The -Glucosidases Responsible for Bioactivation of Hydroxynitrile Glucosides in Lotus japonicus

The b-Glucosidases Responsible for Bioactivation ofHydroxynitrile Glucosides in Lotus japonicus1[W]

Anne Vinther Morant, Nanna Bjarnholt, Mads Emil Kragh, Christian Hauge Kjærgaard,Kirsten Jørgensen, Suzanne Michelle Paquette, Markus Piotrowski, Anne Imberty,Carl Erik Olsen, Birger Lindberg Møller, and Søren Bak*

Plant Biochemistry Laboratory, Department of Plant Biology, Center for Molecular Plant Physiologyand VKR Research Centre ‘‘Pro-Active Plants’’ (A.V.M., N.B., M.E.K., C.H.K., K.J., B.L.M., S.B.), andDepartment of Natural Sciences (C.E.O.), University of Copenhagen, DK–1871 Frederiksberg C,Copenhagen, Denmark; Department of Biological Structure, University of Washington, Seattle,Washington 98195–7420 (S.M.P.); Centre de Recherches sur les Macromolecules Vegetales,CERMAV-CNRS, FR–38041 Grenoble cedex 9, France (A.I.); and Lehrstuhl fur Pflanzenphysiologie,Ruhr-Universitat Bochum, D–44801 Bochum, Germany (M.P.)

Lotus japonicus accumulates the hydroxynitrile glucosides lotaustralin, linamarin, and rhodiocyanosides A and D. Upon tissuedisruption, the hydroxynitrile glucosides are bioactivated by hydrolysis by specific b-glucosidases. A mixture of twohydroxynitrile glucoside-cleaving b-glucosidases was isolated from L. japonicus leaves and identified by protein sequencing asLjBGD2 and LjBGD4. The isolated hydroxynitrile glucoside-cleaving b-glucosidases preferentially hydrolyzed rhodiocyano-side A and lotaustralin, whereas linamarin was only slowly hydrolyzed, in agreement with measurements of their rate ofdegradation upon tissue disruption in L. japonicus leaves. Comparative homology modeling predicted that LjBGD2 andLjBGD4 had nearly identical overall topologies and substrate-binding pockets. Heterologous expression of LjBGD2 andLjBGD4 in Arabidopsis (Arabidopsis thaliana) enabled analysis of their individual substrate specificity profiles and confirmedthat both LjBGD2 and LjBGD4 preferentially hydrolyze the hydroxynitrile glucosides present in L. japonicus. Phylogeneticanalyses revealed a third L. japonicus putative hydroxynitrile glucoside-cleaving b-glucosidase, LjBGD7. Reverse transcription-polymerase chain reaction analysis showed that LjBGD2 and LjBGD4 are expressed in aerial parts of young L. japonicus plants,while LjBGD7 is expressed exclusively in roots. The differential expression pattern of LjBGD2, LjBGD4, and LjBGD7corresponds to the previously observed expression profile for CYP79D3 and CYP79D4, encoding the two cytochromes P450that catalyze the first committed step in the biosyntheis of hydroxynitrile glucosides in L. japonicus, with CYP79D3 expressionin aerial tissues and CYP79D4 expression in roots.

b-Glycosidases that belong to the family 1 glycosidehydrolases catalyze hydrolysis of the b-glycosidicbond in b-glycosides consisting of two carbohydratemoieties or a carbohydrate moiety linked to an arylor alkyl aglucone. In plants, b-glycosidases serve anumber of diverse and important functions, includingbioactivation of defense compounds (Nisius, 1988;Poulton, 1990; Morant et al., 2003; Halkier andGershenzon, 2006; Suzuki et al., 2006), cell wall deg-radation in endosperm during germination (Leah et al.,1995), activation of phytohormones (Kristoffersen et al.,

2000; Lee et al., 2006), and lignification (Dharmawardhanaet al., 1995; Escamilla-Trevino et al., 2006). In addition,b-glycosidases play key roles in aroma formation intea, wine, and fruit juice (Mizutani et al., 2002; Fiaet al., 2005; Maicas and Mateo, 2005). Plants producemyriad secondary metabolites involved in defenseagainst pathogens and herbivores. These defense com-pounds are often stored as b-glycosides and bioacti-vated by specific b-glycosidases (Morant et al., 2008).Glycosylation serves to protect the plant against thetoxic effects of its own chemical defense system, toincrease solubility, and to facilitate storage. Examplesof two-component plant defense systems whereinb-glycosidases act as the bioactivator include thea-hydroxynitrile glycosides (cyanogenic glycosides)that are found in numerous different plant species(Poulton, 1990; Hughes, 1993; Bak et al., 2006; Morantet al., 2007; Bjarnholt and Møller, 2008; Bjarnholt et al.,2008), benzoxazinoid glycosides in Zea mays, Triticumaestivum, and Secale cereale (Niemeyer, 1988; Sue et al.,2000a, 2000b), avenacosides in Avena sativa (saponins;Nisius, 1988; Kim et al., 2000), isoflavonoid glycosidesin legumes (Cairns et al., 2000; Chuankhayan et al., 2005,2007a, 2007b; Suzuki et al., 2006), and glucosinolates

1 This work was supported by grants from the Danish NationalResearch Foundation to the Center for Molecular Plant Physiology,from the Villum Kann Rasmussen Foundation to ‘‘Pro-ActivePlants,’’ and from the Faculty of Life Sciences, University ofCopenhagen, for a Ph.D. stipend to A.V.M.

* 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:Søren Bak ([email protected]).

[W] The online version of this article contains Web-only data.www.plantphysiol.org/cgi/doi/10.1104/pp.107.109512

1072 Plant Physiology, July 2008, Vol. 147, pp. 1072–1091, www.plantphysiol.org � 2008 American Society of Plant Biologists

found mainly in Brassicales (Halkier and Gershenzon,2006). All of these different types of glycosides containan O-linked b-glucosidic bond except for the glucosi-nolates, which are thioglucosides. This difference isreflected in the catalytic machinery of the correspond-ing b-glycosidases. In all b-glycosidases except theglucosinolate-degrading myrosinases (b-thioglucosideglucohydrolases), two Glu residues act as the catalyticnucleophile and the catalytic proton donor. In myro-sinases, the proton donor Glu is replaced by a Glnresidue (for review, see Bones and Rossiter, 2006;Morant et al., 2008).

One of the most well characterized plant defense sys-tems is the a-hydroxynitrile glucoside/b-glucosidasesystem generally referred to as the ‘‘cyanide bomb.’’This two-component system, in which each of thecomponents when separated is chemically inert, pro-vides cyanogenic plants with an immediate defenseagainst attacking pathogens and herbivores by hydro-lyzing a-hydroxynitrile glucosides, resulting in therelease of toxic hydrogen cyanide. a-Hydroxynitrileglucosides thus exercise their effect as preformed de-fense compounds in the first line of chemical defenseagainst pathogens or herbivores. a-Hydroxynitrile glu-cosides are derived from amino acids and found inmore than 2,650 different plant species (Bak et al.,2006), including several important crops (Jones, 1998).a-Hydroxynitrile glucosides and their degradingb-glucosidases are stored separately in different sub-cellular compartments and/or in different cell types.The a-hydroxynitrile glucosides accumulate in thevacuole (Saunders et al., 1977; Saunders and Conn,1978; Gruhnert et al., 1994). a-Hydroxynitrile glucosidecleaving b-glucosidases from monocotyledenous plantscontain an N-terminal chloroplast transit peptide andare hence deposited in the chloroplast (Thayer and Conn,1981). Eudicotyledenous a-hydroxynitrile glucoside-cleaving b-glucosidases contain an N-terminal signalpeptide directing them through the secretory pathway,which results in protein glycosylation and depositionin the apoplast (Kakes, 1985; Frehner and Conn, 1987;Mkpong et al., 1990) or retention of the glycosylatedproteins before secretion, resulting in intracellular de-position in protein bodies (Swain et al., 1992; Poultonand Li, 1994).

In some cyanogenic plant species, including the le-gumes Trifolium repens (white clover) and Lotus cornic-ulatus (common bird’s foot trefoil), cyanogenesis isa polymorphic trait (Hughes, 1991; Gebrehiwot andBeuselinck, 2001; Olsen et al., 2007, 2008). AcyanogenicL. corniculatus and T. repens plants have lost their abil-ity either to synthesize a-hydroxynitrile glucosides,to produce a functional a-hydroxynitrile glucosidehydrolyzing b-glucosidase, or both. The polymorphictrait is supposedly retained in populations of whiteclover by the action of two opposing selective forcesconstituted of herbivory (giving an evolutionary ad-vantage of cyanogenesis through reduction of her-bivory) and temperature (giving an evolutionarydisadvantage of cyanogenesis in cold climates where

plant tissue exposed to frost is disrupted, causingcyanogenesis to occur with resulting exposure of theplant to the toxic effects of its own chemical defensesystem; Till, 1987). In addition, the growth rate hasbeen shown to be higher in low-cyanogen versus high-cyanogen varieties of the same plant species (Gleadowand Woodrow, 2002; Goodger et al., 2004).

The enzymes and corresponding genes encoding theentire a-hydroxynitrile glucoside biosynthetic path-way are known from the monocotyledenous cropSorghum bicolor (great millet), which produces thea-hydroxynitrile glucoside dhurrin with Tyr as theparent amino acid. Tyr is converted into dhurrin viathe concerted action of two endoplasmic reticulum-anchored cytochromes P450, CYP79A1 and CYP71E1,and a soluble UDPG-glucosyltransferase, UGT85B1(Sibbesen et al., 1995; Kahn et al., 1997; Bak et al.,1998; Jones et al., 1999), which function as a metabolonto secure efficient channeling of the intermediates(Møller and Conn, 1980; Jørgensen et al., 2005b;Nielsen et al., 2008). Lotus japonicus (bird’s foot trefoil)contains the Ile- and Val-derived a-hydroxynitrileglucosides lotaustralin and linamarin as well as theIle-derived g- and b-hydroxynitrile glucosides rhodio-cyanoside A and rhodiocyanoside D (Forslund et al.,2004; Bjarnholt and Møller, 2008; Bjarnholt et al., 2008),collectively referred to as hydroxynitrile glucosides.While a-hydroxynitrile glucosides are known to beimplicated in plant defense, the biological function ofthe g- and b-hydroxynitrile glucosides remains un-known. In apical L. japonicus leaves, the lotaustralin-linamarin and rhodiocyanoside A-rhodiocyanoside Dratios are high (Forslund et al., 2004; Bjarnholt et al.,2008). In the same plant species, the two isoenzymesCYP79D3 and CYP79D4 catalyze the first and rate-limiting step in hydroxynitrile glucoside biosynthesis,the conversion of Ile and Val into the correspondingoximes (Forslund et al., 2004; Morant et al., 2007;Bjarnholt et al., 2008). The enzymes that catalyze theremaining steps remain elusive. CYP79D3 is expressedin aerial parts of L. japonicus, whereas CYP79D4 isexpressed exclusively in the roots, as determinedby semiquantitative reverse transcription (RT)-PCR(Forslund et al., 2004). Hydroxynitrile glucosidesare generally only detected in the aerial parts of L.japonicus (Forslund et al., 2004). This questions the invivo function of CYP79D4.

In this work, we have identified and characterizedtwo b-glucosidases, LjBGD2 and LjBGD4, responsiblefor bioactivation of the hydroxynitrile glucosides inL. japonicus leaves. The two isoenzymes share 85%sequence identity at the amino acid level. LjBGD2and LjBGD4 are coexpressed with the biosyntheticCYP79D3 in aerial parts of L. japonicus. Staining forb-glucosidase activity demonstrated a preferentiallocalization in the palisade tissue of the leaves. Heter-ologous expression of LjBGD2 and LjBGD4 in Arabi-dopsis (Arabidopsis thaliana) confirmed that LjBGD2and LjBGD4 both hydrolyze hydroxynitrile glucosidesand have very similar substrate specificity profiles.

