Hydroxylation of Geranylhydroquinone in Shikonin

Plant Physiology Preview. Published on November 29, 2018, as DOI:10.1104/pp.18.01056

2 CYP76B74 catalyzes the 3´´-hydroxylation of geranylhydroquinone

1 3 in shikonin biosynthesis

6 a a a a a 7 Sheng Wang , Ruishan Wang , Tan Liu , Chaogeng Lv , Jiuwen Liang , a a a a a 8 Chuanzhi Kang , Liangyun Zhou , Juan Guo , Guanghong Cui , Yan Zhang , b a,2 a,2 9 Daniele Werck-Reichhart , Lanping Guo , Luqi Huang

10 a 11 State Key Laboratory Breeding Base of Dao-di Herbs, National Resources

12 Center of Chinese Materia Medica, China Academy of Chinese Medical

13 Sciences, 100700, Beijing, China b 14 Institute of Plant Molecular Biology, Centre National de la Recherche

15 Scientifique (CNRS), University of Strasbourg, 67084, Strasbourg, France

17 Short title:

18 A key enzyme in shikonin biosynthesis

20 One sentence summary: CYP76B74 encodes geranylhydroquinone

21 3´´-hydroxylase involved in the shikonin biosynthetic pathway and is necessary

22 for the accumulation of shikonin derivatives in Arnebia euchroma. 23

Downloaded from on November 29, 2018 - Published by www.plantphysiol.org Copyright © 2018 American Society of Plant Biologists. All rights reserved. Copyright 2018 by the American Society of Plant Biologists 24 Footnotes: 1 25 This work was supported by National Natural Science Foundation of China

26 (No.81473307, No.81703648, No.81603239), the National Key Research and

27 Development Program of China (2017YFC1700701 and 2017YFC1701405)

28 and the Fundamental Research Funds for the Central Public Welfare

29 Research Institutes (ZZXT201701).

30 2 31 To whom correspondence should be addressed: China Academy of Chinese

32 Medical Sciences, 100700, No.16 Nanxiaojienei, Dongzhimen, Dongcheng

33 District, Beijing, China; Tel: +86-10-8404-4340; Fax: +86-10-8402-7175;

34 E-mail: [email protected], or Tel: +86-10-8404-4340; Fax:

35 +86-10-8402-7175; E-mail: [email protected], Huang Luqi and Guo Lanping

36 contributed equally as corresponding authors.

38 The author responsible for distribution of materials integral to the findings

39 presented in this article in accordance with the policy described in the

40 Instructions for Authors (www.plantphysiol.org) is: Huang Luqi

41 ([email protected]).

43 G.L. and H.L. conceived the project; G.J., W.R.D., G.L. and H.L. supervised

44 the project; W.S., W.R., L.T., L.C., L.J., K.C. and Z.L. conducted experiments;

45 W.S., W.R., L.T., L.C., L.J., K.C., Z.L., G.J., C.J., W.R.D., G.L. and H.L.

46 analyzed the data; W.S., W.R., L.T., L.C., L.J., K.C., Z.L., G.J., C.J., G.L. and

47 H.L. wrote the article; all authors read and approved the final manuscript.

48 49 50 51 52

54 Shikonin and its derivatives are the most abundant naphthoquinone pigments

55 formed in species of the medicinally and economically valuable Boraginaceae.

56 A key step in the shikonin biosynthetic pathway, namely the C-3´´

57 hydroxylation of the prenylated phenolic intermediate geranylhydroquinone, is

58 expected to be catalyzed by a cytochrome P450. To find identify cytochrome

59 P450 candidates with transcription profiles similar to those of genes known to

60 be involved in shikonin biosynthesis, we carried out co-expression analysis of

61 transcriptome datasets of shikonin-proficient and shikonin-deficient cell lines

62 and examined the spatial expression of candidate genes in different organs of

63 Arnebia euchroma. In biochemical assays using geranylhydroquinone as the

64 substrate, CYP76B74 exhibited geranylhydroquinone 3´´-hydroxylase activity

65 and produced 3´´-hydroxy-geranylhydroquinone. In CYP76B74 RNAi A.

66 euchroma hairy roots, shikonin derivative accumulation decreased

67 dramatically, which demonstrated that CYP76B74 is required for shikonin

68 biosynthesis in the plant. Phylogenetic analysis confirmed that CYP76B74

69 belonged to the CYP76B subfamily and was most likely derived from an

70 ancestral geraniol 10-hydroxylase. In a subcellular localization analysis, a

71 GFP-CYP76B74 fusion localized to endoplasmic reticulum membranes. Our

72 results demonstrate that CYP76B74 catalyzes the key hydroxylation step in

73 shikonin biosynthesis with high efficiency. The characterization of the

74 CYP76B74 described here paves the way for further exploration of the ring

75 closure reactions generating the naphthoquinone skeleton, as well as for the

76 alternative metabolism of geranylhydroquinone 3´´-hydroxylase to

77 dihydroechinofuran. 78

80 Arnebia euchroma (Royle) Johnst, a perennial herbaceous boraginaceaeous

81 plant, is found in Pamirs, Tian-Shan Mountains, Himalayas, and western Tibet

82 at an altitudinal range of 3,700–4,200 m above sea level (Manjkhola et al.,

83 2005). A. euchroma is an important commodity in food, cosmetics, and modern

84 pharmaceutical industries, mostly due to its high content of red

85 naphthoquinone pigments (shikonin derivatives) in the root bark (cork layers)

86 (Papageorgiou et al., 1999; Yazaki, 2017). Recent studies have demonstrated

87 that shikonin derivatives exhibit diverse biological activities, such as

88 antioxidant, antibacterial and anticancer activities, which endow them with a

89 high potential for use in drug development (Papageorgiou et al., 2006; Andujar

90 et al., 2013).

91 The naphthoquinone derivatives are a group of prevalent natural products that

92 include juglone, plumbagin, shikonin, lapachol, vitamin K1, vitamin K2, and

93 vitamin K3 et. al. (Kumagai et al., 2012). Most of their naphthoquinone

94 skeletons are proposed to be generated either by the acetate-malonate

95 pathway involving polyketide synthases, as in the case of fusarubin from

96 Fusarium fujikuroi (Studt et al., 2012), or the phenylalanine pathway as for

97 menaquinone biosynthesis (Meganathan and Bentley, 1979). Shikonins,

98 however, are a special sub-group synthesized via the 4-hydroxybenzoic acid

99 (4-HBA)/geranyl diphosphate (GPP) pathway with subsequent ring closure

100 reactions (Figure 1). This special biosynthetic pathway of shikonins is an

101 expanding understanding of biosynthetic pathways of naphthoquinone

102 derivatives in nature, especially in plants. Confirmation of this route for the

103 formation of the naphthoquinone skeleton would delineate a new metabolic

104 pathway of naphthoquinone derivatives. For these reasons, in vitro

105 biochemical assays and reverse genetics have been employed in the study of

106 shikonin biosynthesis and its regulation (Widhalm and Rhodes, 2016; Yazaki,

107 2017).

