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FEBS Letters 586 (2012) 1055–1061

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UGT75L6 and UGT94E5 mediate sequential glucosylation of to in jasminoides

Mai Nagatoshi a, Kazuyoshi Terasaka a, Miki Owaki a, Makiko Sota a, Tatsunori Inukai a, Akito Nagatsu b, ⇑ Hajime Mizukami a,

a Graduate School of Pharmaceutical Sciences, Nagoya City University, Japan b School of Pharmacy, Kinjo-Gakuin University, Japan

article info abstract

Article history: Crocin is an glycosyl ester accumulating in fruits of and used as a Received 9 February 2012 food coloring and nutraceutical. For the first time, the two glucosyltransferases UGT75L6 and UGT94E5 Revised 3 March 2012 that sequentially mediate the final glucosylation steps in crocin biosynthesis in G. jasminoides have Accepted 5 March 2012 been identified and functionally characterized. UGT75L6 preferentially glucosylates the carboxyl group Available online 10 March 2012 of crocetin yielding crocetin glucosyl esters, while UGT94E5 glucosylates the 60 hydroxyl group of the Edited by Ulf-Ingo Flügge glucose moiety of crocetin glucosyl esters. The expression pattern of neither UGT75L6 nor UGT94E5 correlated with the pattern of crocin accumulation in G. jasminoides. Ó 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Apocarotenoid Crocin Glucosyltransferase Gardenia jasminoides

1. Introduction cultured C. sativus cells [7] and a cDNA clone (UGTCs2) encoding a crocetin glucosyltransferase was isolated from [8]. How- Crocin is a digentiobiosyl ester of crocetin, an apocarotenoid ever, the recombinant UGTCs2 produced unnatural products with aglycone, and is present [1] in saffron (stigmas of sativus) more than 9 glucose molecules attached to crocetin, suggesting as a yellow pigment together with a series of crocetin glucosyl esters that UGTCs2 is not involved in crocin biosynthesis in planta. (Fig. 1). Crocetin glucosyl esters have been used as a water-soluble Gardenia jasminoides accumulates crocetin glucosyl esters, food coloring and have attracted attention as nutraceuticals owing mostly crocin, in its fruits [1]. In the present investigation, we used to their pharmacological benefits [2,3]. The first step specific to homology-based cloning to isolate and identify UGT75L6 and crocin biosynthesis is the position-specific cleavage of UGT94E5 as apocarotenoid glucosyltransferases involved in the to yield crocetin dialdehyde [4]. Crocetin dialdehyde is then oxi- sequential glucosylation of crocetin to crocin in G. jasminoides. dized to crocetin. Sequential glucosylation of crocetin leads to a final Our finding not only sheds light on novel glucosyltransferases product, crocin (crocetin-di-(b-gentiobiosyl)-ester; crocin-1) as responsible for crocin biosynthesis, but may lead to metabolic shown in Fig. 1. engineering of this commercially important pigment. Among the enzymes participating in crocin biosynthesis, a cDNA clone (CsZCD) encoding zeaxanthin 7,8 (70,80)-cleavage diox- 2. Materials and methods ygenase was isolated from C. sativus [5]. As for the enzymes cata- lyzing glucose conjugation of crocetin, their activity was first 2.1. Plant materials detected in a cell-free extract prepared from callus tissues of C. sat- ivus [6]. Subsequently, a glucosyltransferase, catalyzing the ester Cultured G. jasminoides cells were originally obtained from bond formation between the carboxyl residues of crocetin and seedlings and subcultured as described previously [9]. the glucosyl moiety of UDP-glucose, was partially purified from 2.2. Chemicals Abbreviation: PSPG, plant secondary product glycosyltransferase ⇑ Corresponding author. Address: 3-1 Tanabe-dori, Mizuho-ku, Nagoya 467-8603, 4-Coumaroyl-glucose, caffeoyl-glucose and feruloyl-glucose Japan. Fax: +81 52 836 3415. were kindly provided by Professor Y. Ozeki, Tokyo University of Agri- E-mail address: [email protected] (H. Mizukami).

