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Two Oxidosqualene Cyclases Responsible for Biosynthesis of Tomato Fruit Cuticular 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 British Columbia, Vancouver, British Columbia, Canada V6T 1Z1; and Department of Plant Biology, Cornell University, 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 alcohol isomers, 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 this investigation was to clone and characterize OSCs from tomato (Solanum lycopersicum), a species known to accumulate d-amyrin in its fruit cuticular wax, in order to gain insights into the enzymatic formation of this particular triterpenoid. We used a homology-based approach to isolate two tomato OSCs and tested their biochemical properties by heterologous expression in yeast as well as overexpression in tomato. One of the enzymes was found to be a product-specific b-amyrin synthase, while the other one was a multifunctional OSC synthesizing 48% d-amyrin and six other products. The product spectra of both OSCs together account for both the range and the relative amounts of the triterpenoids found in the fruit cuticle. Both enzymes were expressed exclusively in the epidermis of the tomato fruit, indicating that their major function is to form the cuticular triterpenoids. The relative expression levels of both OSC genes, determined by quantitative reverse transcription-polymerase chain reaction, were consistent with product profiles in fruit and leaves of the tomato cultivar MicroTom. However, the transcript ratios were only partially consistent with the differences in amounts of product triterpenoids between the tomato cultivars MicroTom, M82, and Ailsa Craig; thus, transcriptional control of the two OSCs alone cannot explain the fruit triterpenoid profiles of the cultivars. Triterpenoids are a very diverse group of natural products with wide distribution and particularly high chemical diversity in plants. They include compounds such as betulinic acid, the avenacins, and glycyrrhizin, which have important biological functions and medicinal properties (Reichardt et al., 1984; Papadopoulou et al., 1999; Hayashi et al., 2001). The biosynthetic pathway toward triterpenoids proceeds by joining six 1 This work was supported by Natural Sciences and Engineering Research Council of Canada Special Research Opportunity and Strategic Grants, the Canada Foundation for Innovation, the British Columbia Knowledge Development Fund, and the Canada Research Chairs Program, by the National Science Foundation Plant Genome Research Program (grant no. DBI–0606595 to J.K.C.R.), and by the National Institutes of Health (chemistry/biology interface training grant no. T32 GM008500 to T.H.Y.). * Corresponding author; e-mail [email protected]. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described 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 in black 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 subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.110.162883 540 isoprene units together to form the branched long-chain hydrocarbon squalene (Eschenmoser et al., 1955). In prokaryotes, squalene is directly cyclized into hopanoid triterpenes, whereas in eukaryotes, it is first activated into 2,3-epoxysqualene and then cyclized (Abe, 2007). The cyclizations are highly regiospecific and stereospecific, establishing the final carbon structure of the triterpenoid products. The overall cyclization reaction comprises (1) an initial protonation step, (2) a polyene addition cascade forming the up to five carbon cycles, (3) a series of 1,2shifts of hydride and/or methyl groups, and (4) a final deprotonation (Fig. 1; Xu et al., 2004; Phillips et al., 2006). The entire sequence of steps is catalyzed by single enzymes that are designated as triterpenoid synthases after their preferred products or as oxidosqualene cyclases (OSCs) after their common substrate (Abe et al., 1993). The great diversity of triterpenoid structures, with more than 100 different carbon skeletons, is due to different OSCs and, in particular, to the numbers of rearrangement steps the different enzymes can catalyze in the third stage of the reaction (Xu et al., 2004). Approximately 50 OSCs have been cloned from various plant species and have been characterized, typically using heterologous expression in yeast. Many of the plant OSCs were found to form predominantly one triterpenoid product, but some were also Plant PhysiologyÒ, January 2011, Vol. 155, pp. 540–552, www.plantphysiol.org Ó 2010 American Society of Plant Biologists Downloaded from on June 18, 2017 - Published by www.plantphysiol.org Copyright © 2011 American Society of Plant Biologists. All rights reserved. Biosynthesis of Tomato Fruit Triterpenoids Figure 1. Mechanism for the cyclization of epoxysqualene into pentacyclic triterpenoids. The reaction starts with the protonation of oxidosqualene (step 1), then involves a series of carbocationic intermediates that first undergo cyclization (step 2) and various rearrangements (step 3), before deprotonation (step 4) yields the various natural products. reported to be multifunctional (Kushiro et al., 2000; Morita et al., 2000; Basyuni et al., 2006). For a few