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First published online April 2, 2004; 10.1104/pp.103.038133 Plant Physiology 134:1401-1413 (2004) © 2004 American Society of Plant Biologists Functional Analysis of the Final Steps of the 1-Deoxy-D-xylulose 5-phosphate (DXP) Pathway to Isoprenoids in Plants Using Virus-Induced Gene Silencing1Leibniz-Institut für Pflanzenbiochemie (J.E.P., J.S., T.M.K.), 06120 Halle (Saale), Germany; and Biozentrum der Universität Halle, 06120 Halle (Saale), Germany (G.H., W.G., M.R., M.H.Z.)
Isoprenoid biosynthesis in plant plastids occurs via the 1-deoxy-D-xylulose 5-phosphate (DXP) pathway. We used tobacco rattle virus (TRV) to posttranscriptionally silence the expression of the last two enzymes of this pathway, the IspG-encoded (E)-4-hydroxy-3-methylbut-2-enyl diphosphate synthase (HDS) and the IspH-encoded isopentenyl/dimethylallyl diphosphate synthase (IDDS), as well as isopentenyl/dimethylallyl diphosphate isomerase (IDI), the enzyme that interconverts IPP and DMAPP. TRV-IspG and TRV-IspH infected Nicotiana benthamiana plants had albino leaves that contained less than 4% of the chlorophyll and carotenoid pigments of control leaves. We applied [13C]DXP and [14C]DXP to silenced leaves and found that 2-C-methyl-D-erythritol 2,4-cyclodiphosphate accumulated in plants blocked at HDS while DXP, (E)-4-hydroxy-3-methylbut-2-enyl phosphate and (E)-2-methylbut-2-ene-1,4-diol accumulated in IDDS-blocked plants. Albino leaves from IspG- and IspH-silenced plants displayed a disorganized palisade mesophyll, reduced cuticle, fewer plastids, and disrupted thylakoid membranes. These findings demonstrate the participation of HDS and IDDS in the DXP pathway in plants, and support the view that plastid isoprenoid biosynthesis is metabolically and physically segregated from the mevalonate pathway. IDI-silenced plants had mottled white-pale green leaves with disrupted tissue and plastid structure, and showed an 80% reduction in pigments compared to controls. IPP pyrophosphatase activity was higher in chloroplasts isolated from IDI-silenced plants than in control plant chloroplasts. We suggest that a low level of isoprenoid biosynthesis via the DXP pathway can occur without IDI but that this enzyme is required for full function of the DXP pathway.
Isoprenoids (also called terpenoids or terpenes) form one of the largest and most structurally diverse groups of metabolites in nature. Members of the plant kingdom alone are estimated to produce >30,000 isoprenoid compounds (Gershenzon, personal communication), many of them secondary metabolites with defensive or signaling properties (Croteau et al., 2000  ). Plants synthesize all isoprenoids from two C5 precursors, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), with DMAPP serving as a starter unit for chain elongation reactions that yield C10, C15, C20, C30, and C40 diphosphate esters. Such prenyl diphosphates are further metabolized to give monoterpenes (C10), sesquiterpenes (C15), diterpenes, and the phytol side chain of chlorophylls (C20), sterols (C30), and carotenoids (C40). In plants, IPP and DMAPP are synthesized by independent metabolic pathways, which are active in different cellular compartments. The mevalonate (MVA) pathway, which provides C5 precursors for the synthesis of some sesquiterpenes, sterols and the side chain of ubiquinone (in higher plants), is localized in the cytoplasm while the plastid localized 1-deoxy-D-xylulose 5-phosphate (DXP) pathway (Fig. 1
) produces the IPP and DMAPP used to synthesize monoterpenes, some sesquiterpenes and diterpenes. The DXP pathway is alternatively known as the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. Perhaps most significantly for plant function, the two major classes of photosynthetic pigments, chlorophylls and carotenoids, are formed in the plastid with IPP and DMAPP from the DXP pathway (Arigoni et al., 1997; Eisenreich et al., 2001).
The enzymatic steps of the DXP pathway and the genes encoding them have been completely elucidated in Escherichia coli and for most steps, also in plants (for reviews, see Rodríguez-Concepción and Boronat, 2002 ; Rohdich et al., 2003; Rohmer, 2003). The penultimate enzyme of the DXP pathway is (E)-4-hydroxy-3-methylbut-2-enyl diphosphate synthase (HDS), encoded in E. coli by the IspG/GcpE gene (Cunningham et al., 2000; Altincicek et al., 2001), which has been shown to catalyze the formation of (E)-4-hydroxy-3-methylbut-2-enyl diphosphate (HMBPP) from 2-C-methyl-D-erythritol 2,4-cyclodiphosphate (cMEPP; Hecht et al., 2001). An Arabidopsis IspG-like cDNA complements an IspG-defective E. coli strain (Querol et al., 2002), providing evidence that the plant HDS has a similar function to its bacterial homolog. The final step in the DXP pathway was recently shown to be catalyzed by isopentenyl/dimethylallyl diphosphate synthase (IDDS, encoded by the E. coli IspH/LytB gene and its homologs in other organisms), which converts HMBPP to IPP and DMAPP (Rohdich et al., 2002). While clarified both genetically (Cunningham et al., 2000; McAteer et al., 2001) and biochemically in bacteria, the role of IspH has yet to be functionally analyzed in plants. IPP and DMAPP are interconverted by isopentenyl/dimethylallyl diphosphate isomerase (IDI; EC 5.3.3.2), the protein product of the IDI gene. IDI plays a central role in terpene biosynthesis, apparently regulating the ratio of C5 precursors from the DXP and mevalonate pathways in the plastid and cytosol, respectively. IDI is a nonessential gene in E. coli (Hahn et al., 1999) and it is not clear if it is absolutely required in plants.
