Functional Divergence of Diterpene Syntheses in the · separately combining each SmCPS with SmKSL1 or SmKSL2 and feeding GGPP as substrate. As expected, combining SmCPS1 and SmKSL1

Functional Divergence of Diterpene Syntheses in theMedicinal Plant Salvia miltiorrhiza1[OPEN]

Guanghong Cui, Lixin Duan, Baolong Jin, Jun Qian, Zheyong Xue, Guoan Shen, John Hugh Snyder,Jingyuan Song, Shilin Chen, Luqi Huang, Reuben J. Peters*, and Xiaoquan Qi*

Key Laboratory of Plant Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing100093, China (G.C., L.D., Z.X., G.S., J.H.S., X.Q.); State Key Laboratory Breeding Base of Dao-di Herbs,National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing100700, China (G.C., B.J., L.H.); Institute of Medicinal Plant Development, Chinese Academy of MedicalSciences and Peking Union Medical College, Beijing 100193, China (J.Q., J.S., S.C.); and Department ofBiochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, Iowa 50011 (R.J.P.)

ORCID IDs: 0000-0003-4691-8477 (R.J.P.); 0000-0001-7175-115X (X.Q.).

The medicinal plant Salvia miltiorrhiza produces various tanshinone diterpenoids that have pharmacological activities such asvasorelaxation against ischemia reperfusion injury and antiarrhythmic effects. Their biosynthesis is initiated from the generalditerpenoid precursor (E,E,E)-geranylgeranyl diphosphate by sequential reactions catalyzed by copalyl diphosphate synthase(CPS) and kaurene synthase-like cyclases. Here, we report characterization of these enzymatic families from S. miltiorrhiza, whichhas led to the identification of unique pathways, including roles for separate CPSs in tanshinone production in roots versus aerialtissues (SmCPS1 and SmCPS2, respectively) as well as the unique production of ent-13-epi-manoyl oxide by SmCPS4 andS. miltiorrhiza kaurene synthase-like2 in floral sepals. The conserved SmCPS5 is involved in gibberellin plant hormonebiosynthesis. Down-regulation of SmCPS1 by RNA interference resulted in substantial reduction of tanshinones, andmetabolomics analysis revealed 21 potential intermediates, indicating a complex network for tanshinone metabolism definedby certain key biosynthetic steps. Notably, the correlation between conservation pattern and stereochemical product outcome ofthe CPSs observed here suggests a degree of correlation that, especially when combined with the identity of certain key residues,may be predictive. Accordingly, this study provides molecular insights into the evolutionary diversification of functionalditerpenoids in plants.

Salvia miltiorrhiza, a Lamiaceae species known as redsage or tanshen, is a traditional Chinese medicinal herbthat is described in Shen Nong Ben Cao Jing, the oldestclassical Chinese herbal book, which dates from

between 25 and 220 C.E. The lipophilic pigments fromthe reddish root and rhizome consist of abietanequinone diterpenoids (Nakao and Fukushima, 1934),largely tanshinone IIA, cryptotanshinone, and tan-shinone I (Zhong et al., 2009). These are highly bioac-tive. For example, tanshinone IIA exerts vasorelaxativeactivity, has antiarrhythmic effects, provides protectionagainst ischemia reperfusion injury (Zhou et al., 2005;Gao et al., 2008; Sun et al., 2008), and exhibits anticanceractivities (Efferth et al., 2008; Lee et al., 2008; Wanget al., 2008; Gong et al., 2011). In addition, tanshinoneshave been reported to have a broad spectrum of anti-microbial activities against various plant pathogens,including rice (Oryza sativa) blast fungus Magnaportheoryzae (Zhao et al., 2011). Although tanshinones aremainly accumulated in the roots, trace amounts oftanshinones have been detected in aerial organs as well(Hang et al., 2008).

Diterpenoid biosynthesis is initiated by diterpenesynthases (diTPSs), which catalyze cyclization and/orrearrangement of the general acyclic precursor (E,E,E)-geranylgeranyl diphosphate (GGPP) to form varioushydrocarbon backbone structures that are precursors tomore specific families of diterpenoids (Zi et al., 2014).Previous work has indicated that tanshinone biosyn-thesis is initiated by cyclization of GGPP to copalyl

1 This work was supported by the Key Project of Chinese NationalPrograms for Fundamental Research and Development (grant no.2013CB127000 to X.Q.), the National Science Fund for DistinguishedYoung Scholars (grant no. 81325023 to L.H.), the National NaturalScience Foundation of China (grant no. 81001604 to G.C.), the Inde-pendent Studies Supported by China Academy of Chinese MedicalSciences (grant no. ZZ20090301 to G.C.), and the National Institutesof Health (grant no. GM076324 to R.J.P.).

* Address correspondence to [email protected] and [email protected].

The author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Xiaoquan Qi ([email protected]).

X.Q., R.J.P., G.C., and L.H. designed the research; G.C. performedexperiments in cooperation with L.D. for metabolomics analyses, B.J.for enzyme purification and mutagenesis, Z.X. for molecular evolu-tion analysis, and J.Q., J.S., G.S., and S.C. for genomic sequence anal-ysis; G.C., G.S., and J.H.S. prepared the article; X.Q. and R.J.P. wrotethe article.

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diphosphate (CPP) by a CPP synthase (SmCPS1)and subsequent further cyclization to the abietanemiltiradiene by a kaurene synthase-like cyclase (SmKSL1),so named for its homology to the ent-kaurene syn-thases (KSs) required for GA plant hormone biosyn-thesis (Gao et al., 2009). Miltiradiene is a precursor to atleast cryptotanshinone (Guo et al., 2013), and RNA in-terference (RNAi) knockdown of SmCPS1 expressionreduces tanshinone production, at least in hairy rootcultures (Cheng et al., 2014). The identification ofSmCPS1 and SmKSL1 has been followed by that ofmany related diTPSs from other Lamiaceae plant spe-cies (Caniard et al., 2012; Sallaud et al., 2012; Schalket al., 2012; Brückner et al., 2014; Pateraki et al., 2014).These largely exhibit analogous activity, particularlythe CPSs, which produce CPP or the stereochemicallyrelated 8a-hydroxy-labd-13E-en-15-yl diphosphate(LDPP) rather than the enantiomeric (ent) CPP relevantto GA biosynthesis.

To further investigate diterpenoid biosynthesis inS. miltiorrhiza, we report here a more thorough char-acterization of its diTPS family. A previously reportedwhole-genome shotgun sequencing survey (Ma et al.,2012) has indicated that there are at least fiveCPSs, although only two KSL genes in S. miltiorrhiza(Supplemental Table S1). Intriguingly, based on acombination of biochemical and genetic (RNAi genesilencing) evidence, we find that these diTPSs never-theless account for at least four different diterpenoidbiosynthetic pathways, each dependent on a uniqueCPS, with the KS presumably involved in GA biosyn-thesis seeming to be responsible for alternative diter-penoid metabolism as well. In addition, our studiesclarify the evolutionary basis for the observed func-tional diversity, with investigation of gene structure,positive selection, molecular docking, and mutationalanalysis used to explore the driving force for the func-tional divergence of these diTPSs. Moreover, we reportmetabolomic analysis, also carried out with SmCPS1RNAi lines, which enables prediction of the down-stream steps in tanshinone biosynthesis.