Bioactivation of Hydroxynitrile Glucosides in Lotus

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A third b-glucosidase, LjBGD7, with approximately 82%amino acid sequence identity to LjBGD2 and LjBGD4,is coexpressed with the biosynthetic CYP79D4 in L.japonicus roots. This implies that two parallel path-ways for the biosynthesis and turnover of hydroxyni-trile glucosides exist in L. japonicus.

RESULTS

Hydrolysis of Lotaustralin and Rhodiocyanosides uponCell Disruption

Upon cell disruption, both a-hydroxynitrile glu-cosides and rhodiocyanosides are hydrolyzed byb-glucosidases in L. japonicus, as measured usingcrude L. japonicus leaf extracts (Figs. 1 and 2). Theaglucone resulting from hydrolysis of a-hydroxynitrileglucosides dissociates either spontaneously or by theaction of a-hydroxynitrilases to release toxic HCN anda ketone or an aldehyde (Fig. 1A). In contrast, hy-drolysis of rhodiocyanoside A and D affords g- andb-hydroxynitriles, respectively, which do not resultin HCN release (Fig. 1B). To examine the rate ofhydrolysis of the individual hydroxynitrile glucosidesupon cell disruption, leaves from a L. japonicus35STCYP79D2 line (Forslund et al., 2004) were em-ployed. This transgenic L. japonicus line accumulatesapproximately 20-fold higher amounts of linamarincompared with the wild type and was obtained byintroduction of the CYP79D2 gene of Manihot esculenta(cassava) under the control of the constitutive cauli-flower mosaic virus (CaMV) 35S promoter. Comparedwith L. japonicus CYP79D3 and CYP79D4, M. esculentaCYP79D2 shows enhanced activity toward Val andthus favors the formation of linamarin (Forslund et al.,2004). The use of the L. japonicus 35STCYP79D2 linethus allows for a careful examination of changes inlotaustralin, rhodiocyanoside A and D, and linamarinlevels in response to tissue disruption. The rhodiocya-nosides are most rapidly hydrolyzed, followed bylotaustralin, while linamarin hydrolysis proceeds at avery slow rate (Fig. 2). When the corresponding ex-periment was conducted with L. japonicus wild-typeleaves, the same relative pattern of rhodiocyanosideand lotaustralin degradation was observed (data notshown), while degradation of linamarin could not bedetected due to the very low amounts of linamarininitially present. These results show that L. japonicusleaves contain b-glucosidase activity, which upon celldisruption preferentially catalyzes the hydrolysis ofrhodiocyanosides and lotaustralin compared withlinamarin.

Identification of Two b-Glucosidases Involved

in Hydroxynitrile Glucoside Hydrolysis inL. japonicus Leaves

To identify the enzymes responsible for hydrolysisof hydroxynitrile glucosides in L. japonicus, the en-zymes showing activity with these substrates were

purified from crude soluble protein extracts of youngL. japonicus leaves by a simple two-step procedureusing cation exchange followed by gel filtration chro-matography. Purification using the cation exchangeresin was carried out by batch elution to prevent

Figure 1. Bioactivation of lotaustralin, linamarin, and rhodiocyanosideA in L. japonicus leaves. A, Cyanogenic glucosides are b-glucosides ofa-hydroxynitriles. Upon cell disruption, b-glucosidases catalyze the hy-drolysis of the O-b-glucosidic bond to yield Glc and an a-hydroxynitrileaglucone. The a-hydroxynitrile either spontaneously or enzymaticallybreaks down to liberate a ketone and toxic HCN. B, Rhodiocyanosidesare b-glucosides of b- or g-hydroxynitriles. In contrast to a-hydroxynitrileglucosides, aglucone formation is not accompanied by the releaseof HCN.

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aggregation and immediately followed by gel filtra-tion to remove Rubisco (Fig. 3). The fractions from thegel filtration column that showed hydrolytic activitytoward hydroxynitrile glucosides were enriched inproteins with an apparent molecular mass of 107 kD,as monitored by comparison with known molecularmass standards coapplied to the gel filtration chro-matography column (data not shown). SDS-PAGEanalysis and Coomassie Blue staining revealed thatthe most active gel filtration fraction contained threedistinct protein bands, with apparent molecularmasses of 60 kD (band A), 55 kD (band B), and 48kD (lower band; Fig. 3). In the course of fractionation,no lotaustralin- or linamarin-hydrolyzing activity wasfound in the discarded fractions (Fig. 3). Hence, noindications of the occurrence of hydroxynitrile gluco-side-cleaving b-glucosidases that exhibited a differentfractionation pattern were observed. The cation ex-change and gel filtration chromatography resulted in8- and 18-fold purification, respectively, of the hydroxy-nitrile glucoside-cleaving b-glucosidases, as mea-sured by HCN release with lotaustralin as substrate(data not shown). The relatively low purification foldis a reflection of the high abundance of the enzymes inthe crude leaf protein extract.

The proteins migrating with apparent molecularmasses of 60, 55, and 48 kD (Fig. 3, lane 5) weresubjected to in-gel trypsin digestion, and the obtainedpeptides were sequenced by mass spectrometry. Pep-tide fingerprints of the 60- and 55-kD protein bandswere nearly identical, and subsequent alignment of the

13 peptide sequences obtained from the 60- and 55-kDprotein bands with the L. japonicus b-glucosidasesequences provided by the Kazusa DNA ResearchInstitute revealed that both sequenced protein bandscontained a mixture of two different L. japonicusb-glucosidases, LjBGD2 and LjBGD4 (SupplementalFig. S1). Of the 13 peptide sequences obtained, sevenmatched perfectly to LjBGD2, three matched perfectlyto LjBGD4, and three matched perfectly to both se-quences. An overall amino acid sequence coverage of24% and 14% was obtained for LjBGD2 and LjBGD4,respectively. The deduced LjBGD2 and LjBGD4 poly-peptide sequences are 514 and 518 amino acids long,respectively, and share 85% amino acid sequenceidentity and 91% similarity. PSORT and TargetP anal-ysis of the full-length LjBGD2 and LjBGD4 amino acidsequences predicted that the polypeptides both con-tain an N-terminal signal peptide consisting of 27amino acids (Supplemental Fig. S1) and are destinedfor the secretory pathway, in agreement with otherknown dicotyledenous a-hydroxynitrile glucoside-cleaving b-glucosidases (Kakes, 1985; Frehner andConn, 1987; Mkpong et al., 1990; Swain et al., 1992;Poulton and Li, 1994). No amino acid sequencescorresponding to the N terminus were obtained. Se-quencing of peptides obtained from the 48-kD proteinband revealed a perfect match to Rubisco (data notshown).

The b-Glucosidases Isolated from L. japonicusLeaves Preferentially Hydrolyze Rhodiocyanosides

and Lotaustralin

To characterize the biochemical activity of the L.japonicus leaf b-glucosidases, a range of b-glucosides(Fig. 4) were tested as substrates for the isolatedmixture of LjBGD2 and LjBGD4. Km and Vmax valueswere determined for a number of different aliphaticand aromatic hydroxynitrile glucosides. Of thosetested, the L. japonicus b-glucosidases exhibited thelowest Km for rhodiocyanoside A, followed by dhur-rin, prunasin, and lotaustralin (Table I). In agreementwith the data obtained using leaf extracts, linamarinproved to be a poor substrate, with a Km 10-fold higherthan that for rhodiocyanoside A (Table I). The Vmaxvalues obtained using different hydroxynitrile gluco-sides as substrates were similar. Accordingly, the lowturnover of linamarin observed (Fig. 2) is primarilydue to a high Km value and not a lower Vmax (Table I).The kinetic data obtained with the purified mix ofLjBGD2 and LjBGD4 correspond well with the degra-dation profile observed using crude L. japonicusleaf extracts (Fig. 2) and support the idea that thepurified b-glucosidases catalyze the hydrolysis ofhydroxynitrile glucosides in planta. As expected, thea-hydroxynitrile diglucoside amygdalin and the thio-glucoside p-hydroxybenzylglucosinolate (pOHBG) werenot hydrolyzed (data not shown). Hydrolysis of theflavonoid glucoside kuromanin was not detected us-ing concentrations up to 100 mM. Daidzin was cleaved,

Figure 2. Degradation of lotaustralin, linamarin, and rhodiocyanosidesin leaves of L. japonicus following cell disruption. The rate of degra-dation of endogenous hydroxynitrile glucosides in transgenic L. japon-icus 35STCYP79D2 leaves following tissue disruption was monitoredby LC-MS and analysis of the amounts of metabolites remaining at timepoints between 0 and 60 min following cell disruption. The total amountof hydroxynitrile glucosides (linamarin 1 lotaustralin 1 rhodiocyano-sides) at time 0 was defined as 100. Rhodiocyanosides are most rapidlydegraded, followed by lotaustralin, which is degraded at a moderate rate;only negligible degradation of linamarin is observed.



and the Km value was lower than that observed forrhodiocyanoside A (Table I). The Vmax for daidzin waslow compared with the Vmax values obtained for thehydroxynitrile glucosides (Table I).

Heterologous Expression of LjBGD2 and LjBGD4 in

Arabidopsis and Analysis of Their IndividualSubstrate Specificities

LjBGD2 and LjBGD4 could not be separated by theprotein purification methods applied in this study.This may reflect the co-occurrence of differently glyco-sylated forms of each protein. To examine the substratespecificity of the two b-glucosidases independently,LjBGD2 and LjBGD4 were heterologously expressed intransgenic Arabidopsis. Arabidopsis was chosen asexpression host because of the straightforward trans-formation protocol, the absence of endogenous hy-droxynitrile and isoflavonoid glucosides, and becausecrude extracts from this plant were shown not to pos-sess any endogenous b-glucosidase activity towardhydroxynitrile and isoflavonoid glucosides (Fig. 5). Asa dicotyledenous plant, Arabidopsis would be ex-pected to properly process LjBGD2 and LjBGD4 withrespect to signal peptide recognition, targeting of theenzymes through the secretory pathway, and cotrans-lational signal peptide processing and glycosylation,as required for enzyme stability (Keresztessy et al.,1996; Cicek and Esen, 1999; Zhou et al., 2002).

The LjBGD4 cDNA was cloned, including the firstintron, to overcome difficulties in PCR amplification ofthe full-length cDNA. Moreover, the use of Escherichia

coli SURE cells as recipients for transformation withthe ligation product was critical for successful cloning.These observations suggested that the LjBGD4 nu-cleotide sequence formed secondary structures thatinterfered with PCR and standard cloning proce-dures. Analysis of the LjBGD4 nucleotide sequenceusing the Arabidopsis intron splice site predictionserver NetPlantGene (http://www.cbs.dtu.dk/services/NetPGene/) predicted that Arabidopsis would recog-nize the L. japonicus-derived intron.

Six independent kanamycin-resistant Arabidopsis35STLjBGD2 transformants and five independentgentamycin-resistant Arabidopsis 35STLjBGD4 trans-formants that showed b-glucosidase activity towardlotaustralin were obtained. The ability of Arabidopsis35STLjBGD2 and 35STLjBGD4 to hydrolyze lotaus-tralin confirmed that both L. japonicus b-glucosidaseswere successfully expressed in an active form and thatthe intron included in the LjBGD4 construct wasproperly recognized and excised in Arabidopsis.

