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Biochem. J. (2013) 449, 729–740 (Printed in Great Britain) doi:10.1042/BJ20120988 729

Functional characterization of long-chain prenyl diphosphate synthasesfrom tomatoMatthew O. JONES, Laura PEREZ-FONS, Francesca P. ROBERTSON, Peter M. BRAMLEY1 and Paul D. FRASERSchool of Biological Sciences, Royal Holloway, University of London, Egham, Surrey TW20 0EX, U.K.

The electron transfer molecules plastoquinone and ubiquinone areformed by the condensation of aromatic head groups with long-chain prenyl diphosphates. In the present paper we report thecloning and characterization of two genes from tomato (Solanumlycopersicum) responsible for the production of solanesyl anddecaprenyl diphosphates. SlSPS (S. lycopersicum solanesyldiphosphate synthase) is targeted to the plastid and both solanesoland plastoquinone are associated with thylakoid membranes.A second gene [SlDPS (S. lycopersicum solanesyl decaprenyldiphosphate synthase)], encodes a long-chain prenyl diphosphatesynthase with a different subcellular localization from SlSPS andcan utilize geranyl, farnesyl or geranylgeranyl diphosphates inthe synthesis of C45 and C50 prenyl diphosphates. When expressedin Escherichia coli, SlSPS and SlDPS extend the prenyl chain

length of the endogenous ubiquinone to nine and ten isopreneunits respectively. In planta, constitutive overexpression of SlSPSelevated the plastoquinone content of immature tobacco leaves.Virus-induced gene silencing showed that SlSPS is necessaryfor normal chloroplast structure and function. Plants silenced forSlSPS were photobleached and accumulated phytoene, whereassilencing SlDPS did not affect leaf appearance, but impacted onprimary metabolism. The two genes were not able to complementsilencing of each other. These findings indicate a requirement fortwo long-chain prenyl diphosphate synthases in the tomato.

Key words: isoprenoid, plastoquinone, solanesol, tomato,ubiquinone, virus-induced gene silencing (VIGS).

INTRODUCTION

The prenylquinones PQ (plastoquinone) and UQ (ubiquinone) areelectron carriers involved in numerous biochemical processes.In the chloroplast PQ is a component of the electron-transportchain, mediating the flow of electrons from photosystem II to thecytochrome b6f complex [1]. PQ is a component of the redoxchain associated with the desaturation of phytoene [2], whereasthe redox state of the PQ pool has been shown to regulate theexpression of nuclear- and plastid-encoded genes involved inphotosynthesis [3]. UQ acts in the mitochondria where it hasa central role in the mitochondrial respiratory chain, mediatingelectron transfer from the NADH dehydrogenase complex throughto the cytochrome bc1 complex [4]. Its reduced form, ubiquinol, isan antioxidant preventing the initiation of lipid peroxidation andphoto-oxidation of mitochondrial lipids.

Structurally PQ and UQ comprise an aromatic head groupwith an isoprenoid (prenyl) side chain to confer lipid solubility.Isoprenoids are derived from a five carbon precursor, IPP(isopentenyl diphosphate), and its isomer DMAPP (dimethylallyldiphosphate). In plants these are produced by the cytosolicMVA (mevalonic acid) pathway and in plastids via theMEP (methylerythritol 4-phosphate) pathway [5]. Labellingexperiments have demonstrated that the IPP utilized in theformation of the UQ side chain is synthesized from MVA, whereasPQ originates from IPP formed via the MEP pathway [6,7].Thus these two prenylquinones not only function in separate

organelles, but also use IPP formed in different subcellularcompartments. Prenyl chains are assembled from the consecutivecondensation of IPP and its allylic isomer DMAPP, involving theformation of short chains, such as FPP (farnesyl diphosphate,C15) and GGPP (geranylgeranyl diphosphate, C20), which in turnare elongated further by a class of enzymes known as long-chain prenyl diphosphate synthases. The length of the chain isdetermined by the activity of these enzymes and is known to varyamong different organisms and is determined by the specificityof the long-chain prenyl diphosphate synthases present [8].SPSs (solanesyl diphosphate synthases) and DPSs (decaprenyldiphosphate synthases) are responsible for the production of theC45 and C50 prenyl chains respectively (Figure 1A).

To produce PQ, solanesyl diphosphate is attached to thearomatic ring of homogentisate by the prenyl transferase [9],known as HST (homogentisate solanesyl transferase). Thisreaction marks the first committed step in PQ formation and resultsin the formation of 2-methyl-6-solanesyl-1,4-benzoquinol [10].This is then methylated by MSBQ MT (2-methyl-6-solanesyl-1,4-benzoquinone methyltransferase) to produce PQ. These reactionsare believed to occur exclusively in the plastid [11]. In themitochondrion, the condensation of the aromatic intermediatePHB (p-hydroxybenzoic acid) with the polyprenyl diphosphateis catalysed by PPT (PHB polyprenyltransferase), leading toUQ formation (Figure 1B and [12]). However, little is knownabout their functional equivalents in crop species, includingtomato.

Abbreviations used: ABTS, 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid); ACN, acetonitrile; APCI, atmospheric pressure chemical ionization;APPI, atmospheric pressure photoionization; AtPPPS, Arabidopsis thaliana trans-type polyprenyl pyrophosphate synthase; CaMV, cauliflower mosaicvirus; DET1, DE-ETIOLATED1; DMAPP, dimethylallyl diphosphate; DPS, decaprenyl diphosphate synthase; DTT, dithiothreitol; DW, dry mass; FPP, farnesyldiphosphate; GFP, green fluorescent protein; GGPP, geranylgeranyl diphosphate; GGPPS, GGPP synthase; GPP, geranyl diphosphate; hp, high-pigment;IPP, isopentenyl diphosphate; LC, liquid chromatography; MALDI, matrix-assisted laser-desorption ionization; MDA, malonaldehyde; MEP, methylerythritol4-phosphate; MS/MS, tandem MS; MVA, mevalonic acid; nESI, nano electrospray ionization; PDA, photodiode array; PDS, phytoene desaturase; PHB,p-hydroxybenzoic acid; PPT, PHB prenyltransferase; PQ, plastoquinone; qRT–PCR, quantitative reverse-transcription PCR; SlDPS, Solanum lycopersicumDPS; SPS, solanesyl diphosphate synthase; AtSPS, A. thaliana SPS; OsSPS, Oryza sativa SPS; SlSPS, S. lycopersicum SPS; TBARS, 2-thiobarbituricacid-reacting substance; TEAC, Trolox equivalent antioxidant capacity; TFA, trifluoroacetic acid; TOF, time-of-flight; UQ, ubiquinone; VIGS, virus-inducedgene silencing.

1 To whom correspondence should be addressed (email [email protected]).

c© The Authors Journal compilation c© 2013 Biochemical Society

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730 M. O. Jones and others

Figure 1 Biosynthesis of PQ-9 and UQ-10 in the tomato

The long-chain prenyl diphosphate tails are produced from isoprenoid precursors (A) and subsequently combined with aromatic intermediates to produce PQ-9 and UQ-10 (B). HST, homogentisatesolanesyl transferase; OPP, diphosphate moiety.

The hp (high-pigment) tomato mutants are characterized bysimultaneous elevation of multiple classes of phytochemicals andexaggerated responsiveness to light. The hp phenotype resultsfrom defects to genes encoding negative regulators of phyto-chrome signal transduction [13,14] and manipulation of lightsignalling pathways has been seen as an effective strategy toimprove the nutritional value of the tomato [15]. Transgenicsuppression of the gene responsible for the hp2 mutant, DET1(DE-ETIOLATED1) resulted in substantial elevations in thecarotenoid and flavonoid content in fruit [16]. Multi-level ‘omic’characterization of these varieties has revealed that co-ordinatedup-regulation of core metabolic processes, such as the Calvincycle and photorespiration, as well as plastid size are the probableprogenitors of the hp chemotype, rather than transcriptionalactivation of isoprenoid biosynthetic pathway genes [17]. Inthe present study, we have identified a putative trans-long-chainprenyl diphosphate synthase gene that is up-regulated in responseto DET1 silencing in tomato fruit, concomitant with increasedaccumulation of solanesol and PQ. This and a further putativetomato long-chain prenyl lipid synthase has been characterizedin vitro and in vivo, their biochemical role in prenyl lipid formation

elucidated and the effect of modulating PQ and UQ levels on themetabolome determined.

EXPERIMENTAL

Plant material

Tobacco (Nicotiana tabacum cv Wisconsin 38) was used forthe generation of transgenic plants. Plants were grown under a16 h photoperiod at 25 ◦C. The tomato (Solanum lycopersicum)T56 genotype was used for the isolation of long-chain prenyldiphosphate synthases and VIGS (virus-induced gene silencing)treatments. Homozygous TFM7 was used as the DET1 down-regulated genotype and grown as described in [17]. Nicotianabenthamiana was used in the transient expression system.

Vector construction

The tomato SPS full-length coding sequence (1194 bp, GenBank®

accession number DQ889204) was amplified from tomato leafcDNA using KOD Hot Start DNA Polymerase (Novagen) with


Tomato long-chain prenyl diphosphate synthases 731

oligonucleotide primers containing restriction sites (underlined)SPSNdeI_For (5′-CGGACATATGTCTGTGACTTGCCATAA-TC-3′) and SPSBamHI_Rev (5′-CTGAGGATCCCTATTCAAT-TCTCTCCAGAT-3′). The amplified products were cloned intothe NdeI-BamHI sites of the Escherichia coli expression vectorpET14b (Novagen).

For construction of the plant transformation vectorpEXP35SSPS, two oligonucleotide primers (SPSTOPO_For,5′-CACCATGTCTGTGACTTGCCATAATC-3′ and SPSTOPO_Rev, 5′-CTATTCAATTCTCTCCAGATTAT-3′) were used toamplify the complete SPS coding sequence. SPSTOPO_Forincludes the CACC sequence upstream of the ATG start codon(in bold) to allow the amplified product to be introducedinto the Gateway® transformation vector pENTR/D-TOPO(Invitrogen) by the TOPO reaction, following the manufacturer’sinstructions. This produced pENTR-SPS, which was recombinedby the LR reaction (Invitrogen) into the plant transformationvector pK7WG2, which allows strong expression from theCaMV35S (where CaMV is cauliflower mosaic virus) promoter[18]. This yielded vector pEXP35S-SPS and transformantswere selected in LB (Luria–Bertani) broth supplemented with50 mg/l spectinomycin. The SPS sequences in E. coli and planttransformation vectors were verified by sequencing.

