The flavonoid biosynthetic enzyme chalcone isomerase modulates terpenoid production in glandular trichomes of tomato

Abstract

Flavonoids and terpenoids are derived from distinct metabolic pathways but nevertheless serve complementary roles in mediating plant interactions with the environment. Here, we show that glandular trichomes of the anthocyanin free (af) mutant of cultivated tomato (Solanum lycopersicum) fail to accumulate both flavonoids and terpenoids. This pleiotropic metabolic deficiency was associated with loss of resistance to native populations of coleopteran herbivores under field conditions. We demonstrate that Af encodes an isoform (SlCHI1) of the flavonoid biosynthetic enzyme chalcone isomerase (CHI), which catalyzes the conversion of naringenin chalcone to naringenin and is strictly required for flavonoid production in multiple tissues of tomato. Expression of the wild-type SlCHI1 gene from its native promoter complemented the anthocyanin deficiency in af. Unexpectedly, the SlCHI1 transgene also complemented the defect in terpenoid production in glandular trichomes. Our results establish a key role for SlCHI1 in flavonoid production in tomato and reveal a link between CHI1 and terpenoid production. Metabolic coordination of the flavonoid and terpenoid pathways may serve to optimize the function of trichome glands in dynamic environments.

Figure 1.

Schematic overview of terpenoid and flavonoid biosynthetic pathways. The names of compounds and select enzymes (italicized) are as follows: ANS, anthocyanidin synthase; CDP-ME, 4-(cytidine 5′-diphospho)-2-C-methyl-d-erythritol; CDP-MEP, 2-phospho-4-(cytidine 5′-diphospho)-2-C-methyl-d-erythritol; CMK, 4-(cytidine 5′-diphospho)-2-C-methyl-d-erythritol kinase; DAHP, 3-deoxy-d-arabino-heptulosonate 7-phosphate; DFR, dihydroflavonol 4-reductase; DMAPP, dimethylallyl diphosphate; DXP, 1-deoxy-d-xylulose 5-phosphate; DXPS, DXP synthase; DXR, DXP reductoisomerase; F3H, flavanone 3-hydroxylase; FLS, flavonol synthase; FPP, farnesyl diphosphate; FPPS, FPP synthase; GPP, geranyl diphosphate; GPPS, GPP synthase; HDR, 4-hydroxy-3-methylbut-2-enyl diphosphate reductase; IPP, isopentenyl diphosphate; MCT, 2-C-methyl-d-erythritol 4-phosphate cytidylyltransferase; MTS, monoterpene synthase; PAL, Phe ammonia lyase; PEP, phosphoenolpyruvate; STS, sesquiterpene synthase. Note that although some sesquiterpenes in tomato are synthesized via the MEP pathway (as shown for simplicity; Sallaud et al., 2009), most sesquiterpenes are derived from the cytosolic mevalonate pathway. [See online article for color version of this figure.]

Figure 2.

Anthocyanin and trichome phenotypes of the tomato af mutant. A and B, Light microscopy images of the abaxial surface of wild-type (A) and af (B) leaves. C and D, Light microscopy images of wild-type (C) and af (D) stems. E and F, Scanning electron micrographs of the adaxial surface of wild-type (E) and af (F) leaves. Representative type I and type VI trichomes are labeled I and VI, respectively. Three-week-old plants were used for all experiments. Bars = 1,000 µm (A and B), 500 µm (C and D), and 100 µm (E and F).

Figure 3.

af leaves are deficient in the production of flavonoid-related metabolites. Levels of the indicated secondary metabolites were measured in leaf-dip extracts (A) and isolated type VI glands (B). Each data point represents the mean ± se of four biological replicates from wild-type (WT) and af plants. Asterisks represent significant differences between wild-type and af plants (unpaired Student’s t test: *P < 0.05, **P < 0.01, ***P < 0.001). nd, Not detected.

Figure 4.