We used virus-induced gene silencing (VIGS) to analyze the function of IspG, IspH, and IDI in plants. VIGS exploits the RNA silencing pathway to posttranscriptionally block expression of host plant genes (Baulcombe, 1999 In this paper we show that the VIGS knockout of IspG and IspH produces plants with albino leaves due to the impairment of the ability to synthesize chlorophylls and carotenoids via the DXP pathway. Silencing IDI produced plants with a mottled white-pale green leaf phenotype. Microscopic examination of silenced tissue found abnormalities in leaf ultrastructure, including degraded chloroplasts, in IspG-, IspH-, and IDI-silenced plants. We characterized the metabolic blocks in IspG- and IspH-silenced plants by feeding labeled DXP and profiling the intermediates of the DXP pathway. In the case of IspG, albino leaves accumulated cMEPP, the substrate of the IspG-encoded HDS enzyme, and leaves of IspH silenced plants accumulated HMBP and MBDO, which are dephosphorylated congeners of HMBPP, the substrate of the IspH-encoded IDDS enzyme. These results provide the first evidence that selective knockout of IspG and IspH prevents the formation of plant isoprenoids. Furthermore, it confirms the fundamental metabolic and physical separation of the MVA and DXP pathways. Silencing of IDI also led to decreased chlorophyll and carotenoid levels, although somewhat less than that observed with IspG and IspH silencing, demonstrating that IDI, unlike its E. coli homolog, is required for normal functioning of isoprenoid biosynthesis in plant plastids.
Cloning of TRV-IspG, -IspH, and -IDI Silencing Constructs
Full-length sequences for N. benthamiana IspG, IspH, and IDI cDNAs have not been published and we used sequences from tobacco (Nicotiana tabacum) and other Solanaceae to design primers to amplify cDNA fragments. PCR with IspG primers gave an 804-bp product, NbIspG, which showed 90% identity with tomato (Lycopersicon esculentum) IspG cDNA. In the same fashion, a 500-bp NbIspH and a 453-bp NbIDI cDNA fragment were amplified. NbIspH was most closely related (98% identity) to a partial cDNA clone of the tobacco IspH gene (AF159699). NbIDI showed 94% nucleotide identity with the IPP1 gene encoding the plastidic isoform of tobacco IDI, compared with 87% identity with the IPP2 gene encoding the cytosolic isoform (Nakamura et al., 2001
Our strategy for constructing TRV plasmids was to ligate PCR amplified cDNAs into the cloning vector, pGEM-Teasy. cDNA fragments were excised by restriction enzyme digestion and ligated into pTV00, a binary vector that expresses a modified TRV-RNA2 under the control of the 35S promoter (Ratcliff et al., 2001 ). This strategy introduced 4 bp and 46 bp of the pGEM-Teasy multiple cloning site at the 5' and 3' end of the inserted cDNAs, respectively. The IspG fragment was in the sense orientation relative to the TRV coat protein while the IspH and IDI fragments were in the antisense orientation. TRV-IspG, -IspH, and -IDI constructs were coinfiltrated with Agrobacterium tumefaciens containing pBINTRA6, a binary vector construct of TRV-RNA1 (Ratcliff et al., 2001), into N. benthamiana leaves. Mock infected plants infiltrated with the A. tumefaciens resuspension buffer and plants infected with pTV00 plasmid without a cDNA insert (empty TRV) served as controls.
As reported by Ratcliff et al. (2001)
Anatomy of Silenced Leaves To further characterize the IspG-, IspH-, and IDI-silencing phenotypes, we examined leaf ultrastructure using light and transmission electron microscopy (Fig. 4 ). Leaf tissue from mock and empty TRV infected plants was indistinguishable, indicating that TRV infection itself had little effect on tissue or plastid structure. A thick cuticle, organized palisade mesophyll, and numerous large starch granules within chloroplasts of mesophyll cells were characteristic features of control leaves. As well, electron microscopy showed that chloroplasts in such plants had robust grana with intact thylakoid membranes. Albino tissue of TRV-IspG infected plants was characterized by a number of unusual features including epidermal protuberances, a cuticle of reduced thickness, and disorganized palisade mesophyll. Chloroplasts were reduced in number and size in all cell types and starch granules were not visible. In chloroplasts that were present, thylakoid membranes were disrupted and globular lytic vesicles were observed. IspH-silenced tissues also had epidermal protuberances, reduced cuticle, and disorganized palisade mesophyll structure, although these features were less pronounced than in IspG-silenced plants. Although chloroplasts were also reduced in number and had severely disrupted thylakoid membranes in most cell types, chloroplasts in vascular tissue and companion cells of IspH-silenced plants appeared normal. The features that marked the albino tissue of IspG and IspH plants were not as distinct in TRV-IDI infected plants. Such leaves contained a normal number of chloroplasts although starch granules were still absent. A notable feature of IDI-silenced leaf tissue was that chloroplasts contained thylakoid membranes with an unusual fluted structure (Fig. 4).
Semiquantitative RT-PCR Analysis of Gene Expression in Silenced Tissue We measured transcript levels of NbIspG, NbIspH, and NbIDI in silenced tissue using RT-PCR with primers annealing at the 5' end of the silenced genes, outside of the region used for the TRV constructs (Fig. 2). Actin was amplified as a control transcript. The accumulation of each of the three transcripts was reduced by more than 90% in silenced tissue (Fig. 5 ).
