Functional Analysis of the Final Steps of the 1-Deoxy-d-xylulose 5-phosphate (DXP) Pathway to Isoprenoids in Plants Using Virus-Induced Gene Silencing

Abstract

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 [ C]DXP and [14 C]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 IspHsilenced 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. IDIsilenced 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 a...

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“…5), inferring that the substrate entered the chloroplast where it was converted to HMBPP. Our DXP feeding results align with those of Page et al (20), who observed conversion of labeled DXP to the diol [3] in intact leaves of N. benthamiana plants containing gene-silenced HDR. However, it is generally regarded that phosphorylated substrates such as DXP are not taken up by plant cells and that DX is a more suitable substrate for MEP pathway feeding studies (22).…”

“…Previously Page et al (20) using gene silencing in Nicotiana benthamiana showed that deletion of HDR activity caused a diversion of HMBPP to the diol [3]. This was confirmed (20) by feeding labeled DXP to excised leaves.…”

“…Previously Page et al (20) using gene silencing in Nicotiana benthamiana showed that deletion of HDR activity caused a diversion of HMBPP to the diol [3]. This was confirmed (20) by feeding labeled DXP to excised leaves. Using this experimental setup, we fed pathway intermediates (DXP, HMBPP, and diol [3]) to leaves of Arabidopsis, excised from plants that had been subjected to 4 d NO 3 − starvation (via both water only and NO 3 − deplete treatments).…”

Disruption of the 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) gene results in albino, dwarf and defects in trichome initiation and stomata closure in Arabidopsis

“…Previous pathway always show feedback regulation [30,32]. It was thus reasonable to hypothesize that the same kind of metabolic changes might exist in the dxr mutant or tissues with reduced DXR expression levels.…”

“…Exogenous application of GAs to the medium could partially rescue the dwarf phenotype of the pds3 mutant [30]. Other research indicated that levels of not only GAs but also other important compounds were decreased as a result of the metabolic disruption in the mutants of enzymes involved in the MEP pathway [21,[30][31][32][33][34]. However, no detailed analyses have been [30]).…”