plant species, more than one OSC has been characterized, so that the suite of OSCs accounted for at least part of the triterpenoid 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 assumed that the OSC product specificities determined in vivo in yeast would accurately reflect the true enzyme activities; however, in planta data to corroborate the specificities have only rarely been reported (Han et al., 2006) and in vitro results are missing, probably due to problems with handling the lipophilic substrate and the membrane-associated enzymes. A glimpse of the biochemical diversity within OSCs can be seen in the case of Arabidopsis (Arabidopsis thaliana), where the genome was found to contain 13 OSC genes (Fazio et al., 2004). Six of the corresponding gene products were characterized as monofunctional enzymes 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 other Arabidopsis OSC genes were found to encode multifunctional 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; Kolesnikova et al., 2007; Shibuya et al., 2007), while the function of one gene remains unknown. Even though the previous studies provide substantial information about the sequence variability and biochemical specificity within this large gene family, the information on the cyclization mechanism is still fairly limited. This is mainly due to the fact that the large majority of OSCs characterized to date form lupeol and b-amyrin, the two triterpenoids most commonly found throughout the plant kingdom. Only rarely have OSCs forming other pentacyclic triterpenoid products been described. That such OSCs with diverse product profiles exist can be seen from a recent report in which three Kalanchoe daigremontiana OSCs were shown to synthesize taraxerol, glutinol, and friedelin (i.e. products with carbon skeletons requiring relatively many rearrangements in the course of the OSC reaction; Wang et al., 2010). For some of the known triterpenoid products, including d-amyrin, no OSC activities have been described to date. However, information on a broad range of OSCs from various plant species, and with varying product specificities, would help us understand how the different enzymes can catalyze specific numbers of rearrangement steps, stabilize the high-energy intermediates involved, and quench the reaction by deprotonation of a particular carbocation to form specific end products. A number of studies have reported that d-amyrin accumulates to relatively high concentrations in the lipid mixture coating the surface of tomato (Solanum lycopersicum) fruit (Bauer et al., 2004b; Vogg et al., 2004; Hovav et al., 2007; Saladié et al., 2007; Isaacson et al., 2009). Besides, 10 other triterpenoids have also been identified in the fruit cuticular waxes extracted from the surfaces of various tomato cultivars that have been studied in much detail in recent years (Baker et al., 1982; Smith et al., 1996; Bauer et al., 2004a, 2004b; Vogg et al., 2004; Hovav et al., 2007; Leide et al., 2007; Saladié et 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 variety MicroTom contains approximately 25% triterpenoids, and 36% of this fraction is d-amyrin (Vogg et al., 2004). The relative portions of the triterpenoid fraction within the waxes decrease in the course of fruit development, while the relative amounts of very-long-chain aliphatics derived from fatty acid metabolism increase, Plant Physiol. Vol. 155, 2011 541 Downloaded from on June 18, 2017 - Published by www.plantphysiol.org Copyright © 2011 American Society of Plant Biologists. All rights reserved. Wang et al. most importantly unbranched and methyl-branched alkanes (Leide et al., 2007; Mintz-Oron et al., 2008). Thus, the chemical data indicate that triterpenoids are formed during early fruit expansion rather than ripening, so expression of OSC genes should peak relatively early. Various studies have further shown that fruit waxes from different tomato cultivars differ in the relative portions of triterpenoids, especially of b-amyrin, with ratios of 3:3:2 between d-amyrin, b-amyrin, and a-amyrin in MicroTom and ratios of 3:2:2 in most other cultivars, including M82 and Ailsa Craig (Vogg et al., 2004; Mintz-Oron et al., 2008; Isaacson et al., 2009). This suggests that more than one OSC should be involved in formation of the different triterpenoids and that the varying product ratios should reflect different expression levels and/or enzyme activities in the different cultivars. However, it has not been determined how many OSCs are involved in forming the tomato fruit cuticular triterpenoids and which of those OSCs are product-specific or multifunctional enzymes. Based on all the previous evidence, the goal of this investigation was to clone and characterize multiple OSCs from tomato fruit. In particular, one or more enzymes forming d-amyrin were targeted. The primary 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 sequences and expression patterns. With this, we sought to answer the question of which OSCs are responsible for the formation of the triterpenoids accumulating in the tomato fruit cuticle and, thus, contribute to the important ecophysiological functions of the fruit epidermis. RESULTS The goal of this investigation was to