RESULTS

Molecular Analysis of the S. miltiorrhiza diTPSs

Using the working-draft genome sequences ofS. miltiorrhiza (Ma et al., 2012), we identified seven com-plementary DNA (cDNA) sequences encoding diTPSsfrom an inbred line (bh2-7). The deduced amino acidsequences showed that five contain the (D,E)XDDmotifrequired for the protonation-initiated cyclization reac-tions catalyzed by CPSs (Prisic et al., 2004) and aredefined here as SmCPS1 to SmCPS5, while two havethe DDXXD motif involved in binding the divalentmagnesium ions required for heterolytic cleavage/ionization of the allylic diphosphate ester bond cata-lyzed by terpene synthases such as KSs (Christianson,2006) and are defined here as SmKSL1 and SmKSL2,respectively (Supplemental Table S1).

Phylogenetic analysis revealed distinct conservationof these diTPSs. SmCPS1 to SmCPS3 cluster with otherpreviously characterized CPSs from the Lamiaceae, allof which (including SmCPS1) produce CPP or thestereochemically analogous LDPP. On the other hand,SmCPS4 and SmCPS5 cluster with CPSs that have beenpreviously shown to produce ent-CPP, generally for GAbiosynthesis. As previously reported (Hillwig et al.,2011), SmKSL1 has undergone loss of the N-terminalg-domain usually found in KSLs (although not mostother terpene synthases), which has been a character-istic of the KSLs involved in more specialized diterpe-noid metabolism from the Lamiaceae. By contrast,SmKSL2 retains the g-domain and clusters with otherdicot KSLs, many of which have been shown to act asKSs involved in GA biosynthesis (Fig. 1).

Biochemical Characterization of the S. miltiorrhiza diTPSs

To determine the biochemical activity of thesediTPSs, in vitro enzyme assays were carried out usingcrude extracts of recombinantly expressed proteins,separately combining each SmCPS with SmKSL1 orSmKSL2 and feeding GGPP as substrate. As expected,combining SmCPS1 and SmKSL1 led to the previouslyreported production of miltiradiene. Notably, combin-ing SmCPS2 and SmKSL1 also led to production ofmiltiradiene (Fig. 2A; Supplemental Fig. S1A). Kineticanalysis indicates that SmCPS1 has both higher affinity(Km = 0.54 mM versus 0.95 mM) and activity (.18-foldhigher catalytic constant) with GGPP than SmCPS2(Fig. 2A). Nevertheless, this observation raises the po-tential for redundancy between SmCPS1 and SmCPS2.No product was observed when either SmCPS1 orSmCPS2 was combined with SmKSL2. No product wasobserved when SmCPS3 was combined with eitherSmKSL1 or SmKSL2. Intriguingly, combining SmCPS4and SmKSL2 led to production of an unknown com-pound, while combining SmCPS5 and SmKSL2 led tothe production of ent-kaurene, the diterpene precursorto GAs (Fig. 2A; Supplemental Fig. S1A).

To identify the compound produced by SmCPS4and SmKSL2, the observed mass spectra was usedto search a number of publically available data-bases, revealing a close match to that of the knownditerpenoid 13-epi-manoyl oxide (Supplemental Fig.S1B; Demetzos et al., 2002). This presumably arisesfrom cyclization of LDPP, suggesting that SmCPS4produces this intermediate. However, SmCPS4 ismore closely related to ent-CPP-producing CPSs ratherthan the previously characterized LDPP synthasesfrom Lamiaceae, whose products are enantiomeric.Previous mutational analysis has shown that the CPSfromArabidopsis (Arabidopsis thaliana) involved in GAmetabolism (AtCPS), which then produces ent-CPP,can be easily diverted to the production of ent-LDPP(Potter et al., 2014). Thus, the stereochemistry of theSmCPS4 product was further investigated. Among thepreviously identified LDPP synthases is one from

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Coleus forskohlii, CfTPS2 (a CPS homolog), and thisLamiaceae species also encodes a subsequently actingKSL, CfTPS3, that reacts with LDPP to producemanoyl oxide (Pateraki et al., 2014). Both SmCPS4 andCfTPS2 reacted with GGPP to produce LDPP, detectedas labdenediol by gas chromatography (GC)-massspectrometry (MS) following dephosphorylation (Fig.2B; Supplemental Fig. S1B). To determine if these wereenantiomeric, CfTPS2 and SmCPS4 were separatelyincubated with either SmKSL2 or CfTPS3, respec-tively, and GGPP as substrate. Notably, combiningSmCPS4 with CfTPS3 did not lead to the productionof manoyl oxide (Fig. 2C). In addition, combining

CfTPS2 with SmKSL2 led to predominant productionof manoyl oxide with relatively less 13-epi-manoyloxide (Fig. 2C). These results indicate that SmCPS4produces ent-LDPP (8b-hydroxy-ent-CPP). Therefore,the product of SmCPS4 and SmKSL2 appears to beent-13-epi-manoyl oxide (Fig. 2D).

Physiological Roles of S. miltiorrhiza diTPSs

The tissue-specific expression pattern of the S.miltiorrhiza diTPSs was investigated by quantitativereverse-transcription (qRT)-PCR analysis of various

Figure 1. Phylogeny of diTPS genes in S. miltiorrhiza.The phylogenetic relationship was reconstructed us-ing the JTT models by PhyML 3.0 with 68 represen-tative characterized diTPS (Supplemental Table S2).Numbers on branches indicate the bootstrap per-centage values calculated from 100 bootstrap repli-cates. Physcomitrella patens CPS/kaurene (PpCPSKS)was used as outgroup. Blue lines show diTPS genesinvolved in more specialized diterpenoidmetabolismfrom Lamiaceae, and red lines show the loss ofN-terminal g-domain found in KSLs. Red-markedenzymes show diTPS from S. miltiorrhiza.

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organs, including leaves and roots of 3-d-old seedlingsand the root periderm, cortex, and xylem, as well asstem, leaf, sepal, petal, stamen, pistil, and immatureseeds of adult plants at the flowering stage (Fig. 3).SmCPS1 and SmKSL1 exhibit closely coordinated ex-pression, and their transcripts were found at extremelyhigh levels in the root periderm, consistent with theirrole in biosynthesis of the tanshinone pigments, whichaccumulate in this tissue. SmCPS2 and SmCPS3 weremost highly expressed in seedling leaves, althoughSmCPS2 also is highly expressed in the petals of adultplants. SmCPS4wasmost highly expressed in the sepal,and SmCPS5 was most highly expressed in the stem.SmKSL2 was most highly expressed in the root xylem,although this was expressed throughout all tissues ofadult plants to some extent.

