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Vacuole-Localized Berberine Bridge Enzyme-LikeProteins Are Required for a Late Step of NicotineBiosynthesis in Tobacco1[C][W]

Masataka Kajikawa, Tsubasa Shoji, Akira Kato, and Takashi Hashimoto*

Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara 630–0192,Japan

Tobacco (Nicotiana tabacum) plants synthesize nicotine and related pyridine-type alkaloids, such as anatabine, in their roots andaccumulate them in their aerial parts as chemical defenses against herbivores. Herbivory-induced jasmonate signalingactivates structural genes for nicotine biosynthesis and transport by way of the NICOTINE (NIC) regulatory loci. Thebiosynthesis of tobacco alkaloids involves the condensation of an unidentified nicotinic acid-derived metabolite with theN-methylpyrrolinium cation or with itself, but the exact enzymatic reactions and enzymes involved remain unclear. Here, wereport that jasmonate-inducible tobacco genes encoding flavin-containing oxidases of the berberine bridge enzyme family(BBLs) are expressed in the roots and regulated by the NIC loci. When expression of the BBL genes was suppressed in tobaccohairy roots or in tobacco plants, nicotine production was highly reduced, with a gradual accumulation of a novel nicotinemetabolite, dihydromethanicotine. In the jasmonate-elicited cultured tobacco cells, suppression of BBL expression efficientlyinhibited the formation of anatabine and other pyridine alkaloids. Subcellular fractionation and localization of greenfluorescent protein-tagged BBLs showed that BBLs are localized in the vacuoles. These results indicate that BBLs are involvedin a late oxidation step subsequent to the pyridine ring condensation reaction in the biosynthesis of tobacco alkaloids.

Tobacco (Nicotiana tabacum) and other Nicotiana spe-cies synthesize nicotine to mount defensive responsesto insect herbivores. Because the activation of acetyl-choline receptors is inherently toxic to all heterotrophswith neuromuscular junctions, nicotine is thoughtto be a broadly effective plant defense metabolite(Baldwin, 2001). A reduction in the amount of nicotinein the tobacco leaf, due to either natural variation(Jackson et al., 2002) or genetic manipulation (Steppuhnet al., 2004), results in more pronounced damage byinsect herbivores. Tobacco plants sense browsing in-sects on their leaves and increase the de novo synthesisof nicotine by utilizing the general jasmonate signalingpathway and nicotine-specific regulatory components(Shoji and Hashimoto, 2011). The highly bioactiveIle conjugate of jasmonate is perceived by a complexof CORONATINE INSENSITIVE1 and the transcrip-tional repressor JAZ proteins (Sheard et al., 2010),resulting in JAZ degradation and a subsequent release

of transcription repression (Chini et al., 2007). Tobaccoorthologs of these signaling proteins are utilized intobacco to regulate jasmonate-inducible nicotine bio-synthesis (Shoji et al., 2008). As reported recently (Shojiet al., 2010), several tobacco transcription factors, in-cluding ERF189, of an ethylene response factor (ERF)subfamily are encoded in clustered genes at the nico-tine regulatory locus NICOTINE2 (NIC2) and bind tothe GCC box element in the promoter of the putrescineN-methyltransferease gene (PMT; Hibi et al., 1994).ERF189 and related tobacco transcription factors areshown to directly and specifically activate all knownstructural genes in the nicotine pathway (Shoji et al.,2010).

The early enzymatic steps of nicotine biosynthesisare well established (Supplemental Fig. S1). The pyr-rolidine ring of nicotine is derived from putrescine, viaconsecutive reactions involving the enzymes PMTandN-methylputrescine oxidase (Heim et al., 2007; Katohet al., 2007), which together produce the N-methyl-pyrrolinium cation, whereas the pyridine ring usesenzymes involved in the early steps of NAD bio-synthesis, such as Asp oxidase, quinolinic acid syn-thase, and quinolinic acid phosphoribosyl transferase(Sinclair et al., 2000; Katoh et al., 2006). The genes forthese five enzymes are coordinately regulated in to-bacco roots (Shoji et al., 2010). When isotopicallylabeled nicotinic acids were fed to tobacco plants, theisotope labels were incorporated into the pyridine ringof nicotine (Dawson et al., 1956). Interestingly, thetritium label at C-6 of nicotinic acid was specificallylost in isolated nicotine (Dawson et al., 1960; Leete and

1 This work was supported by the Global Center of Excellenceprogram of NAIST (Frontier Biosciences: Strategies for Survival andAdaptation in a Changing Global Environment) from theMinistry ofEducation, Sports, Science, and Technology, Japan.

* Corresponding author; e-mail [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policydescribed in the Instructions for Authors (www.plantphysiol.org) is:Takashi Hashimoto ([email protected]).

[C] Some figures in this article are displayed in color online but inblack and white in the print edition.

[W] The online version of this article contains Web-only data.www.plantphysiol.org/cgi/doi/10.1104/pp.110.170878

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Liu, 1973), suggesting that the C-6 position is firstoxidized and subsequently reduced during the incor-poration of nicotinic acid into nicotine. The putativeoxidoreductases involved in the activation of nicotinicacid and the mechanisms behind the nicotine-formingcondensation reactions between a nicotinic acid-derived intermediate and the N-methylpyrroliniumcation are yet to be clarified. A pinoresinol-lariciresinolreductase/isoflavone reductase/phenylcoumaran ben-zylic ether reductase family oxidoreductase, A622, isrequired for the biosynthesis of tobacco alkaloids, pos-sibly in a step to produce a nicotinic acid-derivedprecursor, but the exact enzymatic reaction catalyzedby A622 is not known (Deboer et al., 2009; Kajikawaet al., 2009).Besides nicotine, other pyridine alkaloids also accu-

mulate at substantial levels in tobacco roots, in elicitedcultured tobacco cells, and in wild Nicotiana species(Supplemental Fig. S1; Saito et al., 1985; Shoji andHashimoto, 2011). Anatabine is synthesized by thedimerization of a metabolite of nicotinic acid (Leeteand Slattery, 1976), whereas the piperidine ring ofanabasine is derived from Lys by way of cadaverine(Watson et al., 1990). Anatalline consists of two pyridylrings and a central piperidine ring (Hakkinen et al.,2004). The biosynthesis of tobacco alkaloids consistingof more than two six-membered heterocyclic ringsmay involve the same ring condensation reactionsas predicted for the production of nicotine. Indeed,suppression of A622, an enzyme proposed to forma coupling-competent nicotinic acid intermediate, intobacco roots and cultured tobacco cells inhibited theformation of not only nicotine but also other pyridinealkaloids (Kajikawa et al., 2009). Nornicotine is formedfrom nicotine by cytochrome P450 monooxygenases ofthe CYP82E subfamily in tobacco leaf tissues (Siminszkyet al., 2005; Xu et al., 2007; Lewis et al., 2010), but thepathway of nornicotine biosynthesis in other cell typesis not established.In this study, we identified vacuole-localized to-

bacco flavoproteins, berberine bridge enzyme-likeproteins (BBLs), that were required for the synthesisof nicotine, anatabine, anabasine, and anatalline. Theaccumulation of a novel pyridine alkaloid in BBL-suppressed tobacco root tissues suggests that BBLs areinvolved in a late, possibly the final, step of tobaccoalkaloid biosynthesis. The dimerization of a pyridinering with an unsaturated pyrrolidine or piperidinering appears to involve multiple oxidoreductases ofdifferent families.

