The tomato terpene synthase gene family

Abstract

Compounds of the terpenoid class play numerous roles in the interactions of plants with their environment, such as attracting pollinators and defending the plant against pests. We show here that the genome of cultivated tomato (Solanum lycopersicum) contains 44 terpene synthase (TPS) genes, including 29 that are functional or potentially functional. Of these 29 TPS genes, 26 were expressed in at least some organs or tissues of the plant. The enzymatic functions of eight of the TPS proteins were previously reported, and here we report the specific in vitro catalytic activity of 10 additional tomato terpene synthases. Many of the tomato TPS genes are found in clusters, notably on chromosomes 1, 2, 6, 8, and 10. All TPS family clades previously identified in angiosperms are also present in tomato. The largest clade of functional TPS genes found in tomato, with 12 members, is the TPS-a clade, and it appears to encode only sesquiterpene synthases, one of which is localized to the mitochondria, while the rest are likely cytosolic. A few additional sesquiterpene synthases are encoded by TPS-b clade genes. Some of the tomato sesquiterpene synthases use z,z-farnesyl diphosphate in vitro as well, or more efficiently than, the e,e-farnesyl diphosphate substrate. Genes encoding monoterpene synthases are also prevalent, and they fall into three clades: TPS-b, TPS-g, and TPS-e/f. With the exception of two enzymes involved in the synthesis of ent-kaurene, the precursor of gibberellins, no other tomato TPS genes could be demonstrated to encode diterpene synthases so far.

Figure 1.

Phylogenetic tree of the proteins encoded by the 29 functional or potentially functional TPS genes in the tomato genome.

Figure 2.

Subcellular localization of TPS14 and TPS36. The complete open reading frame of TPS14 (TPS14-GFP) and the first 60 codons of TPS36 (tpTPS36-GFP) were fused to a downstream GFP and transiently expressed in Arabidopsis leaf protoplasts. GFP fluorescence indicates the location of each fusion protein; the location of the chloroplasts was determined by chlorophyll autofluorescence (shown in red), and the location of mitochondria was determined by the fluorescence of MitoTracker Red dye (shown in artificial blue color to distinguish it from chlorophyll autofluorescence). The column labeled “Merged Signals” provides a view of all fluorescent signals obtained for this sample. A to C, TPS14-GFP. D to G, tpTPS36-GFP. H to J, Expression of a nonfused GFP showing cytosolic (and nuclear) localization, used here for control. K to M, Expression of a Rubisco-transit peptide-GFP fusion gene (Nagegowda et al., 2008) showing plastid localization of the protein, used here for control.

Figure 3.

The organization of TPS genes and other genes in clusters on chromosomes 1, 2, 6, 8, and 10. AAT, Alcohol acyltransferase; AOX, alcohol oxidase; CPT, confirmed or putative cis-prenyltransferase; P450, putative cytochrome P450 oxidoreductase. The P450-2 gene has a large insertion in the first exon, and the AAT-1 gene has multiple deletions and mutations.

Figure 4.

GC analysis of the products formed in the reactions catalyzed by TPS14, TPS36, and TPS32 with z,z-FPP and e,e-FPP as the substrates. The reaction products (peaks) were identified by comparison of their GC-MS profiles with authentic standards and NIST libraries: (1) β-bisabolene, (2) γ-bisabolene (cis), (3) γ-bisabolene (trans), (4) α-bisabolene (trans), (5) nerolidol, (6) unidentified, (7) unidentified, (8) viridiflorene, (9) unidentified, (10) unidentified. TPS14, TPS36, and TPS32 enzymatic products were analyzed on different columns with different programs (see “Materials and Methods”). GC analysis is shown for alternative substrates only when activity of the enzyme with such a substrate was more than 10% compared with the best substrate.

Figure 5.

RT-PCR analysis of transcripts of TPS9 and TPS12 in leaves of tomato (accession M82), S. pennellii (accession LA0716), and the introgression lines IL6-2 and IL10-3.

Figure 6.

Comparison of transcript levels of TPS12 and β-caryophyllene and α-humulene levels in leaves and stems. A, qRT-PCR analysis of TPS12 transcripts. B, GC analysis of sesquiterpene volatiles detected in leaves and stems. RQ, Ratio of qRT-PCR results; TIC, total ion chromatograph.

Figure 7.

Identification of the major products of TPS3 enzymatic activity in vitro as camphene and tricyclene, and correlation with the presence of these compounds and of transcripts of TPS3 in leaf and stem tissues of tomato. A, TPS in vitro activity with GPP as the substrate (top panel) and GC analysis of monoterpenes extracted from leaves and stems by SPME (bottom two panels). B, RT-PCR analysis of TPS3 transcripts in leaves and stems. The reaction products (peaks) were identified by comparison of their GC-MS profiles with authentic standards and NIST libraries: (1) tricyclene, (2) α-pinene, (3) camphene, (4) sabinene, (5) β-pinene, (6) β-myrcene, (7) limonene, (8) unidentified. TIC, Total ion chromatograph.

Figure 8.

GC analysis of the products formed in vitro by the enzymatic activities of TPS7 (A), TPS8 (B), TPS37 (C and F), TPS39 (D and G), and TPS38 (E). Enzymes were incubated with GPP, NPP, e,e-FPP, and z,z-FPP, and products were analyzed as described in “Materials and Methods.” Reaction products (peaks) were identified by comparison of their GC-MS profiles with authentic standards and NIST libraries: (1) α-pinene, (2) sabinene, (3) β-pinene, (4) β-myrcene, (5) limonene, (6) linalool, (7) 1,8-cineole, (8) α-bergamotene, (9) nerolidol. GC analysis is shown for alternative substrates only when activity of the enzyme with such a substrate was more than 10% compared with the best substrate. TIC, Total ion chromatograph.

Figure 9.

RT-PCR analysis of the expression of monoterpene synthase genes from the chromosome 1 gene cluster in leaves of tomato (accession M82), S. pennellii (accession LA0716), and IL1-3 plants, and comparison of the monoterpenes found in these leaves. A, GC analysis. B, RT-PCR analysis. TIC, Total ion chromatograph.

Figure 10.

GC analysis demonstrating that TPS24 catalyzes the formation of ent-kaur-16-ene from CPP. E. coli cells containing the plasmid pGGeC, which carries the A. grandis GGPPS cDNA and the Arabidopsis CPS cDNA, and E. coli cells containing the pGGeC plasmid as well as a second plasmid with the P. glauca ent-kaurene synthase or the tomato TPS24 cDNA were used. The E. coli cells expressing TPS24 produced a peak that had the identical retention time and MS pattern as the peak produced by PgKS, which had previously been authenticated as ent-kaur16-ene (Keeling et al., 2010). TIC, Total ion chromatograph.

Figure 11.

Comparison of the enzymatic activities of the proteins encoded by TPS20 genes from tomato and S. pennellii. A, GC analysis of tomato (accession M82) trichome monoterpenes. B, GC analysis of the products of the in vitro reaction catalyzed by purified SlPHS1 with NPP as the substrate. C, GC analysis of S. pennellii (LA0716) trichome monoterpenes. D, GC analysis of the products of the in vitro reaction catalyzed by purified SpPHS1 with NPP as the substrate. Numbered peaks are as follows: (1) δ-2-carene, (2) limonene, (3) γ-terpinene. TIC, Total ion chromatograph.