108 The first committed step of the shikonin biosynthetic pathway is the

109 condensation of 4-HBA and GPP catalyzed by 4-hydroxybenzoate

110 geranyltransferase (also named p-hydroxybenzoate geranyltransferase, PGT

Downloaded from on November 29, 2018 - Published by www.plantphysiol.org Copyright © 2018 American Society of Plant Biologists. All rights reserved. 111 for short) (Yazaki et al., 2002). After the PGT-catalyzed reaction, little is known

112 about the ring closure reactions that generate the naphthoquinone skeleton. It

Downloaded from on November 29, 2018 - Published by www.plantphysiol.org Copyright © 2018 American Society of Plant Biologists. All rights reserved. 113 is generally accepted that the PGT product 3-geranyl-4-hydroxybenzolic acid

114 (GBA) undergoes decarboxylation and hydroxylation of the C-1 position,

115 leading to the generation of geranylhydroquinone (GHQ). After that,

116 hydroxylation of C-3´´ of the isoprenoid side chain of GHQ provides the key

117 intermediate 3´´-hydroxy-geranylhydroquinone (GHQ-3´´-OH) for cyclization to

118 form quinones. The enzyme catalyzes this reaction, geranylhydroquinone

119 3´´-hydroxylase (GHQ3´´H), was once partially purified from the microsomal

120 fraction of non-pigmented L. erythrorhizon suspension cultures and shown to

121 require NADPH and molecular oxygen, suggesting it is a cytochrome P450

122 (CYP)-dependent monooxygenase (Yamamoto et al., 2000). Until now, the

123 GHQ3´´H has never been characterized at the molecular level. The identity,

124 regulation, and phylogeny of the gene encoding this protein remain to be

125 understood.

126 Previously, we established two types of cultured cells from A. euchroma

127 hypocotyls, a red shikonin-proficient cell line and a white shikonin-deficient cell

128 line, which responded differently to the elicitation by methyl jasmonate.

129 Specialized metabolite contents (shikonins, shikofurans and phenolic acids)

130 and the expression levels of key enzymatic genes involved in their

131 biosynthesis changed differentially for the two cell lines under elicitation (Wang

132 et al., 2014). Herein, we performed transcriptome analysis of the two cell lines,

133 combined with in vitro biochemical assays, subcellular localization analysis,

134 and RNA interference to characterize a candidate cytochrome P450 gene,

135 CYP76B74. Our results indicate that CYP76B74 catalyzes the key

136 3´´-hydroxylation step in the shikonin biosynthetic pathway with high efficiency.

137 The characterization of GHQ3´´H paves the way for further exploration of the

138 ring closure reactions generating the naphthoquinone skeleton in

139 Boraginaceae, and for the alternative metabolization of GHQ-3´´-OH to

140 dihydroechinofuran.

141 142

144 Discovery of CYP candidate genes

145 The differentially expressed transcripts of shikonin-proficient (SP) and

146 shikonin-deficient (SD) A. euchroma suspension culture cell lines were mined

147 in order to discover possible enzymes involved in shikonin biosynthesis, with a

148 main focus on identifying the GHQ3´´H. Two cDNA libraries were prepared

149 from the SP and SD cell lines, and then were sequenced on an Illumina

150 HiSeq™ 2000 platform to obtain an overview of genes associated with the

151 shikonin biosynthetic pathway. After raw data filtration and de novo assembly,

152 103,294 unigenes with N50 length (median length of all non-redundant

153 sequences) of 1110 bp were generated in the SP library; and 100,627

154 unigenes with N50 length of 1267 bp were generated in the SD library. From

155 these populations, a total of 121,239 unigenes with N50 length of 1565 bp

156 were sequenced and bioinformatically processed. The functional annotation of

157 all assembled unigenes was cataloged using six public databases (i.e., NR,

158 NT, SwissProt, KEGG, COG, GO). As a result, a total of 78,947 unigenes

159 (65.12%) in the SP and SD libraries were annotated in at least one of the

160 above databases.

161 Based on their annotation, a series of unigenes were found to be related to

162 shikonin biosynthesis according to previous reports (Figure 1). Most unigenes

163 involved in the shikonin biosynthetic pathway were up-regulated in the SP cell

164 line compared to the SD cell line, such as HMGR, PAL, and PGT genes. This

165 suggested that the transcriptomes could be used to facilitate the discovery of

166 genes involved in shikonin biosynthetic pathway efficiently based on their

167 co-expression patterns. Additionally, all unigenes related to the mevalonate

168 (MVA) pathway and the phenylpropanoid (PP) pathway were up-regulated,

169 while unigenes related to the methylerythritol 4-phosphate (MEP) pathway

170 were not. These results support the MVA pathway as a preferred route of GPP

171 supply for shikonin biosynthesis in A. euchroma.

172 Co-expression analysis of the transcriptome dataset focused on CYP genes

173 whose expression proﬁles matched those of genes known to encode enzymes

174 that catalyze the early steps of shikonin biosynthesis in an attempt to identify

Downloaded from on November 29, 2018 - Published by www.plantphysiol.org Copyright © 2018 American Society of Plant Biologists. All rights reserved. 175 candidates responsible for subsequent steps. Analysis of the annotated

176 transcriptome dataset revealed the presence of approximately 480 CYP

177 contigs, which included both full-length and partial cDNA sequences. Analysis

178 of differentially expressed unigenes (DEG) using a stringent threshold (i.e.,

179 |Log2(ratio)| ≥ 1 and FDR < 0.001 and Max(FPKM) ≥ 1) led to the detection of

180 99 differentially expressed CYPs, of which 78 were up-regulated in the SP cell

181 line compared to the SD cell line. This population of CYP unigenes was

182 selected for investigation on the basis of their signiﬁcant increase in transcript

183 levels and integrity of open reading frame. The search was limited to CYP

184 unigenes up-regulated in the SP cell line due to the previous properties

185 (require NADPH and molecular oxygen) of LeGHQ3´´H described by

186 Yamamoto, suggesting it is a cytochrome P450 (CYP)-dependent

187 monooxygenase (Yamamoto et al., 2000). Unigenes with an open reading

188 frame less than 1200 bp were abandoned, given the average length of plant

189 CYPs. This led to 21 CYP candidates that may be involved in shikonin

190 biosynthesis. For each initial candidate, detailed information and BLAST

191 search results are displayed in Supplemental Table S1.

192 Given the accumulation of shikonin in the root, as well as the previously

193 demonstrated tissue-speciﬁc transcription of other secondary metabolites, we

194 hypothesized that shikonin biosynthetic enzymes would be speciﬁcally

195 expressed in the root. The relative expression of each of the 21 up-regulated

196 candidates was thus analyzed in the roots and leaves of A. euchroma by

197 reverse transcription quantitative PCR (RT-qPCR) (Figure 2). Transcripts for

198 12 of the 21 candidates were found to be more abundant in the root, four in the

199 leaf, and five showed no significant differential expression. The 12 unigenes

200 showing higher expression in root (framed in Figure 2) were selected as

201 candidates for further functional screening.

202 Gene-specific primers were derived and led to the amplification of transcripts

203 from SP cells of A. euchroma. The 12 CYP candidates, boldly marked in

204 Supplemental Table S2, were then cloned to enable further biochemical

205 assays using GHQ as the substrate. The proteins were named based on the

206 standardized CYP nomenclature system (Nelson, 2009), i.e., CYP76A74,

Downloaded from on November 29, 2018 - Published by www.plantphysiol.org Copyright © 2018 American Society of Plant Biologists. All rights reserved. 207 CYP706G18, CYP734A49, CYP76A46,9 CYP82AR1, CYP707A137,

Downloaded from on November 29, 2018 - Published by www.plantphysiol.org Copyright © 2018 American Society of Plant Biologists. All rights reserved. 208 CYP73A155, CYP73A64, CYP76A48, CYP76B75, CYP76B74, and

209 CYP88A76.

210 In vitro enzyme activity assays for CYP candidates

211 CYP candidates were heterologously expressed in the S. cerevisiae BY-T20

212 (BY4742) strain. The ability of these CYP candidates to react with GHQ was

213 examined by in vitro assay using microsomal preparations from the resulting

214 recombinant yeast.

