Two Oxidosqualene Cyclases Responsible for Biosynthesis · PDF fileTwo Oxidosqualene Cyclases Responsible for Biosynthesis of Tomato Fruit ... in order to gain insights into the enzymatic

Two Oxidosqualene Cyclases Responsiblefor Biosynthesis of Tomato FruitCuticular Triterpenoids1[C][W][OA]

Zhonghua Wang, Ortwin Guhling, Ruonan Yao, Fengling Li, Trevor H. Yeats,Jocelyn K.C. Rose, and Reinhard Jetter*

Department of Botany (Z.W., O.G., R.Y., F.L., R.J.) and Department of Chemistry (R.J.), University of BritishColumbia, Vancouver, British Columbia, Canada V6T 1Z1; and Department of Plant Biology, CornellUniversity, Ithaca, New York 14853 (T.H.Y., J.K.C.R.)

The first committed step in triterpenoid biosynthesis is the cyclization of epoxysqualene into various triterpene alcoholisomers, a reaction catalyzed by oxidosqualene cyclases (OSCs). The different OSCs have characteristic product specificities,which are mainly due to differences in the numbers of high-energy intermediates the enzymes can stabilize. The goal of thisinvestigation was to clone and characterize OSCs from tomato (Solanum lycopersicum), a species known to accumulate d-amyrinin its fruit cuticular wax, in order to gain insights into the enzymatic formation of this particular triterpenoid. We used ahomology-based approach to isolate two tomato OSCs and tested their biochemical properties by heterologous expression inyeast as well as overexpression in tomato. One of the enzymes was found to be a product-specific b-amyrin synthase, while theother one was a multifunctional OSC synthesizing 48% d-amyrin and six other products. The product spectra of both OSCstogether account for both the range and the relative amounts of the triterpenoids found in the fruit cuticle. Both enzymes wereexpressed exclusively in the epidermis of the tomato fruit, indicating that their major function is to form the cuticulartriterpenoids. The relative expression levels of both OSC genes, determined by quantitative reverse transcription-polymerasechain reaction, were consistent with product profiles in fruit and leaves of the tomato cultivar MicroTom. However, thetranscript ratios were only partially consistent with the differences in amounts of product triterpenoids between the tomatocultivars MicroTom, M82, and Ailsa Craig; thus, transcriptional control of the two OSCs alone cannot explain the fruittriterpenoid profiles of the cultivars.

Triterpenoids are a very diverse group of naturalproducts with wide distribution and particularly highchemical diversity in plants. They include compoundssuch as betulinic acid, the avenacins, and glycyrrhizin,which have important biological functions and medic-inal properties (Reichardt et al., 1984; Papadopoulouet al., 1999; Hayashi et al., 2001). The biosyntheticpathway toward triterpenoids proceeds by joining six

isoprene units together to form the branched long-chainhydrocarbon squalene (Eschenmoser et al., 1955). Inprokaryotes, squalene is directly cyclized into hopanoidtriterpenes, whereas in eukaryotes, it is first activatedinto 2,3-epoxysqualene and then cyclized (Abe, 2007).The cyclizations are highly regiospecific and stereo-specific, establishing the final carbon structure of thetriterpenoid products.

The overall cyclization reaction comprises (1) aninitial protonation step, (2) a polyene addition cascadeforming the up to five carbon cycles, (3) a series of 1,2-shifts of hydride and/or methyl groups, and (4) a finaldeprotonation (Fig. 1; Xu et al., 2004; Phillips et al.,2006). The entire sequence of steps is catalyzed bysingle enzymes that are designated as triterpenoidsynthases after their preferred products or as oxido-squalene cyclases (OSCs) after their common substrate(Abe et al., 1993). The great diversity of triterpenoidstructures, with more than 100 different carbon skel-etons, is due to different OSCs and, in particular, to thenumbers of rearrangement steps the different enzymescan catalyze in the third stage of the reaction (Xu et al.,2004). Approximately 50 OSCs have been cloned fromvarious plant species and have been characterized,typically using heterologous expression in yeast.Many of the plant OSCs were found to form predom-inantly one triterpenoid product, but some were also

1 This work was supported by Natural Sciences and EngineeringResearch Council of Canada Special Research Opportunity andStrategic Grants, the Canada Foundation for Innovation, the BritishColumbia Knowledge Development Fund, and the Canada ResearchChairs Program, by the National Science Foundation Plant GenomeResearch Program (grant no. DBI–0606595 to J.K.C.R.), and by theNational Institutes of Health (chemistry/biology interface traininggrant no. T32 GM008500 to T.H.Y.).

* 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:Reinhard Jetter ([email protected]).

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

[W] The online version of this article contains Web-only data.[OA] Open Access articles can be viewed online without a sub-

scription.www.plantphysiol.org/cgi/doi/10.1104/pp.110.162883

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reported to be multifunctional (Kushiro et al., 2000;Morita et al., 2000; Basyuni et al., 2006). For a few plantspecies, more than one OSC has been characterized, sothat the suite of OSCs accounted for at least part of thetriterpenoid profile in those species (Morita et al., 1997,2000; Hayashi et al., 2000, 2001; Iturbe-Ormaetxe et al.,2003; Sawai et al., 2006a, 2006b). It is generally as-sumed that the OSC product specificities determinedin vivo in yeast would accurately reflect the trueenzyme activities; however, in planta data to corrob-orate the specificities have only rarely been reported(Han et al., 2006) and in vitro results are missing,probably due to problems with handling the lipophilicsubstrate and the membrane-associated enzymes.A glimpse of the biochemical diversity within OSCs

can be seen in the case of Arabidopsis (Arabidopsisthaliana), where the genome was found to contain 13OSC genes (Fazio et al., 2004). Six of the correspondinggene products were characterized as monofunctionalenzymes forming cycloartenol, thalianol, marneral,arabidiol, lanosterol, or b-amyrin (Corey et al., 1993;Fazio et al., 2004; Suzuki et al., 2006; Xiang et al., 2006;Xiong et al., 2006; Shibuya et al., 2009). Six otherArabidopsis OSC genes were found to encode multi-functional enzymes (Herrera et al., 1998; Kushiro et al.,2000; Segura et al., 2000; Husselstein-Muller et al.,2001; Ebizuka et al., 2003; Lodeiro et al., 2007; Kolesnikovaet al., 2007; Shibuya et al., 2007), while the function ofone gene remains unknown. Even though the previ-ous studies provide substantial information about thesequence variability and biochemical specificitywithin this large gene family, the information on thecyclization mechanism is still fairly limited. This ismainly due to the fact that the large majority of OSCscharacterized to date form lupeol and b-amyrin, thetwo triterpenoids most commonly found throughoutthe plant kingdom. Only rarely have OSCs forming