Analysis of Photosynthetic Pigments in Silenced Leaf Tissue The effect of knocking out IspG, IspH, and IDI expression on isoprenoid biosynthesis was measured by quantifying total chlorophylls and carotenoids in silenced leaves (Fig. 6 ). Silenced tissue was separated from green tissue and extracted with organic solvent. For mock and empty TRV infected plants, tissue from an equivalent location and leaf age to that used for silenced samples was used. The content of total chlorophylls and carotenoids was slightly higher in leaves of empty TRV infected plants compared to mock infected, an observation that was consistent throughout our experiments. Silenced tissue in TRV-IspG infected plants had total chlorophyll and carotenoid levels that were reduced by 98% and 96% of mock-infected plants, respectively. In TRV-IspH infected leaves pigment levels were reduced by 98% of mock infected for total chlorophylls and 96% for total carotenoids. IDI silencing produced a less dramatic reduction in photosynthetic pigment levels compared to the other TRV constructs; a result in agreement with mottled green appearance of TRV-IDI infected leaves. Total chlorophylls in TRV-IDI leaves were reduced 80%, and total carotenoids by 78%, compared to mock infected leaves.
Metabolic Profiles After [13C]/[14C]DXP Feeding to TRV-IspG and TRV-IspH Silenced Plants
To examine the effect that silencing IspG and IspH has on the intermediates of the DXP pathway, we fed [14C]DXP to detached leaves of mock and empty TRV infected control plants and to albino leaves of TRV-IspG and TRV-IspH silenced plants. This feeding study assumed that DXP is dephosphorylated and then rephosphorylated after entry into cells by D-xylulokinase (Wungsintaweekul et al., 2001
Incorporation of [14C]DXP by IspH-silenced leaves led to the accumulation of two new 14C-labeled compounds (Rt approximately 13 and 18 min) not found in either control or TRV-IspG silenced plants, together with a substantial amount of [14C]DXP (Rt, approximately 16 min; Fig. 7C). Based on the comparison of retention times with reference compounds, we tentatively identified the 13 min peak as MBDO. For conclusive identification, we fed detached leaves with [3,4,5-13C3]DXP mixed with a small amount of [3,4,5-14C3]DXP. The leaves were worked up as described above, the aqueous fraction treated with alkaline phosphatase, and the putative MBDO peak purified using TLC. Subsequent mass spectrometric analysis unequivocally demonstrated it to be MBDO, as evidenced by the three 13C-atoms from the applied [3,4,5-13C3]DXP. The 18-min peak shared exact retention time characteristics with an authentic standard of HMBP, a compound not previously reported. Its identification as such was further supported by its conversion to MBDO upon alkaline phosphatase treatment (data not shown). MBDO and HMBP are both dephosporylated congeners of HMBPP, the substrate of IDDS.
To examine the effect of silencing IDI on the metabolism of IPP, chloroplasts isolated from green leaf tissue from mock infected and white-pale green tissue from TRV-IDI infected plants were incubated with [1-14C]IPP. RadioHPLC analysis found that IPP was dephosphorylated to isopentenyl monophosphate (IP) by chloroplasts of both plants. This conversion was significantly higher in TRV-IDI leaf tissue compared to mock infected tissue. Kinetic analysis (Fig. 8 ) showed that after a 180-min incubation, less than 20% of the applied [1-14C]IPP was converted to IP by chloroplasts from mock infected plants while more than 50% was converted by chloroplasts from tissue in which IDI was silenced. We also detected significant conversion of IP further to isopentenol in IDI-silenced plants (data not shown).
We analyzed the function of IspG, IspH, and IDI cDNAs from N. benthamiana using a VIGS approach. The albino leaves of IspG- and IspH-silenced plants, which contained less than 4% of the isoprenoid-derived chlorophyll and carotenoid pigments of control plants, provide evidence for the participation of the IspG-encoded HDS and IspH-encoded IDDS in the DXP pathway. Neither HDS nor IDDS have been previously characterized by knockout in plants; indeed, this is the first report to our knowledge to prove the role of a plant IDDS in the DXP pathway. We analyzed the metabolic blocks caused by silencing using feeding studies with [13C]DXP/[14C]DXP. Leaves in which HDS expression was silenced converted labeled DXP to DX, ME, and cMEPP, the latter of which was purified and identified spectroscopically. cMEPP is the substrate of HDS and has been shown to accumulate in E. coli cells overexpressing a synthetic operon containing the genes (DXS-DXR-IspD-IspE-IspF) in the DXP pathway up to the HDS catalyzed step (Hecht et al., 2001 ). This compound also builds up in gram-negative bacteria under "oxidative stress" conditions (Ostrovsky et al., 1992; Ostrovsky et al., 1998) and therefore serves as a stable metabolic sink in the DXP pathway. Metabolites that occur after the HDS-catalyzed step in the pathway were not detected in IspG-silenced plants despite the sensitivity afforded by the incorporation of [14C]DXP. In addition to the desphorylated intermediates DX and ME detected in IspG-silenced plants, leaves blocked at the IspH-encoded IDDS step showed incorporation of [14C]DXP into MBDO and HMBP, as identified by comparison of their retention times with standards and mass spectroscopic analysis. A possible explanation for the accumulation of MBDO and HMBP in IspH-silenced plants is that HMBPP is metabolically unstable, and is dephosphorylated first to HMBP (Fig. 1, 13) and finally to the metabolic inert MBDO (Fig. 1, 14).