0014-5793/$36.00 Ó 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.febslet.2012.03.003 1056 M. Nagatoshi et al. / FEBS Letters 586 (2012) 1055–1061

OH

HO zeaxanthin

CHO OHC crocetin dialdehyde

O HO OH O crocetin UGT75L6

O HO HO OH O OH O O OH crocin-5 UGT94E5 UGT75L6 O OH HO HO O O O OH HO HO OH HO O O OH O O HO OH OH O OH O O OH OH crocin-4 O crocin-3 OH UGT75L6 UGT94E5 OH O O HO HO HO O O OH OH OH O O HO O OH OH crocin-2 O OH OH O UGT94E5 HO HO O OH O O HO HO HO O O OH OH OH O O HO O OH OH crocin-1 (crocin) O OH

Fig. 1. Biosynthetic pathway of crocin (crocin-1). UGT75L6 and UGT94E5 were identified in the present investigation. culture and Technology. Crocetin was prepared by sulfuric acid NMR spectroscopy using a JNM a-500 spectrometer (JEOL, Tokyo, hydrolysis of crocin obtained from San-Ei Gen F.F.I., Inc. (Osaka, Ja- Japan). The LC-MS analyses were carried out using a QUATTRO Pre- pan). All other chemicals were of commercial reagent-grade quality. mier XE mass spectrometer (Waters, Milford, MA, USA).

2.3. Biotransformation of crocetin 2.5. PCR cloning and heterologous expression of glycosyltransferase cDNAs Crocetin (3.0 lmol) was dissolved in DMSO and added to cell suspension cultures of G. jasminoides through membrane filters. Full-length cDNA clones encoding glycosyltransferases, their het- Cells were collected by vacuum filtration at an appropriate time, erologous expression, and purification of the N-terminal fusion pro- immediately frozen in liquid nitrogen, and stored at 70 °C until teins with a His6 tag were described in the previous paper [9].AC- use. Extraction and HPLC analysis of the biotransformation product terminal fusion protein was obtained by subcloning an open reading is described in the Supplementary Material. frame (ORF) of a cDNA clone into the pET22b vector (Novagen, Mad- ison, WI, USA) and an Escherichia coli strain, Rosetta-gami2 (Nova- 2.4. Isolation and identification of glucosylation products of crocetin gen), was used as the expression host. Protein content in the enzyme preparations was estimated using the Bradford method [10]. To identify the glucosylation products, the methanol extracts were concentrated to dryness in vacuo. These extracts were sepa- 2.6. Enzyme assay rated by DIAION HP20 (Mitsubishi Chemicals, Tokyo, Japan) and Sephadex LH20 (GE Healthcare, Buckinghamshire, UK) column Glucosyltransferase activities toward crocetin and crocetin chromatographies and the products obtained were further purified glucosyl-esters were screened as described in the Supplementary by preparative HPLC. The isolated products were analyzed by 1H- Material. M. Nagatoshi et al. / FEBS Letters 586 (2012) 1055–1061 1057

2.7. Analysis of gene expression by RT-PCR 3.2. Identification of UDP-glucose:crocetin glucosyltransferase

Total RNA was extracted from cultured cells and organs of G. In addition to 13 full-length cDNAs (GjUGT1–GjUGT13) of family jasminoides using an RNeasy plant mini kit (Qiagen, Hilden, Ger- 1 plant secondary product glycosyltransferases (PSPGs) described many) and a Fruit-mate (Takara, Shiga, Japan). First-strand cDNAs previously [9], we obtained two additional PSPG cDNAs (GjUGT14 for RT-PCR were synthesized from 0.5 lg total RNA using Super- and GjUGT15) by homology-based cloning from cultured G. jasmi- Script III RNase HReverse Transcriptase (Invitrogen, Carlsbad, noides cells. CA, USA). Gene-specific primer sets used for RT-PCR are shown in ORFs of the 15 cDNAs were expressed in E. coli as N-terminal fu-