isolate and characterize OSCs from tomato fruit and to test their involvement in the formation of cuticular triterpenoids accumulating in the fruit skin. To this end, a set of four experiments was carried out centered on the tomato cv MicroTom. First, a homology-based PCR approach was used to clone the gene(s), initially targeting a core segment of the sequence and then extending it by RACE experiments at the 5# and 3# termini. Second, the biochemical characterization of the enzyme(s) was carried out by heterologous expression in yeast and by overexpression in tomato. Third, the expression of OSC enzyme(s) in the epidermis 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 conserved QW motifs thought to stabilize OSC structure are highlighted by single underlines, while the highly conserved DCTAE motif involved in substrate binding and protonation is marked by a double underline. [See online article for color version of this figure.] 542 Plant Physiol. Vol. 155, 2011 Downloaded from on June 18, 2017 - Published by www.plantphysiol.org Copyright © 2011 American Society of Plant Biologists. All rights reserved. Biosynthesis of Tomato Fruit Triterpenoids Table I. Identity levels in pairwise comparisons between the SlTTS1 and SlTTS2 cDNAs and alleles in the tomato cultivars MicroTom, M82, and Ailsa Craig Nucleotide identities are given as percentages in normal font, and percentages of amino acid identity are shown in italics. cDNA Cultivar TTS1 MicroTom M82 TTS2 Ailsa Craig MicroTom M82 Ailsa Craig 89.2 89.1 89.1 – 99.4 99.4 89.2 89.1 89.1 99.8 – 100 89.2 89.1 89.1 99.8 100 – % TTS1 TTS2 MicroTom M82 Ailsa Craig MicroTom M82 Ailsa Craig – 99.6 99.5 87.8 87.7 87.7 99.7 – 100 87.7 87.5 87.5 was used to determine the relative expression levels of the OSC genes, both in the leaves and fruit of MicroTom. The expression studies were extended to the tomato cultivars M82 and Ailsa Craig and complemented by chemical analyses of the triterpenoids in the M82 and MicroTom cuticular waxes. Cloning and Sequence Analysis of OSCs from Tomato In order to obtain a core sequence, PCR was performed with a set of degenerate primers designed using conserved OSC amino acid sequences and cDNAs isolated from the epidermis of growing fruit. The primary PCR product corresponded to the expected size of approximately 1,000 bp. A second PCR 99.6 100 – 87.5 87.4 87.4 was used to extend the fragment, the product was isolated and cloned into Escherichia coli, and sequencing inserts from five bacterial colonies revealed the presence of only two different cDNAs. Both had substantial similarity to other OSC sequences and were subjected to 5# and 3# RACE, resulting in two full-length cDNAs that were designated as SlTTS1 and SlTTS2. The open reading frames (ORFs) of SlTTS1 and SlTTS2 are predicted to encode proteins of 761 and 763 amino acids with masses of 89.7 and 88.1 kD, respectively (Fig. 2). The two protein sequences are 88% identical, and both contain the DCTAE motif thought to be involved in substrate binding and the four QW motifs characteristic of the OSC superfamily. Figure 3. Phylogenetic analysis comparing the two new OSC cDNAs cloned from tomato cv MicroTom with previously known OSCs from other plant species. The gene names and sequences as well as the full names of species are given in “Materials and Methods.” Plant Physiol. Vol. 155, 2011 543 Downloaded from on June 18, 2017 - Published by www.plantphysiol.org Copyright © 2011 American Society of Plant Biologists. All rights reserved. Wang et al. Figure 4. Gene structure of the tomato cv MicroTom OSCs in comparison with those of other plant species. Exons are represented by boxes and introns as lines. Nucleotide numbers of exons are shown above the boxes, exons with common length between most OSC genes are shaded in light gray, and exons with a characteristic length in only a few species are highlighted in dark gray. Furthermore, SlTTS1 and SlTTS2 are highly similar to previously reported b-amyrin synthases and multifunctional triterpene synthases (Table I). Phylogenetic analysis using neighbor-joining methods showed that SlTTS1 and SlTTS2 are more closely related to each other than to any other OSC and that they, together with the Panax ginseng b-amyrin synthases (Kushiro et al., 1998a, 1998b), form a subclade within a group of OSC enzymes that were all characterized as b-amyrin synthases from various plant species (Fig. 3). In order to analyze the gene structure of SlTTS1 and SlTTS2, the tomato genome sequence database (www. solgenomics.net) was queried using the two corresponding cDNA sequences. One bacterial artificial chromosome clone, Hba0131G17, was found to contain both OSC genes; accordingly, SlTTS1 and SlTTS2 are arranged in tandem in a 23-kb region on chromosome 12. The intron patterns and exon lengths of the two SlTTS genes are very similar to those of other OSCs (Fig. 4), with SlTTS2 gene organization most closely resembling OSC3 of Lotus japonicus (Sawai et al., 2006b). However, SlTTS2 differs from L. japonicus OSC3 in the length of the first and last exons. The two 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 expressing the 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. 