RESULTS

Isolation of NIC-Regulated BBL Genes from Tobacco

To identify novel genes that are controlled by theNIC regulatory loci, we compared comprehensivecDNA expression profiles of wild-type tobacco rootsand nic1nic2 double mutant roots obtained using to-

bacco oligonucleotide microarrays (Katoh et al., 2007;Shoji et al., 2010). One tobacco gene (BBLa) encoding aBBL was identified as a putative target of the NICregulatory loci. After searching the tobacco genomedatabase (http://solgenomics.net/) and extensive PCRscreening and sequencing, we recovered four tobaccoBBL cDNAs encoding full-length proteins (BBLa, BBLb,BBLc, and BBLd). BBLb has been reported as ajasmonate-inducible gene in cultured tobacco BY-2 cells(Goossens et al., 2003). BBLa is 94%, 83%, and 63%identical in amino acid sequence to BBLb, BBLc, andBBLd, respectively (Supplemental Fig. S2). These tobaccoBBLs constitute a distinct clade in the FAD-containingoxidoreductase family that includes berberine bridgeenzymes (BBEs), carbohydrate oxidases, cannabinoidsynthases, and 6-hydroxynicotine oxidases (Fig. 1).Tobacco BBL genes contained no introns. Genomic PCRusing primers specific to each BBL gene amplified BBLa

Figure 1. Phylogenetic tree of BBE-like proteins. Shown is an unrootedneighbor-joining phylogenetic tree of tobacco BBE-like proteins (NtBBLs)and of related proteins belonging to four subgroups: BBEs, 6-hydroxy-nicotine oxidases, cannabinoid synthases, and carbohydrate oxidases.The tree was generated using MEGA4 software (Tamura et al., 2007)with the neighbor-joining algorithm. Bootstrap values (1,000 replicates)are indicated at branch nodes, and the scale bar indicates the numberof amino acid substitutions per site. Species are as follows: NtBBLa(N. tabacum), NtBBLb (N. tabacum), NtBBLc (N. tabacum), NtBBLd(N. tabacum), EcBBE (Eschscholzia californica), BsBBE (Berberis stolo-nifera), PsBBE (Papaver somniferum), TfBBE (Thalictrum flavum),AoHDNO (Arthrobacter oxidans), AnHDNO (Arthrobacter nicotino-vorans), CsCBDAS (C. sativa), CsTHCAS (C. sativa), HaCHOX (Heli-anthus annuus), LsCHOX (Lactuca sativa), and NspNEC5 (Nicotianalangsdorffii 3 Nicotiana sanderae).

Novel Tobacco Proteins Required for Nicotine Biosynthesis

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and BBLc from the genomic DNA of N. tabacumand Nicotiana sylvestris and BBLb and BBLd from thatof N. tabacum and Nicotiana tomentosiformis (Fig. 2A).Therefore, the diploid progenitors of N. tabacum pos-sess two related BBL genes: BBLa and BBLc probablyoriginate from N. sylvestris, whereas BBLb and BBLdmay be derived from N. tomentosiformis.

Expression profiles of BBLswere analyzed by reversetranscription (RT)-PCR. By using specific primers toamplify each BBL gene, we showed that BBLa, BBLb,and BBLc are expressed in wild-type tobacco roots,but BBLd mostly is not (Fig. 2B). Levels of BBLa to -cexpression were markedly low in the nic1nic2 mutantroots, indicating that they were positively regulatedby the NIC loci. Quantitative RT-PCR using a commonprimer set that amplified all BBL genes was nextconducted to examine tissue-specific and jasmonate-inducible expression patterns. BBLs were expressedstrongly in the root but at very low or negligible levelsin the stem and leaf of wild-type tobacco plants (Fig.2C). In the nic1nic2mutant roots, the abundance of theBBL transcripts was reduced by 80% (Fig. 2C). Theapplication of 50 mM methyl jasmonate (MeJA) in-creased BBL expression 4- to 5-fold in the wild-typetobacco roots (Fig. 2D) and strongly induced BBL ex-pression from an initially very low level within 30 minin cultured tobacco BY-2 cells (Fig. 2E).When the expres-

sion of the NIC2-locus ERF genes, which specificallyactivated all known structural genes of nicotine bio-synthesis (Shoji et al., 2010), was suppressed by theconstitutive expression of a dominant repressive formof ERF189 in two transgenic tobacco root lines (D1 andD2; Shoji et al., 2010), expression of the BBL genes waseffectively reduced by more than 80% (Fig. 2F). Fur-thermore, overexpression of ERF189 in three trans-genic tobacco root lines (OE9, OE10, and OE11; Shojiet al., 2010) resulted in 2- to 3-fold increases in thelevels of the BBL transcripts (Fig. 2G). These expres-sion patterns of BBLs are highly similar to those ofstructural genes involved in the biosynthesis and trans-port of nicotine (Hibi et al., 1994; Reed and Jelesko,2004; Cane et al., 2005; Katoh et al., 2007; Kajikawa et al.,2009; Shoji et al., 2009, 2010).

Constitutive BBL Knockdown in Tobacco Hairy Roots

To investigate their possible roles in the biosynthe-sis of tobacco alkaloids, we first suppressed the ex-pression of BBL genes constitutively with RNAinterference (RNAi) in tobacco hairy roots, using thecauliflower mosaic virus 35S promoter. Three inde-pendent transgenic lines (KR1, KR2, and KR3) wereobtained in which the transcript levels of BBLa, BBLb,and BBLc were highly reduced (Fig. 3A). When the

Figure 2. Expression profiles of tobacco BBL genes. A and B, PCR primers specific to each BBL member were used, and thetobacco a-Tubulin gene was amplified as a control. A, Genomic PCR analysis of N. tabacum (Nta) and its probable progenitors,N. sylvestris (Nsy) and N. tomentosiformis (Nto). B, Transcript levels of each BBL gene were assessed by RT-PCR in the roots ofwild-type (WT) and nic1nic2 mutant (nic) tobacco plants. C to G, Quantitative RT-PCR analysis of BBL genes using BBL-consensus PCR primers. Transcript levels are shown as relative values. C, Organ-specific expression pattern in the wild-type plantand expression level in the nic root. NT, Not detectable. D, Treatment of tobacco roots with 100 mMMeJA for 24 h. E, Treatment ofcultured tobacco cells with 50 mM MeJA for the periods indicated. F, BBL transcript levels in cultured tobacco roots of the VC lineand two transgenic lines expressing a dominant-negative ERF189 form (ERF189-EAR, D1 and D1; Shoji et al., 2010). G, BBLtranscript levels in cultured tobacco roots of the VC line and three transgenic lines overexpressing ERF189 (OE9, OE10, andOE11; Shoji et al., 2010).