Plant transformation

The vector pEXP35S-SPS was transformed into electrocompetentAgrobacterium tumefaciens strain LBA4404 and selected onYEB (yeast extract broth) plates (50 mg/l spectinomycin,100 mg/l streptomycin and 50 mg/l rifampicin). N. tobaccumwas transformed as described by Horsch et al. [19]. A totalof 30 independent transformants were screened by PCR usingthe primers 35SFor (5′-CAATCCCACTATCCTTCGC-3′) andSPSTOPO_Rev (5′-CTATTCAATTCTCTCCAGATTAT-3′) todetect the presence of the inserted T-DNA.

Subcellular localization

The vectors pEXP35S-SPS:GFP, pEXP35SDPS:GFP and thehelper plasmid P19K [20] were separately transformed intoAGL1 Agrobacterium containing the virG expression enhancer[21]. Cultures of Agrobacterium containing P19K or theGFP (green fluorescent protein) vectors were pelleted bycentrifugation (5000 g for 20 min at 4 ◦C) and resuspendedto a D600 of 0.1 in infiltration medium (as in [22]).The GFP and P19K cultures were mixed 1:1 and usedto inoculate the abaxial surface of mature leaves ofN. benthamiana using a needleless syringe. Leaves were collected4 days after infiltration and imaged immediately. Confocalmicroscopy was performed with a FluoView FV1000 (Olympus).GFP and chlorophyll were excited using the 488 nm line fromthe argon laser, and emission signals were collected in separatechannels, at wavelengths between 495 and 526 nm, and between631 and 729 nm respectively. Transmitted light was collected ina separate channel. The GFP signal was false coloured green andchlorophyll autofluorescence was false coloured red.

VIGS

The TRV (tobacco rattle virus)-based silencing vectors pTRV1,pTRV2-MCS and pTRV2-LePDS were obtained from theArabidopsis Biological Resources Centre (Ohio State University,Colombus, OH, U.S.A.). For the construction of pTRV2-SlSPS, a 409 bp fragment of the SlSPS (S. lycopersicum SPS)

gene was PCR amplified from tomato leaf cDNA using theprimers SPS VIGS forward (5′-CGGTCTAGACAAGAACTT-GCATAATATTG-3′) and SPS VIGS reverse (5′-CGGGGATCC-CACCACTTGCAAAGTCTTTAA-3′). For the pTRV2-SlDPSconstruction a 409 bp fragment of the SlDPS (S. lycopersicumDPS) gene was PCR amplified using DPS VIGS forward (5′-CGGTCTAGAGTTGCAGAGTAATTCATTC-3′) and DPS VIGSreverse (5-CGGGGATCCCAGTACATCATCATGAAGTA-3′).The resulting PCR products were cloned into the XbaI and BamHIsites of pTRV2-MCS to form pTRV2-SPS and pTRV2-DPSrespectively. For pTRV2-SPS:DPS construction, the SlSPS andSlDPS fragments above were fused together by PCR using primersSPS VIGS forward, DPS VIGS reverse and the SPS:DPS fusion(5′-TAAAGACTTTGCAAGTGGTGGTTGCAGAGTAATTCA-TTCA-3′) and subsequently cloned into pTRV2-MCS. As acontrol pTRV2-PDS was constructed using a 409 bp fragmentof the tomato PDS (phytoene desaturase) gene (GenBank®

accession number X71023), using primers PDS VIGS forward(5′-CGGTCTAGAGGCACTCAACTTTATAAACC-3′) and PDSVIGS reverse (5′-GCTGGATCCCTTCAGTTTTCTGTCAA-ACC-3′) cloned into the XbaI and BamHI sites of pTRV2MCS. pTRV1 and the pTRV2-derived vectors were separatelytransformed into A. tumefaciens strain GV3101.

Plant infiltration was performed as described previously [22],using 2-week-old tomato seedlings. Plants were incubated indarkness overnight after infiltration. A visible phenotype wasobserved 10 days after inoculation and leaf material was harvestedin liquid nitrogen 12 days later.

Protein purification and digestion

pET14b-SPS was transformed into BL21 Star (DE3) pLysSE. coli cells (Sigma–Aldrich). For high level expression therecombinant proteins were expressed by adding isopropyl β-D-thiogalactopyranoside to a final concentration of 0.4 mMovernight at 24 ◦C. Cells were pelleted by centrifugation at 5000 gfor 20 min at 4 ◦C and pellets were resuspended in 10 ml ofprotein-binding solution [20 mM Tris/HCl (pH 8.0), 0.5 M NaCland 5 mM imidazole) and stored at − 20 ◦C. Harvested cellswere defrosted and lysed by sonication using a Sonic VibracellUltrasonic processor (Sonics). The lysate was centrifuged at5000 g for 15 min at 4 ◦C and supernatants transferred at roomtemperature (20 ◦C) to an Amersham Biosciences ChelatingSepharoseTM Fast Flow column to purify His-tagged protein fromthe soluble fraction.

Purified proteins were visualized by SDS/PAGE and foridentification of purified proteins by MS, bands correspondingto SlSPS or SlDPS (by size estimation) were excised frompolyacrylamide gels prior to destaining and trypsin digestion asdescribed in [23]. Peptides were dried down under vacuum andresuspended in 40 μl of 0.1% TFA (trifluoroacetic acid) and 2 %(v/v) ACN (acetonitrile) and filtered through a 0.45 μm nylonmembrane.

MALDI (matrix-assisted laser-desorption ionization)–TOF(time-of-flight)-MS

Digested peptides, desalted and concentrated using Zip Tipsand eluted in 1:1 H2O/ACN with 0.1 % TFA were spotted(0.5 μl) on to a 600 μm Anchor chip 384 format MALDI targetplate (Bruker Daltonics) and overlaid with 0.5 μl of DHB (2,5-dihydroxybenzoic acid) matrix (ACN + 0.1% TFA) and airdried. Mass spectra were obtained on a Bruker Autoflex MALDI –TOF/TOF-MS (Bruker Daltonics) equipped with a nitrogen-pulsed laser (377 nm). Operating conditions were as follows:



positive reflectron mode, ion source 1 = 19.1 kV, ion source2 = 16.74 kV, lens voltage = 8.85 kV, reflectron voltage 1 = 21.11kV, reflectron voltage 2 = 9.7 kV, laser power = 14%, laserfrequency = 200 Hz and matrix suppression = 400 Da. Massspectra were collected in the range 500–4500 m/z. At total often random points per sample were each obtained with 30 lasershots per point. Peptides from mass spectra were analysedin Flex Analysis 3.3 and Biotools v.3.2 (Bruker Daltonics)and matched against the databases NCBInr Viridiplantae andSwissProt Viridiplantae using an in-house Mascot search engine(Matrix Science). Search parameters were: mass tolerance of0.5 Da; enzyme, trypsin; modifications, deamidation; and missedcleavages, 1.

nESI (nano electrospray ionization)-LC (liquidchromatography)-MS/MS (tandem MS) identification of purifiedSlSPS and SlDPS

Identification of trypsin-digested SlSPS and SlDPS peptideswas carried out using the Amazon ETD (Bruker Daltonics)on-line with a UHPLC UltiMate 3000 (Dionex Softron).Chromatographic separations were performed at 35 ◦C using anAcclaim PepMap RSLC nano C18 2 μm 100 Å (1 Å = 0.1 nm)column (75 μm×15 cm, LC Packings/Dionex), coupled to anAcclaim PepMap C18 3 μm 100 Å (75 mm×2 cm) nano-trapcolumn (LC Packings/Dionex). The mobile phases consisted of(A) 0.1 % formic acid in water and (B) 80 % (v/v) ACN and0.1% formic acid. The gradient used was 96 % (A) isocraticallyfor 5 min at a flow rate of 0.25 μl/min, followed by a lineargradient over 20 min to 50% (A), and another linear gradient for5 min to 90%. This last condition was held in isocratic modefor 5 min. The ionization mode used was nESI operating inpositive mode. Vaporization temperatures were set at 200 ◦C anda full MS scan was performed from 100 to 2000 m/z.

MS/MS spectra were exported to Biotools v. 3.2 (BrukerDaltonics) and submitted for Mascot search of Viridiplantaeentries with the following parameters: one missed cleavage, masserror MS 0.4 Da, search for single, double and triple chargedions, and searching the decoy database. The MS/MS spectrawere also compared with the deduced amino acid sequencesof SlSPS and SlDPS following theoretical digestion (performedusing trypsin, with two missed cleavages and the followingmodifications: oxidation to methionine, carbamidomethylationand deamidations) and the percentage coverage of observedfragments was determined.

Isoprenoid analyses

The extraction, HPLC separation, PDA (photodiode array)detection and quantification of carotenoids were performed asdescribed previously [24]. Three extractions were performed fromeach biological replicate. For quantification of solanesol, aliquotsof 20 mg of ground freeze dried plant tissue were saponified in200 μl of methanol, 500 μl of dH2O (distilled water) and 6%(v/v) KOH and incubated for 1 h at 40 ◦C. Solanesol was extractedin 500 μl of chloroform and the organic hypophase was removedand re-extracted in 500 μl of chloroform. Pooled extracts weredried under nitrogen and resuspended in 100 μl of ethyl acetate.Samples were analysed by HPLC according to [24], with threeextractions performed from each biological replicate. Solanesolwas identified by co-chromatography with an authentic standard(Sigma–Aldrich) and quantified at 210 nm by comparison withdose–response curves (0.2–1.0 mg).

Ratios of UQ-8/UQ-9/UQ-10 were calculated from theircorresponding peak areas obtained from 275 nm chromatograms.

Under these chromatographic conditions [24] retention times forUQ-8, UQ-9 and UQ-10 were 20.6 min, 23.3 min and 25.5 minrespectively. Quantification of ratios was performed using thissystem as it provided better separation of UQ-10 (25.5 min)and Mk-8 (24.6 min) than the LC-PDA-MS system (Figure 4)described in the section below.