other pentacyclic triterpenoid products been de-scribed. That such OSCs with diverse product pro-files exist can be seen from a recent report in whichthree Kalanchoe daigremontiana OSCs were shown tosynthesize taraxerol, glutinol, and friedelin (i.e. pro-ducts with carbon skeletons requiring relativelymany rearrangements in the course of the OSC reac-tion; Wang et al., 2010). For some of the knowntriterpenoid products, including d-amyrin, no OSCactivities have been described to date. However,information on a broad range of OSCs from variousplant species, and with varying product specificities,would help us understand how the different enzymescan catalyze specific numbers of rearrangement steps,stabilize the high-energy intermediates involved, andquench the reaction by deprotonation of a particularcarbocation to form specific end products.

A number of studies have reported that d-amyrinaccumulates to relatively high concentrations in thelipid mixture coating the surface of tomato (Solanumlycopersicum) fruit (Bauer et al., 2004b; Vogg et al., 2004;Hovav et al., 2007; Saladie et al., 2007; Isaacson et al.,2009). Besides, 10 other triterpenoids have also beenidentified in the fruit cuticular waxes extracted fromthe surfaces of various tomato cultivars that have beenstudied in much detail in recent years (Baker et al.,1982; Smith et al., 1996; Bauer et al., 2004a, 2004b; Vogget al., 2004; Hovav et al., 2007; Leide et al., 2007; Saladieet al., 2007; Mintz-Oron et al., 2008; Adato et al., 2009;Isaacson et al., 2009; Kosma et al., 2010). In particular,the surface wax on mature fruit of the tomato varietyMicroTom contains approximately 25% triterpenoids,and 36% of this fraction is d-amyrin (Vogg et al., 2004).The relative portions of the triterpenoid fractionwithin the waxes decrease in the course of fruit devel-opment, while the relative amounts of very-long-chainaliphatics derived from fatty acid metabolism increase,

Figure 1. Mechanism for the cyclization of epoxysqualene into pentacyclic triterpenoids. The reaction starts with the pro-tonation of oxidosqualene (step 1), then involves a series of carbocationic intermediates that first undergo cyclization (step 2) andvarious rearrangements (step 3), before deprotonation (step 4) yields the various natural products.

Biosynthesis of Tomato Fruit Triterpenoids

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most importantly unbranched and methyl-branchedalkanes (Leide et al., 2007; Mintz-Oron et al., 2008).Thus, the chemical data indicate that triterpenoids areformed during early fruit expansion rather than rip-ening, so expression of OSC genes should peak rela-tively early. Various studies have further shown thatfruit waxes from different tomato cultivars differ inthe relative portions of triterpenoids, especially ofb-amyrin, with ratios of 3:3:2 between d-amyrin,b-amyrin, and a-amyrin in MicroTom and ratios of3:2:2 in most other cultivars, including M82 and AilsaCraig (Vogg et al., 2004; Mintz-Oron et al., 2008;Isaacson et al., 2009). This suggests that more thanone OSC should be involved in formation of thedifferent triterpenoids and that the varying productratios should reflect different expression levels and/orenzyme activities in the different cultivars. However, ithas not been determined how many OSCs are in-volved in forming the tomato fruit cuticular triterpe-noids and which of those OSCs are product-specific ormultifunctional enzymes.

Based on all the previous evidence, the goal of thisinvestigation was to clone and characterize multipleOSCs from tomato fruit. In particular, one or moreenzymes forming d-amyrin were targeted. The pri-mary focus of the investigation was on cv MicroTom;

however, other cultivars, such as M82 and Ailsa Craig,were also included in order to compare OSC sequencesand expression patterns.With this, we sought to answerthe question of which OSCs are responsible for theformation of the triterpenoids accumulating in thetomato fruit cuticle and, thus, contribute to the impor-tant ecophysiological functions of the fruit epidermis.

RESULTS

The goal of this investigation was to isolate andcharacterize OSCs from tomato fruit and to test theirinvolvement in the formation of cuticular triterpe-noids accumulating in the fruit skin. To this end, a setof four experiments was carried out centered on thetomato cv MicroTom. First, a homology-based PCRapproach was used to clone the gene(s), initiallytargeting a core segment of the sequence and thenextending it by RACE experiments at the 5# and 3#termini. Second, the biochemical characterization ofthe enzyme(s) was carried out by heterologous ex-pression in yeast and by overexpression in tomato.Third, the expression of OSC enzyme(s) in the epider-mis and/or in inner parts of the fruit was evaluated.Finally, quantitative reverse transcription (qRT)-PCR

Figure 2. Amino acid sequences of the two OSCs, SlTTS1 and SlTTS2, isolated from the tomato cv MicroTom. The conservedQW motifs thought to stabilize OSC structure are highlighted by single underlines, while the highly conserved DCTAE motifinvolved in substrate binding and protonation is marked by a double underline. [See online article for color version of this figure.]

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was used to determine the relative expression levels ofthe OSC genes, both in the leaves and fruit of Micro-Tom. The expression studies were extended to thetomato cultivars M82 and Ailsa Craig and comple-mented by chemical analyses of the triterpenoids inthe M82 and MicroTom cuticular waxes.

Cloning and Sequence Analysis of OSCs from Tomato

In order to obtain a core sequence, PCR was per-formed with a set of degenerate primers designedusing conserved OSC amino acid sequences andcDNAs isolated from the epidermis of growing fruit.The primary PCR product corresponded to the ex-pected size of approximately 1,000 bp. A second PCR

was used to extend the fragment, the product wasisolated and cloned into Escherichia coli, and sequenc-ing inserts from five bacterial colonies revealed thepresence of only two different cDNAs. Both hadsubstantial similarity to other OSC sequences andwere subjected to 5# and 3# RACE, resulting in twofull-length cDNAs that were designated as SlTTS1 andSlTTS2.