544 Plant Physiol. Vol. 155, 2011 Downloaded from on June 18, 2017 - Published by www.plantphysiol.org Copyright © 2011 American Society of Plant Biologists. All rights reserved. Biosynthesis of Tomato Fruit Triterpenoids 16 and 17, which are distinct in SlTTS2, are fused into a single exon 16 in SlTTS1, which indeed distinguishes SlTTS1 from all other OSC genes studied to date. Biochemical Characterization of the Tomato SlTTS Proteins In order to biochemically characterize SlTTS1 and SlTTS2, two sets of experiments using heterologous expression in yeast and overexpression in tomato fruit were carried out. For yeast expression, the full-length cDNAs of the two putative OSCs were cloned into the expression vector pYES2, and this construct was transformed into the yeast mutant GIL77 (gal2 hem3-6 erg7 ura3-167). This host strain lacks lanosterol synthase and so accumulates 2,3-oxidosqualene, which can serve as a substrate for heterologously expressed OSCs (Morita et al., 1997). Yeast transformants harboring one of the pYES-TTS constructs or the empty pYES vector were grown, gene expression was induced with Gal for 24 h, and then lipophilic compounds were extracted with hexane. After purification by thin-layer chromatography (TLC), the triterpenoids were identified by comparing their gas chromatography-mass spectrometry (GC-MS) characteristics with those of authentic standards and literature data (Figs. 5 and 6). Yeast cells expressing SlTTS1 cDNA were found to produce a single triterpenoid product that was identified as b-amyrin (Fig. 7). In contrast, heterologous expression of SlTTS2 led to the formation of a mixture of triterpenoids, comprising 48% 6 0.3% d-amyrin, 13% 6 0.1% b-amyrin, 18% 6 0.8% a-amyrin, and 3% to 7% each of multiflorenol, C-taraxasterol, taraxasterol, and one unidentified triterpene alcohol isomer. The yeast expression experiments were repeated three times, always with very similar results. Overall, they indicate that SlTTS1 is a monofunctional b-amyrin synthase, while SlTTS2 is a multifunctional OSC enzyme catalyzing the formation of seven different triterpenoid isomers. In a second experiment, the potential OSC activities of the two tomato SlTTS proteins were further assessed by overexpression in tomato and chemical analysis of the resulting fruit surface waxes. Both SlTTS cDNAs were cloned into the pGWB5 vector, transformed into tomato cv MicroTom, and expressed under transcriptional control of the 35S promoter. Two sets of 20 independent lines constitutively expressing SlTTS1 or SlTTS2 were recovered, and the surface materials of fruit from the F1 generation were extracted for wax analysis. The SlTTS1 overexpressor lines were found Figure 6. Mass spectra of pentacyclic triterpenoids. Compounds 3 and 5 to 7, formed by yeast strains heterologously expressing the two tomato cv MicroTom OSCs (left), have fragmentation patterns identical to those of triterpenoids found in the cuticular wax of tomato fruit (right). Wax constituents 5 to 7 were identified as multiflorenol, C-taraxasterol, and taraxasterol in accordance with the literature, while compound 3 remained unidentified. Spectra are shown for the trimethylsilyl derivatives of the triterpenoid alcohols. Plant Physiol. Vol. 155, 2011 545 Downloaded from on June 18, 2017 - Published by www.plantphysiol.org Copyright © 2011 American Society of Plant Biologists. All rights reserved. Wang et al. Figure 7. GC analysis of triterpenoids in transgenic yeast. Yeast was transformed with the vectors indicated in the various panels, grown, and extracted. Triterpenoid alcohols were separated from other neutral lipids by TLC and converted into trimethylsilyl ethers prior to GC analysis. In the empty vector control, no triterpenoids were detected. In contrast, the yeast strains harboring SlTTS1 and SlTTS2 constructs were found to contain the triterpenoid compound 2 and a series of compounds, 1 to 7, respectively. All seven compounds 1 to 7 were also detected in the MicroTom fruit cuticular wax, together with very-longchain fatty acid derivatives a to e (a, n-nonacosane; b, n-triacontane; c, n-hentriacontane; d, n-dotriacontane; e, n-tritriacontane). to have amounts of d-amyrin and a-amyrin similar to those on wild-type tomato fruit surfaces (Fig. 8). In contrast, the amounts of b-amyrin were doubled from 0.7 mg cm22 in the wild type to 1.4 mg cm22 in SlTTS1 overexpressors. All the non-amyrin triterpenoids remained unchanged after SlTTS1 overexpression (data not shown). The transgenic lines harboring the SlTTS2 overexpression construct showed highly variable triterpenoid amounts in their fruit cuticular waxes, while the very-long-chain fatty acid derivatives were present at constant levels identical to those in the wild type. The transgenic fruit could be classified into three distinct categories with significantly different triterpenoid profiles (Student’s t test, P , 0.01). One of them, found for four of the lines, had triterpenoid amounts very similar to wild-type levels. Three of the lines showed a gain-of-function phenotype, characterized by significant increases of all seven fruit wax triterpenoids. Notably, the amounts of d-, b-, and a-amyrin were all approximately doubled while the ratios between them