215 In the presence of NADPH, only one of the proteins (designated CYP76B74 by

216 the Cytochrome P450 Nomenclature Committee) was found to catalyze the

217 conversion of GHQ to its hydroxide (Figure 3), as confirmed by liquid

218 chromatography-mass spectrometry (LC-MS). No product formation was found

219 in assays that contained microsomes from yeast cells harboring the empty

220 vector, and none of the other 11 CYPs were able to react with GHQ under the

221 identical assay conditions (Supplemental Figure S1). After large-scale

222 enzymatic reactions, the oxidized product was purified by preparative HPLC 1 13 223 and verified to be GHQ3´´H by mass spectrometry and H NMR, C NMR

224 spectroscopy (Supplemental Figure S2, S3).

225 Results of steady-state kinetic analysis, in the presence of excess NADPH,

226 demonstrated that CYP76B74 efficiently catalyzed the conversion of geranyl

227 hydroquinone to 3´´-hydroxy-geranyl hydroquinone with Km = 23 ± 6 μM, and

228 Vmax = 8.3 ±0.8 μmols product/mmol protein/min (Figure 4). This Km is in the

229 same range as the Km of the CYP76B6 homolog from Catharanthus roseus for

230 its substrate geraniol (15.81 μM) (Sung et al., 2011). The results suggest GHQ

231 might be the natural substrate of this hydroxylase, CYP76B74, in A. euchroma.

232 The ability of CYP76B74 to react with GBA was assayed under the same

233 condition as GHQ (Supplemental Figure S4). CYP76B74 was found to weakly

234 catalyze the conversion of GBA to its hydroxide. Only a trace amount of

235 hydroxylated GBA can be detected by HPLC, and there was less than 10% of

236 the substrate can be metabolized when the reaction was left overnight on a

237 150 rpm shaking incubator at 28°C. While under the same conditions, almost

238 all GHQ were metabolized. Without an authentic reference product, the Km

239 and Vmax of recombinant CYP76B74 for GBA were too difficult to determine.

Downloaded from on November 29, 2018 - Published by www.plantphysiol.org Copyright © 2018 American Society of Plant Biologists. All rights reserved. 240 This is consistent with the previous findings when microsomes from L.

241 erythrorhizon cell suspension cultures were used (Yamamoto et al., 2000).

242 Physiological function of CYP76B74 in vivo

243 To provide more deﬁnitive evidence for CYP76B74 involvement in shikonin

244 biosynthesis, an RNAi approach was used to knock down its gene expression

245 in A. euchroma hairy roots. A less conserved and unique fragment in the

246 upstream coding region (from nucleotide 83 to 492 of the cDNA) of CYP76B74

247 was cloned into the plant expression vector pK7GWIWG2D(II), which

248 expresses self-complementary 'hairpin' RNA fragments that induce silencing.

249 The pK7GWIWG2D-CYP76B74 and pK7GWIWG2D-control were transfected

250 via Agrobacterium rhizogenes into explants of A. euchroma to produce

251 recombinant hairy roots.

252 Independent hairy root lines generated on plates were transferred to triangular

253 flasks for liquid culture, and further processed for gene expression and

254 metabolite analysis. RT-qPCR indicated that the expression of CYP76B74 was

255 efﬁciently suppressed in transformed hairy root lines (Figure 5A). Interestingly,

256 the expression of AeHMGR, Ae4CL, AeC4H, and AePGT6 involved in shikonin

257 biosynthesis was also repressed in RNAi-cyp76b74 lines (Supplemental

258 Figure S5), suggesting a critical role of CYP76B74 in the regulation of shikonin

260 Because of shikonin accumulation, the roots and the hairy roots of A.

261 euchroma appear red, the characteristic color of shikonin and its derivatives.

262 The transformed hairy roots grown on plates were observed under a

263 stereoscopic microscope. The readily perceivable color difference between

264 RNAi-cyp76b74 (Figure 5Ca) and control lines (Figure 5Cb) indicated that the

265 accumulation of shikonins was indeed suppressed in RNAi-cyp76b74 lines.

266 Given the auto-fluorescence of shikonin derivatives, root tips were also

267 observed under two-photon confocal microscopy. The resulting

268 photomicrographs demonstrated that accumulation of shikonin derivatives was

269 high in the control lines, and almost absent in RNAi-cyp76b74 lines (Figure

270 5Cc-h).

271 After individual hairy root lines were cultured in liquid medium for 10 days

Downloaded from on November 29, 2018 - Published by www.plantphysiol.org Copyright © 2018 American Society of Plant Biologists. All rights reserved. 272 (Figure 5B), shikonin derivative contents were quantified in root tissues (Figure

273 5D) and culture medium (Figure 5E) by HPLC. Suppression of CYP76B74

274 seems to alter the root growth: roots are much shorter, thicker and have fewer

275 branches, but there is no change in biomass based on the weight of fresh

276 roots, because of its surrounding spongy tissue. The total shikonin content

277 compared to the control decreased about 8-fold in RNAi-cyp76b74 hairy roots,

278 and 9-fold in the culture medium. Among the shikonin derivatives quantified,

279 isovalerylshikonin (IVS), isobutyrylshikonin(IBS), acetoxylisovalerylshikonin

Downloaded from on November 29, 2018 - Published by www.plantphysiol.org Copyright © 2018 American Society of Plant Biologists. All rights reserved. 280 (AOIVS), acetylshikonin(AS), β-hydroxyisovalerylshikonin (HIVS) were

281 reduced by an average of about 4 to 7-fold in RNAi-cyp76b74 hairy roots. And

282 the accumulation of acetoxylisovalerylshikonin (AOIVS) was strongly reduced

283 to almost absent by an average of 36-fold in RNAi-cyp76b74 hairy roots

284 compared to the control and 476-fold in the culture medium. The positive

285 association between expression of CYP76B74 and shikonin accumulation thus

286 supports the requirement of this CYP for the production of shikonins in the

287 plant.

288 CYP76B74 is anchored to the ER

289 In silico analysis of CYP76B74 using protein-targeting prediction software

290 (TMHMM) revealed the presence of a 17-amino acid N-terminal membrane

291 anchor (Krogh et al., 2001). Expression of an in-frame fusion of GFP to the

292 N-terminus of CYP76B74 under the control of the CaMV 35S promoter in rice

293 (Oryza sativa) protoplasts was used to identify the subcellular localization of

294 the protein (Figure 6A). The chimeric PIN5::MKATE protein was used as an

295 endoplasmic reticulum (ER) marker control (Figure 6B). Image overlay

296 demonstrated identical localization patterns for CYP76B74 and marker

297 proteins, indicating that CYP76B74 is localized to ER membranes (Figure 6D).

