-tomatine was detected 161

by monitoring the m/z 1037 ion [M+H]+ (Fig. 2G and H), while non-labeled α-tomatine was 162

detected by monitoring the m/z 1034 ion [M+H]+ in seedlings that were fed with 163

[15,15,16α,17α-2H4]cholesterol and [15,15,17α-2H3]cholesterol. By contrast, when 164

[15,15,16β,17α-2H4]cholesterol was administered, labeled α-tomatine was detected by 165

monitoring the m/z 1038 ion [M+H]+ (Fig. 2I), which is one mass higher than the results 166

obtained with [15,15,16β,17α-2H4]cholesterol and [15,15,17α-2H3]cholesterol. These results 167

indicate that 16β-hydrogen was retained but 16α-hydrogen was eliminated in tomato SGA 168

biosynthesis. These results are consistent with the previous observation reported by Canonica 169



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et al. (1977). Taken together, these findings suggest that oxidation (dehydrogenation) occur at 170

both the C-16α and C-16β positions in potato, and at the C-16α position in tomato during 171



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SGA biosynthesis. 172

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Identification of the candidate 16DOX gene in potato and tomato 174

To identify the candidate genes that are involved in the oxidative modification of 175

cholesterol for SGA biosynthesis, we surveyed several enzyme superfamilies that are included 176

in the EST databases of potato from the DFCI Plant Gene Indices 177

(http://compbio.dfci.harvard.edu/tgi/plant.html) and the tomato databases from MiBASE 178

(http://www.pgb.kazusa.or.jp/mibase/) and Sol Genomics (http://solgenomics.net). We 179

initially selected CYP genes based on the correlation between the read numbers of the EST 180

contigs and the tissue specific accumulation of SGAs in potato and tomato. This led to the 181

identification of the CYPs PGA1 (Sotub06g021140, CYP72A208) and PGA2 182

(Sotub07g016580, CYP72A188) as a steroid 26-hydroxylase and a steroid 22-hydroxylase, 183

respectively, for potato SGA biosynthesis (Umemoto et al., 2016). However, we were unable 184

to identify a 16-hydroxylase among the candidate CYPs. The 2OGD superfamily is also 185

associated with the oxygenation reactions of various specialized metabolites (Kawai et al., 186

2014). Therefore, we surveyed the unigenes encoding 2OGDs from the potato database as a 187

second focus. 188

A total of 256 2OGD transcripts were extracted by Basic Local Alignment Search 189

Tool X (BLASTX) search of nucleotide sequences of all transcript sequences obtained from 190

Spud DB potato genomics resource (http://solanaceae.plantbiology.msu.edu/) against protein 191

sequences of 130 2OGDs from Arabidopsis (Kawai et al., 2014). One of the 2OGD transcripts 192

(PGSC0003DMT400030676) showed the highest FPKM value in tuber sprout among the 256 193

2OGD transcripts in RNA-Seq Gene Expression Data obtained from Spud DB (Supplemental 194



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Table S1), and we selected the transcript as a candidate gene encoding a 16-hydroxylase, 195

which was designated as St16DOX. Quantitative reverse transcription polymerase chain 196



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reaction (RT-PCR) analysis showed that potato St16DOX was highly expressed in the tuber 197

sprouts (Fig. 3), where large amounts of SGAs are accumulated (Friedman and Dao, 1992; 198

Smith et al., 1996; Milner et al., 2011). BLAST searches against the genome databases of 199

Solanum species (Sol Genomics Network: http://solgenomics.net) showed that St16DOX is 200

identical to Sotub07g016570 in the Potato ITAG protein database and shows 93% amino acid 201

identity to tomato Solyc07g043420 (designated as Sl16DOX) in the Tomato ITAG protein 202

database. Tomato Sl16DOX was highly expressed in the flowers, which accumulate large 203

amounts of α-tomatine (Supplemental Fig. S1). These results suggest that 16DOX genes are a 204

candidate for 16-hydroxylase in SGA biosynthesis (Fig. 3 and Supplemental Fig. S1). 205

In a recent phylogenetic classification of the plant 2OGD superfamily (Kawai et al., 206

2014), 16DOXs were found to belong to clade DOXC41 which includes hyoscyamine 207

6β-hydroxylase (H6H), which is involved in the biosynthesis of the tropane alkaloid 208

scopolamine (Matsuda et al., 1991). The 16DOXs share around 44% amino acid sequence 209

identity to H6H in Hyoscyamus niger and contain several sequence motifs that are highly 210

conserved among 2OGDs (Supplemental Fig. S2). The St16DOX and Sl16DOX proteins 211

contain the Fe(II)-binding motif His-X-Asp-Xn-His (His-217, Asp-219, and His-272 in 212

16DOX), which is conserved in the 2OGD superfamily (Bugg, 2003; Lukačin and Britsch, 213

1997; Wilmouth et al., 2002). The Arg–X–Ser motif (Arg-282 and Ser-284), which binds to 214

the C-5 carboxy group of 2-oxoglutarate, is also conserved in the 16DOX proteins (Lukačin et 215

al., 2000; Wilmouth et al., 2002). 216

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In vitro functional analysis of the recombinant 16DOX protein 218

To investigate the catalytic functions of 16DOX, recombinant St16DOX protein 219



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was prepared with a bacterial expression system in Escherichia coli and an in vitro enzyme 220

assay was performed with (22S)-22-hydroxycholesterol as a substrate. The reaction products 221



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were analyzed using gas chromatography-mass spectrometry (GC-MS). St16DOX 222

metabolized (22S)-22-hydroxycholesterol to a product with a retention time of 20.0 min and a 223

major mass fragment ion at m/z 173 (Fig. 4). This product was identical to the authentic 224

compound (22S)-16α,22-dihydroxycholesterol in terms of both the retention time and the 225

mass spectrum, but different from the authentic compound (22S)-16β,22-dihydroxycholesterol, 226

indicating that St16DOX catalyzes the 16-hydroxylation of (22S)-22-hydroxycholesterol 227

specifically in the 16α-configuration. The catalytic activity of tomato Sl16DOX was 228

consistent with that of St16DOX (Fig. 4). 229

We next determined the substrate specificity of St16DOX toward cholesterol and 230

several oxygenated cholesterols (Fig. 5). St16DOX showed the highest activity toward 231

(22S,25S)-22,26-dihydroxycholesterol, reaching a rate that was approximately 50 times higher 232

than that with (22S)-22-hydroxycholesterol (Fig. 5 and Supplemental Fig. S3). LC-MS 233

analysis showed a fragment ion at m/z 435 in the product, which corresponds with the 234

deduced parent ion of 16,22,26-trihydroxycholesterol (Supplemental Fig. S4). St16DOX 235

weakly metabolized (22R)-22-hydroxycholesterol to a new product (Supplemental Fig. S5), 236

but the structure of the metabolite was unknown. By contrast, the assays with the other 237

substrates did not give any product peaks. In terms of kinetic parameters, the Michaelis 238

constant (Km value) for St16DOX toward (22S,25S)-22,26-dihydroxycholesterol was 239

determined to be 4.19 ± 0.17 μM (Supplemental Fig. S6). These results strongly suggest that 240

St16DOX functions as a 16α-hydroxylase of (22S,25S)-22,26-dihydroxycholesterol in SGA 241

biosynthesis. 242

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SGA analysis of St16DOX-silenced transgenic potato plants. 244



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To confirm the contribution of St16DOX to SGA biosynthesis in potato, we 245

transformed potato plants with an RNA interference vector to create St16DOX-silenced 246



