TY - JOUR
AU - Jang, In-Cheol
AB - Abstract Stevia (Stevia rebaudiana) produces not only a group of diterpenoid glycosides known as steviol glycosides (SGs), but also other labdane-type diterpenoids that may be spatially separated from SGs. However, their biosynthetic routes and spatial distribution in leaf tissues have not yet been elucidated. Here, we integrate metabolome and transcriptome analyses of Stevia to explore the biosynthetic capacity of leaf tissues for diterpenoid metabolism. Tissue-specific chemical analyses confirmed that SGs were accumulated in leaf cells but not in trichomes. On the other hand, Stevia leaf trichomes stored other labdane-type diterpenoids such as oxomanoyl oxide and agatholic acid. RNA sequencing analyses from two different tissues of Stevia provided a comprehensive overview of dynamic metabolic activities in trichomes and leaf without trichomes. These metabolite-guided transcriptomics and phylogenetic and gene expression analyses clearly identified specific gene members encoding enzymes involved in the 2-C-methyl-d-erythritol 4-phosphate pathway and the biosynthesis of steviol or other labdane-type diterpenoids. Additionally, our RNA sequencing analysis uncovered copalyl diphosphate synthase (SrCPS) and kaurene synthase1 (SrKS1) homologs, SrCPS2 and KS-like (SrKSL), which were specifically expressed in trichomes. In vitro and in planta assays showed that unlike SrCPS and SrKS1, SrCPS2 synthesized labda-13-en-8-ol diphosphate and successively catalyzed the formation of manoyl oxide and epi-manoyl oxide in combination with SrKSL. Our findings suggest that Stevia may have evolved to use distinct metabolic pathways to avoid metabolic interferences in leaf tissues for efficient production of diverse secondary metabolites. Stevia (Stevia rebaudiana), a perennial shrub belonging to the Asteraceae family, has been used for centuries in South America as a sweetener for herbal teas and foods (De et al., 2013). In addition to being a sweetening agent, Stevia has been used as a cardiotonic for hypertension and heartburn and also to lower uric acid levels. For reasons of food and medicine, there has been wide-spread cultivation of this plant throughout Europe, Asia, and North America (Philippe et al., 2014). The sweetness of Stevia leaves is attributed to a group of diterpenoid derivatives known as steviol glycosides (SGs), which are 150 to 300 times sweeter than cane sugar (Saccharum officinarum). In contrast to artificial sweeteners, SGs are natural and can be used primarily as a noncalorie sweetener and/or flavor enhancer. Stevia accumulates SGs in its leaves to as high as 20% of the dry weight, which are comprised of approximately 10 major components in varying levels (Geuns, 2003). All of these compounds share a common backbone, a steviol aglycone, but with a different number of sugar moieties (Glc, rhamnose, and Xyl; Obtani and Yamasaki, 2002; Starratt et al., 2002). The quality and intensity of the taste are determined by the C-position (C-13 or C-19) of steviol being modified and the number of the sugar moieties (Obtani and Yamasaki, 2002). As diterpenoid glycosides, SGs are synthesized from a 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway in plastids, which produces two isoprene units, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) from pyruvate and glyceraldehyde-3-P through a series of seven enzyme-catalyzed steps (Totté et al., 2000; Kumar et al., 2012). The MEP pathway is also important for the synthesis of many other terpenoids, in particular mono-, di-, and tetraterpenes, because IPP and DMAPP are their common precursors (McGarvey and Croteau, 1995). Once the 5-carbon unit IPP is synthesized in plastids by the MEP pathway, IPP isomerase (IDI) catalyzes the reversible isomerization of IPP to form DMAPP, and this enzyme might play a regulatory role in determining cellular DMAPP levels (Adam et al., 2002; Hunter, 2007). Among terpenoids, diterpenoids are classically defined by 20-carbon isoprenoids derived from the common precursor geranylgeranyldiphosphate (GGPP), which is catalyzed by GGPP synthase (GGPPS) inside plastids (Zi et al., 2014). Being required for the production of photosynthetic pigments and phytohormones (e.g., GA3 and abscisic acid), diterpenoid biosynthesis is essential for plant growth and development. In addition, plants also produce over 10,000 different diterpenoids as secondary metabolites, which may function as direct defense compounds against herbivores and microorganisms (Zerbe et al., 2013; Zi et al., 2014). The most commonly found version of diterpenoids is labdane-type compounds such as GA3s, phytoalexins, sclareol, forskolin, taxol, and steviol, many of which are of significant value to human life as biobased pharmaceuticals, flavors, and fragrances (Zerbe and Bohlmann, 2015). The structural diversity of different diterpenoids arises from divergent evolution of specialized diterpenoid biosynthetic pathways. These pathways share a common precursor GGPP and use a few types of enzymes, including different classes of diterpene synthases (diTPSs), cytochrome P450-dependent monooxygenases, and various modifying transferases (e.g., acyl-, methyl-, or glycosyltranferases), which may be unique for a given plant species or family (Zerbe et al., 2013; Zi et al., 2014; Zerbe and Bohlmann, 2015). In angiosperms, specialized diterpenoids are characterized by their common biosynthetic origins in the initial dual cyclization and/or rearrangement reactions by a sequential pair of class II and class I diTPSs (Peters, 2010). Class II diTPSs contain a DxDD motif, which is required for the protonation-initiated cationic cyclization and rearrangement of GGPP to distinct bicyclic diphosphate intermediates (Peters, 2010; Chen et al., 2011). Subsequently, class I diTPSs possessing DDxxD and NSE/DTE motifs for the binding of the substrate diphosphate catalyze cleavage of the phosphate group of intermediates and further cyclize or rearrange the resulting carbocations (Peters, 2010; Chen et al., 2011). In Stevia, SG biosynthesis shares three steps in common with GA3 biosynthesis pathway for the formation of kaurenoic acid, which requires two diTPSs, copalyl diphosphate synthase (CPS) and kaurene synthase (KS), and one member of the cytochrome P450 family, kaurene oxidase (KO; Richman et al., 1999; Humphrey et al., 2006). Because GA3 is an important phytohormone for plant growth and elongation of cells, it is not surprising that these genes are conserved among higher plants (Hedden and Phillips, 2000). Moreover, an additional P450 monooxygenase, kaurenoic acid hydroxylase (KAH), is involved in the biosynthesis of steviol, which provides the backbone for all SGs (Brandle and Telmer, 2007). The last stage of SG biosynthesis is the glycosylation of the diterpenoid steviol by UDP-glycosyltransferases, which transfer sugar residues from activated sugars to an aglycone acceptor (Richman et al., 2005; Brandle and Telmer, 2007). The chemical complexity of lipophilic components produced in Stevia leaves has been previously examined by gas chromatography (GC)-mass spectrometry (MS) showing the presence of diverse mono- and sesquiterpenes and fatty acids (Marković et al., 2008). Interestingly, apart from kaurene as a diterpenoid hydrocarbon precursor for GA3s or steviol, other labdane-type diterpenoids such as manoyl oxide, sclareol, austroinulin, and sterebins are also detected in Stevia leaf extracts, which may confer biological and pharmacological properties (Ibrahim et al., 2007; Marković et al., 2008; Cho et al., 2013). However, the biosynthetic pathways or terpene synthases for these diterpenoids in Stevia have not yet been reported. The biosynthesis and accumulation of many plant diterpenoids are restricted to specialized tissues or specific cell types, which enables plants to effectively synthesize specific natural chemicals and to avoid metabolic interference. For example, a diterpenoid forskolin accumulates in specialized root cork cells of Coleus forskohlii (Pateraki et al., 2014), diterpene resin acids in resin ducts of conifers (Zulak and Bohlmann, 2010), and Z-abienol in tobacco (Nicotiana tabacum) leaf trichomes (Sallaud et al., 2012). This cell/tissue-specific accumulation of diterpenoids may be associated with differential gene expression rather than their translocation and accumulation in specific cell/tissues. Although Stevia genes encoding CPS, KS, and KO related to GA3 or steviol biosynthesis have been shown to express in leaf parenchyma (Humphrey et al., 2006), it is unclear where in the leaf tissues SGs are accumulated. In plants, specialized metabolites are stored in the place of their synthesis, or in some cases, they can be transported to other tissues (Alvarez, 2014). The experimental approach of combining bioinformatics and metabolite analysis has facilitated the characterization of biosynthetic pathways and related genes for plant secondary metabolism (Bleeker et al., 2011; Zerbe et al., 2013; Jin et al., 2015; Zerbe and Bohlmann, 2015). As the Stevia genome has not yet been sequenced, only limited sources of gene information are available from randomly selected ESTs (Brandle et al., 2002). Recently, Chen et al. (2014) reported a RNA sequencing (RNA-seq) data set of three Stevia genotypes comparing expression patterns of previously reported genes involved in SG biosynthesis in these genotypes, but there were no additional new genes identified related to the MEP pathway, SG biosynthesis, or the synthesis of other terpenoids. Detailed or enriched transcriptome data sets from different tissues of terpenoid-producing nonmodel plants not only provide opportunities for the discovery of new genes and enzymes, but also explain the biosynthetic ability of specialized terpene metabolism that may be spatially restricted to organs, tissues, or cell types (Zerbe et al., 2013; Jin et al., 2014; Zerbe and Bohlmann, 2015). Here, we performed comparative analysis of RNA-seq transcriptomes of two different tissues, namely, trichomes and leaves without trichomes (leaf-trichomes). Comparison of transcripts between these two tissues showed that more than 20% of the assembled unigenes were