298 Subcellular localization of CYP76B74 is thus consistent with the cytosolic

299 localization of the 4-hydroxybenzoate geranyltransferase in Lithospermum

300 erythrorhizon cell cultures that generates the expected substrate of the

301 GHQ3´´H (Yamaga et al., 1993).

302 Phylogenetic analysis of CYP76B74

303 Using the full-length amino acid sequences, a phylogenetic analysis was

304 carried out focused on the evolutionary relationship between CYP76B74 and

305 CYP76 family sequences with CYP71AV1 as an outgroup (Figure 7).

306 CYP76B74 was used to query the oneKP database

307 (https://db.cngb.org/onekp/search/) in Boraginaceae and de novo assembled

308 transcriptomes of Arnebia euchroma, Echium plantagineum and

309 Lithospermum erythrorhizon with raw RNA sequencing data downloaded from

310 the NCBI’s Short Read Archive to obtain homologous sequences from

311 Boraginaceae and possibly related taxa. Figure 7 shows sequences from

Downloaded from on November 29, 2018 - Published by www.plantphysiol.org Copyright © 2018 American Society of Plant Biologists. All rights reserved. 312 Boraginaceae, which formed a subclade of the larger CYP76B subfamily

313 clade. The other major subclade comprised geraniol 10-hydroxylases (G10H)

Downloaded from on November 29, 2018 - Published by www.plantphysiol.org Copyright © 2018 American Society of Plant Biologists. All rights reserved. 314 from various angiosperm species. The resulting neighbor-joining tree

315 confirmed that CYP76B74 specifically evolved in Boraginaceae and belongs to

316 the CYP76B subfamily, which includes CYP76B1 from Helianthus tuberosus,

Downloaded from on November 29, 2018 - Published by www.plantphysiol.org Copyright © 2018 American Society of Plant Biologists. All rights reserved. 317 CYP76B6 from C. roseus, CYP76B10 from Swertia mussotii and CYP76B9

318 from Petunia x hybrida. 319

321 Advances in DNA sequencing technology have enabled plant genome

322 sequencing projects to reveal a vast number of CYPs, leading to the

323 annotation of more than 184,522 sequences in the databases. More than

324 16,219 CYPs were named and divided into 277 CYP families (Nelson, 2018).

325 CYPs are among the largest plant gene superfamilies and constitute around

326 1% of all the protein coding genes in plants. In plants, CYPs play a critical role

327 in secondary metabolic product diversity, e.g. flavonoids, coumarins, alkaloids,

328 terpenoids, and other biological substances. It is a remarkable fact that studies

329 of medicinal plant secondary metabolism genes have shown rapid evolution of

330 CYPs, especially the CYPs genes in a specific downstream metabolic pathway

331 such as the seco-iridoid pathway from C. roseus (Miettinen et al., 2014) and

332 the opioids pathway form opium poppies (Galanie et al., 2015). However, the

333 biochemical functions of the majority of CYP proteins remain unknown.

334 Shikonin and its derivatives are the most abundant naphthoquinones formed in

335 species of the medicinally and economically valuable Boraginaceae. Shikonin

336 biosynthesis was initially proposed to start from geranyl pyrophosphate and

337 4-hydroxybenzoate (Mühlenweg et al., 1998), followed by a series of oxidative

338 transformations mediated by the prenylated phenolic GHQ. To identify CYPs

339 that are responsible for the early hydroxylation step(s) in shikonin

340 biosynthesis, we carried out co-expression analysis using the transcriptomes

341 of shikonin-proficient and deficient cell lines and the spatial distribution of

342 candidate gene expression across different organs to find candidates whose

343 expression patterns matched those of known upstream genes in the shikonin

344 biosynthestic pathway. We constructed yeast strains that expressed these

345 CYP candidates. Using GHQ as the substrate, microsomes from the

346 CYP76B74 expressing yeast strain produced GHQ-3´´-OH. Further, RNAi

347 knockdown of CYP76B74 expression in hairy root cultures of A. euchroma

348 demonstrated that CYP76B74 was necessary for shikonin biosynthesis.

349 Phylogenetic analysis confirmed that CYP76B74 belongs to the CYP76B

350 subfamily and subcellular localization indicated it was anchored to ER

351 membranes. Taken together, the gene identity, regulation, and phylogeny of

Downloaded from on November 29, 2018 - Published by www.plantphysiol.org Copyright © 2018 American Society of Plant Biologists. All rights reserved. 352 GHQ3´´H was finally elucidated nearly two decades after it was found in L.

353 erythrorhizon (Yamamoto et al., 2000).

354 There are currently several CYP76B subfamily members that have been

355 identified, but their physiological functions remain uncertain, except for those

356 of CYP76B6 (Collu et al., 2001) and CYP76B10 (Wang et al., 2010), both

357 referred to G10H. The natural substrate of CYP76B1 in the Jerusalem

358 artichoke (Helianthus tuberosus) tuber has still not been characterized and its

359 monoterpenol oxidation activity is low (Batard et al., 1998, Höfer et al., 2014).

360 CYP76B9 was reported to be able to ω-hydroxylate medium-chain fatty acids

361 in vitro, but it has not been confirmed as a physiological function in the plant

362 (Imaishi and Petkova-Andonova, 2007; Wang et al., 2010). In the phylogenetic

363 tree, the CYP76B subfamily is most closely related to the CYP76F subfamily,

364 the members of which catalyze the formation of the santalol and bergamotol

365 components of sandalwood fragrance (Diaz-Chavez et al., 2013) via santalene

366 and bergamotene hydroxylation at the C-12 position. In the same way,

367 shikonin is hydroxylated on its terpenoid side chain, although not at the

368 terminal position. Involvement of more distantly related CYP76 family

369 members in isoprenoid metabolism has also been functionally established, for

370 example, for CYP76Cs from Arabidopsis (Arabidopsis thaliana) (Höferer et al.,

371 2014; Boachon et al., 2015) or CYP76Ms from rice (Swaminathan et al., 2009;

372 Wang et al., 2012). Altogether, our data thus support a predominant role of the

373 CYP76 family members in terpenoid-derived metabolism.

374 Phylogenetic reconstruction suggests that the group to which CYP76B4

375 belongs is a single evolutionary subclade of a Boraginaceae-specific clade of

376 CYP76Bs, which are most likely all devoted to the biosynthesis of the same

377 class of compounds, i.e. geranyl-hydroxybenzoic acids. Boraginaceae is one

378 of the few families with uncertain taxonomic status in the APG IV classification

379 system. In APG IV, they recognize an order, Boraginales, to accommodate the

380 family, and consider Boraginales to comprise a single family, Boraginaceae

381 s.l., including Boraginaceae s.s., Codonaceae, Cordiaceae, Ehretiaceae, etc.,

382 and A. euchroma, E. plantagineum and L. erythrorhizon belonging to

383 Boraginaceae s.s. (The Angiosperm Phylogeny Group, 2016). The

Downloaded from on November 29, 2018 - Published by www.plantphysiol.org Copyright © 2018 American Society of Plant Biologists. All rights reserved. 384 phylogenetic tree indicated that CYP76B74 specifically evolved in

385 Boraginaceae s.s.. Recently, unigenes annotated as G10H, which have a

386 similar function to the GHQ3´´H enzyme and belong to the P450

387 monooxygenase family, have been found up-regulated in red roots compared

388 with green leaves/stems of three Boraginaceae plants, i.e., L. erythrorhizon, A.

389 euchroma and E. plantagineum (Wu et al., 2017). Our results are consistent

390 with this previous work and indicate CYP76Bs are expanded within

391 Boraginaceae s.s.. The fossil record of the Boraginaceae is restricted to the

392 Tertiary starting in the Early Eocene (Martínez-Millán, 2010), and the fruit of

393 Lithospermum dakotense was dated to Late Miocene, which was found in Ash

394 Hollow, Bennett, SD, USA (Gabel, 1987). Based on the fossil record, the

395 duplication event leading to the novel activity of CYP76B74 should be dated to

396 the Early Eocene to Late Miocene.