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transgenic potato plants. Among 41 lines of St16DOX-silenced transgenic plants, the in 247

vitro-grown shoots of five independent lines (#15, #16, #28, #39, and #41; Fig. 6) had 248



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significantly lower St16DOX transcript levels than the control (Fig. 6A) and consistently had 249

a much lower SGA content (Fig. 6B). All of the silenced lines grew normally in a greenhouse, 250

and the potato tubers were harvested. The SGA contents in the tuber peel and cortex of the 251

five silenced lines were significantly lower than the control (Fig. 6C and D), indicating that 252

St16DOX is involved in SGA biosynthesis in potato. Similarly, the SGA contents in 253

Sl16DOX-silenced transgenic tomato plants were also severely reduced (Supplemental Fig. 254

S7). 255

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Phenotype of St16DOX-silenced transgenic potato plants 257

The St16DOX-silenced transgenic potato plants had similar tuber yields to the 258

control (Fig. 6E), however, the tuber sprouts of these plants stopped the elongation under dark 259

and light conditions. Although sprout initiation seemed to be normal, but the sprouts did not 260

grow even after more than three months at 20 ºC or for a one year at 4 ºC after the cessation 261

of usual dormancy in the control plants (Supplemental Fig. S8A). Interestingly, tiny sprouts 262

could start to grow after the tubers were planted in soil (Supplemental Fig. S8B), but not in 263

water under light or dark conditions. In addition, after placing separated tops of the tiny 264

sprouts on tissue culture media, the sprouts could grow (Supplemental Fig. S8C). The 265

phenotype is completely same to those of PGA1- and PGA2-silenced potato plants (Umemoto 266

et al., 2016). PGA1- and PGA2-silenced potato plants did not bloom and are sterile. Unlike to 267

PGA1- and PGA2-silenced potato plants, the St16DOX-silenced transgenic potato plants 268

bloomed normally. The Sl16DOX-silenced transgenic tomato plants are confirmed to be fertile 269

and did not demonstrate any outward difference compared with the control. 270

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Endogenous metabolite analysis in St16DOX-silenced transgenic potato plants. 272

To examine the effects of St16DOX gene silencing on the endogenous metabolites, 273

we analyzed changes in steroidal compounds in the St16DOX-silenced plants using LC-MS. 274

This analysis detected seven distinctive peaks that were presumed to be steroidal saponins, 275

which had retention times of 17.44, 17.94, 20.02, 20.56, 23.36, 24.05, and 28.45 min, and 276

gave putative masses of parental ions at m/z 1236.0, 1220.0, 1074.0, 1058.0, 911.8, 895.8, and 277

603.7, respectively (Supplemental Fig. S9). These peaks had a major mass fragment ion at m/z 278

383.6, which corresponds to [dihydroxycholesterol – 2H2O + H+]+, and therefore were 279

assumed to be the glycosides (1-5 saccharides) of dihydroxycholesterol. 280

To determine the aglycone structure of these steroidal glycosides that were 281

accumulated in the St16DOX-silenced plants, we extracted the endogenous metabolites and 282

hydrolyzed them with 1N HCl, and then analyzed the trimethylsilylated metabolites using 283

GC-MS. Compared with non-transgenic plants, the St16DOX-silenced plants had three new 284

peaks in the extracted ion chromatogram targeting the ion m/z 171 (Fig. 7A). One of these 285

peaks had a retention time of 23.8 min, and was identical to the authentic 286

(22S,25S)-22,26-dihydroxycholesterol in terms of both the retention time and the mass 287

spectrum (Fig. 7A and B). The other two peaks, which had retention times of 19.1 min and 288

19.3 min, were identical to the artifacts that occur during the processes of acid hydrolysis and 289

trimethylsilylated derivatization of authentic (22S,25S)-22,26-dihydroxycholesterol (Fig. 7A). 290

These results indicate that St16DOX gene silencing results in the accumulation of the 291

glycosides of (22S,25S)-22,26-dihydroxycholesterol, which was determined to be the best 292

substrate for St16DOX in the in vitro assay. Similarly, Sl16DOX-silenced transgenic tomato 293

plants also accumulated the glycosides of (22S,25S)-22,26-dihydroxycholesterol 294



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(Supplemental Fig. S10). 295

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DISCUSSION 299

16DOX is a dioxygenase that shows steroid 16α-hydroxylase activity in SGA biosynthesis 300

SGA biosynthesis in potato and tomato is thought supposed to require the oxidation 301

of cholestrol at the C-16, C-22, and C-26 positions, followed by transamination and 302

glycosylation (Petersen et al., 1993; Friedman, 2002; Ginzberg et al., 2009). We recently 303

identified two CYPs (PGA1 and PGA2) that are involved in SGA biosynthesis, which 304

catalyze the hydroxylation of cholesterol at C-26 and C-22, respectively (Umemoto et al., 305

2016). In the present study, we investigated the role of a 2OGD gene named 16DOX, which 306

belongs to clade DOXC41. An in vitro functional analysis of the recombinant 16DOX protein 307

confirmed that 16DOX catalyzes hydroxylation of steroids at C-16α (Fig. 4). In addition, 308

16DOX-silenced transgenic plants contained significantly lower amounts of SGAs (Fig. 309

6B-D). These results clearly demonstrate that 16DOX is a steroid 16α-hydroxylase that is 310

involved in SGA biosynthesis in Solanum species. Although many CYPs from various 311

organisms have been characterized as catalyzing the oxidative modification of steroid 312

compounds, this is the first 2OGD to be identified as functioning in the oxidation of steroids. 313

314

Reaction orders during steroidal alkaloid biosynthesis in Solanum and Veratrum species 315

In this study, we found that 16DOX-silenced transgenic plants accumulated the 316

glycosides of dihydroxycholesterol (Supplemental Fig. S8). Similarly, Itkin et al. (2013) also 317

reported that transgenic tomato plants in which GAME11, a gene coding a putative 318

dioxygenase, was silenced by virus-induced gene silencing accumulated dihydroxycholesterol 319

saponins of an unknown structure. This GAME11 corresponds to Sl16DOX and their results 320

were consistent with our observations. In this study, we determined the aglycone structure of 321



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the glycoside product as (22S,25S)-22,26-dihydroxycholesterol (Fig. 7) and, furthermore, 322

functional characterization revealed that this was the best substrate for the recombinant 323

St16DOX (Fig. 5). Therefore, we conclude that 16DOX catalyzes the C-16 hydroxylation of 324

(22S,25S)-22,26-dihydroxycholesterol, which is biosynthesized via the C-22 and C-26 325

hydroxylation of cholesterol by PGA2 and PGA1, respectively. 326

Veratrum species are known to contain the steroidal alkaloid cyclopamine, which 327

exhibits potent pharmacological activities, and Veratrum alkaloids are proposed to be 328

biosynthesized from cholesterol via the predicted intermediates verazine and solanidine 329

(Kaneko et al., 1976, 1977). Therefore, since solanidine is a common precursor for the 330

biosynthesis of SGA as well as Veratrum alkaloids, verazine has also been proposed as a 331

precursor in potato SGA biosynthesis (Freidman, 2006). Recently, Augustin et al. (2015) 332

reported the functional characteristic of the four genes (CYP90B27, CYP94N1, GABAT1, 333

and CYP90G1) that catalyze the first six reactions in the production of verazine from 334

cholesterol in V. californicum. In this pathway, oxidation and transamination at C-26, 335

subsequent oxidation at C-22, and F-ring closure occurred in the absence of the hydroxylation 336

at C-16 (Augustin et al., 2015), and previous phytochemical studies have also supported this 337

reaction order (Kaneko et al., 1970, 1975, 1976, 1977). By contrast, our study demonstrated 338

that 16DOX hydroxylated (22S,25S)-22,26-dihydroxycholesterol at C-16 (Fig. 5) and this was 339

supported by the observation that the glycosides of 22,26-dihydroxycholesterol accumulated 340

in 16DOX-silenced transgenic plants (Supplemental Fig. S9) whereas verazine-type 341

compounds were not detected. These different reaction orders may be explained by the 342

independent origin of the biosynthetic genes in Solanum and Veratrum species (Fig. 8). Potato 343