LC-PDA-MS analysis of isoprenoids and prenylquinones

Identification of solanesol, decaprenol, α-tocopherol, γ -tocopherol and UQ-10 was carried out using the high-resolutionQ-TOF mass spectrometer UHR-MAXIS (Bruker Daltonics),on-line with a UHPLC UltiMate 3000 equipped with a PDAdetector (Dionex Softron). Chromatographic separations wereperformed in a similar manner to [24], with the exception thata reverse-phase C30 3 μm column [150 mm×2.1 mm i.d. (internaldiameter)] coupled to a 20 mm×4.6 mm C30 guard column wasused (YMC). The mobile phase was altered to facilitate ionizationand was composed of (i) methanol, containing 0.1% formicacid and (ii) tert-butyl methyl ether, containing 0.1% formicacid. These solvents were used in a gradient mode starting at100% (i) for 2 min, then stepped to 80% (i) for 1 min, heldfor 3 min and followed by a linear gradient over 4 min to 30%(i). This last condition was kept for 10 min in isocratic modeand after that initial conditions (100% i) were restored for2 min. The column was then re-equilibrated for 5 min. The flowrate used was 0.2 ml/min and the injection volume was 10 μl.The positive ionisation mode was APCI (atmospheric pressurechemical ionization). The capillary and APCI vaporizationtemperatures were 250 ◦C and 450 ◦C respectively and the drygas (nitrogen) and nebulizer were set at 4 l/min and 2 bar (1bar = 100 kPa) respectively. The APCI source settings were:corona discharge voltage at 4000 nA and a capillary voltageof 4 kV. A full MS scan was performed from 200–1000 m/zand MS/MS spectra were recorded at an isolation width of 0.5m/z. A collision energy ramp from 40 to 80 eV was applied fortarget masses between 650 and 900 m/z. Instrument calibrationwas performed externally prior to each sequence with APPI(atmospheric pressure photoionization)/APCI calibrant solution(Fluka). Automated post-run internal calibration was performedby injecting the same APPI/APCI calibrant solution at the end ofeach sample run via a six port divert valve equipped with a 20 μlloop.

GLC-MS analyses of metabolites

Extraction and analysis of polar metabolites was performedas described by [17], with slight modifications. Freeze driedpowder (10 mg) was extracted in 1 ml of methanol/0.21 M HCl(80:20 by volume), containing the internal standard ribitol (finalconcentration 0.04 mg/ml). A 20 μl aliquot was removed frompelleted samples and dried under nitrogen gas. Four extractionswere performed on each biological replicate. Derivatizationwas performed by the addition of 30 μl of methoxyamine-HCl (Sigma–Aldrich) at 20 mg/ml in pyridine. Samples wereincubated at 40 ◦C for 1 h, after which 80 μl of MSTFA (N-methyl-N-trimethylsilyltrifluoroacetamide; Sigma–Aldrich) wasadded and the samples incubated for 2 h at 40 ◦C before analysis.GLC-MS was performed as described previously [17], using a20:1 split injector. Metabolites were quantified using ChemStationsoftware (Agilent), facilitating integrated peak areas for specificcompound targets (qualifier ions) relative to the internal standard(ribitol) peak. Heat maps were generated using Qlucore software,using mean metabolite levels for each treatment.



In vitro solanesol diphosphate synthase assays

Solanesol diphosphate synthase activity was determined in vitro asdescribed [25], with slight modifications. The incubation mixturecontained, in a total volume of 200 μl, 50 mM Tris/HCl buffer(pH 7.5), 5 mM MgCl2, 1 mM DTT (dithiothreitol), 46 μM [1-14C]IPP (1.85 GBq/mmol), 0.5% Triton X-100 and 38.6 μgof purified protein, and GPP (geranyl diphosphate; 13.7 μM),GGPP (11.1 μM) or FPP (11.5 μM). Samples were incubatedfor 1 h at 30 ◦C and reactions stopped by cooling on ice andthe addition of 200 μl of 1 M NaCl. Samples were extracted in2 ml of water-saturated butanol overnight at − 20 ◦C. Butanolextractable products were dephosphorylated according to [26].Potato acid phosphatase (1 unit; Sigma–Aldrich) was added to1 ml of extract and incubated in 700 μM 0.1% Triton X-100 and4 ml of methanol in a total volume of 10 ml at 37 ◦C overnight ona shaking platform water bath. Samples were extracted in 10 mlof 10:90 (v/v) diethyl ether/petroleum ether (boiling point 40–60 ◦C) and the organic phase dried under nitrogen. Solanesol wasidentified by TLC using reverse-phase C18 F254S plates (Merck),with a mobile phase of 19:1 (v/v) acetone/water and visualized byexposure to iodine vapour. Comparison of the effect of differentdivalent cations was performed as described above with slightmodifications. The incubation mixture (100 μl) contained 50 mMTris/HCl buffer (pH 7.5), 177.6 nM GGPP (for the SlSPS assay)or 184.6 nM FPP (for the SlDPS assay), 1 mM DTT, 36 μM [1-14C]IPP, 0.5% Triton X-100 and 2.5 μg of purified protein and10 mM CaCl2, 10 mM MgCl2, 10 mM MnCl2 or 10 mM ZnCl2.Samples were incubated for 15 min at 30 ◦C and then the reactionsstopped by cooling on ice and the addition of 100 μl of 1 MNaCl. Samples were extracted in 1 ml of water-saturated butanoland after centrifugation the amount of [1-14C]IPP incorporatedinto butanol-extractable polyprenyl diphosphate was measuredin triplicate by scintillation counting. For kinetic studies, theconcentration of the allylic substrate (FPP/GPP) was varied,while the co-substrate ([1-14C]IPP) was at 36 μM. Kineticparameters and their standard errors were estimated by non-linearregression analysis using GraphPad Prism software v.5 (GraphPadSoftware).

Solanesol isomers were identified using LC-MS. The solanesolstandard (1 mg, Sigma, tobacco extract, 90% pure) wasresuspended in chloroform and separated by reverse-phase TLC[solvent 19:1 (v/v) acetone/water]. Two solanesol bands werevisualized by exposure to iodine vapour, heated to 50 ◦C andsubsequently scraped from the TLC plate. The compoundswere extracted with 1 ml of chloroform, dried under nitrogen,redissolved in 100 μl of chloroform and analysed by LC-MS, asdescribed above.

TEM (transmission electron microscopy)

Leaf tissues from representative VIGS-treated plants were fixed in2.5% (w/v) glutaraldehyde in 50 mM sodium cacodylate bufferovernight at 4 ◦C. Samples were rinsed twice in the samebuffer prior to fixing in 1% (w/v) osmium tetroxide in 50 mMsodium cacodylate buffer for 1 h at room temperature. Sampleswere dehydrated in a graded ethanol series, then embedded in LV(low viscosity) resin (Agar Scientific) and polymerized at 60 ◦Cfor 24 h. Sections (75 nm) were prepared with a RMC-MTXLultramicrotome (Diatome) and counterstained with 4.5 % (w/v)uranyl acetate in 1% (v/v) acetic acid for 45 min and Reynoldslead citrate stain for 7 min. Microscopy was performed on a Jeol1230 transmission electron microscope and images captured witha Gatan digital camera.

Lipid peroxidation

Lipid peroxidation in tomato leaf tissue was measured as TBARS(2-thiobarbituric acid-reacting substances) formation, accordingto the method described in [27], using approximately 100 mg ofintact leaflets from five plants per genotype. TBARS values areexpressed as MDA (malonaldehyde) equivalents, using the molarabsorption coefficient 155 mM− 1 · cm− 1.

TEAC (Trolox equivalent antioxidant capacity) total antioxidantactivity assays

A non-polar extract was generated using the caroten-oid/isoprenoid extraction procedure described above, with 10 mgof homogenized freeze-dried tobacco leaf tissue. TEAC assayswere performed as described in [28] by generating two ABTS[2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid); Sigma–Aldrich] free radical cations, ABTS+ . Results are expressed as aTEAC in mmol of Trolox/g of DW (dry mass).

Measurement of gene expression by real-time qRT–PCR(quantitative reverse-transcription PCR)

RNA was extracted from fresh-frozen plant material extractedusing RNeasy reagents (Qiagen). The QuantiFast SYBR Greenone-step real time qRT–PCR kit (Qiagen) was used to determinegene expression levels using gene specific primers. Melt-curve analyses verified product specificity and the CT valuecalculations were performed by Rotor-Gene software (Qiagen),calibrated against a dilution series of cloned gene products,run simultaneously with experimental samples. The tomato andtobacco actin genes served as references for normalization.

RESULTS AND DISCUSSION

The effect of DET1 down-regulation on the transcription of SlSPSand SlDPS in the tomato

Previous transcriptomic studies using microarrays havedemonstrated that the increases in isoprenoids in tomato plantssilenced for DET-1 are generally not achieved through increasesin expression of isoprenoid biosynthetic genes, but by post-transcriptional regulation [16]. However, transcripts of a SlSPS(GenBank® accession number DQ889204) were found to beelevated at the mature green stage of fruit development in theDET-1 silenced variety TFM7, relative to the wild-type control(T56) [17]. In the present study, we have used qRT–PCR toaccurately analyse transcript levels of SlSPS. The SlSPS transcriptwas detected in immature green fruit of T56 and TFM7 (10-and 15-mm diameter fruit, Figure 2A) and remained virtuallyconstant through development into ripe fruit in T56, but inTFM7 fruit levels were elevated at the mature green stageonwards compared with those in T56 until a significant 3-foldincrease in the transcript levels was found within ripe fruit tissue(Figure 2A). These elevated transcript levels in TFM7 mirroredthe increases in solanesol and PQ (Figures 2B and 2C). TFM7fruit accumulated 5-fold greater levels at the B + 7 stage than didT56 fruit (Figure 2C). Similarly, solanesol, the alcohol derivativeof solanesyl diphosphate, was more abundant in TFM7 fruit thanT56 fruit at all developmental stages and both varieties displayeda similar profile through to the mature green stage (Figure 2B). InTFM7, however, the levels became dramatically increased at thebreaker (B) and 7 days post-breaker (B + 7) stages, to produce a12-fold difference between the two varieties. These increases insolanesol were in same order of magnitude as the elevations in



Figure 2 Changes in SPS gene expression and solanesol and PQ contentsresulting from down-regulation of the endogenous DET1 gene in RNAinterference tomato fruit

(A) Expression profile of SlSPS in TFM7 relative to T56 over four development time points.Fruit were collected at four developmental stages, 15-mm diameter (15 mm), 30-mm diameter(30 mm), mature green (MG) and 7 days post-breaker (B + 7). For each developmental stage,two to three fruit were collected from three individual plants for each genotype and pooled.qRT–PCR was performed on total RNA, with primers specific for SlSPS and expression levelsdetermined by normalizing to actin. Solanesol (B) and PQ (C) were quantified in extracts fromthe same starting material. Each genotype is represented by three biological replicates (threeplants), from which multiple fruit were pooled. Three extractions were performed from eachbiological replicate and analysed by HPLC as described in the Experimental section. Results aremeans +− S.E.M. obtained from the three biological replicates.

carotenoids, xanthophylls, chlorophylls and tocopherols reportedpreviously [17]. Since the DET1 phenotype is characterized by theoverproduction of both the photosynthetic apparatus, and phenolicand ascorbate antioxidants, it is plausible that up-regulation ofSlSPS in DET1 mutants may serve to allow for greater productionof PQ to function in photosynthesis and as an antioxidant. Thereason for this difference in the mechanism for regulating levelsof these compounds in not yet understood.