397 A shikonin biosynthetic pathway has been proposed wherein cyclization of

398 GHQ-3´´-OH to a naphthoquinone skeleton occurs through oxidation of the

399 C-3´´ to an aldehyde group capable of forming the C-C bond with the

400 aromatic-stabilized carbon via an electrophilic reaction (Yamamoto et al.,

401 2000). It is important to note that there are many CYP proteins in different

402 pathways catalyzing consecutive oxidations into alcohols, aldehydes, and

403 acids at the same position. Examples of this activity include C20Ox

404 (CYP76AK6-8) of Rosmarinus ofﬁcinalis and Salvia fruticosae, which ensures

405 sequential oxidation at the C-20 position leading to miltiradien-20-al, carnosic

406 acid, and pisiferic acid (Scheler et al., 2016). CYP76C1 and

407 CYP76B6-mediated acyclic monoterpene linalool and geraniol metabolism has

408 also been shown to involve successive oxidation steps (Höfer et al., 2013;

409 Boachon et al., 2015). Therefore, we tested yeast-expressed CYP76B74 to

410 determine if it catalyzed further oxidation of GHQ-3´´-OH. However, no further

411 conversion of GHQ-3´´-OH was observed. This is consistent with the

412 Michaelis-Menten kinetics observed for CYP76B74 towards GHQ-3´´-OH. In

413 the case of the seco-iridoid pathway from C. roseus, geraniol 8-hydroxylase

414 (G8H, CYP76B6) can catalyze the two consecutive oxidation steps of geraniol

415 to 8-oxogeraniol, while another soluble enzyme, 8-hydroxygeraniol

Downloaded from on November 29, 2018 - Published by www.plantphysiol.org Copyright © 2018 American Society of Plant Biologists. All rights reserved. 416 oxidoreductase (8-HGO) was found to catalyze the stepwise conversion of

417 8-OH-geraniol into 8-oxogeraniol or 8-OH-geranial and then into 8-oxogeranial

418 (Miettinen et al., 2014). Considering the catalytic similarity between CYP76B6

419 and CYP76B74, we speculated that other CYPs or soluble NADPH-dependent

420 oxidoreductase were oxidizing the allylic primary alcohol at the C-3´´ position,

421 as is the case in C. roseus. In this study, we also cloned another cytochrome

422 P450 enzyme belonging to the CYP76B family, CYP76B75, that shares 53%

423 identity with CYP76B74 and 68% identity with G10H form Ophiorrhiza pumila.

424 In an analysis of tissue-specific expression, CYP76B75 was found to be more

425 abundant in the root, similar to CYP76B74 (Figure 2). However, when tested

426 via in vitro enzyme activity assay, CYP76B75 showed no catalytic activity

427 towards GHQ or GHQ-3´´-OH (Supplemental Figure S1).

428 In spite of recent studies regarding the biosynthesis of shikonin and its

429 controlling factors (Malik et al., 2016), the site of shikonin biosynthesis in cells

430 is still an open question. Electron microscopy studies suggested that shikonin

431 production was closely associated with the development of elongated rough

432 ER and the subsequent formation of electron-dense vesicles (0.1-0.2 pm),

433 which appeared to transport shikonin derivatives to the outside of the cell wall

434 by a process of exocytosis. However, direct evidence for ER-associated

435 shikonin metabolism remained to be demonstrated (Yamaga et al., 1993;

436 Papageorgiou et al., 1999). We used protoplast transformation in combination

437 with GFP imaging to localize the GHQ3´´H step of shikonin biosynthesis in A.

438 euchroma. This approach allowed us to demonstrate that like most other plant

439 CYP enzymes, CYP76B74 is associated with the ER membranes and may

440 contribute to anchoring the terminal segment of the shikonin metabolism to

441 these membranes. In this study, we also established a direct observation

442 method to observe shikonin localization via two-photon laser confocal

443 microscopy, taking advantage of the spontaneous fluorescence of shikonins.

444 This novel experimental system may enable further biochemical investigation

445 of the kinetics and mechanism of shikonin accumulation and secretion.

446 In summary, our results conclusively demonstrated that the cytochrome P450

447 enzyme CYP76B74 catalyzes the regio-specific C-3´´ hydroxylation of GHQ,

Downloaded from on November 29, 2018 - Published by www.plantphysiol.org Copyright © 2018 American Society of Plant Biologists. All rights reserved. 448 which is a critical step in the biosynthesis of shikonins, the major active

449 components of Boraginaceae medicinal plants. To date, CYP76B74 is the first

450 cytochrome P450 enzyme functionally characterized in the shikonin

451 biosynthetic pathway. It will pave the way for functional investigation of other

452 CYPs potentially involved in shikonin biosynthesis. In addition, the ability to

453 generate GHQ-3´´-OH, the CYP76B74 product, will provide an intermediate for

454 testing the next steps of the pathway. We have recently established a platform

455 for the production of GBA in yeast (Wang et al., 2017). CYP76B74 can now be

456 used in further synthetic biology approaches towards heterologous production

457 of shikonins in yeast.

458

459 Materials and Methods

460 Plant materials and chemicals

461 The shikonin-proficient (SP) and shikonin-deficient (SD) suspension culture

462 cell lines were derived from hypocotyls of Arnebia euchroma (Royle) Johnst

463 and were grown in improved Linsmaier & Skoog liquid medium as previously

464 described (Wang et al., 2014). A. euchroma plants for organ-speciﬁc

465 expression analysis and seeds for aseptic seedling transformation were

466 collected in Xinjiang, China. Geranyl hydroquinone (GHQ) was purchased

467 from WuXi AppTec (Shanghai, China). GBA (3-geranyl-4-hydroxybenzoic acid)

468 was isolated from engineered yeast in our lab (Wang et al., 2017). Shikonin

469 derivatives were purchased from Tokyo Chemical Industry (Tokyo, Japan). The

470 purity of these chemicals exceeded 95%.

471 RNA sequencing and bioinformatic processing

472 The cDNA libraries of SP and SD cells were sequenced on an Illumina HiSeq TM 473 2000 platform, and the raw sequencing data were deposited in the NCBI’s

474 Short Read Archive (SRA) under accession number SRP137782. The

475 transcriptome was de novo assembled using the Trinity program (Grabherr et

476 al., 2011), and the functional annotation of all assembled unigenes were

477 performed with Nonredundant Protein Sequence (NR), Nucleotide Sequence

478 (NT), SwissProt, InterPro, Kyoto Encyclopedia of Genes and Genomes

479 (KEGG), Clusters of Orthologous Groups of Proteins (COG) and Gene

Downloaded from on November 29, 2018 - Published by www.plantphysiol.org Copyright © 2018 American Society of Plant Biologists. All rights reserved. 480 Ontology (GO) databases successively. The expression level of each unigene

481 was calculated using RNA-Seq quantification analysis with the fragments per

482 kb per million mapped fragments (FPKM) method by DESeq2 (Mortazavi et al.,

483 2008; Love et al., 2014).

484 Isolation of Total RNA and RT-qPCR Analysis

485 Total RNA from different tissues and organs of A. euchroma was extracted

486 using TRIzol reagent (Invitrogen, U.S.A.) following the manufacturer’s

487 instructions. After treatment with DNases, samples of total RNA (2 μg) were

488 fractionated on a 1% agarose gel in TBE buffer and stained with ethidium

489 bromide to analyze RNA integrity and genomic DNA contamination. RNA is

490 considered to be of high quality when the ratio of eukaryotic 28S:18S band

491 intensity is about 2.0. First-strand cDNAs were synthesized with PrimerScript

492 1st Strand cDNA Synthesis Kit with random primers and oligo-(dT) at the same

493 time (TaKaRa, Japan). Reverse transcription quantitative polymerase chain

494 reaction (RT-qPCR) was performed using the Power SYBR Green PCR Master

495 Mix (Applied Biosystems, U.S.A.) and an Applied Biosystems 7500 Real-time

496 instrument (Applied Biosystems, U.S.A.). Real-time PCR was performed as

497 previously described (Wang et al., 2014). The primers used are listed in

498 Supplemental Table S3. The 18S ribosomal RNA (18S rRNA) was used as the

499 endogenous control to normalize expression data (Kuchipudi et al., 2012). At

500 least three independent experiments were performed for each analysis.