The open reading frames (ORFs) of SlTTS1 andSlTTS2 are predicted to encode proteins of 761 and 763amino acids with masses of 89.7 and 88.1 kD, respec-tively (Fig. 2). The two protein sequences are 88%identical, and both contain the DCTAE motif thoughtto be involved in substrate binding and the fourQW motifs characteristic of the OSC superfamily.

Figure 3. Phylogenetic analysis com-paring the two new OSC cDNAscloned from tomato cv MicroTomwith previously known OSCs fromother plant species. The gene namesand sequences as well as the fullnames of species are given in “Mate-rials and Methods.”

Table I. Identity levels in pairwise comparisons between the SlTTS1 and SlTTS2 cDNAs and allelesin the tomato cultivars MicroTom, M82, and Ailsa Craig

Nucleotide identities are given as percentages in normal font, and percentages of amino acid identityare shown in italics.

cDNA CultivarTTS1 TTS2

MicroTom M82 Ailsa Craig MicroTom M82 Ailsa Craig

%

TTS1 MicroTom – 99.7 99.6 89.2 89.2 89.2M82 99.6 – 100 89.1 89.1 89.1Ailsa Craig 99.5 100 – 89.1 89.1 89.1

TTS2 MicroTom 87.8 87.7 87.5 – 99.8 99.8M82 87.7 87.5 87.4 99.4 – 100Ailsa Craig 87.7 87.5 87.4 99.4 100 –





Furthermore, SlTTS1 and SlTTS2 are highly similar topreviously reported b-amyrin synthases and multi-functional triterpene synthases (Table I). Phylogeneticanalysis using neighbor-joining methods showed thatSlTTS1 and SlTTS2 are more closely related to eachother than to any other OSC and that they, togetherwith the Panax ginseng b-amyrin synthases (Kushiroet al., 1998a, 1998b), form a subclade within a group ofOSC enzymes that were all characterized as b-amyrinsynthases from various plant species (Fig. 3).

In order to analyze the gene structure of SlTTS1 andSlTTS2, the tomato genome sequence database (www.

solgenomics.net) was queried using the two corre-sponding cDNA sequences. One bacterial artificialchromosome clone, Hba0131G17, was found to con-tain both OSC genes; accordingly, SlTTS1 and SlTTS2are arranged in tandem in a 23-kb region on chromo-some 12. The intron patterns and exon lengths of thetwo SlTTS genes are very similar to those of otherOSCs (Fig. 4), with SlTTS2 gene organization mostclosely resembling OSC3 of Lotus japonicus (Sawaiet al., 2006b). However, SlTTS2 differs from L. japoni-cus OSC3 in the length of the first and last exons. Thetwo tomato genes differ from each other in that exons

Figure 5. Mass spectra of pentacyclic triterpenoids. Compounds 1, 2, and 4, formed by yeast strains heterologously expressingthe two tomato cv MicroTom OSCs (left), have fragmentation patterns identical to those of authentic standards of d-amyrin,b-amyrin, and a-amyrin (right). Spectra are shown for the trimethylsilyl derivatives of the triterpenoid alcohols.

Figure 4. Gene structure of the tomatocv MicroTom OSCs in comparisonwith those of other plant species. Exonsare represented by boxes and intronsas lines. Nucleotide numbers of exonsare shown above the boxes, exons withcommon length between most OSCgenes are shaded in light gray, andexons with a characteristic length inonly a few species are highlighted indark gray.

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16 and 17, which are distinct in SlTTS2, are fused intoa single exon 16 in SlTTS1, which indeed distin-guishes SlTTS1 from all other OSC genes studied todate.

Biochemical Characterization of the TomatoSlTTS Proteins

In order to biochemically characterize SlTTS1 andSlTTS2, two sets of experiments using heterologousexpression in yeast and overexpression in tomato fruitwere carried out. For yeast expression, the full-lengthcDNAs of the two putative OSCs were cloned into theexpression vector pYES2, and this construct was trans-formed into the yeast mutant GIL77 (gal2 hem3-6 erg7ura3-167). This host strain lacks lanosterol synthaseand so accumulates 2,3-oxidosqualene, which can serveas a substrate for heterologously expressed OSCs(Morita et al., 1997). Yeast transformants harboringone of the pYES-TTS constructs or the empty pYESvector were grown, gene expression was induced withGal for 24 h, and then lipophilic compounds wereextracted with hexane. After purification by thin-layerchromatography (TLC), the triterpenoids were identi-fied by comparing their gas chromatography-massspectrometry (GC-MS) characteristics with those

of authentic standards and literature data (Figs. 5and 6).

Yeast cells expressing SlTTS1 cDNA were found toproduce a single triterpenoid product thatwas identifiedas b-amyrin (Fig. 7). In contrast, heterologous expressionof SlTTS2 led to the formation of a mixture of triterpe-noids, comprising 48% 6 0.3% d-amyrin, 13% 6 0.1%b-amyrin, 18% 6 0.8% a-amyrin, and 3% to 7% each ofmultiflorenol, C-taraxasterol, taraxasterol, and one un-identified triterpene alcohol isomer. The yeast expres-sion experimentswere repeated three times, alwayswithvery similar results. Overall, they indicate that SlTTS1 isa monofunctional b-amyrin synthase, while SlTTS2 is amultifunctional OSC enzyme catalyzing the formation ofseven different triterpenoid isomers.

In a second experiment, the potential OSC activitiesof the two tomato SlTTS proteins were further assessedby overexpression in tomato and chemical analysis ofthe resulting fruit surface waxes. Both SlTTS cDNAswere cloned into the pGWB5 vector, transformed intotomato cv MicroTom, and expressed under transcrip-tional control of the 35S promoter. Two sets of 20independent lines constitutively expressing SlTTS1 orSlTTS2 were recovered, and the surface materials offruit from the F1 generation were extracted for waxanalysis. The SlTTS1 overexpressor lines were found

Figure 6. Mass spectra of pentacyclic triterpenoids. Compounds 3 and 5 to 7, formed by yeast strains heterologously expressingthe two tomato cv MicroTom OSCs (left), have fragmentation patterns identical to those of triterpenoids found in the cuticularwax of tomato fruit (right). Wax constituents 5 to 7 were identified as multiflorenol, C-taraxasterol, and taraxasterol inaccordance with the literature, while compound 3 remained unidentified. Spectra are shown for the trimethylsilyl derivatives ofthe triterpenoid alcohols.





to have amounts of d-amyrin and a-amyrin similar tothose on wild-type tomato fruit surfaces (Fig. 8). Incontrast, the amounts of b-amyrin were doubled from0.7 mg cm22 in the wild type to 1.4 mg cm22 in SlTTS1overexpressors. All the non-amyrin triterpenoids re-mained unchanged after SlTTS1 overexpression (datanot shown).