The albino phenotype caused by IspG- and IspH-silencing parallels that of loss-of-function mutants of the DXP pathway. T-DNA insertions in DXS were found to be responsible for the albino Arabidopsis cla1-1 mutant (Mandel et al., 1996
VIGS seems an ideal method for knockout of these genes as it allowed us to overcome the problem of seedling lethality or dwarfism that characterizes plants blocked in the DXP pathway. N. benthamiana plants infected with TRV silencing constructs of PDS (Ratcliff et al., 2001
Plants infected with TRV-IspG and TRV-IspH plants displayed somewhat different leaf phenotypes. Plants infected with TRV-IspG developed albino leaves earlier than TRV-IspH plants and IspG silencing typically resulted in a greater proportion of fully white leaves than IspH plants, which often had white tissue only along leaf veins. Although some IspH-silenced plants developed fully white leaves after virus infection had proceeded for 2 to 3 weeks, the ability of the TRV-IspH construct to spread was impaired relative to TRV-IspG. Insertion of heterologous sequences into a viral genome may alter replication or infectivity. The observed differences in silencing phenotype may therefore be due to intrinsic properties, such as RNA secondary structure, of the IspG and IspH cDNA fragments used for silencing. It is interesting to note that Ham et al. (1999)
A body of evidence from feeding studies shows that precursor exchange (metabolic crosstalk) occurs between the DXP and MVA pathways. Examples of plant metabolites formed from both DXP and MVA precursors include monoterpenes and sesquiterpene volatiles in Phaseolus lunatus (Piel et al., 1998
We observed profound changes in the tissue and plastid structure of IspG, IspH, and IDI-silenced leaves. The most notable perturbations at the tissue level were the disorganization of the palisade mesophyll and reduction in chlorophyll number; characteristic changes in plastids included the disruption (or degradation) of thylakoids and the appearance of lytic vesicles. Such changes in structure were most obvious in IspG-silenced plants, followed by IspH- and IDI-silenced plants. Similar changes in plastid ultrastructure have been reported in Arabidopsis cla1-1, which was first isolated as an altered chloroplast mutant, and immutans mutants (Aluru et al., 2001
The mottled white-pale green leaves of IDI-silenced plants were strikingly different from the albino leaves of IspG and IspH-silenced plants. Chlorophyll levels in IDI-silenced leaves were reduced by about 80% compared to empty virus controls, but were 10-fold higher than in TRV-IspG and TRV-IspH infected plants. This difference was probably not due to the ability of TRV-IDI to mediate silencing since RT-PCR analysis showed that VIGS reduced IDI expression to a similar extent as for IspG and IspH transcripts in TRV-IspG and TRV-IspH plants, respectively (Fig. 5). IDI plays a role in both the DXP and MVA pathways with plastid (DXP pathway) and cytosol (MVA pathway) targeted isoforms of IDI reported in tobacco (Nakamura et al., 2001
We suggest that an explanation for the IDI-silenced leaf phenotype lies in the physiological role of IDI in plastid isoprenoid biosynthesis. Loss-of-function IDI mutants have not previously been reported in plants and this study represents the first attempt to knockout IDI activity. Insertional disruption of IDI in E. coli has no effect on the organism's growth rate (Hahn et al., 1999
Cloning of IspG, IspH, and IDI Gene Fragments
Total RNA was isolated from Nicotiana benthamiana Domin leaves using guanidium thiocyanate-phenol-chloroform. Five micrograms were reverse transcribed using Superscript II reverse transcriptase and an oligo(dT) primer. PCR for all three genes was performed using 2.5 units Taq polymerase, 200 µM dNTPs, and 200 nM of each primer in a 50-µL volume. PCR conditions were 94°C, 3 min followed by 94°C, 30 s; 50°C, 30 s; 72°C, 1 min for 30 cycles and finally 72°C, 7 min. Primer pairs were 5'-ATGCCATTCAAGGATCTGGC-3' and 5'-CGTCTTTCCGACATAAAGGTC-3', 5'-TGTGAAGAACATGGCAGAGG-3' and 5'-TCTCAACCAACTCACCATGC-3' and 5'-GGAAAGTGGGGAGAACATGA-3' and 5'-TCGACAGAAAGCCACAGCTA-3' designed to amplify fragments of IspG, IspH, and IDI cDNAs, respectively. PCR products were purified, ligated into pGEM-T easy (Promega, Madison, WI), and sequenced. Plasmid DNA was digested with Apa1 and Spe1 and the released DNA fragments purified before ligation into Apa1 and Spe1 digested pTV00. Constructs were confirmed by sequencing before transformation into Agrobacterium tumefaciens GV3101/pSa_RepA (Hellens et al., 2000
N. benthamiana plants were grown in a controlled environment chamber with 16 h/22°C days and 8 h/20°C nights. Cultures of A. tumefaciens containing pBINTRA6 or pTV constructs were grown overnight. After centrifugation, bacteria were resuspended in buffer containing 1 mM MES (pH 5), 10 mM MgCl2, and 100 µM acetosyringone to OD600 = 1 and allowed to stand at room temperature for 2 to 4 h. Suspensions of pBINTRA6 and pTV containing cultures were mixed 1:1 and infiltrated into the underside of two or three leaves of 4- to 5-week-old plants using a 2-mL syringe. Mock-infected control plants were infiltrated with Agrobacterium resuspension buffer. Empty TRV control plants were infiltrated with pBINTRA6 and pTV00 without cDNA insert.