To investigate the diterpenoid natural productarsenal of S. miltiorrhiza, both untargeted and tar-geted metabolomics analyses were carried out usingliquid chromatography (LC)-quadrupole time-of-flight(qTOF)-MS and GC-triple quadrupole (QqQ)-MS, re-spectively. Notably, although S. miltiorrhiza has notbeen previously reported to produce (ent-)13-epi-manoyl oxide, this diterpenoid was found here,mainly in sepals, with lower amounts found in pet-als, leaves, and stems, but not in roots (Fig. 2E;Supplemental Fig. S1B). This tissue-specific accumula-tion pattern matches SmCPS4mRNA expression with acorrelation coefficient of 0.97, indicating that the pro-duction of ent-13-epi-manoyl oxide in S. miltiorrhiza

depends on expression of SmCPS4 (as well as the moreubiquitous expression of SmKSL2).

As previously reported (Hang et al., 2008), tan-shinones were found not only in the root and rhizome,but also in aerial tissues of S. miltiorrhiza. Given thedistinct tissue-specific expression patterns of SmCPS1and SmCPS2, both of which can produce the relevantCPP intermediate, it seemed possible that these are notredundant but, instead, contribute separately to tan-shinone biosynthesis in the roots and aerial tissues, re-spectively. Moreover, as the only identified ent-CPPsynthase, it also seemed likely that SmCPS5 is involvedin GA metabolism.

Genetic Evidence for Distinct Diterpenoid Pathways inS. miltiorrhiza

To further investigate the distinct roles of the variousSmCPSs in S. miltiorrhiza diterpenoid biosynthesis,RNAi gene silencing was carried out targeting eitherSmCPS1 or SmCPS5 separately. RNAi knockdown ofSmCPS5 (Supplemental Fig. S2A) resulted in dwarftransgenic plants with significantly shorter pinnatelycompound leaves, shorter and narrower top leaves, andsmaller flowers compared with wild-type plants andcould be rescued (i.e. normal growth restored) by ap-plying GA3 to the T0 generation plants (SupplementalFig. S2, B–D). Further, T1 generation plants showed a3:1 segregation ratio for the dwarf phenotype, with

Figure 2. Functional identification of diTPS in S. miltiorrhiza. A, Total ion chromatography of diterpene products from in vitroassays with kinetic constants of SmCPS1 and SmCPS2. kcat, Catalytic constant. B, Total ion chromatography of the assay withpurified SmCPS4 and CfTPS2. The assay product was extracted directly by hexane (no treatment) or after the treatment of alkalinephosphatase (treatment). C, Total ion chromatography of the assay combines SmCPS4 with SmKSL2, together with the identifiedenzyme CfTPS2 (CPS) and CfTPS3 (KSL) from C. forskohlii. Assays all with 30 mM GGPP as substrate. D, Proposed pathway toent-13-epi-manoyl oxide in S. miltiorrhiza. OH, Hydroxyl; OPP, diphosphate. E, Total ion chromatography of hexane extracts fromdifferent organs in S. miltiorrhiza. The compounds are labdenediol (1), manoyl oxide (2), and ent-13-epi-manoyl oxide (3).


Cui et al.







severe stunting of shoots as well as dark-green leaves(Supplemental Fig. S2E). These phenotypes are associ-ated with GA deficiency (Margis-Pinheiro et al., 2005),indicating that SmCPS5, presumably together withSmKSL2, function in the GA biosynthetic pathway inS. miltiorrhiza.To investigate the potential redundancy between

SmCPS1 and SmCPS2, RNAi targeting SmCPS1 wascarried out, generating five lines exhibiting silencing ofapproximately 90% (Fig. 4A). These SmCPS1-RNAiplants exhibited an obvious white-color root phenotypecompared with wild-type roots, which had the char-acteristic reddish color associated with tanshinones(Fig. 4B). Root-directed metabolite analysis demon-strated that cryptotanshinone, tanshinone IIA, andtanshinone I were dramatically reduced, from 14,4656995, 2,311 6 13, and 2,010 6 117 mg g–1 in wild-typeplants to 0.106 0.01, 1.26 0.2, and 146 1 mg g–1 in theSmCPS1-RNAi plants, respectively (Fig. 4C). As a con-trol, the water-soluble polyphenolic acid content of theplants, including lithospermic acid B and rosmarinicacid, also was investigated. The lack of any statisti-cally significant differences between the wild-type andSmCPS1-RNAi samples (Fig. 4C) indicates that down-regulation of SmCPS1 expression specifically affectstanshinone biosynthesis (i.e. this did not interfere withthe production of at least these polyphenolic acidmetabolites).Notably, tanshinone levels in the aerial tissues were

not affected in the SmCPS1-RNAi plants (Table I).Given that SmCPS2 is predominantly expressed inthese tissues (Fig. 3), these results suggest that SmCPS2mediates tanshinone biosynthesis in aerial organs of

S. miltiorrhiza independently of the SmCPS1-dependentpathway in the root periderm, such that SmCPS1 andSmCPS2 are not redundant.

Metabolomic Analysis Clarifies Tanshinone Biosynthesis

Metabolomics analysis was carried out with not onlythe wild-type plant roots, but also SmCPS1-RNAi plantroots, with LC-electrospray ionization-qTOF-MS re-vealing 39 and GC-electron impact (EI)-QqQ-MS 19metabolites with significantly reduced accumulation(i.e. a .2-fold change with P , 0.05; SupplementalTables S3 and S4). On the other hand, four metabolites(by using GC-EI-QqQ-MS) with elevated accumulationalso were identified (Supplemental Table S4). By com-parison to known compounds, 21 of the metaboliteswith reduced accumulation in the roots of SmCPS1-RNAi plants were identified as diterpenoids, all ofwhich were predominantly accumulated (.94.5%) inthe periderm (Fig. 4D; Supplemental Tables S3 and S4).Of the metabolites exhibiting increased accumulation,three were identified as diterpenoids (geranylgeraniol,a-springene, and b-springene), and these were notdetected in the periderm, cortex, and xylem of wild-type plant roots (Supplemental Table S4). These com-pounds presumably appear due to the down-regulationof SmCPS1, with accumulation of GGPP leading tohydrolysis to geranylgeraniol, with subsequent de-hydration leading to a-springene and b-springene(Fig. 4E).

Of the 21 identified SmCPS1-dependent metabolites,19 are tanshinones or plausible biosynthetic interme-diates, while two are rearranged abietane diterpenoids(i.e. przewalskin and salvisyrianone; Fig. 4E). The 19tanshinones and biosynthetically relevant metabolitescould be further divided into five main groupsaccording to the progressive modification of their car-bon skeletons (Fig. 4E). Group I is simply composed ofmiltiradiene, with its planar cyclohexan-1,4-diene Cring. Group II compounds are dehydro-abietanes, witha characteristic aromatic C ring (i.e. abietatriene, fer-ruginol, and sugiol). Group III compounds are nor-abietatetraen-11,12-diones, with conversion of theC ring to an ortho-quinone (i.e. keto groups at C-11 andC-12), as well as aromatization of the B ring, along withloss of C-20 (e.g. miltirone and 4-methylene-miltirone).The 11 metabolites comprising group IV all are vari-ously named tanshinones, with addition of the(dihydro)furan ring D. Group V contains three metab-olites that have been additionally modified by the lossof one of the geminal methyl groups from the C-4 po-sition, along with the presence of at least one doublebond in the A ring.

