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Characterization of the rice carotenoid cleavagedioxygenase 1 reveals a novel route for geranialbiosynthesisAndrea Ilg, Peter Beyer and Salim Al-BabiliFaculty of Biology, Institute of Biology II, Albert-Ludwigs University of Freiburg, GermanyCarotenoids are isoprenoid pigments synthesized by allphotosynthetic organisms and some nonphotosyntheticbacteria and fungi. In plants, carotenoids are essentialin protecting the photosynthetic apparatus fromphoto-oxidation, and represent essential constituents ofthe light-harvesting and of the reaction center com-plexes [1–4]. Carotenoids are also the source of apoca-rotenoids [5–7], which are physiologically activecompounds, including the ubiquitous chromophoreretinal, the chordate morphogen retinoic acid and thephytohormone abscisic acid as the best-known exam-ples. Further carotenoid-derived signaling moleculesare represented by strigolactones, a group of C15apoc-arotenoids attracting both symbiotic arbuscular mycor-rhizal fungi and parasitic plants [8,9] and, as recentlyshown, exerting functions as novel plant hormones reg-ulating shoot branching [10,11]. In addition, the devel-opment of arbuscular mycorrhiza is also accompaniedby accumulation of cyclohexenone (C13) and mycor-radicin (C14) derivatives [12], all of which are apoca-rotenoids conferring a yellow pigmentation to infectedroots [13]. Apocarotenoids, such as bixin in Bixa orell-ana [14] and saffron in Crocus sativus [15], are plantpigments of economic value.The synthesis of apocarotenoids is initiated bythe oxidative cleavage of double bonds in carotenoidKeywordsapocarotenoids; carotenoid cleavage;carotenoid dioxygenase; geranial; lycopenecleavageCorrespondenceS. Al-Babili, Institute for Biology II, CellBiology, Albert-Ludwigs University ofFreiburg, Schaenzlestr. 1, D-79104 Freiburg,GermanyFax: +49 761 203 2675Tel: +49 761 203 8454E-mail: salim.albabili@biologie.uni-freiburg.de(Received 19 September 2008, revised 25November 2008, accepted 26 November2008)doi:10.1111/j.1742-4658.2008.06820.xCarotenoid cleavage products – apocarotenoids – include biologically activecompounds, such as hormones, pigments and volatiles. Their biosynthesisis initiated by the oxidative cleavage of C–C double bonds in carotenoidbackbones, leading to aldehydes and ⁄ or ketones. This step is catalyzed bycarotenoid oxygenases, which constitute an ubiquitous enzyme family,including the group of plant carotenoid cleavage dioxygenases 1 (CCD1s),which mediates the formation of volatile C13ketones, such as b-ionone, bycleaving the C9–C10 and C9¢–C10¢ double bonds of cyclic and acycliccarotenoids. Recently, it was reported that plant CCD1s also act on theC5–C6 ⁄ C5¢–C6¢ double bonds of acyclic carotenes, leading to the volatileC8ketone 6-methyl-5-hepten-2-one. Using in vitro and in vivo assays,we show here that rice CCD1 converts lycopene into the three differentvolatiles, pseudoionone, 6-methyl-5-hepten-2-one, and geranial (C10),suggesting that the C7–C8 ⁄ C7¢–C8¢ double bonds of acyclic carotenoidends constitute a novel cleavage site for the CCD1 plant subfamily. Theresults were confirmed by HPLC, LC-MS and GC-MS analyses, andfurther substantiated by in vitro incubations with the monocyclic caroten-oid 3-OH-c-carotene and with linear synthetic substrates. Bicyclic carote-noids were cleaved, as reported for other plant CCD1s, at the C9–C10 andC9¢–C10¢ double bonds. Our study reveals a novel source for the widelyoccurring plant volatile geranial, which is the cleavage of noncyclic ends ofcarotenoids.AbbreviationsCCD, carotenoid cleavage dioxygenase; GST, glutathione S-transferase; NIST, National Institute of Standards and Technology; OsCCD1,Oryza sativa carotenoid cleavage dioxygenase 1; SPME, solid-phase microextraction.736 FEBS Journal 276 (2009) 736–747 Journal compilation ª 2008 FEBS. No claim to original German government worksbackbones, generally catalyzed by carotenoid oxygen-ases, nonheme iron enzymes that are common in alltaxa [5–7,16]. VP14 (viviparous14) from maize, whichcatalyzes the formation of the abscisic acid precursorxanthoxin by cleaving 9-cis-epoxy carotenoids [17], isthe first identified member of this enzyme class. On thebasis of their substrate specificity, VP14 and its ortho-logs have been termed 9-cis-epoxy-carotenoid dioxy-genases. Plants possess a second group of carotenoidoxygenases, carotenoid cleavage dioxygenases (CCDs),which act on different carotenoid substrates. TheCCDs of higher plants contribute to diverse physio-logical processes, including the regulation of the out-growth of lateral shoot buds [18–20] and plastiddevelopment [21].Plants release volatile apocarotenoids, including C13ketones such as b-ionone and damascone [22], whichconstitute