High-throughput quantitative expression analysis of S. fruticosa genes.

For gene expression studies using the Biomark HD system, total S. fruticosa RNA was isolated from trichomes and leaves without trichomes from three different developmental stages of leaves (starting from the first expanded pair of leaves—stage 1, following with stage 4 and 7). Additionally, total RNA was prepared from trichomes and leaves without trichomes of mechanically wounded leaves belonging to the stage 1. Wounding was performed by cutting the leaves in uniform stripes with scissors in planta, and samples were collected after 0, 3, and 6 hours. Trichome isolation was performed using a combination of dry ice and synthetic brush abrasion methods, as explained in the Supplementary Information (S1 Method). Total RNA was isolated using Spectrum Plant Total RNA Kit (Sigma, USA) from three independent biological replicates for the gene expression studies of different developmental stages, while for the wounding assays, two biological replicates were used.

To remove traces of genomic DNA contamination, the RNA samples (1 μg) were treated with DNase I (DNA-free Kit, Ambion, USA) in a final reaction volume of 25 μl. After removal of genomic DNA, RNA quality was determined using Bioanalyzer 2100 (Agilent, USA). For RT-PCR, cDNA was synthesized from 400 ng of total RNA (DNA free) using Superscript III Reverse Transcriptase (Life Technologies, USA) and oligo-(dT) primers. Gene expression was quantified by real-time PCR using the Biomark HD instrument (Fluidigm, USA) and 2x SsoFast EvaGreen supermix with low Rox (Biorad, USA) dye. The cDNA samples were diluted to 6 ng/μl and pre-amplified using TaqMan PreAmp Master mix (Life Technologies, USA). Primers were used at a final concentration of 500 nM. After pre-amplification cDNAs were treated with Exonuclease I to remove leftover primers. The qPCR analysis was performed in technical duplicates following manufacturer’s instructions. Primer pairs for each gene candidate were designed based on the final isolated nucleotide sequences using Primer3 (http://bioinfo.ut.ee/primer3-0.4.0/) and PrimerQuest (http://eu.idtdna.com/PrimerQuest/Home/Index). The PCR efficiency for each primer pair used was calculated according to a dilution series from a pooled cDNA sample including all biological treatments. Expression fold was calculated using an efficiency-corrected delta-Ct method [50]. The pp2a gene was selected as endogenous reference for normalization of transcript levels.

qPCR of S. fruticosa CPS and KSL genes.

For the analysis of gene expression, young (1–2 cm) and aged (3–4 cm) leaves were collected. Leaf material was frozen in liquid N₂ immediately after the collection and stored at -80°C until further processing. Isolation of trichomes was performed using the dry-ice abrasion method (S1 Method).

High quality total RNA was isolated using the commercial Spectrum Plant Total RNA Kit (Sigma, USA), according to the manufacturers’ specifications with additional ethanol washes. cDNA was constructed from 1 μg total RΝΑ using the Superscript III kit (Life Technologies, USA) according to manufacturers’ specifications with 1 μg random primers (Life Technologies, USA).

For gene expression analysis, primer pairs were manually designed (S3 Table). The endogenous control selected and used for normalizing all quantitative real-time PCR (qPCR) analyses was the housekeeping gene elongation factor 4a (elf4a) [27]. Samples were prepared using the KAPA SYBR® FAST qPCR Kit (KAPA Biosystems, USA) and qPCR analysis for each sample was performed with three technical replicates in an Applied Biosystems 7500 Real-Time PCR System (Life Technologies, USA). General thermocycler conditions were 95°C for 2 min; then 6 cycles of 95°C for 35 s; 64°C for 30 s; 72°C for 30 s; followed by 35 cycles of 95°C for 20 s; 62°C for 20 s; 72°C for 15 s; a final extension at 72°C for 10 min and plate read at 76°C. To identify and verify the PCR products a melting curve was performed from 70°C to 95°C with readings every 0.1°C and a 10 s hold between observations. Relative expression values were calculated by the expression data of isolated trichomes relative to those from leaves without trichomes. qPCR data and the correct relative quantitation of results (RQ), and therefore the relative expression for genes, was calculated based on the efficiency-adjusted delta Ct method, where the fold change in the target gene was determined with the following formula:

Fold change = E-^ΔΔCT, where ΔΔCT = (Ct_{target gene}- Ct_Ref) at Point X—(Ct_{target gene}- Ct_Ref) a Point Y, and E is the efficiency of the reaction [27].