Positive Selection for Divergent CPS Activity

Given the ease with which diTPSs can be diverted toalternative activity (Wilderman and Peters, 2007; Xu

Figure 3. qRT-PCR analysis of transcript levels of seven diTPS genes in12 organs including leaves and roots of 3-d-old seedlings, and the rootperiderm, cortex, and xylem, as well as stem, leaf, sepal, petal, stamen,pistil, and immature seeds of adult plants at the flowering stage. Theexpression level was normalized to that of Actin. Data are means fromthree technical replicates of at least three biological replicates.











et al., 2007; Keeling et al., 2008; Morrone et al., 2008;Criswell et al., 2012; Potter et al., 2014), it is not clearwhat underlies the expanded nature and divergent ac-tivity observed in the S. miltiorrhiza diTPS family (i.e.selective pressure or genetic drift). Gene structure,specifically the number and placement of introns, hasbeen associated with evolutionary descent in the ter-pene synthase gene family (Trapp and Croteau, 2001).Accordingly, the CPS and KSL genes of S. miltiorrhiza,Arabidopsis, and rice can be divided into three groups.Group I genes associated with GA metabolism eachhave the typical 15 exons and 14 introns for CPS genesand 14 exons and 13 introns forKS genes (SupplementalFig. S3), which corresponds to the ancestral plant diTPSgene structure (Trapp and Croteau, 2001). Group IICPS/KSL genes, including SmCPS1, SmCPS3, SmCPS4,

SmKSL1, OsCPS2, OsCPS4, OsKSL5, OsKSL6, OsKSL8,and OsKSL10, show diverged sequences and genomicarchitecture. In particular, intron loss (Zhang et al.,2014) relative to the conserved group I genes are oftenobserved. For example, SmCPS1 has lost the 10th and12th introns, SmCPS4 the 5th intron, and SmCPS3 thefirst four introns, whileOsCPS2 has lost the 2nd and 3rdintrons, and OsKSL5, OsKSL6, OsKSL8, and OsKSL10all have lost the last intron (Supplemental Fig. S3).These architecturally divergent group II genes are in-volved in the biosynthesis of specialized metabolites inS. miltiorrhiza and rice. On the other hand, the group IIIgenes SmCPS2,OsKSL4, andOsKSL7 exhibit conservedgenomic architecture but divergent sequences and func-tions relative to the group I genes associated with GAbiosynthesis (Supplemental Fig. S3).

Figure 4. Phenotype andmetabolic profiles caused by down-regulation of SmCPS1 in T0 generation plants. A, qRT-PCR analysis oftranscript levels of five CPS genes in root of SmCPS1-RNAi and the wild type (WT). Expression was normalized to that of Actin. Theerror bars show the SDs frommean value (n = 3 experiments). SmCPS2 is not expressed in these samples. B, The phenotype of down-regulation of SmCPS1. C, Quantitative analysis of five major compounds including cryptotanshinone (CT), tanshinone IIA (T-IIA),tanshinone I (T-I), lithospermic acid B (LAB), and rosmarinic acid (RA) in root of SmCPS1-RNAi lines and thewild type. The error barsshow the SDs frommean value (n = 3 experiments). Asterisks indicate significant difference at P, 0.01 compared with the wild typeby Student’s t test. D, Cross section of the root. E, Summary of metabolite flux caused by down-regulation of SmCPS1. The red arrowshows the accumulated metabolites, and the blue arrow shows the reduced metabolites in root of SmCPS1-RNAi lines comparedwith the wild type. Underlined metabolites are identified by standard reference. FW, Fresh weight; OH, hydroxyl; OPP,diphosphate; R1, R2, and R3, substituent group. Bar = 100 mm.


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To investigate whether the observed functional di-vergence of the CPS genes involved in biosynthesis ofthe tanshinones and other more specialized labdane-related diterpenoids is a function of positive selection,phylogenetic analysis of the protein-coding DNA se-quences of CPS members from an array of angiospermswas carried out. The resulting phylogenetic tree (Fig.5A) provided the basis for branch site model analysisconducted with the PAML package (Zhang, 2004;Zhang et al., 2005; Yang and dos Reis, 2011). Thisrevealed a statistically significant signature for positiveselection associated with functional divergence fromthe production of ent-CPP required for GA biosynthesisto the enantiomeric (i.e. normal) CPP involved in pro-duction of the tanshinones, as well as stereochemicallyrelated LDPP for other more specialized diterpenoidbiosynthesis (branch c, nonsynonymous to synony-mous substitution ratio = 5.64, P, 0.01; Fig. 5, A and B;Supplemental Table S5).To more closely examine the basis for the observed

functional diversification, the analysis was extended toindividual codons in SmCPS1 versus SmCPS5. Thepositively selected sites identified by Bayes EmpiricalBayes analysis correspond to Ser-362, Asp-389, Ser-415,and Pro-416 in SmCPS1, which are Trp-364, Ser-391,Thr-416, and Gly-417 in SmCPS5 (Fig. 5C). Models forboth SmCPS1 and SmCPS5 were generated andrevealed that three of these residues, Ser-362:Trp-364(S-W) and Ser-415/Pro-416:Thr-416/Gly-417 (SP-TG)are part of the active site cavity, whereas Asp-389:Ser-391 (D-S) is more than 6 Å away from the active sitecavity (Supplemental Fig. S4, A and B). Site-directedmutagenesis was performed to swap these residuesbetween SmCPS1 and SmCPS5, except Thr-416 inSmCPS5, as this corresponds to Thr-421 in AtCPS,which has already been reported to be involved in ca-talysis (Köksal et al., 2014). Assays using the mutantenzymes reacting with GGPP alone or in combinationwith SmKSL1 or SmKSL2 showed that the mutantSmCPS1:S362W, in which Ser-362 (S) of SmCPS1 wasreplaced by the Trp (W) found at the same position inSmCPS5, was more than 100-fold less efficient in theproduction of CPP, and miltiradiene when assayedwith SmKSL1, relative to the wild-type SmCPS1.Moreover, this mutant also did not yield any detectableproduct when incubated with SmKSL2 and GGPP

(Supplemental Fig. S5), indicating that no ent-CPP isproduced. The mutant SmCPS5:W364S also showedabout 80-fold lower efficiency in production of ent-CPP,and ent-kaurenewhen assayedwith SmKSL2 comparedwith wild-type SmCPS5. Similarly, this mutant also didnot yield any product when incubated with SmKSL1.The other four mutant enzymes SmCPS1:D389S,SmCPS1:P416G, SmCPS5:S391D, and SmCPS5:G417Pdid not cause significant change in enzymatic activityrelative to the parental/wild-type CPSs (SupplementalFig. S5). Thus, while no change in product stereo-chemistry was observed, it does seem that the residuecorresponding to the S-W position is important for ca-talysis in both SmCPS1 and SmCPS5 (SupplementalFig. S4, C and D), despite their difference in productoutcome, suggesting that this substitution has a role inenabling the production of normal versus ent-CPP and,hence, biosynthesis of the derived tanshinones.