an essential aroma note in tea, grapes, roses,tobacco and wine [23]. Such compounds may arise byunspecific oxidative degradation or lipid co-oxidationprocesses, involving lipoxygenases [24]. Alternatively,they are produced by double bond-specific cleavagereactions mediated by peroxidases [25] or by CCDssuch as members of the plant CCD1 subfamily. PlantCCD1s cleave numerous cyclic and linear all-trans-car-otenoids at the C9–C10 and C9¢–C10¢ double bondsinto C14dialdehydes, which are common to all carot-enoid substrates, and two variable end-group-derivedC13ketones [6,7,16]. The wide substrate specificity ofplant CCD1s allows the production of divergent vola-tile C13compounds, including b-ionophores, a-ionones,pseudoionone and geranylacetone. The first member ofthe CCD1 subfamily was identified from Arabidopsisthaliana [26], and was later shown to act as adioxygenase [27]. Sequence homology then allowed theidentification and charcterization of orthologs fromseveral plant species, such as crocus, tomato, grape,melon, petunia and maize [15,28–32].The biological function of CCD1s was confirmed byloss-of-function experiments in tomato fruits and petu-nia flowers, leading to decreased emission of b-ionone[27,31]. Moreover, recent studies on the CCD1 frommaize indicated its involvement in the formation ofcyclohexenone and mycorradicin in mycorrhizal roots[33]. Underscoring a role of CCD1 in carotenoidcatabolism, seeds of Arabidopsis ccd1 mutants con-tained elevated carotenoid levels [34]. This suggestedthat the modification of CCD1 expression is instru-mental for altering volatile production contributing totaste, or in increasing the carotenoid content in cropplant tissues where elevated provitamin A carotenoidlevels are aimed for, such as high-b-carotene tomato[35,36], canola [37], golden rice [38] or golden potato[39]. Hence, the identification of substrates and cleav-age sites of CCD1s from crop plants is considered tobe a worthwhile approach.It has recently been discovered that plant CCD1sexert additional activity at the C5–C6 and ⁄ or the C5¢–C6¢ double bonds of acyclic carotenoids, leading to theformation of the C8ketone 6-methyl-5-hepten-2-one[32]. In this study, we investigated the enzymatic activi-ties of the sole CCD1 [Oryza sativa CCD1 (OsCCD1)]occurring in rice. Our study revealed the C7¢–C8¢ dou-ble bond of linear and monocyclic carotenoids to bean additional novel cleavage site of OsCCD1, leadingto geranial and indicating a novel plant route for theformation of this widespread volatile compound.ResultsPurified OsCCD1 cleaves acyclic apolycopenals atthe C7–C8 double bondTo investigate the activity of OsCCD1, the correspond-ing cDNA was cloned and expressed as a glutathioneS-transferase (GST)-fusion protein in Escherichia colicells. However, the insolubility of the fusion protein,which could not be improved by modulating theexpression conditions, hampered its purification.Therefore, the GST–OsCCD1 fusion was expressed inBL21(DE3) E. coli cells harboring the vector pGro7,which encodes the chaperones groES–groEL, enhanc-ing correct folding. This resulted in a striking improve-ment of the GST–OsCCD1 solubility, allowing proteinpurification (Fig. S1).It has been shown that CCD1s from Arabidopsisand maize maintain their regional specificity in cleav-ing the C9–C10 double bond with the synthetic apoca-rotenoid b-apo-8¢-carotenal (C30), forming b-ionone(C13) and the C17dialdehyde apo-8,10¢-carotendial[27,32]. To test the cleavage activity of OsCCD1,in vitro assays were performed with this substrate,using purified enzyme. Subsequent HPLC analyses(data not shown) revealed a cleavage pattern identicalto that of the plant CCD1s mentioned above. Todetermine the impact of the chain length and of the io-none ring on the cleavage pattern, purified OsCCD1was incubated with b-apo-10¢-carotenal (C27), which isshorter than b-apo-8¢-carotenal (C30), and the acyclicsubstrates apo-10¢-lycopenal (C27) and apo-8¢-lycopenal(C30). HPLC analyses of the incubation with the cyclicb-apo-10¢-carotenal (Fig. 1; substrate I) revealed analmost complete conversion of this substrate(Table S1) and the formation of the C14dialdehydeapo-10,10¢-carotendial (rosafluene dialdehyde; prod-uct 1) and b-ionone (C13; product 2), as confirmed byA. Ilg et al. A novel route for geranial formationFEBS Journal 276 (2009) 736–747 Journal compilation ª 2008 FEBS. No claim to original German government works 737LC-MS and GC-MS analyses, respectively (data notshown). The relatively low amount of the C14dialde-hyde is probably a result of instability. The formationof b-ionone from b-apo-8¢-carotenal (C30) and b-apo-10¢-carotenal (C27) suggested that the cleavage of theC9–C10 double bond occurs independently of thechain length of