LjBGD2 and LjBGD4 activity in the transgenicArabidopsis leaves was measured using excised leafdiscs and discs from wild-type plants as controls.LjBGD2 and LjBGD4 were found to possess verysimilar hydrolytic activities toward the differentb-glucosides tested (Fig. 5). Both aliphatic and aromatica-hydroxynitrile monoglucosides were hydrolyzed,whereas the a-hydroxynitrile diglucoside amygdalinwas not (Fig. 5). Due to the presence of endogenousmyrosinases in Arabidopsis, glucosinolates were notapplied as substrates. The background activity ob-served in Arabidopsis wild-type plants was due tohydrolysis of endogenous indole glucosinolates,which results in the formation of SCN2 (for review,see Bones and Rossiter, 2006). SCN2 is known to reactin the colorimetric assay for CN2 determination, asdescribed previously (Bak et al., 1999, 2001; Bak andFeyereisen, 2001). The background activity observedin wild-type Arabidopsis with p-nitrophenyl b-D-glucopyranoside (pNPG) is most likely due to pNPGbeing a general b-glucosidase substrate that is hy-drolyzed by endogenous Arabidopsis b-glucosidases.These results show that despite their co-occurrence(Figs. 3 and 8 [below]), LjBGD2 and LjBGD4 are indi-vidually able to hydrolyze the same range of aliphaticand aromatic a-hydroxynitrile monoglucosides andrhodiocyanoside A as observed for the partially puri-fied mixture of the two enzymes. Accordingly, hetero-mer formation is not required for activity.

The assays with leaf discs expressing either LjBGD2or LjBGD4 showed the ability of each of theseb-glucosides to hydrolyze daidzin (data not shown).This cross-reactivity is consistent with previous obser-vations that almond (Prunus dulcis) b-glucosidase hy-drolyzes daidzin (Ismail and Hayes, 2005; Chuankhayanet al., 2007a), whereas b-glucosidases hydrolyzingisoflavonoid glucosides have not been found to hy-drolyze cyanogenic glucosides (Ketudat Cairns et al.,2000; Chuankhayan et al., 2005). The isoflavonoidglucoside profile in L. japonicus is currently not known,

Figure 3. Purification of hydroxynitrile glucoside-cleaving b-glucosidasesfrom L. japonicus leaves. Protein fractions obtained during the purifi-cation procedure were analyzed by 12% SDS-PAGE, and the proteincomposition was visualized by Coomassie Blue staining. The lanesshow protein composition in the total crude leaf extract (1), solublecrude leaf extract (2), cation exchange supernatant (3), cation exchangeeluate (4), and fractions from gel filtration chromatography containingb-glucosidase activity against hydroxynitrile glucosides (5). The pres-ence (1) or absence (O) of b-glucosidase activity toward lotaustralinand linamarin as measured by HCN release is indicated at the top ofeach lane. Protein sequencing identified the protein bands indicated byA and B as a mixture of LjBGD2 and LjBGD4, while the faster migratingprotein band was identified as Rubisco.

Morant et al.


and daidzin has not been reported to be present (Faraget al., 2007; Suzuki et al., 2008). This argues that the inplanta function of LjBGD2 and LjBGD4 is to hydrolyzehydroxynitrile glucosides and not isoflavonoid glu-cosides. The data obtained using leaf discs to inde-pendently characterize the substrate specificity ofLjBGD2 and LjBGD4 are in accordance with the re-sults obtained with the purified mixture of the twob-glucosidases obtained from L. japonicus leaves (TableI). As in the experiments with the mixture of the two

purified BGDs, hydrolysis of kuromanin could not bedetected in the leaf disc assay using transgenic Arabi-dopsis expressing LjBGD2 or LjBGD4 (Table I).

No or limited activities were observed with extractsof transgenic Arabidopsis leaves obtained by macera-tion or freeze/thaw cycles. This apparent inactivationprevented the isolation of active LjBGD2 and LjBGD4from the transgenic Arabidopsis lines. An obviousexplanation for the observed lack of activity of L.japonicus b-glucosidase in Arabidopsis leaf extractsfollowing homogenization would be the inhibition byendogenous glucosinolates or breakdown productsthereof liberated in the course of the maceration pro-cess. However, no inhibitory effect of glucosinolateson LjBGD2 and LjBGD4 was observed upon the addi-tion of a 5-fold molar excess of aliphatic or aromaticglucosinolates [4-(methylthio)butyl glucosinolate andpOHBG, respectively] or NaSCN to purified LjBGD2and LjBGD4 (data not shown).

Exploration of the Active Site Architecture of LjBGD2

and LjBGD4 by Homology Modeling

To gain insight into the active site architecture ofLjBGD2 and LjBGD4 and their respective abilities toaccommodate the L. japonicus hydroxynitrile gluco-sides, models of the protein structures were builtbased on three-dimensional structures of T. repenslinamarase (TrCBG), S. bicolor dhurrinase 1 (SbDhr1),and Z. mays DIMBOA-Glc-hydrolase (Zm-Glu-1), whichhave all been solved at high resolution by x-raychrystallography (Barrett et al., 1995; Czjzek et al.,2001; Verdoucq et al., 2004). TrCBG, SbDhr1, andZm-Glu-1 show high amino acid sequence identityto LjBGD2 and LjBGD4 (approximately 55%, 45%,and 46%, respectively) and hence represent excellenttemplates for modeling of the two L. japonicusb-glucosidases. The models obtained of the three-dimensional structures of the LjBGD2 and LjBGD4monomers, with closeup views of the active sites intowhich lotaustralin, linamarin, and rhodiocyanoside Aare docked, are presented in Figure 6. The predictedstructures of LjBGD2 and LjBGD4 are highly similar tothose of TrCBG (Fig. 6A), SbDhr1, and Zm-Glu-1 (datanot shown). In Figure 6B, the amino acids within theLjBGD2 and LjBGD4 aglucone-binding sites and thetwo catalytic glutamates (indicated by magenta andblue arrowheads in Supplemental Fig. S1) are dis-played with lotaustralin docked in the active site. Onlythe amino acids corresponding to those located withinthe aglucone-binding pocket of SbDhr1 (Verdoucqet al., 2004) plus Gly-211/Val-215 and Trp-210/Trp-214, which in this study were also predicted tolie within the aglucone-binding pocket of LjBGD2and LjBGD4, are shown. Studies on Zm-Glu-1 andSbDhr1 have demonstrated the importance of addi-tional amino acid residues in determining agluconespecificity. Substitution of single amino acids in Zm-Glu-1with their SbDhr1 counterparts by site-directed muta-genesis demonstrated that Ala-467, Tyr-473, and Asp-

Figure 4. Structures of the main b-glucosides used to examine the sub-strate specificity of the hydroxynitrile glucoside-cleaving b-glucosidasesfrom L. japonicus. Val-derived linamarin and Ile-derived lotaustralin andrhodiocyanoside A are synthesized by L. japonicus. The a-hydroxynitrileglucosides dhurrin and prunasin are derived from Tyr and Phe, respec-tively. Amygdalin is the diglucoside derived from glucosylation ofprunasin. pNPG is an artificial chromogenic b-glucoside degraded by awide range of b-glycosidases. Daidzin and kuromanin are isoflavonoidand flavonoid glucosides, respectively. All glucosides tested contain anO-b-glucosidic bond except for the glucosinolates, which contain anS-b-glucosidic bond.



261 (Zm-Glu-1 amino acids and numbering), whichare located in close proximity of the aglucone-bindingpocket, played key roles in aglucone specificity inZm-Glu-1 and SbDhr1 (Verdoucq et al., 2003). Thecorresponding amino acids in LjBGD2 and LjBGD4(marked by ‘‘Z’’ in Supplemental Fig. S1) are replacedby Ser, Phe, and Val, respectively, and conservedbetween LjBGD2 and LjBGD4. In Prunus serotina (blackcherry), two additional amino acids at positions cor-responding to Val-219 and Thr-391 in LjBGD2 (indi-cated by ‘‘P’’ in Supplemental Fig. S1) were shown tobe crucial for the aglucone specificity of amygdalinhydrolase 1 and the prunasin hydrolases (Zhou et al.,2002). These were likewise located in proximity to theaglucone-binding pocket, but interestingly, the corre-sponding amino acids differ between LjBGD2 andLjBGD4 (Supplemental Fig. S1). The Thr-391 (LjBGD2)and Asp-395 (LjBGD4) immediately preceded a highlyconserved Trp residue located in the aglucone-bindingpocket (shown in gray in Fig. 6B). According to Czjzeket al. (2001), the amino acid residue at this positioncould be responsible for determining the particularconformation of the Trp in the active site. Interestingly,this Trp is preceded by a Pro in Zm-Glu-1 (Czjzeket al., 2001) and by a Gly in P. serotina amygdalinhydrolase 1 (Zhou et al., 2002).

The lipophilic surface representations of the activesite pockets in Figure 6C are shown using the sameorientations as in Figure 6B to allow comparison ofthe amino acid residues that form the aglucone-accommodating part of the active site pocket. The aminoacids involved in glucone binding are highly con-served in all b-glucosidases belonging to the family1 glycoside hydrolases (Czjzek et al., 2001; Supple-mental Fig. S2). This is reflected in the identical topol-ogies of the glucone-binding pockets that form theupper parts of the active sites (Fig. 6C). As a startingmodel for docking of the L. japonicus substrates inLjBGD2 and LjBGD4, Zm-Glu-1 complexed with theinhibitor p-nitrophenyl b-D-thioglucoside was used(Czjzek et al., 2001). The glucone part of this complexis poorly defined, probably due to the glucone takingmultiple conformations during catalysis; accordingly,it was decided to model the hydroxynitrile glucosideswith the glucone in a chair conformation. The hydroxy-nitrile glucoside glucones docked into the predictedactive sites of LjBGD2 and LjBGD4 were in hydrogen-bonding distance to the same amino acids as thosereported in the Zm-Glu-1-inhibitor template (Czjzeket al., 2001), except His-162/His-166, which was not inhydrogen-bonding distance to the glucone in the con-formation chosen. The predicted LjBGD2 and LjBGD4

aglucone-binding pockets have similar overall topol-ogies. Only two amino acid differences betweenLjBGD2 and LjBGD4 are found within the aglucone-binding pockets (Gly-211/Val-215 and His-281/Tyr-285). The relatively bulkier residues of LjBGD4 arepredicted to result in a narrower aglucone-bindingpocket in LjBGD4 compared with LjBGD2. The three L.japonicus hydroxynitrile glucosides are equally accom-modated into the predicted active sites. Based on thenearly identical substrate binding pockets, it waspredicted that LjBGD2 and LjBGD4 would have sim-ilar catalytic activities and substrate specificities.

The predicted similar active site topologies ofLjBGD2 and LjBGD4 (Fig. 6) agree well with thesimilar substrate specificities and activities (Fig. 5).

Phylogenetic Analysis Reveals Three Putative

Hydroxynitrile Glucoside-Cleaving b-Glucosidasesin L. japonicus

Several genes encoding putative b-glucosidaseshave been sequenced from the L. japonicus genome aspart of the L. japonicus genome sequencing project(Kazusa DNA Research Institute). Figure 7 shows theresults of a phylogenetic analysis that focuses onplant b-glucosidases involved in the bioactivation of

Table I. Catalytic parameters of a mixture of LjBGD2 and LjBGD4 isolated from L. japonicus leaves

Parameter Lotaustralin Linamarin Rhodiocyanoside A Prunasin Dhurrin pNPG Daidzin

Km (mM) 0.69 6 0.06 6.6 6 1.1 0.19 6 0.03 0.5 6 0.1 0.5 6 0.1 0.65 6 0.02 0.03 6 0.02Vmax (nmol min21) 2.47 6 0.06 1.6 6 0.2 3.4 6 0.3 1.8 6 0.1 1.1 6 0.1 3.78 6 0.03 0.5 6 0.1r 2 0.99 0.99 0.96 0.97 0.97 0.99 0.77

Figure 5. Substrate specificity profiles of LjBGD2 and LjBGD4 expressedin Arabidopsis. Arabidopsis leaf discs producing recombinant LjBGD2and LjBGD4 were assayed for the ability to hydrolyze a range ofb-glycosides. Rhod A, Rhodiocyanoside A. All incubation assays (10 min)were conducted using 1 mM substrate corresponding to a total of 200nmol. Arabidopsis wild-type leaf discs served as a negative control.