501 cDNA cloning and heterologous expression of CYPs candidates in yeast

502 Full-length cDNAs of CYP candidates were cloned from cDNA isolated from

503 SP cells, using PrimeStar DNA polymerase (Takara Bio, Dalian). The ORF

504 region of CYP candidates were subcloned into yeast epitope-tagging vector

505 pESC-Trp by the restriction endonuclease free (RF) cloning method (Unger et

506 al., 2010). Primers for PCR ampliﬁcation are shown in Supplemental Table S4,

507 S5. The clones with correct insert were confirmed by sequencing. Constructed

508 expression vectors were transformed into the yeast strain BY-T20 (Dai et al.,

509 2013), a high yield diterpene strain derived from BY4742

510 (ΔTrp1,Trp1::HIS3-PPGK1-BTS1/ERG20-TADH1-PTDH3-SaGGPS-TTPI1-PTEF1-tHM

511 G1-TCYC1). The transformants were selected on yeast synthetic dropout

Downloaded from on November 29, 2018 - Published by www.plantphysiol.org Copyright © 2018 American Society of Plant Biologists. All rights reserved. −1 512 medium –Trp (SD–Trp) containing 20 g L glucose and grown at 28°C for 48 −1 513 h. The isolated recombinant strain was initially grown in SD–Trp with 20 g L

514 glucose at 30°C for about 48 h. Cells were centrifuged and washed with sterile

515 water to remove any residual glucose. Cells were then resuspended in SD–Trp −1 516 with 20 g L galactose and grown for 24 h at 30°C to induce recombinant

517 protein expression.

518 In order to produce a sufﬁcient amount of microsomal protein for kinetic assays

519 of CYP76B74 and a sufﬁcient amount of CYP76B74 product, fermentation of

520 yeast expressing CYP76B74 was scaled up to 5 L in Minifors (MCGS,

521 GuangShi Bio). Collected cells were homogenized by continuous

522 high-pressure cell disruption (APV2000, APV), and microsomes were prepared

523 by differential centrifugation as previously described (Yamamoto et al., 2000).

524 All these procedures were performed at 4°C.

525 In vitro enzyme activity assay

526 The in vitro enzyme activity assays with microsomal fractions of yeast

527 expressing CYPs were conducted as previously described (Guo et al., 2016).

528 The microsomes were prepared by the glass beads method (Pompon et al.,

529 1996). Microsomes were suspended in storage buffer containing 50 mM

530 Tris·HCl (pH 7.5), 1 mM EDTA, and 20% (v/v) glycerol. Initial in vitro

531 hydroxylation assays were conducted in a total volume of 500 μL of 90 mM

532 Tris·HCl (pH 7.5), containing 250 μg of microsomal protein, 100 μM GHQ and

533 0.5 mM NADPH along with a regenerating system (consisting of 5 μM FAD, 5

534 μM FMN, 5 mM glucose-6-phosphate, 0.5 U glucose-6-phosphate

535 dehydrogenase, and 2 mM DTT). The assay samples were incubated with

536 shaking for 4 h at 28°C, and the reactions were terminated by three extractions

537 with an equal volume of ethyl acetate. The products were dried by N2, and

538 redissolved in methanol for liquid chromatography-mass spectrometry

539 (LC-MS) analysis. Substrates structurally related to GHQ were assayed under

540 the same condition except for 100 μM of specific substrate in place of GHQ.

541 In order to produce enough enzymatic product for chemical structure

542 characterization, the enzymatic assay containing the same composition as

543 above was scaled up to 80 mL and performed overnight on a 150 rpm shaking

Downloaded from on November 29, 2018 - Published by www.plantphysiol.org Copyright © 2018 American Society of Plant Biologists. All rights reserved. 544 incubator at 30°C. The incubation products were extracted with ethyl acetate,

545 dried by rotary evaporator and resuspended in methanol. The CYP76B74

546 product was puriﬁed by preparative high-performance liquid chromatography

547 (HPLC) for structure characterization by nuclear magnetic resonance (NMR)

548 spectroscopy.

549 Kinetic analysis

550 For kinetic analysis, the concentration of CYP450 protein was estimated using

551 the reduced CO-binding difference spectra detection kit (GMS18003v.A,

552 GENMED SCIENTIFICS INC. U.S.A). Experiments were carried out against

553 GHQ in a serial concentration of 0, 5, 10, 15, 30, 40, 60 and 100 μM in 1 mL

554 enzyme assays using 1 mg microsomal protein prepared in the same batch.

555 Enzymatic assay experiments contained the buffer and NADPH regeneration

556 system described earlier. Reactions were initiated by adding substrate and

557 incubating at 30°C for 30 min with shaking (150 rpm), and terminated by

558 extraction three times with 1 mL ethyl acetate. The products were dried by N2

559 and redissolved in methanol for ultra-performance liquid chromatography

560 (UPLC) analysis. KM and Vmax values were calculated by nonlinear regression

561 of Michaelis-Menten enzyme kinetics using GraphPad Prism 6, and data

562 reported are the means ± SD from triplicate analyses.

563 A time series experiment was carried out in a time series of 0, 10, 21, 31, 40

564 and 50 min to confirm that the 30 min time-point for the steady-state kinetics

565 experiments stayed within the linear range of the assay (Supplemental Figure

566 S6). The enzymatic assay contained the same composition as the kinetic

567 analysis descried above with the addition of 100 uM GHQ in the 1 mL enzyme

568 assays, and the products were analyzed using UPLC.

569 UPLC, LC-MS, and NMR analysis

570 UPLC was carried out with a Waters ACQUITY UPLC-PDA system equipped

571 with a Waters C18 1.8 μm 2.1×100 mm T3 HHS column with an absorbance

572 range of 210-400 nm. The column temperature was set at 40°C. For in vitro

573 enzymatic product determination, the mobile phase comprised acetonitrile (A) -1 574 and water (formic acid 0.1%, B) at 0.5 mL min with a gradient program (0 min,

575 10% A; 4 min, 35% A; 4.3 min, 60% A; 8 min, 72% A; 8.5 min, 98% A; 10.5

Downloaded from on November 29, 2018 - Published by www.plantphysiol.org Copyright © 2018 American Society of Plant Biologists. All rights reserved. 576 min, 98% A; 11 min, 10% A; 13 min, 10% A). To determine contents of shikonin

577 derivatives in hairy roots, about 20 mg of lyophilized hairy root was extracted

578 by ultrasonication in 1 mL methanol. The ﬁltered extract then underwent UPLC

579 analysis with a gradient program (0 min, 2% A; 1 min, 20% A; 4 min, 25% A;

580 4.5 min, 75% A; 9.5 min, 75% A; 10 min, 100% A; 11 min, 100% A; 11.5 min,

581 2% A; 13.5 min, 2% A).

582 LC-MS was carried out using an Acquity UPLC system (Waters Corp, U.S.A.)

583 with column and separation conditions as described above. Time-of-ﬂight MS

584 detection was performed with a Xevo G2-S MS system (Waters Corp, UK) with

585 the data acquisition range set from 50 to 1000 Da. The source temperature

586 was set at 100°C, and the desolvation temperature was set at 450°C, with -1 587 desolvation gas ﬂow at 900 L h . The lock mass compound used was leucine -1 588 enkephalin at a concentration of 200 pg μL . The capillary voltage was set at

589 2500 V. The cone voltage was set at 40 V. The collision energy was set as 6

590 eV for low-energy scan, and 25-40 eV ramp for high-energy scan. The

591 instrument was controlled by MassLynx 4.1 software.