Supplementary Table S1. The specific annealing of each primer sion proteins with His6 tags. The crude enzyme extracts corre- set to the target cDNA was confirmed by PCR using cDNA clones sponding to GjUGT1–GjUGT14 were used for the glucosyl transfer GjUGT1–GjUGT15 as templates. The 60S acidic ribosomal protein assay using crocetin as a glucose acceptor substrate in the presence P0C primer pair was used as an internal control. Signal intensity of UDP-glucose. The crude enzyme containing the recombinant was analyzed using the Image J software (http://vsb.info.nih.gov/ GjUGT1 or GjUGT14 converted crocetin to two more polar metabo- ij). lites (Fig. 3). The 1H-NMR spectrum of the major product CG5 iso- lated by preparative HPLC was completely identical to that of 2.8. Quantitative determination of crocetin glucosides crocetin-mono-(b-glucosyl)-ester (crocin-5) described previously [1]. The 1H-NMR data are shown in Supplementary Table S2. The Plant tissues of G. jasminoides were ground to a fine powder in minor product was presumably identified as crocin-3 because the liquid nitrogen using a mortar and pestle, and the powdered tis- UV–visible spectrum and the retention time were identical with sues (0.5 g) were sonicated in 1 ml of 70% (v/v) methanol for those of CG3. Neither the other recombinant proteins nor the crude leaves, stems, and flowers, or 70% (v/v) ethanol for fruits. The ex- enzyme preparation from E. coli harboring the control vector pro- tracts were subjected to HPLC analysis by the same procedure as duced glucosylation products from crocetin. described in Section 2.3. The GjUGT1 cDNA contains an ORF corresponding to a protein (UGT75L6) of 474 amino acids with a predicted molecular mass of 3. Results 53.0 kDa. The deduced amino acid sequence of GjUGT14 is 99% iden- tical with that of GjUGT1. UGT75L6 shares the highest (69%) amino 3.1. Crocetin glucosylation by cultured G. jasminoides cells acid identity with NtGT2 from Nicotiana tabacum [11] and UGT73A10 from Lycium barbarum [12], but exhibits only 33% identity with Crocetin was added to cell suspension cultures of G. jasminoides UGTCs2 reported as a crocetin glucosyltransferase in C. sativus [8]. to a final concentration of 0.1 mM at 4 days after cell inoculation, and the cells were cultured for an additional 8 h. At least four 3.3. Functional characterization of UGT75L6 potentially glycosylated products (CG1–CG4) were detected when the methanol extract of G. jasminoides cells supplemented with To investigate the catalytic function of UGT75L6, we tried to crocetin was analyzed by HPLC (Fig. 2). All the products yielded purify the N-terminal His6-tagged protein of UGT75L6 by nickel crocetin when digested with almond b-glucosidase. affinity chromatography. However, UGT75L6 could not be purified These products were purified by column chromatography and in any condition because of the low expression level. We also tried 1 finally isolated by preparative HPLC. The H-NMR spectra of CG1 to create and purify C-terminal His6-tagged UGT75L6 using a and CG2 were completely identical to those of crocetin-di-(b- pET22b vector and the E. coli strain Rosetta-gami2. Although the gentiobiosyl)-ester (crocin-1) and crocetin-(b-glucosyl)-(b-gentio- expression level of UGT75L6 was increased compared with that biosyl)-ester (crocin-2), respectively [1]. The 1H-NMR data are of the N-terminal tagged protein, purification was yet unsuccess- shown in Supplementary Table S2. In the LC–ESI-MS analysis, the ful. Therefore, we used the crude enzyme preparation containing peaks corresponding to CG3 and CG4 exhibited the same dominant the C-terminal-tagged UGT75L6 for further investigation. The glu- ions at m/z 675 (crocetin + glucose 2 + Na). Based on their dom- cosyl-acceptor specificity of UGT75L6 was examined using various inant ions in MS and retention times in HPLC, CG3 and CG4 were substrates shown in Supplementary Fig. S1. HPLC analysis revealed tentatively identified as crocetin-di-(b-glucosyl)-ester (crocin-3), and crocetin-mono-(b-gentiobiosyl)-ester (crocin-4), respectively, A crocetin because the polarity of crocin-3 is higher than that of crocin-4. 5 min 0.15

0.10 0.04 crocetin 0.05 CG5

0.03 0.00 B 0.02 CG2 CG3 30 min 0.15 0.01 CG4 CG1 0.10 0 Absorbance at 440 nm (AU) at 440 Absorbance Absorbance at 440 nm (AU) at 440 Absorbance 0 10 20 [min] 0.05 CG3