DISCUSSION

The combined biochemical and genetic work repor-ted here has defined the roles of the diTPS family inS. miltiorrhiza. Of the five SmCPSs, while SmCPS3 ap-pears to be inactive, each of the other SmCPSs definesseparate diterpenoid pathways. Rather than reflectingredundancy, the similar biochemical activity of SmCPS1and SmCPS2 is coupled to their distinct roles in tan-shinone biosynthesis in the roots versus aerial tissues,respectively. This discovery provides the possibility ofusing metabolic engineering strategies to enhance theproduction of tanshinones in aerial organs. Cultivationof the resulting plants could then provide annually re-newable source materials for extraction of tanshinonesby harvesting aerial tissues without destroying theentire plant.

Both SmCPS1 and SmCPS2 react with GGPP to formnormal CPP in S. miltiorrhiza. Our phylogenic analysisindicates that SmCPS1 and SmCPS2 are in the sameclade that also contains other CPSs from the Lamiaceaeand closely related Solanaceae (Fig. 1) that similarlyproduce CPP and LDPP with analogous stereochemis-try. These CPSs were likely derived from an ancestralCPS that produced ent-CPP for GA biosynthesis viaearly gene duplication and neofunctionalization that

Table I. Main compounds detected in aerial part and seedling of the wild type and SmCPS1-RNAi

Organ

Concentrationa Relative Concentrationb

Cryptotanshinone Tanshinone IIA Miltirone Trijuganone B Tanshinone I

Wild Type RNAi Wild Type RNAi Wild Type RNAi Wild Type RNAi Wild Type RNAi

ng g–1 fresh wtPetal 0.2 0.2 2.2 2.2 12.1 12.0 19.9 20.2 10.6 11.0Sepal 1.2 1.2 22.1 23.0 72.2 73.0 143.0 144.4 14.4 15.0Young leaf 0.5 0.5 7.3 7.6 25.9 26.1 67.1 68.0 13.5 13.0Young root 0.8 0.8 8.9 9.0 61.5 62.3 59.3 60.2 20.3 21.0

aThe concentration obtained by calibration of standards. b The relative concentration obtained by comparison with internal standardumbelliferone.












occurred at least before the divergence of the Lamiaceaeand Solanaceae (Figs. 1 and 5), which has been estimatedto have been 64 million years ago (48–75 million years;Zhang et al., 2012). During the evolution of Lamiaceae,two more gene duplication events likely happened,producing SmCPS3 as well as SmCPS1 and SmCPS2,which individually are representative of two morewidespread clades (e.g. homologs to both are found inC. forskohlii), respectively. SmCPS2 retained the ancestralgene architecture (Supplemental Fig. S3) and exhibits asimilar gene expression pattern as the SmCPS5 in-volved in GA biosynthesis (Fig. 3), whereas SmCPS1 hasundergone more divergence, including intron loss(Supplemental Fig. S3) and altered transcriptional reg-ulation (Fig. 3), alongwith exhibiting significantly highercatalytic activity than SmCPS2. These data suggest thattanshinone biosynthesis may have evolved early in theLamiaceae, and the gene duplication leading to SmCPS1and SmCPS2 enabled further localization and up-regulation of tanshinone biosynthesis in root peridermcells in some Salvia spp., including S. miltiorrhiza. Thepresence of distinct SmCPS1- and SmCPS2-dependenttanshinone pathways in the root periderm and aerialtissues, respectively, indicates that these labdane-relatedditerpenoids may play important roles in plant devel-opment and in adaptation to different stress conditions,which is a topic worth future investigation.

The ability of SmCPS4 to produce the enantiomericform of LDPP was suggested by the ability of SmKSL2to react with both ent-CPP (to produce ent-kaurene) andthis ent-LDPP, but not the CPP product of SmCPS1 (orSmCPS2). The observed production of ent-13-epi-manoyl oxide in S. miltiorrhiza then suggests dualfunction for SmKSL2 (i.e. in GA and this more special-ized diterpenoid biosynthesis). Regardless, identifica-tion of the ability of SmCPS4 and SmKLS2 providesaccess to this unique diterpenoid. In addition, theability of SmCPS4 to produce ent-LDPP was presagedby its phylogenetic relationship to ent-CPP-producingCPSs. Based on the similar clustering of a previouslyidentified LDPP synthase from Grindelia robusta (Zerbeet al., 2013), as well as the ability of a KS to selectivelyreact with its product, it is suggested here that thisGrTPS1 may produce ent-LDPP (Fig. 2D).

It is interesting to note that certain previously iden-tified residues are consistent with the CPS enzymaticactivity observed here. SmCPS5, shown here to playa role in GA biosynthesis, contains the His (His-326)associated with susceptibility to inhibition by Mg2+

(Mann et al., 2010), which has been suggested to serve aregulatory role in such CPSs (Prisic and Peters, 2007). Inaddition, SmCPS5 contains theHis-Asn dyad (His-258/Asn-317; Fig. 5C) that has been suggested to act as thecatalytic base and is conserved in CPSs that produce

Figure 5. Molecular evolution of CPS genes. A, Bold lines illustrate branches or genes evolved under positive selection withsignificant statistical support at P, 0.05 both in branch site model tests 1 and 2. Branch c shows the divergence of normal-CPPandnormal-LDPP synthase from the ent-CPP synthase. dN/dS, Nonsynonymous to synonymous substitution ratio. B, Different stereo-isomers of CPP and LDPP. C, Alignment of conserved residues. The asterisks indicate positive selection sites with posteriorprobability greater than 95% by Bayes Empirical Bayes analysis.


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ent-CPP (Potter et al., 2014). By contrast, the ent-LDPP-producing SmCPS4 contains a Ser (Ser-299) in place ofthe corresponding Asn (Fig. 5C), consistent with theability of such substitution of smaller residues to enableproduction of hydroxylated CPP (Potter et al., 2014). Inaddition, the investigation of residues showing signs ofpositive selection here indicates other positions im-portant for altering stereochemical product outcome,although further experiments are required to determinehow many and which residues are required. Never-theless, altogether, the results reported here suggestthat combining phylogenetic relationships with theidentity of residues at positions of known catalyticrelevance may be predictive for CPS catalytic activity.

MATERIALS AND METHODS

Plant Materials

The Salvia miltiorrhiza species has two different flower colors, purple andwhite.Varieties with purple flowers are distributed throughout China. The varietywith white flowers (S. miltiorrhiza f. alba.) is only found in Shandong Province.S. miltiorrhiza f. alba is a rare and thusmore valuable variety of Danshen. Thewhiteflower line bh2-7, inbred for 5 generations, was used in our study.

Plant Growth and Culture Conditions

bh2-7 seeds were surface sterilized with 5% (v/v) sodium hypochlorite andcultured on solid hormone-freeMurashige and Skoog basal medium containing30 g L–1 Suc and 8 g L–1 agar. Cultures were maintained at 25°C under a16-h-light/8-h-dark photoperiod. Seedlings were transferred to pots filled withsoil:vermiculite (3:1) mix and grown under the same temperature and lightregime in a plant growth room. Dwarf plants of SmCPS5-RNAi were sprayedwith 150 mM GA3 solution to complement the phenotype in the T0 generation.