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defense compounds. In the phylogenetic tree, theb-glucosidases known from the literature to hydrolyzea-hydroxynitrile glucosides form separate clades inmonocotyledons and eudicotyledons. This argues thatthe ability to hydrolyze a-hydroxynitrile glucosideshas evolved independently in monocotyledons andeudicotyledons. A more elaborate phylogenetic anal-ysis of plant b-glycosidases, including those presentedin Figure 7, is available at http://www.p450.kvl.dk/BGD.shtml. The phylogenetic analysis identified threeL. japonicus b-glucosidases within the group of eudicot-yledenous a-hydroxynitrile glucoside b-glucosidases:LjBGD2 and LjBGD4, which were isolated from L.japonicus leaves, and a third b-glucosidase, LjBGD7,with approximately 82% amino acid sequence iden-tity and approximately 90% similarity to LjBGD2and LjBGD4. LjBGD2, LjBGD4, and LjBGD7 form acluster with TrCBG, a b-glucosidase from white cloverthat hydrolyzes linamarin. In agreement with the phy-logenetic analysis provided by Chuankhayan et al. (2007b),the isoflavonoid glucoside-cleaving b-glucosidases fromGlycine max and Dalbergia cochinchinensis were foundto cluster between hydroxynitrile glucoside-specificb-glucosidases from legumes and P. serotina. Based onthe phylogenetic analysis, isoflavonoid glucoside-cleaving b-glucosidases in legumes appear to haveevolved from the b-glucosidases involved in the hy-drolysis of hydroxynitrile glucosides.

LjBGD2 and LjBGD4 Are Expressed in Aerial Parts

of L. japonicus, while LjBGD7 Is Expressed in Roots

To determine the expression profiles of the threeb-glucosidase-encoding genes from L. japonicus iden-tified in the phylogenetic analysis (Fig. 7), semiquan-titative RT-PCR was performed using cDNA preparedfrom leaves, stems, and roots of 21-d-old L. japonicus

Figure 6. Models of the three-dimensional architecture of the substrate-binding pockets of LjBGD2 and LjBGD4 with docking of lotaustralin,

linamarin, and rhodiocyanoside A. A, Comparison of the backboneconfigurations of the modeled LjBGD2 and LjBGD4 monomers and theknown structure of TrCBG. A superimposition of the three structuresillustrates the highly conserved structure of a-hydroxynitrile glucoside-cleaving b-glucosidases. B, Stick models of the amino acids liningthe aglucone-binding pockets of LjBGD2 and LjBGD4. Lotaustralin(green) is docked with the two catalytic Glu residues (E208 and E420[LjBGD2 numbering], shown in blue) positioned on both sides of theb-glucosidic bond. Four highly conserved amino acids (N349, Y350,Y351, and W392 [LjBGD2 numbering], shaded in gray) line one side ofthe aglucone-binding pocket, while the amino acids lining the oppositeside (magenta) are highly variable. Only the unconserved amino acidsare specified. The LjBGD2 and LjBGD4 aglucone-binding pocketsdiffer at two positions (G211/V215 and H281/Y285). The amino acidnumbering corresponds to that applied in Supplemental Figure S1. Theamino acids that define the glucone-binding sites are highly conservedin all b-glucosidases belonging to the family 1 glycoside hydrolases andare not shown for reasons of simplicity. C, Lipophilic surface repre-sentations of the active sites of LjBGD2 and LjBGD4. Lipophilic areasare shown in brown, hydrophilic areas in blue, and neutral areas ingreen. The orientation of the active site is the same as in B. The threeendogenous L. japonicus substrates lotaustralin, linamarin, and rho-diocyanoside A are docked into the active sites.



Figure 7. Phylogenetic analysis of selected plant b-glucosidases involved in the bioactivation of defense compounds. Thephylogenetic tree includes hydroxynitrile and isoflavonoid glucoside-cleaving b-glucosidases from eudicotyledons, glucosinolate-degrading myrosinases (Brassicales), and selected b-glucosidases involved in the bioactivation of defense compounds inmonocotyledons. The defense compounds degraded are indicated for the different groups of b-glucosidases. ‘‘S’’ indicatesenzymes for which the crystal structures have been solved. LjBGD2, LjBGD4, and LjBGD7, L. japonicus b-glucosidases (thisstudy); TrCBG, T. repens cyanogenic b-glucosidase (Barrett et al., 1995); GmICHG, G. max isoflavone conjugate-hydrolyzingb-glucosidase (Suzuki et al., 2006); DcBDGLU, D. cochinchinensis dalcochinase (Cairns et al., 2000); PsAH1, PsPH1, PsPH4,and PsPH5, P. serotina amygdalin hydrolase and prunasin hydrolase isoenzymes (Kuroki and Poulton, 1987; Zheng and Poulton,1995; Zhou et al., 2002); VaVH, Vicia angustifolia vicianin hydrolase (Ahn et al., 2007); MeLinamarase, M. esculenta linamarase(Hughes et al., 1992; Keresztessy et al., 2001); HbLinamarase, H. brasiliensis linamarase (Selmar et al., 1987); AtTGG1 andAtTGG2, Arabidopsis myrosinases (Barth and Jander, 2006); SaMYR, Sinapis alba (white mustard) myrosinase (Burmeister et al.,1997); BjMYR and BjMYR1, Brassica juncea (mustard greens) myrosinases (Heiss et al., 1999); RsRMB1 and RsRMB2, Raphanussativus (radish) myrosinases (Hara et al., 2000); BnMYR1, Brassica napus (rape) myrosinase (Chen and Halkier, 1999); As-Glu-1 and As-Glu-2, A. sativa avenacosidases (Gusmayer et al., 1994; Kim et al., 2000); ScBxGlcGLU, S. cereale DIBOA-Glcb-glucosidase (Nikus et al., 2003); Zm-Glu-1, Z. mays glucosidase 1 (Czjzek et al., 2001); SbDhr1 and SbDhr2, S. bicolordhurrinases (Hosel et al., 1987; Verdoucq et al., 2004); PcCBG, Pinus contorta (lodgepole pine) coniferin b-glucosidase(Dharmawardhana et al., 1995, 1999). The bootstrapped neighbor-joining tree was built in MEGA 4.0 (Tamura et al., 2007). Thetree was bootstrapped with 1,000 iterations (node cutoff value, 50%). The underlying amino acid sequences in fastA format andthe multiple alignment can be accessed at http://www.p450.kvl.dk/VintherMorant_etal_Figure7.tfa and http://www.p450.kvl.dk/VintherMorant_etal_Figure7_Alignment.pdf, respectively. The phylogenetic tree was rooted using PcCBG as an outgroup. For thebootstrap analysis, 1,000 trials were performed, and the bootstrap values are shown in percentages; bootstrap node values below50% are not shown. A more elaborate phylogenetic analysis of plant b-glycosidases, including those presented here, is availableat http://www.p450.kvl.dk/BGD.shtml.

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seedlings (Forslund et al., 2004). Based on the RT-PCRanalysis, LjBGD2 and LjBGD4 are expressed in aerialparts of L. japonicus, with an apparent highest expres-sion level in young leaves, and LjBGD7 is expressedexclusively in roots (Fig. 8). If LjBGD7 is a hydroxyni-trile glucoside-cleaving b-glucosidase, as suggested,the different expression profiles explain why LjBGD2and LjBGD4 and not LjBGD7 were obtained from L.japonicus leaves. The expression profile of LjBGD2 andLjBGD4 is in agreement with results from T. repens, alegume closely related to L. japonicus, which show thatthe a-hydroxynitrile glucoside-cleaving b-glucosidase(linamarase) is present in young leaves and not inroots (Dunn et al., 1988).

Localization of b-Glucosidase Activity in L. japonicusand Transgenic Arabidopsis by in Tissue6-Bromo-2-Naphtyl b-D-Glucopyranoside Staining

In order to determine the tissue localization ofb-glucosidase activity in L. japonicus wild type andtransgenic Arabidopsis expressing LjBGD2 and LjBGD4,tissue sections were stained with the chromogenic sub-strate 6-bromo-2-naphtyl b-D-glucopyranoside (BNG)in the presence of 4-benzoylamino-2,5-diethoxybenzene-diazonium chloride hemi[zinc chloride] salt (FastBlue BB salt). Upon hydrolysis of BNG, the agluconeadheres to proteins (Cohen et al., 1952) and forms aninsoluble complex with the Fast Blue BB salt, resultingin red/brown staining at the site of hydrolysis. BNGis an artificial b-glucosidase substrate cleaved bysome b-glucosidases and has previously been usedto monitor the localization of a-hydroxynitrile glucosideb-glucosidase activity in L. corniculatus (Rissler and Millar,1977) and almond (Sanchez-Perez et al., 2008). Thereason that BNG may be used to specifically show thelocalization of hydroxynitrile-cleaving b-glucosidasesis that these are abundant.

In L. japonicus wild-type leaves, color developmentrepresenting b-glucosidase activity was observed innearly all mesophyll cells, while none was detectedin epidermal cells (Fig. 9, A and B). The strongestb-glucosidase activity was observed in palisade cellsand in the spongy cells adjacent to these (Fig. 9, A and B).At the cellular level, distinct areas within the symplastshowed strong staining (Fig. 9D). No or very weakcolor development was observed upon the addition ofFast Blue BB salt in the absence of BNG, demonstratingthat the chromogenic reaction is dependent on the pres-ence of the BNG aglucone formed by b-glucosidasehydrolytic activity (Fig. 9C).

The transgenic Arabidopsis expressing eitherLjBGD2 or LjBGD4 showed strong b-glucosidase ac-tivity upon the addition of Fast Blue BB salt in thepresence of BNG (Fig. 9, F and G). In contrast, no BNG-specific b-glucosidase activity was observed in Arabi-dopsis wild-type leaves (Fig. 9H). Thus, the observedstaining in the transgenic leaves reflects the expressionof LjBGD2 or LjBGD4 and demonstrates that theseenzymes are active after fixation.

The color development and, hence, BNG hydrolysisproceeded at a slower rate compared with L. japonicus.This is most likely due to a more abundant accumu-lation of the b-glucosidases in L. japonicus and/or thepresence of an inhibitor in Arabidopsis. The presenceof an inhibitor is in agreement with the observed lackor low levels of enzyme activity in Arabidopsis leafhomogenates. In agreement with the results observedin L. japonicus leaves, both LjBGD2 and LjBGD4 arelocalized within distinct areas in the symplast uponheterologous expression in Arabidopsis (Fig. 9, I andJ). No or very weak color development was observedin the apoplast, demonstrating that only a weak back-ground reaction takes place in the absence of BNG(Fig. 9, E and K) in L. japonicus wild type and in bothwild-type and transgenic Arabidopsis.

In all transgenic Arabidopsis lines, LjBGD2 andLjBGD4 activity was concentrated in the phloem pa-renchyma, and the activity was very low in other celltypes (Fig. 9, F and G). This is unexpected, as expres-sion of LjBGD2 an LjBGD4 is under the control of thegenerally regarded constitutive CaMV 35S promoter.These results suggest that the activity of the heterol-ogously expressed LjBGD2 and LjBGD4 could be specif-ically inhibited in Arabidopsis leaf cells except thosesurrounding the vascular tissue. This is in agreementwith the observed ability to measure hydroxynitrileglucoside b-glucosidase activity only when assayingleaf discs and not upon tissue disruption caused bymaceration or freezing/thawing. The data obtainedwith the Arabidopsis transgenic lines complement thesymplastic localization of hydroxynitrile glucoside-cleaving b-glucosidase observed in L. japonicus (com-pare Fig. 9, D and I).

