The Tomato cis-Prenyltransferase Gene Family.

Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, Michigan, 48109, USA.
The Plant Journal (Impact Factor: 5.97). 11/2012; 73(4). DOI: 10.1111/tpj.12063
Source: PubMed


cis-Prenyltransferases (CPTs) are predicted to be involved in the synthesis of long-chain polyisoprenoids, all with >5 isoprene (C5) units. Recently, we identified a short-chain CPT, neryl diphosphate synthase (NDPS1), in tomato (Solanum lycopersicum). Here, we searched the tomato genome and identified and characterized its entire CPT gene family, which is composed of seven members (SlCPT1-7, with NDPS1 designated as SlCPT1). Six SlCPT genes encode proteins with N-terminal targeting sequences, which, when fused to the green fluorescent protein (GFP), mediated GFP transport to the plastids of Arabidopsis protoplasts. The SlCPT3-GFP fusion protein was localized to the cytosol. Enzymatic characterization of recombinant SlCPT proteins demonstrated that SlCPT6 produces Z,Z-FPP and SlCPT2 catalyzes the formation of nerylneryl diphosphate, while SlCPT4, SlCPT5, and SlCPT7 synthesize longer chain products (C25-C55). While no in vitro activity could be demonstrated for SlCPT3, its expression in the Saccharomyces cerevisiae dolichol biosynthesis mutant (rer2) complemented the temperature sensitive growth defect. Transcripts of SlCPT2, SlCPT4, SlCPT5, and SlCPT7 are present at low levels in multiple tissues, SlCPT6 is exclusively expressed in red fruit and roots, and SlCPT1, SlCPT3 and SlCPT7 are highly expressed in trichomes. RNA interference-mediated suppression of NDPS1 led to a large decrease in β-phellandrene (which is made from neryl diphosphate), with greater reductions achieved with the general 35S promoter compared to the trichome-specific MKS1 promoter. Phylogenetic analysis revealed CPT gene families in both eudicots and monocots and that all the short-chain CPTs from tomato (SlCPT1, SlCPT2, and SlCPT6) are closely linked to terpene synthase gene clusters. © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.

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    • "The cis-IDSs generally produce longer prenyl diphosphates than the trans-IDSs, with up to 5000 C 5 units in natural rubber. However recently, a group of short-chain cis-IDSs was described in several tomato species (Sallaud et al., 2009; Schilmiller et al., 2009; Akhtar et al., 2013), and a medium and longchain trans-IDS that makes C 25 -C 45 products was reported from Arabidopsis thaliana (Hsieh et al., 2011). "
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    ABSTRACT: Isoprenyl diphosphate synthases (IDS) catalyze some of the most basic steps in terpene biosynthesis by producing the prenyl diphosphate precursors of each of the different terpenoid classes. Most plants investigated have distinct enzymes producing each of the short-chain, all-trans (E) prenyl diphosphates, geranyl (C10 ), farnesyl (C15 ) or geranylgeranyl (C20 ) diphosphate (GGDP). In the genome of Arabidopsis thaliana 15 trans-product forming IDS are present. Ten of these have recently been shown to produce GGDP by genetic complementation of a carotenoid pathway engineered into E. coli. In verifying the product pattern of IDS producing GGDP by a new LC-MS/MS procedure, we found that five of these IDS produce geranylfarnesyl diphosphate (GFDP, C25 ) instead of GGDP as their major product in enzyme assays performed in vitro. Overexpression of one of the GFDP synthases in A. thaliana confirmed the production of GFDP in vivo. Enzyme assays with A. thaliana protein extracts made from the roots, but not other organs, showed the formation of GFDP. Furthermore, GFDP itself was detected in root extracts. Sub-cellular localization studies in leaves indicated that four of the GFDP synthases were targeted to the plastoglobules of the chloroplast and one to the mitochondria. Sequence comparison and mutational studies showed that the size of the R group of the 5(th) amino acid residue in front of the first aspartate rich motif is responsible for C25 vs. C20 product formation, with smaller R groups (Ala, Ser) giving GGPP (C20 ) as a product and a larger R group (Met) giving GFPP (C25 ). This article is protected by copyright. All rights reserved.
    The Plant Journal 10/2015; DOI:10.1111/tpj.13064 · 5.97 Impact Factor
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    • "In higher plants, such as Arabidopsis thaliana (Kera et al. 2012; Surmacz and Swiezewska 2011) and Solanum lycopercicum (Akhtar et al. 2013), cis-prenyltransferases are encoded by a multigene family, and their transcripts are induced in response to various abiotic stimuli. These reports suggest that some cis-prenyltransferases and their products functions in different tissues and/or participates in different cellular processes in response to various environmental stimuli. "
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    • "In contrast, plants usually express several CPTs. Thus, seven genes encoding CPT producing almost exclusively short-chain polyisoprenoids have been identified in tomato (SlCPT1–7) [16]. Interestingly, only one CPT (LAA66) has been described in Lilium logiflorum [17] which in fact does not preclude the existence of a family of CPT encoding genes in this plant. "
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    ABSTRACT: cis-Prenyltransferases (CPTs) comprise a numerous enzymes synthesizing isoprenoid hydrocarbon skeleton with isoprenoid units in the cis (Z) configuration. The chain-length specificity of a particular plant CPT is in most cases unknown despite the composition of the accumulated isoprenoids in the tissue of interest being well established. In this report AtCPT6, one of the nine Arabidopsis thaliana CPTs, is shown to catalyze the synthesis of a family of very short-chain polyisoprenoid alcohols of six, seven, and eight isoprenoid units, those of seven units dominating. The product specificity of AtCPT6 was established in vivo following its expression in the heterologous system of the yeast Saccharomyces cerevisiae and was confirmed by the absence of specific products in AtCPT6 T-DNA insertion mutants and their overaccumulation in AtCPT6 - overexpressing plants. These observations are additionally validated in silico using an AtCPT6 model obtained by homology modeling. AtCPT6 only partially complements the function of the yeast homologue of CPT - Rer2 since it restores the growth but not protein glycosylation in rer2Δ yeast. This is the first in planta characterization of specific products of a plant CPT producing polyisoprenoids. Their distribution suggests that a joint activity of several CPTs is required to produce the complex mixture of polyisoprenoid alcohols found in Arabidopsis roots.
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