Genomic Sequence of diTPS Gene Family Members

To show the structural divergence that occurred between SmCPS1, SmCPS3,andSmCPS4, we cloned the full genomic sequence of each gene frombh2-7. Cetyl-trimethyl-ammoniumbromidemethodwasused to extract the genomicDNAandamplified with specific primers (Supplemental Table S6). Intron/exon structureswere predicted using the Gene Structure Display Server (Guo et al., 2007).

Gene Expression Analysis

Plant samples were harvested and immediately frozen in liquid nitrogen.Total RNAwas extracted using amodified cetyl-trimethyl-ammoniumbromideprotocol and treated with RNase-Free DNase I (Takara) to remove residualgenomic DNA. RNA integrity and quality were checked by denaturing gelelectrophoresis, and the absence of genomic DNAwas confirmed by PCR usingprimers for Smactin, prior to reverse transcription. One to five micrograms oftotal RNA was reverse transcribed into cDNA using the SuperScript III reversetranscriptase and oligo(dT)12–18 primer (Invitrogen), according to the manu-facturer’s instructions. The synthesized cDNA was then diluted 10-fold. Onemicroliter of this diluted template was used for subsequent qRT-PCR analysiswith a total PCR reaction volume of 20 mL. qRT-PCR was performed with aSuperReal PreMix for SYBR Green Kit (TIANGEN) on a Corvett Rotor-Gene3000 real-time PCR detection system. All reactions were performed using thefollowing PCR conditions: initial denaturation step of 95°C for 10min, followedby 40 cycles each of 95°C for 5 s, 60°C for 15 s, and 72°C for 20 s, with a finalmelting stage from 55°C to 95°C. A final dissociation step was performed toassess the quality of the amplified product. cDNA from a series of 5-fold di-lutions were used for calibration, and the efficiency of the PCR amplificationswas found to be in the range of 90% to 110%, which is considered desirable forquantitative PCR (Taylor et al., 2010). Relative expression levels were calculatedas the ratio of the target gene transcript level to the transcript level of thehousekeeping gene Actin (Smactin; Yang et al., 2010). Primer specificity was

confirmed by direct cloning and sequencing of individual PCR amplificationproducts. qRT-PCR was performed with three technical replicates of at leastthree biological replicates for each tissue or transformed plant line. The hot mapof gene expression data was generated in the R software package.

Phylogenetic Analysis

Sixty-eight diTPSs with characterized functions were included in the phy-logenetic analysis (Supplemental Table S2). To carry out the phylogenic re-construction, multiple protein sequence alignments were performed withMAFFT version 7.012 employing the E-INS-I method (Katoh et al., 2005).Maximum likelihood trees were built using PhyML version 3.0 (Guindon et al.,2010). Specifically, PhyML analyses were conducted with the JTT substitutionmodel, four rate substitution categories, and 100 or 1,000 bootstrap replicateanalyses. The phylogeny was displayed using FigTree software (http://tree.bio.ed.ac.uk/software/figtree/).

Hairy Root and Plant Transformation for Knockdown ofSmCPS1 and SmCPS5 by RNAi

A 289-bp gene-specific sequence including the 39 untranslated region ofSmCPS1 and a 399-bp gene-specific sequence in the 39 end of SmCPS5 wereamplified by PCR using cDNA as a template and then cloned using Gatewaytechnology into the pK7GWIWG (II) binary vector (Limpenset al., 2005). Posi-tive plasmids of pK7GWIWG-CPS1 and pK7GWIWG-CPS5 were identifiedwith sequencing and restriction enzyme analysis and then introduced intoAgrobacterium tumefaciens strain EHA105 and Agrobacterium rhizogenes strainACCC 10060 by electroporation. Transformation of leaf explants from S. miltiorrhizabh2-7 plants was carried out following previously described methods (Yan andWang, 2007) with minor modifications. Single colonies of A. tumefaciens strainEHA105 cells harboring the various RNAi vectors were inoculated into 10mL ofliquid Luria-Bertani medium with 50 mg L–1 spectinomycin and 100 mg L–1 ri-fampicin and then grown on a shaker (180 rpm) at 28°C for 16 to 18 h. Cells werecollected by centrifugation when the optical density at 600 nm reached 0.6 andwere resuspended in 20 mL of liquid Murashige and Skoog medium. Leavesor petioles were cut into 0.5- 3 0.5-cm pieces discs and precultured for2 d on Murashige and Skoog basal medium supplemented with 2.0 mg L–1

6-benzyladenine. The discs were then submerged with shaking in a bacterialsuspension for 15 min and cocultured on the Murashige and Skoog basal me-dium for 2 d. The leaf discs were then transferred to selection Murashige andSkoog basal medium supplementedwith 2.0 mg L–1 6-benzyladenine, 50 mg L–1

kanamycin, and 225 mg L–1 timentin. After two to three rounds of selection(10 d each), the regenerated buds with expression of GFP were transferred toMurashige and Skoog basal medium supplemented with 25 mg L–1 kanamycinfor root formation and elongation. Rooted plantlets were further cultured onMurashige and Skoog basal medium for about 1 month. The plantlets (7–8 cmtall, with roots 5–6 cm long) were transplanted to soil and vermiculite (3:1) andcovered by beakers to maintain humidity for 1 week and then graduallyhardened off in pots in a greenhouse for further growth.

Hairy root cultures can be successfully obtained with S. miltiorrhiza, and weused both hairy root cultures and full plants to analyze the silencing effect ofSmCPS5. Though hairy root cultures with silencing of SmCPS5 showed no sig-nificant phenotype, we found that it was an easy and fast system to analysis theeffect of different silencing vectors. The A. rhizogenes-based transformation had asimilar procedure as the A. tumefaciens-based transformation described above.When the hairy roots were 2 to 3 cm in length, expression of GFP was observedunder a fluorescencemicroscope to identify positive lines. The positive hairy rootswere excised and cultured on solid, hormone-free Murashige and Skoog basalmedium containing 50 mg L–1 kanamycin and reduced timentin from 225 mg L–1

to zero in two or three selection cycles (15 d each). The rapidly growingkanamycin-resistant and GFP-visible lines with no bacterial contamination werethenmaintained at 25°C in the dark with Murashige and Skoogmediumwithoutammonium nitrate and routinely subcultured every 25 to 30 d.

Metabolomics Profiling Using LC-qTOF-MS andGC-QqQ-MS

The metabolomics profiling data were acquired using a combination of twoindependent analytical platforms. LC-qTOF-MS analysis of methanol extractswas used for global unbiased metabolite detection. GC-QqQ-MS analysis ofhexane extracts, optimized for detection of targeted intermediates, was used for






http://tree.bio.ed.ac.uk/software/figtree/

http://tree.bio.ed.ac.uk/software/figtree/


the analysis of metabolites that were not readily detectable with the LC-qTOF-MS platform.