Carotenoids and chlorophylls were extracted from N. benthamiana leaves 3 to 4 weeks after infiltration using a method modified from Fraser et al. (2000)
Total RNA was isolated from 100-mg portions of silenced leaf tissue from three plants using guanidium/ammonium thiocyanate-phenol-chloroform. First-strand cDNA was reverse transcribed from 5 µg total RNA using Superscript II reverse transcriptase and random primers. PCR for all genes was performed using 1 unit Taq polymerase, 200 µM dNTPs, and 200 nM of each primer in a 20-µL volume. To control for an equal amount of cDNA in each reaction, the amount of cDNA required to produce Actin amplification products of similar intensity for all samples was titered using 5'-ATGGCAGACGGTGAGGATATTCA-3' and 5'-GCCTTTGCAATCCACATCTGTTG-3' designed to amplify a 1.1-kb Actin cDNA fragment (Romeis et al., 2001
Fully white leaves of TRV-IspG and TRV-IspH infected plants or equivalent leaves from mock or empty TRV plants were detached at the petiole and placed in an aqueous solution of 0.5 µCi [3,4,5-14C3]DXP (specific activity 155 µCi/µmol) and 2.3 µmol [3,4,5-13C3]DXP. After most of the solution had been taken up, 500 µL of water was added in 100 µL aliquots over 7 to 8 h after which the excised leaf was provided with water ad libitum for 16 h. Leaves were weighed and leaf frozen in liquid nitrogen before extraction twice with 10 mL boiling 80% (v/v) ethanol for 20 min. The extract was concentrated under reduced pressure to give a residue that was partitioned between 1 mL water and 2 x 750 µL ethyl acetate. Total radioactivity in the ethanolic extract, and aqueous and organic phases, was measured by scintillation counting. The aqueous phase was lyophilized, redissolved in 250 µL water, and analyzed by ion-pair radioHPLC performed using a 4.6 mm x 250 mm Luna 5-µm C8 column (Phenomenex, Torrance, CA) with an ion-pair solvent system consisting of solvent A (10 mM tetra-n-butylammonium hydrogen sulfate in water) and solvent B (10 mM tetra-n-butylammonium hydrogen sulfate in 70% [v/v] methanol) and a gradient elution of 0 min (100% A), 20 min (100% A), 80 min (40% A:60% B), 85 min (100% A), and 100 min (100% A). Radioactivity was measured with a Ramona 2000 detector (Raytest, Straubenhardt, Germany). Alkaline phosphatase treatment was performed in a 1-mL reaction containing 50 mM Tris-HCl pH 8, 10 mM MgCl2, and 5 units alkaline phosphatase at 37° for 2 h. MBDO, DXP, HMBP, and cMEPP were identified by comparison with authenticate standards.
Detached albino leaves from TRV-IspG infected plants were fed 0.5 µCi [3,4,5-14C3]DXP and 2.3 µmol [3,4,5-13C3]DXP and extracted as described above. The aqueous phase was applied to a DEAE Sephadex (formate form) column (1 x 18 cm), which was eluted with a linear gradient of 0.06 M to 0.56 M ammonium formate (pH 8) at a flow-rate of 1.1 mL/min. Fractions were analyzed by scintillation counting and those containing the cyclic diphosphate were combined and lyophilized in vacuum. HRMS and 13C-NMR data of the residue so obtained showed that it was [1,3,4-13C3]cMEPP as follows. ESI-FT-ICRMS (negative ion mode) m/z: 276.98749 (14.4%, cMEPP, calc. for C5H11O9P2– 276.98838) and 279.99782 (100%, [1,3,4-13C3]cMEPP, calc. for C213C3H11O9P2– 279.99844); 13C-NMR data (500 MHz, H2O): 69.57 (d, J = 42.7 Hz, C-3), 68.14 (d, JCP = 4.1 Hz, C-1), 66.82 (dd, JCC = 42.7 Hz, JCP = 6.1 Hz, C-4).
Detached albino leaves from TRV-IspH infected plants were fed [3,4,5-14C3]DXP and [3,4,5-13C3]DXP and extracted as described for TRV-IspG plants. The resulting aqueous fraction was incubated with 3 units alkaline phosphatase for 2 h at 37°C. After centrifugation, the supernatant was separated on a silica plate (SIL-G/UV254, Macherey-Nagel, Duren, Germany) developed in chloroform-methanol-H2O (4:1:0.1). The band corresponding to [1,3,4-13C3]MBDO, identified by comparison of its Rf with an authenticate standard, was extracted with methanol and analyzed by radioHPLC and gas chromatography-mass spectrometry (GC-MS). The isolated compound and authentic MBDO showed the same behavior by ion-pair radioHPLC (RT: 13 min) and GC-MS. The 70 eV-EI mass spectrum of [1,3,4-13C3]MBDO was RT = 15.76 min, (m/z [rel. int., %]): 235 ([M-CH3]+, 0.4), 192 (45), 191 (13), 161 (5.6), 160 (7.4), 159 (3.6), 156 (2.9), 148 (22), 147 (78), 146 (42), 145 (23), 144 (8.6), 143 (19), 141 (2.2), 117 (5.0), 75 (43), and 73 (100). The isotopic distribution of 13C in the labeled MBDO was calculated using the [M-TMSiOH]+-ion (m/z 156 in nonlabeled MBDO) with values of 20% 13C0, 1% 13C1, 3% 13C2, 21% 13C3, 37% 13C4, and 17% 13C5. The 70 eV-EI mass spectrum of MBDO was RT = 15.68 min, (m/z [rel. int., %]): 231 ([M-CH3]+, 0.4), 191 (32), 156 ([M-TMSiOH]+, 9.6), 148 (10), 147 (66), 144 (8.8), 143 (70), 141 (7.7), 115 (9.1), 75 (27), and 73 (100). The GC-MS of MBDO and [1,3,4-13C3]MBDO were performed with a GC-MS system (Voyager, ThermoQuest, Dreieich, Germany). The following conditions were used: 70 eV EI, source temperature 200°C, column DB-5MS (J&W, 30 m x 0.25 mm, 0.25 µm film thickness), injection temperature 250°C, interface temperature 300°C, carrier gas He, flow rate 1.0 mL/min, constant flow mode, splitless injection. The column temperature program was 40°C for 5 min, rising to 300°C at a rate of 10°C/min and then held at 300°C for 4 min. The trimethylsilylation of the samples was carried out with N-methyl-N-trimethylsilylfluoroacetamide.