PGA2 (CYP72A188) and PGA1 (CYP72A208) belong to the CYP72 family, and differ from 344



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Veratrum CYP90B27 and CYP94N1, which catalyze C-22 hydroxylation and two steps of 345

C-26 oxidation to form C-26 aldehyde, which can be transaminated by GABAT1 (Augustin et 346



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al., 2015). The identification of a C-22 oxidase in Solanum species and a C-16 hydroxylase in 347

Veratrum species will provide further support for the different reaction orders and origins of 348

the steroloidal alkaloid pathways in these genera. 349

350

16DOX catalyzes C-16 hydroxylation specifically in the 16α-configuration 351

Feeding with the three labeled cholesterols indicated that the 16α-hydrogen of 352

cholesterol was eliminated but the 16β-hydrogen was retained in tomato SGA biosynthesis, 353

(Fig. 2G-I), which supported the finding that 16DOX catalyzes C-16 hydroxylation 354

specifically in the 16α-configuration. However, the structure of the final product α-tomatine is 355

not consistent with this, as the oxygen atom in the E-ring of α-tomatine is in the 356

16β-configuration and 16α-hydrogen is also present. Curiously, our tracer experiments 357

suggested that the 16α-hydrogen of α-tomatine is derived from the 16β-hydrogen of 358

cholesterol. In the proposed biosynthetic pathway of α-tomatine (Itkin et al., 2013), 359

16,22,26-trihydroxycholesterol is likely re-oxidized at C-22, followed by E-ring closure to 360

form furostanol-type aglycone, in which the oxygen atom is in the 16β-configuration. 361

Therefore, this apparent discrepancy may be explained by the process of E-ring closure, 362

during which the inversion of 16β-hydrogen to the C-16α position occurs, although a 2nd step 363

of C-22 oxidase and a E-ring cyclase that are involved in tomato SGA biosynthesis have not 364

yet been characterized. 365

Our tracer experiments also suggested that both the 16α- and 16β-hydrogen atoms 366

of cholesterol were eliminated during potato SGA biosynthesis while St16DOX calalyzes 367

only 16α-hydroxylation (Fig. 2A-F). These results suggest that loss of 16β-hydrogen atom is 368

catalyzed by an additional unknown C-16 oxidase and that this oxidation step is specifically 369



25

occurred in potato SGA biosynthesis. In addition, 16α-hydrogen was subsequently added to 370

form solanidine in potato SGA biosynthesis (Fig. 2A-F), suggesting that an unknown C-16 371

reductase which introduces 16α-hydrogen may be involved in the final stage of solanidine 372

biosynthesis. Taken together, these findings indicate that further investigation is required to 373

elucidate the mechanism by which the E- and F-rings of spirosolane and solanidane are 374

formed during SGA biosynthesis. 375

376

Co-expression of 16DOX with SGA biosynthetic genes 377

Quantitative RT-PCR analysis in various tissues of potato showed that the 16DOX 378

gene was co-expressed with the PGA1 and PGA2 genes as well as the UGT genes that are 379

responsible for the glycosylation steps in SGA biosynthesis (Fig. 3). Itkin et al. (2013) 380

reported that SGA biosynthetic genes including GAME11 (which corresponds to 16DOX) 381

exist as gene clusters on chromosomes 7 and 12 of tomato and potato. Such physical 382

clustering of functionally related genes may enable the coordinated regulation of gene 383

expression at the chromatin level (Field et al., 2011). Previously, Sl16DOX was reported as 384

the pistil-expressed dioxygenase that has been variously named TPP1, GAD2, SPP2, and 385

H6H-like protein in tomato (Milligan and Gasser, 1995; Jacobsen and Olszewski, 1996; 386

Sylviane, 1999; Nakane, 2003). The findings of these studies were consistent with the present 387

results that Sl16DOX showed high expression levels in flowers (Fig. 3). Recently, Thagun et 388

al. (2016) reported that the overexpression and suppression of jasmonate-responsive ERF 389

(JRE) genes (JRE3, JRE4, and JRE5) in tomato significantly influenced SGA accumulation 390

and expression of the genes encoding the SGA biosynthetic enzymes, including those 391

involved in the upstream mevalonate pathway. JRE4 was found to bind a GCC box-like 392



26

element that is enriched in the promoters of JRE-regulated genes and also controlled the 393

coordinated expression of the SGA biosynthetic genes. Similarly, Cárdenas et al. (2016) 394

revealed that GAME9 (which is identical to JRE4) in tomato and potato regulates the 395

expression of the SGA biosynthetic genes by directly or indirectly acting on the promoters of 396

downstream target genes. Thus, co-expression analysis of the SGA biosynthetic genes that are 397

regulated by JRE4/GAME9 will help to identify novel genes that are involved in the 398

formation of the E- and F-rings of spirosolane and solanidane, including a C-16 oxidase in 399

potato. 400

401

Phenotype of 16DOX-silenced transgenic plants 402

16DOX-silenced transgenic potato plants contained significantly lower amounts of 403

α-solanine and α-chaconine, and the silenced lines grew normally in a greenhouse with 404

comparable yields of tubers to the wild type. Interestingly, 16DOX-silenced transgenic potato 405

plants did not sprout (Supplemental Fig. S8A), but the sprouts could grow by planting the 406

tubers in soil or placing the excised sprout tips on tissue culture media (Supplemental Fig. 407

S8B and C). These phenotypes are similar to those of PGA1- and PGA2-silenced plants, 408

except that latter also exhibited abnormal flowers (Umemoto et al., 2016). By contrast, 409

GAME4/PGA3- or SSR2-silenced transgenic potato plants were phenotypically identical to 410

control plants, despite containing much lower SGA contents (Itkin et al., 2013; Umemoto and 411

Sasaki, 2013; Sawai et al., 2014). These findings demonstrate that the amounts of SGA are 412

unrelated to sprouting morphogenesis, and so 16DOX-silenced plants may accumulate 413

unknown compounds that inhibit sprouting and can be removed by planting the tubers in soil 414

(Umemoto et al., 2016). We found that 16DOX-silenced transgenic plants accumulated 415



27

dihydroxycholesterol and/or its glycosides (Supplemental Fig. S9), and PGA1- and 416

PGA2-silenced plants were also found to accumulate 22- or 26-hydroxycholesterols and/or 417

their glycosides (Umemoto et al., 2016). Therefore, it is possible that the suppression of 418

sprouting morphogenesis is caused by the accumulation of hydroxylated cholesterol or its 419

glycosides in the sprout clusters of the silenced potatoes. 420

Our observations demonstrate that it may be possible to breed SGA-free potato, as 421

reported by Umemoto et al. (2016). 16DOX is a valuable target for the development of a 422

loss-of-function mutant because it occurs as a single copy gene in the genome of potato and 423

tomato. Such a loss-of-function mutant for 16DOX could be obtained thought mutation 424

breeding using targeting induced local lesions in genomes (TILLING; Elias et al., 2009) and 425

genome editing techniques (Sawai et al., 2014; Nicolia et al., 2015). 426

427



28

MATERIALS AND METHODS 428

Chemicals 429

Authentic samples of α-solanine, α-chaconine, α-tomatine, 430

(22R)-22-hydroxycholesterol, and (22S)-22-hydroxycholesterol were purchased from 431