Recombinant expression, purification and in vitro properties ofSlSPS and SlDPS

The tomato Unigene library (http://www.solgenomics.net)contains a candidate DPS (SGN-U573523), exhibiting significantsimilarity to the putative tomato solanesol diphosphate synthase.The ORF (open reading frame) encoded by SGN-U573523shares 71% amino acid identity with the rice mitochondrial

SPS [OsSPS (Oryza sativa SPS) 1] and 40 % identity withthe plastidial SPS (OsSPS2) [29] and has been designatedSlDPS. Conversely, SlSPS shares 67% amino acid identitywith OsSPS2 and 40% identity with OsSPS1. Accordingly,SlSPS possesses a putative transit peptide sequence for plastidlocalization, whereas SlDPS contains a putative N-terminalmitochondrial signal peptide sequence, as predicted by theprotein localization programs TargetP and ChloroP [30]. Thededuced amino acid sequences of SlSPS and SlDPS each containtwo conserved aspartate-rich motifs [DD(X)nD; SupplementaryFigure S1 at http://www.biochemj.org/bj/449/bj4490729add.htm]and phylogenetic analysis positioned these two tomatogenes among known long-chain prenyl diphosphate synthasesfrom other species and distinct from the tomato GGPPSs(geranylgeranyl diphosphate synthases; Figure 3).

In order to functionally characterize these prenyl diphosphatesynthase gene products, the full-length coding regions wereamplified from tomato leaf cDNA and separately cloned intothe E. coli expression vector pET-14b. Expression in DPSBL21star (DE3) pLysS E. coli cells and subsequent purificationyielded His-tagged SlSPS and SlDPS proteins of 45446 and45443 Da respectively. The identity of the purified proteinswas verified by MS/MS of proteolytic digests, with observedamino acid coverage of 58.5 and 41.6% for the SlSPS andSlDPS sequences respectively (Supplementary Table S1 athttp://www.biochemj.org/bj/449/bj4490729add.htm).

When purified SlSPS was assayed with either FPP or GGPP,two prominent compounds were observed on TLC (Rf values0.52 and 0.46, Figure 4A). Authentic solanesol also producedtwo bands on TLC with these Rf values. Analysis by LC-MSindicated that the two products, which had different retentiontimes (18.17 and 18.38 min), both have the appropriate molecularmass for solanesol (m/z 613.57 [M + H] +− H2O). Purified SlDPSwas able to use FPP, GPP or GGPP as its allylic substrate andthe resulting products included a major component with an Rf of0.37 (Figure 4B). In the presence of FPP, bands co-migrating withthe products obtained with SlSPS were also visible. The productsof both enzyme activities were confirmed by LC-MS analyses assolanesol, whereas SlDPS was shown to produce solanesol anddecaprenol (Supplementary Figure S1).

Direct involvement of SlSPS and SlDPS in polyprenyl diphos-phate synthase activity in vivo was determined by LC-MS analysisof UQ species in E. coli DH5a BL21 cells (Supplementary TableS2 at http://www.biochemj.org/bj/449/bj4490729add.htm). Cellstransformed with an empty pET-14b vector were shown by LC-MS to produce UQ-8 (m/z 727.57 [M + H]+ ) which is synthesizedby the endogenous E. coli octaprenyl diphosphate synthase andeluted with a retention time (Rt) of 10.3 min (Supplementary Fig-ure S2 at http://www.biochemj.org/bj/449/bj4490729add.htm).Menaquinone-8 (m/z 717.56 [M + H]+ ) was also detected. Incontrast, transformation with pET14-b SPS produced a distinctquinone profile resulting from accumulation of the C45 compoundUQ-9 (m/z 795.63 [M + H]+ ), which eluted at an Rt of 11.2 min(Figure 4C). LC-MS analysis of extracts of E. coli harbouringpET14-b DPS demonstrated that recombinant SlDPS expressionhad resulted in production of UQ-9 as well as UQ-10 (m/z863.69 [M + H]+ ), which eluted after menaquinone-8 at an Rt

of 11.7 min. These findings demonstrate that SlDPS encodesa functioning DPS. Therefore expression of SlSPS or SlDPSalone in E. coli was sufficient to modify the chain length of theendogenous UQ species, demonstrating that like the long-chainprenyl diphosphates characterized in other plant species [29,31–33] those in tomato function as single gene products. SlSPSexpression provided for UQ prenyl side chains consisting strictlyof nine isoprene units, whereas SlDPS provided side chains with


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Figure 3 Phylogenetic analysis of the tomato SPS and DPS clones withother plant trans-long-chain prenyl diphosphate synthases, GGPPSs andSPSs

The full-length amino acid sequences were aligned using MEGA (version 4.01 Beta 3, [49]) withthe default settings. A phylogenetic tree was constructed with the neighbour-joining methodusing the default settings of MEGA. Bootstrap values were calculated from 1000 replicates.Amino acid sequences used for the analysis are listed as follows with the GenBank® accessionnumbers shown in parentheses: AtGPS1, A. thaliana GPS1 (NP_001031483.1); AtGGPPS1, A.thaliana GGPPS1 (NM_119845); AtGGPPS2, A. thaliana GGPPS2 (NM_127943); AtGGPPS3,A. thaliana GGPPS3 (NM_112315); AtGGPPS4, A. thaliana GGPPS4 (NM_127420);AtGGPPS6, A. thaliana GGPPS6 (NM_103841); A. thaliana FPPS (Q09152); AtSPS1, A.thaliana SPS1 (AB188497); AtSPS2, A. thaliana SPS2 (AB188498); COQ1, hexaprenyldiphosphate synthase of S. cerevisiae (J05547); ddsA, DPS of Gluconobacter suboxydans(AB006850); dps1, subunit 1 of DPS from S. pombe (D84311); dlp1, subunit 2 ofDPS from S. pombe (NM_001019856.1); HbSPS, H. brasiliensis SPS (DQ437520);ispB, octaprenyl diphosphate synthase of E. coli (NP_417654.1); OsFFPS1, O. sativaFPPS1 (D85317); OsFPPS2, O. sativa FPPS2 (AB021979); OsSPS1, O. sativa SPS1(AK071299); OsSPS2, O. sativa SPS2 (AK065579); Nt SGN-U432530, N. tabacum unigene;Pt, P. trichocarpa predicted protein (XM_002300406.1); SlSPS, S. lycopersicum SPS(DQ889204); SlDPS, S. lycopersicum DPS (SGN-U573523); TcSPS, Trypanosoma cruziSPS (AF28277.1); and Zm, Zea mays predicted protein (ACN27420).

nine or ten isoprene units. Thus the tomato genome, unlike thatof Arabidopsis and rice which produce only single species of sidechain (C45), contains genes allowing the production of C45 and C50

side chains.The divalent cation requirements of purified tomato SlSPS

and SlDPS (Supplementary Table S3 at http://www.biochemj.org/bj/449/bj4490729add.htm) were shown to be similar to thosefrom the homodimer type enzymes found in rice [OsPPT (O.sativa PPT) 1] and S. cerevisiae (COQ2, [34]). Purified SlSPS wasmost active in the presence of MgCl2, whereas MnCl2 and MgCl2

were both effective at stimulating SlDPS activity. In addition the

apparent Km values for SlSPS and SlDPS indicate a preference forGGPP as a substrate rather than FPP for both enzymes (0.46 and1.51 μM respectively, Table 1), in agreement with Arabidopsisenzymes with SPS activity, AtSPS (Arabidopsis thaliana SPS)OsSPS1 and AtSPS2 [32,33]. It is unclear why those enzymesnot targeted to the plastid (SlDPS and AtSDS1) do not show apreference for the cytosolic substrate FPP, although here the Vmax

value of SlDPS for FPP was higher than that for GGPP (Table 1).Long-chain prenyl diphosphate synthases exhibit a requirementfor divalent metal cations, particularly Mg2 + , to allow binding ofthe substrates to the active site [35], as do the aromatic prenyltransferases [36,37].

Subcellular localization of tomato long-chain prenyl transferasesand solanesol

To experimentally confirm the subcellular localization of SlSPSand SlDPS, their full length coding regions were transcriptionallyfused to the GFP gene, placed under the control of theCaMV35S promoter and the vectors expressed in N. benthamiana.SlSPS was shown to be localized in chloroplasts as judgedby chlorophyll autofluorescence in the confocal microscope(Figure 5). The fluorescence signal of SlDPS–GFP was morediffuse and it was not possible to determine precisely itssubcellular location, although it may resemble the mitochondriallocalization previously reported for the rice OsSPS1 [29]. Thefirst Arabidopsis long-chain diphosphate synthase to be isolated,AtSPS1, was shown to be targeted to the ER [32,33] and presumedto be responsible for the production of solanesol moieties for UQbiosynthesis. Recently, however, a further enzyme, AtPPPS (A.thaliana trans-type polyprenyl pyrophosphate synthase), whichpossesses SPS activity in vitro, was shown to be targeted to themitochondrion and plastid and identified as the main contributorto SPS activity in UQ biosynthesis [38]. Since the Arabidopsis p-hydroxybenzoate prenyl transferase AtPPT1 (A. thaliana PPT1)is also localized to the mitochondrion, this is likely to be the mainsite of UQ synthesis [12]. Silencing AtPPPS did not affect PQaccumulation and hence, despite plastid targeting, does not appearto participate in production of PQ [39]. A further ArabidopsisSPS, AtSPS2, is believed to provide the prenyl diphosphate forthis purpose and is also targeted to the plastid [31,32], where allof the enzymes involved in the final steps of PQ synthesis arelocalized [40,41].