It must be noted that each time, the statistical analysis takes into account the efficiencies of the reactions. Normalized expressions of the SfCPS and SfKSL genes were calculated using the software program Q-gene [51].

Isolation of SfCPS and SfKSL genes.

For gene isolation, a leaf trichome cDNA library of S. fruticosa provided by previous work [27] was initially used. Two partial sequences of potential diterpene synthase genes (contig 66 and contig 195) were identified by sequence homology to known diterpene synthase coding genes. As a first step to obtaining the 5’ and 3’ region of the candidate genes, PCR amplification with Platinum Taq DNA Polymerase (Life Technologies, USA), was performed using the above cDNA library as a template. The PCR conditions were as follows: 1 cycle of 2 min at 94°C; 35 cycles of 30 sec at 94°C, 30 sec at 55°C, and 2 min and 30 sec at 72°C; and a final extension of 7 min at 72°C.

For contig 195 (putative SfCPS gene), combinations of gene-specific (793–03_F22_F1 and 04_K19_R) and library plasmid-specific primers (M13F and pDNRLib reverse 2) were used (S4 Table). In this way, additional but incomplete information about the SfCPS sequence was obtained, with the 5’ end of the gene still not revealed. To isolate the missing sequence of SfCPS, 5´ RACE System for Rapid Amplification of cDNA Ends (Life Technologies, USA) was further applied using S. fruticosa trichome total RNA as a template. Trichomes from young leaves were extracted using the dry-ice abrasion method (S1 Method). RNA extraction was performed using the Spectrum Plant Total RNA Kit (Sigma, USA). For the reverse transcription reaction, gene specific primer 5CopR172_GSP1 was used and the cDNA was (dC)-tailed. Amplification was performed using the two rounds of PCR, with a combination of the RACE kit primers (Abridged Anchor Primer—AAP, Abridged Universal Amplification Primer—AUAP) and gene specific primers (first round- 5CopR140_GSP2, nested- 5CopR83_GSP3) (S4 Table), using the described PCR conditions. The 5´ RACE has resulted in identification of the SfCPS complete sequence.

PCR amplification using the cDNA library did not result in isolating any additional sequence based on contig 66. It has resulted, though, in the isolation of 5’ and 3’ region of a similar gene, with even higher homology to the family of KSLs. The primers were 424_02_A10F and RCA02_A10_R (contig 66-specific), and M13F and pDNRLib reverse 2 (library plasmid-specific). Using the cDNA library, the sequence of this gene, named SfKSL, was fully identified. Finally, full-length cDNA of both SfCPS and SfKSL was amplified from trichome cDNA using Platinum Taq DNA Polymerase (Life Technologies, USA), ligated into pCRII-TOPO vector using the Invitrogen TA cloning system (Life Technologies, USA) and verified by sequencing. ORF sequences for both isolated genes have been deposited in the GenBank with the following accession numbers: KP091840 (SfCPS) and KP091841 (SfKSL).

Isolation of cytochrome P450 genes.

The cDNA sequences coding for candidate P450s of S. fruticosa and R. officinalis were obtained based on trichome EST databases (http://www.terpmed.eu/). Two non-overlapping S. fruticosa singleton sequences (Sfru.N01.C016156 and Sfru.N01.C021415), appeared to be transcripts of the same gene, according to a BLAST analysis. Sfru.N01.C016156 sequence represented the 5′ end of the gene and contained the START codon, while Sfru.N01.C021415 sequence comprised the 3′ region with the STOP codon. Primers for the full length sequence were designed based on the two singletons. For R. officinalis, two sequences with high similarity to each other (92,4% similarity of nucleotides), Roff.N01.C021640 and Roff.N01.C021643, were identified as candidates. The genes were amplified from the cDNA using Phusion Hot Start Flex DNA Polymerase (New England Biolabs, USA), cloned into pCRII-TOPO vector and sequenced.