0.00 Fig. 2. HPLC profiles of biotransformation products of crocetin from cultured G. 0102030[min] jasminoides cells. Crocetin was added to cell suspension cultures of G. jasminoides and incubated for 8 h. A methanol extract of the collected cells was subjected to Fig. 3. Glucosylation of crocetin by GjUGT14 (UGT75L6). Crocetin was incubated HPLC separation. CG1, CG2, CG3, and CG4 are glucosylation products and identified with GjUGT14 in the presence of UDP-glucose for 5 min (A) and 30 min (B), and the as crocin-1, crocin-2, crocin-3, and crocin-4, respectively. assay mixture was subjected to HPLC analysis. CG5 was identified as crocin-5. 1058 M. Nagatoshi et al. / FEBS Letters 586 (2012) 1055–1061

Fig. 4. Un-rooted molecular phylogenetic tree of PSPGs. PSPGs isolated from G. jasminoides are shown in boldface. UGT75L6 and UGT94E5 are shown in red. The tree was constructed by the neighbor-joining method following multiple alignment using the CLUSTALW program. The bar indicates 0.1 amino acid substitution per site. The accession numbers of PSPGs in the tree are given in Supplementary Table S3. the formation of a product with higher water solubility than the ucts whose UV spectra and retention times were identical to those substrate when 4-coumaric acid, caffeic acid, or ferulic acid was of crocetin gentiobiosyl esters (crocin-1, crocin-2, and crocin-4) in- used as the acceptor substrate. The UV–visible spectra and reten- creased in a time-dependent manner, and crocin-1 accumulated as tion times of the products were identical to those of authentic sam- the sole product after prolonged incubation. No further elongation ples of glucosyl esters of the corresponding phenolic carboxylic of glucose residues was observed. This conversion of crocetin glu- acid (data not shown). No products corresponding to their O-gluco- cosyl esters to crocetin gentiobiosyl esters was not observed when sides were detected from any of the substrates. No activity was de- the recombinant GjUGT15 was added to the reaction mixture. tected when indole-3-acetic acid, , or norbixin was used as the The GjUGT9 cDNA encodes a protein (UGT94E5) of 444 amino glucosyl acceptor. acids with a predicted molecular weight of 49.8 kDa. The amino Kinetic parameters of UGT75L6 for acceptor substrates using acid sequence of UGT94E5 shares 54% identity with a quercetin UDP-glucose as a sugar-donor substrate were determined. The glucoside 1,6-glucosyltransferase from Catharanthus roseus [14]. apparent Km value for crocetin was 0.46 ± 0.19 mM (mean ± stan- dard deviation from triplicate measurements). The apparent Km 3.5. Functional characterization of UGT94E5 values for phenolic carboxylic acids (1.5 ± 0.38 mM for 4-coumaric acid, 2.5 ± 0.80 mM for caffeic acid, and 1.7 ± 0.22 mM for ferulic Kinetic parameters of UGT94E5 for crocetin glucosyl esters acid) were at least threefold higher than that for crocetin. UGT75L6 using UDP-glucose as the sugar-donor substrate were determined

(GjUGT1) exhibited the highest specific activity toward crocetin, using the affinity-purified protein (Table 1). The apparent Km val- followed by 4-coumaric acid (26% of crocetin), ferulic acid (17%), ues for crocin-5 (crocetin monoglucosyl ester) and crocin-3 (croce- and caffeic acid (13%). tin diglucosyl ester) were 0.072 and 0.023 mM, respectively. The

kcat/Km ratio for crocin-3 was sixfold higher than that for crocin- 3.4. Identification of UDP-glucose: crocetin glucosyl ester 5. We also tested the glucosylation activity of UGT94E5 on various glucosyltransferase naturally occurring glucosides such as flavonoid glucosides and coumarin glucosides as well as unnatural glucosides such as curcu- Molecular phylogenetic analysis of 15 PSPGs isolated from G. min glucosides and 4-nitrophenyl glucoside. The chemical struc- jasminoides indicated that GjUGT9 and GjUGT15 belong to Group tures of these compounds are shown in Supplementary Fig. S2. A(Fig. 4). Since PSPGs in this group catalyze sugar chain elongation Except that weak glucosylation activity toward curcumin gluco- of flavonoid glycosides or anthocyanins [13], we predicted that sides was detected by HPLC analysis, none of these compounds GjUGT9 and/or GjUGT15 exhibit glucosyltransferase activity to- served as substrates for UGT94E5. ward crocetin glucosyl esters. We examined the catalytic activity of GjUGT9 and GjUGT15 to- 3.6. Expression of the UGT75L6 and UGT94E6 gene in G. jasminoides ward crocetin glucosyl esters produced by UGT75L6 (Fig. 5). When recombinant GjUGT9 was added to the reaction mixture, the peaks To compare the expression levels of UGT75L6 and UGT94E5 corresponding to crocetin glucosyl esters decreased rapidly. Prod- among various G. jasminoides tissues, we examined the mRNA lev- M. Nagatoshi et al. / FEBS Letters 586 (2012) 1055–1061 1059