differentially expressed in trichomes, whereas genes specifically expressed in the leaf-trichomes were less than 12%. Most genes involved in SG biosynthesis were predominantly expressed in leaf-trichomes where SGs accumulated. Moreover, we found that SrCPS2 and KS-like (SrKSL), homologs of SrCPS and SrKS1, were specifically expressed in trichomes. Taken together, our results show that comparative transcriptomic analysis along with metabolite profiling provided useful information on the distinct biosynthetic pathways for specialized diterpenoids that preferentially accumulate in different tissues of the Stevia leaves. RESULTS Metabolite Profiles in Stevia Leaves SGs are known to accumulate mainly in Stevia leaves (Brandle and Rosa, 1992; Kumar et al., 2012); however, it is unclear whether SGs are synthesized and stored in specialized tissue such as trichomes or oil cells in Stevia leaf tissues. We first investigated trichomes on the surface of Stevia leaves using scanning electron microscopy (SEM). Figure 1 shows that there were three different types of trichomes: glandular, long nonglandular and short nonglandular. These three types of trichomes could be distinguished by their differences in morphology, structure, and size (Fig. 1). Among these, glandular trichomes in other plants have been shown to produce, store, and secrete large amounts of diverse secondary metabolites such as terpenoids, phenylpropanoids, and acyl sugars (Glas et al., 2012). To examine if there is any differential distribution of terpenes in different cell/tissues of Stevia leaves, we first purified trichomes (T) from young leaves using a standard glass bead abrasion method (Lange et al., 2000). We also prepared leaf tissues stripped of trichomes (leaves minus trichomes [L-T]) by cold brushing (Wang et al., 2001). Both SEM and light microscopy confirmed the purity of the trichome preparation. Note that the preparation still contained a mix of the three types of trichomes, which are difficult to separate (Supplemental Fig. S1). Figure 1. Open in new tabDownload slide Glandular and filamentous trichomes of Stevia. A, SEM images of Stevia leaves showing three types of trichomes. a, Glandular trichome. b, Long nonglandular filamentous trichome. c, Short nonglandular filamentous trichome. B, A detailed view of the three types of trichomes of Stevia. Scale bars are included on all images. Figure 1. Open in new tabDownload slide Glandular and filamentous trichomes of Stevia. A, SEM images of Stevia leaves showing three types of trichomes. a, Glandular trichome. b, Long nonglandular filamentous trichome. c, Short nonglandular filamentous trichome. B, A detailed view of the three types of trichomes of Stevia. Scale bars are included on all images. Next, we used HPLC and GC-MS to examine SG content and other diterpenoids in T and L-T samples. Figure 2A shows that most SGs were exclusively found in L-T but not in T. In this HPLC analysis, each SG was identified by comparison with the retention time of known standards. Whereas SGs were almost absent in T, GC-MS analysis of T extract revealed a wider range of terpenes, including mono, sesqui-, and diterpenes, compared with the extract derived from L-T (Fig. 2B). Metabolites identified in T are listed in Table I. Figure 2. Open in new tabDownload slide Distribution of diterpenoids in Stevia leaf tissues. Tissue-specific abundance of metabolites was evaluated by ultra high performance liquid chromatography (A) and GC-MS (B). A, Comparison of SG content between leaf-trichomes and trichomes. Standards contain a mixture of nine known SGs. Peak a, Rebaudioside (Reb) D. Peak b, Reb A. Peak c, stevioside. Peak d, Reb F. Peak e, Reb C. Peak f, dulcoside A. Peak g, rubusoside. Peak h, Reb B. Peak i, steviobioside. mAU, Milliabsorbance units. B, GC traces showing the difference between leaf-trichomes and trichomes. The arrow indicates the peak of camphor (10 µg µL–1), the internal standard used in the assay. The peaks numbered in GC traces were identical to those listed in Table I. Figure 2. Open in new tabDownload slide Distribution of diterpenoids in Stevia leaf tissues. Tissue-specific abundance of metabolites was evaluated by ultra high performance liquid chromatography (A) and GC-MS (B). A, Comparison of SG content between leaf-trichomes and trichomes. Standards contain a mixture of nine known SGs. Peak a, Rebaudioside (Reb) D. Peak b, Reb A. Peak c, stevioside. Peak d, Reb F. Peak e, Reb C. Peak f, dulcoside A. Peak g, rubusoside. Peak h, Reb B. Peak i, steviobioside. mAU, Milliabsorbance units. B, GC traces showing the difference between leaf-trichomes and trichomes. The arrow indicates the peak of camphor (10 µg µL–1), the internal standard used in the assay. The peaks numbered in GC traces were identical to those listed in Table I. Chemical composition of trichomes from Stevia Table I. Chemical composition of trichomes from Stevia No.a . Compounds . RTb . RIc . Formula . RAd . min % 1 α-Pinene 9.07 922 C10H16 0.13 2 (+)-Sabinene 9.74 972 C10H16 0.15 3 β-Pinene 9.84 979 C10H16 1.10 4 β-Linalool 11.82 1,126 C10H18O 0.17 5 l-α-Terpineol 13.49 1,186 C10H18O 0.06 6 β-Elemene 16.82 1,389 C15H24 1.84 7 Caryophyllene 17.37 1,424 C15H24 2.15 8 α-Bergamotene 17.45 1,432 C15H24 0.94 9 α-Humulene 17.89 1,452 C15H24 1.18 10 Germacrene D 18.29 1,484 C15H24 1.10 11 β-Selinene 18.40 1,489 C15H24 0.29 12 Elixene 18.52 1,492 C15H24 1.27 13 γ-Cadinene 18.74 1,513 C15H24 0.24 14 δ-Cadinene 18.82 1,524 C15H24 0.17 15 (±)-trans-Nerolidol 19.27 1,561 C15H26O 0.93 16 Farnesol 19.47 1,620 C15H26O 0.17 17 Germacrene d-4-ol 19.69 1,660 C15H26O 0.56 18 α-epi-Cadinol 20.58 1,638 C15H26O 0.27 19 α-Cadinol 20.79 1,652 C15H26O 0.28 20 Isoaromadendrene epoxide 21.08 1,579 C15H24O 0.40 21 Ledene oxide-(II) 22.18 1,683 C15H24O 0.09 22 [2-(4-Methoxy-phenyl)-[1,3]dioxolan-2-yl]-acetic acid 23.46 1,930 C12H14O5 0.15 23 Manoyl oxide 25.32 1,988 C20H34O 1.07 24 n-Octadecanol 25.95 2,053 C18H38O 2.39 25 Ethanol, 2-[2-[4-(1,1,3,3-tetramethylbutyl)phenoxy]ethoxy]- 26.93 2,126 C18H30O3 0.28 26 Stearyl acetate 27.30 2,160 C20H40O2 0.33 27 3-Oxomanoyl oxide 27.90 2,219 C20H32O2 2.73 28 (+)-Agathadiol 28.80 2,357 C19H30O2 4.80 29 (+)-Copaiferic acid 28.96 2,369 C20H32O2 4.62 30 4,8,13-Duvatriene-1,3-Diol 29.28 2,400 C21H28O2 0.72 31 4-(3-Hydroxy-3-methylpentyl)-3,4a,8,8-tetramethyldecahydro-1-naphthalenol 29.57 2,415 C20H34O2 1.30 32 Androstan-17-one, 3-methoxy-16,16-dimethyl-, (3β,5α)- 29.86 2,418 C22H36O2 1.90 33 Labda-8(20),13-dien-15-oic acid, 19-hydroxy-, methyl ester, (E)- 30.45 2,429 C21H32O3 29.64 34 Pregnane-18,20-diol, (5α)- 31.09 2,449 C21H36O2 5.63 35 Allopregnane-3α,20α-diol 31.47 2,475 C21H36O2 12.20 36 17-α-Hydroxypregnenolone 31.57 2,480 C20H32O2 9.97 37 2-[5-(2,2-Dimethyl-6-methylene-cyclohexyl)-3-methyl-pent-2-enyl]-[1,4]benzoquinone 31.84 2,489 C21H28O2 2.91 38 Isophthalic acid, 3,7-dimethyloct-6-enyl propyl ester 31.96 2,492 C21H30O4 3.30 39 3-Ethyl-5-(2'-ethylbutyl)octadecane 32.13 2,599 C26H54 0.76 40 Allopregnane-7α,11α-diol-3,20-dione 32.36 2,613 C21H32O4 0.39 41 Ethanol, 2-[2-[2-[2-[p-(1,1,3,3-tetramethylbutyl)phenoxy]ethoxy]ethoxy]ethoxy]- 32.64 2,676 C22H38O 0.97 42 n-Heptacosane 33.06 2,700 C27H56 0.45 No.a . Compounds . RTb . RIc . Formula . RAd . min % 1 α-Pinene 9.07 922 C10H16 0.13 2 (+)-Sabinene 9.74 972 C10H16 0.15 3 β-Pinene 9.84 979 C10H16 1.10 4 β-Linalool 11.82 1,126 C10H18O 0.17 5 l-α-Terpineol 13.49 1,186 C10H18O 0.06 6 β-Elemene 16.82 1,389 C15H24 1.84 7 Caryophyllene 17.37 1,424 C15H24 2.15 8 α-Bergamotene 17.45 1,432 C15H24 0.94 9 α-Humulene 17.89 1,452 C15H24 1.18 10 Germacrene D 18.29 1,484 C15H24 1.10 11 β-Selinene 18.40 1,489 C15H24 0.29 12 Elixene 18.52 1,492 C15H24 1.27 13 γ-Cadinene 18.74 1,513 C15H24 0.24 14 δ-Cadinene 18.82 1,524 C15H24 0.17 15 (±)-trans-Nerolidol 19.27 1,561 C15H26O 0.93 16 Farnesol 19.47 1,620 C15H26O 0.17 17 Germacrene d-4-ol 19.69 1,660 C15H26O 0.56 18 α-epi-Cadinol 20.58 1,638 C15H26O 0.27 19 α-Cadinol 20.79 1,652 C15H26O 0.28 20 Isoaromadendrene epoxide 21.08 1,579 C15H24O 0.40 21 Ledene oxide-(II) 22.18 1,683 C15H24O 0.09 22 [2-(4-Methoxy-phenyl)-[1,3]dioxolan-2-yl]-acetic acid 23.46 1,930 C12H14O5 0.15 23 Manoyl oxide 25.32 1,988 C20H34O 1.07 24 n-Octadecanol 25.95 2,053 C18H38O 2.39 25 Ethanol, 2-[2-[4-(1,1,3,3-tetramethylbutyl)phenoxy]ethoxy]- 26.93 2,126 C18H30O3 0.28 26 Stearyl acetate 27.30 2,160 C20H40O2 0.33 27 3-Oxomanoyl oxide 27.90 2,219 C20H32O2 2.73 28 (+)-Agathadiol 28.80 2,357 C19H30O2 4.80 29 (+)-Copaiferic acid 28.96 2,369 C20H32O2 4.62 30 4,8,13-Duvatriene-1,3-Diol 29.28 2,400 C21H28O2 0.72 31 4-(3-Hydroxy-3-methylpentyl)-3,4a,8,8-tetramethyldecahydro-1-naphthalenol 29.57 2,415 C20H34O2 1.30 32 Androstan-17-one, 3-methoxy-16,16-dimethyl-, (3β,5α)- 29.86 2,418 C22H36O2 1.90 33 Labda-8(20),13-dien-15-oic acid, 19-hydroxy-, methyl ester, (E)- 30.45 2,429 C21H32O3 29.64 34 Pregnane-18,20-diol, (5α)- 31.09 2,449 C21H36O2 5.63 35 Allopregnane-3α,20α-diol 31.47 2,475 C21H36O2 12.20 36 17-α-Hydroxypregnenolone 31.57 2,480 C20H32O2 9.97 37 2-[5-(2,2-Dimethyl-6-methylene-cyclohexyl)-3-methyl-pent-2-enyl]-[1,4]benzoquinone 31.84 2,489 C21H28O2 2.91 38 Isophthalic acid, 3,7-dimethyloct-6-enyl propyl ester 31.96 2,492 C21H30O4 3.30 39 3-Ethyl-5-(2'-ethylbutyl)octadecane 32.13 2,599 C26H54 0.76 40 Allopregnane-7α,11α-diol-3,20-dione 32.36 2,613 C21H32O4 0.39 41 Ethanol, 2-[2-[2-[2-[p-(1,1,3,3-tetramethylbutyl)phenoxy]ethoxy]ethoxy]ethoxy]- 32.64 2,676 C22H38O 0.97 42 n-Heptacosane 33.06 2,700 C27H56 0.45 a Compound listed in order of elution in an HP-5MS Ultra Inert (UI) column. b Retention time. c Retention indices calculated against C7-C30 n-alkanes on the HP-5MS UI column. d Relative amount. Ratio expressed against the sum of all peaks. Open in new tab Table I. Chemical composition of trichomes from Stevia No.a . Compounds . RTb . RIc . Formula . RAd . min % 1 α-Pinene 9.07 922 C10H16 0.13 2 (+)-Sabinene 9.74 972 C10H16 0.15 3 β-Pinene 9.84 979 C10H16 1.10 4 β-Linalool 11.82 1,126 C10H18O 0.17 5 l-α-Terpineol 13.49 1,186 C10H18O 0.06 6 β-Elemene 16.82 1,389 C15H24 1.84 7 Caryophyllene 17.37 1,424 C15H24 2.15 8 α-Bergamotene 17.45 1,432 C15H24 0.94 9 α-Humulene 17.89 1,452 C15H24 1.18 10 Germacrene D 18.29 