Sigma-Aldrich. Cholesterol was purchased from Tama Biochemical Co. Authentic compounds 432

of (25R)-26-hydroxycholesterol, (25S)-26-hydroxycholesterol, 433

(22S)-16α,22-dihydroxycholesterol, (22S)-16β,22-dihydroxycholesterol, 434

(22S,25S)-22,26-dihydroxycholesterol, (22R,25S)-22-hydroxy-26-oxocholesterol, 435

(22S,25RS)-22-hydroxy-26-oxocholesterol, [15,15,16α,17-2H4]-cholesterol, 436

[15,15,16β,17-2H4]-cholesterol, and [15, 15, 17-2H3]-cholesterol were synthesized as 437

described in the Supplemental Materials and Methods. 438

439

RNA extraction and reverse transcription 440

Total RNA extraction was performed using the RNeasy Plant Mini Kit (QIAGEN, 441

Hilden, Germany) and the RNase-Free DNase Set (QIAGEN). Total RNAs of potato and 442

tomato were prepared from the leaves, flowers, tuber peels, stems, roots, stolons and tuber 443

sprouts of S. tuberosum cv. Sassy, and the leaves, flowers, mature green fruits, yellow fruits, 444

orange fruits, and red fruits of S. lycopersicum cv. Micro-Tom, respectively. The extracted 445

total RNAs of potato and tomato were used to synthesize the first strand cDNAs using the 446

SuperScript® First-Strand Synthesis System for RT-PCR (Life Technologies) and the 447

Transcriptor First Strand cDNA Synthesis Kit (TOYOBO), respectively. 448

449

Cloning of 16DOX cDNAs 450



29

The cDNA fragments that contained the open reading frame of each 16DOX gene 451

were amplified by RT-PCR with primers 1 and 2 for St16DOX, and primers 3 and 4 for 452

Sl16DOX, respectively (Supplemental Table S2), which were designed from the potato and 453

tomato unigene sequences (Sotub07g016570 in the Potato ITAG protein database and 454

Solyc07g043420 in the Tomato ITAG protein database, respectively). The PCR products were 455

cloned into the pENTR/D-TOPO plasmid (Life Technologies). 456

457

Expression of the recombinant 16DOX protein in E. coli 458

The coding sequences for each 16DOX gene were amplified from the 459

pENTR/D-TOPO plasmid using primers 5 and 6 for St16DOX, and primers 7 and 8 for 460

Sl16DOX (Supplemental Table S2), which contained restriction sites. The amplified DNA 461

fragments were ligated into the pMD19 vector (TaKaRa) and digested with BamHI and SalI. 462

The DNA fragments were then ligated into the BamHI-SalI sites of pGEX4T-1. E. coli strain 463

BLR (DE3) (Clontech) transformed with the constructed plasmid was grown at 37°C in 464

lysogeny broth (LB) with 50 μg/ml ampicillin until its OD600 reached 0.5. Recombinant 465

protein expression was induced by adding 0.1 mM isopropyl β-D-1-thiogalactopyranoside 466

(IPTG) and was continued for 20 h at 18°C. The culture was then centrifuged at 3,500 rpm for 467

30 min at 4°C and the cell pellets were resuspended in 5 ml of cold sonication buffer 468

containing 50 mM sodium phosphate (pH 7.4), 300 mM NaCl, and 20% (v/v) glycerol. The 469

solution was then sonicated three times for 30 sec each on ice using a Bandelin Sonopuls HD 470

2070 ultrasonic homogenizer typeMS73 (Sigma) at a sound intensity of 200 W/cm2 and 471

centrifuged at 15,000 rpm for 10 min at 4°C. The GST-tagged proteins present in the 472

supernatant were purified using GST Spin Trap columns (GE Healthcare) according to the 473



30

manufacturer’s instructions. After two column washes, the adsorbed proteins were eluted 474

twice in 200 μl of a solution, containing 50 mM Tris-HCl (pH 8.0), 20 mM reduced 475

glutathione and 20% (v/v) glycerol, and the elution was mixed. The concentration of the 476

purified proteins was determined by the Bradford system. The purified recombinant proteins 477

were visualized by sodium dodecyl sulfate polyacrylamide gel electropholesis (SDS–PAGE). 478

The proteins were revealed by staining the gel with Coomassie brilliant blue R-250 and were 479

then used for further analyzes. 480

481

In vitro enzyme activity assay 482

An in vitro enzyme activity assay was performed using 100 μl of reaction mixture that 483

consisted of 100 mM Bistris-HCl (pH 7.2), 5 mM 2-ketoglutaric acid, 10 mM sodium 484

ascorbate, 0.2 mM FeSO4, 25 μM (22S)-22-hydroxycholesterol as a substrate, and each of the 485

purified recombinant 16DOX proteins of potato and tomato as an enzyme. The reaction was 486

initiated by the addition of the enzyme being tested and was carried out at 30ºC for 3 h. The 487

reaction was then stopped by the addition of 100 μl ethyl acetate, followed by the addition of 488

0.2 μg 25-hydroxycholesterol in 100% ethanol as an internal standard. The reaction products 489

were extracted three times with an equal volume of ethyl acetate, and the organic phase was 490

collected and evaporated. The residue was trimethylsilylated with the TMS-HT Kit (Tokyo 491

Chemical Industry Co., Ltd.) at 80ºC for 30 min. 492

GC-MS analysis of the reaction products was performed as previously described by 493

Seki et al. (2008) with minor modifications. GC-MS was conducted using a GC-MS-QP2010 494

Ultra (Shimadzu) with a DB-5MS (30 m × 0.25 mm, 0.25 μm film thickness; J&W Scientific) 495

capillary column. The injection temperature was 250ºC and the following column temperature 496



31

program was used: 80ºC for 1 min, followed by a rise to 300ºC at a rate of 20ºC/min and a 497

hold at 300ºC for 20 min. The carrier gas was He and the flow rate was 1.0 ml/min. The 498

interface temperature was 300ºC, with a splitless injection. 499

500

Biochemical analysis of recombinant St16DOX 501

To determine the substrate specificity of St16DOX, its activity was assayed using 25 502

μM cholesterol, (22R) and (22S)-22-hydroxycholesterols, 22-oxocholesterol, (25R) and 503

(25S)-26-hydroxycholesterols, (22S,25S)-22,26-dihydroxycholesterol, (22R,25S), and 504

(22S,25RS)-22-hydroxy-26-oxocholesterols. Each reaction was carried out at 30°C for 3 h. 505

Extraction and GC-MS analysis of the reaction product were performed as described above. 506

In addition, the reaction product for (22S,25S)-22,26-dihydroxycholesterol was also analyzed 507

by LC-MS by extracting and evaporating the product as described above, and dissolving the 508

residue in 200 μl ethanol. LC-MS analysis was performed using a system consisting of an 509

ACQUITY UPLC H-Class System (Waters, Milford, MA, USA) and an SQ Detector 2 510

(Waters), and data acquisition and analysis were performed using MassLynx 4.1 software 511

(Waters). Each sample (5 μl) was injected into an ACQUITY UPLC BEH C-18 512

chromatographic column (50 × 2.1 mm, 1.7 μm; Waters), in which the column temperature 513

was set at 40°C and the flow rate was set at 0.2 ml/min. The mobile phases were water with 514