592 Preparative HPLC separation was performed using a LC-6AD instrument

593 (SHIMADZU，Japan), with a YMC-Pack ODS-A column (250×10 mm, S-5 μm,

594 12 nm). The ﬂow rate was kept at 4 mL/min. The mobile phase was a 6:4

595 mixture of water and acetonitrile (v/v). The target fraction was collected

596 manually, identified by UPLC, dried and resuspended in deuterated chloroform

597 (CDCl3) for structural analysis by NMR spectroscopy. 1 598 Chemical structure characterization of GHQ and GHQ-3´´OH utilizied H NMR 13 599 (400 MHz), C NMR (100 MHz), and two-dimensional (2D) NMR spectra

600 collected on a Bruker DRX 400 spectrometer. Tetramethylsilane was used as

601 the internal standard. The observed chemical shift values are reported in ppm.

602 RNA interference in hairy root of A. euchroma

603 Gene-speciﬁc fragments of CYP76B74 were cloned into the pENTR vector

604 using the Directional TOPO Cloning Kit (Invitrogen, U.S.A.; primers shown in

605 Supplemental Table S6), and further subcloned into the binary vector

606 pK7GWIWG2D(II) using Gateway LR Clonase Enzyme Mix (Invitrogen).

607 Constructs, including a CYP76B74 RNAi vector and an empty vector used as a

Downloaded from on November 29, 2018 - Published by www.plantphysiol.org Copyright © 2018 American Society of Plant Biologists. All rights reserved. 608 negative control, were introduced into Agrobacterium rhizogenes C58C1 using

609 the freeze-thaw transformation method (Cheng et al., 2014). These

610 recombinant A. rhizogenes were then transfected into explants of A. euchroma

611 as described (Cheng et al., 2014). In contrast to other plants, only the

612 cotyledons of two-week-old A. euchroma seedling can be used as explants to

613 generate hairy roots. The transformed hairy root lines were cultured in

614 half-strength Murashige and Skoog (MS) solid medium without ammonium

615 nitrate at 25°C in the dark for 6-8 weeks. The rapidly growing

616 kanamycin-resistant lines in which green fluorescent protein (GFP) was visible

617 and that had no bacterial contamination were then maintained at 25°C in the

618 dark with half-strength MS solid medium without ammonium nitrate and

619 routinely sub-cultured every 10-15 days. Tissues were then collected for

620 RT-qPCR, metabolite analysis and microscopy.

621 Sub-cellular localization of CYP76B74 in rice protoplasts

622 A transmembrane helical domain was predicted in the first 40 residues

623 (N-terminal) of CYP76B74 using TMHMM server analysis

624 (http://www.cbs.dtu.dk/services/ TMHMM/).

625 In order to determine subcellular localization of CYP76B74, the complete open

626 reading frame (ORF) sequence was subcloned into the BsaI/Eco31I restriction

627 sites of the pBWA(V)HS-ccdb-GLosgfp plasmid. This fusion expression vector,

628 CYP76B74-GFP, is driven by CaMV 35S promoter. The GFP was

629 codon-optimized for high expression in rice (Oryza sativa) (Genscript, China).

630 We also used mKATE, which is a far-red fluorescent protein, fused to PIN5 to

631 stain ER membranes as a control protein for endoplasmic reticulum (ER)

632 targeting (pBWA(V)HS-PIN5-mKATE) (Mravec et al., 2009). Rice seedlings in

633 plastic pots filled with soil and grown in the dark at 28°C for 1-2 weeks were

634 used for the isolation of leaf protoplasts without chlorophyll. The protoplasts

635 were isolated and transformed as described previously with modifications

636 (Zhang et al., 2011). The transformed protoplasts were examined under a light

637 microscope as well as a laser confocal microscope (Nikon C2-ER, Nikon). For

638 GFP detection, excitation at 488 nm and detection at 510 nm was used. For

639 mKATE detection, excitation at 588 nm and detection at 635 nm was used.

Downloaded from on November 29, 2018 - Published by www.plantphysiol.org Copyright © 2018 American Society of Plant Biologists. All rights reserved. 640 The images acquired from the confocal microscope were processed using

641 NIS-Elements Viewer 4.20.

642 Microscopy and confocal microscopy

643 The phenotype of hairy roots was examined under a stereomicroscope

644 (SZX10, OLYMPUS). CYP76B74 RNAi-derived hairy roots and the empty

645 vector control were imaged using a LSM 880 (Zeiss) confocal microscope

646 equipped with an AxioObserver. Slides were prepared by root-tip squashing

647 method. Collected images were processed using Zeiss Efficient Navigation 2

648 (Zen 2) and Adobe Photoshop CS3. Shikonin auto-ﬂuorescence was detected

649 using a 543 nm excitation wavelength laser and a 568-712 nm emission ﬁlter

650 for the 640 nm emission wavelength. Fluorescence intensity was analyzed by

651 ImageJ software (n=3) (Tatsumi et al., 2016).

652 Phylogenetic sequence analysis

653 Phylogenetic and molecular evolutionary analyses were conducted using

654 MEGA version 6 (Tamura et al., 2013). Protein sequences were aligned using

655 the MUSCLE program (Edgar, 2004). Phylogenetic relationships were

656 reconstructed by the maximum-likelihood (ML) method based on the JTT/+G

657 model (five categories) and a bootstrap of 1,000 replicates. All protein

658 sequences used for sequence alignment and phylogenetic tree construction

659 are listed in Supplemental Table S1.

660

661

662 Accession numbers

663 Sequence data from this article can be found in the GenBank/EMBL data

664 libraries under accession numbers MH124060(CYP76A74),

665 MH124061(CYP706G18), MH124062(CYP734A49), MH124063(CYP76A46),

666 MH124064(CYP82AR1), MH124065(CYP707A137),

667 MH124066(CYP73A155), MH124067(CYP73A64), MH124068(CYP76A48),

668 MH124069(CYP76B75), MH077962(CYP76B74) and MH124070(CYP88A76).

669

670 Supplemental Data

671 The following Supplementary materials are available.

Downloaded from on November 29, 2018 - Published by www.plantphysiol.org Copyright © 2018 American Society of Plant Biologists. All rights reserved. 672 Supplemental Figure S1. HPLC analysis of the enzyme assays using the 12

673 recombinant CYP candidates and geranyl hydroquinone as the substrate. 1 674 Supplemental Figure S2. H NMR spectra of

675 3´´-hydroxy-geranylhydroquinone. 13 676 Supplemental Figure S3. C NMR spectra of

677 3´´-hydroxy-geranylhydroquinone.

678 Supplemental Figure S4. UPLC analysis of the recombinant CYP76B74

679 enzyme assays using geranylhydroquinone (GHQ) and

680 3-geranyl-4-hydroxybenzoic acid (GBA) as substrates.

681 Supplemental Figure S5. Relative expression level of genes involved in

682 shikonin biosynthesis in RNAi-cyp76b74 roots and the control roots.

683 Supplemental Figure S6. Time series curve of catalytic reaction of CYP76B74

684 within 50 minutes.

685 Supplemental Table S1. Protein sequences used for phylogenetic analysis with

686 their corresponding accession numbers.

687 Supplemental Table S2. Details of 21 CYP candidate unigenes that might be

688 involved in shikonin biosynthesis.

689 Supplemental Table S3. Primers for RT-qPCR of CYP candidates.

690 Supplemental Table S4. Primers for full-length cloning of CYP candidates.

691 Supplemental Table S5. Primers for RF cloning of CYP candidates.

692 Supplemental Table S6. Primers for gene-speciﬁc fragment cloning of

693 CYP76B74 for RNA interference.

694

695 Figure legends

696 Figure 1. Expression of unigenes involved in shikonin biosynthesis in

697 shikonin-proficient and shikonin-deficient cell lines of Arnebia euchroma. Data

698 are from comparative transcriptome analysis of the shikonin-proficient and

699 deficient lines. The up-regulated genes in the SP cell line compared to the SD

700 cell line are underlined and marked in red, and genes that are down-regulated

701 or not significantly different are marked in green. Uncharacterized steps are

702 shown with dotted arrows. A “?” indicates that one step is unknown and “?*”

703 indicates several steps are unknown.