Phylogenetic analysis.

Multiple sequence alignments were generated using the ClustalW program. A Neighbor—Joining tree was constructed using MEGA version 5 [52] with a bootstrap of 1000 replicates, and rooted with Physcomitrella patens PpCPS/KS as an outgroup. Sequence analysis with TargetP 1.1 methodology has been used to predict the length of the transit peptide-coding sequences of the isolated genes (http://www.cbs.dtu.dk/services/TargetP/) [53]. A protein alignment using ClustalW (MegAlign, DNAStar) was done to calculate sequence distances in S12 Fig.

E. coli expression.

Based on the predictions performed using the TargetP 1.1 algorithm, the region coding for putative transit peptide of SfCPS was 90 bp, and for the SfKSL, 147 bp long. Using these results, pseudomature versions (m in the construct name) of the two genes were cloned without the N-terminal transit peptide by PCR amplification from the pCRII-TOPO vector using Platinum Taq DNA Polymerase with the addition of restriction sites. PCR products were cloned into pCRII-TOPO vector. Upon sequence confirmation, the pseudomature fragments were subcloned into ptacHA expression vector, yielding the final plasmids ptacHA-SfCPSm and ptacHA-KSLm. ptacHA expression vector was created from pGEX-5X vector (Pharmacia, USA) by replacing a GST-tag with N-terminal polyhistidine (6xHis) tag and a Human influenza hemagglutinin (HA) tag.

The mature versions of SfCPS and SfKSL ligated into ptacHA vector were expressed in E. coli BL21-CodonPlus (DE3)-RIL competent cells. Liquid cultures of the bacteria harbouring the expression construct were grown to appropriate OD₆₀₀ in Luria-Bertani medium supplemented with antibiotics for selection (50 μg/ml Ampicillin, 50 μg/ml Kanamycin, 17 μg/ml Chloramphenicol) with shaking at 37°C prior to induction with isopropyl thio-galactopyranoside (IPTG). After induction, cultures were grown at room temperature. In order to define the optimal conditions for bacterial expression of the mature SfCPS, combinations of different induction and growth parameters were tested (data not shown). Incubation at room temperature for 4 h of the culture induced with 0.4 mM IPTG, at the moment when the OD₆₀₀ reached 0.5, gave the highest protein yields. These conditions were, thus, applied for producing the protein that was further purified and used in the enzymatic assays. For SfKSL, induction was performed in the same way as for SfCPS, with the only difference being that, after induction, cultures were incubated at room temperature for 16 h. Cell pellets collected after expression were either resuspended in SDS-PAGE sample buffer directly for SDS-PAGE analysis of crude extract, or stored at -20°C to be purified by Ni-NTA affinity chromatography.

Bacterial expression vector, ptacHA, carries an N-terminal polyhistidine (6xHis) tag, which has enabled the purification of overexpressed protein by Ni-NTA affinity chromatography. Cell pellets were resuspended in ice-cold lysis buffer (50 mM NaH₂PO₄, 300 mM NaCl, 10 mM Imidazole [pH 8.0]) with the addition of lysozyme (1 mg/ml) and 1 mM phenylmethylsulfonyl fluoride (PMSF). The amount of lysis buffer added was 1 ml per 10–20 ml of starting bacterial culture. The cell suspension was left on ice for 30 min, after which it was disrupted by sonication on ice until becoming slightly translucent. The lysate was cleared by centrifugation at 13000 g for 20 min at 4°C. 50 μl of 50% Talon metal affinity resin (Clontech, USA) equilibrated in lysis buffer was added per 1 ml of the cleared lysate. The bead suspension was incubated with rotation at 4°C for 60 min to allow the polyhistidine-tagged protein to bind the resin. The beads were then collected with centrifugation at 1400 rpm at 4°C in a benchtop cooled centrifuge. The beads were washed three times with washing buffer (50 mM NaH₂PO₄, 300 mM NaCl, 20 mM Imidazole [pH 8.0]). The amount of washing buffer was 1 ml per 100 μl of 50% Ni²⁺ agarose slurry. The polyhistidine-tagged protein was eluted by adding 100 μl of elution buffer (50 mM NaH₂PO₄, 300 mM NaCl, 250 mM Imidazole [pH 8.0]) per 100 μl of 50% Ni²⁺ agarose slurry. Three separate elution fractions were collected. Aliquots of the washing and elution fractions were analysed by SDS-PAGE, and the first elution fraction was stored in 50% glycerol at -80°C to be assayed for activity later.