The transgenic lines harboring the SlTTS2 overex-pression construct showed highly variable triterpe-noid amounts in their fruit cuticular waxes, while thevery-long-chain fatty acid derivatives were present atconstant levels identical to those in the wild type. Thetransgenic fruit could be classified into three distinctcategories with significantly different triterpenoid pro-files (Student’s t test, P, 0.01). One of them, found forfour of the lines, had triterpenoid amounts very sim-ilar to wild-type levels. Three of the lines showed again-of-function phenotype, characterized by signifi-cant increases of all seven fruit wax triterpenoids.Notably, the amounts of d-, b-, and a-amyrin were allapproximately doubled while the ratios between themwere unchanged in comparison with the wild type(Fig. 8). The remaining four lines recovered in thecourse of the SlTTS2 overexpression experiment hadfruit surface waxes containing increased levels ofb-amyrin and drastically reduced levels of d-amyrinand a-amyrin when compared with the wild type (Fig.8). Similarly, the quantities of the non-amyrin triterpe-noids were also decreased, in these cases to trace levelsthat were detectable only by single ion monitoring GC-MS (data not shown).

Investigation of SlTTS Expression Patterns inTomato Fruit

RT-PCR analyses were employed to test the tissuespecificity of OSC transcription within the green to-mato fruit. To this end, two pairs of primers weredesigned based on the SlTTS1 and SlTTS2 sequencesand proved to be gene specific when tested withplasmid templates harboring either of the two OSCcDNAs (data not shown). To determine whether theSlTTS genes were expressed differentially betweeninner parts of the tomato fruit and the epidermis layer,total RNA was isolated from both tissues. RT-PCRanalysis using subsequently derived cDNA templatesshowed that both SlTTS transcripts are expressed inthe epidermal cells but not in the inner tissues of thefruit (Fig. 9). Parallel metabolite analyses revealed thatthe major part of the triterpenoids in the tomato fruitreside in the cuticle, with 74%, 79%, and 74% of thetotal d-amyrin, b-amyrin, and a-amyrin aglyconeamounts present in the surface wax, respectively,and the remaining 21% to 26% in the underlyingepidermal cell layer and the internal tissues of fruit.

Relative Expression Levels of SlTTS1 and SlTTS2 in Fruit

and Leaves of Different Tomato Cultivars

Finally, qRT-PCR analyses were carried out to de-termine the relative expression levels of SlTTS1 andSlTTS2 within leaves and fruit. Since the ratios ofdifferent triterpenoid products vary not only between

Figure 7. GC analysis of triterpenoidsin transgenic yeast. Yeast was trans-formed with the vectors indicated inthe various panels, grown, and extrac-ted. Triterpenoid alcohols were sepa-rated from other neutral lipids by TLCand converted into trimethylsilyl ethersprior to GC analysis. In the emptyvector control, no triterpenoids weredetected. In contrast, the yeast strainsharboring SlTTS1 and SlTTS2 con-structs were found to contain the tri-terpenoid compound 2 and a series ofcompounds, 1 to 7, respectively. Allseven compounds 1 to 7 were alsodetected in the MicroTom fruit cu-ticular wax, together with very-long-chain fatty acid derivatives a to e (a,n-nonacosane; b, n-triacontane; c,n-hentriacontane; d, n-dotriacontane;e, n-tritriacontane).

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these two organs but also between fruit of differenttomato cultivars (Vogg et al., 2004; Mintz-Oron et al.,2008; Isaacson et al., 2009), this experiment was to alsocompare expression patterns of both SlTTS genesbetween MicroTom and the cultivars M82 and AilsaCraig. The same primers originally designed for thecloning of the full-length sequence from MicroTomwere used to amplify corresponding cDNAs fromM82and Ailsa Craig. One allele each of SlTTS1 and SlTTS2was cloned and sequenced from both cultivars, andthe ORFs of the orthologous genes were found to bevery similar or identical between M82 and Ailsa Craig(Supplemental Fig. S1). However, both SlTTS1 andSlTTS2 differed between these cultivars and Micro-Tom, with up to eight nucleotide differences betweencultivars resulting in up to four amino acid changesand more than 99.6% DNA and more than 99.4%protein sequence identity (Table I). The similaritiesbetween both genes were approximately 89% on thenucleotide level across cultivars and 87% to 88% on theamino acid level.The expression levels of SlTTS1 and SlTTS2 were

found to be similar to each other in the leaves and fruitof cv MicroTom (Fig. 10). Both genes were also ex-pressed at similar levels to each other in leaves of M82,whereas in the fruit of this cultivar, SlTTS2 was ex-pressed at a much higher level than SlTTS1. AilsaCraig differed further, with higher expression levels ofSlTTS2 in fruit and leaves.

Terpenoid Profiles in Fruit and Leaves of DifferentTomato Cultivars

The terpenoid profiles of the cuticular wax mixturesof leaves and fruits from the MicroTom, M82, and

Ailsa Craig cultivars were assessed, with particularfocus on the relative compositions of amyrin isomers.The compositions of fruit waxes from the three culti-vars (Vogg et al., 2004;Mintz-Oron et al., 2008; Isaacsonet al., 2009) and also the leaf wax composition ofMicroTom had been reported before (Vogg et al., 2004).However, exact data on the leaf triterpenoid profiles ofall three cultivars were missing. Therefore, a detailedstudy of the leaf wax from M82 and MicroTom wascarried out. It was found that theM82 leaf was coveredby 4.8 6 0.5 mg cm22 cuticular wax and that the waxmixture contained 3.0%6 1.0% triterpenoids, 64.3%612.5% very-long-chain fatty acid derivatives, and32.7% 6 13.5% unidentified compounds (Fig. 11).n-Alkanes (C27–C33) were the dominating compoundclass, at 45.6% 6 8.9%, accompanied by 16.6% 6 3.1%branched alkanes (total carbon nos. C29–C33) and20.5% 6 7.4% alcohols (C28 and C32). The triterpenoidfraction was dominated by amyrin isomers, compris-ing 29%, 33.4%, and 14.4% d-amyrin, b-amyrin, anda-amyrin, respectively. Other triterpenoid alcoholswere detected in the form of lupeol (23.3%) and traceamounts of multiflorenol, C-taraxasterol, and tarax-asterol. MicroTom leaf wax had a similar composition,with a conspicuous ratio of 2:3:1 between d-amyrin,b-amyrin, and a-amyrin, respectively.