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accumulation of BBL proteins was analyzed by im-munoblotting using the BBL-specific antiserum, thewild-type and two vector control (VC) hairy root linesshowed signals for BBLs of 58 kD (isoform I) and 53 kD(isoform II), whereas the three KR lines did not (Fig.3B). Since the predicted full-length forms of BBLa,BBLb, and BBLc are 62, 63, and 62 kD, respectively,BBLs are processed in tobacco cells to yield twosmaller protein species. Suppression of BBL expres-sion did not affect the growth of the hairy roots.Tobacco alkaloids consisted mostly of nicotine, in

addition to a smaller amount of nornicotine, in thewild-type and VC hairy roots (Fig. 3C). Nicotine levelswere much lower in the KR lines (1–3 mmol g21 dryweight) than the wild-type and VC lines (13–16 mmolg21 dry weight). In contrast, nornicotine levels wereslightly higher in the KR lines (4–6 mmol g21 dryweight) than the wild-type and VC lines (2 mmol g21

dry weight). Interestingly, an unknown compoundwas detected in the KR lines at a retention time of 19.7min in the gas-liquid chromatograms (Fig. 4A). Thisnovel metabolite was identified as dihydrometanico-tine (DMN; Fig. 4C) by comparing its retention time inthe gas-liquid chromatograms (Fig. 4A), its mass spectra(Fig. 4B), and its mobility shift in the thin-layer chro-matograms (Fig. 5C), with the authentic compound.DMN has been isolated as a catabolite of nicotinein mammals (De Clercq and Truhaut, 1962; Neurathet al., 1966) but had not been found in plants. DMNwas only detectable in the KR lines, where it accumu-lated at levels comparable to nicotine (2–3 mmol g21

dry weight; Fig. 3C).

Inducible BBL Knockdown in Tobacco Hairy Roots

To examine the time courses of the reduction innicotine and the accumulation of DMN after suppres-sion of the BBL genes, we used an inducible expressionsystem (the XVE-b-estradiol system; Zuo et al., 2000)to drive RNAi-based gene suppression. Two indepen-dent tobacco hairy root lines (iKR1 and iKR2) wereobtained in which expression of the BBL genes wasefficiently suppressed after the addition of the inducerb-estradiol to the culture medium. In the iKR1 line, thelevels of the BBL proteins began to decrease after 2 d oftreatment and were very low by day 3 (Fig. 5A). Wedid not observe inhibitory effects of the inducer onroot growth in the iKR lines or on the accumulation oftobacco alkaloids in wild-type and vector control roots(Kajikawa et al., 2009). When the iKR line was treatedwith the inducer, the formation of nicotine continuedfor 2 d and then stopped, whereas the untreated iKRroots continued to synthesize nicotine (Fig. 5B). DMNbecame detectable after 2 d of treatment and continuedto increase thereafter. It should be noted, however, thatthe accumulation of DMN was very small comparedwith the suppressed level of nicotine synthesis. Forexample, at day 7, the production of as much as 17mmol g21 dry weight of nicotine was estimated to beinhibited by the suppression of BBLs, but DMNmerely accumulated to a level of 0.5 mmol g21 dryweight. Similar results were obtained in another in-ducible BBL-knockdown line, iKR2 (Supplemental Fig.S3). When the alkaloid extracts from iKR hairy rootswere analyzed by thin-layer chromatography, twometabolites with RF values of 0.01 and 0.07, in addition

Figure 3. Down-regulation of BBL genes in trans-genic tobacco roots. Tobacco hairy roots of thewild type (WT), two vector-transformed controllines (VC1 and VC2), and three RNAi lines (KR1,KR2, and KR3) were analyzed. A, RT-PCR analysisof BBLa, BBLb, and BBLc as well as the controla-Tubulin gene. B, Immunoblot analysis using theantisera against BBL and PEPC (loading control).BBLs existed in two forms with different molec-ular masses (I, 60 kD; II, 53 kD). C, Alkaloidcontents of hairy roots. Data indicate mean values6 SD from three biological replicates. ND, Notdetected.





to DMN (RF of 0.11), were detected using Dragen-dorff’s reagent after the suppression of BBLs (Fig. 5C).Although the chemical structures of these metaboliteswere not determined, their reactivity toward Dragen-dorff’s reagent suggests that they contain nitrogen.These two compounds also accumulated in the BBL-suppressed lines KR1 to -3 but not in the untreated iKRlines, VC lines, or wild-type tobacco roots (data notshown). Thus, at least three metabolites, includingDMN, accumulate in tobacco cells as a consequence ofthe BBL suppression.

BBL Knockdown in Transgenic Tobacco Plants

To evaluate the consequences of the BBL suppres-sion in tobacco plants, we constitutively suppressedthe expression of the BBL genes by RNAi in transgenictobacco plants. Six independent transgenic lines (KP1,KP2, KP3, KP4, KP5, and KP6) showed little accumu-lation of BBL proteins in the immunoblot analysis (Fig.6A). Suppression of BBLs did not affect the growth ordevelopment of the KP tobacco plants. We analyzedtobacco alkaloids in the leaves and roots of 1-month-old plants. Nicotine was the predominant alkaloid inboth parts in wild-type plants (Fig. 6, B and C). In theleaves, the amounts of nicotine and nornicotine (mmolg21 dry weight) were much lower in the KP plants (1–8and 0.3–3 mmol g21 dry weight, respectively) thanwild-type plants (22 and 5 mmol g21 dry weight,respectively). We did not detect DMN in the leavesof either wild-type or KP plants. In the roots, thenicotine content was considerably lower in the KPplants (1–3 mmol g21 dry weight) than wild-typeplants (4 mmol g21 dry weight), whereas the nornico-tine content was somewhat higher in the KP plants(0.6–1 mmol g21 dry weight) than in the wild-type

plants (0.4 mmol g21 dry weight). Interestingly, DMNwas the most abundant alkaloid in the roots of the KPplants, reaching 12 to 21 mmol g21 dry weight, but wasabsent in the wild-type roots (Fig. 6C). DMN mayaccumulate in the root tissue, possibly because of itsinability to be transported to the aerial tissues via thexylem.