Affinity purified SfCPS was assayed for activity by using 5 μl recombinant protein in a buffer containing 10 mM MOPS, pH 7.0, 20 mM MgCl₂, 0.2 mM MnCl₂, 1 mM dithiothreitol and 60 μM geranylgeranyl pyrophosphate (GGDP) as a substrate, in 0.25 ml final volume. Assay mixtures were incubated overnight at room temperature and the resulting mixtures were incubated at 37°C for 4 h with 0.025 units/μl of bacterial alkaline phosphatase (Takara, Japan) with the addition of equal volumes of hexane overlay. Hydrolyzed products were hexane extracted and traces of water were removed by adding a small amount of Na₂SO₄, which was next removed by centrifugation. The extracts were concentrated to 50 μl by rotary evaporation prior to analysis by GC-MS.

To identify SfKSL products, enzyme reactions contained 3 μl of SfKSL affinity purified protein in assay buffer, as above, containing 2 μl of purified SfCPS, 200 μM GGDP (substrate for SfCPS) in 50 μl final volume. Reaction mixtures were immediately carefully overlaid with equal volume of hexane, and left to incubate at room temperature overnight. Reaction products were hexane extracted and treated as above to remove traces of water. The extracts were stored at 4°C to be analysed by GC-MS.

Expression assays in Saccharomyces cerevisiae.

SfCPSm and SfKSLm were both subcloned from ptacHA into yeast (Saccharomyces cerevisiae) expression vectors, pUTDH3 (P_TDH3 2 μ URA3) for the SfCPSm and pHTDH3myc (P_TDH3 2 μ HIS3) for SfKSLm, yielding the final plasmids pUTDH3-SfCPSm and pHTDH3-SfKSLm. The plasmids were co-transformed into AM104 yeast strain and the clones containing both plasmids were selected on glucose CM media lacking uracil and histidine. The final yeast strains were cultivated in 50 ml liquid media at 30°C and 250 rpm.

The strain AM104 was developed from the previously described AM102 strain [54], harboring heterozygous deletions in the genes UBC7/ubc7, ssm4/SSM4, erg9/ERG9 and chromosomally integrated expression cassettes overexpressing a stabilized variant of HMG2 was used the parental strain for further modification. The strain was modified with COD7/CcGGDPS1 cassette, which was developed by PCR amplifying the ORF of CcGGDPS1 [29] using primers CcGGDPS1-BamHI and CcGGDPS1-XhoI (S5 Table). The amplified ORF was cloned into the COD7 integration cassette vector [54] (P_TDH3-MCS-ts-LoxP-HIS5-LoxP) using BamHI and XhoI restriction enzymes generating the construct COD7/CcGGDPS1 (P_TDH3-CcGGDPS1-ts-LoxP-HIS5-LoxP). The COD7/CcGGDPS1 cassette was then PCR amplified using primers 5-FLO8-COD7 and 3-FLO8-COD7 (S5 Table) which incorporate flanking sequences that are homologous to the 3’UTR region of FLO8 gene. The cassette was transformed into AM102 cells and colonies growing in the absence of histidine were selected. The transformed colonies were tested by co-expressing CrtI and CrtYB genes of carotenoid biosynthesis. The strain with most intense coloration due to carotenoid production was selected. The HIS5 selection marker was excised by transiently expressing Cre recombinase, as previously described [55], generating strain AM104 (S6 Table).