monocyclic apocarotenals, pointing tothe ionone ring as a determinant governing the reac-tion site.The two acyclic substrates were cleaved almost com-pletely within 30 min (Table S1), proving them to beas suitable as the monocyclic apocarotenals. However,as shown in the HPLC analyses (Fig. 1), the cleavageof apo-10¢-lycopenal (C27; substrate II) led to a morecomplex mixture of products, including three com-pounds (products 1, 4 and 5) identified as dialdehydeson the basis of the fine structure of the correspondingUV–visible spectra. The three dialdehydes differed intheir chain lengths, as indicated by their retentiontimes and absorption maxima. Products 1 and 5 weresupposed to represent a C14and a C19dialdehyde,respectively. These are expected to arise upon cleavageat the known plant CCD1 sites (C9–C10 and C5–C6).The retention time of product 4 indicated a chainlength between C14and C19. This pointed to the recog-nition of a novel cleavage site, at the C7–C8 doublebond, between the above mentioned C9–C10 andC5–C6 positions, yielding a C17dialdehyde. To con-firm their nature, the dialdehyde products, 1, 4 and 5,were purified and analyzed by LC-MS. In order to sta-bilize the C14dialdehyde (product 1), it was derivatizedwith O-methyl-hydroxylamine-hydrochloride prior toLC-MS analyses. As shown in Fig. 2, derivatizedABCFig. 1. (A) HPLC analyses of OsCCD1 in vitro assays with syntheticapocarotenoids. The cyclic b-apo-10¢-carotenal (C27, I) was cleavedinto apo-10,10¢-carotendial (C14, 1) and b-ionone (C13, 2); the loweramount of the former is probably a result of its instability. The acy-clic substrates apo-10¢-lycopenal (C27, II) and apo-8¢-lycopenal (C30,III) were converted into pseudoionone (3) and three dialdehydes ofdifferent chain lengths (1, 4 and 5 from II; 4, 6 and 7 from III), asindicated by their UV ⁄ visible-spectra (shown in the insets) andretention times. (B) Structures of the synthetic substrates showingthe cleavage. I, b-apo-10¢-carotenal; II, apo-10¢-lycopenal; III, apo-8¢-lycopenal. The substrates were cleaved at three different doublebonds, including the novel site (b, shaded). Cleavage of the C9–C10double bond (a) results in products 1 and 2 from I, products 1 and3 from II, and products 3 and 4 from III. The cleavage of (b) leadsto product 4 from II, and product 6 from III. The cleavage of theC5–C6 double bond (c) leads to product 5 from II, and prod-uct 7 from III. (C) Structures of the cleavage products detected: 1,apo-10,10¢-carotendial (C14dialdehyde); 2, b-ionone (C13); 3,pseudoionone (C13); 4, apo-8,10¢-carotendial (C17dialdehyde); 5,apo-6,10¢-carotendial (C19dialdehyde); 6, apo-8,8¢-carotendial (C20dialdehyde); 7, apo-6,8¢-carotendial (C22dialdehyde).A novel route for geranial formation A. Ilg et al.738 FEBS Journal 276 (2009) 736–747 Journal compilation ª 2008 FEBS. No claim to original German government worksproduct 1 exhibited an [M + (NCH3)2+H]+molec-ular ion of mass 275.17 (substrate I), consistent withthe C14dialdehyde structure, and the molecular ionsfor products 4 and 5 (Fig. 2; substrates II and III)proved their identities as C17and C19dialdehydes,respectively.HPLC analyses of the incubation with apo-8¢-lyco-penal (C30; substrate III in Fig. 1) confirmed novelcleavage of the C7–C8 double bond. As shown inFig. 1, the reaction led to an equivalent series of C17,C20and C22dialdehydes (products 4, 6 and 7), corre-sponding to the cleavage of the C9–C10, C7–C8 andC5–C6 double bonds, respectively. The nature of thesedialdehyde products was confirmed by LC-MS anal-yses (data not shown).Cleavage of the three double bonds described abovemust also lead to the three different mono-oxygenatedproducts pseudoionone (C13), geranial (C10) and6-methyl-5-hepten-2-one (C8) (for structures, seeFig. 4). Pseudoionone was found by HPLC analysis(Fig. 1; product 3) and its presence was further demon-strated by GC-MS analyses, which also showed theformation of geranial and and 6-methyl-5-hepten-2-one(data not shown).OsCCD1 mediates double cleavage ofdifferent site combinations in 3-OH-c-caroteneand lycopeneTo determine the cleavage sites in natural substrates,purified OsCCD1 was incubated with the bicycliczeaxanthin, the monocyclic 3-OH-c-carotene and theacyclic lycopene. As shown in Fig. 3, OsCCD1 con-verted zeaxanthin (substrate I) into the two products3-OH-b-ionone (C13; product 1) and rosafluene-dialde-hyde (C14; product 2), as confirmed by LC-MS andGC-MS analyses, respectively (data not shown). Thissuggested that OsCCD1 cleaves the C9–C10 andC9¢–C10¢ double bonds of bicyclic carotenoids, likeother plant orthologs.Although it occurred at lower conversion rates thanwith the synthetic substrates (Table S1), we clearlyobserved the accumulation of the C17and C19dialde-hydes (Fig. 3; products 