A 0.06 0 min crocin-3

0.04 crocin-5

0.02

0.00 B 0.03 15 min

0.02 crocin-2

0.01 crocin-1 crocin-4

0.00

Absorbance at 440 nm (AU) at 440 Absorbance 0.03 C 60 min

0.02

0.01

0.00 0102030[min]

Fig. 5. Glucosylation of crocetin glucosyl-esters by GjUGT9 (UGT94E5). First, crocin-3 and crocin-5 were generated by incubating crocetin and UDP-glucose with UGT75L6 for 30 min (A). Subsequently, recombinant UGT94E5 was added to the reaction mixture which was further incubated for 15 min (B), and 60 min (C). The reaction was terminated by adding methanol and glucosylation products were analyzed by HPLC.

Table 1 ases (PSPGs) involved in modification of secondary metabolites Kinetic parameters of recombinant UGT94E5 toward crocetin glucosyl esters.a are characterized by the presence of a 44-amino acid C-terminal 1 1 1 Km (mM) kcat (s ) kcat/Km (mM s ) signature motif designated as the PSPG box [17]. In a previous Crocin-5 0.072 ± 0.025 0.036 ± 0.0068 0.51 ± 0.071 investigation [9], we obtained 13 cDNA clones designated Crocin-3 0.023 ± 0.001 0.071 ± 0.0043 3.1 ± 0.11 GjUGT1–GjUGT13 encoding PSPGs by homology-based cloning a Data represent means ± standard deviations from triplicate measurements. using the consensus sequences within the PSPG box from cultured G. jasminoides cells, and identified GjUGT2 as an iridoid-specific glu- cosyltransferase involved in geniposide biosynthesis. G. jasminoides accumulates crocetin glycosyl esters in the fruits together with irid- els of GjUGT1, GjUGT14, and GjUGT9 by semi-quantitative RT-PCR oid glucosides, and cultured G. jasminoides cells efficiently convert (Fig. 6A and B). mRNA expression of the three PSPGs was detected crocetin to its glucosides. Therefore, we screened the glucosyltrans- in all the organs of G. jasminoides. The expression level of GjUGT1 ferase activity of the recombinant proteins obtained from the cDNA was highest in the cultured cells and lowest in the stem, whereas clones GjUGT1–GjUGT13 and two additional clones GjUGT14 and that of GjUGT14 was lowest in the cultured cells. GjUGT15 toward crocetin and crocetin glucosyl esters, and identi- Crocetin glucosyl ester contents in G. jasminoides fruits at vari- fied two glucosyltransferases, UDP-glucose:crocetin glucosyltrans- ous developmental stages were quantitatively determined ferase (GjUGT1 and GjUGT14; UGT75L6) and UDP-glucose:crocetin (Fig. 6C). Apocarotenoid content gradually increased in parallel glucosyl ester glucosyltransferase (GjUGT9; UGT94E5) that cata- with fruit development. Crocin-1 was the predominant crocetin lyze the sequential glucosylation of crocetin to crocin (crocin-1). glucosyl ester in G. jasminoides fruits irrespective of the develop- Molecular phylogenetic analysis revealed that UGT75L6 be- mental stage. There was no significant correlation between the longed to Group L of PSPGs. UGT75L6 catalyzed the transfer of a mRNA levels of the three PSPGs and the pigment accumulation glucose moiety of UDP-glucose to the free carboxyl group of croce- during fruit development. Crocetin glucosyl esters were detected tin to yield crocetin monoglucosyl ester (crocin-5) and crocetin in neither flowers, leaves, nor stems. diglucosyl ester (crocin-3), but no product was detected when crocetin diglucosyl ester (crocin-3) was used as the acceptor sub- 4. Discussion strate. UGT75L6 failed to glucosylate the closely related apocarot- enoid aglycones bixin and norbixin but showed weak activity Glycosyltransferases constitute an enzyme superfamily respon- toward some phenolic carboxylic acids such as 4-coumaric acid, sible for attaching sugar moieties to a wide array of acceptor sub- caffeic acid, and ferulic acid. The glucosylation products were strates including carbohydrates, proteins, lipids and secondary exclusively glucosyl esters of these phenolic carboxylic acids and metabolites [15,16]. Plant secondary metabolite glycosyltransfer- no O-glucosides were formed. The regioselectivity of UGT75L6 is 1060 M. Nagatoshi et al. / FEBS Letters 586 (2012) 1055–1061