DISCUSSION

In this study, a homology-based approach was usedto clone two tomato OSCs, SlTTS1 and SlTTS2, whichwere found to encode highly similar amino acid se-quences. Our principal goal was to characterize theproduct spectrum of the enzymes, with the expecta-tion that a d-amyrin synthase might be uncovered.Furthermore, we aimed to elucidate the role of theenzymes in the formation of the triterpenoids accu-mulating in the fruit cuticular wax.

A phylogenetic analysis showed that SlTTS1 andSlTTS2 fall into a clade consisting entirely of b-amyrin

Figure 8. Relative amounts of amyrin isomers in the fruit cuticular waxesof various lines ofMicroTom. The percentages of d-amyrin, b-amyrin, anda-amyrin are given for the wild type (WT), transgenic fruit overexpressingthe OSCs [SlTTS1(+) and SlTTS2(+)], and overexpressors with cosup-pressed SlTTS2 [SlTTS2(2)]. Amounts of each triterpenoid were quanti-fied as percentages relative to the level of wild-type d-amyrin and aregiven as averages of three independent lines and analyses with SD.Asterisks denote significant differences from the wild type.

Figure 9. RT-PCR analysis of the expression patterns of the two OSCgenes in MicroTom fruit. Transcripts corresponding to the SlTTS1 andSlTTS2 enzymes generating pentacyclic triterpenoids were found ex-pressed only in the epidermis layers but not in the internal tissues ofthe fruit.





synthases. Thus, the primary sequence of the newOSCs is in accordance with the prediction that thetomato enzymes catalyze the formation of amyrins.However, based on genetic information alone, it wasnot possible to predict the product specificities of thetomato enzymes. It should also be noted that, incontrast to the similarity of exon sequences withamyrin synthases, the intron pattern of SlTTS2 wasidentical to that of OSC3 from L. japonicus, a lupeolsynthase that had fairly dissimilar sequence (Sawaiet al., 2006b).

The product profiles of SlTTS1 and SlTTS2 weredetermined by heterologous expression in yeast. Itwas found that SlTTS1 forms b-amyrin as its soleproduct, while SlTTS2 catalyzes the formation of sevendifferent triterpenoids, with d-amyrin as the majorproduct. The in planta overexpression of SlTTS1 andSlTTS2 led to increased accumulation of cuticulartriterpenoids, in both cases of the same products thathad also been found in yeast. This finding is veryimportant, since most previous reports on biochemicalcharacterizations of OSCs had relied entirely on yeastexpression systems, and it had only rarely been testedwhether the heterologous environment might alter theenzyme specificity (Han et al., 2006). The match be-tween yeast and in planta expression results in thisstudy confirms the validity of the yeast expressionsystem, at least for the two tomato OSCs studied.Overall, we conclude that the two enzymes are asingle-product b-amyrin synthase and a multifunc-tional OSC, respectively. SlTTS2, to our knowledge, isthe first enzyme reported to synthesize predominantlyd-amyrin.

Interestingly, some of the tomato lines harboring theSlTTS2 construct exhibited a wax triterpenoid pheno-type opposite to that of the SlTTS2 overexpressors,suggesting that in these lines the native gene wasdown-regulated by cosuppression. The observed in-crease in b-amyrin may be the result of additional

2,3-oxidosqualene substrate availability for SlTTS1upon silencing of SlTTS2. The SlTTS2 loss-of-functionlines thus not only confirm the biochemical function ofthe enzyme but also indicate that this enzyme mustplay a central role in the formation of the tomato fruitwax triterpenoids. We conclude that SlTTS2 is crucialfor the biosynthesis of six of the seven triterpenoidisomers found in the wax and partially also contrib-utes to the formation of the seventh component,b-amyrin. Another part of the latter compound isformed by the closely related enzyme SlTTS1. Bothenzyme product profiles taken together completelymatch the wax triterpenoid composition; therefore,SlTTS1 and SlTTS2 are likely the two major enzymesforming the triterpenoids found in the tomato fruitcuticular wax. The involvement of additional OSCs can-not be excluded at this point, but it seems unlikely thatany OSC other than SlTTS1 and SlTTS2 would contributesubstantially to the triterpenoid amounts or spectrum.

Two apparently orthologous OSCs were identifiedfrom each of the tomato cultivars M82 and Ailsa Craig,with a high degree of sequence identity to those fromMicroTom. The differences between the two geneswithin one cultivar were much greater than the differ-ences between the alleles in the cultivars. As differ-ences between orthologous proteins were restricted tochanges in three or four amino acids, it is very likelythat both SlTTS1 and SlTTS2 were functional enzymesin all three cultivars and that their biochemical func-tions should be very similar in MicroTom, M82, andAilsa Craig. We conclude that all three cultivars haveat least one b-amyrin synthase (SlTTS1) and onemultifunctional OSC producing d-amyrin and otherisomers (SlTTS2).

The relative expression levels of the SlTTS1 andSlTTS2 genes varied between fruit of the three tomatocultivars investigated, with transcripts of both genesshowing similar abundance in MicroTom but SlTTS2

Figure 10. qRT-PCR analysis of the twoOSC genes in fruit and leaves ofthe three tomato cultivars MicroTom (MT), M82, and Ailsa Craig (AC).The relative expression levels were determined in green immature fruitand normalized for each mRNA sample (n = 3; error bars indicate SD).

Figure 11. Chemical composition of cuticular wax on leaves of tomatocultivars M82 and MicroTom. The absolute amounts of all identifiedcompounds are given as averages of three independent parallel exper-iments with SD.