One and one-half grams of silenced tissue from TRV-IDI infected leaves or equivalent mock infected tissue were homogenized in 60 mL ice-cold buffer A (50 mM HEPES, pH 8.0, 1 mM EDTA, 1 mM DTE, and 0.4 M Suc) with an Ultraturrax at 9500 rpm for 20 s. The homogenate was filtered through four layers of nylon gauze (50 µm) and the filtrate was centrifuged at 3,076g for 10 min at 4°C. The resulting green pellet was carefully resuspended in buffer A and centrifuged as above. The pellet was taken up in 200 µL buffer B (50 mM HEPES, pH 7.6, and 1 mM dithioerythritol) and filtered through one layer of nylon gauze. The resulting chloroplast suspension contained approximately 2.5 mg chlorophyll/ml as determined using the method of Joyard et al. (1987)
Plant material was prepared for microscopic investigations as described in Hause and Hahn (1998) Upon request, all novel materials described in this publication will be made available in a timely manner for noncommercial research purposes, subject to the requisite permission from any third-party owners of all or parts of the material. Obtaining any permissions will be the responsibility of the requestor. Sequence data from this article have been deposited with the EMBL/GenBank data libraries under accession numbers AY497303 (NbIspG), AY497304 (NbIspH), and AY497305 (NbIDI).
We are grateful to David Baulcombe (Sainsbury Laboratory, Norwich, UK) and Plant Biosciences Limited (Norwich, UK) for making available the TRV vectors. At the IPB-Halle, we thank Verona Dietl for technical assistance, Ursula Schäfer for help with plant cultivation, Christine Kuhnt for performing the GC-MS measurements of the MBDO samples, and Dr. Andrea Porzel for measuring the NMR spectra. Received December 21, 2003; returned for revision January 28, 2004; accepted January 28, 2004.
1 This work was supported in part by grants from the Deutsche Forschungsgemeinschaft (to M.H.Z.) and the Fonds der Chemischen Industrie, Frankfurt (to T.M.K. and M.H.Z.). 
2 Present address: Plant Biotechnology Institute, National Research Council Canada, Saskatoon, SK Canada S7N 0W9.
3 Present address: Department of Chemistry, University of Utah, Salt Lake City, UT 84112. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.038133. * Corresponding author; e-mail jon.page{at}nrc-cnrc.gc.ca; fax 1–306–975–4839.
Adam P, Hecht S, Eisenreich W, Kaiser J, Grawert T, Arigoni D, Bacher A, Rohdich F (2002) Biosynthesis of terpenes: studies on 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase. Proc Natl Acad Sci USA 99: 12108–12113 Adam KP, Thiel R, Zapp J (1999) Incorporation of 1-[1-(13)C]deoxy-D-xylulose in chamomile sesquiterpenes. Arch Biochem Biophys 369: 127–132[CrossRef][Web of Science][Medline]
Altincicek B, Kollas A-K, Sanderbrand S, Wiesner J, Hintz M, Beck E, Jomaa H (2001) GcpE is involved in the 2-C-methyl-D-erythritol 4-phosphate pathway of isoprenoid biosynthesis in Escherichia coli J Bacteriol 183: 2411–2416 Altincicek B, Duin EC, Reichenberg A, Hedderich R, Kollas AK, Hintz M, Wagner S, Wiesner J, Beck E, Jomaa H (2002) LytB protein catalyzes the terminal step of the 2-C-methyl-D-erythritol-4-phosphate pathway of isoprenoid biosynthesis. FEBS Lett 532: 437–440[CrossRef][Medline]
Aluru MR, Bae H, Wu D, Rodermel SR (2001) The Arabidopsis immutans mutation affects plastid differentiation and the morphogenesis of white and green sectors in variegated plants. Plant Physiol 127: 67–77
Arigoni D, Sagner S, Latzel C, Eisenreich W, Bacher A, Zenk MH (1997) Terpenoid biosynthesis from 1-deoxy-D-xylulose in higher plants by intramolecular skeletal rearrangement. Proc Natl Acad Sci USA 94: 10600–10605 Baulcombe DC (1999) Fast forward genetics based on virus-induced gene silencing. Curr Opin Plant Biol 2: 109–113[CrossRef][Web of Science][Medline] Bick JA, Lange BM (2003) Metabolic cross talk between cytosolic and plastidial pathways of isoprenoid biosynthesis: unidirectional transport of intermediates across the chloroplast envelope membrane. Arch Biochem Biophys 415: 146–154[Medline]
Budziszewski GJ, Lewis SP, Glover LW, Reineke J, Jones G, Ziemnik LS, Lonowski J, Nyfeler B, Aux G, Zhou Q, McElver J, Patton DA, et al (2001) Arabidopsis genes essential for seedling viability: isolation of insertional mutants and molecular cloning. Genetics 159: 1765–1778 Campbell M, Hahn FM, Poulter CD, Leustek T (1997) Analysis of the isopentenyl diphosphate isomerase family from Arabidopsis thaliana. Plant Mol Biol 36: 323–328 Croteau R, Kutchan TM, Lewis NG (2000) Natural products (secondary metabolites). In B. Buchanan, W. Gruissem, R. Jones, eds, Biochemistry and Molecular Biology of Plants. American Society of Plant Physiologists, Rockville, MD, pp 1250–1268