For LC-qTOF-MS, fresh plant root samples were frozen in liquid nitrogenand ground to a fine powder under continuous cooling. One hundred milli-grams fresh weight of the powder was extracted in 2 mL of methanol, con-taining an internal standard (umbelliferone, 20 mg mL–1). The extracts weresonicated twice for 15 min, centrifuged (1,500g) for 10 min, and then filteredthrough a 0.2-mm polytetrafluoroethylene syringe filter (Agilent). An aliquot ofeach filtrate (5 mL) was separated using an Agilent 1290 Infinity ultra highperformance liquid chromatography system consisting of a binary pump, anautosampler, a column temperature controller, and a variable wavelength de-tector at 285 nm. The chromatography was performed using a ZORBAX RRHDSB-C18 column from Agilent Technologies (2.13 100 mm, 1.8 mm). The mobilephase consisted of 0.01% (v/v) formic acid in acetonitrile (A) and water con-taining 0.01% (v/v) formic acid (B). A gradient program was used as follows:linear gradient from 10% to 20%A (0–5min), linear gradient from 20% to 40%A(5–7 min), linear gradient from 40% to 100% A (7–10 min), isocratic at 100% A(10–14 min), and linear gradient from 100% to 10% A (14–15 min). The mobilephase flow rate was 0.25 mL min–1, and the column temperature was set at30°C. Five reference standard compounds, namely salvianolic B, rosmarinicacid, crypotanshinone, tanshinone IIA, and tanshinone I were dissolved inmethanol to create standard curves for use in absolute quantification calcu-lations. To ensure that analytes were in the linear range and to exclude someartifactual peaks, samples were also diluted by 10- and 50-fold andreanalyzed.

MS was performed using an Agilent 6540 qTOF equipped with an electro-spray ionization source operating in positive ion mode. The nebulization gaswas set to 40 pounds per square inch. The drying gas was set to 10 L min–1 at atemperature of 350°C, and the sheath gas was set to 11 L min–1 at a temperatureof 350°C. The capillary voltage was set to 4,000 V. The qTOF acquisition ratewas set to 0.5 s. For full-scanMS analysis, the spectra were recorded in the rangeof mass-to-charge ratio (m/z) 100 to 1,000. Chromatographic separation, fol-lowed by full-scan mass spectra, was performed to record retention time andm/z values of all detectable ions present in the samples.

For GC-QqQ-MS, plant material was lyophilized for 48 h. One hundredmilligrams of the lyophilized powder was extracted in 2 mL of hexane thatcontained two internal standards (tricosane, 6.5 mg mL–1; and tetracosane,0.8 mg mL–1). The extracts were sonicated twice for 15 min and centrifuged (3,000g)for 10 min. The supernatant was evaporated under nitrogen, resuspended in50 mL of hexanes or derivatized with 80 mL of N-methyl-N-(trimethylsilyl)trifluoroacetamide and 8 mL of pyridine at 80°C for 40min, and then analyzedby GC-MS.

GC-MS analyses were performed on anAgilent 7890AGC system connectedto an Agilent 7000B triple quadrupole mass spectrometer with EI ionization. A1-mL portion of the extract was injected in splitless mode onto the column. Thecolumn used was a DB-5ms (30-m 3 0.25-mm i.d., 0.25-mm film thickness;Agilent J&W Scientific) fused silica capillary column. Helium was used as thecarrier gas for GC at a flow rate of 1.0 mL min–1. The injector temperature was280°C. The oven programwas as follows: 50°C for 2min, linear ramp at a rate of20°Cmin–1 to 200°C, and then followedwith a linear ramp at a rate of 5°Cmin–1

to 300°C, held at 300°C for 10 min. The transfer line temperature was 280°C.Raw data were processed with Mass Hunter Qualitative Analysis software

(Agilent). Mass Profiler Professional (Agilent) software was used to identifysignificantly different ion features. A series of filtration steps was performed tofurther filter the initial results. First, only features with abundances above 1,000ion countswere selected. Second, featureswere passed through a quality controltolerance window of 0.1% plus 0.15 min and 5 mg mL21 plus 2.0 mD chosen foralignment of retention time and m/z values, respectively. Third, features thatwere not present in all biological replicates of any single sample group wereremoved. The data were normalized to the detected values for the internalstandard peak; GC-QqQ-MS data were normalized to tetracosane. After align-ment and normalization of the peaks of each sample, a single data set stored as amatrix was prepared. Fold change values were calculated as the ratio of meanSmCPS1-RNAi line feature values compared with the mean values for thesefeatures in the wild-type lines. Student’s t tests were then used to determinewhether each feature was increased or decreased significantly. The aligned datawere exported to SIMCA-P+ 12.0 for multivariate analyses (partial least-squares discriminant analysis).

Differentially accumulated features detected by LC-qTOF-MS were identi-fied by automatic comparison to a personalized metabolite database estab-lished with METLIN software (Sana et al., 2008). Seven hundred sixty-twoditerpenoids were collected from different Saliva spp., among them, 86 tan-shinones and biosynthetically related metabolites came from S. miltiorrhiza. The

putative metabolite peaks were tentatively identified by comparison withMS/MS spectra (Yang et al., 2006; Zhou et al., 2009) and reference standardcompounds including sugiol, cryptotanshinone, tanshinone IIA, tanshinoneIIB, tanshinone I, dihydrotanshinone I, przewalskin, salvisyrianone (BioBiopha),and miltirone (Faces Biochemical). Differentially accumulated features detectedby GC-QqQ-MS were compared with the NIST 05 standard mass spectral data-bases and four reference standard compounds, including geranylgeraniol (Sigma),miltiradiene (Luqi Huang’s lab), and ferruginol and sugiol (BioBiopha).

In Vitro Assays

For in vitro functional assays, the full coding sequence of S.miltiorrhizadiTPSgenes with specific restriction enzyme sites (Supplemental Table S6) was clonedinto the pGEM-T vector (Promega), digested with corresponding restrictionenzymes, and subcloned into the expression plasmid pET32a (Merck) to createpET32-CPSs and pET32-KSLs. AtCPS (AAA53632) and AtKS (AAC39443) werecloned from Arabidopsis (Arabidopsis thaliana). CfTPS2 (KF444507) and CfTPS3(KF444508) were full synthesized. The expression, purification, and kineticanalysis of the recombinant proteins were performed as described previously(Hillwig et al., 2011). The constructs were transformed into Tuner (DE3) orOrigami B (DE3) competent cells (Merck). Three to five positive colonies werecultured in Luria-Bertani medium with 50 mg L–1 carbenicillin, and 0.1 to 0.4 mM

isopropyl b-D-thiogalactopyranoside was added to induce the expression of theprotein. Subsequently, cell pellets were collected and resuspended in assay buffer(50 mM phosphate, pH 7.4, 10% (v/v) glycerol, 2 mM dithiothreitol, and 10 mM