Wang et al.




accumulating to higher levels than SlTTS1 in M82 andAilsa Craig. This expression pattern is in accord withthe amyrin profiles in the corresponding fruit waxes,where the latter two cultivars had lower levels of theSlTTS1 product b-amyrin than MicroTom (Vogg et al.,2004; Mintz-Oron et al., 2008; Isaacson et al., 2009).However, it should be noted that the differences intriterpenoid profiles between cultivars may further bedue to factors other than differential gene expression,possibly including differences in enzyme activitiesand/or additional OSCs being present.Our qRT-PCR results showed that the SlTTS1 and

SlTTS2 genes are expressed not only in tomato fruitbut also in the leaves of MicroTom, M82, and AilsaCraig. Therefore, it seems likely that both genes alsocontribute to the formation of leaf cuticular triterpe-noids. However, it must be noted that the leaf waxeshave triterpenoid profiles differing from those in thefruit in quantitative and in qualitative terms, as shownby our detailed analyses of M82 leaf wax in compar-ison with literature data. The most prominent quanti-tative difference is the shift in amyrin ratios from 3:2:2for d-amyrin, b-amyrin, and a-amyrin in the fruit to2:2:1 in the leaf wax. This trend correlates with thedifferences in expression levels of SlTTS1 and SlTTS2between both organs of M82 and can thus, at least inpart, be explained by transcriptional control. It shouldbe noted that Ailsa Craig leaves showed a very con-spicuous expression pattern with particularly low

levels of SlTTS2 transcript. Based on this finding, itmay be expected that the leaf waxes of this cultivarshould contain relatively little b-amyrin; however, theamyrin profiles of the leaf wax of Ailsa Craig have notbeen determined to date.

One outstanding qualitative difference between thetriterpenoid compositions of fruit and leaves, at leastof the cultivars MicroTom and M82, is the presence oflupeol in the latter organ, a product that is not formedby either SlTTS1 or SlTTS2. Therefore, one or moreother OSCs must be involved in the formation of leafcuticular triterpenoids. More detailed investigationsare needed to determine the contribution of SlTTS1and SlTTS2 to the biosynthesis of tomato leaf cuticles.

Our results on the tissue-specific expression ofSlTTS1 and SlTTS2 showed that these enzymes arelocalized exclusively in the epidermis of tomato fruit.We further found that the triterpenoid products arealso restricted to the fruit skin, as they accumulate tohigh concentrations in the cuticular waxes coating theepidermis but not in the internal parts of the fruit. Theclose match in localization of transcripts and metabo-lites makes it very likely that SlTTS1 and SlTTS2 arededicated entirely to making the triterpenoids des-tined for the cuticular wax of the fruit surface. Withthis, a major biological function can be assigned tothese two OSCs, as the cuticular triterpenoids contrib-ute significantly to the chemical composition and tothe ecophysiological properties of the fruit cuticle

Table II. Accession data for the OSCs used in the phylogenetic analyses

Accession No. Species Function

At4g15340 Arabidopsis Arabidiol synthase PEN1At4g15370 Arabidopsis Baruol synthase BARS1/PEN2At5g48010 Arabidopsis Thalianol synthase PEN4At5g42600 Arabidopsis Marneral synthase PEN5At1g78500 Arabidopsis Multifunctional triterpene synthase PEN6At3g45130 Arabidopsis Lanosterol synthase LAS1/PEN7At1g78970 Arabidopsis Multifunctional triterpene synthase LUP1At1g78960 Arabidopsis Multifunctional triterpene synthase LUP2At1g78950 Arabidopsis b-Amyrin synthase AtBAS LUP4At1g66960 Arabidopsis Multifunctional triterpene synthase LUP5At2g07050 Arabidopsis Cycloartenol synthase CAS1AB263204 Rhizophora stylosa Multifunctional triterpene synthase RsM2AB257507 Kandelia candel Multifunctional triterpene synthase KcMSAB037203 Glycyrrhiza glabra b-Amyrin synthase GgbAS1AB181244 Lotus japonicus b-Amyrin synthase OSC1AB034802 Pisum sativum b-Amyrin synthase PSYAJ430607 Medicago truncatula b-Amyrin synthase AMY1AF478455 L. japonicus Multifunctional triterpene synthase LjAMY2AB034803 P. sativum Multifunctional triterpene synthase PSMAB289585 Bruguiera gymnorhiza b-Amyrin synthase BgbASAB014057 P. ginseng b-Amyrin synthase PgbAS/PNY2AB009030 P. ginseng b-Amyrin synthase PNYAB263203 R. stylosa Multifunctional triterpene synthase RsM1AB206469 Euphorbia tirucalli b-Amyrin synthase EtbASAB181245 L. japonicus Lupeol synthase OSC3AB116228 G. glabra Lupeol synthase GgLUS1AB181246 L. japonicus Cycloartenol synthase QSC5AB244671 L. japonicus Lanosterol synthase OSC7





(Vogg et al., 2004; Isaacson et al., 2009). It should benoted that similar biological functions had previouslybeen attributed to a few other OSCs, for example, aglutinol synthase and a friedelin synthase from K.daigremontiana (Wang et al., 2010).

The sequences of SlTTS1 and SlTTS2 are relativelysimilar to each other, containing only 92 amino acidchanges. On the other hand, the two enzymes werefound to have fairly distinct product profiles, withSlTTS1 yielding exclusively b-amyrin whereas SlTTS2is catalyzing the formation of d-amyrin (among otherproducts). It has been predicted that the formation ofd-amyrin requires only one rearrangement step lessthan b-amyrin in the course of the OSC-catalyzedcyclization (Fig. 1; Xu et al., 2004; Phillips et al., 2006).This relatively subtle difference in the mechanism offormation of both triterpenoids must be due to smalldifferences in protein architecture, possibly involvingspecific changes in amino acids lining the active sitecavity. The sequence differences between SlTTS1 andSlTTS2 give the first information on the candidateresidues that may be involved in determining theamyrin isomer specificity.

