CYP76C1 (Cytochrome P450)-Mediated Linalool Metabolism and the Formation of Volatile and Soluble Linalool Oxides in Arabidopsis Flowers: A Strategy for Defense against Floral Antagonists

Abstract

The acyclic monoterpene alcohol linalool is one of the most frequently encountered volatile compounds in floral scents. Various linalool oxides are usually emitted along with linalool, some of which are cyclic, such as the furanoid lilac compounds. Recent work has revealed the coexistence of two flower-expressed linalool synthases that produce the (S)- or (R)-linalool enantiomers and the involvement of two P450 enzymes in the linalool oxidation in the flowers of Arabidopsis thaliana. Partially redundant enzymes may also contribute to floral linalool metabolism. Here, we provide evidence that CYP76C1 is a multifunctional enzyme that catalyzes a cascade of oxidation reactions and is the major linalool metabolizing oxygenase in Arabidopsis flowers. Based on the activity of the recombinant enzyme and mutant analyses, we demonstrate its prominent role in the formation of most of the linalool oxides identified in vivo, both as volatiles and soluble conjugated compounds, including 8-hydroxy, 8-oxo, and 8-COOH-linalool, as well as lilac aldehydes and alcohols. Analysis of insect behavior on CYP76C1 mutants and in response to linalool and its oxygenated derivatives demonstrates that CYP76C1-dependent modulation of linalool emission and production of linalool oxides contribute to reduced floral attraction and favor protection against visitors and pests.

Figure 1.

Scheme Summarizing CYP76C1-Catalyzed Reactions. Arrows indicate CYP76C1- and NADPH-dependent reactions in vitro. Mainstream CYP76C1-dependent pathway in vivo is shown in bold. Dashed arrows indicate that the formation of the compounds is not dependent on CYP76C1 in vivo. The minor product of primary linalool oxidation, 9-OH-linalool, was not available and thus not further tested as a substrate.

Figure 2.

CYP76C1 Expression and Coexpression Patterns in Arabidopsis Flowers. (A) Expression heat map of the top 40 genes most closely coregulated with CYP76C1. Coexpression analysis was performed using the Expression Angler tool and the AtGenExpress Tissue Compendium data set (Toufighi et al., 2005). Heat map shows the expression levels in selected flowers tissues: flower stages 9, 10/11, 12, and 15 and petals, stamens, pedicels, and carpel at flower stages 12 and 15. Coexpressed genes are ranked according to their Pearson correlation coefficients with CYP76C1 (R values). Genes highlighted in red are known to be involved or putatively involved in linalool metabolism. (B) Relative transcripts levels of CYP76C1, CYP71B38, TPS10, and TPS14 in flower organs (left panel) and during flower development (right panel). Relative transcript levels were determined by qRT-PCR using the EΔCt method, and the specific efficiency of each primer pair and normalization with four reference genes for which the stable expression level are known (Czechowski et al., 2005). Results represent the mean ± se of three biological replicates for the flower parts and four biological replicates for flower stages. (C) to (E) GUS staining showing spatiotemporal floral expression of CYP76C1. Staining was 19 h for inflorescence (C), flowers at different stages (D), and parts of fully opened flowers (E).

Figure 3.

Gas Chromatography Analysis of the Products Resulting from Yeast-Expressed CYP76C1 Activity on Linalool and Linalool Oxides. GC-MS chromatograms of a mix of authentic standards (A) and of ethyl acetate extracts of the products from the conversion of a racemic mix of (R/S)-linalool (B), 8-OH-linalool (C), 8-oxo-linalool (D), and lilac aldehydes (E) by yeast-expressed CYP76C1. Microsomal membranes from the recombinant yeast expressing CYP76C1 (final [P450] ∼50 nM) or transformed with an empty vector (Empty control) were incubated for 15 min with 200 µM of substrate in presence or absence (neg. control) of NADPH. Chromatograms show the relative abundance of total ion current and the selected ions m/z 111 + 153 + 155 in the inserts. Compared with control, CYP76C1-dependent 8-oxo-linalool metabolism results in a decrease of 8-oxo-linalool and lilac aldehydes and a simultaneous increase in lilac alcohols (D). This suggested that the lilac aldehydes formed via conversion of 8-oxo-linalool might be converted rapidly into alcohols by CYP76C1 (E). Formation of lilac aldehydes from 8-oxo-linalool was confirmed performing a time-course experiment using low substrate and P450 concentrations (Supplemental Figure 7). (1) Racemic R/S-linalool, (2) 8-OH-linalool, (3) 9-OH-linalool, (4) 8-oxo-linalool, (5) 8-OH-6,7-dihydrolinalool, (6) 8-oxo-6,7-dihydrolinalool, (7) lilac aldehydes, and (8) lilac alcohols. IS, nonyl acetate used as internal standard for normalization. (2), (4), (7), and (8) were identified by comparison of RT and MS with those of authentic standards (Supplemental Figure 4). (3) was identified by comparison of its MS with MS of 8-OH-linalool (2) when separated from (4) on a HP-35ms column (Supplemental Figure 3). (5) and (6) were identified by NMR after reaction upscaling and purification by preparative GC (Supplemental Figure 6).