BBL Knockdown in Cultured Tobacco Cells

Next, we examined whether BBLs are required forthe synthesis of anatabine, anabasine, and anatalline,which consist entirely of the pyridine moiety. Culturedtobacco BY-2 cells mainly produce these pyridine-typealkaloids upon elicitation by MeJA, since they do notsynthesize the N-methylpyrrolinium cation, due tovery inefficient expression of the N-methylputrescineoxidase genes (Shoji and Hashimoto, 2008). The met-abolic impact of BBL suppression was thus examinedin the MeJA-elicited BY-2 cells. Two control cell lines(VC1 and VC2) were transformed with an emptyvector, and two KB cell lines (KB1 and KB2) weretransformed with the BBL RNAi vector. An immuno-blot analysis showed that 50 mM MeJA induced theexpression of BBL proteins in the wild-type and VCcell lines but not in the KB line cells (Fig. 7A).Anatabine was the major alkaloid in the MeJA-elicitedwild-type and VC cells; nicotine and anatalline werethe next most abundant alkaloids, and anabasine wasa minor alkaloid (Fig. 7B). The amount of anatabine inthe two KB cell lines was 1% to 3% of that in the wild-type and VC cell lines. Other tobacco alkaloids (nico-tine, anatalline, and anabasine) also accumulated athighly reduced levels in the KB cell lines comparedwith the wild-type and VC cell lines (Fig. 7C). DMNand other novel metabolites were not detected in the

Figure 4. Identification of DMN in theBBL-suppressed tobacco roots. A, Gas-liquid chromatograms of alkaloid fractionsfrom the cell extracts of the wild type (WT)and the KR2 line, along with the DMNstandard. DMN (retention time, 19.7 min)accumulated in the KR2 roots but not inwild-type roots. B, Mass fragment patternsof the DMN peak in the KR2 roots and ofthe authentic DMN. C, Chemical struc-tures of DMN and nicotine.

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MeJA-elicited KB cell lines when analyzed by gas-liquid chromatography.

Subcellular Localization of BBL Proteins

To examine the subcellular localization of the BBLproteins, we expressed a fusion protein comprising thefull-length BBLa and GFP in cultured tobacco BY-2cells and analyzed its distribution by confocal lasermicroscopy. The cultured cells contained large centralvacuoles, which could be labeled by the vacuole-targeting marker SP-GFP-2SC (Mitsuhashi et al., 2000;Fig. 8A). BBLa-GFP was consistently observed in thecentral vacuoles (Fig. 8B). Accordingly, secretion sig-nal peptides were predicted at the N termini of BBLa(21 amino acids), BBLb (22 amino acids), and BBLc (22amino acids) by the motif prediction program SignalP(Emanuelsson et al., 2007; Supplemental Fig. S2). To

test the functions of these N-terminal sequences, wefused the N-terminal 50 amino acids of BBLs to GFPand expressed BBL(1-50)-GFP proteins in culturedtobacco cells. BBLa(1-50)-GFP was transported to thevacuole, which was demarked by the FM4-64-labeledtonoplast (Fig. 8C). Similarly, BBLb(1-50)-GFP andBBLc(1-50)-GFP were also located in the central vac-uoles (Supplemental Fig. S4). These results indicate thatthe N-terminal region of BBLs contains the vacuolarsorting determinants.

To further support the vacuolar localization of BBLproteins, vacuole-rich vesicles and cytoplasm-rich mini-protoplasts were purified by Percoll gradient centrifu-gation of protoplasts prepared from the BBLa-expressingBY-2 cells (Hamada et al., 2004; Fig. 8D). Immunoblottingwith an antiserum against an established vacuole-resident protein, class I chitinase (Matsuoka et al., 1995),confirmed the purity of the prepared fractions. A BBL

Figure 5. Time courses of inducible BBLsuppression and alkaloid accumulation intransgenic tobacco roots. A, Immunoblotanalysis of BBL and PEPC proteins in theinducible BBL RNAi root line (XN1) afterthe roots were treated with b-estradiol forthe period indicated. B, Accumulation ofnicotine and DMN in XN1 roots culturedin the absence or presence of b-estradiol.Data indicate mean values6 SD from threebiological replicates. C, Metabolite anal-ysis by thin-layer chromatography (TLC).Nitrogen-containing compounds weredetected using Dragendorff’s reagent. Atleast two unidentified compounds (RF val-ues of 0.01 and 0.07), in addition to DMN(RF of 0.11), were detectable after the in-ducer treatment.

Figure 6. BBL down-regulation in tobacco plants.Tobacco plants of the wild type (W) and sixindependent BBL RNAi lines (KP1–KP6) weregrown for 1 month after germination and ana-lyzed for gene expression and alkaloids. A, Im-munoblot analysis of BBL and PEPC (control)proteins in the root. B and C, Levels of nicotine,DMN, and nornicotine in the fourth newest leaf(B) and the root (C). Data indicate mean values6SD from three replicates. ND, Not detected.





of 58 kD (isoform I) was detected by the antiserumin the protoplasts and the vacuole-rich vesicles but notin the miniprotoplasts, indicating that BBLs are highlyenriched in the vacuole-rich vesicles. The signal inten-sity of a smaller BBL, isoform II (53 kD), was below thedetection limit.

Biochemical Properties of Recombinant BBL ProteinsProduced in Yeast

To gain insights into the biochemical properties ofBBLs, we expressed recombinant BBL proteins assecreted forms in the methyrotrophic yeast Pichiapastoris and purified them from the culture medium.The predicted N-terminal signal sequence of BBLs wassubstituted with the signal sequence of yeast a-factor,and the Strep tag was added to the C terminus of BBLs.Since we obtained the highest expression levelfor BBLa, compared with BBLb and BBLc, we purifiedthe recombinant BBLa from the culture medium. Therecombinant BBLa (95 kD) was much larger thanthe theoretical size of BBLa (60 kD), which lacked thepredicted secretion signal sequence, and was found tobe glycosylated, as revealed by periodic acid-Schiff(PAS) staining (Fig. 9A). When the purified BBLaprotein was treated by an endoglycosidase, EndoHf,the deglycosylated form had a theoretical molecularmass of 60 kD and was insensitive to PAS staining (Fig.9, A and B), indicating that the secreted recombinantBBLa protein was highly N-glycosylated by the en-dogenous glycosyltransferases of the yeast host. Thetwo endogenous BBL isoforms (53 and 58 kD; lane 2 inFig. 9B) found in the MeJA-treated tobacco BY-2 cellswere smaller than the 60-kD deglycosylated BBLaexpressed in yeast (lane 1 in Fig. 9B).

The concentrated solution of the recombinant BBLaprotein that had been purified from the yeast culturemedium and subsequently deglycosylated was yel-lowish and showed absorbance maxima at 350 and 439

nm, which resembled the absorbance spectrum of freeFAD (Fig. 9C). The fluorescence emission of the BBLaprotein exhibited a maximum at 514 nm, and treat-ment of the enzyme solution with the flavin-reducingreagent sodium dithionite effectively quenched thefluorescence (Fig. 9D). These spectrophotometric prop-erties suggest that the recombinant BBLa protein has aflavin molecule. All the known members of the BBEfamily are flavoproteins, containing an enzyme-boundFAD (Brandsch et al., 1987; Dittrich and Kutchan, 1991;Carter and Thornburg, 2004; Sirikantaramas et al.,2004). Indeed, a putative flavin-binding site is found inthe BBL protein sequences (indicated by a dashed linein Supplemental Fig. S2).