were unchanged in comparison with the wild type (Fig. 8). The remaining four lines recovered in the course of the SlTTS2 overexpression experiment had fruit surface waxes containing increased levels of b-amyrin and drastically reduced levels of d-amyrin and a-amyrin when compared with the wild type (Fig. 8). Similarly, the quantities of the non-amyrin triterpenoids were also decreased, in these cases to trace levels that were detectable only by single ion monitoring GCMS (data not shown). Investigation of SlTTS Expression Patterns in Tomato Fruit RT-PCR analyses were employed to test the tissue specificity of OSC transcription within the green tomato fruit. To this end, two pairs of primers were designed based on the SlTTS1 and SlTTS2 sequences and proved to be gene specific when tested with plasmid templates harboring either of the two OSC cDNAs (data not shown). To determine whether the SlTTS genes were expressed differentially between inner parts of the tomato fruit and the epidermis layer, total RNA was isolated from both tissues. RT-PCR analysis using subsequently derived cDNA templates showed that both SlTTS transcripts are expressed in the epidermal cells but not in the inner tissues of the fruit (Fig. 9). Parallel metabolite analyses revealed that the major part of the triterpenoids in the tomato fruit reside in the cuticle, with 74%, 79%, and 74% of the total d-amyrin, b-amyrin, and a-amyrin aglycone amounts present in the surface wax, respectively, and the remaining 21% to 26% in the underlying epidermal 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 determine the relative expression levels of SlTTS1 and SlTTS2 within leaves and fruit. Since the ratios of different triterpenoid products vary not only between 546 Plant Physiol. Vol. 155, 2011 Downloaded from on June 18, 2017 - Published by www.plantphysiol.org Copyright © 2011 American Society of Plant Biologists. All rights reserved. Biosynthesis of Tomato Fruit Triterpenoids Figure 8. Relative amounts of amyrin isomers in the fruit cuticular waxes of various lines of MicroTom. The percentages of d-amyrin, b-amyrin, and a-amyrin are given for the wild type (WT), transgenic fruit overexpressing the OSCs [SlTTS1(+) and SlTTS2(+)], and overexpressors with cosuppressed SlTTS2 [SlTTS2(2)]. Amounts of each triterpenoid were quantified as percentages relative to the level of wild-type d-amyrin and are given as averages of three independent lines and analyses with SD. Asterisks denote significant differences from the wild type. these two organs but also between fruit of different tomato cultivars (Vogg et al., 2004; Mintz-Oron et al., 2008; Isaacson et al., 2009), this experiment was to also compare expression patterns of both SlTTS genes between MicroTom and the cultivars M82 and Ailsa Craig. The same primers originally designed for the cloning of the full-length sequence from MicroTom were used to amplify corresponding cDNAs from M82 and Ailsa Craig. One allele each of SlTTS1 and SlTTS2 was cloned and sequenced from both cultivars, and the ORFs of the orthologous genes were found to be very similar or identical between M82 and Ailsa Craig (Supplemental Fig. S1). However, both SlTTS1 and SlTTS2 differed between these cultivars and MicroTom, with up to eight nucleotide differences between cultivars resulting in up to four amino acid changes and more than 99.6% DNA and more than 99.4% protein sequence identity (Table I). The similarities between both genes were approximately 89% on the nucleotide level across cultivars and 87% to 88% on the amino acid level. The expression levels of SlTTS1 and SlTTS2 were found to be similar to each other in the leaves and fruit of cv MicroTom (Fig. 10). Both genes were also expressed at similar levels to each other in leaves of M82, whereas in the fruit of this cultivar, SlTTS2 was expressed at a much higher level than SlTTS1. Ailsa Craig differed further, with higher expression levels of SlTTS2 in fruit and leaves. Terpenoid Profiles in Fruit and Leaves of Different Tomato Cultivars The terpenoid profiles of the cuticular wax mixtures of leaves and fruits from the MicroTom, M82, and Ailsa Craig cultivars were assessed, with particular focus on the relative compositions of amyrin isomers. The compositions of fruit waxes from the three cultivars (Vogg et al., 2004; Mintz-Oron et al., 2008; Isaacson et al., 2009) and also the leaf wax composition of MicroTom had been reported before (Vogg et al., 2004). However, exact data on the leaf triterpenoid profiles of all three cultivars were missing. Therefore, a detailed study of the leaf wax from M82 and MicroTom was carried out. It was found that the M82 leaf was covered by 4.8 6 0.5 mg cm22 cuticular wax and that the wax mixture contained 3.0% 6 1.0% triterpenoids, 64.3% 6 12.5% very-long-chain fatty acid derivatives, and 32.7% 6 13.5% unidentified compounds (Fig. 11). n-Alkanes (C27–C33) were the dominating compound class, at 45.6% 6 8.9%, accompanied by 16.6% 6 3.1% branched alkanes (total carbon nos. C29–C33) and 20.5% 6 7.4% alcohols (C28 and C32). The triterpenoid fraction was dominated by amyrin isomers, comprising 29%, 33.4%, and 14.4% d-amyrin, b-amyrin, and a-amyrin, respectively. Other triterpenoid alcohols were detected in the form of lupeol (23.3%) and trace amounts of multiflorenol, C-taraxasterol, and taraxasterol. 