Distortion of trichome morphology by the hairless mutation of tomato affects leaf surface chemistry

Abstract

Trichomes are specialized epidermal structures that function as physical and chemical deterrents against arthropod herbivores. Aerial tissues of cultivated tomato (Solanum lycopersicum) are populated by several morphologically distinct trichome types, the most abundant of which is the type VI glandular trichome that produces various specialized metabolites. Here, the effect of the hairless (hl) mutation on trichome density and morphology, chemical composition, and resistance to a natural insect herbivore of tomato was investigated. The results show that the major effect of hl on pubescence results from structural distortion (bending and swelling) of all trichome types in aerial tissues. Leaf surface extracts and isolated type VI glands from hl plants contained wild-type levels of monoterpenes, glycoalkaloids, and acyl sugars, but were deficient in sesquiterpene and polyphenolic compounds implicated in anti-insect defence. No-choice bioassays showed that hl plants are compromised in resistance to the specialist herbivore Manduca sexta. These results establish a link between the morphology and chemical composition of glandular trichomes in cultivated tomato, and show that hl-mediated changes in these leaf surface traits correlate with decreased resistance to insect herbivory.

Fig. 1.

Light micrographs of trichomes on the leaf, stem, and hypocotyl of wild-type (WT) and hl plants. The edge of the adaxial leaf surface of WT (A) and hl (B) plants. Stem of WT (C) and hl (D) plants. Hypocotyl region of WT (E) and hl (F) plants. Scale bars represent 250 μm in (A) and (B), and 500 μm in (C–F). Three-week-old plants were used for all photographs. The arrows indicate various trichome types.

Fig. 2.

Cryoelectron micrographs of leaf trichomes on wild-type (WT) and hl plants. (A and B) Adaxial surface of a WT leaf. (C–E) Adaxial leaf surface of hl plants. Three-week-old plants were used for all images. Various trichome types, including type IV-like trichomes (A), are indicated by arrows.

Fig. 3.

hl has minor effects on trichome density. (A) Density of glandular trichomes (types I, VI, and VII) in the base, middle, and tip region of leaflets from 4-week-old wild-type (WT) plants. Data show the mean (±SE) trichome number of six replicate leaves taken from position L3 (see Supplementary Fig. S4 at JXB online). Asterisks represent significant differences between trichome density in the tip region compared with either the middle or base region (unpaired t-test: *P <0.05; **P <0.01; ***P <0.001). (B) Effect of leaf developmental age on trichome density in WT plants. Trichome counts were performed on the middle region of lateral leaflets (Supplementary Fig. S4C at JXB online). Data show the mean (±SE) trichome number of six replicate leaves. Asterisks represent significant differences between the oldest leaf (leaf 4) and each of three developmentally younger leaves (unpaired t-test: *P <0.05; **P <0.01; ***P <0.001). Leaf 1 corresponds to the newly emerging, youngest leaf. (C) Leaf trichome density in WT and hl plants. Mean (±SE) trichome number of six replicate leaves on 4-week-old WT and hl plants. Asterisks represent significant differences between WT and hl plants (unpaired t-test: *P <0.05; **P <0.01).

Fig. 4.

Terpene profiles in leaf dips and isolated type VI glands. (A and B) Detached leaflets were immersed in MTBE and the resulting extracts were analysed by GC-MS. (C and D) Terpenes were extracted from isolated type VI glands collected with a glass pipette. For the latter analysis, the amount of material injected for each GC-MS run was equivalent to 200 type VI glands. The indicated monoterpene peaks (A and C) correspond to the following compounds: 1, α-pinene; 2, 2-carene; 3, α-phellandrene; 4, α-terpinene; 5, β-phellandrene. Under the GC conditions used, minor amounts of limonene co-eluted with β-phellandrene. The sesquiterpene peaks (B and D) correspond to δ-elemene (6), β-caryophyllene (7), and α-humulene (8).

Fig. 5.

Comparison of terpene levels in wild-type (WT) and hl leaves. (A) Analysis of monoterpenes (left panel) and sesquiterpenes (right panel) extracted from WT (filled bar) and hl (open bar) plants (4 weeks old) using the leaf dip method. (B) Analysis of monoterpenes (left panel) and sesquiterpenes (right panel) obtained from collected type VI glands. Peak areas for each terpene compound were normalized to leaflet weight (A) or to 200 type VI glands (B). Under the GC conditions used, minor amounts of limonene co-eluted with β-phellandrene. Each mean represents data from four replicates. Asterisks denote significant differences between wild-type and hl (unpaired t-test: *P <0.05; **P <0.01; ***P <0.001). nd, not detected.

Fig. 6.

LC-MS analysis of non-volatile leaf metabolites. Extracted ion chromatograms were obtained for various metabolites extracted from wild-type leaves using either the leaflet dip procedure (Leaf) or collected type VI glands (t VI). For analysis of isolated type VI glands, the amount of material injected for each LC-MS run was equivalent to 1000 type VI glands. Chromatograms are for the respective [M–H]^– ions with the exception of G–I (α-tomatine, dehydrotomatine, and acyl sugar), which correspond to the [M+HCOO]^– ion. The identified acyl sugar contains a sucrose tetraester substituted with three C5 and one C2 fatty acyl chain, and is the major acyl sugar derivative identified in S. lycopersicum.

Fig. 7.

Relative levels of non-volatile metabolites in wild-type (WT) and hl leaves. (A) Results obtained from analysis of compounds in leaf dip extracts. (B) Results obtained from analysis of compounds in isolated type VI glands. Peak areas for each compound were normalized to leaflet weight (A) or to 200 type VI glands (B). Each data point represents the mean ±SE of four replicates. Asterisks represent significant differences between WT and hl plants (unpaired t-test: *P <0.05; **P <0.01; ***P <0.001). nd, not detected.

Fig. 8.

hl plants are compromised in resistance to feeding by Manduca sexta larvae. (A) Mean (±SE) mass of M. sexta larvae (n=14) reared for 8 d on either wild-type (WT) or hl plants. Each larva was grown on a single plant. (B) Mean (±SE) mass of M. sexta larvae (n=24) reared for 10 d on either WT or hl plants. Each plant was challenged with three larvae. Asterisks represent significant differences between WT and hl plants (unpaired t-test: *P <0.05; ***P <0.001).