A

3.0 GjUGT1 2.5 GjUGT14

2.0

1.5

1.0 UGT75L6 mRNA level mRNA UGT75L6 0.5 (Relative value: value: Stage (Relative 1=1.0)

0.0 S1 S2 S3 S4 S5 S6 L F St C B 3.5 3.0

2.5

2.0

1.5

1.0 UGT94E5 mRNA mRNA level UGT94E5

(Relative value: value: (Relative Stage 1=1.0) 0.5

0.0 S1 S2 S3 S4 S5 S6 L F St C C 1.4 crocin-1 1.2 crocin-2 1.0 /g)

6 crocin-3

10 0.8 × × crocin-4 0.6 crocin-5

0.4 (Peak Area

Crocetin glucosyl Crocetin glucosyl esters 0.2

ND ND ND ND 0 S1 S2 S3 S4 S5 S6 L F S C

Fig. 6. Temporal and tissue distribution of UGT75L6 (GjUGT1 and GjUGT14) and UGT94E5 (GjUGT9) mRNA (A and B) and crocetin glucosyl esters (C) in G. jasminoides. Fruits, leaves, and stems were collected from a single G. jasminoides plant on the same day, powdered in liquid nitrogen, and used for mRNA preparation and HPLC analysis of crocetin glucosyl esters. The fruits were classified into six maturation stages based on their red coloration. Each box and bar represent an average value with standard deviation from triplicate measurements. consistent with the previous finding that many PSPGs belonging to the PSPGs responsible for the glucosylation of crocetin during Group L catalyze the regioselective glycosylation of carboxylic crocin biosynthesis in saffron remain unidentified, though no sim- acids [18]. The Km value of UGT85A24 for crocetin (0.46 mM) is ilar sequence was found using UGT75L6 sequence as a query in the same range of the Km values of other glucosyltransferases against the EST database constructed from saffron [21]. belonging to Group L such as indole-3-acetic acid glucosyltransfer- UGT94E5 is a new member of sugar–sugar glycosyltransferases ase of Arabidopsis (0.24 mM) and cinnamic acid glucosyltransferase and catalyzes the b1 ? 6 glucosylation of the sugar moiety of from strawberry (0.36 mM) [19,20]. UGT75L6 shares only 35% crocetin glucosyl esters. Among naturally occurring glucoside sub- identity with C. sativus UGTCs2, which produces unnatural crocetin strates, UGT94E5 specifically catalyzed glucosyl chain elongation glucosyl esters with at least 9 glucose residues. This suggests that toward crocetin glucosyl esters. The kinetic analysis exhibited that M. Nagatoshi et al. / FEBS Letters 586 (2012) 1055–1061 1061 crocin-3 is a more preferable glucose accepting substrate of References UGT94E5 than crocin-5. Crocin-2 was also efficiently converted to crocin-1 by UGT94E5 although the kinetic parameters could [1] Pfister, S., Meyer, P., Steck, A. and Pfander, H. (1996) Isolation and structure elucidation of glycosyl esters in Gardenia fruits (Gardenia not be determined because of the limited supply of the substrate jasminoides Ellis) and saffron (Crocus sativus Linne). J. Agric. Food Chem. 44, (data not shown). To our interest, the activity of UGT94E5 was 2612–2615. inhibited by crocin-5 above 0.2 mM while such substrate inhibi- [2] Yamauchi, M., Tsuruma, K., Imai, S., Nakanishi, T., Umigai, N., Shimazawa, M. tion was not observed for crocin-3. All these results may indicate and Hara, H. (2011) Crocetin prevents degeneration induced by oxidative and endoplasmic reticulum stresses via inhibition of caspase that most of crocin-5 is converted not to crocin-4 but to crocin-3 activity. Eur. J. Pharmacol. 650, 110–119. which is then rapidly glucosylated to the final product crocin-1. [3] Ghadrdoost, B., Vafaei, A.A., Rashidy-Pour, A., Hajisoltani, R., Bandegi, A.R., Although UGT75L6 and UGT94E5 were isolated from cell sus- Motamedi, F., Haghighi, S., Sameni, H.R. and Pahlvan, S. (2011) Protective effects of saffron extract and its active constituent crocin against oxidative pension cultures of G. jasminoides, RT-PCR experiments clearly stress and spatial learning and memory deficits induced by chronic stress in showed that both genes are expressed in various organs of intact rats. Eur. J. Pharmacol. 