SfFS, RoFS1 and RoFS2 were subcloned from pCRII-TOPO constructs into the yeast expression vector pWTDH3 (P_TDH3 2 μ TRP1). SfFS was transformed into the above mentioned yeast strain AM104, which already contained the SfCPSm and SfKSLm gene constructs. RoFS1 and RoFS2 were individually transformed into the AM113 yeast strain containing the RoCPS1m and RoKSL1f gene constructs [24]. The AM113 strain harbors heterozygous deletions for ERG9, UBC7, SSM4, MCT1, WHI2, and expresses chromosomal inserted GGDPS1 from Cistus creticus, the FDPS1 from Salvia fruticosa and further contains two copies of a stabilized HMG2 variant [24]. All the yeast strains were additionally transformed with a PtCPR2 gene [32], cloned into the pYX143 vector. Produced volatiles by the transformed yeast cells were sampled by Solid Phase Micro-extraction (SPME) by using 2 cm-50/30 μm VB/Carboxen/PDMS StableFlex Fiber for Manual Sampling (Supelco, USA). Yeast cultures were grown in 50 ml flasks to saturation achieved after 24–48 h and volatiles were collected by SPME fibre for 30 min. The exposed SPME fibre was withdrawn into the outer septum-piercing needle and removed from the flask, which made it ready for analysis using GC-MS.

Liquid-liquid extraction of the 25 ml yeast cultures was performed with 3×25 ml of pure hexane. After overlaying with hexane, the cultures were sonicated for 15 min in an ultrasonic water bath at 40°C. Hexane extracts were combined, dried using anhydrous MgSO₄, evaporated under a stream of air to a final volume of 100 μl and analysed by GC-MS. Extraction for the structural characterization by NMR was performed by overlaying yeast cultures (750 ml) with equal volumes of hexane followed by vigorous shaking by hand. The extraction was repeated three times.

Transient expression of SfCPS, SfKSL, SfFS, RoFS1 and RoFS2 in Nicotiana benthamiana.

The entire open reading frame (ORF) of each gene (SfCPS, SfKSL, SfFS, RoFS1 and RoFS2) was PCR-amplified from pCRII-TOPO constructs with the addition of restriction sites. SfCPS, SfKSL, RoFS1 and RoFS2 sequences were subcloned into the expression vector pGA643 [56], while in the case of SfFS, pART7/pART27 binary vector system was used [57]. These expression vectors contain the CaMV 35S promoter and the nptII kanamycin resistance gene. Upon sequencing, constructs pGA643-SfCPS, pGA643-SfKSL, pART27-SfFS, pGA643-RoFS1 and pGA643-RoFS2 were introduced into A. tumefaciens GV3101 (pMP90) strain using the freeze-thaw method. An additional transformation was performed with cytochrome P450 reductase from A. thaliana (AtCPR1) cloned in pICH47732 vector. Transformed cells were grown on LB agar plates supplemented with antibiotics for selection of transformants until the appearance of visible colonies (rifampicin 50 μg/ml, gentamycin 25 μg/ml, and tetracycline 15 μg/ml for pGA643 constructs; rifampicin 50 μg/ml, gentamycin 25 μg/ml and spectinomycin 25 μg/ml for pART27 construct). LB broth containing appropriate antibiotics was inoculated from single colonies and the cultures were grown for 48 h at 28°C with vigorous shaking (250 rpm). Cells were harvested by centrifugation for 20 min at 4000×g. In addition, Agrobacterium strain harbouring a gene encoding the viral p19 silencing suppressor was also prepared [58]. For SfFS, RoFS1 and RoFS2 transient expression, Nicotiana benthamiana plants stably expressing the p19 silencing suppressor gene kindly provided by Dr. Kriton Kalantidis (Univ. of Crete, Heraklion, Greece) were used for the infiltration experiments.