Functional characterization of SlSPS and SlDPS in planta

VIGS was used to gain further insight into the functionof SlSPS and SlDPS in tomato vegetative tissues. A mixture ofAgrobacterium cultures containing the pTRV2-derived vectorsand pTRV1, a plant binary transformation plasmid containinga cDNA clone of tobacco mosaic virus RNA 1, was infiltratedinto tomato seedlings. pTRV1, mixed with an empty vector(pTRV2-MCS), was infiltrated as a negative control (TRV) anda vector (pTRV2-PDS), containing a 409 bp fragment of thetomato phytoene desaturase gene was used as a positive control(TRV2-PDS, [22]). Tomato plants infected with pTRV2-PDSdeveloped a bleaching phenotype in the upper leaves 4 weeks afterAgrobacterium infiltration. Plants infected with either pTRV2-SPS or pTRV2-SPS-DPS also became photobleached, althoughless dramatically than pTRV2-PDS. The leaflets contained palegreen sectors at the base, giving a variegated appearance andwere abnormal (Figure 6A). There was no visible phenotype inplants infected with TRV2-DPS. The mottled phenotype is similarto that observed by phytoene desaturase silencing, although less





Figure 4 In vitro and in vivo activities of SlSPS and SlDPS(A and B) In vitro production of solanesol and decaprenol by purified SlSPS and SlDPS proteins respectively. TLC autoradiogram of the prenyl alcohols obtained by enzymatic hydrolysis of theproducts formed by the incubation of purified SlSPS (A) and SlDPS (B) with [14C]IPP and IPP, DMAPP, FPP, GPP or GGPP as indicated. C, control (no enzyme); C15 indicates the position of authenticfarnesol (R f = 0.79–0.82); C45

a (R f = 0.51) and C45b (R f = 0.44–0.46) indicate two authentic solanesol isomers; C50, decaprenol (R f = 0.37). Ori., origin; S.F., solvent front. (C) Chromatogram

trace at 270 nm of prenyl lipids with different chain lengths produced in E. coli DH5α cells harbouring empty vector pET-14b (top), pET-14b SPS (middle) and pET-14b DPS (bottom) and detectedby LC-MS. (D) Mass spectral identities of each identified quinone species. Co8, ubiquinone-8; Co9, ubiquinone-9; Co10, ubiquinone-10. Menaquinone, Mk-8, is present in all samples and elutesbefore ubiquinone-10 (Co10) in (C) (bottom panel).



Table 1 Kinetic parameters of purified SlSPS and SlDPS

The kinetic parameters of SlSPS and SlDPS in the presence of GGPP or FPP, with a countersubstrate of 36 μM [1-14C]IPP. Reactions were incubated at 30◦C for 15 min as described inthe Experimental section. Results are means +− S.E.M. from two assays measured in duplicate.V max is presented as pmol of [1-14C]IPP incorporated per mg of protein per min. Values shownin bold indicate significant differences between GGPP and FPP as substrates at 95 % confidencelevel.

Enzyme Co-substrate K m (μM) V max (pmol/mg of protein/min) V max/K m

SlSPS GGPP 0.46 +− 0.14 962.4 +− 59.1 2104.1FPP 1.51 +− 0.28 865.2 +− 40.0 571.5

SlDPS GGPP 4.18 +− 1.05 1013.6 +− 70.5 242.7FPP 8.63 +− 3.00 1480.0 +− 172.0 171.4

pronounced, and presumably results from inhibited productionof PQ since plants silenced for SlSPS were found to containsignificantly decreased levels of PQ (reduced 42 % in TRV2-SPSand 38% in TRV2-SPS-DPS, although not statistically significantin the latter case compared with the control; SupplementaryTable S4 at http://www.biochemj.org/bj/449/bj4490729add.htm).PQ acts as a cofactor in carotenoid biosynthesis by acceptingthe hydrogen atoms released during phytoene desaturation, re-oxidizing the reduced flavin (FADH2) and it is subsequentlyreoxidized by the plastid terminal oxidase [42]. Leaf bleachingis observed in mutants defective in PQ reoxidation [42] orbiosynthesis [2,40]. We propose that as a result of SlSPS silencingreduced biosynthesis of PQ inhibits carotenoid biosynthesis,making plastids vulnerable to photooxidative damage andpreventing their normal development. Plastids in bleached tissue(Figure 6B, ii) resembled those resulting from treatment with thePDS inhibitor norflurazon [43], having a modified morphologyand stroma largely void of thylakoid membranes. It is noteworthythat in our experiments SlDPS was not able compensate for lossesof SlSPS activity in TRV2-SPS-treated plants. The TRV2-SPSplastids also contained small aggregates of electron dense globulias well as large less dense membrane bound bodies.

Transcripts levels of SlSPS and SlDPS genes were measuredin the treated leaf tissue. In pTRV2-SlSPS-infected plants thetranscripts of SlSPS were reduced by 69% and, similarly,SlDPS expression was reduced to 55 % in pTRV2-SlDPSplants (Supplementary Figure S3 at http://www.biochemj.org/bj/449/bj4490729add.htm). In the TRV2 SPS-DPS-treated plantsthe SlSPS and SlDPS transcripts were shown to be reducedby 62 and 52% respectively (Supplementary Figure S4at http://www.biochemj.org/bj/449/bj4490729add.htm). Thesedecreases in transcript levels are relatively modest, but this isthought to reflect the uneven tissue distribution of the VIGSphenotype penetration observed previously in the tomato [44].

Chlorophyll and carotenoid analyses of VIGS-treated tissuesshowed that the predominant effect was the accumulation ofphytoene in plants silenced for SlSPS (TRV2-SlSPS, 49-foldand TRV2-SPS-DPS, 40-fold over TRV controls; SupplementaryTable S4), whereas phytoene levels in plants treated with TRV2-DPS alone were not significantly altered. TRV2-SPS plants alsohad lower levels of chlorophylls a and b, as well as lutein and thexanthophylls neoxanthin and violaxanthin.

TRV2-SPS plants were further analysed for accumulation ofother prenyl lipid species, since the tocopherols and PQ sharethe precursor homogentisate. Compared with the TRV controls,TRV2-SPS-treated plants were found to accumulate elevatedlevels of α-tocopherol (2-fold), but there was no change in thelevels of γ -tocopherol. Interestingly, levels of UQ-10 were alsoincreased 3-fold in plants silenced for SlSPS (SupplementaryFigure S4A; see Supplementary Table S5 at http://www.biochemj.org/bj/449/bj4490729add.htm for compound identifica-tion). Since quinones and carotenoids have a role in the protectionof membranes against oxidative stress, leaves from glasshouse-grown TRV2 and TRV2-SPS were analysed for their levels of lipidperoxidation. TRV2-SPS plants contained 50% greater levels oflipid peroxides (MDA/g of fresh mass, Supplementary FigureS4), indicating increased susceptibility to reactive oxygen speciesgenerated as products of photosynthesis and that the chloroticphenotype observed is a consequence of oxidative damage tolipids. In agreement with the photobleached appearance andperturbed plastid ultrastructure of the TRV2-SPS plants, the

Figure 5 Subcellular localization of SlSPS and SlDPS GFP fusions

The GFP-fusion constructs of SlSPS–GFP and SlDPS–GFP were used to inoculate leaves of N. benthamiana by Agrobacterium infiltration. Constructs were designed for the constitutive overexpressionof SlSPS (SlSPS–GFP) and SlDPS (SlDPS–GFP) transcriptionally fused to GFP to enable visualization. Uninoculated cells were imaged as a negative control. Leaves were imaged 4 days afterinfiltration with a confocal laser-scanning microscope. Transmission, transmission microscopy images; chlorophyll and chlorophyll autofluorescence false are coloured in red; GFP and GFPfluorescence false are coloured in green. Merged, overlaid images of chlorophyll and GFP fluorescence images. Co-localization of GFP and chlorophyll appears as orange. Scale bar = 50 μm.









Figure 6 Silencing of SlSPS and SlDPS in tomato using VIGS

(A) Representative leaves of tomato plants photographed 4 weeks after infection with emptyvector control TRV (TRV2-MCS, i) or carrying a fragment of PDS (ii) or both SlSPS and SlDPSfused together to silence both genes (iii). Infection with pTRV2-PDS silences endogenousPDS in tomato plants and causes a photobleached phenotype due to inhibition of carotenoidbiosynthesis. Infection with pTRV2-SPS-DPS causes a photobleached effect less severe than inpTRV2-PDS plants, accompanied by a mild distortion of leaf morphology. Scale bar = 10 mm.(B) Representative plastids from palisade cells of control (TRV2-MCS, i) and SlSPS silenced(TRV2-SPS, ii) leaves. Scale bar = 1 μm. m, membrane bound body; pl, plastoglobules; t,thylakoid membranes.

maximum quantum yield of photosystem II was also reduced.This is estimated from the Fv/Fm ratio (Fm − Fo/Fm, where Fo

and Fm are minimum and maximum chlorophyll a fluorescencerespectively of dark-adapted leaves) and is a reliable indicator ofphotosynthetic performance. TRV2 leaves had a mean Fv/Fm of0.824 +− 0.001 and TRV2-SPS bleached leaves had a mean Fv/Fm

of 0.462 +− 0.109.To provide insight into the sectors of metabolism affected by

the VIGS treatments, metabolite profiling was undertaken. Aheatmap, incorporating metabolites extracted in polar and non-polar fractions, is shown in Figure 7 and the data presentedin full in Supplementary Table S6 (at http://www.biochemj.org/bj/449/bj4490729add.htm). For comparative purposes, eachmetabolite is presented as variance from its mean abundanceacross all genotypes. Noteworthy are the similarities in clustersbetween those plants treated with TRV2-PDS and TRV2-SPS-DPS, marked by relative reductions in multiple classes ofmetabolites, including sugars, organic acids and isoprenoids, andincreases in a number of amino acids. TRV2-SlDPS plants aredifferentiated from TRV controls by a cluster containing elevatedlevels of sugars glucose, fructose, mannose and arabinose,whereas a cluster containing reduced levels of chlorophylls,lutein and PQ is present in the TRV2-SlSPS plants. Silencingof either SlSPS or SlDPS alone thus produced metabolite profilesthat were distinct from one another and the TRV2-PDS, TRV2-SlSPS-SlDPS cluster. The metabolic profile of TRV2-SlSPS-SlDPS plants does not appear to represent a combination ofthe profiles resulting from silencing the two long-chain prenyltransferases individually, but clustering of TRV2-SlSPS-SlDPSand TRV2-PDS away from the control does reflect the severity ofthe phenotype resulting from VIGS treatment. The difference inprofiles between the PDS- and SlSPS-silenced phenotype may

Figure 7 Hierarchical clustering of primary and secondary metabolites inleaves of VIGS-treated plants

Clustering is based on mean values for each variety, derived from four biological replicates andanalysed with three to five technical replications as described in the Experimental section. Eachhorizontal bar represents the mean content of a single metabolite in each treatment, normalized tothe mean abundance in all samples (mean = 0 and S.D. = 1) and shown on a scale from − 2 to+ 2 as indicated in the scale bar. For each metabolite, red and green indicate the extent of increaseand decrease relative to the mean value, respectively. The exact values for each metabolite areprovided in Supplementary Table S6 (at http://www.biochemj.org/bj/449/bj4490729add.htm).

be explained by the differences in the effectiveness of VIGStreatment. Alternatively, TRV2-SPS plants may compensate forlack of solanesyl diphosphate by incorporating shorter prenyldiphosphate chains into PQ. These were not detected in the presentstudy, but have been reported previously [45]. Furthermore,increased accumulation in SlSPS-silenced plants of UQ-10 andα-tocopherol may be a compensatory antioxidant mechanism or,in the case of α-tocopherol, owing to reduced competition withPQ biosynthesis for homogentisate.