4 and 5, respectively) from3-OH-c-carotene (Fig. 3; substrate II). Owing to itsinstability, the third dialdehyde (C14), expected fromthe cleavage of the C9–C10 and C9¢–C10¢ doublebonds, occurred at low levels only (Fig. 3; product 2).The formation of the C14,C17and C19dialdehydes(for structures, see Fig. 3C) suggested a single cut atthe C9–C10 double bond of the ring-bearing side of3-OH-c-carotene in combination with several cleavageoptions in the linear half of the molecule, namely atthe C9¢–C10¢,C7¢–C8¢ and C5¢–C6¢ double bond. Theoccurrence of the C17dialdehyde confirmed the novelsite at the C7¢–C8¢ double bond observed with apoly-copenals and implied the formation of geranial.Accordingly, the GC-MS analyses of the in vitro assayspointed to the conversion of 3-OH-c-carotene intoFig. 2. LC-MS identification of the dialdehydes produced in vitro.The three dialdehydes formed from apo-10¢-lycopenal in vitro(Fig. 1; products 1, 4 and 5 ) represent a C14,aC17and a C19dialde-hyde, respectively, as suggested by their molecular ions of mass275.17 (I, [M + (NCH3)2+H]+; product 1), 257.13 (II,[M+H]+;product 4) and 283.14 (III,[M+H]+; product 5).A. Ilg et al. A novel route for geranial formationFEBS Journal 276 (2009) 736–747 Journal compilation ª 2008 FEBS. No claim to original German government works 739geranial (Fig. 4; substrate IV), as indicated by thedetection of the expected [M]+molecular ion of mass152.3 and the fragmentation pattern (Fig. 4;substrate IV), which was correctly recognized by theNational Institute of Standards and Technology(NIST) library (mass spectral search program Ver-sion 2.0). In addition, these GC-MS analyses (data notshown) revealed the formation of the known plantCCD1 mono-oxo products 6-methyl-5-hepten-2-one,3-OH-b-ionone and pseudoionone, the latter two ofwhich had already been detected in the HPLC analyses(Fig. 3; products 1 and 3).The high lipophilicity of lycopene did not allow theuse of octyl-b-glucoside as detergent in the correspond-ing in vitro assays. Therefore, lycopene micelles wereproduced with a Triton X mixture. The productsformed from this substrate (Fig. 3; substrate III, prod-ucts 3, 4 and 5 ) suggested the cleavage of the doublebond combinations C9–C10 ⁄ C7¢–C8¢, C9–C10 ⁄ C5¢–C6¢and their symmetrical counterparts. The C14dialde-hyde formed by cleavage of the C9–C10 ⁄ C9¢–C10¢double bond combination was only detectable inlonger incubations (data not shown). GC-MS analysesof the lycopene incubations demonstrated the forma-tion of pseudoionone and 6-methyl-5-hepten-2-one.However, geranial could not be detected, although theformation of the C17dialdehyde confirmed the cleav-age of lycopene at the novel C7–C8 or C7¢–C8¢ site.OsCCD1 catalyzes the formation of three differentvolatiles from lycopene in vivoTo confirm cleavage at the C7–C8 or C7¢–C8¢ doublebond in vivo, OsCCD1 was expressed in lycopene-ABCFig. 3. (A) HPLC analyses of OsCCD1 in vitro incubations withthree natural carotenoids. UV–visible spectra of the products areshown in the insets. The bicyclic zeaxanthin (I) was converted into3-OH-b-ionone (1) and apo-10,10¢-carotendial (C14, 2). The products3-OH-b-ionone (C13, 1), apo-10,10¢-carotendial (C14dialdehyde, 2),pseudoionone (C13, 3), apo-8,10¢-carotendial (C17dialdehyde, 4) andapo-6,10¢-carotendial (C19dialdehyde, 5) were obtained from 3-OH-c-carotene (II). The cleavage of lycopene (III) led to pseudoionone(C13, 3), apo-8,10¢-carotendial (C17dialdehyde, 4) and apo-6,10¢-caro-tendial (C19dialdehyde, 5). Longer incubations with lycopene alsoresulted in the accumulation of the C14dialdehyde apo-10,10¢-caro-tendial corresponding to compound 2 (not shown). (B) Structures ofthe substrates showing the cleavage sites, including the novelC7–C8 ⁄ C7¢–C8¢ double bonds (b, b¢, shaded). I, zeaxanthin; II,3-OH-c-carotene; III, lycopene. OsCCD1 cleaves zeaxanthin at theC9–C10 ⁄ C9¢–C10¢ double bonds (a ⁄ a¢). 3-OH-c-Carotene (II)iscleaved at the C9–C10 double bond (a) in combination with theC9¢–C10¢ double bond (a¢), the C7¢–C8¢ double bond or the C5¢–C6¢(c¢) double bond. Lycopene (III) is cleaved either at the C9–C10 dou-ble bond (a) in combination with one of the three double bondsC9¢–C10¢ (a¢), C7¢–C8¢ (b¢)orC5¢–C6¢ (c¢), or at the C9¢–C10¢ doublebond (a¢) in combination with one of the a, b or c sites. (C)Structures of the cleavage products detected.A novel route for geranial formation A. Ilg et al.740 FEBS Journal 276 (2009) 736–747 Journal compilation ª 2008 FEBS. No claim to original German government worksaccumulating E. coli cells, and volatile compoundswere collected from the medium and analyzed by GC-MS. As shown in Fig. 4, the activity of the enzymeresulted in the accumulation of 6-methyl-5-hepten-2-one (I), the reduced form of geranial, geraniol (II), andpseudoionone (III). The three compounds, undetect-able in the controls, were identified by their correct[M]+molecular ions and by comparing the mass spec-A B Fig. 5. Determination of the relative amounts of dialdehyde prod-ucts. (A) Incubations with apo-8¢-lycopenal (C30). The peak areas ofthe three dialdehydes (C17,C20and C22) formed from apo-8¢-lyco-penal were calculated at a max. plot of 350–550 nm. The valuesrepresent the proportion of each dialdehyde in the sum of the threepeak areas. (B) Incubations with apo-10¢-lycopenal (C27), 3-OH-c-car-otene and lycopene. The values represent ratios of the C17and C19dialdehydes in the sum of their peak areas calculated by integratingeach peak at its individual kmax. Data represent the average of sixindependent incubations.Fig. 4. GC-MS analyses of volatile OsCCD1 products. Volatile com-pounds produced in lycopene-accumulating and OsCCD1-expres-sing cells were collected using SPME and subjected to GC-MS(I–III). As suggested by the [M]+molecular ions (bold) and compari-son of mass spectra with the NIST library, the in vivo activity ofOsCCD1 led to the volatiles 6-methyl-5-hepten-2-one (C8, I), gera-niol (C10, II) and pseudoionone (C13, III). IV represents the detectionof geranial (C10) produced from 3-OH-c-carotene in vitro.A. Ilg et al. A novel route for geranial formationFEBS Journal 276 (2009) 736–747 Journal compilation ª 2008 FEBS. No claim to original German government works 741tra with the NIST library. HPLC analyses of the corre-sponding cell pellets revealed the accumulation of acomplex mixture of compounds, tentatively identifiedas dialcohols corresponding to three dialdehydesdescribed above (data not shown).OsCCD1 exhibits different preferences for thethree cleavage sitesTo gain insights into the preference of OsCCD1 forthe three cleavage sites, we determined the relativeamounts of the C17,C20and C22dialdehyde productsformed upon incubation with apo-8¢-lycopenal (C30),corresponding to C9–C10, C7–C8 and C5–C6 doublebond recognition, respectively (Fig. 1B; substrate III).The enzyme exhibited by far the highest preference forthe C9–C10 double bond in vitro, as suggested by thepredominance of the C17dialdehyde, which accountedfor about 90% of the total dialdehyde products(Fig. 5A). The relative amount (about 7%) of the C20dialdehyde was much higher than of the C22dialde-hyde (about 2%), indicating a higher affinity for thenovel C7–C8 double bond than for the C5–C6 doublebond. The instability of the C14dialdehyde arisingfrom the double cleavage at the C9–C10 ⁄ C9¢–C10¢double bonds in lycopene and 3-OH-c-carotene ham-pered determination of the preference for these sites,and allowed only a comparison of the C9–C10 ⁄C7¢–C8¢ and C9–C10 ⁄ C5¢–C6¢ cleavage products corre-sponding to the C17and C19dialdehydes, respectively.The higher relative amount of the C17dialdehyde indi-cated a higher preference for the C7–C8 than for theC5–C6 double bond in 3-OH-c-carotene, whereas theopposite tendency was observed with lycopene andapo-10¢-lycopenal (Fig. 5B).DiscussionPlant CCD1s are known to catalyze the cleavage ofthe C9–C10 and the C9¢–C10¢ double bonds of severalcarotenoids. Recently, it was shown that these enzymescan also cleave at the C5–C6 and ⁄ or the C5¢–C6¢ dou-ble bonds in lycopene [32]. This was deduced fromGC-MS analyses of lycopene-accumulating E. coli cellsexpressing different plant CCD1s and from in vitroincubations with a CCD1 from maize (ZmCCD1),showing in both cases the formation of 6-methyl-5-hepten-2-one (C8), besides pseudoionone. Geranial(C10) was not detected, and cleavage of the C7–C8 ⁄ C7¢–C8¢ double bonds was therefore excluded [32].On the basis of our recent work on a Nostoc caroten-oid oxygenase producing geranial and derivativesthereof from monocyclic carotenoids [40], we assumedthat plant CCD1s may also be able to cleave theC7–C8 ⁄ C7¢–C8¢ double bonds. Here, we demonstratethat the rice enzyme OsCCD1 cleaves linear ends ofcarotenoids at three different double bond positions,including the C7–C8 ⁄ C7¢–C8¢ double bonds, leading togeranial.To avoid further metabolization of products thatcan occur in vivo, we relied first on in vitro incubationsusing purified enzyme, which allowed clear identifica-tion of the products formed. In a first approach, wechecked the site specificities using synthetic apocarote-nals packed in octyl-b-glucoside micelles. This enabledus to observe the cleavage of the C7–C8 double bond.However, the confirmation of this novel activity withthe highly lipophilic lycopene and c-carotene requiredthe use of different detergents. The best activities wereobtained with micelles produced with a Triton X mix-ture, following the protocol of Prado-Cabrero et al.