MgCl2) and sonicated for 10 s six times on ice. Lysate from the samples wascentrifuged at 12,000g, and the resulting supernatant was used for the assays. Theconversion of GGPP to CPP or LDPP was carried out by incubating 500 mg ofpET32-CPS sample protein extract with 20 to 50 mM GGPP (Sigma) in a finalvolume of 250 mL of assay buffer for 2 to 4 h at 30°C. Assay mixtures were hy-drolyzed (dephosphorylated) with 75 units of bacterial alkaline phosphatase atpH8 for 16h at 37°C toproduce hexane-soluble products.GGPPwas converted tokaurene, miltiradiene, or manoyl oxide by mixing 250 mg of pET32-CPS proteinextract and 250 mg of pET32-KSL protein extraction with 20 to 50 mM of GGPPand incubated for 2 to 4 h at 30°C. Assay mixtures were extracted three timeswith an equal volume of hexane. The hexane fractions were pooled, evaporatedunder nitrogen, resuspended in 50 mL of hexanes or derivatized with 80 mL ofN-methyl-N-(trimethylsilyl) trifluoroacetamide and 8 mL of pyridine at 80°Cfor 40 min, and then analyzed by GC-MS. To identify possible products, weobtained and analyzed a series of standard reference compounds includinggeranylgeraniol (Sigma), geranyllinalool (Sigma), (13E)-labda-8a,15-diol(BioBiopha), 13-epi-manool (BioBiopha), and sclareol (Sigma).

Purification of Recombinant CPS and KSL

Cell pellets were resuspended in 16 mL of protein lysis buffer (50 mM

phosphate, pH 7.4, 300 mM NaCl, 10% (v/v) glycerol, 10 mM MgCl2, and 20 mM

imidazole) and sonicated for 10 s, six times, on ice. Lysate from the samples wascentrifuged at 12,000g for 20 min at 4°C. The cleared lysate was transferred toprewashed nickel-nitrilotriacetic acid agarose beads and incubated for 30min at4°C. Thereafter, the nickel-nitrilotriacetic acid agarose beads were rinsed threetimes with 15 mL of washing buffer (20 mM phosphate, pH 7.4, 300 mM NaCl,and 100 mM imidazole). The tagged protein was then eluted by the addition of6 mL of elution buffer (20 mM phosphate, pH 7.4, 300 mM NaCl, and 500 mM

imidazole) to the bead bed. The buffer of the eluted proteins was then ex-changed using a PD-10 column equilibrated with assay buffer (50 mM phos-phate, pH 7.4, 10% [v/v] glycerol, 2 mM dithiothreitol, and 10 mM MgCl2). Thepurified proteins were then identified by SDS-PAGE gel and quantified byBradford assays (Genstar).

Enzyme Kinetic Analysis of SmCPS1 and SmCPS2

For kinetic assays, 20 nM purified SmCPS1 and SmCPS2 was used, with a2-min reaction time at 25°C. Single-vial assays were used as described above.Assayswere completed in triplicate with 0.5 to 20mMGGPP for SmCPS1 and 0.5to 8 mM GGPP for SmCPS2. Enzymes were deactivated at the end of the 2-minreactions by incubating the reaction vial at 80°C for 3 min, followed byquenching on ice. Three hundred molar purified SmKSL1 enzyme was thenadded to the vial in the single-vial assay for another reaction at 30°C for 2 h.Assayswere analyzed via GC-QqQ using selected ionmonitoring ofm/z 57 (forinternal standard tetracosane) and m/z 134 for the miltiradiene product.


Cui et al.




Miltiradiene concentrations were determined relative to the internal standardby using excess SmCPS1 and SmKSL1 in single-vial assays and allowing thereaction to proceed to completion (2 h). Kinetic parameters were determinedby nonlinear regression using a Michaelis-Menten model implemented inGraphPad Prism 6.03.

Positive Selection Analysis

For molecular evolution analysis, 29 protein-coding DNA sequences forCPSs were used to construct phylogenetic trees, using PhyML3.0 (Guindonet al., 2010) under the general time-reversible nucleotide substitution modelwith four rate substitution categories. The branches with bootstrap valueshigher than 700 were used for branch site model tests 1 and 2 in the PAMLpackage to detect whether positive selection had acted on particular aminoacid sites within specific lineages (Zhang, 2004; Zhang et al., 2005; Yang anddos Reis, 2011). Branch site model uses a maximum-likelihood approach tocalculate nonsynonymous to synonymous rate ratios. Likelihood ratio testswere performed, and the values of twice the difference between the loglikelihood of different models were posteriorly transformed into exactP values using PAML 4.6 (Yang, 2007). The x2 distributions with the degreeof freedom = 2 and degree of freedom = 1, which have been shown to beconservative under conditions of positive selection (Zhang, 2004), wereused to perform tests 1 and 2, respectively. Probabilities of sites underpositive selection were obtained using Bayesian approaches (Yang et al.,2005) implemented in PAML.

Homology Modeling, Molecular Docking,and Mutagenesis

Homology models were constructed within the SWISS-MODELWorkspaceusing the automatic alignment algorithm (Arnold et al., 2006). The crystalstructures of CPS from Arabidopsis (Protein Data Bank nos. 3pya and 3pyb)were used as the templates (Köksal et al., 2011, 2014). The ligand dockingmodeling was performed with AutoDock Vina (Trott and Olson, 2010). Modelvisualization and binding site analysis were performed using PyMOL (http://www.pymol.org). Mutants were generated by whole-plasmid PCR amplifica-tion with overlapping mutagenic primers of the pGEM-T vector (Promega)clones and verified by complete gene sequencing prior to subcloning into theexpression vector pET32a (Merck). The resulting constructs were heterologousexpressed and analyzed as described above.

Sequence data from this article can be found in the GenBank/EMBL datalibraries with the following accession numbers: SmCPS1 (KC814639), SmCPS2(KC814640), SmCPS3 (KC814641), SmCPS4 (KP063138), SmCPS5 (KC814642),and SmKSL2 (KC814643).

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. EI mass spectrum of diterpenes in this study.

Supplemental Figure S2. The dwarf phenotype caused by down-regulation of SmCPS5.

Supplemental Figure S3. The intron/exon structure divergence of diTPSgenes involved in labdane-related diterpenoid biosynthesis in Arabidopsis,rice, and S. miltiorrhiza.

Supplemental Figure S4. Homology modeling and molecular docking re-sults show the positive selection sites.

Supplemental Figure S5. GC-MS analysis of the six mutant enzymes ac-tivity of SmCPS1 and SmCPS5.

Supplemental Table S1. Information of diTPS gene family inS. miltiorrhiza.

Supplemental Table S2. List of plant diTPS used in this paper.

Supplemental Table S3. Different changed metabolites obtained frommetabolomics analysis of wild-type and SmCPS1-RNAi lines byLC-qTOF-MS.

Supplemental Table S4. Different changed metabolites obtained frommetabolomics analysis of wild-type and SmCPS1-RNAi lines byGC-QqQ-MS.

Supplemental Table S5. Summary of statistics for detection of positiveselection for the CPS group.

Supplemental Table S6. Information about primers used in this study.

Received May 7, 2015; accepted June 10, 2015; published June 15, 2015.

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