In order to assess the potential of SlSPS as a tool to elevate thesynthesis of solanesol and its role in prenyl quinone formation,transgenic tobacco plants overexpressing tomato SlSPS wereproduced. From a total of 30 plants displaying resistance tokanamycin, 90% were found to be PCR positive for SlSPS. Each






Table 2 Carotenoid, chlorophyll, PQ and solanesol contents found in leaves of transgenic 35S::SlSPS varieties compared to the wild-type (WT) background

For each genotype, three mature and immature leaves from single T1 generation plants were pooled and three determinations made per sample. Results are means +− S.E.M. Values that weredetermined by Student’s t test to be significantly different from the TRV MCS control are shown in bold. *P < 0.05, **P < 0.01 and ***P < 0.001.

Isoprenoid content (μg/g of DW)

Leaves WT 1 WT 2 WT 3 S-9-19 (azygous) S-9-6 S-9-16 S-29-2 S-29-6

Immature leavesSolanesol 13.3 +− 1.1 26.0 +− 2.0 20.4 +− 1.1 19.3 +− 4.3 52.7 +− 1.5*** 19.4 +− 1.1 7.3 +− 0.2* 21.1 +− 1.8PQ 469.6 +− 39.5 538.4 +− 14.8 497.5 +− 17.5 535.8 +− 10.4 771.9 +− 38.8*** 778.2 +− 44.4*** 780.4 +− 11.6*** 684.4 +− 17.8***

Mature leavesSolanesol 196.3 +− 3.3 115.2 +− 5.2 926.1 +− 21.9 1022.2 +− 27.4* 1216.7 +− 110.8** 1160.0 +− 40.0** 1255.7 +− 146.8** 1383.6 +− 69.3**PQ 2406.4 +− 55.5 1528.1 +− 67.1 1733.1 +− 52.6 2242.0 +− 17.7 2207.0 +− 96.2 2105.5 +− 158.6 1858.4 +− 75.8 1596.7 +− 81.9

of these plants was regenerated in the glasshouse and analysedfor their total solanesol content. The levels of solanesol in thevegetative tissues were highly variable and a number of plantsdisplayed unusual phenotypic characteristics, such as reducedstature and mottled leaf appearance (Supplementary Table S7at http://www.biochemj.org/bj/449/bj4490729add.htm). A totalof four lines (S-9, S-11, S-19 and S-29), selected on the basis ofsolanesol content, were self-pollinated to create the T1 generationplants. PCR screening of T1 generation plants from these selectedlines identified that the majority (89%) of the T1 progenycontained the SlSPS transgene with azygous plants only identifiedfor lines 9 and 19, indicating that S-11 and S-29 contained multipletransgene inserts. Of the 48 T1 plants screened, 31 individuals,including three azygous individuals from line 9, showed solanesolcontent above the mean wild-type level (412.5 μg/g of DW) infully expanded mature leaves. The solanesol content was greatestin the line S-29-6, at 1383.6 μg/g of DW, 3.4-fold greater thanthe wild-type mean content (Table 2). The four transgenic plantswith the highest solanesol content and a single azygous plant werechosen to investigate the effects of high solanesol content on thebiosynthesis of other isoprenoids. Only line 9-6 showed alteredpigment contents, with an increased abundance of β-carotene,lutein and chlorophyll a of 33%, 39% and 34% respectively.The PQ levels in the transgenic lines were no different fromthose determined for the wild-type plants (Table 2). The samecompounds were then screened in immature leaves to see whetheroverexpression of SlSPS had any effects in developing tissue. Thesolanesol content in immature leaves was one order of magnitudelower than that found in mature leaves and in only one transgenicline measured (S9-6) was the solanesol content elevated relativeto the wild-type levels (Table 2). However, immature leaves ofall transgenic lines showed a consistent elevation between 36and 55% in PQ levels above that in the wild-type. Line 9-16also showed small, but significant, elevations in lutein (12 %),β-carotene (13%) and chlorophyll (18%) levels compared withthe wild-type plants, whereas line 29-6 contained amounts oflutein, violaxanthin, β-carotene and chlorophylls reduced by26%, 39%, 41% and 30% respectively.

Since plastoquinol (PQ-H2) is known to be an effectiveantioxidant, TEAC analysis was performed on non-polar extractsfrom immature leaf tissue to assess whether overexpressionof SlSPS had resulted in elevated total antioxidant capacityin tobacco. Statistically significant increases in antioxidantcapacity of 15 and 24% were observed in lines S-9(P < 0.01) and S-29 (P < 0.001) respectively, over the wild-typecontrol (Supplementary Figure S5 at http://www.biochemj.org/bj/449/bj4490729add.htm).

These data suggest that expression of SlSPS and supply ofsolanesol is limiting for the production of PQ in immature leaves.The increase in PQ content may also explain the enhanced

antioxidant activity in non-polar extracts of immature transgenictobacco leaves. Like tocopherols, PQ levels are elevated in planttissues in response to a range of abiotic and biotic stresses andthere is strong evidence that in its reduced form it has an importantfunction as an antioxidant [46]. Furthermore, solanesol levels arealso induced in tobacco leaves following viral infection and areimplicated as part of the natural response to pathogens [47,48].Whether plants that accumulate greater solanesol levels are moreresistant to pathogens has yet to be determined.

In summary, we have shown that two enzymes from tomato(S. lycopersicum) with different subcellular distributions, areresponsible for the production of solanesyl and decaprenyldiphosphates, namely SlSPS and SlDPS. The two enzymes cannotcomplement each other and have different substrate specificities.In silenced DET-1 lines their expression is up-regulated, unlikeother genes encoding isoprenoid biosynthetic enzymes. Whenexpressed in E. coli, SlSPS and SlDPS extend the prenyl chainlength of the endogenous UQ to nine and ten isoprene unitsrespectively.

AUTHOR CONTRIBUTION

Matthew Jones performed the experimental work with the assistance of Laura Perez-Fonsand Francesca Robertson, especially related to the MS analysis of metabolites and proteinsrespectively. Matthew Jones, Paul Fraser and Peter Bramley contributed to the design ofthe experimental approach. All authors contributed to data interpretation, preparation andwriting of the paper. Peter Bramley and Paul Fraser obtained the funding and Peter Bramleyedited the paper prior to submission.

ACKNOWLEDGEMENTS

We thank Chris Gerrish for excellent technical support.

FUNDING

This work was supported by the U.K. Biotechnology and Biological Sciences ResearchCommittee [grant number BB/F005644/1].

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Received 19 June 2012/2 November 2012; accepted 5 November 2012Published as BJ Immediate Publication 5 November 2012, doi:10.1042/BJ20120988


Biochem. J. (2013) 449, 729–740 (Printed in Great Britain) doi:10.1042/BJ20120988

SUPPLEMENTARY ONLINE DATAFunctional characterization of long-chain prenyl diphosphate synthasesfrom tomatoMatthew O. JONES, Laura PEREZ-FONS, Francesca P. ROBERTSON, Peter M. BRAMLEY1 and Paul D. FRASERSchool of Biological Sciences, Royal Holloway, University of London, Egham, Surrey TW20 0EX, U.K.

Figure S1 Multiple sequence alignment of solanesyl and decaprenyl diphosphate synthases

The conserved functional motifs, DD(X)nD, are boxed in blue. Identical and similar amino acid residues are shaded in black and grey respectively. The alignment of complete deduced aminoacid sequences was performed by MultiAlign [1] and shaded using BOXSHADE 3.21 (http://www.ch.embnet.org/software/BOX_form.html). AtSPS1, A. thaliana SPS1 (AB188497); A. thalianaSPS2 (AB188498); HbSPS, H. brasiliensis SPS (DQ437520); OsSPS1, O. sativa SPS1 (AK071299); OsSPS2, O. sativa SPS2 (AK065579); SlSPS, S. lycopersicum SPS (DQ889204); SlDPS, S.lycopersicum DPS (SGN-U572523); TcSPS, Trypanosoma cruzi SPS (AF28277.1).

1 To whom correspondence should be addressed (email [email protected]).


http://www.ch.embnet.org/software/BOX_form.html

M. O. Jones and others

Figure S2 Validation by LC-MS of in vitro production of solanesol and decaprenol by purified SlSPS and SlDPS proteins.

Extracted ion chromatograms of masses 613.5 +− 0.1 (A) corresponding to solanesol and 681.6 +− 0.1 (B) corresponding to decaprenol. Panels show products of negative control (top) with noenzyme; purified SlSPS (middle) and purified SlDPS (bottom).


Tomato long-chain prenyl diphosphate synthases

Figure S3 Expression levels of long-chain prenyl diphosphate synthasesin VIGS-treated tomato plants determined by qRT–PCR

Upper panel, relative transcript abundance of SlSPS in plants silenced for the gene byintroduction of pTRV2-SPS or pTRV2-SPS-DPS shown relative to control plants treated withpTRV2-MCS. Lower panel, relative transcript abundance of SlDPS in plants silenced forthe gene by introduction of pTRV2-DPS or pTRV2-SPS-DPS relative to plants treated withpTRV2-MCS. Transcripts were normalized within each treatment to the tomato actin gene.Results are means +− S.E.M. for three independent experiments.

Figure S4 Phenotypic analysis of tomato plants silenced for SlSPS usingVIGS

Upper panel, accumulation of α-tocopherol, γ -tocopherol and UQ10 in control (TRV2) andTRV2-SlSPS tomato plants. Leaf material was collected from four seedlings 4 weeks afterinfection with Agrobacterium hosting the TRV1- and TRV2-based vectors. The leaves werepooled and three determinations made per sample. Results are means + S.E.M. Student t testswere used to determine significant differences between respective wild-type backgrounds and thetransgenic varieties. Lower panel, lipid peroxidation levels in control (TRV2) and TRV2-SlSPStomato plants, represented as mmol of MDA/g of FW (fresh mass). Determinations are from fiveplants per treatment, with a single leaflet (60–170 mg) used for each reaction.

Figure S5 Correlation between solanesol content and expression of SlSPSin mature leaves of transgenic tobacco varieties

Isoprenoid contents are given as μg/g of DW and determined as described in the Experimentalsection of the main text. Three representative leaves from a single T1 generation plant were used,the leaves were pooled and three determinations made per sample. qRT–PCR was performedusing three determinations per genotype from the same tissue, using primers specific for SlSPSnormalized to tobacco actin. Results are means +− S.E.M.

