The tomato odorless-2 mutant is defective in trichome-based production of diverse specialized metabolites and broad-spectrum resistance to insect herbivores

Abstract

Glandular secreting trichomes of cultivated tomato (Solanum lycopersicum) produce a wide array of volatile and nonvolatile specialized metabolites. Many of these compounds contribute to the characteristic aroma of tomato foliage and constitute a key part of the language by which plants communicate with other organisms in natural environments. Here, we describe a novel recessive mutation called odorless-2 (od-2) that was identified on the basis of an altered leaf-aroma phenotype. od-2 plants exhibit pleiotrophic phenotypes, including alterations in the morphology, density, and chemical composition of glandular trichomes. Type VI glandular trichomes isolated from od-2 leaves accumulate only trace levels of monoterpenes, sesquiterpenes, and flavonoids. Other foliar defensive compounds, including acyl sugars, glycoalkaloids, and jasmonate-regulated proteinase inhibitors, are produced in od-2 leaves. Growth of od-2 plants under natural field conditions showed that the mutant is highly susceptible to attack by an indigenous flea beetle, Epitrix cucumeris, and the Colorado potato beetle, Leptinotarsa decemlineata. The increased susceptibility of od-2 plants to Colorado potato beetle larvae and to the solanaceous specialist Manduca sexta was verified in no-choice bioassays. These findings indicate that Od-2 is essential for the synthesis of diverse trichome-borne compounds and further suggest that these compounds influence host plant selection and herbivore community composition under natural conditions.

Figure 1.

Volatile profiles of wild-type (WT) and od-2 leaves. A and B, GC traces of volatiles released from detached wild-type (A) and od-2 (B) leaflets. C and D, GC traces of volatiles released from detached wild-type (C) and od-2 (D) leaflets that were mechanically damaged prior to collection of head space-containing volatiles with a SPME fiber. Numbers and letters correspond to the following compounds: C, CO₂; 1, hexanal; 2, cis-3-hexenal (coeluting with hexanal); 3, α-pinene; 4, 2-carene; 5, α-phellandrene; 6, α-terpinene; 7, limonene; 8, β-phellandrene. Sesquiterpenes (β-caryophyllene and α-humulene) eluted at later retention times (data not shown).

Figure 2.

Genetic map of Od-2. Od-2 was mapped in a BC1 population (98 plants) to a genetic interval between C2_At1g44790 and TG400 on chromosome 11. Molecular markers are indicated above the line. Numbers in parentheses indicate the number of recombination events identified between that marker and the target gene. Numbers under the line indicate genetic distances relative to the top (0 centimorgan) and bottom (100 centimorgan) of chromosome 11, according to the Tomato-EXPEN 2000 map (http://solgenomics.net/).

Figure 3.

Trichome morphology on leaves in wild-type (WT) and od-2 plants. A and B, Light microscopic images of the adaxial surface of wild-type (A) and od-2 (B) leaves. C and D, Scanning electron micrographs of the adaxial surface of wild-type (C) and od-2 (D) leaves. E to I, Cryoscanning electron micrographs of the adaxial surface of wild-type (E and H) and od-2 (F, G, and I) leaves. All images were taken from plants at the seedling (approximately 3-week-old) stage. Type I, VI, and VII trichomes are indicated by arrows and uppercase characters. I* in B denotes abnormal rod-shaped type I trichomes.

Figure 4.

Comparison of terpene levels in isolated type VI glands from wild-type (WT) and od-2 leaves. A, The data show the amount of each of the indicated monoterpene (left panel) and sesquiterpene (right panel) compounds present in 200 type VI glands manually collected (into MTBE) from the adaxial leaf surface of 3-week-old plants. B, Measurement of the same compounds in leaf-dip extracts obtained by briefly immersing detached leaflets in MTBE. Under the GC conditions used, minor amounts of limonene coeluted with β-phellandrene. Each data point represents the mean + se of four biological replicates. Asterisks denote significant differences between the wild type and od-2 (unpaired t test: * P <0.05, ** P <0.01, *** P < 0.001). nd, Not detected.

Figure 5.

Comparison of nonvolatile secondary metabolite levels in type VI glands from wild-type (WT) and od-2 leaves. A, The data show the amount of each of the indicated compounds present in 200 type VI glands collected from the adaxial leaf surface of 3-week-old plants. B, Measurement of the same compounds in leaf-dip extracts obtained by briefly immersing detached leaflets in a solution containing isopropanol-acetonitrile-water. Each data point represents the mean + se of five biological replicates. Asterisks represent significant differences between wild-type and od-2 plants (unpaired t test: * P < 0.05, ** P < 0.01, *** P < 0.001). nd, Not detected.

Figure 6.

Field-grown od-2 plants are susceptible to natural populations of insect herbivores. A, Mean ± se number of flea beetles on wild-type (WT) and od-2 plants. B, Mean ± se number of flea beetle feeding sites (as measured by hole number) on each host genotype. Data in A and B were determined for 20 replicate plants per genotype 9 d after transplantation of seedlings to the field plot. C, Mean ± se number of CPB on each host genotype. Beetles were counted on 18 replicate wild-type and od-2 plants 40 d after plants were transplanted to the field plot. Asterisks represent significant differences between the wild type and od-2 (unpaired t test: * P < 0.05, *** P < 0.001).

Figure 7.

Effect of od-2 on host resistance to CPB larvae. No-choice bioassays were performed by placing newly hatched larvae on wild-type and od-2 mutant plants. A, Photograph of representative wild-type (left) and od-2 (right) plants taken 5 d after initiation of the feeding trial. B, CPB larvae recovered after 5 d of feeding on 12 replicate wild-type (left dish) and od-2 (right dish) plants.

Figure 8.

Effect of od-2 on host resistance to M. sexta larvae. No-choice bioassays were performed by placing first instar M. sexta larvae on wild-type (WT) and od-2 mutant plants. A, Photograph taken 12 d after initiation of the feeding trial. B, Mean ± se weight of M. sexta larvae (n = 16) reared for 12 d on either wild-type or od-2 plants. Each plant was challenged with two larvae. C, Results from an independent bioassay in which three M. sexta larvae were reared for 10 d on each of eight wild-type and od-2 plants. Data show the mean ± se weight of larvae (n = 24). Asterisks represent significant differences between wild-type and od-2 plants (unpaired t test: ** P < 0.01).

Figure 9.

od-2 plants are not defective in PI-II accumulation. A, Leaves of 15-d-old wild-type (WT) and od-2 plants (five replicates per genotype) were mechanically wounded (W) with a hemostat, and PI-II levels were measured 2 d after wounding. Control (C) plants were not wounded. Data show mean ± se PI-II levels in leaves from five replicate plants of each genotype. B, Mean ± se PI-II levels in leaf tissue from field-grown wild-type and od-2 plants. PI-II levels were measured 14 d after transplantation of plants to the field site. Asterisks represent significant differences between wild-type and od-2 plants (unpaired t test: * P < 0.05).