0.1% (v/v) formic acid (A) and acetonitrile (B), using a gradient elution of 10%-90% B at 515

0-30 min, 90%-100% B at 30-40 min and 100% B at 40-46 min (0-30 min and 30-40 min, 516

linear gradient). The mass spectra were obtained in positive electrospray ionization (ESI), 517

with a capillary voltage of 3 kV and a sample cone voltage of 60 V. MS scan mode with a 518

mass range of m/z 250-1400 was used. 519



32

Next, we determined the kinetic parameters of recombinant St16DOX in triplicate 520

assays. The activity was assayed using (22S,25S)-22,26-dihydroxycholesterol at a 521

concentration ranging from 1 to 50 μM. The reaction was carried out at 30°C for 30 min. 522

Extraction and GC-MS analysis of the reaction product were performed as described above. 523

Kinetic parameters were determined by non-linear regression using the ANEMONA program 524

(Hernandez and Ruiz, 1998). 525

526

Generation of transformation vectors, plant transformation, and growth conditions 527

A 370-bp fragment of St16DOX cDNA was PCR amplified using primers 9 and 10 528

(Supplemental Table S2), which contained restriction sites. An RNAi binary vector that 529

targeted the St16DOX gene (pKT258) was constructed from the binary vector pKT11 530

(Umemoto et al., 2001) by locating two 370-bp fragments of St16DOX in opposite directions 531

that interposed the third intron of the Arabidopsis thaliana At4g14210 gene under the control 532

of the cauliflower mosaic virus 35S (CaMV35S) promotor in the T-DNA region as previously 533

described (Umemoto et al., 2016). pKT258 was then electroporated into Agrobacterium 534

tumefaciens GV3110 mp90. Potatoes (S. tuberosum cv. Sassy) were transformed using 535

Agrobacterium GV3110 mp90 cells with pKT258, as previously reported (Monnma, 1990). In 536

vitro-grown plants were cultured at 20°C under a 16 h light/8h dark cycle, following which 41 537

transformants were individually selected by genomic PCR of in vitro-grown shoots using 538

primers 11 and 12 (Supplemental Table S2), which targeted the kanamycin resistance gene in 539

the T-DNA region that was integrated into the potato genome. Quantitative RT-PCR analysis 540

of St16DOX was performed using primers 13 and 14 (Supplemental Table S2) as described in 541

the Real-time quantitative RT-PCR analysis section. Total RNA was then prepared from the 542



33

stems of five independent lines of in vitro-cultured plants: #5, #16, #28, #39, and #41. Primers 543

15 and 16 (Supplemental Table S2), which targeted the potato elongation factor 1α gene 544

(EF1α) (Nicot et al., 2005), were used as a control. Tomato plants (S. lycopersicum cv. 545

Micro-Tom) were also transformed using Agrobacterium GV3110 mp90 cells with pKT258, 546

as previously reported (Sun et al., 2006) 547

548

LC-MS analysis of SGAs in 16DOX-silenced transgenic plants 549

The SGAs that were contained in 16DOX-silenced transgenic plants were extracted 550

and quantified as previously described (Umemoto et al., 2016). LC-MS analysis of the 551

steroidal compounds accumulated in the 16DOX-silenced plants was performed as described 552

in the Biochemical analysis of recombinant St16DOX section with minor modifications: 553

LC-MS was conducted with an ACQUITY UPLC HSS T3 chromatographic column (100 × 554

2.1 mm, 1.8 μm; Waters); water with 0.1% (v/v) formic acid (A) and acetonitrile (B) were 555

used as the mobile phases, using a gradient elution of 10% B at 0-2 min and 10%-55% B at 556

2-32 min (linear gradient); and MS scan mode was used with a mass range of m/z 300-1250. 557

558

GC-MS analysis of steroids in 16DOX-silenced transgenic plants 559

The steroid compounds that accumulated in the in vitro-grown shoots of 560

16DOX-silenced transgenic potato plants and the leaves of 16DOX-silenced transgenic tomato 561

plants were extracted and analyzed using a similar method to that previously described 562

(Ohyama et al., 2013). The extracted residue was trimethylsilylated with 563

N-methyl-N-trimethylsilyltrifluoroacetamide (Sigma-Aldrich) at 80ºC for 30 min. GC-MS 564

analysis was performed as described above with minor modifications: a DB-1MS (30 m × 565



34

0.25 mm, 0.25 μm film thickness; J&W Scientific) capillary column was used. 566

567

Real-time quantitative RT-PCR analysis 568

Quantitative RT-PCR was performed with a LightCycler®Nano (Roche) using 569

THUNDERBIRDTM SYBR® qPCR Mix (TOYOBO) with the following primers sets: 17 570

and 18 for St16DOX, 19 and 20 for PGA1, 21 and 22 for PGA2, 23 and 24 for SGT1, 25 and 571

26 for SGT3 and 15 and 16 for EF1α (Nicot et al., 2005) using the cDNA of various potato 572

tissues as templates; and 17 and 18 for Sl16DOX, 27 and 28 for PGA1-homolog, 29 and 30 573

for PGA2-homolog, 31 and 32 for GAME1, and 33 and 34 for ubiquitin using the cDNA of 574

various tomato tissues as templates (Supplemental Table S2). Cycling was carried out at 95˚C 575

for 10 min, 45 cycles at 95°C for 10 sec, 60°C for 10 sec, and 72°C for 15 sec for 576

amplification, followed by holding at 95°C for 30 sec and ramping up from 60°C to 95°C at 577

0.1°C/sec to perform a melting curve analysis. Three biological replicates were analyzed in 578

duplicate. The gene expression levels were normalized against the values obtained for the 579

EF1α and ubiquitin genes, which were used as an internal reference in potato and tomato, 580

respectively. Data acquisition and analysis were performed using LightCycler®Nano software 581

(Roche). 582

583

Tracer experiments using stable isotope-labeled compounds 584

Tracer experiments were performed according to a previously reported method 585

(Ohyama et al., 2013) with minor modifications: each stable isotope-labeled compound (1.0 586

mg) was dissolved in acetone (25 μl)–Tween 80 (25 μl). In vitro-grown potato (S. tuberosum 587

cv. Sassy) shoots and tomato (S. lycopersicum cv. Micro-Tom) seedlings were prepared for 588



35

tracer experiments and fed with the stable isotope-labeled compounds 589

[15,15,16α,17-2H4]cholesterol, [15,15,16β,17-2H4]cholesterol, and [15,15,17-2H3]cholesterol. 590

After 7 days, the SGAs that accumulated in the harvested potato shoots and tomato seedlings 591

were extracted and analyzed by LC-MS as described above. 592

593

Accession Numbers 594

St16DOX, LC222743; Sl16DOX, LC222744 595

596

Supplemental Data 597

Supplemental Fig. S1. Quantitative RT-PCR analysis of the expression pattern of SGA 598

biosynthetic genes in various organs of tomato. 599

Supplemental Fig. S2. Amino acid sequence alignment of St16DOX, Sl16DOX, and 600

hyoscyamine 6β-hydroxylase from Hyoscyamus niger (HnH6H). 601

Supplemental Fig. S3. GC-MS analysis of the reaction products obtained from the 602

recombinant St16DOX protein with (22S,25S)-22,26-dihydroxycholesterol as a substrate. 603

Supplemental Fig. S4. LC-MS analysis of the reaction products from the recombinant 604

St16DOX protein with (22S,25S)-22,26-dihydroxycholesterol as a substrate. 605

Supplemental Fig. S5. GC-MS analysis of the reaction products from the recombinant 606