Cunningham FX Jr, Lafond TP, Gantt E (2000) Evidence of a role for lytB in the nonmevalonate pathway of isoprenoid biosynthesis. J Bacteriol 182: 5841–5848 Eisenreich W, Rohdich F, Bacher A (2001) Deoxyxylulose phosphate pathway to terpenoids. Trends Plant Sci 6: 78–84[CrossRef][Web of Science][Medline] Ershov Y, Gantt RR, Cunningham FX, Gantt E (2000) Isopentenyl diphosphate isomerase deficiency in Synechocystis sp. strain PCC6803. FEBS Lett 473: 337–340[CrossRef][Medline]
Estévez JM, Cantero A, Romero C, Kawaide H, Jimenez LF, Kuzuyama T, Seto H, Kamiya Y, Leon P (2000) Analysis of the expression of CLA1, a gene that encodes the 1-deoxyxylulose 5-phosphate synthase of the 2-C-methyl-D-erythritol-4-phosphate pathway in Arabidopsis. Plant Physiol 124: 95–104 Fellermeier M, Raschke M, Sagner S, Wungsintaweekul J, Schuhr CA, Hecht S, Kis K, Radykewicz T, Adam P, Rohdich F, et al (2001) Studies on the nonmevalonate pathway of terpene biosynthesis: the role of 2C-methyl-D-erythritol 2,4-cyclodiphosphate in plants. Eur J Biochem 268: 6302–6310[Medline] Fraser PD, Pinto ME, Holloway DE, Bramley PM (2000) Technical advance: application of high-performance liquid chromatography with photodiode array detection to the metabolic profiling of plant isoprenoids. Plant J 24: 551–558[CrossRef][Medline]
Hahn FM, Hurlburt AP, Poulter CD (1999) Escherichia coli open reading frame 696 is IDI, a nonessential gene encoding isopentenyl diphosphate isomerase. J Bacteriol 181: 4499–4504 Ham BK, Lee TH, You JS, Nam YW, Kim JK, Paek KH (1999) Isolation of a putative tobacco host factor interacting with cucumber mosaic virus-encoded 2b protein by yeast two-hybrid screening. Mol Cells 9: 548–555[Web of Science][Medline] Hause G, Hahn H (1998) Cytological characterization of multicellular structures in embryogenic microspore cultures of Brassica napus L. Bot Acta 111: 204–211[Web of Science] Hecht S, Eisenreich W, Adam P, Amslinger S, Kis K, Bacher A, Arigoni D, Rohdich F (2001) Studies on the nonmevalonate pathway to terpenes: the role of the GcpE (ispG) protein. Proc Natl Acad Sci USA 26: 14837–14842 Hellens RP, Edwards EA, Leyland NR, Bean S, Mullineaux PM (2000) pGreen: a versatile and flexible binary Ti vector for Agrobacterium-mediated plant transformation. Plant Mol Biol 42: 819–832[CrossRef][Web of Science][Medline] Joyard J, Dorne AJ, Douce R (1987) Use of thermolysin to probe the cytosolic surface of the outer envelope membranes from plastids. In L Packer, R Douce, eds, Plant Cell Membranes. Academic Press, San Diego, pp 195–206 Kajiwara S, Fraser PD, Kondo K, Misawa N (1997) Expression of an exogenous isopentenyl diphosphate isomerase gene enhances isoprenoid biosynthesis in Escherichia coli. Biochem J 324: 421–426
Kasahara H, Hanada A, Kuzuyama T, Takagi M, Kamiya Y, Yamaguchi S (2002) Contribution of the mevalonate and methylerythritol phosphate pathways to the biosynthesis of gibberellins in Arabidopsis. J Biol Chem 277: 45188–45194
Laule O, Furholz A, Chang HS, Zhu T, Wang X, Heifetz PB, Gruissem W, Lange M (2003) Crosstalk between cytosolic and plastidial pathways of isoprenoid biosynthesis in Arabidopsis thialiana. Proc Natl Acad Sci USA 100: 6866–6871 Lichtenthaler H (1987) Chlorophylls and carotenoids: pigments of photosynthetic biomembranes. In L Packer, R Douce, eds, Plant Cell Membranes. Academic Press, San Diego, pp 350–382 Liu Y, Schiff M, Marathe R, Dinesh-Kumar SP (2002) Tobacco Rar1, EDS1 and NPR1/NIM1 like genes are required for N-mediated resistance to tobacco mosaic virus. Plant J 30: 415–429[CrossRef][Web of Science][Medline]
Mahmoud SS, Croteau RB (2001) Metabolic engineering of essential oil yield and composition in mint by altering expression of deoxyxylulose phosphate reductoisomerase and menthofuran synthase. Proc Natl Acad Sci USA 98: 8915–8920