When the recombinant BBLa protein and crude cellextracts from the tobacco root or elicitor-treated cul-tured tobacco cells were used in the in vitro enzymeassay, we did not detect oxidative conversion of DMNto nicotine or other alkaloids (data not shown). Theexact enzymatic reaction catalyzed by BBLs needs tobe studied further.

DISCUSSION

Here, we identified novel BBL genes required for thebiosynthesis of tobacco alkaloids. Spectrophotometricanalyses of a recombinant BBL protein that had beenproduced and purified from the culture medium of ayeast cell culture suggest that BBLs contain FAD. BBLsbelong to an oxidoreductase subfamily that includesBBE (Fig. 1) within a larger vanillyl-alcohol oxidaseflavoprotein superfamily (Leferink et al., 2008). Thethree-dimensional x-ray crystal structures of BBE, glu-cooligosaccharide oxidase, 6-hydroxy-D-nicotine oxidase,and aclacinomycin oxidoreductase showed that the fla-voproteins of the BBE subfamily comprise two domains:a conserved FAD-binding domain and an a/b-domainwith a seven-stranded, antiparallel b-sheet forming the

Figure 7. BBL down-regulation in cultured to-bacco cells. Cultured BY-2 cells of the wild type(WT), two vector-transformed control cell lines(VC1 and VC2), and two BBL RNAi lines (KB1 andKB2) were treated with 50 mM MeJA for 48h (+),and their protein levels and alkaloid contentswere analyzed. Wild-type BY-2 cells were alsocultured in the absence of MeJA (2). A, Immu-noblot analysis of BBL and PEPC (control) pro-teins. B, Anatabine content. C, Levels of nicotine,anabasine, and anatalline. Note that the scale isdifferent from that in B. Data indicate meanvalues 6 SD from three replicates. ND, Notdetected.

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less-conserved substrate-binding domain. BBE (Winkleret al., 2008), glucooligosaccharide oxidase (Huang et al.,2005), and aclacinomycin oxidoreductase (Alexeevet al., 2007) bicovalently attach FAD to the protein viatwo amino acid residues, His and Cys, whereas6-hydroxy-D-nicotine oxidase covalently binds the fla-vin cofactor only via a His residue (Koetter and Schulz,2005). In BBLs, the His residue, which forms the co-valent linkage of FAD in these four enzymes, is con-served, but the Cys residue, which is used for thebicovalent linkage, is missing, except for BBLd, whoseexpression was not detected in the tobacco roots (Fig.2B), as in the case of 6-hydroxy-D-nicotine oxidase(Supplemental Fig. S2). Besides these flavoproteins, forwhich crystal structures have been solved, many otherBBE subfamily oxidoreductases catalyze the oxidationof a variety of metabolites, with the consumption ofmolecular oxygen and the production of hydrogenperoxide, and generally contain covalently tetheredFAD (Leferink et al., 2008). Covalent flavinylation isthought to increase the redox potential of the cofactorand thus its oxidation power.Metabolic functions of BBLs were analyzed by

suppressing their expression in transgenic tobaccosystems that normally synthesize pyridine-type alka-loids. Suppressed expression of BBL genes in tobaccohairy roots and tobacco plants effectively inhibited theformation of nicotine, whereas in jasmonate-elicitedcultured tobacco cells, BBL suppression severely in-

hibited the formation of a major alkaloid, anatabine, aswell as of minor tobacco alkaloids, nicotine, anabasine,and anatalline. These pyridine-type alkaloids are theproducts of condensation between a pyridine ring andanother pyridine ring (anatabine, anabasine, and ana-talline) or a pyrrolidine ring (nicotine). Therefore, BBLsare required for a presumed step in the activationof nicotinic acid or a condensation reaction yieldingbicyclic pyridine alkaloids.

Expression profiles of BBL genes are also consistentwith their involvement in the biosynthesis of tobaccoalkaloids. Nicotine is formed almost exclusively in theroots, and its synthesis is enhanced by herbivory onleaves by way of the general jasmonate signaling path-way and the specific nicotine regulatory loci, NIC1 andNIC2. Tobacco BBL genes are specifically expressed inthe roots, induced by jasmonate treatment, and posi-tively regulated by the NIC loci. Dominant negativesuppression and overexpression of a NIC2-locus ERFgene showed that the NIC2 locus regulates the ex-pression of BBL genes. These expression patterns areshared by all known tobacco structural genes involvedin nicotine biosynthesis (Shoji et al., 2010).

To narrow down the enzymatic step catalyzed byBBL oxidoreductases, it is informative to analyzemetabolites that accumulate in otherwise alkaloid-synthesizing tobacco cells when the BBL reaction isinhibited. A novel alkaloid, DMN, began to accumu-late as soon as the expression of BBLs was inducibly

Figure 8. Subcellular localization of BBLa protein in cultured tobacco cells. A, Full-length BBLa protein was fused at its Cterminus to GFP and stably expressed in cultured BY-2 cells. Fluorescence was observed with a laser scanning confocalmicroscope. B, Confocal image of BY-2 cells expressing SP-GFP-2SC, which had been shown to be located in the vacuoles(Mitsuhashi et al., 2000). C, An N-terminal 50-amino acid fragment of BBLawas fused to GFPand stably expressed in the tobaccocells. The transformed tobacco cells were pulse labeled by the fluorescent dye FM4-64 and inspected 10 h later when the dye hadbeen shown to primarily label the vacuolar membrane (Shoji et al., 2009). Bars = 50 mm. D, Immunoblots of subcellularfractions. Vacuoles and cytoplasm-rich miniprotoplasts were prepared from protoplasts of a BBLa-overexpressing tobacco cellline by Percoll gradient centrifugation. Vacuoles were stained with a 10 mg mL21 neutral red solution for 10 min. Immunoblotsprobed with antisera against BBL and class I chitinase (vacuolar resident protein) are shown, together with a Coomassie BrilliantBlue (CBB)-stained gel as a loading control. Bars = 100 mm. [See online article for color version of this figure.]





suppressed in tobacco hairy roots. DMN has beenidentified as a metabolite of nicotine in the rat (DeClercq and Truhaut, 1962) and was reported in ciga-rette smoke (Neurath et al., 1966) but has not beenreported in tobacco plants, indicating that the BBL-catalyzed reaction normally proceeds very efficientlyin planta. In the BBL-suppressed tobacco plants, DMNwas retained in the roots, constituting the major alka-loid in this organ, but was totally absent in the leaves,suggesting that DMN is not transported from the rootsto the aerial parts. The root-to-leaf transport system fortobacco alkaloids, possibly involving putative trans-porters, may not recognize DMN. Alternatively, DMNmay not be exported from a subcellular compartment,possibly the vacuole, in the root cells for the long-distance transport. The chemical structure of DMN, inwhich the N-(methylamino)butyl moiety is attached tothe C-3 position of the pyridine ring, shows that thecondensation of the pyridine ring and the N-methyl-pyrroline ring has been completed before the BBL-catalyzed oxidation acts on an unknown reactionintermediate. Possibly, DMN may be an in vivo sub-strate of BBLs. Our inability to demonstrate the con-version of DMN to nicotine by a yeast-produced BBLprotein might be caused by a lack of putative post-