Figure 8. Expression profiles of LjBGD2, LjBGD4, and LjBGD7 in 21-d-old L. japonicus seedlings. cDNA produced from mRNA isolatedfrom different tissues was obtained from Forslund et al. (2004), and PCRwas performed with specific primers amplifying LjBGD2, LjBGD4,LjBGD7, and ACTIN. LjBGD2 and LjBGD4 are detected in aerialtissues, while LjBGD7 is detected exclusively in roots.



DISCUSSION

LjBGD2 and LjBGD4 Are Hydroxynitrile Glucosideb-Glucosidases Present in L. japonicus Leaves

Purification, identification by protein sequencing,and biochemical characterization of LjBGD2 andLjBGD4 showed that these two enzymes are thehydroxynitrile glucoside-cleaving b-glucosidases inL. japonicus leaves. Heterologous expression in Arabi-dopsis verified these conclusions and demonstratedthat LjBGD2 and LjBGD4 possess very similar activityprofiles and that the activity is not dependent on het-erodimer formation. The localization of LjBGD2 andLjBGD4 in wild-type leaves of L. japonicus and in trans-genic Arabidopsis leaves was determined using thechromogenic b-glucosidase substrate BNG. The dataindicate that the hydroxynitrile glucoside-cleavingb-glucosidases are localized intracellularly, possibly inprotein bodies, as observed for the P. serotina prunasinand amygdalin hydrolases (Swain et al., 1992).

The predicted molecular masses of the mature LjBGD2and LjBGD4 proteins are 55.9 and 56.9 kD, respec-tively. Analysis of the primary sequences revealed thepresence of three and five potential N-glycosylationsites in LjBGD2 and LjBGD4, respectively. Other dicot-yledenous a-hydroxynitrile glucoside b-glucosidasesare known to be glycosylated (Hughes et al., 1992; Liet al., 1992; Ahn et al., 2007), and heterogeneity in theoverall glycosylation pattern within LjBGD2 andLjBGD4 may account for the observed presence ofboth proteins in the 60- and 55-kD protein bandsfollowing SDS-PAGE (Fig. 3, lane 5). The apparentmolecular mass of 107 kD observed by gel filtrationchromatography suggests that the native LjBGD2 andLjBGD4 proteins exist as dimers, as reported previ-ously for a selection of other a-hydroxynitrile gluco-side b-glucosidases (Hughes and Dunn, 1982; Kurokiand Poulton, 1987; Verdoucq et al., 2004). It cannot beexcluded that a third b-glucosidase specific for rho-

Figure 9. Localization of b-glucosidase activity in leaves of L. japoni-cus wild type (A–E) and transgenic Arabidopsis expressing LjBGD2 andLjBGD4 (F–K). b-Glucosidase activity is represented by red/brownstaining resulting from the hydrolysis of the artificial substrate BNG andsubsequent aglucone complex formation with Fast Blue BB salt. Crosssections (80 mm) of apical leaves (fresh tissue) of L. japonicus wild typeshow strong color development in leaf palisade tissue in the presence ofBNG and Fast Blue BB salt (A and B), while no staining is observed inthe absence of BNG (C). A represents a 203 magnification, while B andC show 53 magnifications. Visualization of the subcellular localization

of b-glucosidase activity is possible at 403 magnification of 6-mm crosssections of fixed apical leaf tissue. Application of BNG results in strongstaining (indicated by white arrows) localized to the symplast (D),whereas only diffuse and weak apoplastic background color develop-ment is observed in the absence of BNG (E). BNG staining forb-glucosidase activity in 80-mm cross sections (203 magnification,fresh tissue) of rosette leaves of Arabidopsis expressing LjBGD2 (F),LjBGD4 (G), and the wild type (H) show that LjBGD2 and LjBGD4 bothhydrolyze BNG but no BNG hydrolysis is observed in Arabidopsis wildtype. I, J, and K are 403 magnifications of 10-mm sections of fixedArabidopsis rosette leaves. I shows the subcellular localization ofLjBGD2 in a cross section (black arrows). J illustrates the subcellularlocalization of LjBGD4 in a longitudinal section (black arrows). Veryweak apoplastic background staining is observed around the xylem inArabidopsis wild type (K) as well as in transgenic Arabidopsis. Identicalsubcellular and tissue localizations of LjBGD2 and LjBGD4 activity areobserved upon heterologous expression of the enzymes in Arabidopsis,with activity localized in the symplast at the subcellular level (inagreement with L. japonicus) and in and around the vascular tissue atthe tissue level (in contrast to L. japonicus).

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diocyanoside A cleavage was lost during purification,because protein fractions that did not show an abilityto hydrolyze lotaustralin or linamarin were not sepa-rately analyzed for hydrolytic activity toward rhodio-cyanoside A due to a shortage of this substrate.However, this is unlikely to have happened, becauseLjBGD2 and LjBGD4 both showed high activity towardrhodiocyanoside A. The hydroxynitrile glucoside-cleaving b-glucosidases were purified 18-fold fromyoung L. japonicus leaves. The low purification foldreflects the high concentration of these enzymesamong the soluble leaf proteins and correspondsto the levels of other abundantly expressed plantb-glucosidases from T. repens, Hevea brasiliensis (rubbertree), Z. mays, Prunus avium (sweet cherry), and S.bicolor (Hughes and Dunn, 1982; Selmar et al., 1987;Pocsi et al., 1989; Esen, 1992; Gerardi et al., 2001;Verdoucq et al., 2004). Purification of linamarase fromM. esculenta and prunasin and amygdalin hydrolasesfrom P. serotina seeds provided significantly higherpurification folds (Kuroki and Poulton, 1986, 1987;Li et al., 1992; Yeoh and Woo, 1992) because of theminor abundance of these a-hydroxynitrile glucoside-cleaving b-glucosidases.

Linamarin Is a Poor Substrate for L. japonicusHydroxynitrile Glucoside-Cleaving b-Glucosidases in

Contrast to Lotaustralin and Rhodiocyanoside A

Upon tissue disruption of L. japonicus leaves, therhodiocyanosides and lotaustralin were rapidly hy-drolyzed, whereas linamarin was not (Fig. 2). Thispattern is reflected in the catalytic parameters ob-served for the partially purified LjBGD2 and LjBGD4,with the Km values for rhodiocyanoside A, lotaustra-lin, and linamarin being 0.2, 0.7, and 6.7 mM, respec-tively (Table I). The Km values for rhodiocyanosideA and lotaustralin are similar to those obtained forother purified a-hydroxynitrile glucoside b-glucosidases(Fan and Conn, 1985; Hosel et al., 1987; Kuroki andPoulton, 1987; Mkpong et al., 1990; Li et al., 1992).Although the Km of the L. japonicus enzymes towardlinamarin appeared unphysiologically high, it remainscomparable to the reported Km values of 4.3, 5.6, and7.6 mM toward linamarin for linamarases purifiedfrom T. repens, Phaseolus lunatus (butter bean), andH. brasiliensis (Itoh-Nashida et al., 1987; Selmar et al., 1987;Pocsi et al., 1989), respectively. It has been suggestedthat the high abundance of some a-hydroxynitrileglucoside b-glucosidases compensates for their lowturnover (Esen, 1992), the most significant examplebeing H. brasiliensis linamarase, which constitutes ap-proximately 30% of the total soluble leaf protein(Selmar et al., 1987).

The low abundance of linamarin in L. japonicus wild-type leaves (less than 5% of the total hydroxynitrileglucosides; Forslund et al., 2004; Bjarnholt et al., 2008)could suggest that linamarin is not a relevant physi-ological substrate for LjBGD2 and LjBGD4. In L.japonicus cotyledons, however, linamarin constitutes

a higher proportion (20%) of the total a-hydroxynitrileglucoside content (Forslund et al., 2004). It is pos-sible that an a-hydroxynitrile glucoside-cleavingb-glucosidase that more efficiently degrades linamarinis specifically expressed in cotyledons. However, amongthe b-glucosidase sequences obtained from the L.japonicus genome sequencing project, only LjBGD2,LjBGD4, and LjBGD7 fall within the hydroxynitrile-cleaving b-glucosidase cluster in the phylogeneticanalysis (Fig. 7; http://www.p450.kvl.dk/BGD.shtml),which argues against this possibility. Alternatively,rapid degradation of lotaustralin results in an imme-diate release of HCN to provide a cyanide bomb-typedefense to instantly repel chewing insects, whereasslow degradation of linamarin would result in a lesspronounced but long-term release of HCN, signalingongoing cyanogenesis as a warning to potential at-tackers.

Docking of the hydroxynitrile glucosides in thepredicted LjBGD2 and LjBGD4 active sites (Fig. 6C)suggested that the small aglucone of linamarin wouldenable relatively free movement within the active site.This could prevent orientation of the b-glucosidicbond in a fixed position between the two catalyticGlu residues, as required for hydrolysis, and thusresult in a higher Km. The bulkier aglucones of rho-diocyanoside A and lotaustralin are expected to directa more rigid binding of these hydroxynitrile gluco-sides in the active site and thus favor a more efficienthydrolysis of the b-glucosidic bond.

The in Planta Function of LjBGD2 and LjBGD4 Is toHydrolyze Hydroxynitrile Glucosides

The substrate specificity of plant b-glucosidases hasbeen a matter of much debate (Hosel and Conn, 1982;Conn, 1993). The S. bicolor dhurrinases show verynarrow substrate specificity and only hydrolyze thenatural substrate dhurrin, the Phe-derived sambuni-grin, and, in the case of dhurrinase 2, the artificialfluorescent substrate 4-methylumbelliferyl b-glucoside(Hosel et al., 1987). P. serotina prunasin hydrolases like-wise show high substrate specificity toward prunasinand no activity against aliphatic a-hydroxynitrile glu-cosides, but in contrast to S. bicolor dhurrinase 1 and 2,they do hydrolyze pNPG (Kuroki and Poulton, 1987).As opposed to these highly specific a-hydroxynitrileglucoside b-glucosidases, linamarases isolated fromLinum ussitassimum (flax) and P. lunatus efficientlyhydrolyze aromatic a-hydroxynitrile glucosides andpNPG in addition to their endogenous aliphatic sub-strates, linamarin and lotaustralin (Fan and Conn,1985; Itoh-Nashida et al., 1987). The broad substratespecificity of LjBGD2 and LjBGD4 toward hydroxyni-trile glucosides resembles those of the L. ussitassimumand P. lunatus linamarases.

