Herbivore-induced and floral homoterpene volatiles are biosynthesized by a single P450 enzyme (CYP82G1) in Arabidopsis

Abstract

Terpene volatiles play important roles in plant-organism interactions as attractants of pollinators or as defense compounds against herbivores. Among the most common plant volatiles are homoterpenes, which are often emitted from night-scented flowers and from aerial tissues upon herbivore attack. Homoterpene volatiles released from herbivore-damaged tissue are thought to contribute to indirect plant defense by attracting natural enemies of pests. Moreover, homoterpenes have been demonstrated to induce defensive responses in plant-plant interaction. Although early steps in the biosynthesis of homoterpenes have been elucidated, the identity of the enzyme responsible for the direct formation of these volatiles has remained unknown. Here, we demonstrate that CYP82G1 (At3g25180), a cytochrome P450 monooxygenase of the Arabidopsis CYP82 family, is responsible for the breakdown of the C(20)-precursor (E,E)-geranyllinalool to the insect-induced C(16)-homoterpene (E,E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene (TMTT). Recombinant CYP82G1 shows narrow substrate specificity for (E,E)-geranyllinalool and its C(15)-analog (E)-nerolidol, which is converted to the respective C(11)-homoterpene (E)-4,8-dimethyl-1,3,7-nonatriene (DMNT). Homology-based modeling and substrate docking support an oxidative bond cleavage of the alcohol substrate via syn-elimination of the polar head, together with an allylic C-5 hydrogen atom. CYP82G1 is constitutively expressed in Arabidopsis stems and inflorescences and shows highly coordinated herbivore-induced expression with geranyllinalool synthase in leaves depending on the F-box protein COI-1. CYP82G1 represents a unique characterized enzyme in the plant CYP82 family with a function as a DMNT/TMTT homoterpene synthase.

Fig. 1.

Proposed biosynthetic pathways for the formation of volatile homoterpenes in plants. GGPP, geranylgeranyl diphosphate; FPP, farnesyl diphosphate; TMTT, 4,8,12-trimethyltrideca-1,3,7,11-tetraene; DMNT, 4,8-dimethyl-1,3,7-nonatriene; GES, geranyllinalool synthase; NES, nerolidol synthase; P450, cytochrome P450 monooxygenase.

Fig. 2.

Emission of TMTT and expression of CYP82G1 in wild type and CYP82G1 knockout plants in response to mock- and alamethicin-treatment. (A) Schematic of the CYP82G1 gene and position of the T-DNA insertion in the GK377A01 line. Gray boxes represent exons, and introns are shown by the black line. The position of the T-DNA insertion in intron 1 is indicated. (B) Semiquantitative RT-PCR analysis of CYP82G1 and GES (geranyllinalool synthase) gene transcripts in rosette leaves of Col-0 and GK377A01 plants in response to 30 h of mock- and alamethicin (Ala)-treatment. Actin8 transcripts were analyzed as a control. (C) Quantitative analysis of geranyllinalool and TMTT emission from wild-type and CYP82G1 mutants treated as described in B. Numbers are means ± SEM (n = 3).

Fig. 3.

Complementation analysis of the CYP82G1 T-DNA insertion line GK377A01. (A) Quantitative RT-PCR analysis of relative CYP82G1 and GES transcript levels in Col-0 and Pro_35S:CYP82G1 plants with and without alamethicin (Ala) treatment. Transcript levels of Col-0 mock controls were arbitrarily set to 1. The average GES mRNA level in mock controls of transgenic plants was 11.1 (not visible at the selected y-axis scale). Different letters above bars indicate significant differences among the means calculated with one-way analysis of variance (ANOVA) and Tukey-Kramer HSD test for CYP82G1 (P < 0.01) and GES (P < 0.05). Values were normalized to transcript levels of protein phosphatase 2A and are means ± SEM of three individual wild type plants or transgenic plants of three independent lines (#1–3). (B) Quantification of TMTT and geranyllinalool emission from wild type and Pro_35S:CYP82G1 plants of two independent lines. Lines carrying the empty expression vector or an expression construct with a truncated CYP82G1 gene were used as controls (Fig. S1E). Numbers are means ± SEM from at least three individual plants. Differences in TMTT emission between Pro_35S:CYP82G1 plants and wild-type plants were not statistically significant.

Fig. 4.

Histochemical analysis of CYP82G1 promoter activity. A 2.6-kb fragment of the CYP82G1 promoter was cloned upstream of the GUS reporter gene. Pro_CYP82G1:GUS gene expression pattern in inflorescences (A and B), and stems (C). GUS activity was induced locally at sites of feeding damage by P. xylostella (D), and thrips (E, Inset F), as well as by mechanical wounding (G). The arrow indicates the part of the shoot, which is closest to the inflorescence. Images are representative for at least five independent transgenic lines.

Fig. 5.

Position of (3S)-(E,E)-geranyllinalool (purple) and (3S)-(E)-nerolidol (orange) in the active site of the CYP82G1 homology model. The main interacting residues including hydrophobic active site residues are illustrated. According to the best-ranked docking mode for both substrates, a strong hydrogen bond is formed between the hydroxyl groups at C-3 and the carbonyl oxygen of Thr313 with a distance of 2.1 Å (black dashed line). A hydrogen at C-5 and the hydroxyl group at C-3 have equal distances of 5.3 Å (red dashed lines) to the Fe atom of the heme group.