Next, the cells were resuspended in the buffer containing 10 mM MES, 10 mM MgCl₂ and 100 μM acetosyringone (Sigma, USA) to a final OD₆₀₀ of approximately 0.5 and left to incubate at room temperature with gentle shaking (50 rpm) for 2 h. Equal volumes of A. tumefaciens cells from each gene construct resuspended in the buffer were used to infiltrate leaves of N. benthamiana, with a needleless 1 ml syringe. Plants were grown in the growth chamber at 23°C with 16 h light/8 h dark cycle for five days. After five days, infiltrated leaves were harvested and pulverized in liquid N₂ with mortar and pestle. One gram of fine powder was extracted with 2×3 ml pure hexane after a brief vortexing followed by sonication for 15 min in an ultrasonic water bath. Combined hexane phases were separated from the precipitated leaf tissue after centrifugation for one min at 5000 rpm, dried using anhydrous MgSO₄, evaporated under a stream of air to a final volume of 50 μl and analysed by GC-MS.

GC-MS analysis.

GC-MS analyses for the E. coli, yeast and N. benthamiana extracts were carried out using a Shimadzu GC/MS-QP2010 system (Shimadzu, Germany) connected to a model QP-2010 electron impact (EI) mass spectrometer. Data acquisition and control of the GC/MS-QP2010 system was carried out using the GC-MS solution software. Chromatographic separation was achieved on an HP-5-MS capillary column (Agilent, USA) (30 m×0.25 mm inner diameter; 0.25 μm film thickness) for the yeast and N. benthamiana extract analysis, and a ZB5 (Phenomenex, USA) (0.25 mm×30 m, 0.25 μm film thickness) column for the E. coli expression extract. The carrier gas was ultra-high purity helium at a flow rate of 1.03 ml/min. For the enzymatic assay products (E. coli expression) the hexane extract was manually injected into the heated injector in the splitless mode. The inlet temperature was set to 230°C. The column temperature was raised from 60°C to 240°C with a rate of 3°C/min and maintained at this temperature for 5 min to equilibrate. For the yeast and N. benthamiana extract analyses, splitless mode was also used for the injector with the inlet temperature set to 230°C. The column temperature was initially held at 60°C for 1 min, then raised at 240°C with a rate of 3°C/min and held for 15 min. The extracts (1 μl) were injected automatically via the AOC20i-AOC20s autosampler. For the analysis of SPME sampled volatiles, manual injection of the device needle into the heated injection port of the gas chromatogram was performed.

Chromatographs in Fig 11 were generated using a 2010 Plus Shimadzu gas chromatograph with GCMS-QP2010 Ultra gas chromatograph mass spectrometer. Chromatographic separation was achieved on a MEGA-5 MS capillary column (MEGA s.n.c., Italy) (30 m×0.25 mm inner diameter; 0.25 μm film thickness). The column temperature was initially held at 50°C for 3 min, then raised at 300°C with a rate of 14°C/min and held for 3 min. The extracts (2 μl) were injected automatically via the AOC-20 Dual autosampler.

Structure elucidation.

For structural characterization by NMR, yeast culture extracts were purified by silica gel flash column chromatography in hexane. This resulted in obtaining a fraction that contained 20 mg of the compound of interest, which was a satisfactory amount for the analysis by NMR.

NMR spectra for miltiradiene were recorded at 25°C on a Bruker Avance III at 300 MHz for ¹H and 75 MHz for ¹³C, respectively, using CDCl₃ as solvent. Chemical shifts are expressed in δ values (parts per million) relative to TMS as internal standard for ¹H and relative to TMS (0.00 ppm) or to CDCl₃ (77.05 ppm) for ¹³C NMR spectra. Coupling constants ⁿJ are reported in Hertz. Moreover, the complete assignment of ¹H and ¹³C NMR signals was possible using 1D and 2D experiments (COSY H-H, HMQC, and HMBC) using the standard commercial pulse programs.

The first structure elucidation for miltiradiene was reported earlier by Gao et al. (2009) followed by Sugai et al. (2011) [18,59]. The mass spectrum of the product showed a molecular ion at m/z 272. The 20 different carbon signals from the ¹³C-NMR in conjunction with the molecular ion led to a possible diterpene structure without any symmetry. The four alkenyl carbon signals at 140.0, 135.5, 124.0, and at 116.5 ppm revealed two C = C double bonds. Further, 2D NMR data analysis suggested that the synthesized product was the miltiradiene. The proton signal at 1.26 ppm corresponding to the carbon resonated at 29.8 ppm is attributed to an impurity from hexane used for extractions. The complete assignment of proton and carbon chemical shifts is presented in S7 Table and in S4 Fig.