Figure S6 Trolox equivalent antioxidant activity (TEAC) determinations oftobacco-overexpressing SlSPS under control of the CaMV35S promoter

Immature leaf tissue was harvested from six wild-type (WT) and six separate T1 generationplants derived from two independent transformants. Samples were freeze dried and analysedin duplicate as described the Experimental section of the main text. Error bars show S.E.M.Student’s t test was used to determine significant differences between untransformed controland the transgenic variety.



Table S1 Characterization of purified SlSPS and SlDPS proteins by MS

Proteins were excised by SDS/PAGE and digested with trypsin as described in the Experimental section of the main text. Two separate digests were performed for each protein and analysed separatelyby both MALDI–TOF-MS and ESI.

(a) SlSPS

ESI MALDI

Position Parent ion Sequence ESI Ion Ion species MALDI Ion Species

14–27 1555.4362 TPLESLACGCSFSK Yes 519.486 3 +14–27 1555.5054 TPLESLACGCSFSK Yes 778.76 2 +56–69 1410.6874 ASLTGLAPVLDLNK Yes 706.351 2 +93–118 2636.7674 NLHNIVGAENPVLMSAAEQIFGAGGK Yes 1319.391 2 +93–118 2637.0742 NLHNIVGAENPVLMSAAEQIFGAGGK Yes 880.032 3 +93–118 2638.0102 NLHNIVGAENPVLMSAAEQIFGAGGK + 2 DEAMIDATION Yes 880.344 3 +93–118 2653.9862 NLHNIVGAENPVLMSAAEQIFGAGGK + OXIDATION (M) Yes 885.336 3 +93–118 2792.0549 NLHNIVGAENPVLMSAAEQIFGAGGKR Yes 699.021 4 +93–118 2792.9869 NLHNIVGAENPVLMSAAEQIFGAGGKR + DEAMIDATION Yes 699.254 4 +119–130 1411.812 RVRPALVFLVSR Yes 1412.819 SINGLE120–130 1255.5442 VRPALVFLVSR Yes 419.522 3 + Yes 1256.777 SINGLE131–139 906.3354 ATAEMSGLK Yes 454.175 2 +140–146 869.2474 ELTTNHR Yes 435.631 2 + Yes 870.4306 SINGLE174–185 1401.5154 GKETIHQLYGTR Yes 701.765 2 + Yes 1402.752 SINGLE174–185 1401.5302 GKETIHQLYGTR Yes 468.184 3 +176–185 1216.4194 ETIHQLYGTR Yes 609.217 2 + Yes 1217.623 SINGLE213–219 799.3414 LISQVIK Yes 400.678 2 +213–227 1646.9794 LISQVIKDFASGEIK Yes 824.497 2 +220–227 865.2474 DFASGEIK Yes 433.631 2 +228–245 2098.6552 QASNLFDCDVGLDEYLLK Yes 700.559 3 + Yes 2099.972 SINGLE228–245 2098.7174 QASNLFDCDVGLDEYLLK Yes 1050.366 2 +250–259 961.3894 TASLIAASTK Yes 481.702 2 +260–280 2291.6614 GAAIFSEVGSDISEQMFQYGR + DEAMIDATED Yes 1146.838 2 +260–280 2291.7682 GAAIFSEVGSDISEQMFQYGR Yes 764.93 3 + Yes 2292.04 SINGLE313–325 1371.4672 GNLTAPVLFALEK Yes 458.196 3 +313–325 1371.6314 GNLTAPVLFALEK Yes 686.855 2 +313–330 1981.8394 GNLTAPVLFALEKEPNLR Yes 991.427 2 +313–330 1981.7572 GNLTAVLFALEKEPNLR + DEAMIDATION Yes 661.593 3 +331–351 2327.6469 NIIESEFHDAGSLEEAINLVK Yes 582.919 4 + Yes 2328.145 SINGLE331–351 2327.7741 NIIESEFHDAGSLEEAINLVK Yes 1164.893 2 +331–351 2327.9422 NIIESEFHDAGSLEEAINLVK Yes 776.989 3 +365–375 1259.4514 EKADLAMQNLK Yes 630.733 2 +365–375 1275.4834 EKADLAMQNLK + OXIDATION (M) Yes 638.749 2 +367–375 1002.3494 ADLAMQNLK Yes 502.182 2 +376–391 1787.6512 CLPSSPFQAALEEIVK Yes 596.891 3 +376–391 1787.7474 CLPSSPFQAALEEIVK Yes 894.881 2 +392–398 935.3214 YNLERIE Yes 468.668 2 + Yes 936.5386 SINGLE

(b) SlDPS

ESI MALDI

Position Parent ion Sequence ESI Ion Ion species MALDI Ion Ion species

6–12 743.3902 GLAQISR Yes 744.3975 SINGLE13–17 678.3382 NRFSR Yes 679.3455 SINGLE42–46 603.2875 VLGCR Yes 604.2948 SINGLE94–102 958.3334 SMVVAEVPK Yes 480.174 2 +94–102 974.2954 SMVVAEVPK + OXIDATION M Yes 488.155 2 +103–112 1145.3574 LASAAEYFFK Yes 573.686 2 +139–153 1591.5854 SAPQVDVDSFSGDLR Yes 796.8 2 +139–153 1591.6802 SAPQVDVDSFSGDLR Yes 1592.688 SINGLE185–196 1236.4074 GIGSLNFVMGNK + DEAMIDATION NQ Yes 619.211 2 +185–196 1251.4734 GIGSLNFVMGNK + OXIDATION M Yes 626.744 2 +197–208 1273.5934 LAVLAGDFLLSR Yes 637.804 2 +197–208 1273.6774 LAVLAGDFLLSR Yes 1274.685 SINGLE248–256 1238.2854 CSMEYYMQK + OXIDATION M Yes 620.15 2 +248–260 1793.7424 CSMEYYMQKTYYK Yes 1794.75 SINGLE261–270 1079.3374 TASLISNSCK 540.676 2 +261–270 1079.668 TASLISNSCK Yes 540.668 2 +316–322 746.3529 GSLSDIR Yes 747.3601 SINGLE323–341 2184.0033 HGIVTAPILYAMEEFPQLR Yes 2185.011 SINGLE323–341 2200.0041 HGIVTAPILYAMEEFPQLR + OXIDATION (M) Yes 2201.011 SINGLE347–363 1863.8268 GFDDPVNVEIALDYLGK Yes 1864.834 SINGLE



Table S1 Continued

ESI MALDI

Position Parent ion Sequence ESI Ion Ion species MALDI Ion Ion species

376–398 2467.7549 KHASLASAAIDSLPESDDEEVQR Yes 617.946 4 +376–398 2467.8082 KHASLASAAIDSLPESDDEEVQR + DEAMIDATION NQ Yes 823.61 3 +376–398 2467.1311 KHASLASAAIDSLPESDDEEVQR Yes 2468.138 SINGLE377–398 2339.0202 HASLASAAIDSLPESDDEEVQR Yes 2340.028 SINGLE401–409 1093.4674 RALVELTHR 547.741 2 +401–409 1093.5807 RALVELTHR Yes 1094.588 SINGLE402–409 937.4872 ALVELTHR Yes 938.4945 SINGLE402–409 937.3654 ALVELTHR Yes 469.69 2 +

Table S2 Quinone species identified in E. coli extract harbouring the plasmids pET14b-SPS and pET14b-DPS

Compounds were identified by LC-MS(/MS) and for UQ9 and UQ10 were verified by authentic standards.

Compound Rt (min) Formula Monoisotopic neutral mass [M + H]+ (calculated) [M + H]+ (measured)

Menaquinone 8 11.6 C51H72O2 716.553232 717.560508 717.5525UQ8 10.3 C49H74O4 726.558711 727.565987 727.5285UQ9 C54H82O4 794.621311 795.628588

Standard 11.3 795.6285Sample 11.2 795.6198

UQ10 C59H90O4 862.683911 863.691188Standard 11.8 863.6912Sample 11.7 863.6804

Table S3 Effect of divalent cation requirement of purified SlSPS and SlDPSproteins in vitro

Diphosphate synthase activity was measured in the presence of the indicated divalent cations at afinal concentration of 10 mM in a reaction containing 50 mM Tris/HCl buffer (pH 7.5), 177.6 nMGGPP (for purified SlSPS assay or 184.6 nM FPP for SlDPS assay), 1 mM DTT, [1-14C]IPP,0.5 % Triton X-100 and 2.5 μg of purified protein as described in the Experimental sectionof the main text. Values show the amount of [1-14C]IPP incorporated into butanol-extractablepolyprenyl diphosphate as DPM/min per μg of purified protein +− S.E.M. from three assaysmeasured in duplicate.

DPM/min per μg of protein

Cation added SlSPS SlDPS

MgCl2 388.8 +− 73.5 249.5 +− 19.6MnCl2 274.7 +− 55.0 284.4 +− 7.5ZnCl2 160.5 +− 2.3 195.3 +− 4.5CaCl2 104.9 +− 37.8 82.1 +− 10.4MgCl2 (no enzyme) 69.5 +− 4.8 129.2 +− 5.4No cation 120.2 +− 42.0 82.5 +− 23.0



Table S4 Carotenoid, chlorophyll and PQ contents found in seedlings infected with TRV2 DPS, TRV2 SDS, TRV2 SDS-DPS and TRV2 PDS compared withTRV2 MCS controls

Isoprenoid contents are given as μg/g of DW and determined as described in the Experimental section of the main text. For each treatment, leaf material was collected from five seedlings 4 weeksafter infection with Agrobacterium hosting the TRV1- and TRV2-based vectors. The leaves were pooled and three determinations made per sample. Results are +− S.E.M. Values that were determinedby Student’s t test to be significantly different from the TRV MCS control are shown in bold. *P < 0.05, **P < 0.01 and ***P<0.001.