16DOX proteins with (22R)-22-hydroxycholesterol as a substrate. 607

Supplemental Fig. S6. Kinetic analysis of the recombinant St16DOX with (22S,25S)-22, 608

26-dihydroxycholesterol. 609

Supplemental Fig. S7. LC-MS analysis of α-tomatine levels in the leaves of 610

Sl16DOX-silenced transgenic tomato plants. 611



36

Supplemental Fig. S8. Phenotypes of St16DOX-silenced transgenic potato plants. 612

Supplemental Fig. S9. LC-MS analysis of the accumulated compounds in the leaves of 613

St16DOX-silenced transgenic potato plants. 614

Supplemental Fig. S10. GC-MS analysis of the accumulated compounds in leaves of 615

Sl16DOX-silenced transgenic tomato plants. 616

Supplemental Table S1. The list of a total of 256 2OGD transcripts extracted from Spud DB 617

potato genomics resource. 618

Supplemental Table S2. The oligonucleotides that were used in the current study. 619

620

621

The following materials are available in the online version of this article. 622

Supplemental Fig. S1. Quantitative RT-PCR analysis of the expression pattern of SGA 623

biosynthetic genes in various organs of tomato. Transcript levels of the SGA biosynthetic 624

genes are shown relative to that of the ubiquitin gene, which was used as an internal reference. 625

Bars indicate one standard deviation from the mean (n = 3). 626

627

Supplemental Fig. S2. Amino acid sequence alignment of St16DOX, Sl16DOX, and 628

hyoscyamine 6β-hydroxylase from Hyoscyamus niger (HnH6H). Multiple sequence 629

alignment was performed using the ClustalW multiple alignment analysis tool of BioEdit. 630

Identical and similar amino acid residues are shaded in black and gray, respectively. 631

Conserved Fe(II)-binding motifs among the 2OGD superfamily are indicated with solid 632

arrows. Conserved motifs binding to the C-5 carboxy group of 2-oxoglutarate in the 2OGD 633

superfamily are indicated with dashed arrows. 634



37

635

Supplemental Fig. S3. GC-MS analysis of the reaction products obtained from the 636

recombinant St16DOX protein with (22S,25S)-22,26-dihydroxycholesterol as a substrate. (A) 637

The extracted ion chromatograms targeting ion m/z 99 of the reaction products. (B) Mass 638

spectra of the substrate and the product peaks from St16DOX shown in (A) with retention 639

times of 23.8 min and 25.8 min, respectively. 640

641

Supplemental Fig. S4. LC-MS analysis of the reaction products from the recombinant 642

St16DOX protein with (22S,25S)-22,26-dihydroxycholesterol as a substrate. (A) Total ion 643

chromatograms of the reaction products. (B) Mass spectra of the substrate and the product 644

peaks from St16DOX shown in (A) with retention times of 18.16 min and 15.16 min, 645

respectively. 646

647

Supplemental Fig. S5. GC-MS analysis of the reaction products from the recombinant 648

16DOX proteins with (22R)-22-hydroxycholesterol as a substrate. (A) The extracted ion 649

chromatograms targeting ion m/z 171 of the reaction products. (B) Mass spectrum of the 650

product peak from St16DOX shown in (A) with a retention time of 21.8 min. 651

652

Supplemental Fig. S6. Kinetic analysis of the recombinant St16DOX with (22S,25S)-22, 653

26-dihydroxycholesterol. Enzyme activities were measured with substrate concentrations up 654

to 50 μM (22S,25S)-22,26-dihydroxycholesterol. A Michaelis–Menten curve (featuring a Km 655

value of 4.19 μM) was fitted to the values obtained. Kinetic parameters were determined by 656

non-linear regression with ANEMONA (Hernandez and Ruiz, 1998). 657



38

658

Supplemental Fig. S7. LC-MS analysis of α-tomatine levels in the leaves of 659

Sl16DOX-silenced transgenic tomato plants. Bars indicate one standard deviation from the 660

mean (n = 3). FW, fresh weight; NT, non-transgenic control line; #9, #17, #29, and #32, 661

independent transgenic lines. 662

663

Supplemental Fig. S8. Phenotypes of St16DOX-silenced transgenic potato plants. (A) 664

Sprouted St16DOX-silenced (#28 and #41, left and middle) and control (right) potato plants 665

18 weeks after the cessation of plant dormancy in the control. (B) Sprouted 666

St16DOX-silenced potato (#28) planted into soil. (C) In vitro growth of the sprout tips on 667

tissue culture media-sprout tips were cut from tubers of the St16DOX-silenced plant (#28) and 668

placed on tissue culture media without plant hormones. (D) Flowers of an St16DOX-silenced 669

plant (#16). 670

671

Supplemental Fig. S9. LC-MS analysis of the accumulated compounds in the leaves of 672

St16DOX-silenced transgenic potato plants. (A) Total ion chromatogram of the accumulated 673

compounds. (B) Mass spectra of the peaks shown in (A) with retention times of 17.44 min, 674

17.94 min, 20.02 min, 20.56 min, 23.36 min, 24.05 min, and 28.45 min. 675

676

Supplemental Fig. S10. GC-MS analysis of the accumulated compounds in leaves of 677

Sl16DOX-silenced transgenic tomato plants. (A) The extracted ion chromatograms targeting 678

ion m/z 171 of the accumulated compounds and the authentic compound. (B) Mass spectra of 679

the peaks shown in (A) with a retention time of 23.8 min. NT, Non-transgenic control line; 680



39

#17, a transgenic line. 681

682

Supplemental Table S1. The list of a total of 256 2OGD transcripts extracted from Spud DB 683

potato genomics resource. 684

685

Supplemental Table S2. The oligonucleotides that were used in the current study. 686

687

ACKNOWLEDGMENTS 688

We thank Masako Otsuka, Noriko Yasuno and Ryo Negoya for technical assistance. Parts of 689

the experimental measurements were carried out using the Bruker and JEOL 600 MHz NMR 690

spectrometers, as well as the JEOL JMS-700 mass spectrometer in the Joint Usage/Research 691

Center (JURC) at the Institute for Chemical Research, Kyoto University. The synthetic 692

research was supported by 2nd Seiken Academic Incentive Fund provided by Research 693

Instituted for Production Development, Kyoto, Japan. 694

695

Figure legends 696

Fig. 1. The putative biosynthetic pathway for SGAs in potato and tomato. Thick solid 697

arrow indicates the reaction step characterized in this work. Thin solid arrows indicate the 698

reaction steps reported by Umemoto et al. (2016). White arrows represent multiple reaction 699

stages. 700

701

Fig. 2. LC-MS analysis of SGAs upon feeding stable isotope-labeled compounds to in 702

vitro-grown potato shoots and tomato seedlings. The two traces on the left are 703



2

mass-chromatograms for the indicated m/z ions. The right mass spectrum was obtained at the 704

retention time indicated by the arrow. Plants were fed with [15,15,17α-2H3]cholesterol (A-C), 705

[15,15,16α, 17α-2H4]cholesterol (D-F), or [15,15,16β,17α-2H4]cholesterol (G-I). (A, D, and 706

G) LC-MS analysis of α-chaconine in potato shoots. (B, E, and H) LC-MS analysis of 707

α-solanine in potato shoots. (C, F, and I) LC-MS analysis of α-tomatine in tomato seedlings. 708

709

Fig. 3. Quantitative RT-PCR analysis of the expression patterns of SGA biosynthetic 710

genes in various organs of potato plants. Transcript levels of SGA biosynthetic genes are 711

shown relative to that of EF1α as an internal reference gene. Bars indicate one standard 712

deviation from the mean (n = 3). 713

714

Fig. 4. GC-MS analysis of the reaction products from the recombinant St16DOX and 715

