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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
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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
217
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
243
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
256
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
271
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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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298
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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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23
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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24
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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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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