translational processing of the nascent BBL polypep-tide in the yeast. The two native BBL isoforms (I and II)detected in alkaloid-producing tobacco cells are con-siderably smaller in molecular mass than the yeast-produced deglycosylated BBL protein, indicative ofposttlanslational processing of BBLs in tobacco cells,which might be important for their catalytic activation.Alternatively, DMN might be derived from an unsta-ble BBL substrate. At least two metabolites accumu-lated, in addition to DMN, when the BBL reaction wasinhibited in the tobacco cells that otherwise producednicotine. These unidentified metabolites likely containnitrogen, based on their reactivity toward Dragen-dorff’s reagent. The accumulation of multiple metab-olites suggests that the intermediate accumulatingimmediately after the blockage of the BBL reactionis unstable and readily metabolized in tobacco cells.An analogous situation is found for the tobacco or-phan reductase A622 (Kajikawa et al., 2009). Whenthe expression of A622 was suppressed in alkaloid-producing tobacco cells, the formation of tobaccoalkaloids was effectively inhibited, with a concomitantaccumulation of nicotinic acid N-glucoside. The glu-coside, however, is not a substrate for A622 but isthought to be a detoxification product of nicotinic acid.

Figure 9. Biochemical characterization of the recombinant BBLa protein produced in the Pichia cell culture. A, BBLa purifiedfrom the culture medium of P. pastoris was glycosylated. The purified protein was treated with (+) the endoglycosidase EndoHfand analyzed by SDS-PAGE. Separated proteins were stained with Coomassie Brilliant Blue (CBB) or the carbohydrate-stainingPAS reagent after being transferred to a polyvinylidene difluoride membrane. Approximately 1 mg of the native protein andapproximately 5 mg of the deglycosylated protein were loaded in the lanes. B, Immunoblot analysis of the deglycosylatedrecombinant BBLa protein produced from the Pichia culture (lane 1) and BBL proteins present in MeJA-treated wild-type BY-2cells (lane 2). The antiserum against BBL was used for detection. C, Absorbance spectra of the deglycosylated recombinant BBLaprotein (0.23 mg mL21 in 10 mM sodium phosphate buffer, pH 7.0) and a standard solution of FAD. D, Fluorescence emissionspectra of the deglycosylated recombinant BBLa protein before (control) and after the treatment with sodium dithionite. Theprotein samples (0.23 mg mL21 in 10 mM sodium citrate buffer, pH 4.0) were irradiated at 450 nm.

Kajikawa et al.




Although the results presented here strongly suggestthat BBLs catalyze a late oxidation step in tobaccoalkaloid biosynthesis, we cannot rule out the possibil-ity that BBLs are accessory factors required for the fullactivities of biosynthetic enzymes.BBLs are recruited to the vacuole when analyzed as

GFP-fused constructs or by subcellular fractionation.The N-terminal 50 amino acid residues of BBLs aresufficient for targeting GFP to the vacuole, probably byway of the endoplasmic reticulum (ER), and containpredictable signal peptides. Interestingly, the charac-terized BBE family proteins of plant origin all containN-terminal cleavable signal peptides (Dittrich andKutchan, 1991; Bird and Facchini, 2001; Carter andThornburg, 2004; Sirikantaramas et al., 2004; Tauraet al., 2007b). The cannabidiolic acid synthase andD1-tetrahydrocannabinolic acid synthase of Cannabissativa are probably glycosylated and recruited to thevacuole (Sirikantaramas et al., 2004; Taura et al.,2007b). The nectarin V protein of ornamental tobacco(Nicotiana langsdorffii 3 Nicotiana sanderae) possessesGlc oxidase activity and is secreted into the floralnectar (Carter and Thornburg, 2004). The BBE ofPapaver somniferum is transported to the vacuole, al-though it is presumed to be active in the lumen of theER or an ER-derived vesicle that is destined for thevacuole (Bird and Facchini, 2001). The subcellularlocalization of BBLs in the vacuole indicates that alate step of nicotine biosynthesis is catalyzed in thisorganelle or in the lumen of the ER or an ER-derived,vacuole-targeted vesicle. Determining the subcellulardistribution of other enzymes involved in nicotinebiosynthesis will clarify the compartmentation of thepathway. There may be unexpected transmembranetrafficking of intermediates.It should be noted that, in tobacco hairy roots, the

nornicotine level did not decrease, or actually increased,after knockdown of the BBL genes (Fig. 3). Previously,we observed that levels of nicotine and nornicotine didnot correlate in tobacco hairy roots when the regulatoryNIC loci were mutated in combinations (Hibi et al.,1994). In the tobacco leaf, nornicotine is synthesizedfrom nicotine in an oxidative demethylation reactioncatalyzed by nicotineN-demethylases of the cytochromeP450 monooxygenase subfamily CYP82E (Siminszkyet al., 2005). When three known nicotine N-demeth-ylase genes were inactivated by mutations, the tri-ple mutant tobacco plants still contained a smallamount of nornicotine (Lewis et al., 2010). Theremight be an alternative pathway of nornicotine bio-synthesis that does not utilize nicotine as the sole,direct intermediate. In addition to its preferred sub-strate N-methylputrescine, tobacco N-methylputres-cine oxidase converts putrescine to D1-pyrroline(Heim et al., 2007; Katoh et al., 2007). If D1-pyrrolinecondenses with a derivative of nicotinic acid in to-bacco plants, nornicotine could be generated by aroute independent of nicotine. BBLs might be dis-pensable for such a nicotine-independent pathway ofnornicotine formation.

MATERIALS AND METHODS

Plant Materials and Genetic Transformation

Binary vectors were introduced into Agrobacterium rhizogenes by electro-

poration. To generate transgenic hairy roots, leaf discs were prepared from

4- to 6-week-old tobacco (Nicotiana tabacum ‘Petit Havana SR1’) plants, were

sterilized, and were inoculated with A. rhizogenes ATCC15834 as described by

Kanegae et al. (1994). After selection and disinfection on solid Murashige and

Skoog (MS) medium containing 250 mg L21 cefotaxime and 15 mg L21

hygromycin (for pXVE-BBLRNAi) or 50 mg L21 kanamycin (for pHANNI-

BAL-BBLRNAi), hairy roots were subcultured in liquid MS medium with 3%

Suc every 2 weeks. Transgenic tobacco plants were generated with Agro-

bacterium tumefaciens strain EHA105, as described by Horsch et al. (1985).