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 used to clone two tomato OSCs, SlTTS1 and SlTTS2, which were found to encode highly similar amino acid sequences. Our principal goal was to characterize the product spectrum of the enzymes, with the expectation that a d-amyrin synthase might be uncovered. Furthermore, we aimed to elucidate the role of the enzymes in the formation of the triterpenoids accumulating in the fruit cuticular wax. A phylogenetic analysis showed that SlTTS1 and SlTTS2 fall into a clade consisting entirely of b-amyrin Figure 9. RT-PCR analysis of the expression patterns of the two OSC genes in MicroTom fruit. Transcripts corresponding to the SlTTS1 and SlTTS2 enzymes generating pentacyclic triterpenoids were found expressed only in the epidermis layers but not in the internal tissues of the fruit. Plant Physiol. Vol. 155, 2011 547 Downloaded from on June 18, 2017 - Published by www.plantphysiol.org Copyright © 2011 American Society of Plant Biologists. All rights reserved. Wang et al. Figure 10. qRT-PCR analysis of the two OSC genes in fruit and leaves of the three tomato cultivars MicroTom (MT), M82, and Ailsa Craig (AC). The relative expression levels were determined in green immature fruit and normalized for each mRNA sample (n = 3; error bars indicate SD). synthases. Thus, the primary sequence of the new OSCs is in accordance with the prediction that the tomato enzymes catalyze the formation of amyrins. However, based on genetic information alone, it was not possible to predict the product specificities of the tomato enzymes. It should also be noted that, in contrast to the similarity of exon sequences with amyrin synthases, the intron pattern of SlTTS2 was identical to that of OSC3 from L. japonicus, a lupeol synthase that had fairly dissimilar sequence (Sawai et al., 2006b). The product profiles of SlTTS1 and SlTTS2 were determined by heterologous expression in yeast. It was found that SlTTS1 forms b-amyrin as its sole product, while SlTTS2 catalyzes the formation of seven different triterpenoids, with d-amyrin as the major product. The in planta overexpression of SlTTS1 and SlTTS2 led to increased accumulation of cuticular triterpenoids, in both cases of the same products that had also been found in yeast. This finding is very important, since most previous reports on biochemical characterizations of OSCs had relied entirely on yeast expression systems, and it had only rarely been tested whether the heterologous environment might alter the enzyme specificity (Han et al., 2006). The match between yeast and in planta expression results in this study confirms the validity of the yeast expression system, at least for the two tomato OSCs studied. Overall, we conclude that the two enzymes are a single-product b-amyrin synthase and a multifunctional OSC, respectively. SlTTS2, to our knowledge, is the first enzyme reported to synthesize predominantly d-amyrin. Interestingly, some of the tomato lines harboring the SlTTS2 construct exhibited a wax triterpenoid phenotype opposite to that of the SlTTS2 overexpressors, suggesting that in these lines the native gene was down-regulated by cosuppression. The observed increase in b-amyrin may be the result of additional 2,3-oxidosqualene substrate availability for SlTTS1 upon silencing of SlTTS2. The SlTTS2 loss-of-function lines thus not only confirm the biochemical function of the enzyme but also indicate that this enzyme must play a central role in the formation of the tomato fruit wax triterpenoids. We conclude that SlTTS2 is crucial for the biosynthesis of six of the seven triterpenoid isomers found in the wax and partially also contributes to the formation of the seventh component, b-amyrin. Another part of the latter compound is formed by the closely related enzyme SlTTS1. Both enzyme product profiles taken together completely match the wax triterpenoid composition; therefore, SlTTS1 and SlTTS2 are likely the two major enzymes forming the triterpenoids found in the tomato fruit cuticular wax. The involvement of additional OSCs cannot be excluded at this point, but it seems unlikely that any OSC other than SlTTS1 and SlTTS2 would contribute substantially to the triterpenoid amounts or spectrum. Two apparently orthologous OSCs were identified from each of the tomato cultivars M82 and Ailsa Craig, with a high degree of sequence identity to those from MicroTom. The differences between the two genes within one cultivar were much greater than the differences between the alleles in the cultivars. As differences between orthologous proteins were restricted to changes in three or four amino acids, it is very likely that both SlTTS1 and SlTTS2 were functional enzymes in all three cultivars and that their biochemical functions should be very similar in MicroTom, M82, and Ailsa Craig. We conclude that all three cultivars have at least one b-amyrin synthase (SlTTS1) and one multifunctional OSC producing d-amyrin and other isomers (SlTTS2). The relative expression levels of the SlTTS1 and SlTTS2 genes varied between fruit of the three tomato cultivars