1,484 C15H24 1.10 11 β-Selinene 18.40 1,489 C15H24 0.29 12 Elixene 18.52 1,492 C15H24 1.27 13 γ-Cadinene 18.74 1,513 C15H24 0.24 14 δ-Cadinene 18.82 1,524 C15H24 0.17 15 (±)-trans-Nerolidol 19.27 1,561 C15H26O 0.93 16 Farnesol 19.47 1,620 C15H26O 0.17 17 Germacrene d-4-ol 19.69 1,660 C15H26O 0.56 18 α-epi-Cadinol 20.58 1,638 C15H26O 0.27 19 α-Cadinol 20.79 1,652 C15H26O 0.28 20 Isoaromadendrene epoxide 21.08 1,579 C15H24O 0.40 21 Ledene oxide-(II) 22.18 1,683 C15H24O 0.09 22 [2-(4-Methoxy-phenyl)-[1,3]dioxolan-2-yl]-acetic acid 23.46 1,930 C12H14O5 0.15 23 Manoyl oxide 25.32 1,988 C20H34O 1.07 24 n-Octadecanol 25.95 2,053 C18H38O 2.39 25 Ethanol, 2-[2-[4-(1,1,3,3-tetramethylbutyl)phenoxy]ethoxy]- 26.93 2,126 C18H30O3 0.28 26 Stearyl acetate 27.30 2,160 C20H40O2 0.33 27 3-Oxomanoyl oxide 27.90 2,219 C20H32O2 2.73 28 (+)-Agathadiol 28.80 2,357 C19H30O2 4.80 29 (+)-Copaiferic acid 28.96 2,369 C20H32O2 4.62 30 4,8,13-Duvatriene-1,3-Diol 29.28 2,400 C21H28O2 0.72 31 4-(3-Hydroxy-3-methylpentyl)-3,4a,8,8-tetramethyldecahydro-1-naphthalenol 29.57 2,415 C20H34O2 1.30 32 Androstan-17-one, 3-methoxy-16,16-dimethyl-, (3β,5α)- 29.86 2,418 C22H36O2 1.90 33 Labda-8(20),13-dien-15-oic acid, 19-hydroxy-, methyl ester, (E)- 30.45 2,429 C21H32O3 29.64 34 Pregnane-18,20-diol, (5α)- 31.09 2,449 C21H36O2 5.63 35 Allopregnane-3α,20α-diol 31.47 2,475 C21H36O2 12.20 36 17-α-Hydroxypregnenolone 31.57 2,480 C20H32O2 9.97 37 2-[5-(2,2-Dimethyl-6-methylene-cyclohexyl)-3-methyl-pent-2-enyl]-[1,4]benzoquinone 31.84 2,489 C21H28O2 2.91 38 Isophthalic acid, 3,7-dimethyloct-6-enyl propyl ester 31.96 2,492 C21H30O4 3.30 39 3-Ethyl-5-(2'-ethylbutyl)octadecane 32.13 2,599 C26H54 0.76 40 Allopregnane-7α,11α-diol-3,20-dione 32.36 2,613 C21H32O4 0.39 41 Ethanol, 2-[2-[2-[2-[p-(1,1,3,3-tetramethylbutyl)phenoxy]ethoxy]ethoxy]ethoxy]- 32.64 2,676 C22H38O 0.97 42 n-Heptacosane 33.06 2,700 C27H56 0.45 No.a . Compounds . RTb . RIc . Formula . RAd . min % 1 α-Pinene 9.07 922 C10H16 0.13 2 (+)-Sabinene 9.74 972 C10H16 0.15 3 β-Pinene 9.84 979 C10H16 1.10 4 β-Linalool 11.82 1,126 C10H18O 0.17 5 l-α-Terpineol 13.49 1,186 C10H18O 0.06 6 β-Elemene 16.82 1,389 C15H24 1.84 7 Caryophyllene 17.37 1,424 C15H24 2.15 8 α-Bergamotene 17.45 1,432 C15H24 0.94 9 α-Humulene 17.89 1,452 C15H24 1.18 10 Germacrene D 18.29 1,484 C15H24 1.10 11 β-Selinene 18.40 1,489 C15H24 0.29 12 Elixene 18.52 1,492 C15H24 1.27 13 γ-Cadinene 18.74 1,513 C15H24 0.24 14 δ-Cadinene 18.82 1,524 C15H24 0.17 15 (±)-trans-Nerolidol 19.27 1,561 C15H26O 0.93 16 Farnesol 19.47 1,620 C15H26O 0.17 17 Germacrene d-4-ol 19.69 1,660 C15H26O 0.56 18 α-epi-Cadinol 20.58 1,638 C15H26O 0.27 19 α-Cadinol 20.79 1,652 C15H26O 0.28 20 Isoaromadendrene epoxide 21.08 1,579 C15H24O 0.40 21 Ledene oxide-(II) 22.18 1,683 C15H24O 0.09 22 [2-(4-Methoxy-phenyl)-[1,3]dioxolan-2-yl]-acetic acid 23.46 1,930 C12H14O5 0.15 23 Manoyl oxide 25.32 1,988 C20H34O 1.07 24 n-Octadecanol 25.95 2,053 C18H38O 2.39 25 Ethanol, 2-[2-[4-(1,1,3,3-tetramethylbutyl)phenoxy]ethoxy]- 26.93 2,126 C18H30O3 0.28 26 Stearyl acetate 27.30 2,160 C20H40O2 0.33 27 3-Oxomanoyl oxide 27.90 2,219 C20H32O2 2.73 28 (+)-Agathadiol 28.80 2,357 C19H30O2 4.80 29 (+)-Copaiferic acid 28.96 2,369 C20H32O2 4.62 30 4,8,13-Duvatriene-1,3-Diol 29.28 2,400 C21H28O2 0.72 31 4-(3-Hydroxy-3-methylpentyl)-3,4a,8,8-tetramethyldecahydro-1-naphthalenol 29.57 2,415 C20H34O2 1.30 32 Androstan-17-one, 3-methoxy-16,16-dimethyl-, (3β,5α)- 29.86 2,418 C22H36O2 1.90 33 Labda-8(20),13-dien-15-oic acid, 19-hydroxy-, methyl ester, (E)- 30.45 2,429 C21H32O3 29.64 34 Pregnane-18,20-diol, (5α)- 31.09 2,449 C21H36O2 5.63 35 Allopregnane-3α,20α-diol 31.47 2,475 C21H36O2 12.20 36 17-α-Hydroxypregnenolone 31.57 2,480 C20H32O2 9.97 37 2-[5-(2,2-Dimethyl-6-methylene-cyclohexyl)-3-methyl-pent-2-enyl]-[1,4]benzoquinone 31.84 2,489 C21H28O2 2.91 38 Isophthalic acid, 3,7-dimethyloct-6-enyl propyl ester 31.96 2,492 C21H30O4 3.30 39 3-Ethyl-5-(2'-ethylbutyl)octadecane 32.13 2,599 C26H54 0.76 40 Allopregnane-7α,11α-diol-3,20-dione 32.36 2,613 C21H32O4 0.39 41 Ethanol, 2-[2-[2-[2-[p-(1,1,3,3-tetramethylbutyl)phenoxy]ethoxy]ethoxy]ethoxy]- 32.64 2,676 C22H38O 0.97 42 n-Heptacosane 33.06 2,700 C27H56 0.45 a Compound listed in order of elution in an HP-5MS Ultra Inert (UI) column. b Retention time. c Retention indices calculated against C7-C30 n-alkanes on the HP-5MS UI column. d Relative amount. Ratio expressed against the sum of all peaks. Open in new tab RNA-seq Data of Stevia, de Novo Assembly, and Annotation of Transcriptome To compare transcriptomes of different parts of Stevia leaves, we sequenced RNA-seq libraries derived from T and L-T samples using an Illumina HiSeq 2000. Illumina sequencing runs generated more than 160 million high-quality strand-specific single-end reads of 101 bp from each of the two tissues. The quality of Illumina sequencing outputs was high, as evaluated by FastQC (Supplemental Fig. S2). Our RNA-seq reads were de novo assembled into 53,793 nonredundant unigenes, spanning a total of 95 Mbp of sequence with a GC content of 37.3%. The sequence length of the assembled unigenes was at least longer than 200 bp. The resulting assembly had a N50 value of 1,482 bp. As a result, the assemblies produced 32,336 contigs for L-T and 45,918 contigs for T, giving a total of 53,793 unigenes (Table II). Overview of the assembly results of RNA-seq Table II. Overview of the assembly results of RNA-seq Tissue . Total Reads . No. of Unigenes . No. of Annotations . % of Annotations . Leaf-trichomes 75,097,166 32,336 24,041 74.3 Trichomes 85,902,623 45,918 32,692 71.2 Tissue . Total Reads . No. of Unigenes . No. of Annotations . % of Annotations . Leaf-trichomes 75,097,166 32,336 24,041 74.3 Trichomes 85,902,623 45,918 32,692 71.2 Open in new tab Table II. Overview of the assembly results of RNA-seq Tissue . Total Reads . No. of Unigenes . No. of Annotations . % of Annotations . Leaf-trichomes 75,097,166 32,336 24,041 74.3 Trichomes 85,902,623 45,918 32,692 71.2 Tissue . Total Reads . No. of Unigenes . No. of Annotations . % of Annotations . Leaf-trichomes 75,097,166 32,336 24,041 74.3 Trichomes 85,902,623 45,918 32,692 71.2 Open in new tab To predict accurate gene annotations and to maximize gene annotation percentages, the assembled unigenes were blasted against the National Centre for Biotechnology Information (NCBI) nonredundant protein database and protein databases from Arabidopsis (Arabidopsis thaliana), Vitis vinifera, Glycine max, and Oryza sativa. Among the 53,793 unigenes, 40,243 (74.8%) unigenes were annotated through BLASTX search with E-value ≤ 1e-3. We mapped RNA-seq reads onto the assembled transcripts to calculate their expression levels using Bowtie (Langmead and Salzberg, 2012). RNA-seq by Expectation-Maximization was used to estimate the abundance of assembled transcripts and to determine the expression levels (Li and Dewey, 2011). Differential Gene Expression between L-T and T From the RNA-seq data, 32,336 and 45,918 unigenes were observed to be expressed in L-T and T, respectively. The two expression patterns were clearly distinct, as shown by the heat map (Supplemental Fig. S3). Among the unigenes, 3,807 unigenes were preferentially expressed in L-T, whereas 10,529 unigenes were T specific; the minimum differential expression level was at least 4 times between the two samples (Supplemental File S1). Note that more than 20% of the assembled unigenes were specifically expressed in T. Among the differential expressed genes, about 40% encoded either hypothetical proteins or were unannotated. These might be unique genes with sequences divergent from those of other plants but nevertheless essential for trichome development in Stevia and/or partly be a result of misassembled transcripts. To further analyze possible functions of the differentially expressed unigenes from each sample, we assessed their Gene Ontology (GO) classifications by Trinotate (Quevillon et al., 2005). Figure 3 shows the GO terms for the more abundant unigenes in L-T or T. GO terms related to defense responses to fungus and virus and responses to salicylic acid stimulus were highly represented in T, reflecting the overall defense function of trichomes. However, unigenes from the GO terms of photosynthesis and chlorophyll biosynthesis were enriched in L-T but not detectable in T. Moreover, other terms for cellular components related to chloroplast envelope, thylakoid membrane, and stroma were only found in L-T. These results were not unexpected because the L-T sample contained mesophyll cells where photosynthesis takes place. At the same time, our results showed that the T sample preparation was not contaminated by leaf tissues because Stevia trichomes are not implicated in photosynthesis by GO annotation. Although genes encoding enzymes and proteins related to photosynthesis are significantly expressed in glandular trichomes of tomato (Solanum lycopersicum) and tobacco (Harada et al., 2010; Cui et al., 2011), they are not expressed in peppermint (Mentha × piperita) glandular trichomes (Lange et al., 2000), indicating species differences between trichome types. Figure 3. Open in new tabDownload slide GO analysis of more abundantly expressed unigenes in leaf-trichomes (A) or trichomes (B). x Axis, log(1/P value). P value is the hypergeometric test result for each GO term. Figure 3. Open in new tabDownload slide GO analysis of more abundantly expressed unigenes in leaf-trichomes (A) or trichomes (B). x Axis, log(1/P value). P value is the hypergeometric test result for each GO term. Because SGs were found in the L-T sample (Fig. 2A), we hypothesized that the 3,807 unigenes that were preferentially expressed in this sample may provide an important resource for the identification of unique genes involved in SG biosynthesis and its regulation. Expression and Molecular Analysis of the MEP Pathway and IDI Genes Fourteen genes encoding seven enzymes in the MEP pathway and IDI were identified from our Stevia RNA-seq data sets, including genes for four 1-deoxy-d-xylulose 5-phosphate synthases (DXSs), two each of 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR), 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (HDR), and IDI, and one each of 