In rings A and B the methylene protons are arranged as axial or equatorial and resonated at about 1.1–1.3 and 1.5–1.7 ppm, respectively. The methylene carbons C7, C11 and C14 are attached to alkenyl carbons of ring C having an almost coplanar conformation. Therefore, the protons corresponding to these methylene groups are not diversified and give an almost identical pattern of multiplets. The almost identical methyl signals for C16 and C17 show two doublets with coupling of 7.0 Hz with the methine proton at C15. The differentiation of the two C4 methyl groups is achieved on the basis of NOE and ¹³C chemical shift data. Namely, there is a positive NOE between methyl protons resonated at 0.99 and at 0.86, corresponding to C20 and C19, respectively. There is also a significant difference in the ¹³C chemical shift data of the two C4 methyl groups attributed to their axial and equatorial configuration. C19 methyl and C20 methyl groups both being in axial configuration have 1,3-diaxial steric interaction resulting thus for both methyl carbons upfield resonances around 20 ppm, whereas the C18 methyl being in equatorial configuration resonates at 33.3 ppm.

The structure of miltiradiene, with numbering of skeleton as well as with assignments of proton and carbon NMR chemical shifts is depicted in S4 Fig. Moreover, in S5 Fig–S8 Fig the ¹H and ¹³C NMR spectra are presented, whereas in S9, S10 and S11 Figs some diagnostic HMBC correlations between carbons and protons via ²J_CH and ³J_CH couplings are illustrated. In the case of cyclohexadiene ring C correlations are observed also via ⁴J_CH couplings through the double bonds.

LC-PDA-MS analysis of phenolic diterpenes.

Phenolic diterpenes were monitored in S. fruticosa a) whole leaves of all stages of development from the 3 different genotypes mentioned in “Plant Material” section, b) from young and old leaves of the genotype Kavoussi and c) from isolated trichomes and leaves without trichomes of very young leaves (appr. 1 cm long) of the genotype Kavoussi. The pots had 2 l capacity, containing peat and perlite in ratio 3:1. Plants were watered with a complete balance fertilizer every 20 days. The supplement light followed the natural photoperiod (10 h) and the distance between plants and lamps was 120–160 cm, with a light volume measured at 16–12 Klux. Trichome isolation was performed using the bead-beater abrasion method (S1 Method). The leaves' material was frozen in liquid N₂ and ground to a fine powder with mortar and pestle. For each experiment, three independent biological samples were processed.

Next, compounds were extracted by adding 75% methanol containing 0.1% formic acid in a ratio of 5:1 (ml: g FW), followed by sonication for 15 min. Extracts were centrifuged and filtered over a 0.45 μm PTFE (Teflon) filter into an HPLC vial with glass insert. Phenolic diterpenes were separated, identified and quantified by using a LC-PDA-LTQ-Orbitrap FTMS system (Thermo Scientific) consisting of an Accela HPLC with photodiode array detector (240–600 nm) connected to an LTQ/Orbitrap hybrid mass spectrometer equipped with an ESI source. Injection volume was 5 μl. Chromatographic separation was on a reversed phase column (Luna C18/2, 3 μ, 2.0×150 mm; Phenomenex, USA) at 40°C, using a linear gradient from 5 to 75% acetonitrile (acidified with 0.1% formic acid) in 45 min and a flow rate of 0.19 ml/min. FTMS full scans (m/z 90–1200) were recorded in negative ionization mode with a resolution of 60,000. Phenolic diterpenes were identified based on similarity with the authentic standards of carnosic acid and carnosol, with respect to both absorbance spectrum, accurate mass, allowing a maximum of 3 ppm deviation from the calculated masses, and co-elution. Both compounds were quantified based on the chromatographic area of their specific mass.

Statistical analysis.

Values are expressed as means ± SD or ± SE, and statistical significance was analyzed by Student’s t-test. All probability (p) values were based on two-tailed tests, and p < 0.05 was considered significant.