The observed ability of LjBGD2 and LjBGD4 (ourdata) and almond b-glucosidase (Chuankhayan et al.,2007b) to hydrolyze daidzin is in agreement with thehypothesis that the isoflavonoid glucoside-cleaving



b-glucosidases have evolved from the hydroxynitrileglucoside-cleaving b-glucosidases, as suggested bythe phylogenetic relationship in Figure 7 and byChuankhayan et al. (2007b). Legumes are character-ized by containing cyanogenic glucosides as well asisoflavonoid glucosides. It remains to be investigatedto what extent isolated b-glucosidases are able tohydrolyze cyanogenic glucosides as well as isoflavo-noid glucosides. The a-hydroxynitrile diglucoside-specific b-glucosidases amygdalin hydrolase andlinustatinase, purified from P. serotina and L. ussitassi-mum, catalyze the hydrolysis of the terminal O-linkedGlc residue of endogenously occurring amygdalin andlinustatin, respectively, to provide the correspondingmonoglucosides prunasin and linamarin (Fan andConn, 1985; Kuroki and Poulton, 1986). In addition,amygdalin hydrolase shows activity toward the ali-phatic counterparts linustatin and neolinustatin andvice versa for linustatinase (Fan and Conn, 1985; Kurokiand Poulton, 1986). The lack of ability of LjBGD2 andLjBGD4 to degrade the diglucoside amygdalin, whilethe corresponding monoglucoside is readily hydro-lyzed, is a common trait for all of the above-mentionedhydroxynitrile glucoside-cleaving b-glucosidases: allare highly specific for either monoglucosides or di-glucosides, except for a single report stating thatlinustatinase is able to hydrolyze the a-hydroxynitrilemonoglucoside dhurrin (Fan and Conn, 1985). Thea-hydroxynitrile diglucosides have been proposed tobe nonhydrolyzable transport forms of the correspond-ing monoglucosides (Selmar et al., 1988). Absolutespecificity of the b-glucosidases toward either mono-glucosides or diglucosides would enable tight controlof a-hydroxynitrile glucoside transport through com-partmentalization and specific developmental expres-sion of the b-glucosidases. Until now, neither linustatinnor neolinustatin, the glucosylated forms of linamarinand lotaustralin, have been detected in L. japonicus (A.V.Morant, C.E. Olsen, B.L. Møller, and S. Bak, unpub-lished data). This may be due to their occurrence invery low concentrations and/or their exclusive pres-ence at defined developmental stages and in specificcell types. In H. brasiliensis, linustatin is detected onlyduring a 48-h period in seedling development (Selmaret al., 1988).

The Biological Implications of Rhodiocyanoside

Hydrolysis Is Unknown

The L. japonicus hydroxynitrile glucoside-cleavingb-glucosidases exhibit a low Km and high Vmax towardrhodiocyanoside A, and this compound is rapidlyhydrolyzed upon tissue disruption of apical L. japoni-cus leaves (Fig. 2). L. corniculatus (Zagrobelny et al.,2004, 2007a), T. repens (data not shown), and M.esculenta (data not shown) all accumulate linamarinand lotaustralin but do not contain rhodiocyanosides.Nevertheless, leaf extracts of L. corniculatus and T.repens hydrolyze rhodiocyanoside A as rapidly aslinamarin, whereas M. esculenta extracts hydrolyze

rhodiocyanide A at a somewhat lower rate (A.V.Morant, N. Bjarnholt, K. Jørgensen, B.L. Møller, andS. Bak, unpublished data). Hence, the ability to de-grade g-hydroxynitrile glucosides in addition to thea-hydroxynitrile glucosides might be an intrinsic char-acteristic of linamarases.

The biological function of rhodiocyanosides remainsunknown (Bjarnholt and Møller, 2008). Hydrolysisof rhodiocyanosides does not result in the liberationof toxic HCN. The observed parallel hydrolysis ofrhodiocyanosides and a-hydroxynitrile glucosides sug-gests that the corresponding aglucones have an anti-microbial or herbicidal effect and act in concert with thea-hydroxynitrile glucosides to deter pathogens andherbivores. The Ile-derived rhodiocyanosides mighthave emerged as a side product of lotaustralin bio-synthesis in L. japonicus. Because the hydroxynitrileglucoside-cleaving b-glucosidases hydrolyze rhodio-cyanosides, the biosynthesis of rhodiocyanosides couldrepresent an evolutionary adaptation of L. japonicus tospecialized insects that are attracted rather than repelledby the a-hydroxynitrile glucosides and their hydrolysisproducts. In free-choice laboratory experiments, larvaeof Zygaena filipendulae (burnet moth) prefer feeding onL. japonicus to L. corniculatus, which does not containrhodiocyanosides. However, the larvae raised on L.japonicus suffer during pupation and the transitioninto an imago, as evidenced by reduced growth andincreased mortality rates. While a-hydroxynitrile glu-cosides are transferred from the larval instars to theimago, the rhodiocyanosides are not (Zagrobelny et al.,2007a, 2007b, 2008).

Expression Profiles of Genes Encoding Enzymes

Involved in the Biosynthesis and Bioactivation ofHydroxynitrile Glucosides Suggest the Evolution ofParallel Pathways in Leaves and Roots of L. japonicus

LjBGD2 and LjBGD4 are expressed in L. japonicusaerial parts, as revealed by purification of the encodedenzymes from young leaves and by RT-PCR analysis.In contrast, LjBGD7 is expressed exclusively in L.japonicus roots. This differential expression patterncorresponds well to that observed for the genes en-coding the hydroxynitrile glucoside biosynthetic en-zymes (Forslund et al., 2004), with coexpression ofCYP79D3 with LjBGD2 and LjBGD4 in aerial tissuesand coexpression of CYP79D4 with LjBGD7 in roots.Although the biochemical function of LjBGD7 has notbeen demonstrated, its position within the eudicotyle-denous hydroxynitrile glucoside-specific b-glucosidasecluster in the phylogenetic tree (Fig. 7) and its veryhigh amino acid sequence identity to LjBGD2 andLjBGD4 imply that LjBGD7 is a hydroxynitrile gluco-side-cleaving b-glucosidase. Hydroxynitrile glucosidesare absent, or present only in minute amounts, in wild-type roots of L. japonicus, but upon ectopic expressionof M. esculenta CYP79D2 under the control of theconstitutive CaMV 35S promoter, linamarin and lotaus-tralin but no rhodiocyanosides do accumulate in

Morant et al.


roots (Forslund et al., 2004). Unless this is amere consequence of the transport of a-hydroxynitrileglucosides produced in aerial parts to the roots,these results indicate that L. japonicus roots containthe biosynthetic machinery for the production ofa-hydroxynitrile glucosides. In accordance, LjBGD7could represent the corresponding hydroxynitrileglucoside-cleaving b-glucosidase. The physiologicalconditions required to induce hydroxynitrile gluco-side synthesis in wild-type roots of L. japonicus remainunknown, as does the possible biological significanceof the presence of hydroxynitrile glucosides in thistissue (Bjarnholt and Møller, 2008). Apart from anobvious function in defense, a-hydroxynitrile gluco-sides have been suggested to serve as nitrogen storagecompounds (Selmar et al., 1988; Jenrich et al., 2007). Inroots, the a-hydroxynitrile glucosides or the agluconeor HCN formed after hydrolysis may serve as impor-tant signaling molecules secreted into the rhizosphere.Preliminary results from the expression of a GFP-GUSreporter construct under the control of the CYP79D3and CYP79D4 promoters in L. japonicus indicate that,in addition to being constitutively expressed in roots,CYP79D4 is wound inducible in aerial parts. Theseresults are in agreement with metabolite analysesshowing that the concentration of hydroxynitrile glu-cosides in L. japonicus leaves increases in responseto mechanical wounding (A.V. Morant, N. Bjarnholt,M.E. Kragh, C.H. Kjærgaard, K. Jørgensen, S.M.Paquette, M. Piotrowski, A. Imberty, C.E. Olsen, B.L.Møller, and S. Bak, unpublished data). Further exper-iments are required to determine whether LjBGD7 isable to hydrolyze hydroxynitrile glucosides and, inparallel to CYP79D4, whether it is expressed in aerialparts of L. japonicus in response to wounding. If thehydroxynitrile glucoside/b-glucosidase system in L.japonicus is truly wound inducible, this would extendthe classification of a-hydroxynitrile glucosides in L.japonicus to phytoalexins (defense compounds synthe-sized in response to pathogen or herbivore attack) inaddition to phytoanticipins (preformed defense com-pounds). Insect-induced changes in the profile ofsecondary metabolites otherwise characterized as phy-toanticipins were previously observed for glucosino-lates in Arabidopsis (Mewis et al., 2005).

Why Do L. japonicus Leaves Contain Two

Apparently Highly Similar HydroxynitrileGlucoside b-Glucosidases?

The presence of LjBGD2 and LjBGD4 in L. japonicusleaves raises the question of why L. japonicus producestwo apparently redundant hydroxynitrile glucoside-cleaving b-glucosidases. LjBGD2 is located on chro-mosome 3 and LjBGD4 on chromosome 5. This, incombination with the approximately 85% amino acidsequence identity shared between the encoded en-zymes, argues against LjBGD2 and LjBGD4 as result-ing from a recent gene duplication and as being trulyredundant. The chromosomal location rather suggests

that both paralogues have been retained throughevolution due to different biochemical activities orexpression profiles. Furthermore, pairwise alignmentof a 1,000-nucleotide region immediately upstreamfrom the ATG start codon revealed that the promoterregions of LjBGD2 and LjBGD4 share only approxi-mately 50% sequence identity, with no increase insequence identity in the region approaching the startcodon (data not shown). Combined, these data indi-cate that LjBGD2 and LjBDG4 are not redundantenzymes but have acquired separate biological func-tions in the process of subfunctionalization. The chro-mosomal location of LjBGD7 is currently not known.Future experiments using in tube in situ PCR andpromoter fusion constructs will reveal whetherLjBGD2 and LjBGD4 are differentially expressed atthe cellular or developmental level and might beinvolved in different phases of cyanogenesis, depend-ing on the cellular location or nature of pathogen orinsect attack.

One potential reason for the coaccumulation ofLjBGD2 and LjBGD4 in L. japonicus leaves could bethat heterodimer formation could lead to a change insubstrate specificity or activity. However, the highlysimilar predicted active site topologies and the nearlyidentical substrate specificity profiles obtained whenLjBGD2 and LjBGD4 were separately expressed intransgenic Arabidopsis and when the two copurifyingb-glucosidases were tested together argue against thisscenario.

The presence of two or more isoforms of b-glucosi-dases has been observed in several other cyanogenicand noncyanogenic plants. P. serotina contains severalisoforms of both prunasin and amygdalin hydrolases(Kuroki and Poulton, 1986, 1987; Li et al., 1992), andmultiple isoforms of linamarase are found in M.esculenta (Mkpong et al., 1990). Likewise, Z. mays, S.bicolor, and A. sativa each contain two isoenzymes ofthe b-glucosidases that bioactivate the DIMBOA-Glc,dhurrin, and avenacosides found in these three crops,respectively (Hosel et al., 1987; Kim and Kim, 1998;Cicek and Esen, 1999, Morant et al., 2008). As-Glu-1and As-Glu-2, the two avenacoside b-glucosidases inA. sativa (Fig. 7), exist as homooligomers of As-Glu-1as well as heterooligomers of both isoenzymes (Kimand Kim, 1998; Kim et al., 2000). Whether the homo-oligomers and the heterooligomers display differentbiological functions in vivo is unknown, except thatthe encoding genes are differentially expressed (Kimand Kim, 1998), as are the Z. mays and S. bicolor pa-ralogues (Hosel et al., 1987; Cicek and Esen, 1999). Inaddition, two apparently redundant myrosinases,TGG1 and TGG2 (Fig. 7), with nearly identical expres-sion patterns are found in the aerial parts of Arabi-dopsis (Barth and Jander, 2006). Hence, it appears thatplant b-glucosidases constitute a highly fine-tunedsystem in which even subtle differences in biochemicalactivity, localization, or expression are important forthe bioactivation of defense compounds in responseto diverse biotic stresses at different stages of plant



development. A number of interacting factors maycontrol and modulate b-glucosidase activity in accor-dance with demands (Morant et al., 2008). Recently,the complexity of the pathways responsible for theinduction of isoflavonoid synthesis and b-glucosidase-mediated activation was documented by thoroughstudies in Medicago truncatula (Naoumkina et al., 2007;Farag et al., 2008) and in G. max (Suzuki et al., 2006).