Sl16DOX proteins with (22S)-22-hydroxycholesterol as a substrate. (A) The extracted ion 716

chromatogram targeting ion m/z 173 of the reaction products and the authentic compounds. 717

(B) Mass spectrum of each peak shown in (A) at a retention time of 20.0 min. 718

719

Fig. 5. Relative activities of recombinant St16DOX toward cholesterol and several 720

oxygenated cholesterols. N.D. indicates not detected. 721

722

Fig. 6. SGA contents and yields of tubers from St16DOX-silenced transgenic potato 723

plants. (A) Quantification RT-PCR analysis of St16DOX transcript levels in the in 724

vitro-grown shoots of St16DOX-silenced plants. (B) LC-MS analysis of SGA levels in the in 725

vitro-grown shoots of St16DOX-silenced plants. (C and D) LC-MS analysis of SGA levels in 726



3

the peel (C) and cortex (D) of harvested tubers from St16DOX-silenced plants. (E) Yields of 727

tubers from St16DOX-silenced plants. Bars indicate one standard deviation from the mean (n 728

= 3). FW, fresh weight; NT, non-transgenic control plants; #15, #16, #28, #39, and #41, 729

independent transgenic lines. 730

731

Fig. 7. GC-MS analysis of the accumulated compounds in leaves of St16DOX-silenced 732

transgenic potato plants. (A) The extracted ion chromatogram targeting ion m/z 171 of the 733

accumulated compounds and the authentic compound. (B) Mass spectrum of each peak shown 734

in (A) at a retention time of 23.8 min. NT, non-transgenic control line; #15, a transgenic line. 735

736

Fig. 8. The putative reaction orders in the biosynthetic pathways of steroidal alkaloids in 737

Solanum and Veratrum species. Thick arrows indicate the reaction steps that were 738

characterized in this work. Black-filled arrow indicates the multiple reaction stages reported 739

by Umemoto et al. (2016) and Augustin et al. (2015). White arrows represent unclear multiple 740

reaction stages. 741

742

743



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Bugg TDH (2003) Dioxygenase enzymes: catalytic mechanisms and chemical models. Tetrahedron 59: 7075-7101Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Canonica L, Ronchetti F, Russo G (1977) Fate of the 16ß-hydrogen atom of cholesterol in the biosynthesis of tomatidine andsolanidine. J Chem Soc Chem Commun: 286-287


Cárdenas PD, Sonawane PD, Pollier J, Vanden Bossche R, Dewangan V, Weithorn E, Tal L, Meir S, Rogachev I, Malitsky S, et al (2016)GAME9 regulates the biosynthesis of steroidal alkaloids and upstream isoprenoids in the plant mevalonate pathway. Nat Commun 7:10654


Elias R, Till BJ, Mba C, Al-Safadi B (2009) Optimizing TILLING and Ecotilling techniques for potato (Solanum tuberosum L). BMC ResNotes 2: 141


Field B, Fiston-Lavier AS, Kemen A, Geisler K, Quesneville H, Osbourn AE (2011) Formation of plant metabolic gene clusters withindynamic chromosomal regions. Proc Natl Acad Sci USA 108: 16116-16121


Friedman M (2002) Tomato Glycoalkaloids: Role in the Plant and in the Diet. J Agric Food Chem 50: 5751-5780Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Friedman M (2006) Potato glycoalkaloids and metabolites: roles in the plant and in the diet. J Agric Food Chem 54: 8655-8681Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Friedman M, Dao L (1992) Distribution of glycoalkaloids in potato plants and commercial potato products. J. Agric. Food Chem 40: 419-423


Ginzberg I, Tokuhisa J, Veilleux R (2009) Potato Steroidal Glycoalkaloids: Biosynthesis and Genetic Manipulation. Potato Res 52: 1-15Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Harrison DM (1990) Steroidal alkaloids. Nat Prod Rep 7: 139-147Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Helmut R (1998) Solanum steroid alkaloids - an update. Alkaloids: Chem Biol Perspect 12: 103-185Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Hernandez A, Ruiz MT (1998) An EXCEL template for caluculation of enzyme kinetics parameters by non-linear regression.Bioinformatics 14: 227-228



http://www.ncbi.nlm.nih.gov/pubmed?term=Augustin MM%2C Ruzicka DR%2C Shukla AK%2C Augustin JM%2C Starks CM%2C O%27Neil%2DJohnson M%2C McKain MR%2C Evans BS%2C Barrett MD%2C Smithson A%2C et al %282015%29 Elucidating steroid alkaloid biosynthesis in Veratrum californicum%3A production of verazine in Sf9 cells%2E Plant J 82%3A 991%2D1003&dopt=abstract

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http://www.ncbi.nlm.nih.gov/pubmed?term=Bugg TDH %282003%29 Dioxygenase enzymes%3A catalytic mechanisms and chemical models%2E Tetrahedron 59%3A 7075%2D7101&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Bugg TDH (2003) Dioxygenase enzymes: catalytic mechanisms and chemical models. Tetrahedron 59: 7075-7101

http://scholar.google.com/scholar?as_q=&num=10&as_occt=any&as_sauthors=Bugg&hl=en&c2coff=1

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http://www.ncbi.nlm.nih.gov/pubmed?term=Canonica L%2C Ronchetti F%2C Russo G %281977%29 Fate of the 16�%2Dhydrogen atom of cholesterol in the biosynthesis of tomatidine and solanidine%2E J Chem Soc Chem Commun%3A 286%2D287&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Canonica L, Ronchetti F, Russo G (1977) Fate of the 16�-hydrogen atom of cholesterol in the biosynthesis of tomatidine and solanidine. J Chem Soc Chem Commun: 286-287

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http://www.ncbi.nlm.nih.gov/pubmed?term=Field B%2C Fiston%2DLavier AS%2C Kemen A%2C Geisler K%2C Quesneville H%2C Osbourn AE %282011%29 Formation of plant metabolic gene clusters within dynamic chromosomal regions%2E Proc Natl Acad Sci USA 108%3A 16116%2D16121&dopt=abstract

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http://www.ncbi.nlm.nih.gov/pubmed?term=Friedman M %282002%29 Tomato Glycoalkaloids%3A Role in the Plant and in the Diet%2E J Agric Food Chem 50%3A 5751%2D5780&dopt=abstract

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http://www.ncbi.nlm.nih.gov/pubmed?term=Itkin M%2C Heinig U%2C Tzfadia O%2C Bhide AJ%2C Shinde B%2C Cardenas PD%2C Bocobza SE%2C Unger T%2C Malitsky S%2C Finkers R%2C et al %282013%29 Biosynthesis of antinutritional alkaloids in solanaceous crops is mediated by clustered genes%2E Science 341%3A 175%2D179&dopt=abstract

http://search.crossref.org/?page=1&rows=2&q=Itkin M, Heinig U, Tzfadia O, Bhide AJ, Shinde B, Cardenas PD, Bocobza SE, Unger T, Malitsky S, Finkers R, et al (2013) Biosynthesis of antinutritional alkaloids in solanaceous crops is mediated by clustered genes. Science 341: 175-179

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http://search.crossref.org/?page=1&rows=2&q=Jacobsen SE, Olszewski NE (1996) Gibberellins regulate the abundance of RNAs with sequence similarity to proteinase inhibitors, dioxygenases and dehydrogenases. Planta 198: 78-86

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Moehs CP, Allen PV, Friedman M, Belknap WR (1997) Cloning and expression of solanidine UDP-glucose glucosyltransferase frompotato. Plant J 11: 227-236