2-C-methyl-d-erythritol 4-phosphate cytidylyltransferase (MCT), 4-(cytidine 5′-diphospho)-2-C-methyl-d-erythritol kinase (CMK), 2-C-methyl-d-erythritol 2,4-cyclodiphosphate synthase (MDS), and 4-hydroxy-3-methylbut-2-enyl diphosphate synthase (HDS; Supplemental Fig. S4). We found four DXS genes (designated as SrDXS1–SrDXS4) containing the complete open reading frame (ORF). Because SrDXS that is designated here as SrDXS4 has been previously reported (accession no. AJ429232; Totté et al., 2003), three additional DXS genes, SrDXS1, SrDXS2, and SrDXS3 are newly identified in this study. Among four SrDXS proteins, SrDXS1 is the only member of the DXS1 clade, which is probably involved in primary metabolism, whereas SrDXS2 and SrDXS4, which belong to the DXS2 clade, may be related to secondary metabolism (Fig. 4A; Supplemental Fig. S5; Walter et al., 2002; Phillips et al., 2007). Some plant species have a third DXS protein cluster referred to as the DXS3 clade, which has been proposed to participate in the synthesis of some products essential for plant survival (Cordoba et al., 2009, 2011). We found that SrDXS3 was a member of the DXS3 clade (Fig. 4A). We confirmed differential expression of SrDXS genes by quantitative real-time (qRT)-PCR using the L-T and T samples. Figure 4B shows that SrDXS1 transcripts were 4 to 5 times more abundant in L-T than T. On the other hand, SrDXS4, which was previously reported (Totté et al., 2003), was predominantly expressed in T. Interestingly, SrDXS2 transcripts were rarely detectable in L-T but were highly expressed more than 200 times in T. Figure 4. Open in new tabDownload slide Analysis of genes involved in the MEP pathway from Stevia. A, Phylogenetic analysis of DXSs from Stevia. The maximum likelihood tree was constructed by the MEGA 6 program from an alignment of full-length SrDXSs with other plant DXSs. Abbreviations and accession numbers of proteins are listed in Supplemental Table S2. B, Relative expression levels of SrDXS genes in leaf-trichomes (L-T) and trichomes (T). C, Relative expression levels of SrDXR genes, SrMCT, SrCMK, SrMDS, SrHDS, SrHDR genes, and SrIDI genes in L-T and T. Transcript levels of genes expressed in L-T and T were measured by qRT-PCR. Amplification of Actin mRNA was used for normalization. Figure 4. Open in new tabDownload slide Analysis of genes involved in the MEP pathway from Stevia. A, Phylogenetic analysis of DXSs from Stevia. The maximum likelihood tree was constructed by the MEGA 6 program from an alignment of full-length SrDXSs with other plant DXSs. Abbreviations and accession numbers of proteins are listed in Supplemental Table S2. B, Relative expression levels of SrDXS genes in leaf-trichomes (L-T) and trichomes (T). C, Relative expression levels of SrDXR genes, SrMCT, SrCMK, SrMDS, SrHDS, SrHDR genes, and SrIDI genes in L-T and T. Transcript levels of genes expressed in L-T and T were measured by qRT-PCR. Amplification of Actin mRNA was used for normalization. From our RNA-seq data set, we were able to identify transcripts for all previously reported DXR, MCT, CMK, MDS, HDS, and HDR (Totté et al., 2003; Kumar et al., 2012). Moreover, we found transcripts for an additional copy of DXR and HDR, designated here as DXR2 and HDR2, respectively. Note that SrDXR1 (accession no. AJ429233) and SrHDR1 (accession no. DQ269451) are previously reported genes (Totté et al., 2003; Kumar et al., 2012). Although SrDXR1 and SrDXR2 share 89.4% high amino acid identity (Supplemental Fig. S6), the expression pattern of SrDXR1 and SrDXR2 was clearly distinct in leaf tissues. Figure 4C showed that SrDXR1 expression was slightly higher in T compared with L-T by about 2.5-fold, whereas SrDXR2 transcripts were 20-fold more abundant in T. The expression of other intermediate genes such as SrMCT, SrCMK, SrMDS, and SrHDS was detected in both L-T and T samples (Fig. 4C). All genes except SrMDS were slightly more expressed in T by about 2- to 4-fold, but SrMDS expression was comparable in both samples. SrHDR2 showed 86.7% amino acid identity with SrHDR1 (Supplemental Fig. S7). However, genes for the two related proteins showed opposite expression patterns in L-T and T samples, but the differential expression level of these genes was not significant (Fig. 4C). Transcripts for two IDI genes, SrIDI1 and SrIDI2, were found in our Stevia RNA-seq data set. Although SrIDI1 appeared to be the same as the previously reported SrIDI (accession no. DQ989585; Kumar et al., 2012), the protein encoded by SrIDI1 is 64 amino acids longer than SrIDI at the N terminus, indicating that SrIDI might be a partial sequence (Supplemental Fig. S8). On the other hand, SrIDI2 is, to our knowledge, a new sequence not reported before. Our gene expression analysis showed that SrIDI1 was predominantly expressed more than 6 times in trichomes (Fig. 4C). By contrast, SrIDI2 was expressed both in L-T and T. SrIDI1 and SrIDI2 share 68.4% identity at the amino acid level, and both possess a transit peptide sequence for plastidic targeting (Supplemental Fig. S8). Characterization of the GGPPS Gene Family Analysis of our Stevia RNA-seq uncovered three GGPPS genes designated as SrGGPPS1, SrGGPPS2, and SrGGPPS3. All three SrGGPPSs are clearly distinct from geranyl diphosphate synthase or farnesyl diphosphate synthase family members and have two conserved Asp-rich domains, first aspartate rich motif (DDx9RR) and second aspartate rich motif (DDxxD), which are required for IPP and DMAPP substrate binding and catalysis (Fig. 5A; Supplemental Fig. S9; Liang et al., 2002). SrGGPPS1 transcripts were almost undetectable in the L-T sample (Fig. 5B); rather, this gene was predominantly expressed more than 400 times in T. By contrast, transcripts for SrGGPPS2 and SrGGPPS3 were more abundantly expressed in the L-T sample. All three SrGGPPSs were predicted by the ChloroP program (http://www.cbs.dtu.dk/services/ChloroP) to be plastid localized. SrGGPPS1 and SrGGPPS3 had a putative N-terminal plastid transit peptide sequence of 33 and 45 amino acids, respectively, whereas SrGGPPS2 was predicted to have only an extra six amino acids at the N terminus, which may be too short to function as a plastidic transit peptide sequence (Supplemental Fig. S9). This was confirmed by subcellular localization of each SrGGPPS- yellow fluorescent protein (YFP) fusion protein in Nicotiana benthamiana leaves using Agrobacterium tumefaciens-mediated infiltration. Figure 5C shows that SrGGPPS1 and SrGGPPS3 were clearly localized in chloroplasts, whereas SrGGPPS2 was distributed throughout the cytosol. Figure 5. Open in new tabDownload slide Analysis of GGPPSs from Stevia. A, Phylogenetic analysis of GGPPSs from Stevia. The maximum likelihood tree was constructed by the MEGA 6 program from an alignment of full-length SrGGPPSs with other plant GGPPSs, geranyl diphosphate synthases (GPPSs), small subunit I (GPPS.SSUIs), small subunit II (GPPS.SSUIIs), large subunit (GPPS.LSU), and farnesyl diphosphate synthases (FPPSs). Abbreviations and accession numbers of proteins are listed in Supplemental Table S2. B, Relative expression levels of SrGGPPS genes in leaf-trichomes (L-T) and trichomes (T). Transcript levels of genes expressed in L-T and T were measured by qRT-PCR. Amplification of Actin mRNA was used for normalization. C, Subcellular localization of SrGGPPs. YFP-fused SrGGPPSs (SrGGPPS1-YFP, SrGGPPS2-YFP, and SrGGPPS3-YFP) were transiently expressed in N. benthamiana leaves by A. tumefaciens-mediated infiltration and visualized 3 d postinfiltration using YFP channel of a confocal microscope. Auto, Chlorophyll autofluorescence; YFP, YFP channel image; Light, light microscope image; Merged, merged image between Auto and YFP. Bars = 10 µm. Figure 5. Open in new tabDownload slide Analysis of GGPPSs from Stevia. A, Phylogenetic analysis of GGPPSs from Stevia. The maximum likelihood tree was constructed by the MEGA 6 program from an alignment of full-length SrGGPPSs with other plant GGPPSs, geranyl diphosphate synthases (GPPSs), small subunit I (GPPS.SSUIs), small subunit II (GPPS.SSUIIs), large subunit (GPPS.LSU), and farnesyl diphosphate synthases (FPPSs). Abbreviations and accession numbers of proteins are listed in Supplemental Table S2. B, Relative expression levels of SrGGPPS genes in leaf-trichomes (L-T) and trichomes (T). Transcript levels of genes expressed in L-T and T were measured by qRT-PCR. Amplification of Actin mRNA was used for normalization. C, Subcellular localization of SrGGPPs. YFP-fused SrGGPPSs (SrGGPPS1-YFP, SrGGPPS2-YFP, and SrGGPPS3-YFP) were transiently expressed in N. benthamiana leaves by A. tumefaciens-mediated infiltration and visualized 3 d postinfiltration using YFP channel of a confocal microscope. Auto, Chlorophyll autofluorescence; YFP, YFP channel image; Light, light microscope image; Merged, merged image between Auto and YFP. Bars = 10 µm. Expression Pattern of Genes for Diterpenoid Steviol Biosynthesis and Their Homologs In addition to Stevia diTPSs, SrCPS, and SrKS (Richman et al., 1999), analysis of our Stevia RNA-seq uncovered a homolog each for both SrCPS and SrKS, which were designated as SrCPS2 and SrKSL, respectively. Because both SrCPS2 and SrKSL were initially identified as partial sequences from the RNA-seq data set, we performed 5′-RACE to clone the full-length complementary DNAs (cDNAs). Phylogenetic comparison of the sequences of the full-length cDNAs with other related sequences assigned SrCPS2 and SrKSL to the terpene synthase (TPS)-c and TPS-e/f subfamilies, respectively (Fig. 6A). These two families contain general or specialized diTPSs (Chen et al., 2011; Caniard et al., 2012). Figure 6. Open in new tabDownload slide Analysis of diTPSs from Stevia. A, Phylogenetic analysis of SrCPS2 and SrKSL from Stevia. The maximum likelihood tree was constructed by the MEGA 6 program from an alignment of full-length SrCPS2 and SrKSL with other plant TPSs. Abbreviations and accession numbers of proteins are listed in Supplemental Table S2. B, Relative expression levels of Stevia diTPS (SrCPS, SrCPS2, SrKS1, and SrKSL) and P450 (SrKO1 and SrKAH) genes in leaf-trichomes (L-T) and trichomes (T). Transcript levels of genes expressed in L-T and T were