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The conifer Picea abies (Norway spruce) defends itself against herbivores and pathogens with a terpenoid-based oleoresin composed chiefly of monoterpenes (C(10)) and diterpenes (C(20)). An important group of enzymes in oleoresin biosynthesis are the short-chain isoprenyl diphosphate synthases that produce geranyl diphosphate (C(10)), farnesyl diphosphate (C(15)), and geranylgeranyl diphosphate (C(20)) as precursors of different terpenoid classes. We isolated a gene from P. abies via a homology-based polymerase chain reaction approach that encodes a short-chain isoprenyl diphosphate synthase making an unusual mixture of two products, geranyl diphosphate (C(10)) and geranylgeranyl diphosphate (C(20)). This bifunctionality was confirmed by expression in both prokaryotic (Escherichia coli) and eukaryotic (P. abies embryogenic tissue) hosts. Thus, this isoprenyl diphosphate synthase, designated PaIDS1, could contribute to the biosynthesis of both major terpene types in P. abies oleoresin. In saplings, PaIDS1 transcript was restricted to wood and bark, and transcript level increased dramatically after methyl jasmonate treatment, which induces the formation of new (traumatic) resin ducts. Polyclonal antibodies localized the PaIDS1 protein to the epithelial cells surrounding the traumatic resin ducts. PaIDS1 has a close phylogenetic relationship to single-product conifer geranyl diphosphate and geranylgeranyl diphosphate synthases. Its catalytic properties and reaction mechanism resemble those of conifer geranylgeranyl diphosphate synthases, except that significant quantities of the intermediate geranyl diphosphate are released. Using site-directed mutagenesis and chimeras of PaIDS1 with single-product geranyl diphosphate and geranylgeranyl diphosphate synthases, specific amino acid residues were identified that alter the relative composition of geranyl to geranylgeranyl diphosphate.

To get some insight into the regulatory mechanisms controlling the sterol branch of the mevalonate pathway, tobacco (Nicotiana tabacum cv Bright Yellow-2) cell suspensions were treated with squalestatin-1 and terbinafine, two specific inhibitors of squalene synthase (SQS) and squalene epoxidase, respectively. These two enzymes catalyze the first two steps involved in sterol biosynthesis. In highly dividing cells, SQS was actively expressed concomitantly with 3-hydroxy-3-methylglutaryl coenzyme A reductase and both sterol methyltransferases. At nanomolar concentrations, squalestatin was found to inhibit efficiently sterol biosynthesis as attested by the rapid decrease in SQS activity and [(14)C]radioactivity from acetate incorporated into sterols. A parallel dose-dependent accumulation of farnesol, the dephosphorylated form of the SQS substrate, was observed without affecting farnesyl diphosphate synthase steady-state mRNA levels. Treatment of tobacco cells with terbinafine is also shown to inhibit sterol synthesis. In addition, this inhibitor induced an impressive accumulation of squalene and a dose-dependent stimulation of the triacylglycerol content and synthesis, suggesting the occurrence of regulatory relationships between sterol and triacylglycerol biosynthetic pathways. We demonstrate that squalene was stored in cytosolic lipid particles, but could be redirected toward sterol synthesis if required. Inhibition of either SQS or squalene epoxidase was found to trigger a severalfold increase in enzyme activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase, giving first evidence for a positive feedback regulation of this key enzyme in response to a selective depletion of endogenous sterols. At the same time, no compensatory responses mediated by SQS were observed, in sharp contrast to the situation in mammalian cells.

1-Deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) catalyzes the first committed step of the 2-C-methyl-D-erythritol 4-phosphate pathway for isoprenoid biosynthesis. In Arabidopsis, DXR is encoded by a single-copy gene. We have cloned a full-length cDNA corresponding to this gene. A comparative analysis of all plant DXR sequences known to date predicted an N-terminal transit peptide for plastids, with a conserved cleavage site, and a conserved proline-rich region at the N terminus of the mature protein, which is not present in the prokaryotic DXR homologs. We demonstrate that Arabidopsis DXR is targeted to plastids and localizes into chloroplasts of leaf cells. The presence of the proline-rich region in the mature Arabidopsis DXR was confirmed by detection with a specific antibody. A proof of the enzymatic function of this protein was obtained by complementation of an Escherichia coli mutant defective in DXR activity. The expression pattern of beta-glucuronidase, driven by the DXR promoter in Arabidopsis transgenic plants, together with the tissue distribution of DXR transcript and protein, revealed developmental and environmental regulation of the DXR gene. The expression pattern of the DXR gene parallels that of the Arabidopsis 1-deoxy-D-xylulose 5-phosphate synthase gene, but the former is slightly more restricted. These genes are expressed in most organs of the plant including roots, with higher levels in seedlings and inflorescences. The block of the 2-C-methyl-D-erythritol 4-phosphate pathway in Arabidopsis seedlings with fosmidomycin led to a rapid accumulation of DXR protein, whereas the 1-deoxy-D-xylulose 5-phosphate synthase protein level was not altered. Our results are consistent with the participation of the Arabidopsis DXR gene in the control of the 2-C-methyl-D-erythritol 4-phosphate pathway.

Isopentenyl diphosphate (IPP), which is produced from mevalonic acid or other nonmevalonic substrates, is the universal precursor of isoprenoids in nature. Despite the presence of several isoprenoid compounds in plastids, enzymes of the mevalonate pathway leading to IPP formation have never been isolated or identified to our knowledge. We now describe the characterization of two pepper (Capsicum annuum L.) cDNAs, CapTKT1 and CapTKT2, that encode transketolases having distinct and dedicated specificities. CapTKT1 is primarily involved in plastidial pentose phosphate and glycolytic cycle integration, whereas CapTKT2 initiates the synthesis of isoprenoids in plastids via the nonmevalonic acid pathway. From pyruvate and glyceraldehyde-3-phosphate, CapTKT2 catalyzes the formation of 1-deoxy-xylulose-5-phosphate, the IPP precursor. CapTKT1 is almost constitutively expressed during the chloroplast-to-chromoplast transition, whereas CapTKT2 is overexpressed during this period, probably to furnish the IPP necessary for increased carotenoid biosynthesis. Because deoxy-xylulose phosphate is shared by the plastid pathways of isoprenoid, thiamine (vitamin B1), and pyridoxine (vitamin B6) biosynthesis, our results may explain why albino phenotypes usually occur in thiamine-deficient plants.

Plant cells synthesize a myriad of isoprenoid compounds in different subcellular compartments, which include the plastid, the mitochondria, and the endoplasmic reticulum cytosol. To start the study of the regulation of these parallel pathways, we used pepper (Capsicum annuum) fruit as a model. Using different isoprenoid biosynthetic gene probes from cloned cDNAs, we showed that only genes encoding the plastid enzymes (geranylgeranyl pyrophosphate synthase, phytoene synthase, phytoene desaturase, and capasanthin-capsorubin synthase) are specifically triggered during the normal period of development, at the ripening stage. This pattern of expression can be mimicked and precociously induced by a simple wounding stress. Concerning the cytosol-located enzymes, we observed that the expression of the gene encoding farnesyl pyrophosphate synthase is constitutive, whereas that of farnesyl pyrophosphate cyclase (5-epi-aristolochene synthase) is undetectable during the normal development of the fruit. The expression of these later genes are, however, only selectively triggered after elicitor treatment. The results provide evidence for developmental control of isoprenoid biosynthesis occurring in plastids and that cytoplasmic isoprenoid biosynthesis is regulated, in part, by environmental signals.