investigated, with transcripts of both genes showing similar abundance in MicroTom but SlTTS2 Figure 11. Chemical composition of cuticular wax on leaves of tomato cultivars M82 and MicroTom. The absolute amounts of all identified compounds are given as averages of three independent parallel experiments with SD. 548 Plant Physiol. Vol. 155, 2011 Downloaded from on June 18, 2017 - Published by www.plantphysiol.org Copyright © 2011 American Society of Plant Biologists. All rights reserved. Biosynthesis of Tomato Fruit Triterpenoids Table II. Accession data for the OSCs used in the phylogenetic analyses Accession No. At4g15340 At4g15370 At5g48010 At5g42600 At1g78500 At3g45130 At1g78970 At1g78960 At1g78950 At1g66960 At2g07050 AB263204 AB257507 AB037203 AB181244 AB034802 AJ430607 AF478455 AB034803 AB289585 AB014057 AB009030 AB263203 AB206469 AB181245 AB116228 AB181246 AB244671 Species Arabidopsis Arabidopsis Arabidopsis Arabidopsis Arabidopsis Arabidopsis Arabidopsis Arabidopsis Arabidopsis Arabidopsis Arabidopsis Rhizophora stylosa Kandelia candel Glycyrrhiza glabra Lotus japonicus Pisum sativum Medicago truncatula L. japonicus P. sativum Bruguiera gymnorhiza P. ginseng P. ginseng R. stylosa Euphorbia tirucalli L. japonicus G. glabra L. japonicus L. japonicus accumulating to higher levels than SlTTS1 in M82 and Ailsa Craig. This expression pattern is in accord with the amyrin profiles in the corresponding fruit waxes, where the latter two cultivars had lower levels of the SlTTS1 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 in triterpenoid profiles between cultivars may further be due to factors other than differential gene expression, possibly including differences in enzyme activities and/or additional OSCs being present. Our qRT-PCR results showed that the SlTTS1 and SlTTS2 genes are expressed not only in tomato fruit but also in the leaves of MicroTom, M82, and Ailsa Craig. Therefore, it seems likely that both genes also contribute to the formation of leaf cuticular triterpenoids. However, it must be noted that the leaf waxes have triterpenoid profiles differing from those in the fruit in quantitative and in qualitative terms, as shown by our detailed analyses of M82 leaf wax in comparison with literature data. The most prominent quantitative difference is the shift in amyrin ratios from 3:2:2 for d-amyrin, b-amyrin, and a-amyrin in the fruit to 2:2:1 in the leaf wax. This trend correlates with the differences in expression levels of SlTTS1 and SlTTS2 between both organs of M82 and can thus, at least in part, be explained by transcriptional control. It should be noted that Ailsa Craig leaves showed a very conspicuous expression pattern with particularly low Function Arabidiol synthase PEN1 Baruol synthase BARS1/PEN2 Thalianol synthase PEN4 Marneral synthase PEN5 Multifunctional triterpene synthase Lanosterol synthase LAS1/PEN7 Multifunctional triterpene synthase Multifunctional triterpene synthase b-Amyrin synthase AtBAS LUP4 Multifunctional triterpene synthase Cycloartenol synthase CAS1 Multifunctional triterpene synthase Multifunctional triterpene synthase b-Amyrin synthase GgbAS1 b-Amyrin synthase OSC1 b-Amyrin synthase PSY b-Amyrin synthase AMY1 Multifunctional triterpene synthase Multifunctional triterpene synthase b-Amyrin synthase BgbAS b-Amyrin synthase PgbAS/PNY2 b-Amyrin synthase PNY Multifunctional triterpene synthase b-Amyrin synthase EtbAS Lupeol synthase OSC3 Lupeol synthase GgLUS1 Cycloartenol synthase QSC5 Lanosterol synthase OSC7 PEN6 LUP1 LUP2 LUP5 RsM2 KcMS LjAMY2 PSM RsM1 levels of SlTTS2 transcript. Based on this finding, it may be expected that the leaf waxes of this cultivar should contain relatively little b-amyrin; however, the amyrin profiles of the leaf wax of Ailsa Craig have not been determined to date. One outstanding qualitative difference between the triterpenoid compositions of fruit and leaves, at least of the cultivars MicroTom and M82, is the presence of lupeol in the latter organ, a product that is not formed by either SlTTS1 or SlTTS2. Therefore, one or more other OSCs must be involved in the formation of leaf cuticular triterpenoids. More detailed investigations are needed to determine the contribution of SlTTS1 and SlTTS2 to the biosynthesis of tomato leaf cuticles. Our results on the tissue-specific expression of SlTTS1 and SlTTS2 showed that these enzymes are localized exclusively in the epidermis of tomato fruit. We further found that the triterpenoid products are also restricted to the fruit skin, as they accumulate to high concentrations in the cuticular waxes coating the epidermis but not in the internal parts of the fruit. The close match in localization of transcripts and metabolites makes it very likely that SlTTS1 and SlTTS2 are dedicated entirely to making the triterpenoids destined for the cuticular wax of the fruit surface. With this, a major biological function can be assigned to these two OSCs, as the cuticular triterpenoids contribute significantly to the chemical composition and to the ecophysiological properties of the fruit cuticle Plant Physiol. Vol. 155, 2011 549 Downloaded from on June 18, 2017 - Published by www.plantphysiol.org Copyright © 2011 American Society of Plant Biologists. All