The list of candidate residues for defining d-amyrinsynthases can be somewhat narrowed down based oncomparisons between the two OSCs characterizedhere in three different tomato cultivars and withb-amyrin synthases from other species. A second ob-servation might further help select candidate residues:many of the amino acid changes between both en-zymes are clustered together, and many involve aro-matic residues. Since it is well established that sucharomatic amino acids play important roles in stabiliz-ing the positive charge of high-energy intermediates ofthe cyclization reaction, it has been speculated that thenumber and exact positions of aromatic residues liningthe active site might limit the number of rearrange-ment steps and, therefore, determine the productoutcome of OSC-catalyzed reactions (Christianson,2006; Phillips et al., 2006). Consequently, the changesbetween SlTTS1 and SlTTS2 involving aromatic aminoacids at positions 48/49, 103, 318, 388, 399, 412, 421,478, 496, 560, 675, 742, and 760 make these residuesespecially interesting candidates for defining the OSCspecificity of d-amyrin and b-amyrin synthases. Itappears very promising to use site-directed mutagen-esis experiments to test this hypothesis in order tofurther our detailed understanding of the enzymemechanisms involved in triterpenoid biosynthesis.

MATERIALS AND METHODS

Plant Material and Surface Wax Analysis

Tomato (Solanum lycopersicum ‘MicroTom’) plants were grown in standard

soil under ambient conditions in a greenhouse at the University of British

Columbia. Fruits and leaves were harvested and immediately immersed in

CHCl3 for 1 min at room temperature to extract the cuticular waxes. For di-

rect GC-MS analyses, the wax mixtures were derivatized using bis-N,O-

(trimethylsilyl)trifluoroacetamide (in pyridine at 70�C for 60 min), dried

under N2, and dissolved in CHCl3. The qualitative composition was studied

using a 6890N gas chromatograph (Agilent) equipped with a mass spectro-

metric detector (Agilent 5973N) and an HP-1 capillary column (Agilent; length

30 m, i.d. 320 mm, 1-mm film thickness). One microliter of each sample was

injected on column into a flow of helium gas held constant at 1.4 mL min21.

The oven temperature was programmed for 2 min at 50�C, followed by a 40�Cmin21 ramp to 200�C, held at 200�C for 2 min, increased by 3�C min21 to

320�C, and held at 320�C for 30 min. Triterpenoids were identified by

comparison with authentic compounds (d-amyrin, b-amyrin, a-amyrin,

lupeol) and with literature data. The quantitative composition was studied

using a similar GC system equipped with a flame ionization detector under

the same GC conditions as above, but H2 carrier gas inlet pressure was

programmed for 2 mLmin21. A known amount of n-tetracosane was added to

the solvent prior to extraction and used as an internal standard for quantifying

compound amounts. The extracted surface areas of leaves were determined

using digital images and ImageJ software.

For the analysis of triterpenoids contained in internal tissues, fruits were

surface extracted with CHCl3 to remove cuticular waxes, frozen in liquid

nitrogen, and then ground to powder. The powder was extracted with CHCl3,

and the resulting lipid mixture was fractionated by TLC (203 20 cm, silica gel,

0.5 mm; Merck) using CHCl3 as the mobile phase. After the plate was stained

with primuline and viewed under UV light, distinct bands were scratched off,

extracted with CHCl3, and analyzed by GC-MS and GC-flame ionization

detection (FID) for identification and quantification of the triterpenoids,

respectively. GC conditions were as described above for wax analyses.

Cloning of OSC Genes from Tomato

Green tomato fruits were harvested, immediately plunged into liquid

nitrogen, and ground thoroughly with a mortar and pestle. Total RNA was

extracted from the powder using the Trizol Reagent (Invitrogen). The RNA

was used for cDNA synthesis by SuperScript II reverse transcriptase (Invi-

trogen) following standard protocols. The resulting cDNA mixture served

directly as a template for the following PCRs.

Sequence alignments of previously characterized plant OSCs revealed

conserved regions, which were used to design degenerate oligonucleotide

primers for the specific amplification of the core fragments of OSCs from the

cDNA mixture. The antisense primer AMYT was derived from the EST clone

TC160438 (The Institute for Genomic Research tomato EST database) suspected

to encode part of an OSC sequence. To obtain the triterpenoid synthase

core sequence, PCR was performed with the degenerate sense primer OGA1S

(5#-TTYGGHAGYCAARMRTGGGAT-3#) combined with antisense primer

AMYT (5#-CGGTATTCAGCCAAACCCCA-3#) with recombinant Taq DNA

Polymerase (Invitrogen) under the following cycling conditions: 2 min at

94�C, 30 cycles of 20 s at 94�C, 40 s at 54�C, and 70 s at 72�C, and a 10-min final

extension at 72�C. The resulting PCR products were separated by gel electro-

phoresis (1% agarose) and extracted using the QIAquick Gel Extraction Kit

(Qiagen). A DNA band of 1 kb was recovered, cloned into the pGEM-T vector

(Promega), and transformed into Top10 Competent Cells. Plasmid DNAwas

purified from transformed cells using the QIAprep Spin Miniprep Kit

(Qiagen) and sequenced. All further PCR products mentioned below were

subcloned and sequenced by the same procedure.

The core fragment was extended in a second PCR (conditions as above)

using the primers OGT4S (5#-CAYCAGAAYGAAGATGGW-3#) and gene-

specific primer OGAS1A (5#-CATCATTCATCTCACTGGC-3#) synthesized

according to the obtained core sequences. Two different core sequences were

obtained, one named SlTTS1 and found to be identical to the database

sequence TC160438, and another one named SlTTS2. For 3#-end amplification

of the SlTTS2 cDNA, first-strand synthesis was carried out for 1 h at

42�C using 5 mg of total RNA, AP primer (5#-GGCCACGCGTCGACTA-

GTACTTTTTTTTTTTTTTTTT-3#), and reverse transcriptase in 20 mL. The

product served as template in a PCR (conditions as above except annealing

temperature of 55�C) with the adapter primer AUAP (5#-GGCCACGCGTC-

GACTAGTAC-3#) and the specific primer LEASB1S (5#-AGGGTTGTGG-

TAGTCAATC-3#). The 5#-ends of both cDNAs were amplified by the 5#RACE system of Invitrogen (version 2.0) with two nested gene-specific

antisense primers, OGAS6A (5#-AGAATCCATTTCCTTGCTCTA-3#) and

OGAS7A (5#-CACGCATTATTTACACCGCC-3#).The corresponding full-length cDNAs were amplified using tomato cDNA

as a template and the specific N- and C-terminal primers, respectively:

LeTTS1F (5#-TTGGAGCTCAGGATGTGGAAATTGAAAATTGCTG-3#; SacI

site underlined), LeTTS1R (5#-CCCGAATTCTTAGTTGTTTTCTAATGGT-

AATAC-3#; EcoRI site underlined), LeTTS2F (5#-TTGGAGCTCAAGAT-

GTGGAAGTTGAAGATTGCAA-3#; SacI site underlined), and LeTTS2R

Wang et al.