MATERIALS AND METHODS

Chemicals and Plant Growth Conditions

Lotus japonicus GIFU B-129-S9 plants were grown hydroponically at ap-

proximately 24�C in a greenhouse fitted with extra light bulbs, ensuring a

minimum photosynthetic flux of 100 to 120 mmol photons m22 s21, as

described by Forslund et al. (2004). Arabidopsis (Arabidopsis thaliana) plants

were grown in 9-cm-diameter plastic pots in autoclaved soil supplemented

with vermiculite (10%, v/v) in an insect-free growth chamber at 22�C, with

70% humidity, a photosynthetic flux of 100 mmol photons m22 s21, and a 16-h-

light/8-h-dark regime, as described by Kristensen et al. (2005).

Linamarin was purchased from AG Scientific, lotaustralin from Toronto

Research Chemicals, and dhurrin from Carl Roth. pNPG, BNG, Fast Blue BB

salt, and amygdalin were from Sigma. Daidzin was purchased from Tauto

Biotech, and kuromanin chloride salt was from Polyphenols. Rhodiocyano-

side A was purified from L. japonicus leaves (Bjarnholt and Møller, 2008).

Prunasin was kindly synthesized by Dr. Mohammed Saddik Motawia.

pOHBG and 4-(methylthio)butyl glucosinolate were from C2 Bioengineering.

Analysis of Hydroxynitrile Glucoside Degradation inCrude L. japonicus Extracts

Apical leaf extracts of L. japonicus 35STCYP79D2 line number 5 (Forslund

et al., 2004) were made by subjecting two leaf discs (diameter, 6 mm) to three

freeze/thaw cycles while submersed in 200 mL of MES (20 mM, pH 6.5). The

apical leaf extract was diluted 10 times and incubated at 30�C (300 rpm).

Aliquots (25 mL) were collected at 0, 10, 20, 30, and 60 min and immediately

transferred to an equal volume of 100% methanol to stop further b-glucosidase

activity. The relative concentrations of linamarin, lotaustralin, and rhodiocya-

nosides were quantified by liquid chromatography-mass spectrometry

(LC-MS; described below) based on the extracted ion chromatograms and

taking advantage of the similar ionization efficiency of all three compounds

(Bjarnholt and Møller, 2008).

Isolation of Hydroxynitrile Glucoside-Cleaving

b-Glucosidases from L. japonicus Leaves andProtein Sequencing

Soluble proteins from apical leaves of L. japonicus were extracted by

grinding the leaves in ice-cold extraction buffer (5 v/w plant material of 100

mM MES [pH 7.0], 250 mM Suc, 50 mM NaCl, 2 mM EDTA, 2 mM DTT, 0.5 mM

phenylmethylsulfonyl fluoride, 10% [w/v] polyvinylpolypyrrolidone, and

0.5% [w/v] 6-capronic acid). After removal of the cell debris by centrifugation,

the soluble protein extract (pH adjusted to 5.5) was incubated (4�C, 30 min,

gentle shaking) with 10% (v/v) cation exchange resin preequilibrated in

100 mM MES (pH 5.5; Source 15S; Amersham Biosciences). The resin was

recovered by centrifugation, and bound protein was extracted with 100 mM

MES (pH 7.0) fortified with 500 mM NaCl. The protein extract was dialyzed

(SpectraPor, 12- to 14-kD molecular mass cutoff [Spectrum Laboratories])

against 100 mM MES (pH 7.0, 4�C) and stored at 4�C. An aliquot of the protein

extract (500 mg of protein; Bradford, 1976) was size fractionated by gel

filtration chromatography (Superdex G200 HR 10/ 30 column; Amersham

Biosciences) using 50 mM Tris-HCl (pH 7.0) and 150 mM NaCl as equilibration

and elution buffer. Elution of proteins with b-glucosidase activity was

monitored using lotaustralin and linamarin as substrates and colorimetric

detection of cyanide release (Halkier and Møller, 1989; Jørgensen et al., 2005a).

The polypeptide composition of the main fraction showing b-glucosidase

activity was analyzed by 12% SDS-PAGE and showed enrichment in two

proteins at 60 and 55 kD. In-gel digestion of the Coomassie Blue-stained

protein bands with trypsin and sequencing of the peptides by mass spec-

trometry were performed as described by Piotrowski and Volmer (2006).

Database searches (nrdb95) were done with MS Blast (Shevchenko et al., 2001),

available at http://dove.embl-heidelberg.de/Blast2/msblast.html. LjBGD2

and LjBGD4 were identified by manual assignment of the peptide sequences

to assembled b-glucosidase sequences from L. japonicus.

Biochemical Characterization of LjBGD2 and LjBGD4

Km and Vmax values were determined using assay mixtures (total volume,

200 mL) containing purified L. japonicus b-glucosidases (2003 diluted) and a

number of different substrates (10 concentrations between 25 mM and 4 mM) in

20 mM MES (pH 6.5). At the end of the incubation period (10 min, 30�C, 300

rpm), the enzyme reaction was stopped and metabolic conversions were

measured. Assay mixtures containing linamarin, lotaustralin, dhurrin, pru-

nasin, amygdalin, and pOHBG hydrolysis were stopped by the addition of

NaOH (40 mL, 6 N), and metabolism was measured by colorimetric detection

of cyanide release (Halkier and Møller, 1989; Jørgensen et al., 2005a) or SCN2

release in the case of pOHBG (Bak et al., 1999). KCN and NaSCN standard

curves were included to enable quantification. Assay mixtures containing

rhodiocyanoside A were stopped by the addition of an equal volume of 100%

methanol, and metabolism was measured by quantification of the remaining

amounts of rhodiocyanoside A by LC-MS based on a rhodiocyanoside A

standard curve. For pNPG, 100 mL of 0.4 M Na2CO3 was added and pNPG

metabolism was measured (optical density at 410 nm) by colorimetric detec-

tion of aglucone release. For quantification, a pNPG standard curve was

produced by reaction of 0 to 50 mM pNPG with an excess of cation exchange-

purified L. japonicus leaf protein. Daidzin hydrolysis was tested at seven

concentrations between 25 and 400 mM (added in 5 mL of dimethyl sulfoxide).

Kuromanin hydrolysis was evaluated at a concentration of 1 mM, and the

reaction was stopped by the addition of 200 mL of methanol containing 4%

formic acid for stabilization of the remaining kuromanin. The daidzin and

kuromanin results were quantified by LC-MS as described below.

Assays to determine the biochemical activity of recombinant LjBGD2 and

LjBGD4 were performed using leaf discs from Arabidopsis T3 transformants

homozygous for the respective transgenes. Relative turnovers of glucosides by

Arabidopsis expressing LjBGD2 and LjBGD4 were determined using assay

mixtures (total volume, 200 mL) containing two 6-mm leaf discs from rosette

leaves of 4- to 5-week-old plants and 1 mM substrate in 20 mM MES (pH 6.5)

and incubation for 10 min (30�C, 300 rpm). Reactions were stopped and

analyzed as described above. Daidzin turnover was determined in the same

way except that the substrate concentration was 0.75 mM added in 10 mL of

dimethyl sulfoxide, due to the limited solubility of the compound in aqueous

medium. This did not affect enzyme activity in the leaf disc assay. Kuromanin

turnover was also determined at a concentration of 0.75 mM for comparison

with daidzin, and the reaction was stopped by the addition of 200 mL of

methanol containing 4% formic acid. The results were quantified by LC-MS as

described below.

LC-MS Analysis

Analytical LC-MS of hydroxynitrile glucosides was carried out using an

Agilent 1100 Series liquid chromatograph (Agilent Technologies) fitted with

an XTerra MS C18 column (Waters; 3.5 mM, 2.1 3 100 mm; flow rate, 0.2 mL

min21) coupled to a Bruker Esquire 30001 ion trap mass spectrometer (Bruker

Daltonics). The mobile phases were as follows: A, water with HCOOH (0.1%,

v/v) and NaCl (50 mM); B, MeCN/water (80%, v/v) with HCOOH (0.1%). The

gradient program was as follows: 0 to 4 min, isocratic 2% B; 4 to 10 min, linear

gradient 2% to 8% B; 10 to 30 min, linear gradient 8% to 50% B; 30 to 35 min,

linear gradient 50% to 100% B; and 35 to 40 min, isocratic 100% B. The mass

spectrometer was run in electrospray mode, and positive ions were observed.

The HPLC solvent contained NaCl and formic acid to facilitate the identifi-

cation of adduct ions (and [M1H1]) as described previously (Tattersall et al.,

2001; Forslund et al., 2004; Kristensen et al., 2005). Analyses of daidzin and

kuromanin were carried out using a Dionex HPLC apparatus with an HPLC

pump (Dionex P680), autosampler (Dionex ASI-100), and UV light detector

(Dionex UVD340U) with the column oven set to 25�C (Dionex STH585).

Separation was performed on a Synergy Fusion column from Phenomenex

(150 3 2 mm, 4-mm particles, 0.3 mL/min), and the mobile phases were as

follows: A, water with 0.1% formic acid and 2% MeCN (v/v); and B, MeCN/

water (50:50, v/v) with 0.1% formic acid. The gradient program for daidzin

Morant et al.


was as follows: 0 to 2 min, 30% B; 2 to 12 min, linear gradient 0% to 100% B;

and 12 to 20 min, 100% B, followed by wash and equilibration. The gradient

program for kuromanin was as follows: 0 to 1 min, 0% B; 1 to 8 min, linear

gradient 0% to 100% B; and 8 to 15 min, 100% B, followed by wash and

equilibration. The flow was passed directly from the UV light detector to the

mass spectrometer, a Thermo Finnigan MSQ single quadrupole device oper-

ated in electrospray mode with positive ionization (ionization temperature,

365�C; cone voltage, 2.5 kV). Daidzin and its aglucone were detected as the

[M1H]1 ion and kuromanin as the [M]1 ion.

Protein Sequence Alignments, Phylogenetic Trees,

Protein Sequence Logos, and Prediction of SubcellularLocalization and Molecular Weight

Protein sequence alignments were made using ClustalX 1.83 for Windows

(http://bips.u-strasbg.fr/fr/Documentation/ClustalX/). ClustalX parameters

were as follows. Pairwise alignment parameters: gap opening penalty, 13.50;

gap extension penalty, 0.75; protein weight matrix, Gonnet 250. Multiple

alignment parameters: gap opening penalty, 15.00; gap extension penalty, 1.00;

protein weight matrix, Gonnet series. All gaps were reset before any align-

ment was run. Alignments were colored using Boxshade (http://www.isrec.

isb-sib.ch/ftp-server/boxshade/3.3.1/) compiled for a Windows 32 environ-

ment. Neighbor-joining phylogenetic trees were constructed using MEGA 4.0

(Tamura et al., 2007; http://www.megasoftware.net/index.html) with 1,000

bootstrap trials performed. Gaps and missing data were handled via pairwise

deletion, and the Poisson substitution model was used to calculate distances.

Protein sequence logos were constructed using the Berkeley WebLogo Gen-

erator (http://weblogo.berkeley.edu/) using default parameters.

Signal sequence cleavage sites and subcellular localizations were predicted

using PSORT (http://psort.ims.u-tokyo.ac.jp/) and TargetP (http://www.cbs.

dtu.dk/services/TargetP/).

Molecular weights of the predicted mature sequences of LjBGD2 and

LjBGD4 were predicted using the ExPASy Compute pI/Mw tool (http://

www.expasy.org/tools/pi_tool.html).

Pairwise amino acid and nucleotide sequence alignments were performed

using BioEdit (Hall, 1999) with default settings.

Comparative Modeling of LjBGD2 and LjBGD4

LjBGD2 and LjBGD4 models were built with the homology-modeling

program COMPOSER (Blundell et al., 1988) of the Sybyl software (SYBYL).