[41]. The accumulation of the C17dialdehyde in thelycopene assays confirmed the cleavage at theC9–C10 ⁄ C7¢–C8¢ double bonds. However, the activitiesdetermined were still low in comparison to the incuba-tions with zeaxanthin, and did not allow clear identifi-cation of geranial. Furthermore, we did not detectsignificant conversion of lycopene in the 30 min incu-bations used to determine the substrate preferences ofthe enzyme (Table S1). This weak activity is probablydue to the use of the Triton X mixture, which was nec-essary to soulibilize lycopene, but led to an overallreduction of enzyme activity (Table S1). Therefore, weused the more polar 3-OH-c-carotene, which could beincorporated into octyl-b-glucoside micelles and whichwas readily cleaved at the double bond combinationsC9–C10 ⁄ C9¢–C10¢, C9–C10 ⁄ C7¢–C8¢ and C9–C10 ⁄C5¢–C6¢.The formation of multiple dialdehyde productsallows some conclusions to be drawn on the site pref-erences of OsCCD1. For instance, the C14,C17andC19dialdehydes formed from lycopene and 3-OH-c-carotene arise from cleavage of the C9–C10 doublebond, which is combined with the C9¢–C10¢,C7¢–C8¢or C5¢–C6¢ double bonds. The C9–C10 ⁄ C9¢–C10¢ dou-ble bonds constitute the main site, as suggested by thedetermination of the relative amounts of the dialdehy-des produced from apo-8¢-lycopenal; the C9–C10 ⁄ C9¢–C10¢ double bonds also constitute the sole cleavage sitein ring-bearing moieties of substrates. This preferencemay explain the absence of the C20,C24and C22dial-dehydes in the lycopene incubation, which would beexpected if the cleavage reactions occurred only at theC7–C8 ⁄ C7¢–C8¢ and ⁄ or C5 ⁄ C6 ⁄ C5¢–C6¢ double bonds.A further conclusion is that the first cleavage site playsa role in the determination of the second one in acyclicA novel route for geranial formation A. Ilg et al.742 FEBS Journal 276 (2009) 736–747 Journal compilation ª 2008 FEBS. No claim to original German government workssubstrates. This is shown by the different preferencesfor the C7–C8 and C5–C6 double bonds (Fig. 5) inapo-10¢-lycopenal (C27) and apo-8¢-lycopenal (C30),which mimic a lycopene molecule cleaved at the C9¢–C10¢ and C7¢–C8¢ double bonds, respectively. More-over, comparison of the relative amounts of the C19and C17dialdehydes accumulated in the incubationswith the natural substrates lycopene and 3-OH-c-caro-tene indicates that the preference of OsCCD1 for theC5¢–C6¢ and C7¢–C8¢ double bonds depends on thenature of the substrate.The cleavage of a sole double bond in the cyclic moi-ety of 3-OH-c-carotene and of monocyclic b-apocarote-nals with different chain lengths indicates that theb-ionone end-group may determine the reaction site inthe polyene chain. This may provide an explanation forthe ‘wobbling’ of the enzyme on linear substratemoieties, where three different double bonds are beingrecognized. Similar results were obtained with thecyanobacterial enzyme SynACO, representing up tonow the only carotenoid oxygenase with a known crys-tal structure [42]. SynACO cleaved b-apocarotenoidswith different chain lengths at a sole site, the C15–C15¢double bond, leading to retinal and derivatives thereof[43]. In contrast, apolycopenals were cleaved at multiplepositions, including the C15–C15¢ double bond,and indicating ‘wobbling’ of the enzyme (S. Ruch,S. Al-Babili and P. Beyer; unpublished data).Owing to the high sequence similarity of plantCCD1s, it appears likely that the cleavage of theC7–C8 ⁄ C7¢–C8¢ double bonds of linear substrates isnot unique to OsCCD1. Apart from the formation ofgeranial, the OsCCD1 cleavage reactions were identicalto those of other plant CCD1s, as supported by theformation of pseudoionone, 6-methyl-5-hepten-2-oneand b-ionones. Geranial is biologically active andknown to exert antifungal and antimicrobial activities[44,45]; it represents a major volatile of tomato fruits[46] and roses [47]. Moreover, citral, the mixture ofgeranial and its cis-isomer neral, is a major componentof the aroma of lemon grass and other lemon-scentedplants. Geranial is synthesized from geranyl diphos-phate by the enzymes geraniol synthase [48] and gera-niol dehydrogenase [49]. The enzymatic cleavage ofmonocyclic and acyclic carotenoids into geranial repre-sents a novel biosynthetic route, and may provide anexplanation for the impact of lycopene accumulationon the emission of geranial, as observed in the fruits ofseveral tomato and watermelon varieties [46], as wellas in transgenic tomato fruits, where elevated caroten-oid levels were accompanied by an increased emissionof citral [50]. The possible synthesis of geranial bytomato CCD1s is now under investigation.Experimental proceduresCloning proceduresFive micrograms of total RNA, isolated from 14-day-oldseedlings (O. sativa var. japonica cv. TP309), was used forcDNA synthesis using SuperScript