667, 222–229. G. jasminoides . Their expression levels vary somewhat [4] Pfander, H. and Schurtenberger, H. (1982) Biosynthesis of C20- in Crocus sativus. Phytochemistry 21, 1039–1042. among organs but are not correlated with the accumulation of cro- [5] Bouvier, F., Suire, C., Mutterer, J. and Camara, B. (2003) Oxidative remodeling cin pigment, suggesting that these are not rate-limiting enzymes in of chromoplast carotenoids; Identification of the carotenoid dioxygenase crocin biosynthesis in the plants. Availability of the apocarotenoid CsCCD and CsZCD genes involved in Crocus secondary metabolite biogenesis. Plant Cell 15, 47–62. substrate (crocetin or crocetin dialdehyde) of the glucosyltransfe- [6] Dufrense, C., Cormier, S. and Dorion, S. (1997) In vitro formation of crocetin rases may limit crocin biosynthesis in planta. In fact, zeaxanthin glucosyl esters by Crocus sativus L. callus extract. Planta Med. 63, 150–153. cleavage dioxygenase (CsZCD) is expressed specifically in [7] Côté, F., Cormier, F., Dufrense, C. and Willemot, C. (2001) A highly specific glucosyltransferase is involved in the synthesis of crocetin glucosylesters in of C. sativus [5]. Crocus sativus cultured cells. J. Plant Physiol. 158, 553–560. The present investigation unambiguously demonstrated that [8] Moraga, A.R., Nohales, P.F., Pérez, J.A. and Gómez-Gómez, L. (2004) crocetin is sequentially glucosylated to crocin in fruits of Glucosylation of the saffron apocarotenoid crocetin by a glucosyltransferase G. jasminoides by two distinct UGTs. It is interesting to note that isolated from Crocus sativus stigmas. Planta 219, 955–966. [9] Nagatoshi, M., Terasaka, K., Nagatsu, A. and Mizukami, H. (2011) Iridoid- the WoLF PSORT algorithm [22] predicted that UGT75L6 is most specific glucosyltransferase from Gardenia jasminoides. J. Biol. Chem. 286, likely localized in the chloroplast and UGT94E5 in the cytoplasm. 32866–32874. Because mature fruits have no chloroplasts, chromoplasts are the [10] Bradford, M.M. (1976) A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. most likely location of UGT75L6 in developing G. jasminoides fruits. Anal. Biochem. 72, 248–254. Recently, CsCCD4, an isoform of carotenoid cleavage dioxygenase [11] Taguchi, G., Ubukata, T., Hayashida, N., Yamamoto, H. and Okazaki, M. (2003) most closely related to the previously isolated CsZCD from C. sati- Cloning and characterization of a glucosyltransferase that reacts on 7- hydroxyl group of flavonol and 3-hydroxyl group of coumarin from tobacco vus, was found to be targeted to the plastoglobules of plastids in cells. Arch. Biochem. Biophys. 420, 95–102. saffron [23]. We assumed that crocetin is formed from zeaxanthin [12] Noguchi, A., Sasaki, N., Nakao, M., Fukami, H., Takahashi, S., Nishino, T. and and converted to crocetin glucosyl esters by UGT75L6 in chromop- Nakayama, T. (2008) cDNA cloning of glycosyltransferases from Chinese wolfberry (Lycium barbarum L.) fruits and enzymatic synthesis of a catechin lasts, the esters are then glucosylated to gentiobiosyl esters glucoside using a recombinant enzyme (UGT73A10). J. Mol. Catal. B Enzyme (mostly crocin-1) in the cytoplasm by UGT94E5, and these product 55, 84–92. esters are finally accumulated in vacuoles. Cellular localization of [13] Bowles, D., Isayenkova, L., Lim, E.K. and Poppenberger, B. (2005) Glycosyltransferases of lipophilic small molecules. Curr. Opin. Plant Biol. 8, these PSPGs awaits further investigation. 