Field-grown af plants are susceptible to natural populations of insect herbivores. A, Number of flea beetles (mean ± se) on wild-type (WT) and af plants. The inset shows a flea beetle feeding on a leaf of the af mutant. B, Number of flea beetle feeding sites (mean ± se; see inset in A) on each host genotype. Data in A and B were determined for 30 replicate plants per genotype 9 d after transplantation of seedlings to the field plot. C, Number of Colorado potato beetles (CPB; mean ± se) on each host genotype. Beetles were counted on 18 replicate wild-type and af plants 40 d after plants were transplanted to the field. The inset shows a Colorado potato beetle feeding on the af mutant. D, Number of blister beetles (mean ± se) on each host genotype. Beetles were counted on 11 replicate wild-type and af plants 30 d after plants were transplanted to the field. The inset shows a blister beetle feeding on the af mutant. Asterisks represent significant differences between wild-type and af plants (unpaired Student’s t test: **P < 0.01, ***P < 0.001). [See online article for color version of this figure.]

Figure 5.

The Af gene encodes CHI. A, Fine genetic mapping of Af delimited the target gene to an interval between markers SGN-U571424 and CT93 on chromosome 5. Numbers in parentheses indicate the number of recombination events identified between markers and the target gene. B, Physical map of the region defined by markers SGN-U571424 and C2_At3g55120. Black boxes indicate exons within three predicted genes (arrows). The asterisk denotes the location of the mutation identified in the last exon of CHI1. C, Wild-type (WT) and af-derived nucleotide sequence (top) encoding the C terminus of CHI1 and the corresponding deduced amino acid sequence (bottom). Stop codons are indicated by boldface letters and depicted in the predicted amino acid sequence by asterisks. D, Enzymatic activity of CHI1^WT and CHI1^af. Purified recombinant CHI1^WT and CHI1^af proteins (0.125 µg) were mixed with substrate (naringenin chalcone), and the amount of the substrate was measured spectrophotometrically (A₃₄₀) at various times thereafter. Control reactions in which enzyme was omitted were performed in parallel and used to correct for nonenzymatic turnover of the substrate. E, Western-blot analysis of CHI protein levels in wild-type and af leaves. Crude leaf extract was separated into supernatant (sup.) and pellet fractions by centrifuging at 21,000g for 25 min at 4°C. The resulting supernatant (10 µg of protein) and a proportional amount of detergent-solubilized protein in the pellet fraction were blotted and probed with a polyclonal antibody against tomato CHI1 (top). The polyvinylidene difluoride membrane was stained with Coomassie blue after western-blot analysis (bottom). Arrows denote CHI1 and the large subunit of Rubisco (RbcL).

Figure 6.

Chemical and genetic complementation of the af mutant. A, Three-week-old af seedlings were supplied through the cut stem with 0.1 mm naringenin (+ Nar) or a mock control (− Mock). Anthocyanin accumulation in leaf veins was visualized 3 d after treatment. Photographs in the bottom row (bar = 1 mm) show magnified views of the images in the top row (bar = 2 mm). B, Anthocyanin accumulation in CHI1::CHI1-complemented transgenic lines. Anthocyanins were extracted from leaf tissue of the wild type (WT), af, four complemented transgenic lines (CHI1-2, CHI1-3, CHI1-4, and CHI1-9), and one noncomplemented transgenic line (CHI1-20). A photograph of the resulting extract is shown above the spectrophotometric quantification of each genotype. FW, Fresh weight. C, Western-blot analysis of CHI protein levels in CHI1::CHI1-complemented transgenic lines. Soluble protein (20 µg) from leaves of the indicated genotype was blotted and probed with a polyclonal antibody against tomato CHI1. The large subunit of Rubisco (RbcL) was used as a loading control as described in the legend to Figure 5E.

Figure 7.

af leaves are deficient in trichome-borne terpenoid production. Levels of the indicated compound were measured in leaf-dip extracts (A) and isolated type VI glands (B). Each data point represents the mean ± se of four biological replicates from wild-type (WT) and af plants. Asterisks represent significant differences between wild-type and af plants (unpaired Student’s t test: *P < 0.05, **P < 0.01, ***P < 0.001). FW, Fresh weight.

Figure 8.

Terpenoid accumulation in type VI glands from CHI1::CHI1 transgenic lines. Levels of β-phellandrene (A) and β-caryophyllene (B) were measured in type VI glands isolated from leaves of the indicated genotypes. Transgenic lines (T2 generation) were homozygous for the transgene and correspond to the same lines described in the legend to Figure 6. Each data point represents the mean ± se of three independent plants per line. Asterisks represent significant differences between af and other genotype plants (unpaired Student’s t test: **P < 0.01, ***P < 0.001). WT, Wild type.