measured by qRT-PCR. Amplification of Actin mRNA was used for normalization. C, Subcellular localization of Stevia diTPSs. YFP-fused SrCPS, SrCPS2, and SrKSL (SrCPS-YFP, SrCPS2-YFP, and SrKSL-YFP) were transiently expressed in N. benthamiana leaves by A. tumefaciens-mediated infiltration and visualized 3 d postinfiltration using the YFP channel of a confocal microscope. Auto, Chlorophyll autofluorescence; YFP, YFP channel image; Light, light microscope image; Merged, merged image between Auto and YFP. Bars = 10 µm (SrCPS-YFP) and 20 µm (SrCPS2-YFP and SrKSL-YFP). Figure 6. Open in new tabDownload slide Analysis of diTPSs from Stevia. A, Phylogenetic analysis of SrCPS2 and SrKSL from Stevia. The maximum likelihood tree was constructed by the MEGA 6 program from an alignment of full-length SrCPS2 and SrKSL with other plant TPSs. Abbreviations and accession numbers of proteins are listed in Supplemental Table S2. B, Relative expression levels of Stevia diTPS (SrCPS, SrCPS2, SrKS1, and SrKSL) and P450 (SrKO1 and SrKAH) genes in leaf-trichomes (L-T) and trichomes (T). Transcript levels of genes expressed in L-T and T were measured by qRT-PCR. Amplification of Actin mRNA was used for normalization. C, Subcellular localization of Stevia diTPSs. YFP-fused SrCPS, SrCPS2, and SrKSL (SrCPS-YFP, SrCPS2-YFP, and SrKSL-YFP) were transiently expressed in N. benthamiana leaves by A. tumefaciens-mediated infiltration and visualized 3 d postinfiltration using the YFP channel of a confocal microscope. Auto, Chlorophyll autofluorescence; YFP, YFP channel image; Light, light microscope image; Merged, merged image between Auto and YFP. Bars = 10 µm (SrCPS-YFP) and 20 µm (SrCPS2-YFP and SrKSL-YFP). SrCPS2 contains 777 amino acids, which is 10 amino acids shorter than SrCPS, and shares 56.1% amino acid identity with the latter (Supplemental Fig. S10). Like SrCPS, SrCPS2 possesses a DxDD motif for protonation-initiated cyclization of GGPP; this motif is a characteristic of class II TPSs (Supplemental Fig. S10). SrCPS transcripts were approximately 5 times more abundant in L-T than in T. By contrast, SrCPS2 was only detectable in T, suggesting that it may function differently in Stevia (Fig. 6B). SrKSL encoded a 782 amino acid-long protein that shares 61.2% identity with SrKS1-1 (hereafter referred to as SrKS1). It contains the conserved DDxxD and NSE/DTE motifs of class I TPS for metal-dependent ionization of the prenyl diphosphate substrate (Supplemental Fig. S11). Similar to the relation between SrCPS and SrCPS2, the expression patterns of SrKS1 and KSL were clearly distinct from each other. Figure 6B shows that SrKS1 was predominantly expressed more than 5 times in L-T than T, but SrKSL expression level was more than 8 times higher in T compared with L-T. These results suggest that SrCPS and SrKS1 are involved in SG biosynthesis in L-T as reported (Richman et al., 1999), whereas SrCPS2 and/or SrKSL are likely to function in the biosynthesis of other diterpenoids in trichomes. Plant diTPSs, CPS, and KS have a transit peptide sequence at their N termini for plastidic targeting (Toyomasu and Sassa, 2010). Similarly, a putative N-terminal plastidic transit peptide sequence of 52 amino acids for SrCPS2 and 62 amino acids for SrKSL was also predicted (Supplemental Figs. S10 and S11). In confirmation of this prediction, both SrCPS2-YFP and SrKSL-YFP were found to be localized in chloroplasts of N. benthamiana leaves (Fig. 6C). Functional Characterization of SrCPS2 and SrKSL The different spatial expression patterns between SrCPS and SrCPS2 suggest that SrCPS2 plays little role in the synthesis of copalyl diphosphate (CPP) from GGPP. To determine the catalytic activity of this diTPS in vitro, N-terminally truncated recombinant proteins that lack the putative transit peptide was expressed in Escherichia coli BL21(DE3). 6His-tagged purified recombinant proteins were used for in vitro assays using GGPP as the common substrate for diTPS activity. Reaction products were then dephosphorylated by alkaline phosphatase for analysis by GC-MS. Using SrCPS as a control, a single peak for copal-15-ol, which is a dephosphorylated form of CPP (Supplemental Fig. S12A; Richman et al., 1999), was detected in the reaction mix. Moreover, SrCPS produced ent-kaurene in a sequential reaction with SrKS1 (Supplemental Fig. S12, B and C; Richman et al., 1999). However, assay with SrCPS2 produced labda-13-en-8,15-diol (labdenediol) as a major component instead, suggesting that SrCPS2 is a labda-13-en-8-ol (or copal-8-ol) diphosphate synthase (LPPS) that catalyzes the formation of labda-13-en-8-ol diphosphate (LPP) from GGPP (Fig. 7A). We also detected additional minor components, a racemic mixture of manoyl oxide and epi-manoyl oxide from the SrCPS2 product profile, but these were previously suggested to be the byproducts of a nonenzymatic reaction (Caniard et al., 2012; Zerbe et al., 2013; Pateraki et al., 2014). SrCPS2-catalyzed reaction products were identified by comparison to authentic standards of labdane-type diterpenes based on both retention time and mass spectra (Fig. 7, A and B; Falara et al., 2010). Moreover, mass spectra of the three compounds were nearly identical to the published fragmentation patterns of labdenediol, manoyl oxide, and epi-manoyl oxide (Falara et al., 2010; Caniard et al., 2012; Zerbe et al., 2013; Pateraki et al., 2014). Figure 7. Open in new tabDownload slide In vitro characterization of diTPSs from Stevia. A, GC trace of reaction product from in vitro assay of recombinant SrCPS2 with GGPP as a substrate. Reaction product was dephosphorylated by alkaline phosphatase (AP) for GC-MS analysis. Peaks were compared with authentic standards. B, Mass spectra of peaks a, b, and c obtained from authentic standards and reaction product in A. C to E, GC traces of reaction products from coupled in vitro assays of recombinant SrCPS2 plus SrKSL (C), SrCPS2 plus SrKS1 (D), or SrCPS plus SrKSL (E) with GGPP as substrate. Peak a, Labdenediol; peak b, manoyl oxide; peak c, epi-manoyl oxide; peak d, unidentified compound; peak e, ent-copalol; peak f, ent-kaurene; m/z, mass-to-charge ratio; TIC, total ion chromatogram; EIC, extracted-ion chromatogram. Figure 7. Open in new tabDownload slide In vitro characterization of diTPSs from Stevia. A, GC trace of reaction product from in vitro assay of recombinant SrCPS2 with GGPP as a substrate. Reaction product was dephosphorylated by alkaline phosphatase (AP) for GC-MS analysis. Peaks were compared with authentic standards. B, Mass spectra of peaks a, b, and c obtained from authentic standards and reaction product in A. C to E, GC traces of reaction products from coupled in vitro assays of recombinant SrCPS2 plus SrKSL (C), SrCPS2 plus SrKS1 (D), or SrCPS plus SrKSL (E) with GGPP as substrate. Peak a, Labdenediol; peak b, manoyl oxide; peak c, epi-manoyl oxide; peak d, unidentified compound; peak e, ent-copalol; peak f, ent-kaurene; m/z, mass-to-charge ratio; TIC, total ion chromatogram; EIC, extracted-ion chromatogram. With the newly identified SrKSL being highly expressed in trichomes, it seemed possible that SrKSL functions downstream of SrCPS2 to cyclize LPP. Therefore, we carried out further in vitro enzyme assays containing both SrCPS2 and SrKSL as enzyme sources and GGPP as substrate. Figure 7C and Supplemental Figure S13A show that the coupled enzyme assay with SrCPS2 and SrKSL produced both manoyl oxide and epi-manoyl oxide, but labdenediol was no longer detected. Similarly, transient expression of SrCPS2 and SrKSL in N. benthamiana using A. tumefaciens infiltration also yielded manoyl oxide and epi-manoyl oxide in the infiltrated leaves (Fig. 8, A and B). These results suggest that SrKSL catalyzes the conversion of LPP to manoyl oxide and epi-manoyl oxide. On the other hand, the expression of SrCPS and SrKS1 in N. benthamiana produced ent-kaurene in the infiltrated leaves, as expected (Fig. 8, C and D). Figure 8. Open in new tabDownload slide In vivo characterization of diTPSs from Stevia. SrCPS, SrCPS2, SrKS1, and SrKSL were transiently expressed in N. benthamiana leaves by A. tumefaciens-mediated infiltration. The compounds were analyzed 3 d postinfiltration by GC-MS. The resulting peaks were identified through comparison with reference mass spectra of the NIST 2014 library. A and C, GC traces of hexane extracts from N. benthamiana leaves expressing SrCPS2, SrCPS2 plus SrKSL, and SrCPS2 plus SrKS1 (A) or SrCPS, SrCPS plus SrKS1, and SrCPS plus SrKSL (C). N. benthamiana leaves expressing p19 were used as a control. B and D, Mass spectra obtained from peak a, b, c, and d and reference spectra from the NIST 2014 library. Peak a, Manoyl oxide; peak b, epi-manoyl oxide; peak c, ent-kaurene; peak d, unidentified compound; m/z, mass-to-charge ratio; EIC, extracted-ion chromatogram. Figure 8. Open in new tabDownload slide In vivo characterization of diTPSs from Stevia. SrCPS, SrCPS2, SrKS1, and SrKSL were transiently expressed in N. benthamiana leaves by A. tumefaciens-mediated infiltration. The compounds were analyzed 3 d postinfiltration by GC-MS. The resulting peaks were identified through comparison with reference mass spectra of the NIST 2014 library. A and C, GC traces of hexane extracts from N. benthamiana leaves expressing SrCPS2, SrCPS2 plus SrKSL, and SrCPS2 plus SrKS1 (A) or SrCPS, SrCPS plus SrKS1, and SrCPS plus SrKSL (C). N. benthamiana leaves expressing p19 were used as a control. B and D, Mass spectra obtained from peak a, b, c, and d and reference spectra from the NIST 2014 library. Peak a, Manoyl oxide; peak b, epi-manoyl oxide; peak c, ent-kaurene; peak d, unidentified compound; m/z, mass-to-charge ratio; EIC, extracted-ion chromatogram. However, in our in vitro enzyme assay combining SrCPS2 and SrKS1, the peaks for manoyl oxide and epi-manoyl oxide were also detected (Fig. 7D; Supplemental Fig. S13B). This result was unexpected, because SrKS1 has been identified as a typical kaurene synthase (Richman et al., 1999). Furthermore, in vivo transient assays in N. benthamiana where SrCPS2 was infiltrated alone or in combination with SrKS1 also resulted in the detection of these two peaks (Fig. 8A). These results suggest that