Studies of a biochemical factory: tomato trichome deep expressed sequence tag sequencing and proteomics

Abstract

Shotgun proteomics analysis allows hundreds of proteins to be identified and quantified from a single sample at relatively low cost. Extensive DNA sequence information is a prerequisite for shotgun proteomics, and it is ideal to have sequence for the organism being studied rather than from related species or accessions. While this requirement has limited the set of organisms that are candidates for this approach, next generation sequencing technologies make it feasible to obtain deep DNA sequence coverage from any organism. As part of our studies of specialized (secondary) metabolism in tomato (Solanum lycopersicum) trichomes, 454 sequencing of cDNA was combined with shotgun proteomics analyses to obtain in-depth profiles of genes and proteins expressed in leaf and stem glandular trichomes of 3-week-old plants. The expressed sequence tag and proteomics data sets combined with metabolite analysis led to the discovery and characterization of a sesquiterpene synthase that produces beta-caryophyllene and alpha-humulene from E,E-farnesyl diphosphate in trichomes of leaf but not of stem. This analysis demonstrates the utility of combining high-throughput cDNA sequencing with proteomics experiments in a target tissue. These data can be used for dissection of other biochemical processes in these specialized epidermal cells.

Figure 1.

Tomato stem and petiole trichomes. A, Tomato stems and petioles are covered by multiple trichome types, including the glandular type I and type VI trichomes. B and C, Mixed-type trichome samples (B) were isolated by scraping frozen tissue in liquid nitrogen, and type VI gland samples (C) were isolated by glass bead abrasion and filtering with nylon mesh screens.

Figure 2.

RT-PCR analysis of gene expression in trichomes. M82 tomato plants were grown to 3 weeks old, and RNA was extracted from isolated stem and petiole trichomes (T), stem and petiole tissue after removal of trichomes (−), and stem and petiole tissue with intact trichomes (+). Expression was analyzed for genes with ESTs enriched in trichome tissue compared with the TIGR TA ESTs. LoxC, Lipoxygenase C; MCPI, putative metallocarboxypeptidase inhibitor. Translation elongation factor-1α (EF-1α) was used as a constitutive control, and small subunit of Rubisco (ssRBC; TA17646_4081) was included as a control not expected to be highly expressed in trichome tissue. All reactions were done for 20 cycles except for WRKY, which was 25 cycles.

Figure 3.

Rutin and chlorogenic acid biosynthetic pathways. A, LC-MS-extracted ion chromatograms showing the presence of rutin (m/z 609) and chlorogenic acid (m/z 353) in isolated total stem and petiole trichome extracts. B, Biosynthetic pathways for the production of rutin and chlorogenic acid. For each pathway step, the percentage of ESTs and normalized spectral abundance factors from the mixed-type trichome data are shown in parentheses. PAL, Phe ammonia-lyase; C4H, cinnamate 4-hydroxylase; CHS, chalcone synthase; CHI, chalcone isomerase; FHT, flavanone 3-hydroxylase; F3H, flavonoid 3′-hydroxylase; FLS, flavonol synthase; 4CL, 4-coumaryl-CoA ligase; HCT, cinnamoyl-CoA shikimate/quinate transferase; C3H, p-coumaryl 3′-hydroxylase; HQT, hydroxycinnamoyl-CoA quinate transferase.

Figure 4.

EST abundance and proteomics-normalized spectral abundance factors (NSAF) for steps of the MEP and mevalonate pathways in type VI trichomes. EST counts (percentage of the total type VI ESTs) and NSAF for genes and proteins of the MEP pathway (A) are more abundant than those of the mevalonate pathway (B) in type VI trichomes. DXS, 1-Deoxy-d-xylulose-5-phosphate synthase; DXR, 1-deoxy-d-xylulose-5-phosphate reductoisomerase; IspD, 4-diphosphocytidyl-2-C-methyl-d-erythritol synthase; IspE, 4-(cytidine 5′-diphospho)-2-C-methyl-d-erythritol kinase; IspF, 2-C-methyl-d-erythritol 2,4-cyclodiphosphate synthase; IspG, 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase; IspH, 4-hydroxy-3-methylbut-2-enyl diphosphate reductase; IDI, isopentenyl/dimethylallyl diphosphate isomerase; IPP, isopentenyl diphosphate; DMAPP, dimethylallyl diphosphate; HMGS, 3-hydroxy-3-methylglutaryl-CoA synthase; HMGR, HMG-CoA reductase; MVK, mevalonate kinase; PMVK, diphosphomevalonate kinase; MPD, diphosphomevalonate decarboxylase.

Figure 5.

In vitro assay with purified recombinant CAHS. A, Sesquiterpenes produced by M82 leaf trichomes were collected from a small leaflet in a sealed vial using a solid-phase microextraction fiber. Peak 1, δ-Elemene; peak 2, β-caryophyllene; peak 3, α-humulene. B, α-Humulene standard. C, β-Caryophyllene standard. D, Reaction products from incubation of purified CAHS with FPP. E, Solid-phase microextraction fiber negative control. All data shown are extracted ion chromatograms for m/z 93. RA, Relative abundance.