III RnaseH)(Invitrogen,Paisley, UK), according to the instructions of the manufac-turer. Two microliters of cDNA was then applied for theamplification of OsCCD1 (accession no. AK066766,encoded by Os12g0640600), using the primers CCD-1 (5¢-ATGGGAGGCGGCGATGGCGATGAG-3¢) and CCD-2(5¢-TCACGCTGATTGTTTTGCCAGTTG-3¢). The PCRreaction was performed with 100 ng of each primer, 200 lmdNTPs and 1 lL of Advantage cDNA Polymerase Mix(BD Biosciences, San Jose, CA, USA) in the buffer pro-vided, as follows: 2 min of initial denaturation at 94 °C,followed by 32 cycles of 30 s at 94 °C, 30 s at 58 °C, and2 min at 68 °C, and 10 min of final polymerization at68 °C. The obtained PCR product was purified using GFXPCR DNA and a Gel Band Purification Kit (AmershamBiosciences, Piscataway, NJ, USA), and cloned into thepCR2.1–TOPO vector and pBAD ⁄ TOPO (Invitrogen) toyield pCR–OsCCD1 and pBAD–OsCCD1, respectively.The nature of the cDNA was verified by sequencing. Toexpress OsCCD1 as a GST-fusion protein, the correspond-ing cDNA was excised as an EcoRI fragment frompCR–OsCCD1, and then ligated into accordingly digestedand alkaline phosphatase-treated pGEX–5X-1 (AmershamBiosciences) to yield pGEX–OsCCD1.Protein expression and purificationpGEX–OsCCD1 was transformed into BL21(DE3) E. colicells harboring the plasmid pGro7 (Takara Bio Inc.; Mobitec,Go¨ttingen, Germany), which encodes the groES–groELchaperone system under the control of an arabinose-induciblepromoter. Overnight cultures (2.5 mL) were inoculated into50 mL of 2 · YT medium containing 0.2% (w ⁄ v) arabinose,grown at 28 °CtoaD600 nmof 0.7, and induced with 0.2 mmisopropyl thio-b-d-galactoside for 4 h. Cells were harvestedby centrifugation (10 min, 6000 g), resuspended in 4 mL ofNaCl ⁄ Pi(pH 7.3), and lysed using a French press. Six millili-ters of NaCl ⁄ Pi(pH 7.3) containing 1% Triton X-100 wasthen added, and the suspension was incubated at roomtemperature for 30 min. After centrifugation for 10 min at12 000 g, the fusion protein was purified using glutathione–Sepharose 4B (Amersham Biosciences), according to themanufacturer’s instructions. OsCCD1 was then released byovernight treatment with the protease factor Xa in NaCl ⁄ Pi(pH 7.3) at room temperature, according to the manu-facturer’s instructions (Amersham Biosciences). The proteineluate contained approximately 50% purified OsCCD1.Purification steps and protein expression were monitored bySDS ⁄ PAGE. The control strain expressed only GST.A. Ilg et al. A novel route for geranial formationFEBS Journal 276 (2009) 736–747 Journal compilation ª 2008 FEBS. No claim to original German government works 743Enzyme assaysSynthetic substrates were kindly provided by BASF (Lud-wigshafen, Germany). Lycopene was obtained from Roth(Karlsruhe, Germany). Zeaxanthin and 3-OH-c-carotenewere isolated from E. coli cells transformed with carotenoidbiosynthetic genes (unpublished data). The substrates werepurified using TLC, and quantified spectrophotometricallyat their individual kmaxvalues, using extinction coefficientscalculated from E1% [51]. Protein concentrations weredetermined using the BioRad protein assay kit (BioRad,Hercules, CA, USA).In vitro assays contained 40 lg of purified enzyme eluateat substrate concentrations of 80 lm (lycopene and 3-OH-c-carotene) or 40 lm (zeaxanthin and synthetic substrates).For the production of lycopene micelles, dried substratewas resuspended in 200 lL of benzene and mixed with150 lL of an ethanolic detergent mixture consisting of0.7% (v ⁄ v) Triton X-100 and 1.6% (v ⁄ v) Triton X-405.The mixture was then dried using a vacuum centrifuge toproduce a carotenoid-containing gel. The gel was resus-pended in 110 lLof2· incubation buffer containing2mm tris(2-carboxyethyl)phosphine, 0.6 mm FeSO4and2mgÆmL)1catalase (Sigma, Deisenhofen, Germany) in200 mm Hepes ⁄ NaOH (pH 7.8). One hundred microlitersof this lycopene suspension was then used in the in vitroassay, which was started by adding water and purified Os-CCD1 to obtain a final volume of 200 lL. 3-OH-c-Caro-tene, zeaxanthin and apocarotenoids were solubilized usingoctyl-b-glucoside at a final concentration of 1% (v ⁄ v). Forthis purpose, substrates were mixed with 50 lLofa4%octyl-b-glucoside ethanolic solution, dried using a vacuumcentrifuge, and resuspended in 100 lL of the 2· incuba-tion buffer mentioned above. Water and purified OsCCD1were then added to obtain the final volume of 200 lL.Depending on the substrates, the incubations were per-formed at 28 °C for 4 h (lycopene and 3-OH-c-carotene),2 h (zeaxanthin) or 30 min (synthetic substrates). Reac-tions were stopped by adding two volumes of acetone.Lipophilic compounds were partitioned against petroleumether ⁄ diethyl ether 1 : 4 (v ⁄ v), vacuum-dried, and dis-solved in 40 lL of