SrKS1 and other endogenous diTPSs of N. benthamiana may carry out this catalytic reaction as well. Nevertheless, because SrCPS2 and SrKSL have similar expression patterns in Stevia leaves, it is perhaps likely that they catalyze consecutive steps in the conversion of GGPP to manoyl oxide and epi-manoyl oxide specifically in trichomes. Then, we also tested the SrCPS and SrKSL combination. Interestingly, a unique peak similar to (-)-copalol (or ent-copalol) according to the National Institute of Standards and Technology (NIST) library was detected in this combination (Fig. 7E). This compound was also observed by coexpression of SrCPS and SrKSL in N. benthamiana (Fig. 8C), confirming a specialized function of these two enzymes. However, it was neither ent-copalol nor ent-kaurene, because its retention time did not correspond to the peaks for ent-copalol obtained from in vitro assay with SrCPS hydrolyzed by alkaline phosphatase or for ent-kaurene standard (Fig. 7E). Thus, the identification of this unique compound remains to be further investigated. Note that we only detected this peak when full-length SrKSL including the putative transit peptide was used in the reaction mix. DISCUSSION Differential Distribution of Diterpenoids in Different Cell Types of Stevia Leaves Stevia accumulates SGs largely in the leaves rather than other tissues such as stems and roots (Brandle and Rosa, 1992; Kumar et al., 2012). Here, we found that most SGs are detected in L-T, where genes encoding CPS, KS, and KO related to steviol biosynthesis are expressed (Figs. 2A and 6B; Humphrey et al., 2006). This implies that SGs are stored in the place of their synthesis and are not transported to other tissues or cells. On the other hand, Stevia leaf trichomes stored a variety of monoterpenes, sesquiterpenes, and other labdane-type diterpenes (Fig. 2B; Table I). In general, compounds that can be detected by GC-MS are relatively volatile, indicating that volatile organic compounds (VOCs) of Stevia mostly accumulate in the trichomes. Our result is consistent with other reports that VOCs such as terpenoids or phenylpropanoids are stored in glandular trichomes with a storage cavity, e.g., peltate trichomes of mint and capitate trichomes of basil (Ocimum basilicum; Turner et al., 2000; Gang et al., 2002). Tobacco glandular trichomes are capable of synthesizing diterpenoids such as cis-abienol and duvatrienediol, and the latter compound could be found in Stevia trichomes as well (Fig. 2B; Table I; Keene and Wagner, 1985; Kandra and Wagner, 1988; Guo et al., 1994). These observations suggest that most VOCs detected by GC-MS might be accumulated in glandular trichomes rather than the other two nonglandular trichome types. Our results demonstrated that there is differential accumulation of diterpenoids in different cell types of the Stevia leaf tissues through a biochemical specialization. RNA-seq Data of Stevia Leaf Tissues Distinguished Gene Members Involved in SG or Other Labdane-Type Diterpenoid Biosynthesis The diterpene steviol, the aglycone of SGs, is known to be synthesized through the MEP pathway from two isoprene units, IPP and DMAPP (Totté et al., 2000, 2003; Supplemental Fig. S4). In addition to eight genes identified previously (Totté et al., 2003; Kumar et al., 2012), our RNA-seq found additionally six genes (SrDXS1, SrDXS2, SrDXS3, SrDXR2, SrHDR2, and SrIDI2) encoding enzymes involved in the MEP pathway and IDI. Among four SrDXS genes, only SrDXS1 transcripts expressed abundantly in the L-T sample (Fig. 4B). It implies that SrDXS1 rather than SrDXS4, which was previously implicated in SG biosynthesis (Totté et al., 2003), may be involved in SG biosynthesis in the L-T sample. On the other hand, two genes, SrDXS2 and SrDXS4, encoding proteins of the DXS2 clade were predominantly expressed in T (Fig. 4B). These results suggest that SrDXS2 and SrDXS4 might be related to the biosynthesis of other volatile terpenoids including labdane-type diterpenoids in trichome cells. As mediators of the MEP pathway and an isomerase of IPP or DMAPP, genes encoding DXR, MCT, CMK, MDS, HDS, HDR, and IDI of Stevia have been previously reported (Totté et al., 2003; Kumar et al., 2012; Supplemental Fig. S4). Most genes except DXR, HDR, and IDI have been found in our RNA-seq data set as a single copy, and the difference of their expression levels was less than 2- to 4-fold in both L-T and T samples (Fig. 4C; Supplemental Fig. S4), implying these genes function both in primary metabolism and terpenoid biosynthesis. SrDXR1, SrHDR1, and SrIDI1 have been suggested to be involved in SG biosynthesis (Kumar et al., 2012). However, our RNA-seq and qRT-PCR analyses showed that SrHDR1 and SrIDI1 transcripts were more abundant in the T sample, suggesting the important role in the biosynthesis of other volatile terpenoids rather than SGs. Based on the expression levels shown in Figure 4C, it is more likely that SrDXR1, SrHDR2, and SrIDI2 are associated with SG production. In plant genomes, GGPPS is usually encoded by a GGPPS gene family comprising of two to 12 members (Beck et al., 2013). Among the three SrGGPPS genes identified here, SrGGPPS1 has been previously implicated in SG biosynthesis (Kumar et al., 2012). However, SrGGPPS1 was in fact predominantly expressed in trichomes. Moreover, it was almost undetectable in the L-T sample where SGs are synthesized (Fig. 5B), which suggests that it is likely to function in the biosynthesis of other diterpenoids rather than SGs. Both SrGGPPS2 and SrGGPPS3 identified here showed similar expression patterns expressing more abundantly in the L-T sample, but SrGGPPS2 was located in cytosol (Fig. 5, B and C). These results propose that the newly identified SrGGPPS3 is solely involved in SG biosynthesis. It has been reported that SrCPS and SrKS1 are highly expressed in mature leaf tissue rather than stems and roots (Richman et al., 1999; Kumar et al., 2012). We confirmed that both transcripts were more than 5 times abundant in L-T where SGs are stored than in T (Fig. 6B). However, their homologs, SrCPS2 and SrKSL, showed opposite expression patterns in L-T and T (Fig. 6B). For this reason, we speculated that they may function differently for the biosynthesis of other diterpenes in trichomes. Phylogenetic analysis further supported a possible functional difference, as it placed SrCPS2 on the same branch as GrTPS1 (Fig. 6A), which was identified as an LPPS from Grindelia robusta (Zerbe et al., 2013). Although SrKSL is closest to SrKS1, it also falls into same subgroup containing GrTPS6 (Fig. 6A), which was characterized as a manoyl oxide synthase working in conjunction with GrTPS1 (Zerbe et al., 2013). Our in vitro and in vivo enzymatic assays demonstrated this speculation, confirming that SrCPS2 is an LPPS (Fig. 7A). Genes encoding LPPS that catalyze the conversion of GGPP to LPP have also been reported in a few other plants. An SsLPPS sequence was detected by deep 454 sequencing of cDNAs from clary sage (Salvia sclarea) calices, where sclareol, a labdane-type diterpene alcohol, is mainly accumulated (Caniard et al., 2012). On the other hand, CfTPS1 was highly expressed in the root cork of C. forskohlii, where another labdane-type diterpenoid forskolin is synthesized (Pateraki et al., 2014). In particular, Cistus creticus Copal-8-ol diphosphate synthase (CcCLS) from C. creticus was highly expressed in leaf trichomes, as was the case with SrCPS2 shown here (Falara et al., 2010). Although the G. robusta GrTPS1 is placed closest to SrCPS2 on the phylogenetic tree, its expression pattern has not yet been investigated (Fig. 6A; Zerbe et al., 2013). Our in vitro and in planta coupled enzyme assays with SrCPS2 and SrKSL newly identified SrKSL as a manoyl oxide synthase. Interestingly, SrKS1 was also capable of producing two manoyl oxide stereoisomers in combination with SrCPS2 in vitro as well as in planta (Figs. 7D and 8A). We postulate that the conversion of LPP to these two manoyl oxide stereoisomers may not require a highly specific enzyme or SrKS1 might have a dual function to use both CPP and LPP as substrates. The latter hypothesis is made more likely by the recent finding that SmKSL2 from Salvia miltiorrhiza reacted with both CPP and LPP to catalyze the formation of ent-kaurene and epi-manoyl oxide in a sequential reaction with SmCPS5 and SmCPS4, respectively (Cui et al., 2015). Another coupled enzyme assay with SrCPS and SrKSL produced an unidentified compound in vitro as well as in planta, which was identified as ent-copalol by the NIST 2014 library (Figs. 7E and 8C). GC-MS has proven to be effective in resolving stereoisomers of many terpenoid compounds (Armstrong et al., 1996; Huang and Armstrong, 2009). Because the peak observed in assays combining SrCPS and SrKSL has a different retention time with a similar mass spectrum as ent-copalol, we postulate that the unidentified compound could be a stereoisomer of ent-copalol. Distinct Functions of diTPSs in Stevia Leaves The extensive differences in the function of Stevia diTPSs were also clearly reflected in the differences in metabolites derived from L-T and T samples (Figs. 2 and 9). We were able to detect a range of SGs in the L-T samples. This result is in agreement with our transcriptome and qRT-PCR data showing that SrCPS and SrKS1, which are involved in SG biosynthesis, are highly expressed in the L-T sample (Figs. 6B and 9). It also suggests that SGs are mostly stored in the same tissue where they are produced. Instead, Stevia trichomes produced a variety of terpenoids rather than SGs, most of which were volatiles. Manoyl oxide was detected among trichome metabolites (Figs. 2B and 9; Table I), correlating with the high transcript levels of SrCPS2 and SrKSL in trichomes. This further suggests that enzymes encoded by these two genes are relevant in the production of manoyl oxide (Figs. 6B, 7C, 8A, and 9). It is also possible that SrKS1 may have a redundant role of SrKSL for manoyl oxide biosynthesis in trichomes, because it catalyzed the formation of manoyl oxide and epi-manoyl oxide in a sequential reaction with SrCPS2 and its transcripts could be detected in the T sample as well (Figs. 6B, 7D, 8A, and 9). In