(5#-CCCGAATTCCTATATGTAGTTGTGTTTTAATGGT-3#; EcoRI site under-

lined). The PCRs were conducted with Phusion High-Fidelity DNA polymer-

ase (New England Biolabs) in a final volume of 50 mL (1 mM of each primer and

1 mL of cDNA) under the following conditions: 30 s at 98�C, 28 cycles of 10 s at98�C, 30 s at 55�C, and 75 s at 72�C, and 10 min at 72�C. The resulting 2.3-kb

PCR products were purified by gel electrophoresis and cloned for sequencing.

Functional Expression of OSC cDNAs in Yeast andProduct Analysis

The full-length cDNAs of putative triterpenoid synthases were double

digested with SacI and EcoRI enzymes and ligated into the yeast expression

vector pYES2 (Invitrogen) under the control of the GAL1 promoter. The

constructs were transformed into Top10 Competent Cells. Plasmid DNAs

were prepared and used to transform the mutant yeast strain GIL77 by the

lithium acetate/single-stranded carrier DNA/polyethylene glycol method

(Gietz and Woods, 2002). After Gal induction and 24 h of incubation in 0.1 M

potassium phosphate-containing Glc and hemin, cells were collected, refluxed

for 5 min in 20% KOH/50% ethanol, and extracted twice with hexane. Both

hexane solutions were combined, the solvent was removed under a gentle

stream of N2, and the residue was redissolved in 0.2 mL of CHCl3. The extracts

were either directly derivatized using bis-N,O-(trimethylsilyl)trifluoroacet-

amide at 70�C for 60 min and then analyzed by GC-FID/MS as described

above or further purified by TLC plate (203 20 cm, silica gel 60 F254, 0.25 mm;

Merck) for GC-FID/MS analysis. Plates were developed using a sandwich

technique and chloroform as the mobile phase, then stained with primuline

and viewed under UV light. The bands potentially containing cyclization

products were scratched from the plates, extracted with CHCl3, filtered, and

prepared for GC analysis.

Tomato Overexpression

SlTTS1 and SlTTS2 cDNAs were cloned into the pGWB5 vector under the

control of a 35S promoter using the Gateway cloning system (Invitrogen). The

vectors were transferred to Agrobacterium tumefaciens GV3301. Plant transfor-

mation was carried out as described by Dan et al. (2006). Transgenic plants

were grown in a greenhouse until the fruits turned to mature red, then

harvested for wax extraction and GC-FID/MS analysis as described before.

RT-PCR Analysis

The epidermal cell layers were peeled from the green fruit surface of

MicroTom, and the remaining inner tissue as well as the epidermis prepara-

tions were immediately frozen in liquid nitrogen, ground into powder using a

mortar and pestle, and used to extract RNAwith Trizol Reagent (Invitrogen).

The RNA samples were used for cDNA synthesis by SuperScript II reverse

transcriptase (Invitrogen) following standard protocols. Gene-specific primers

(SlTTS1, 5#-CGTCGAATGCACTGCCTCAT-3# and 5#-GGACCAAATTGCA-

CTCTATATC-3#; SlTTS2, 5#-TGTTGAGTGCACTAGCTCGG-3# and 5#-ACG-

GACAACTCGATTCACTAAGC-3#) were designed to amplify fragments of

the two OSCs. Additionally, the actin gene fragment was amplified as a

positive control using the primers actinF (5#-CAAGTCATCATCCGTTTG-3#)and actinR (5#-ATACCAGTGGTACGACC-3#). PCR cycle numbers and tem-

plate amounts were optimized to yield products in the linear range of the

reaction. PCR conditions were as follows: denaturing at 94�C for 2 min,

followed by 28 cycles of 94�C for 20 s, 55�C for 30 s, and 72�C for 60 s.

Reactions were maintained at 72�C for 2 min before separation of PCR

products by electrophoresis on a 1% agarose gel.

qRT-PCR Analysis

The RNA samples from tomato leaf or green fruits were used for cDNA

synthesis by SuperScript II reverse transcriptase (Invitrogen) following stan-

dard protocols. Gene-specific primers were designed to amplify a fragment of

SlTTS1 by TTS1F (5#-CGTCGAATGCACTGCCTCAT-3) and T1R2 (5#-TAC-

CATGAACCATCAGGCATT-3#) and a fragment of SlTTS2 by TTS2F

(5#-TGTTGAGTGCACTAGCTCGG-3#) and T2R2 (5#-TACCATGAACCGT-

CAGGCTCC-3#). Quantitative PCR was carried out using the SYBR GreenER

qPCR SuperMix Universal Kit (Invitrogen). The qPCRs were programmed

at 95�C for 9 min, followed by 40 cycles of 94�C for 30 s, 55�C for 30 s, and

72�C for 30 s on a MJ Mini Opticon real-time PCR system.

Phylogenetic Analyses

Sequence alignments and phylogenetic analyses based on a neighbor-

joining method were carried out with the ClustalX program version 1.83

(Thompson et al., 1997) using the amino acid sequences of cloned and

characterized plant OSCs. A phylogenetic tree was created with the MEGA3.1

program. The number of bootstrap replications was 1,000.

Sequence information for the putative triterpenoid synthases SlTTS1 and

SlTTS2 has been deposited in GenBank with accession numbers HQ266579

and HQ266580, respectively. The GenBank accession numbers of the se-

quences used in the analysis are summarized in Table II.

Supplemental Data

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

Supplemental Figure S1. Amino acid sequences of the SlTTS1 and SlTTS2

alleles of tomato cultivars MicroTom and M82.

ACKNOWLEDGMENTS

We thank Bangjun Wang for technical help and Dr. Y. Ebizuka for

providing yeast strain GIL77.

Received July 15, 2010; accepted October 18, 2010; published November 8,

2010.

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Two Oxidosqualene Cyclases Responsible for Biosynthesis · PDF fileTwo Oxidosqualene Cyclases Responsible for Biosynthesis of Tomato Fruit ... in order to gain insights into the enzymatic