Transgenic T0 and T1 plants were selected for resistance against 50 mg L21

kanamycin, and the transgenic plants of the T1 generation were used in this

study. Cultured tobacco BY-2 cells (cv Bright Yellow-2) were grown in liquid

MS medium supplemented with 20 mg L21 KH2PO4, 0.5 g L21 MES, and

0.2 mg L21 2,4-dichlorophenoxyacetic acid. Transgenic tobacco BY-2 cells were

generated with A. tumefaciens strain EHA105, as described by An (1985). Four-

day-old BY-2 cells were elicited with 50 mM MeJA in an auxin-free medium

(Shoji et al., 2008), whereas 1-month-old tobacco plants (cv Burley 21) were

treated with 100 mM MeJA for 1 d, and the root tissues were analyzed for up-

regulated expression of nicotine biosynthesis-related genes (Shoji et al., 2002).

Seeds of Nicotiana sylvestris and Nicotiana tomentosiformis were obtained from

the Leaf Tobacco Research Center of Japan Tobacco, Inc.

Vector Construction

For constitutive expression, the coding region of BBLa cDNA was ampli-

fied by PCR and cloned into pGWB2 (Nakagawa et al., 2007). For subcellular

localization assays, the coding region of BBLa cDNA excluding the stop codon

and the partial fragments (+1–150, with the adenine in the translational start

codon ATG as +1) of BBLa, BBLb, and BBLc cDNAswere amplified by PCR and

cloned into pGWB5 (Nakagawa et al., 2007). For RNAi-mediated constitutive

suppression, a partial fragment of BBLa cDNA (+1,312–1,654) was amplified

by PCR, subcloned into pHANNIBAL (Wesley et al., 2001), and then transferred

to the binary vector pBI121 (Clontech) to provide pHANNIBAL-BBLRNAi,

whereas the inducible RNAi vector pXVE-BBLRNAi was constructed by trans-

ferring the RNAi cassette containing an inverted repeat of the partial BBLa

fragment and the PDK intron from pHANNIBAL-BBLRNAi to a multicloning

site downstream of the OLexA promoter in pER8 (Zuo et al., 2000).

Genomic PCR, RT-PCR, and Quantitative RT-PCR

Genomic DNAwas isolated from root tissues ofN. tabacum cv Petit Havana

SR1, N. sylvestris, and N. tomentosiformis by using the PureLink Plant Total

DNA Purification kit (Invitrogen). The following PCR primers were used to

amplify genomic DNA (10 ng) with ExTaq DNA polymerase (TaKaRa Bio) for

30 cycles of 94�C for 30 s, 55�C for 30 s, and 72�C for 30 s: 5#-AAACTGC-

TACTGGAGCTGTTAC-3# and 5#-TCTTCGCCCATGGCTTTTCGGTCT-3#for BBLa, 5#-ACAAAGAATGATCAAAGTAG-3# and 5#-TCTTCGCCCAT-

GGCTTTTCGGTCT-3# for BBLb, 5#-CTACTAGTGGAGCAGGAGAA-3# and

5#-ACTCCGAATTTTCTGGACAG-3# for BBLc, 5#-AAGGAATCATGCTG-

GTAATAG-3# and 5#-TGCTGGCTCGGGAAATGGCA-3# for BBLd, and

5#-AGTTGGAGGAGGTGATGATG-3# and 5#-TATGTGGGTCGCTCAATGTC-3#for tobacco a-tubulin genes.

Total RNAwas isolated by using an RNeasy Plant Mini kit (Qiagen). cDNA

was synthesized from 1 mg of total RNA by SuperScript II reverse transcrip-

tase (Invitrogen) and amplified using ExTaq DNA polymerase (TaKaRa Bio)

under the following PCR conditions: 94�C for 30 s, 55�C for 30 s, and 72�C for

30 s. The BBL and a-tubulin cDNAs were amplified with the same primers

used for the genomic PCR. The reactions for plant roots were repeated

24 cycles for BBLa and a-tubulin cDNAs and 30 cycles for BBLb, BBLc, and

BBLd cDNAs. The number of reaction cycles for hairy roots was 22 for BBLa,

BBLb, and BBLc cDNAs and 24 for a-tubulin cDNAs.

For quantitative RT-PCR, cDNA was amplified using the LC480 real-time

thermal cycler system (Roche Applied Science) with SYBER Premix ExTaq

(Perfect Real Time; TaKaRa Bio). The thermal program was 5 min at 95�Cfollowed with 45 cycles of 10 s at 95�C, 10 s at 60�C, and 10 s at 72�C. Thespecificity of the reactions was confirmed by the machine’s standard melt

curve method. The primers used were 5#-CTGCTGATAATGTCGTTGAT-





GCTC-3# and 5#-CACCTCTGATTGCCCAAAACAC-3# for BBLs and 5#-AAG-

CCCATGGTTGTTGAGAC-3# and 5#-GTCAACGTTCTTGATAACAC-3# for

the tobacco EF-1a gene as a control.

Production of BBL Antiserum

A partial cDNA fragment of BBLa (+1,006 to the stop codon) was amplified

and cloned into pDONR221 (Invitrogen) by BP reaction and transferred into

an Escherichia coli expression vector, pET-DEST42 (Invitrogen). The recombi-

nant His-tagged BBLa protein was induced in the E. coli strain BL21 (DE3)

(Invitrogen) by the addition of 1 mM isopropylthio-b-galactoside for 3 h at 37�C.The recombinant protein was extracted in denatured extraction buffer (8 M urea,

20 mM sodium phosphate, pH 7.4, 0.5 M NaCl, and 10 mM dithiothreitol) by

sonication and purified by nickel-nitrilotriacetic acid agarose (Qiagen) according

to the manufacturer’s instructions. Eluted fractions containing the recombinant

protein were combined and dialyzed against the extraction buffer containing 4 M

urea. The recombinant protein was further purified by gradient elution (from 0

to 1 M NaCl) from the Q-Sepharose Fast Flow column (GE Healthcare). BBLa

antisera were produced in rabbits by Hokkaido System Science.

Immunoblot Analysis

Root tissues and cultured cells were frozen in liquid nitrogen, homoge-

nized by mortar and pestle, thawed, and then immediately mixed with

CelLytic P Cell Lysis Reagent (Sigma). After centrifugation of the homogenate,

3 mg of soluble protein in the supernatant was separated by SDS-PAGE (10%

T) and transferred onto a BioTrace polyvinylidene difluoride membrane (Pall)

using a Transblot SD Semi-Dry Electrophoretic Transfer Cell (Bio-Rad). The

membrane was blocked in 13 Tris-buffered saline (TBS) buffer containing

3% (w/v) bovine serum albumin for 1 h. Polyclonal anti-BBLa rabbit serum

and polyclonal anti-phosphoenolpyruvate carboxylase (PEPC) rabbit serum

(Chemicon) were diluted 1:1,000 in 13 TBS buffer containing 0.05% Triton

X-100 and incubated with the membranes for 1 h. Similarly, polyclonal anti-

class I chitinase rabbit serum was diluted 1:500 in 13 TBS buffer containing

0.05% Triton X-100 and incubated with the membranes for 1 h. After washing,

horseradish peroxidase-conjugated anti-mouse IgG from sheep and horse-

radish peroxidase-conjugated anti-rabbit IgG from donkey (GE Healthcare)

were used as secondary antibodies to detect target proteins with the ECL-Plus

Western Blotting System (GE Healthcare).