High-resolution crystal structures of TrCBG (1CBG; Barrett et al., 1995),

SbDhr1 (1V02; Verdoucq et al., 2004), and Zm-Glu-1 (1e1f; Czjzek et al., 2001)

were used as templates to build the structurally conserved regions of LjBGD2

and LjBGD4 (Protein Data Bank codes [Berman et al., 2000] are given in

parentheses). Alignments of LjBGD2 and LjBGD4 with TrCBG, SbDhr1, and

Zm-Glu-1 to define structurally conserved regions were performed with

ClustalX and modified manually. Regions between two structurally conserved

regions were defined as loops. Loops were modeled by comparison with

TrCBG and SbDhr1. One loop in LjBGD2 (six amino acids long) and three

loops in LjBGD4 (three to five amino acids long) could not be modeled by

comparison with known b-glucosidase structures and were built manually. To

identify backbone conformations that fell outside the allowed areas of the

Ramachandran plot, the models were analyzed using PROCHECK (Laskowski

et al., 1993) and subsequently optimized. Hydrogen atoms were added to all

atoms of the LjBGD2 and LjBGD4 models, and partial atomic charges were

calculated with the Pulmann method.

Linamarin, lotaustralin, and rhodiocyanoside A were graphically edited in

Sybyl from b-Glc taken from the Glyco-3D monosaccharide database (http://

www.cermav.cnrs.fr/glyco3d/index.php). Atom types and charges were

chosen according to the PIM parameters for carbohydrate (Imberty et al.,

1999), and each compound was docked into the active sites of LjBGD2 and

LjBGD4 using COMPOSER. The Glc moieties were positioned in a chair

conformation in the glucone-binding pockets of the models using the

Zm-Glu-1 structure crystallized as a complex with the inhibitor p-nitrophenyl

b-D-thioglucoside (Czjzek et al., 2001) as a template. While the substrate glucone

was kept fixed in this defined position, the aglucone conformations were fit into

the optimal position of the aglucone-binding sites by several cycles of energy

optimization involving the aglucone and the amino acid side chains surround-

ing the binding site with the use of the Tripos force field (Clark et al., 1989). The

active site pocket topologies were visualized using MOLCAD (Waldherr-

Teschner et al., 1992), applying lipophilic surface representations. Cartoons

showing overall protein conformations and stick representations of the amino

acids lining the aglucone-binding pocket were made using the PyMOL molec-

ular visualization system (http://pymol.sourceforge.net/).

Analysis of the Expression Patterns of LjBGD2,

LjBGD4, and LjBGD7 in L. japonicus by RT-PCR

LjBGD2 cDNA was amplified using primers BGD2RTfor (5#-CATCTCTTT-

TATCTTCGATCTGC-3#) and BGD2RTrev (5#-CAGCAAGATGAAAGCAG-

GATCCTTG-3#). LjBGD4 cDNA was amplified with primers BGD4RTfor

(5#-CATCTCTTTTATCTTCAATCTGC-3#) and BGD4RTrev (5#-CCACTAAAA-

CAACCCAACAAGACG-3#), and LjBGD7 cDNA was amplified using primers

BGD7RTfor (5#-CATATATTATCACACAAGAACAAGC-3#) and BGD7RTrev

(5#-GATATGCTGCGGATGCTGTCCCAA-3#). Primers for the amplification of

cDNA encoding the L. japonicus putative actin homolog were those applied by

Forslund et al. (2004). L. japonicus cDNA obtained from Forslund et al. (2004) was

used as template in PCRs (total volume, 20 mL) performed in Phusion HF PCR

buffer (Finnzymes) containing 250 mM of each dNTP, 1.5 mM MgCl2, 0.5 mM of

forward and reverse primers, and 0.2 unit of Phusion polymerase (Finnzymes).

Thermal cycling parameters were as follows: 98�C for 30 s, followed by 30

(LjBGD2, LjBGD4, and ACTIN) or 35 (LjBGD7) cycles of 98�C for 10 s, 58�C for

30 s, and 72�C for 15 s. RT-PCR products were separated by agarose gel

electrophoresis and visualized with ethidium bromide under UV light. The

authenticity of the RT-PCR amplicons was verified by DNA sequencing.

Heterologous Expression of LjBGD2 and LjBGD4in Arabidopsis

The full-length LjBGD2 cDNA was assembled from a genomic bacterial

artificial chromosome (BAC) clone and a partial cDNA clone. Nucleotides 1 to

174 corresponding to the first exon were PCR amplified from BAC clone

TM0568 (Kazusa DNA Research Institute) using primers 5#-ggtggtggt-

ctgcagATGGCACTCAACACGTTCTTGG-3# (restriction site underlined) and

5#-TCGCGCACCTTCATACTGGTATGCCGAGGATGCTGTC-3#. Nucleotides

175 to 1,545 corresponding to the remaining LjBGD2 cDNA sequence were

amplified using the partial LjBGD2 EST AV423894 (Kazusa DNA Research

Institute) as a template with primers 5#-TCCTCGGCATACCAGTATG-

AAGGTGCGGCAAATAAAGG-3# and 5#-ccaccaccacccgggCTAATATCTTTT-

AAGAAAGTTTG-3#. PCR (total volume, 50 mL) was carried out in 10 mM

Tris-HCl (pH 8.3), 50 mM KCl, and 2 mM MgSO4 containing 2.5 units of Pwo

DNA polymerase (Roche), 50 mM dATP, 50 mM dCTP, 50 mM dGTP, 50 mM dTTP,

and 10 pmol of the forward and reverse primers. Thermal cycling parameters

were as follows: 94�C for 2 min; followed by 30 cycles of 94�C for 30 s, 55�C for

1 min, 0.2�C s21 until 45�C, 45�C for 1 min, and 72�C for 30 s (first exon) or 90 s

(remaining cDNA); and a final 72�C for 5 min. The partial cDNA sequences

were joined by overlap extension PCR using the two outer primers and the

two purified PCR products as templates. Thermal cycling parameters were

the same as for amplification of LjBGD2 cDNA nucleotides 175 to 1,545. The

purified PCR product corresponding to the full-length LjBGD2 cDNA was

ligated into the PstI/XmaI sites of pPS48 (Odell et al., 1985; Kay et al., 1987) to

yield pPSBGD2, and the nucleotide sequence was verified by DNA sequenc-

ing. In pPS48, the cDNA is inserted between the enhanced CaMV 35S

promoter and terminator. The 35STBGD2 sequence was subsequently ligated

into the HindIII site of pCAMBIA2301 to yield pCAMBIABGD2.

The LjBGD4 expression construct was assembled from a genomic BAC

clone and a partial cDNA clone. The first intron was included to circumvent

problems with PCR amplification and the ability to recover the full-length

LjBGD4 cDNA. A HindIII site at nucleotide positions 512 to 517 in the LjBGD4

cDNA was removed by site-directed mutagenesis according to the recom-

mendations from Stratagene to yield pBlueBGD4-3# using primer 5#-CATTGG-

GATATGCCCCAAGCaTTGGAAGATGAGTATGG-3# (changed nucleotide in

lowercase) and its complement, with the partial LjBGD4 ESTAV425071 (Kazusa

DNA Research Institute) as a template to facilitate subsequent cloning steps.

The first two exons including the first intron were amplified from BAC clone

TM0569 (Kazusa DNA Research Institute) using primers 5#-ggtggtggtctgcag-

ATGGCACTCAACACGTTCTTAG-3# and 5#-CTATGCGCTCTGCATATTG-

CACTGAATAGTTGTGGGCGTAG-3#. The remaining cDNA sequence was

amplified using pBlueBGD4-3# as a template and primers 5#-gaagaagaa-

ggatccCTAATATCTTTTGAGAAAGTTCG-3# and 5#-CTACGCCCACAACTA-

TTCAGTGCAATATGCAGAGCGCATAG-3#. The purified PCR products were

joined by overlap extension PCR using the two outer primers. PCR was

performed as described above. The obtained full-length PCR amplicon was



ligated into the PstI/BamHI sites of pBluescript KS2, and the nucleotide

sequence was verified by DNA sequencing. LjBGD4 was subcloned into the

EcoRI/BamHI sites of pRT101 (Topfer et al., 1988) to introduce the enhanced

CaMV 35S promoter and a poly(A) signal. Finally, the 35STBGD4 sequence was

subcloned into the HindIII site of pPZP221 (Hajdukiewicz et al., 1994) to yield

pPZPBGD4 for transformation into Arabidopsis. At each cloning stage, recom-

binant plasmids were transformed into Escherichia coli SURE cells (Stratagene).

The use of E. coli SURE was required to enable cloning of the LjBGD4

nucleotide sequence, as classical E. coli strains did not allow the recovery of

LjBGD4 plasmids. For construction of pCAMBIABGD2, E. coli XL1-Blue cells

(Stratagene) were used.

pCAMBIABGD2 and pPZPBGD4 were transformed into Arabidopsis

ecotype Columbia via Agrobacterium tumefaciens LBA4404 using the floral

dip method (Clough and Bent, 1998), and transformed seeds were selected on

Murashige and Skoog medium (Murashige and Skoog, 1962) supplemented

with 100 mg/mL kanamycin and gentamycin, respectively.

Localization of b-Glucosidase Activity by in

Tissue Staining

Apical leaves of L. japonicus wild type and rosette leaves of approximately

5-week-old Arabidopsis 35STLjBGD2, 35STLjBGD4, and wild type were

embedded in plastic according to the manufacturer’s recommendations for

Technovit 8100 (Heraeus) with minor alterations. The tissues were dehydrated

in a graded series of acetone solutions (25%, 50%, and 100%, 1 h each) and left

overnight in the filtration solution. Sections (6 mm for L. japonicus, 10 mm for

Arabidopsis) were cut on a Reichert-Jung 2030 rotary microtome. Leaves for

80-mm leaf cross sections were embedded in 5% agarose and cut on a Leica VT

1000S microtome.

In tissue staining for b-glucosidase activity was performed by incubation

at 37�C in a solution of 2.6 mM BNG and 1.8 mM Fast Blue BB salt in 50 mM

citrate and 100 mM phosphate buffer (pH 5.8). Incubation times were as

follows: 15 and 30 min for 6- and 10-mm sections of L. japonicus and

Arabidopsis, respectively, and 4 and 15 min (300 rpm) for 80-mm sections of

L. japonicus and Arabidopsis, respectively. Assays excluding BNG were

included as negative controls. Results were analyzed using a Leica DMR

fluorescence microscope fitted with a Leica DC 300F camera.

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

libraries under accession numbers EU10844 to EU10846.

Supplemental Data

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

Supplemental Figure S1. Alignment of peptide sequences obtained from

purified hydroxynitrile glucoside b-glucosidases with full-length

LjBGD2 and LjBGD4 polypeptide sequences.

Supplemental Figure S2. Illustration of the degree of conservation of

amino acids in plant b-glucosidases by sequence logos.

Supplemental Tree Data S1. Additional phylogenetic trees.

ACKNOWLEDGMENTS

We thank Dr. Mohammed Saddik Motawia for providing prunasin and for

helpful discussions, Dr. Henrik Toft Simonsen for help with LC-MS analyses,

and Ph.D. student Sarah Osmani for help with the presentation of protein

structure models. We are grateful to Dr. Rene Mikkelsen for guidance in gel

filtration chromatography. Steen Malmmose is sincerely thanked for taking

great care of the L. japonicus plants. The Kazusa DNA Research Institute is

thanked for providing L. japonicus b-glucosidase sequences.

Received September 28, 2007; accepted May 6, 2008; published May 8, 2008.

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The -Glucosidases Responsible for Bioactivation of Hydroxynitrile Glucosides in Lotus japonicus

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