Isoprenoid content (μg/g of DW)

Compound TRV2 MCS TRV2 DPS TRV2 SDS TRV2 SDS-DPS TRV2 PDS

Phytoene 5.4 +− 0.82 4.0 +− 0.20 262.4 +− 34.07** 213.6 +− 46.23* 1236.6 +− 652.2***Lutein 2203.1 +− 88.6 2134.8 +− 85.0 1704.1 +− 52.7** 1923.2 +− 172.6 1544.0 +− 182.4β-Carotene 682.1 +− 36.5 626.4 +− 17.9 603.7 +− 83.4 540.4 +− 58.1 420.9 +− 55.9*13-cis-β-Carotene 82.5 +− 2.8 78.8 +− 2.7 65.4 +− 8.8 69.1 +− 6.6 55.5 +− 6.8*Neoxanthin 374.1 +− 17.9 358.7 +− 13.8 274.6 +− 15.0** 317.1 +− 29.9 226.1 +− 36.7*Violaxanthin 406.5 +− 17.6 370.0 +− 11.8 293.5 +− 20.1** 351.2 +− 24.7 228.7 +− 34.3*Chlorophyll a 3169.7 +− 108.1 2941.7 +− 106.0 2361.8 +− 2644.4 +− 240.6 2153.1 +− 257.0*Chlorophyll b 3416.2 +− 130.3 3188.9 +− 110.8 2638.5 +− 81.2** 2936.2 +− 254.5 2346.7 +− 284.7*PQ 422.2 +− 65.6 408.9 +− 22.6 246.3 +− 27.8* 262.6 +− 38.13 326.3 +− 33.4

Table S5 Quinone, tocopherol and polyprenol species detected in tomato and tobacco extracts by LC-MS

Compounds were identified by LC-MS(/MS) and confirmed by authentic standards where shown. N.D., not detected.

Compound Rt (min) Formula Monoisotopic neutral mass [M + H]+ (calculated) [M + H]+ (measured)

UQ10 C59H90O4 862.683911 863.691188Standard 11.6 863.9626Sample 11.6 863.6891

Semi-UQ10 (UQ10-H) C59H91O4 863.6917 864.699Standard 10.9 864.9613Sample N.D.

UQ10 (UQ10-H2) C59H92O4 864.6995 865.7068Standard 8.6 865.9742Sample N.D.

PQ9 11.6 C53H80O2 748.615832 749.623108 749.6213PQ-H2 8.8 C53H82O2 750.631482 751.638758 751.6352

631.581243Solanesol C45H74O 630.573967 613.570679 ( − H2O)

Standard 9.8 613.5702Sample 9.8 613.5713

Decaprenol 10.9 C50H82O 698.636567 699.643844 681.6341681.633279 ( − H2O)

α-Tocopherol C29H50O2 430.381081 431.388357Standard 6.2 431.3877Sample 6.2 431.3846

γ -Tocopherol 5.3 C28H48O2 416.365431 417.372707 417.3718



Table S6 Metabolite levels in seedlings infected with TRV2 DPS, TRV2 SDS, TRV2 SDS-DPS and TRV2 PDS compared with the TRV2 MCS controls

For each treatment, leaf material was collected from four seedlings 4 weeks after infection with Agrobacterium hosting the TRV1- and TRV2-based vectors. Data have been normalized to samplemasses and are shown as mean contents +− S.E.M. Values that were determined by Student’s t test to be significantly different from the control are shown in bold. *P < 0.05, **P < 0.01 and***P<0.01. N.D., not detected.

Metabolite content (mg/g of DW) TRV2 MCS TRV2 DPS TRV2 SDS TRV2 SDS-DPS TRV2 PDS

Amino acidsAlanine 0.124 +− 0.006 0.234 +− 0.030* 0.141 +− 0.011 0.259 +− 0.014*** 0.374 +− 0.012***Asparagine 0.240 +− 0.080 0.263 +− 0.018 0.509 +− 0.047* 0.823 +− 0.101** 2.992 +− 0.126***Glutamine 0.627 +− 0.041 0.708 +− 0.057 0.825 +− 0.035* 0.897 +− 0.072* 1.241 +− 0.084***Glycine 0.107 +− 0.018 0.155 +− 0.034 0.052 +− 0.007* 0.056 +− 0.007* 0.065 +− 0.001Isoleucine 0.018 +− 0.003 0.024 +− 0.003 0.023 +− 0.004 0.035 +− 0.003* 0.070 +− 0.007***Leucine 0.001 +− 0.001 0.003 +− 0.001 0.002 +− 0.002 N.D. 0.084 +− 0.006***Proline 0.406 +− 0.083 0.985 +− 0.178* 0.686 +− 0.190 0.602 +− 0.153 0.243 +− 0.080Serine 0.252 +− 0.037 0.297 +− 0.033 0.255 +− 0.019 0.386 +− 0.052 0.708 +− 0.035***Threonine 0.198 +− 0.027 0.191 +− 0.016 0.155 +− 0.013 0.185 +− 0.019 0.342 +− 0.013**Valine 0.040 +− 0.010 0.057 +− 0.007 0.046 +− 0.002 0.074 +− 0.006* 0.134 +− 0.002***

Organic acidsCitric acid 0.809 +− 0.280 0.459 +− 0.066 1.129 +− 0.015 0.634 +− 0.268 0.306 +− 0.041Fumaric acid 0.255 +− 0.040 0.258 +− 0.023 0.309 +− 0.070 0.156 +− 0.024 0.099 +− 0.005**Glucaric acid 0.185 +− 0.052 0.142 +− 0.022 0.260 +− 0.041 0.153 +− 0.033 0.141 +− 0.020Glycolic acid 0.015 +− 0.001 0.012 +− 0.001 0.018 +− 0.003 0.013 +− 0.002 0.010 +− 0.001*Isocitric acid 0.557 +− 0.031 0.741 +− 0.058* 0.455 +− 0.061 0.332 +− 0.033** 0.222 +− 0.009***Isonicotinic acid 0.055 +− 0.008 0.064 +− 0.003 0.082 +− 0.004* 0.073 +− 0.005 0.088 +− 0.011Maleic acid 0.240 +− 0.030 0.280 +− 0.016 0.283 +− 0.013 0.168 +− 0.026 0.095 +− 0.006**Malic acid 4.169 +− 0.608 3.886 +− 0.299 3.678 +− 0.448 2.560 +− 0.484 1.235 +− 0.127**Palmitic acid 0.455 +− 0.065 0.448 +− 0.035 0.468 +− 0.056 0.612 +− 0.484 0.528 +− 0.117Stearic acid 0.188 +− 0.062 0.113 +− 0.018 0.113 +− 0.017 0.157 +− 0.041 0.127 +− 0.054Succinic acid 0.034 +− 0.002 0033 +− 0.005 0.041 +− 0.005 0.036 +− 0.004 0.032 +− 0.004

SugarsArabinose 0.035 +− 0.004 0.071 +− 0.012* 0.071 +− 0.018 0.054 +− 0.009 0.056 +− 0.004*Fructose 6.463 +− 0.510 18.526 +− 2.099** 11.104 +− 2.135 5.791 +− 0.466 5.177 +− 0.233Galactose 0.762 +− 0.061 2.578 +− 1.325 1.935 +− 0.311* 0.695 +− 0.027 0.389 +− 0.017**Glucaric acid 1.537 +− 0.299 0.867 +− 0.059 1.195 +− 0.153 1.016 +− 0.189 0.648 +− 0.089Gluconic acid N.D. 0.006 +− 0.006 0.028 +− 0.001 0.010 +− 0.007 N.D.Glucose 4.917 +− 0.383 12.363 +− 2.027* 8.514 +− 1.903 4.648 +− 0.334 3.585 +− 0.052*Mannose 0.030 +− 0.002 0.062 +− 0.010* 0.182 +− 0.134 0.017 +− 0.004* 0.017 +− 0.001**Xylose 0.025 +− 0.008 0.054 +− 0.006* 0.065 +− 0.008* 0.055 +− 0.010 0.066 +− 0.004**Unknown sugar 0.297 +− 0.085 0.223 +− 0.016 0.320 +− 0.016 0.253 +− 0.035 0.209 +− 0.034

Sugar alcoholsGlycerol 0.566 +− 0.024 0.562 +− 0.026 0.422 +− 0.026** 0.421 +− 0.047* 0.389 +− 0.012***Inositol 7.979 +− 0.587 12.668 +− 0.530** 9.502 +− 1.301 8.311 +− 0.831 6.244 +− 0.269*Mannitol 0.006 +− 0.006 0.500 +− 0.482 0.176 +− 0.176 0.007 +− 0.004 0.026 +− 0.026

Othersα-D-Glucopyranoside 0.246 +− 0.130 0.576 +− 0.056 0.307 +− 0.061 0.185 +− 0.060 0.095 +− 0.025Unknown flavonoid 1 0.164 +− 0.050 0.152 +− 0.016 0.245 +− 0.045 0.110 +− 0.024 0.021 +− 0.009*Unknown flavonoid 2 0.059 +− 0.027 0.054 +− 0.011 0.116 +− 0.013 0.041 +− 0.007 0.039 +− 0.026



Table S7 Characterization of T0 generation tobacco plants overexpressing SlSPS

Solanesol content and SlSPS expression in expanding leaves of T0 generation tobacco plants expressing SlSDS under control of the CaMV35S promoter. Two leaves were pooled for each plant fromwhich RNA was extracted from genotypes with solanesol levels greater than the control (WT) as well as three lines with no detected solanesol. Wild-type plants were regenerated through tissueculture to serve as controls. For solanesol quantification and gene expression there were two and three determinations per plant respectively. Phenotypic abnormalities were recorded in matureglasshouse-grown plants. N.D., not detected.

Line Solanesol content (μg/g of DW) Relative transcript abundance (where determined) Phenotypic observations

WT 1 N.D. 0.00 +− 0.00WT 2 11.19 +− 0.34 0.00 +− 0.00WT 3 1.84 +− 0.30WT 4 7.98 +− 3.34S-1 N.D.S-3 N.D. 0.09 +− 0.01 Bleached young leavesS-5 N.D.S-7 N.D.S-11 N.D. 0.15 +− 0.02 Dark leaf colourS-12 N.D. 0.26 +− 0.03S-15 N.D.S-17 N.D.S-20 N.D.S-24 N.D.S-25 N.D.S-27 N.D.S-28 N.D.S-23 0.26 +− 0.26S-30 0.28 +− 0.28S-4 0.50 +− 0.17S-13 0.90 +− 0.90S-8 1.45 +− 0.66S-10 2.83 +− 2.65S-16 3.17 +− 1.36S-29 3.60 +− 1.78S-6 8.35 +− 1.94S-19 36.73 +− 5.78 1.00 +− 0.15 Young leaves mottled appearance, reduced statureS-14 50.35 +− 36.27 0.42 +− 0.04 Young leaves bleachedS-9 72.07 +− 1.38 0.39 +− 0.06S-21 193.00 +− 76.30 Young leaves mottled appearance

REFERENCE

1 Corpet, F. (1988) Multiple sequence alignment with hierarchical clustering. Nucleic AcidsRes. 16, 10881–10890

Received 19 June 2012/2 November 2012; accepted 5 November 2012Published as BJ Immediate Publication 5 November 2012, doi:10.1042/BJ20120988


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