Preparations of Vacuoles and Miniprotoplasts

Vacuoles and miniprotoplasts were prepared from tobacco protoplasts

according to Hamada et al. (2004). Five-day-old tobacco BY-2 cells were in-

cubated with an enzyme solution containing 2% Sumizyme C, 0.2% Sumi-

zyme AP2 (both from Shin-Nihonkagaku Industries), and 0.45 M sorbitol, at

30�C (pH 5.5) for 2 h, gently homogenized with a Teflon homogenizer, and

fractionated by density gradient centrifugation in a solution containing 37%

Percoll, 6.5 mM HEPES-KOH (pH 7.3), 0.49 M Suc, 0.62 M sorbitol, and 0.04 M

MgCl2, at 25,000g for 30 min. Cytoplasm-rich miniprotoplasts and vacuole-

rich vesicles were separately collected from the corresponding fractions,

washed twice with cold 0.6 M mannitol, and suspended in 10 volumes of an

ice-cold extraction buffer containing 50 mM HEPES-KOH (pH 7.5), 5 mM

EDTA, 0.25 M sorbitol, 2 mM MgCl2, 1 mM phenylmethylsulfonyl fluoride,

complete protease inhibitor (Roche), and 1 mM dithiothreitol. The fractionated

preparations were homogenized, centrifuged at 13,000g at 4�C for 30 min, and

subjected to SDS-PAGE.

Confocal Microscopy

Tobacco BY-2 cells expressing GFP-fused proteins were examined with a

C1-ECLIPSE E600 confocal laser scanning microscope (Nikon) equipped with

an argon laser, the GFP(R)-BP filter, and the HQ-FITC-BP filter for GFP

fluorescence or with a helium-neon laser and the G-2A filter for FM4-64

fluorescence. Tonoplasts of tobacco BY-2 cells were labeled with FM4-64

(Invitrogen) at 32 mM for 12 h.

Metabolite Analyses

Plant samples (50 mg dry weight) were lyophilized, homogenized, and

soaked in 4 mL of 0.1 N sulfuric acid. The homogenate was sonicated for 15

min and centrifuged at 3,100 rpm for 15 min. The supernatant was neutralized

by adding 0.4 mL of 25% ammonia water. The mixture (1 mL) was loaded onto

an Extrelut-1 column (Merck) and eluted with 6 mL of chloroform. The eluent

was dried at 37�C. The dry residues were dissolved in ethanol containing 0.1%

dodecane as an internal standard and analyzed by gas-liquid chromatography

(GC-2010; Shimadzu) or gas-liquid chromatography-mass spectrometry

(Hewlett Packard 5890 SERIES II/JEOL MStation MS700 system; Agilent)

with an Rtx-5 Amine capillary column (i.d. of 0.25 mm, film thickness of

0.50 mm, 30 m; Restek). Nicotine, nornicotine, anatabine, and anabasine were

purchased from Wako or Sigma. The anatalline standard was a gift from Dr.

K.-M. Oksman-Caldentey (Hakkinen et al., 2004). Dihydrometanicotine was

purchased from Toronto Research Chemicals.

Thin-layer chromatography was used to evaluate alkaloid profiles.

Alkaloid extracts (10 mL) prepared as above were spotted on Merck silica

gel 60 F254 plates (20 3 20 cm, 0.2-mm layer) and developed with a solvent

system comprising chloroform:methanol:25% ammonia water (85:15:2, v/v/v).

Nitrogen-containing compounds were detected after spraying with Dragen-

dorff’s reagent (Harborne, 1984).

Expression of BBLs in Yeast

A partial coding region of BBLa cDNA (+64 to the stop codon) excluding

the putative signal peptide region was fused to the Strep tag II sequence

(Schmidt and Skerra, 2007) at the N terminus by PCR and cloned into the PmlI

site of pPICZaB (Invitrogen), which was then transformed to the Pichia pastoris

SMD1168H strain (Invitrogen) by using the Pichia EasyComp kit (Invitrogen).

Pichia cells grown in BMGYmedium were collected and resuspended in 1,000

mL of modified BMMYmedium containing 100 mM sodium citrate buffer (pH

5.5), 1% (v/v) methanol, 0.5% (w/v) casamino acid, and 10 mg L21 riboflavin,

as described by Taura et al. (2007a). The culture was shaken (90 rpm) at 20�Cfor 4 d with the addition of a 0.5% volume of methanol every 24 h. The culture

was centrifuged, and the supernatant was concentrated by the addition of 70%

(w/v) ammonium sulfate for 12 h. The precipitant was dialyzed against 20 mM

sodium acetate buffer (pH 5.5). The EndoHf endoglycosidase (1,000 units;

New England Biolabs), which had been fused to maltose-binding protein,

was added and incubated at 37�C for 3 h. The EndoHf protein was removed

by passing the samples through an amylose resin column (New England

Biolabs), and the eluents were dialyzed against Strep-Tacin buffer W contain-

ing 100 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 1 mM EDTA. Recombinant

BBLa was purified with a Strep-Tacin Sepharose affinity column (IBA)

according to the manufacturer’s instructions and dialyzed against 10 mM

sodium phosphate buffer (pH 7.0) or 10 mM sodium citrate buffer (pH 4.0).

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

libraries under the accession numbers AB604219 (NtBBLa), AM851017 (NtBBLb),

AB604220 (NtBBLc), AB604221 (NtBBLd), P30986 (EcBBE), AF049347 (BsBBE),

P93479 (PsBBE), AY610511 (TfBBE), P08159 (AoHDNO), AJ507836 (AnHDNO),

AB292682 (CsCBDAS), AB057805 (CsTHCAS), AF472609 (HaCHOX), AF472608

(LsCHOX), and AF503442 (NspNEC5).

Supplemental Data

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

Supplemental Figure S1. Biosynthesis of tobacco alkaloids.

Supplemental Figure S2. Alignment of deduced amino acid sequences of

tobacco BBLs and related proteins.

Supplemental Figure S3. Time courses of inducible BBL suppression and

alkaloid accumulation in the transgenic tobacco root line iRK2.

Supplemental Figure S4. Subcellular localization of BBLb and BBLc

proteins in cultured tobacco cells.

ACKNOWLEDGMENTS

We thank Ikuko Nishimura (Kyoto University), Tsuyoshi Nakagawa

(Shimane University), Num Hai Chua (Rockefeller University), Peter Water-

house (Commonwealth Scientific and Industrial Research Organization Plant

Industry), and Ken Matsuoka (Kyushu University) for providing the SP-GFP-

Kajikawa et al.




2SC-expressing tobacco BY-2 cells, pGWB2 and pGWB5 vectors, pER8 vector,

pHANNIBAL vector, and class I chitinase antiserum, respectively. We are

also grateful to Junko Tsukamoto (Nara Institute of Science and Technology)

for gas chromatography-mass spectrometry.

Received December 11, 2010; accepted February 21, 2011; published February

22, 2011.

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