chloroform. HPLC analyses were thenperformed using 20 lL of the extracts. For GC-MS analy-ses, volatile compounds were collected with solid-phasemicroextraction (SPME) fibers (100 lm polydimethylsil-oxane; Sigma-Aldrich) for 30 min.Conversion rates were determined in 30 min incubationassays using 30 lg of purified enzyme eluate. For quantifi-cation, 200 lL of an acetonic solution of a-tocopheroleacetate (1 mgÆmL)1) was added as internal standard to eachassay prior to extraction. The conversion rates weredetermined by calculating the decrease of substrate peakareas measured at their individual kmaxvalues using themax plot function of the software empower pro (Waters,Eschborn, Germany). Peak areas were normalized relativeto the peak area of the internal standard, which was quan-tified at its absorption maximum of 285 nm.In vivo test using lycopene-accumulatingE. coli cellsLycopene-accumulating XL1-Blue E. coli cells (unpublisheddata), harboring the corresponding biosynthetic genes fromErwinia herbicola , were transformed with pBAD–OsCCD1or with pBAD–TOPO as a negative control. Overnight cul-tures were used to inoculate 50 mL of LB medium. Bacteriawere grown at 28 °CtoaD600 nmof 0.5, and induced with0.08% (w ⁄ v) arabinose. Cells were harvested after 6 h, andvolatile compounds were collected by introducing theSPME fiber into the cell-free medium for 30 min. ForHPLC analyses, cells were harvested after 4 h, and carote-noids were extracted and processed as described above.Analytical methodsFor HPLC analyses, a Waters system equipped with aphotodiode array detector (model 996) was used. AC30-reversed phase column (YMC Europe, Schermbeck,Germany) was developed with solvent system B [MeOH ⁄t-butylmethyl ether ⁄ water (60 : 2 : 20, v ⁄ v ⁄ v)] and solventsystem A [MeOH ⁄ t-butylmethyl ether (1 : 1, v ⁄ v)] at a flowrate of 1 mLÆmin)1, using a linear gradient from 100%solvent B to 43% solvent B within 45 min, and then to 0%solvent B within 1 min. The final conditions were main-tained for 26 min at a flow rate of 2 mLÆmin)1, and thiswas followed by re-equilibration.LC-MS analyses of compounds collected from HPLCwere performed using a Thermo Finnigan LTQ mass spec-trometer coupled to a Surveyor HPLC system consisting ofa Surveyor Pump Plus, Surveyor PDA Plus and SurveyorAutosampler Plus (Thermo Electron, Waltham, MA, USA).Separations were carried out using a YMC-Pack C30-reversed phase column (150 · 3 mm internal diameter,3 lm). Separation and identification of the C17and C19dialdehydes was performed as described in [40]. The oximeof the C14dialdehyde was produced by adding 50 lLofO-methyl-hydroxylamine-hydrochloride (15 mgÆmL)1)toanMeOH solution of the HPLC-purified compound, and thenincubating for 20 min at 50 °C. The product was then par-titioned against petroleum ether ⁄ diethyl ether 1 : 4 (v ⁄ v).The identification of the oxime was carried out accordingto [40].GC-MS analyses were carried out with a Finnigan TraceDSQ mass spectrometer coupled to a Trace GC gas chro-matograph equipped with a 30 m Zebron ZB 5 column(5% phenylpolysilanoxane ⁄ 95% dimethylpolysilanoxane,0.25 mm internal diameter, and 0.25 lm film thickness;Phenomenex, Aschaffenburg, Germany). The temperatureprogram used was as follows: 50 °C held isocraticallyfor 5 min, followed by a ramp of 25 °CÆmin)1to a finalA novel route for geranial formation A. Ilg et al.744 FEBS Journal 276 (2009) 736–747 Journal compilation ª 2008 FEBS. No claim to original German government workstemperature of 340 °C, which was maintained for an addi-tional 5 min. The He carrier gas flow was maintained at1mLÆmin)1using a split flow of 1 : 20. The splitless timewas 3 min, and the injector oven temperature was set at220 °C. Standard electrospray ionization was used at anion source potential of 70 eV and with an ion source tem-perature of 200 °C. Identification of compounds was doneby comparing the mass spectra with the NIST database.AcknowledgementsThis work was supported by the HarvestPlus pro-gramme (http://www.harvestplus.org) and by the Deut-sche Forschungsgemeinschaft (DFG), Grant 892 ⁄ 1-3.We are indebted to J. Mayer for valuable discussions.We thank H. Ernst for providing the synthetic sub-strates and E. Scheffer for skilful technical assistance.References1 Cunningham FX & Gantt E (1998) Genes and enzymesof carotenoid biosynthesis in plants. 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Characterization of the rice carotenoid cleavage dioxygenase 1 reveals a novel route for geranial biosynthesis Andrea Ilg, Peter Beyer and Salim Al-BabiliFaculty. linear and monocyclic carotenoids to bean additional novel cleavage site of OsCCD1, leadingto geranial and indicating a novel plant route for the formation
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