Downloaded from on November 29, 2018 - Published by www.plantphysiol.org Copyright © 2018 American Society of Plant Biologists. All rights reserved. 704 Abbreviations: SP, shikonin-proficient; SD, shikonin-deficient; MVA,

705 mevalonate; MEP, 2-C-methyl-D-erythritol-4-phosphate; HMGS,

706 3-hydroxy-3-methylglutaryl-CoA synthase; HMGR,

707 3-hydroxy-3-methylglutaryl-CoA reductase; MVAK, mevalonate

708 5-phosphokinase; PMVK, 5-phosphomevalonate phosphokinase; MVDD,

709 mevalonate diphosphate decarboxylase; DXPS,

710 1-deoxy-D-xylulose5-phosphate synthase; DXR,

711 1-deoxy-D-xylulose5-phosphate reductoisomerase; MCT,

712 2-C-Methyl-D-erythritol 4-phosphate cytidylyltransferase; CDPMEK,

713 4-(Cytidine 5'-diphospho)-2-C-methyl-D-erythritol 2-phosphokinase; MECDPS,

714 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase; HDS,

715 1-hydroxy-2-methyl-2-butenyl-diphosphate synthase; HDR,

716 1-hydroxy-2-methyl-2-(E)-butenyl4-diphosphatereductase; IPPI, isopentenyl

717 pyrophosphate isomerase; GDPS, geranyl-diphosphate synthase; PAL,

718 phenylalanine ammonialyase; C4H, cinnamic acid 4-hydroxylase; 4CL,

719 4-coumaroyl-CoA ligase; PGT, 4-hydroxybenzoate-3-geranyltransferase;

720 GHQH, geranylhydroquinone 3´´-hydroxylase.

721 Figure 2. RT-qPCR analysis of the 21 candidate unigenes in roots and leaves

722 of A. euchroma. The error bars in the RT-qPCR results represent standard

723 error (SE) of three biological replicates consist of different plants. Unigenes

724 with higher abundance in roots than in leaves were framed.

725 Figure 3. LC-MS/MS analysis of the recombinant CYP76B74 enzyme assays

726 using geranyl hydroquinone as the substrate. The assay samples were

727 incubated with shaking for 4 h at 28°C. (A) LC-MS chromatogram of extracts

728 from the reaction containing CYP76B74 microsomes with NADPH

729 (pESC-Trp-CYP76B74), empty vector control (pESC-Trp) and microsome-free

730 control (GHQ). (B) Mass spectra of the reaction product. (C) Direct conversion

731 of geranyl hydroquinone (1) to 3´´-hydroxy-geranyl hydroquinone (1a).

732 Figure 4. Kinetic proﬁle of CYP76B74. Experimental details are included in

733 Materials and Methods. The error bars in the kinetics results represent

734 standard error (SE) of three technical replicates consist of different reactions.

735 Figure 5. Physiological function of CYP76B74 in vivo. (A) Expression level of

Downloaded from on November 29, 2018 - Published by www.plantphysiol.org Copyright © 2018 American Society of Plant Biologists. All rights reserved. 736 CYP76B74 in hairy roots of the 4 independent RNAi-cyp76b74 lines and the

737 control. (B) Phenotype of RNAi-cyp76b74 lines and the control cultured for 10

738 days in liquid medium. (C) Phenotype of RNAi-cyp76b74 lines and the control

739 cultured on a plate under a stereoscopic microscope (Ca-b) and the root tips

740 under two-photon confocal microscopy (Cc-h). (Cc, Cf) Bright field microscopy

741 of root tips; (Cd, Cg) Shikonin auto-fluorescence field microscopy of root tips;

742 (Ce, Ch) Merged fields of bright and shikonin auto-fluorescence. (D)

743 Percentage of main shikonin derivatives in the dry weight of the 4 independent

744 RNAi-cyp76b74 hairy root ines and the control. (E) Contents of main shikonin

745 derivatives in culture mediums of the 4 independent RNAi-cyp76b74 hairy root

746 lines and the control.

747 Abbreviations: IVS, Isovalerylshikonin; IBS, Isobutyrylshikonin; AOIVS,

748 Acetoxylisovalerylshikonin; AS, Acetylshikonin; HIVS,

749 β-Hydroxyisovalerylshikonin.

750 Figure 6. Subcellular localization of CYP76B74 in rice protoplasts.

751 Protoplasts without chlorophyll were co-transformed with CYP76B74-GFP and

752 PIN5-mKATE, a marker for the endoplasmic reticulum (ER), and examined

753 using confocal microscopy. (A) Green fluorescence indicates the presence of

754 CYP76B74; (B) Red fluorescence indicates the presence of ER marker

755 protein; (C) Under white light; Overlay of these three images (D) indicates

756 CYP76B74 localizes to the ER.

757 Figure 7. Phylogenetic analyses of CYP76B74. The neighbor-joining tree was

758 generated using MEGA (version 6) from a multiple protein sequence alignment

759 and illustrates the evolutionary relationship between CYP76B74 and the

760 CYP76 family. Bootstrap values (1000 repeats) are indicated for each branch

761 of the tree. The scale bar=0.2 amino acid substitutions per site. Known

762 geraniol 10-hydroxylase (G10H) are in pink ， protein sequences of

763 Boraginaceae s.l. are in purple, protein sequences of Boraginaceae s.s. are

764 indicated by red.

765 Abbreviations for the 29 species used in Figures 7 are as follows: At,

766 Arabidopsis thaliana; Ae, Arnebia euchroma; Aa, Artemisia annua; Bv, Beta

767 vulgaris; Cr, Catharanthus roseus; Ep, Echium plantagineum; Ea, Ehretia

Downloaded from on November 29, 2018 - Published by www.plantphysiol.org Copyright © 2018 American Society of Plant Biologists. All rights reserved. 768 acuminata; Gm, Gentiana macrophylla; Ht(u), Helianthus tuberosus; Ht(e),

769 Heliotropium tenellum; Lm, Lennoa madreporoides; Le, Lithospermum

770 erythrorhizon; Mp, Mertensia paniculata; Op, Ophiorrhiza pumila; Os, Oryza

771 sativa; Ph(yd), Persicaria hydropiper; Ph(yb), Petunia hybrida; Pc, Phacelia

772 campanularia; Pk, Picrorhiza kurrooa; Pb, Plectranthus barbatus; Ro,

773 Rosmarinus officinalis; Sf, Salvia fruticosa; Sm(i), Salvia miltiorrhiza; Sm(u),

774 Swertia mussotii; Sa(l), Santalum album; Sa(s), Swertia asarifolia; Tp, Thuja

775 plicata; Vv, Vitis vinifera; G10H, geraniol 10-hydroxylase.

776 Acknowledgements

777 We thank the oneKP project for transcriptomes of Borraginaceae, professor Ye

778 Hechun, Dai Jungui and Lin Wenhan for contributions during early stages of

779 the project, Dr. Liu Juan for help with LSM 880 imaging, Dr. Dai Zhubo for the

780 gift of yeast expression strain BY-T20. This work was supported by National

781 Natural Science Foundation of China (No.81473307, No.81703648,

782 No.81603239), the National Key Research and Development Program of

783 China (2017YFC1700701 and 2017YFC1701405) and the Fundamental

784 Research Funds for the Central Public Welfare Research Institutes

785 (ZZXT201701).

786

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