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http://search.crossref.org/?page=1&rows=2&q=McCue KF, Shepherd LVT, Allen PV, Maccree MM, Rockhold DR, Corsini DL, Davies HV, Belknap WR (2005) Metabolic compensation of steroidal glycoalkaloid biosynthesis in transgenic potato tubers: using reverse genetics to confirm the in vivo enzyme function of a steroidal alkaloid galactosyltransferase. Plant Sci 168: 267-273


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Ohyama K, Okawa A, Moriuchi Y, Fujimoto Y (2013) Biosynthesis of steroidal alkaloids in Solanaceae plants: involvement of analdehyde intermediate during C-26 amination. Phytochemistry 89: 26-31


Petersen HW, Mølgaard P, Nyman U, Olesen CE (1993) Chemotaxonomy of the tuber-bearing Solanum species, subsection Potatoe(Solanaceae). Biochem Syst Ecol 21: 629-644


Roddick JG (1989) The acetylcholinesterase-inhibitory activity of steroidal glycoalkaloids and their aglycones. Phytochemistry 28: 2631-2634


Sawai S, Ohyama K, Yasumoto S, Seki H, Sakuma T, Yamamoto T, Takebayashi Y, Kojima M, Sakakibara H, Aoki T, et al (2014) SterolSide Chain Reductase 2 Is a Key Enzyme in the Biosynthesis of Cholesterol, the Common Precursor of Toxic Steroidal Glycoalkaloidsin Potato. Plant Cell 26: 3763-3774


Seki H, Ohyama K, Sawai S, Mizutani M, Ohnishi T, Sudo H, Akashi T, Aoki T, Saito K, Muranaka T (2008) Licorice ß-amyrin 11-oxidase, acytochrome P450 with a key role in the biosynthesis of the triterpene sweetener glycyrrhizin. Proc Natl Acad Sci USA 105: 14204-14209


Seki H, Tamura K, Muranaka T (2015) P450s and UGTs: Key Players in the Structural Diversity of Triterpenoid Saponins. Plant CellPhysiol 56: 1463-1471


Smith DB, Roddick JG, Jones JL (1996) Potato glycoalkaloids: some unanswered questions. Trends in food science & technology 7:126-131


Sun HJ, Uchii S, Watanabe S, Ezura H (2006) A highly efficient transformation protocol for Micro-Tom, a model cultivar for tomatofunctional genomics. Plant Cell Physiol 47: 426-431


Thagun C, Imanishi S, Kudo T, Nakabayashi R, Ohyama K, Mori T, Kawamoto K, Nakamura Y, Katayama M, Nonaka S, et al (2016)Jasmonate-Responsive ERF Transcription Factors Regulate Steroidal Glycoalkaloid Biosynthesis in Tomato. Plant Cell Physiol 57: 961-975


Umemoto N, Nakayasu M, Ohyama K, Yotsu-Yamashita M, Mizutani M, Seki H, Saito K, Muranaka T (2016) Two Cytochrome P450Monooxygenases Catalyze Early Hydroxylation Steps in the Potato Steroid Glycoalkaloid Biosynthetic Pathway. Plant Physiol 171: 2458-2467


Umemoto N, Sasaki K (2013). Protein having glycoalkaloid biosynthetic enzyme activity and gene encoding the same. US PatentApplication No. 20130167271 A1


Umemoto N, Tsukahara M, Yoshioka M (2001) JP Patent Publication (Kokai) 2001-161373 Ahttps://plantphysiol.orgDownloaded on April 26, 2021. - Published by

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http://www.ncbi.nlm.nih.gov/pubmed?term=Petersen HW%2C M�lgaard P%2C Nyman U%2C Olesen CE %281993%29 Chemotaxonomy of the tuber%2Dbearing Solanum species%2C subsection Potatoe %28Solanaceae%29%2E Biochem Syst Ecol 21%3A 629%2D644&dopt=abstract

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http://www.ncbi.nlm.nih.gov/pubmed?term=Roddick JG %281989%29 The acetylcholinesterase%2Dinhibitory activity of steroidal glycoalkaloids and their aglycones%2E Phytochemistry 28%3A 2631%2D2634&dopt=abstract

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http://www.ncbi.nlm.nih.gov/pubmed?term=Sawai S%2C Ohyama K%2C Yasumoto S%2C Seki H%2C Sakuma T%2C Yamamoto T%2C Takebayashi Y%2C Kojima M%2C Sakakibara H%2C Aoki T%2C et al %282014%29 Sterol Side Chain Reductase 2 Is a Key Enzyme in the Biosynthesis of Cholesterol%2C the Common Precursor of Toxic Steroidal Glycoalkaloids in Potato%2E Plant Cell 26%3A 3763%2D3774&dopt=abstract

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http://www.ncbi.nlm.nih.gov/pubmed?term=Seki H%2C Ohyama K%2C Sawai S%2C Mizutani M%2C Ohnishi T%2C Sudo H%2C Akashi T%2C Aoki T%2C Saito K%2C Muranaka T %282008%29 Licorice �%2Damyrin 11%2Doxidase%2C a cytochrome P450 with a key role in the biosynthesis of the triterpene sweetener glycyrrhizin%2E Proc Natl Acad Sci USA 105%3A 14204%2D14209&dopt=abstract

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http://www.ncbi.nlm.nih.gov/pubmed?term=Smith DB%2C Roddick JG%2C Jones JL %281996%29 Potato glycoalkaloids%3A some unanswered questions%2E Trends in food science %26 technology 7%3A 126%2D131&dopt=abstract

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http://www.ncbi.nlm.nih.gov/pubmed?term=Thagun C%2C Imanishi S%2C Kudo T%2C Nakabayashi R%2C Ohyama K%2C Mori T%2C Kawamoto K%2C Nakamura Y%2C Katayama M%2C Nonaka S%2C et al %282016%29 Jasmonate%2DResponsive ERF Transcription Factors Regulate Steroidal Glycoalkaloid Biosynthesis in Tomato%2E Plant Cell Physiol 57%3A 961%2D975&dopt=abstract

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http://www.ncbi.nlm.nih.gov/pubmed?term=Umemoto N%2C Nakayasu M%2C Ohyama K%2C Yotsu%2DYamashita M%2C Mizutani M%2C Seki H%2C Saito K%2C Muranaka T %282016%29 Two Cytochrome P450 Monooxygenases Catalyze Early Hydroxylation Steps in the Potato Steroid Glycoalkaloid Biosynthetic Pathway%2E Plant Physiol 171%3A 2458%2D2467&dopt=abstract

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Varma KR, Wickramasinghe JAF, Caspi E (1969) Biosynthesis of Biosynthesis of plant sterols. IX. The mode of oxygenation at carbonatom 26 in the formation of sapogenins from cholesterol. J Biol Chem 244: 3951-3957


Wilmouth RC, Turnbull JJ, Welford RWD, Clifton IJ, Prescott AG, Schofield CJ (2002) Structure and mechanism of anthocyanidinsynthase from Arabidopsis thaliana. Structure 10: 93-103



http://www.ncbi.nlm.nih.gov/pubmed?term=Varma KR%2C Wickramasinghe JAF%2C Caspi E %281969%29 Biosynthesis of Biosynthesis of plant sterols%2E IX%2E The mode of oxygenation at carbon atom 26 in the formation of sapogenins from cholesterol%2E J Biol Chem 244%3A 3951%2D3957&dopt=abstract

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A Dioxygenase Catalyzes Steroid 16α-Hydroxylation in ......2017/07/28 · 10 Article Title: A...

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