C. forskohlii, manoyl oxide can be found in oil bodies of the root cork cells and is known to be a precursor to forskolin (Pateraki et al., 2014). We suggest that manoyl oxide in Stevia may act as a precursor to other as yet unidentified diterpenoids, because forskolin is not found in Stevia trichomes (Fig. 9). Figure 9. Open in new tabDownload slide The biosynthetic routes of SGs and other labdane-type diterpenoids from GGPP in different parts of Stevia leaves. For these distinct pathways, SrGGPPS1 and SrGGPPS3 may function separately in trichomes and leaf mesophyll cells, respectively. UGTs, UDP-glycosyltransferases. Figure 9. Open in new tabDownload slide The biosynthetic routes of SGs and other labdane-type diterpenoids from GGPP in different parts of Stevia leaves. For these distinct pathways, SrGGPPS1 and SrGGPPS3 may function separately in trichomes and leaf mesophyll cells, respectively. UGTs, UDP-glycosyltransferases. Many diterpenoids are further modified by varying degrees of oxidation. These modifications give rise to a diversity of structural classes, which provide further functional groups for additional modifications, such as glycosylation, acetylation, and methylation. In plants, the cytochrome P450s are mainly responsible for the oxidation of terpenoids (Boutanaev et al., 2015). In Stevia, kaurene is oxidized and hydroxylated to form steviol by KO and KAH, respectively (Humphrey et al., 2006). The latter enzyme performs the first committed step in SG biosynthesis, providing an aglycone, steviol, which is sequentially glycosylated by a series of UDP-glycosyltransferases in Stevia leaves. We found that both P450 genes, SrKO1 and SrKAH, were abundantly expressed in the L-T sample compared with the T sample (Fig. 6B). The results of transcript analysis correlated with those of chemical analysis. However, we did not find a new Stevia KAH homolog in our RNA-seq data set, suggesting a nonredundant role of SrKAH in SG biosynthesis. Recently, functional diterpenoids with biological activities for treatment of a wide range of medical conditions have been reported from diverse medicinal plants (Guo et al., 2013; Zerbe et al., 2013; King et al., 2014; Pateraki et al., 2014). Although manoyl oxide itself is known to be an anticancer compound, it is considered as the molecular core for a large series of bioactive derivatives (Pateraki et al., 2014). In addition to manoyl oxide, we found several labdane-type and oxidized bicyclic diterpenoids such as oxomanoyl oxide and labda-8(20),13-dien-15-oic acid (agatholic acid) from Stevia trichomes (Fig. 2; Table I); the functions of all these terpenoids are unknown. Querying the transcriptome of Stevia trichomes, we found more than 70 candidate P450s that were preferentially expressed in the T samples with a minimum of 4-fold increase in expression level compared with the L-T sample. Future work should be directed toward the identification of cytochrome P450s involved in the enzymatic conversion of manoyl oxide to bioactive labdane diterpenoids in the Stevia trichomes (Fig. 9). MATERIALS AND METHODS Plant Materials Stevia (Stevia rebaudiana) ‘Bertoni’ seeds were germinated on soil and grown in a greenhouse under natural light condition in Singapore. Four-week-old Nicotiana benthamiana plants grown in a greenhouse were used for subcellular localization and in vivo characterization of Stevia genes. Trichome Isolation Trichomes were isolated using a glass bead abrasion method (Lange et al., 2000; Jin et al., 2014). One-month-old Stevia plants grown in a greenhouse were used. Young leaves (20–30 g; 1 to 2 cm in length) were placed in a 50-mL test tube containing ice-cold imbibition buffer (1 mm aurintricarboxylic acid, 5 mm thiourea, and 2 mm dithiothreitol [DTT], pH 6.6) and soaked for 1 h. After removing the imbibition buffer, glass beads (425–600 µm, Sigma-Aldrich) were added to the tube with an extraction buffer (25 mm 3-[N-Morpholino]-2-hydroxypropanesulfonic acid, pH 6.6, 200 mm d-sorbitol, 10 mm Suc, 0.5 mm sodium phosphate, 1% [w/v] polyvinylpyrrolidone-40, 0.6% [w/v] methyl cellulose, 1 mm aurintricarboxylic acid, 5 mm thiourea, and 2 mm DTT), and the content was vigorously vortexed for 1 min. The mixture was first passed through a cell strainer (mesh size, 100 μm) to remove debris, and trichomes were collected using a 20-μm strainer. The collected trichomes were washed several times with the same buffer before being frozen in liquid nitrogen for further use. To obtain leaf tissues from which trichomes were removed (leaf-trichomes), a cold-brushing method was used to completely remove trichomes (Wang et al., 2001). The quality of trichomes or leaf-trichomes was monitored under a dissecting microscope. RNA Isolation for RNA Sequencing Total RNA was extracted from the isolated trichomes and leaf-trichomes using the Spectrum Plant Total RNA Kit (Sigma-Aldrich), which included ribonuclease-free DNaseI treatment to remove genomic DNA. The RNA quantity was determined with Nanodrop spectrophotometer ND-1000 (Thermo Fisher Scientific). The RNA integrity number (RIN) was measured to evaluate the RNA quality using Agilent 2100 bioanalyzer and RNA 6000 Nano Labchip Kit (Agilent Technologies). RNA samples with a RNA integrity number value of 7 < x < 10 were processed for RNA-seq by the Rockefeller University Genomics Resource Center using a HiSeq 2000 (Illumina). RNA-seq de Novo Assembly The RNA libraries prepared using the TruSeq RNA Sample Preparation Kits v2 set A (RS-122-2001, Illumina) were qualified by the Agilent 2200 TapeStation system (Agilent). The qualified libraries were run on single lanes for 100 cycles (strand-specific single-end) on HiSeq 2000 (Illumina). Raw reads were analyzed by FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) for the quality control. The Trinity method was used for de novo assembly of short sequence reads due to the absence of a reference Stevia genome (Grabherr et al., 2011). The assembled unigenes were annotated based on sequence similarities to sequences in the NCBI nr database and also the protein sequence databases from Arabidopsis (Arabidopsis thaliana), Vitis vinifera, Glycine max, and Oryza sativa. qRT-PCR qRT-PCR was performed to investigate gene expression pattern in different Stevia tissues. One microgram of total RNA from each sample was used for cDNA synthesis with M-MLV Superscript II (Promega). cDNA samples were quantified by Applied Biosystems 7900HT Fast Real-Time PCR system and Applied Biosystems Power SYBR Green PCR Master Mix (Life Technologies). Fold change values of target gene transcripts were subsequently normalized by dividing the delta threshold cycle values by the delta threshold cycle values of control gene transcripts. Gene-specific primers for qRT-PCR were designed using the Primer3 program (http://bioinfo.ut.ee/primer3-0.4.0/) and are listed in Supplemental Table S1. Each PCR product obtained from regular PCR was verified by sequencing after cloning into pGEM-T Easy vector (Promega). A melting curve analysis in Applied Biosystems 7900HT Fast Real-Time PCR system was performed to assess the specificity of the amplified PCR product. All reactions were carried out in technical triplicates and biological replicates. A mock reaction containing all the reverse transcription-PCR reagents, except the reverse transcriptase, was used as a negative control. Stevia Actin gene was used as an internal control for normalization. Isolation of Full-Length ORF of Stevia Genes and Vector Construction for Agrobacterium tumefaciens-Mediated Gene Expression Full-length ORFs of four SrDXS genes (SrDXS1–SrDXS4), three SrGPPS genes (SrGPPS1–SrGPPS3), SrCPS, and SrKS1 were amplified by PCR from Stevia leaf-derived cDNA. Primer sets listed in Supplemental Table S1 were used to amplify gene products that were flanked by attB sites for Gateway cloning (Invitrogen). Purified PCR products were cloned into pDONR221 (Invitrogen) and verified by sequencing. Partial sequences for SrCPS2 and SrKSL were obtained from Stevia RNA-seq data. Using these partial sequences, we designed RACE primers (Supplemental Table S1) and performed a 5′ RACE experiment using SMARTer RACE cDNA Amplification Kit (Clontech). The resulting sequences of SrCPS2 and SrKSL obtained by 5′ RACE were confirmed by sequencing after cloning into pDONR221. For yellow fluorescent protein (YFP)-fusion construct, the pDONR221 clone harboring each gene was integrated into the destination vector, pBA-DC-YFP expression vector, which contained a Cauliflower mosaic virus 35S promoter and a C terminus in frame YFP by LR Clonase (Invitrogen). The final constructs were transformed into A. tumefaciens GV3101 by electroporation (Bio-Rad). Sequence Identification, Multiple Sequence Alignments, and Phylogenetic Analysis DNA sequences were edited and assembled using DNASTAR Lasergene 8. Sequence alignments were conducted using Biology WorkBench 3.2 (http://seqtool.sdsc.edu/CGI/BW.cgi). Phylogenetic analysis was performed using the maximum likelihood method in the Molecular Evolutionary Genetics Analysis (MEGA) version 6 program (Tamura et al., 2013). Abbreviations and GenBank accession numbers of proteins in phylograms are listed in Supplemental Table S2. Subcellular Localization To determine the subcellular localization of C-terminal in-frame YFP-tagged proteins, three SrGGPPS genes (SrGGPPS1, SrGGPPS2, and SrGGPPS3), SrCPS, SrCPS2, SrKS1, and SrKSL were transiently expressed in leaves of 4-week-old N. benthamiana plant as described in Jin et al. (2014). Infiltrated plants were incubated in a growth chamber at 24°C under long-day (16-h-light/8-h-dark) conditions. After 3 d, the leaves were excised, mounted onto slides, and imaged by confocal laser scanning microscopy (Carl Zeiss LSM5 Exciter). The 488- and 514-nm lines of an argon laser were used to excite GFP and YFP, respectively. Band pass was set to 500 to 550 nm, and long pass was set to 560 nm. Images were recorded and processed using the Zeiss LSM Image Browser. In Vitro Functional Characterization of Stevia diTPSs To determine the function of Stevia diTPSs, we performed single or coupled enzyme assays with recombinant proteins. PCR-amplified SrCPS, SrCPS2, SrKS1, and