254–263. Carotenoid cleavage dioxygenases with various substrate and [14] Masada, S., Terasaka, K., Oguchi, Y., Okazaki, S., Mizushima, T. and Mizukami, regioselectivities have been isolated from a wide array of higher H. (2009) Functional and structural characterization of a flavonoid glucoside plants including C. sativus [24]. We have recently isolated a cDNA 1,6-glucosyltransferase from Catharanthus roseus. Plant Cell Physiol. 50, 1401– 1415. clone encoding crocetin dialdehyde dehydrogenase from [15] Thibodeaux, C.J., Melançon, C.E. and Liu, H. (2007) Unusual sugar biosynthesis G. jasminoides (unpublished results). The isolation and identifica- and natural product glycodiversification. Nature 446, 1008–1016. tion of UGT75L6 and UGT94E5 as crocetin glucosyltransferases that [16] Gantt, R.G., Peltier-Pain, P., Cournoyer, W.J. and Thorson, J.S. (2011) Using simple donors to drive the equilibria of glycosyltransferases-catalyzed we have described will contribute not only to our basic under- reactions. Nature Chem. Biol. 7, 685–691. standing of the crocin biosynthesis pathway but also, in combina- [17] Hughes, J. and Hughes, M.A. (1994) Multiple secondary plant product UDP- tion with carotenoid dioxygenases and apocarotenoid dialdehyde glucose glucosyltransferase genes expressed in cassava (Manihot esculenta Crantz) cotyledons. DNA Seq. 5, 41–49. dehydrogenase, will open the way for the production of structur- [18] Lim, E.-K., Doucet, C.J., Li, Y., Elias, L., Worrall, D., Spencer, S.P., Ross, J. and ally modified apocarotenoid glucosyl esters of high pharmacologi- Bowles, D.J. (2002) The activity of Arabidopsis glycosyltransferases toward cal and commercial value. salicylic acid, 4-hydroxybenzoates, and other benzoates. J. Biol. Chem. 277, 586–592. [19] Jackson, R.G., Lim, E.-K., Li, Y., Kowalczyk, M., Sandberg, G., Hpggett, J., Ashford, Acknowledgments D.A. and Bowles, D.J. (2001) Identification and biochemical characterization of an Arabidopsis indole-3-acetic acid glucosyltransferase. J. Biol. Chem. 276, 4350–4356. The authors wish to thank Mr. R. Shimizu, San-Ei Gen F.F.I., and [20] Lunkenbein, S., Bwllido, M., Aharoni, A., Salentijn, E.M.J., Kaldenhoff, R., Coiner, Professor Y. Ozeki, Tokyo University of Agriculture and Technology, H.A., Muñoz-Blanco, J. and Schwab, W. (2006) Cinnamate metabolism in for the generous gift of crocin and authentic standards of phenolic ripening fruit. Characterization of a UDP-glucose:cinnamate carboxylic acid glucosyl esters, respectively. The present study glucosyltransferase from strawberry. Plant Physiol. 140, 1047–1058. [21] D’Agostino, N., Pizzichini, D., Chiusano, M.L. and Giuliano, G. (2007) An EST was supported by a Grant-in-Aid for Scientific Research and a database from saffron stigmas. BMC Plant Biol. 7, 53. Grant-in-Aid for Young Scientists from the Ministry of Education, [22] Horton, P., Park, K.-J., Obayashi, T., Fujita, N., Harada, H., Adams-Collier, C.J. and Culture, Sports, Science and Technology of Japan. Nakai, K. (2007) WoLF PSORT: protein localization predictor. Nucleic Acids Res. 35, W585–W587. [23] Rubio, A., Rambla, J.L., Santaella, M., Gómez, M.D., Orzaez, D., Granell, A. and Appendix A. Supplementary data Gómez-Gómez, L. (2008) Cytosolic and plastglobule-tragetted carotenoid dioxygenases from Crocus sativus are both involved in b- release. J. Biol. Chem. 283, 24816–24825. Supplementary data associated with this article can be found, in [24] Ohmiya, A. (2009) Carotenoid cleavage dioxygenases and their apocarotenoid the online version, at doi:10.1016/j.febslet.2012.03.003. products in plants. Plant Biotechnol. 26, 351–358.