Divergent regulation of terpenoid metabolism in the trichomes of wild and cultivated tomato species

Abstract

The diversification of chemical production in glandular trichomes is important in the development of resistance against pathogens and pests in two species of tomato. We have used genetic and genomic approaches to uncover some of the biochemical and molecular mechanisms that underlie the divergence in trichome metabolism between the wild species Solanum habrochaites LA1777 and its cultivated relative, Solanum lycopersicum. LA1777 produces high amounts of insecticidal sesquiterpene carboxylic acids (SCAs), whereas cultivated tomatoes lack SCAs and are more susceptible to pests. We show that trichomes of the two species have nearly opposite terpenoid profiles, consisting mainly of monoterpenes and low levels of sesquiterpenes in S. lycopersicum and mainly of SCAs and very low monoterpene levels in LA1777. The accumulation patterns of these terpenoids are different during development, in contrast to the developmental expression profiles of terpenoid pathway genes, which are similar in the two species, but they do not correlate in either case with terpenoid accumulation. However, our data suggest that the accumulation of monoterpenes in S. lycopersicum and major sesquiterpenes in LA1777 are linked both genetically and biochemically. Metabolite analyses after targeted gene silencing, inhibitor treatments, and precursor feeding all show that sesquiterpene biosynthesis relies mainly on products from the plastidic 2-C-methyl-d-erythritol-4-phosphate pathway in LA1777 but less so in the cultivated species. Furthermore, two classes of sesquiterpenes produced by the wild species may be synthesized from distinct pools of precursors via cytosolic and plastidial cyclases. However, highly trichome-expressed sesquiterpene cyclase-like enzymes were ruled out as being involved in the production of major LA1777 sesquiterpenes.

Figure 1.

Sesquiterpene production in the glandular trichomes of S. lycopersicum and S. habrochaites f. typicum LA1777. A and B, Scanning electron microscopy images of stem surfaces of S. lycopersicum (cv M82; A) and LA1777 (B). The most abundant trichomes (types IV, V ,and VI) are indicated by arrows. Bars = 1 mm. C and D, Major class I (C) and class II (D) sesquiterpene derivatives found in the trichomes of S. lycopersicum and LA1777, respectively. β-car, β-Caryophyllene; α-hum, α-humulene; α-cS, α-cis santalene; α-cSA, α-cis santalenoic acid; α-cB, α-cis bergamotene; α-cBA, α-cis bergamotenoic acid. E, Current model for terpenoid biosynthesis in plants. DXP, Deoxyxylulose phosphate; fos, fosmidomycin, inhibitor of DXR; G3P, glyceraldehyde-3-phosphate; GPP, geranyl diphosphate; MTS, monoterpene synthases; SST, sesquiterpene synthases.

Figure 2.

Developmental profiles of terpenoid accumulation and trichome density in S. lycopersicum and LA1777. A and B, Trichome density of the most abundant types on equivalent stem internodes of S. lycopersicum ‘M82’ type V and VI trichomes (A) and of S. habrochaites LA1777 type IV and VI trichomes (B). C to F, Accumulation of terpenoids in trichomes of equivalent stem internodes of cv M82 monoterpenes (C) and sesquiterpenes (E) and of LA1777 cII-SCAs (D) and cI-S and cII-S (F) hydrocarbons. Columns 1 to 5, First to fifth internodes from the apex; column M, internodes from the midsection of the stem. B, Bergamotene; BA, bergamotenoic acid; c, cis; germ, germacrene; S, santalene; SA, santalenoic acid; t, trans. Values represent averages and sd from nine plants. For metabolite analysis, material was extracted in three pools of three plants each. A repeat experiment gave similar results.

Figure 3.

Developmental profiles of terpenoid pathway gene expression in trichomes of S. lycopersicum and LA1777. A to G, Expression of genes involved in terpenoid biosynthesis in trichomes of stem internodes in S. lycopersicum ‘M82’ (light gray bars) and LA1777 (dark gray bars). Columns 1 to 5, First to fifth internodes from the apex; column M, internodes from the midsection of the stem. H, Expression of selected genes in stem trichomes and underlying tissues in S. lycopersicum ‘M82’. For abbreviations of gene names, see Figure 1 legend. SST1, SSTLE1/SSTLH1. Gene expression was measured by real-time PCR relative to an internal standard (actin); see Supplemental Table S2 for respective Ct values. Each bar represents a pool of trichome RNA derived from three to six plants and sd for three technical replicates. A repeat experiment gave similar results (Supplemental Fig. S3).

Figure 4.

Variations in MEP and MVA pathway activity affect terpenoid accumulation in LA1777 trichomes. A to C, Effects of silencing DXR (light gray bars) and HMGR (dark gray bars) compared with empty vector controls (TRV; white bars) on the accumulation of cII-SCA (A), cII-S (B), or cI-S (C) hydrocarbons in LA1777 trichomes. B, Bergamotene; BA, bergamotenoic acid; c, cis; ele, elemene; germ, germacrene; S, santalene; SA, santalenoic acid; t, trans. Values represent averages and sd of six samples taken from three plants. D to I, Effects of inhibitor applications on the accumulation of cII-SCA (D and G), cII-S (E and H), or cI-S (F and I) hydrocarbons in the trichomes of LA1777 cuttings treated with fosmidomycin (D–F, gray bars) or mevinolin (G–I, gray bars) compared with mock-treated controls (white bars). fos 0 and fos 100, 0 and 100 μm fosmidomycin applied, respectively; FW, fresh weight; mev 0 and mev 50, 0 and 50 μm mevinolin applied, respectively. Values are averages of two samples of one to three pooled plants each. Repeat experiments gave similar results. J and K, Expression levels of DXR (J) and HMGR (K) in trichomes of control and silenced LA1777 plants (same plants as in A–C). Gene expression was measured by real-time PCR relative to an internal standard (istd; actin). TRV, Empty vector controls; DXR1 to -3 and HMGR1 to -3, three DXR- and HMGR-silenced plants, respectively. Bars represent averages and sd for three controls and single values for individual plants.

Figure 5.

Effects of variations in MEP and MVA pathway activity on terpenoid accumulation in S. lycopersicum trichomes. A and B, Effects of silencing DXR (light gray bars) and HMGR (dark gray bars) compared with empty vector controls (TRV; white bars) on the accumulation of monoterpenes (A) and sesquiterpenes (B) in S. lycopersicum trichomes (cv M82). β-car, β-Caryophyllene; β-phell, β-phellandrene. Values represent averages of triplicate determinations and sd. Repeat experiments gave similar results. C and D, Effects of inhibitor applications on the accumulation of monoterpenes (C) and sesquiterpenes (D) in trichomes of cv M82 cuttings treated with fosmidomycin (light gray bars) or mevinolin (dark gray bars) as compared with mock-treated controls (white bars). fos 100, 100 μm fosmidomycin applied; FW, fresh weight; mev 50, 50 μm mevinolin applied; no inhib, no inhibitor applied. Values represent averages of triplicate determinations of three to four pooled plants each and sd. Repeat experiments gave similar results. E and F, Expression levels of DXR (E) and HMGR (F) in control and silenced cv M82 plants (same plants as in A and B). Gene expression was measured by real-time PCR relative to an internal standard (istd; actin). TRV, Empty vector controls; DXR1 to -3 and HMGR1 to -3, three DXR- and HMGR-silenced plants, respectively. Bars represent averages and sd for three controls and single values for individual plants.

Figure 6.

Proposed terpenoid pathway in the trichomes of S. habrochaites f. typicum LA1777 and S. lycopersicum.