SrKSL cDNAs were inserted into pET28b plasmid (Novagen) to construct the vectors for the production of recombinant N-terminal poly-His-tagged proteins. The final constructs were transformed into Escherichia coli BL21(DE3)pLysS (Invitrogen). E. coli culture was treated with 0.2 mm isopropyl β-d-1-thiogalactopyranoside at 16°C for 12 h to induce His-tagged protein expression, and induced cells were then harvested by centrifugation. After adding binding buffer (20 mm HEPES, pH 7.5, 0.5 m NaCl, 25 mm Imidazole, and 5% [v/v] glycerol), one protease inhibitor cocktail tablet per 50 mL (Roche), and 0.1 mg L–1 lysozyme to the cell pellet, cells were subsequently lysed by sonication. The cell lysate was centrifuged for 30 min at 14,000g, and the supernatant was used for purification of the recombinant proteins using nickel-nitrilotriacetic acid agarose affinity chromatography. Proteins bound to nickel-nitrilotriacetic acid agarose (Qiagen) were eluted from the column with elution buffer (50 mm HEPES, pH 7.5, 0.5 m NaCl, and 250 mm Imidazole) and dialyzed against a dialysis buffer (50 mm HEPES, pH 7.5, 0.1 m KCl, 1 mm DTT, and 5% [v/v] glycerol). In vitro enzyme assay for CPS activity was performed in a final volume of 500 µL of reaction buffer (50 mm HEPES pH 7.5, 100 mm KCl, 7.5 mm MgCl2, 5 mm DTT, 5% [v/v] glycerol, and 20 µm GGPP) with about 5 µg of the purified protein. The reaction mixture was incubated at 30°C for 2 h. After incubation, 10 units of FastAP Thermosensitive Alkaline Phosphatase (Thermo Scientific) was added to hydrolyze CPP. The reaction mixture was overlaid with 500 µL of hexane to trap hexane-soluble copalol and then incubated at 37°C for 4 h. The dephosphorylated compounds were then extracted two times with 500 µL of hexane. The hexane fractions were completely dried under a stream of N2 gas at room temperature and redissolved in 100 µL of hexane for GC-MS analysis. Coupled enzyme assay was performed using two recombinant proteins to determine if they catalyze consecutive steps in a catalytic pathway. The enzymatic reaction and compound extraction were as described above except for the alkaline phosphatase treatment. Enzyme reaction with SrCPS and SrKS1 were used as reference (Richman et al., 1999). In vitro enzyme assays were done at least in biological triplicates. In Vivo Functional Characterization of Stevia diTPSs In vivo characterization of diTPSs was carried out by A. tumefaciens-mediated transient gene expression in N. benthamiana leaves. Overnight cultures of A. tumefaciens strains harboring SrCPS, SrCPS2, SrKS1, and SrKSL constructs as described above were used alone or in combinations. Harvested cultures were suspended in a solution containing 10 mm MgCl2, 10 mm MES, pH 5.6, and 100 μm acetosyringone. After 1-h incubation at room temperature, the A. tumefaciens mixture was infiltrated into the underside of N. benthamiana leaves using a needleless syringe. The infiltrated plants were incubated in the growth chamber at 24°C for 3 d. After incubation, four to five infiltrated leaves were frozen in liquid nitrogen and homogenized with a prechilled mortar and pestle. About 500 mg of leaf powder was dissolved in 500 µL of ethyl acetate containing 1 µL (10 mg mL–1) of camphor (Sigma) as an internal standard and incubated on a horizontal shaker at 200 rpm for 2 h. After centrifugation of the slush at 13,000g for 10 min, the ethyl acetate upper layer was transferred into a new Eppendorf tube and mixed with 300 mg of anhydrous Na2SO4 to remove water. Following an additional centrifugation for 1 min to separate the Na2SO4, the extract was transferred into a 2-mL amber glass GC sample vial for GC-MS analysis. In vivo characterization of diTPSs was done at least in biological triplicates. GC-MS Analysis GC-MS analysis was performed on an Agilent 7890A GC (Agilent Technologies) system and an Agilent Technologies 5975C Inert XL Mass Selective Detector equipped with a HP-5MS UI column (30 m × 0.25 mm × 0.25 µm). Helium was used as the carrier gas at a flow rate of 1 mL min–1. Samples (5 µL) were injected onto the column at 250°C in the splitless mode, and a temperature gradient of 8°C min–1 from 50°C (1-min hold) to 300°C (5-min hold) was applied. Data were processed by an MSD ChemStation Data Analysis (Agilent Technologies). The chemical components of Stevia trichomes were identified by comparison of their mass spectra with those in NIST 2014 library data of GC-MS and with literature data (Adams, 2007) along with the retention indices associated with a series of n-alkanes (C7-C30) mix standard (Sigma-Aldrich, 49451-U). Camphor (10 mg mL–1) was used as an internal standard. The amount of each compound was calculated by measuring its peak area related to that of a known amount of camphor. The chemical analysis of Stevia trichomes was done at least in biological triplicates. The identified components along with their retention indices and relative percentage values are listed in Table I. Compound identification by in vitro and in planta assays was done by comparison to authentic standards, reference spectra from literature, and databases (NIST 2014 library). HPLC Analysis of SGs from Stevia Leaves Fresh Stevia leaves, leaf-trichomes, or isolated trichomes were frozen in liquid nitrogen and ground with a prechilled mortar and pestle. About 100 mg of the samples was extracted in 1 mL of water at 70°C for 3 h. The extract was centrifuged at 1,500g for 15 min, and the supernatant was filtered through a 0.45-μm filter. The filtrate was further extracted using a Finisterre SPE Column C2 (Teknokroma), which was pretreated with methanol followed by water. Two milliliters of the filtrate was loaded on the cartridge and allowed to flow through. The cartridge was washed with water followed by acetonitrile:water (20:80, v/v) and air dried for 3 min. Samples were then eluted in 1 mL of methanol:acetonitrile (50:50, v/v) and filtered using a 0.45-µm nylon centrifuge tube (Corning). HPLC analysis of the samples was carried out on a Shidmadzu Nexera X2 ultra high performance liquid chromatography system using a Shim-pack VP-ODS column (250 mm × 4.6 mm; i.d., 5 μm; and particle size, 4.6 µm) and detected by a photodiode array detector (SPD-M30A with high-sensitivity cell). Five microliters of sample was injected, and the elution was performed over 24 min with a 30% to 80% acetonitrile gradient at a flow rate of 1.0 mL min–1 according to protocol by Shimadzu. Column oven was maintained at 40°C. Peak assignment for the absorbance spectrum was based on comparison with elution profile of known standards (complete Stevia standards kit, KIT–00019565–005, ChromaDex) at a wavelength of 210 nm. HPLC analysis of Stevia samples was done at least in biological triplicates. The RNA-seq data supporting the result of this article is available in the DNA Data Bank of Japan (http://www.ddbj.nig.ac.jp/) with accession number DRA003752. Nucleotide sequences from this study were submitted to the NCBI/GenBank using BankIt with accession numbers SrDXS1 (KT276229), SrDXS2 (KT276230), SrDXS3 (KT276231), SrDXS4 (KT276232), SrDXR2 (KT276233), SrHDR2 (KT276234), SrIDI1 (KT276235), SrIDI2 (KT276236), SrGGPPS2 (KT276237), SrGGPPS3 (KT276238), SrCPS2 (KT276239), and SrKSL (KT276240). Supplemental Data The following supplemental materials are available. Supplemental Figure S1. Stevia tissues for RNA-seq. Supplemental Figure S2. Quality of strand-specific RNA-seq derived from two Stevia tissues. Supplemental Figure S3. Heat map of transcript expression in leaf-trichomes and trichomes. Supplemental Figure S4. Biosynthesis of SGs via the MEP pathway. Supplemental Figure S5. Comparison of deduced amino acid sequences of the four-SrDXS small gene family. Supplemental Figure S6. Comparison of deduced amino acid sequences of SrDXRs. Supplemental Figure S7. Comparison of deduced amino acid sequences of SrHDRs. Supplemental Figure S8. Comparison of deduced amino acid sequences of SrIDIs. Supplemental Figure S9. Comparison of deduced amino acid sequences of SrGGPPSs. Supplemental Figure S10. Comparison of deduced amino acid sequences of SrCPS and SrCPS2. Supplemental Figure S11. Comparison of deduced amino acid sequences of SrKS1-1 and SrKSL. Supplemental Figure S12. GC-MS analysis of in vitro assay with diTPSs from Stevia. Supplemental Figure S13. Mass spectra obtained from coupled in vitro assays of recombinant SrCPS2 and SrKSL or SrCPS2 and SrKS1. Supplemental Table S1. Oligonucleotide primers used in this study. Supplemental Table S2. GenBank accession numbers of proteins used in phylograms. Supplemental File S1. Differential gene expression between leaf-trichomes and trichomes. ACKNOWLEDGMENTS We thank Dr. Angelos K. Kanellis (Aristotle University of Thessaloniki) for the purified chromatographic fraction from yeast (Saccharomyces cerevisiae) culture transformed with CcCLS used as a standard, Dr. Hiroshi Kawaide (Tokyo University of Agriculture and Technology) for ent-kaurene, Dr. Rajani Sarojam for help in trichomes isolation, Lay-Peng Tan (Agilent Technologies Singapore) for technical assistance in GC-MS analysis, and the Temasek Life Sciences Laboratory central facilities for support on SEM and confocal microscopy. 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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: In-Cheol Jang (jangi@tll.org.sg). M.J.K. and J.Z. performed the molecular and biochemical experiments; J.J. and L.W. performed the RNA-seq and transcriptome analyses; N.-H.C. coordinated the RNA-seq and transcriptome analyses and revised the article; I.-C.J. conceived the research, designed the experiments, and wrote the article. www.plantphysiol.org/cgi/doi/10.1104/pp.15.01353 © 2015 American Society of Plant Biologists. All Rights Reserved. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - Comparative Transcriptomics Unravel Biochemical Specialization of Leaf Tissues of Stevia for Diterpenoid Production
JF - Plant Physiology
DO - 10.1104/pp.15.01353
DA - 2015-12-09
UR - https://www.deepdyve.com/lp/oxford-university-press/comparative-transcriptomics-unravel-biochemical-specialization-of-leaf-YiIiss9wKd
SP - 2462
EP - 2480
VL - 169
IS - 4
DP - DeepDyve
ER -