The tomato cis–prenyltransferase gene family

Abstract

SUMMARYcis-prenyltransferases (CPTs) are predicted to be involved in the synthesis of long-chain polyisoprenoids, all with five or more 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 comprises seven members (SlCPT1-7, with NDPS1 designated as SlCPT1). Six of the SlCPT genes encode proteins with N-terminal targeting sequences, which, when fused to 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). Although no in vitro activity was 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. RNAi-mediated suppression of NDPS1 led to a large decrease in b-phellandrene (which is produced 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 showed that all the short-chain CPT genes from tomato (SlCPT1, SlCPT2 and SlCPT6) are closely linked to terpene synthase gene clusters.

A Lettuce (Lactuca sativa) Homolog of Human Nogo-B Receptor Interacts with cis-Prenyltransferase and Is Necessary for Natural Rubber Biosynthesis

“…RNA was isolated according to the manufacturer's protocol. First strand cDNA was synthesized using SuperScript III reverse transcriptase (Invitrogen) and oligo(dT) [12][13][14][15][16][17][18] primer (Invitrogen) using 1-5 g of total RNA. Quantitative real time PCR (qRT-PCR) was performed (Step One Real-Time PCR System; Applied Biosystems, Carlsbad, CA) using Power SYBER Green PCR Master Mix (Applied Biosystems), 5 M primer, and cDNA template (equivalent to 5 ng of total RNA) in a reaction volume of 10 l. The qRT-PCR program was 1 cycle of 95°C for 10 min and 40 cycles of 95°C for 15 s and 58°C for 1 min.…”

“…Null mutations of E. coli UPPS and double knock-out of the two yeast CPTs are lethal, confirming that they are essential genes (9,11). In plants, studies of well annotated genomes showed that multiple copies of CPTs are present (12,13), but the exact physiological role of each CPT is not fully understood.…”

Heterologous Expression of Methylketone Synthase1 and Methylketone Synthase2 Leads to Production of Methylketones and Myristic Acid in Transgenic Plants

“…We recently showed that the promoter of ShMKS1 is highly active in type VI trichomes of cultivated tomato ( Akhtar et al, 2013). To test the activity of the ShMKS2 promoter in cultivated tomato, we made a construct that fused the 1.5-kb upstream region of ShMKS2 with GFP and transformed cultivated tomato plants.…”

“…ShMKS1 and ShMKS2 are specifically expressed in the gland cells of type VI trichomes in wild tomato, and both proteins are targeted to the plastids (Yu et al, 2010;Akhtar et al, 2013;G. Yu, A.L.…”

Polyprenols Are Synthesized by a Plastidial cis-Prenyltransferase and Influence Photosynthetic Performance

“…CPTs are not homologous to trans-prenyldiphosphate synthases, and comparatively little is known about these enzymes, particularly in plants. Bioinformatic analysis indicates that animal and prokaryotic genomes typically encode a single CPT, while plants maintain small CPT gene families, containing anywhere between three to nine members (Surmacz and Swiezewska, 2011;Akhtar et al, 2013). Bacterial CPTs synthesize the Pren-11 (C55) polyprenol known as undecaprenyl diphosphate, which as mentioned above, functions in peptidoglycan biosynthesis, while animal CPTs are involved in the synthesis of longer chain dolichols that serve an indispensable role in protein N-glycosylation.…”