rights reserved. Wang et al. (Vogg et al., 2004; Isaacson et al., 2009). It should be noted that similar biological functions had previously been attributed to a few other OSCs, for example, a glutinol synthase and a friedelin synthase from K. daigremontiana (Wang et al., 2010). The sequences of SlTTS1 and SlTTS2 are relatively similar to each other, containing only 92 amino acid changes. On the other hand, the two enzymes were found to have fairly distinct product profiles, with SlTTS1 yielding exclusively b-amyrin whereas SlTTS2 is catalyzing the formation of d-amyrin (among other products). It has been predicted that the formation of d-amyrin requires only one rearrangement step less than b-amyrin in the course of the OSC-catalyzed cyclization (Fig. 1; Xu et al., 2004; Phillips et al., 2006). This relatively subtle difference in the mechanism of formation of both triterpenoids must be due to small differences in protein architecture, possibly involving specific changes in amino acids lining the active site cavity. The sequence differences between SlTTS1 and SlTTS2 give the first information on the candidate residues that may be involved in determining the amyrin isomer specificity. The list of candidate residues for defining d-amyrin synthases can be somewhat narrowed down based on comparisons between the two OSCs characterized here in three different tomato cultivars and with b-amyrin synthases from other species. A second observation might further help select candidate residues: many of the amino acid changes between both enzymes are clustered together, and many involve aromatic residues. Since it is well established that such aromatic amino acids play important roles in stabilizing the positive charge of high-energy intermediates of the cyclization reaction, it has been speculated that the number and exact positions of aromatic residues lining the active site might limit the number of rearrangement steps and, therefore, determine the product outcome of OSC-catalyzed reactions (Christianson, 2006; Phillips et al., 2006). Consequently, the changes between SlTTS1 and SlTTS2 involving aromatic amino acids at positions 48/49, 103, 318, 388, 399, 412, 421, 478, 496, 560, 675, 742, and 760 make these residues especially interesting candidates for defining the OSC specificity of d-amyrin and b-amyrin synthases. It appears very promising to use site-directed mutagenesis experiments to test this hypothesis in order to further our detailed understanding of the enzyme mechanisms 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 direct 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 spectrometric 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°C min21 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 mL min21. 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 (20 3 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 (Invitrogen) 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 electrophoresis (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 DNA was 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 genespecific 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#-GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTTTTT-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#-GGCCACGCGTCGACTAGTAC-3#) and the specific primer LEASB1S (5#-AGGGTTGTGGTAGTCAATC-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#-CCCGAATTCTTAGTTGTTTTCTAATGGTAATAC-3#; EcoRI site underlined), LeTTS2F (5#-TTGGAGCTCAAGATGTGGAAGTTGAAGATTGCAA-3#; SacI site underlined), and LeTTS2R 550 Plant Physiol. Vol. 155, 2011 Downloaded from on June 18, 2017 - Published by www.plantphysiol.org Copyright © 2011 American Society of Plant Biologists. All rights reserved. Biosynthesis of Tomato Fruit Triterpenoids (5#-CCCGAATTCCTATATGTAGTTGTGTTTTAATGGT-3#; EcoRI site underlined). The PCRs were conducted with Phusion High-Fidelity DNA polymerase (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 at 98°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 and Product 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)trifluoroacetamide at 70°C for 60 min and then analyzed by GC-FID/MS as described above or further purified by TLC plate (20 3 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 transformation 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 preparations were immediately frozen in liquid nitrogen, ground into powder using a mortar and pestle, and used to extract RNA with 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#-GGACCAAATTGCACTCTATATC-3#; SlTTS2, 5#-TGTTGAGTGCACTAGCTCGG-3# and 5#-ACGGACAACTCGATTCACTAAGC-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 template 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 standard protocols. Gene-specific primers were designed to amplify a fragment of SlTTS1 by TTS1F (5#-CGTCGAATGCACTGCCTCAT-3) and T1R2 (5#-TACCATGAACCATCAGGCATT-3#) and a fragment of SlTTS2 by TTS2F (5#-TGTTGAGTGCACTAGCTCGG-3#) and T2R2 (5#-TACCATGAACCGTCAGGCTCC-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 neighborjoining 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 sequences 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. 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