McAteer S, Coulson A, McLennan N, Masters M (2001) The lytB Gene of Escherichia coli is essential and specifies a product needed for isoprenoid biosynthesis. J Bacteriol 183: 7403–7407 Mandel MA, Feldmann KA, Herrera-Estrella L, Rocha-Sosa M, Leon P (1996) CLA1, a novel gene required for chloroplast development, is highly conserved in evolution. Plant J 9: 649–658[CrossRef][Web of Science][Medline] Martin VJ, Pitera DJ, Withers ST, Newman JD, Keasling JD (2003) Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Nat Biotechnol 21: 796–802[CrossRef][Web of Science][Medline] Nagata N, Suzuki M, Yoshida S, Muranaka T (2002) Mevalonic acid partially restores chloroplast and etioplast development in Arabidopsis lacking the non-mevalonate pathway. Planta 216: 345–350[CrossRef][Web of Science][Medline] Nakamura A, Shimada H, Masuda T, Ohta H, Takamiya K (2001) Two distinct isopentenyl diphosphate isomerases in cytosol and plastid are differentially induced by environmental stresses in tobacco. FEBS Lett 506: 61–64[CrossRef][Medline] Okada K, Kawaide H, Kuzuyama T, Seto H, Curtis IS, Kamiya Y (2002) Antisense and chemical suppression of the nonmevalonate pathway affects ent-kaurene biosynthesis in Arabidopsis. Planta 215: 339–344[CrossRef][Web of Science][Medline] Ostrovsky D, Diomina G, Lysak E, Matveeva E, Ogrel O, Trutko S (1998) Effect of oxidative stress on the biosynthesis of 2-C-methyl-D-erythritol-2,4-cyclopyrophosphate and isoprenoids by several bacterial strains. Arch Microbiol 171: 69–72[CrossRef][Medline] Ostrovsky D, Shipanova I, Sibeldina L, Shashkov A, Kharatian E, Malyarova I, Tantsyrev G (1992) A new cyclopyrophosphate as a bacterial antistressor? FEBS Lett 298: 159–161[Medline]
Park H, Kreunen SS, Cuttriss AJ, DellaPenna D, Pogson BJ (2002) Identification of the carotenoid isomerase provides insight into carotenoid biosynthesis, prolamellar body formation, and photomorphogenesis. Plant Cell 14: 321–332 Piel J, Donath J, Bandemer K, Boland W (1998) Mevalonate-independent biosynthesis of volatiles in plants: induced and constitutive emission of volatiles. Angew Chem Int Ed Engl 37: 2478–2481[CrossRef] Querol J, Campos N, Imperial S, Boronat A, Rodríguez-Concepción M (2002) Functional analysis of the Arabidopsis thaliana GCPE protein involved in plastid isoprenoid biosynthesis. FEBS Lett 514: 343–346[CrossRef][Medline] Ratcliff F, Martin-Hernandez AM, Baulcombe DC (2001) Tobacco rattle virus as a vector for analysis of gene function by silencing. Plant J 25: 237–245[CrossRef][Web of Science][Medline]
Rohdich F, Hecht S, Gärtner K, Adam P, Krieger C, Amslinger S, Arigoni D, Bacher A, Eisenreich W (2002) Studies on the nonmevalonate terpene biosynthetic pathway: metabolic role of IspH (LytB) protein. Proc Natl Acad Sci USA 99: 1158–1163 Rohdich F, Hecht S, Bacher A, Eisenreich W (2003) The deoxyxylulose phosphate pathway of isoprenoid biosynthesis. Discovery and function of the ispDEFGH genes and their cognate enzymes. Pure Appl Chem 75: 393–405
Rodríguez-Concepción M, Boronat A (2002) Elucidation of the methylerythritol phosphate pathway for isoprenoid biosynthesis in bacteria and plastids. A metabolic milestone achieved through genomics. Plant Physiol 130: 1079–1089 Rohmer M (2003) Mevalonate-independent methylerythritol phosphate pathway for isoprenoid biosynthesis: elucidation and distribution. Pure Appl Chem 75: 375–388 Romeis T, Ludwig AA, Martin R, Jones JD (2001) Calcium-dependent protein kinases play an essential role in a plant defence response. EMBO J 20: 5556–5567[CrossRef][Web of Science][Medline] Schwarz M, Arigoni D (1999) Ginkgolide biosynthesis. In D Cane, ed, Comprehensive Natural Product Chemistry, Vol 2. Pergamon, Oxford, pp 367–399 Voinnet O (2001) RNA silencing as a plant immune system against viruses. Trends Genet 17: 449–459[CrossRef][Web of Science][Medline] Wungsintaweekul J, Herz S, Hecht S, Eisenreich W, Feicht R, Rohdich F, Bacher A, Zenk MH (2001) Phosphorylation of 1-deoxy-D-xylulose by xylulokinase of Escherichia coli. Eur J Biochem 268: 310–316[Web of Science][Medline]
Yoshioka H, Numata N, Nakajima K, Katou S, Kawakita K, Rowland O, Jones JD, Doke N (2003) Nicotiana benthamiana gp91phox homologs NbrbohA and NbrbohB participate in H2O2 accumulation and resistance to Phytophthora infestans. Plant Cell 15: 706–718 Zeidler J, Schwender J, Müler C, Wiesner J, Weidemeyer C, Beck E, Jomaa H, Lichtenthaler HK (1998) Inhibition of the non-mevalonate 1-deoxy-D-xylulose 5-phosphate pathway of plant isoprenoid biosynthesis by fosmidomycin. Z Naturforsch C 53: 980–986 This article has been cited by other articles:
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