Figure 4.

Targeted UPLC-MS/MS Analysis of the Products Resulting from Yeast-Expressed CYP76C1 Activity on 8-Oxo-Linalool. Samples were analyzed by UPLC-MS/MS using MRM. Left panels show a specific channel of MS/MS transition developed for the detection of 8-oxo-linalool (151.2 → 92.8 m/z). Right panels show a specific channel of MS/MS transition developed for the detection of 8-COOH-linalool (167.2 → 92.8 m/z). (A) Mix of authentic standards. (B) Methanol extract of the products from the conversion of 8-oxo-linalool by CYP76C1. Yeast microsomal membranes (final [P450] ∼100 nM) were incubated for 20 min with the substrate (200 µM) in the presence or absence (neg. control) of NADPH. Red panel shows the consumption of the substrate by CYP76C1 in presence of NADPH compared with the negative control. Minor amount of 8-COOH-linalool is detected in the negative control, probably due to 8-oxo-linalool autoxidation. 8-COOH-linalool was identified from its specific MS/MS and retention time compared with the authentic standard. More details and extended results are shown in Supplemental Figure 8. (4) 8-oxo-linalool and (9) 8-COOH-linalool.

Figure 5.

Comparative GC-MS Analysis of the Flower Headspace of Wild-Type Arabidopsis, cyp76c1 Mutant, and 35S:CYP76C1 Lines. (A) Targeted GC-MS chromatograms showing linalool, lilac compounds (inset), and caryophyllene emission based on selected ion currents 93 + 111 + 126 m/z (main panels) and 93 + 153 + 155 m/z (insets). Representative chromatograms are shown for an authentic standard mix of (1) linalool, (7) lilac aldehydes, (8) lilac alcohols, (10) caryophyllene, and (IS) nonyl acetate internal standard (top panel), as well as wild-type (Col-0), cyp76c1-1, and 35S:CYP76C1 lines. (B) Quantification of headspace volatiles. Data are mean ± se of three biological replicates. Statistically significant differences relative to the wild type are indicated (Student’s t test: *P ≤ 0.05; **P > 0.01; ***P < 0.001).

Figure 6.

Targeted UPLC-MS/MS Quantification of Linalool and Linalool Derivatives in the Flowers from Wild-Type and CYP76C1 Insertion and Complemented Lines. Methanol extracts of the flowers were treated with β-glycosidase before UPLC-MS/MS analysis using the MRM method. The total amount of aglycones detected was quantified based on standard curves, except for lynalyl derivatives, which were only detected as conjugates and for 8-OH-6,7-dihydrolinalool, for which no standard was available. Data are mean ± se of three biological replicates. Statistically significant differences relative to the wild type are indicated (Student’s t test: *P ≤ 0.05; **P > 0.01; ***P < 0.001). Representative chromatograms are shown in Supplemental Figure 10.

Figure 7.

Heterologous Reconstitution of the Linalool-Derived Pathway by Transient Coexpression of Linalool Synthases and CYP76C1 in N. benthamiana Leaves. N. benthamiana leaves were infiltrated with Agrobacterium tumefaciens transformed with an empty vector or with vectors driving the expression of CYP76C1, TPS10, or TPS14. Each gene was expressed alone or in the combinations TPS10/CYP76C1 or TPS14/CYP76C1. Five days postinfiltration, headspace volatiles were collected from the transformed leaves and analyzed by GC-MS. Simultaneously, β-glucosidase-treated methanol extracts from the leaves were analyzed by UPLC-MS/MS. (A) Head-space analysis. (B) Quantification by targeted UPLC/MS-MS of 8-OH-linalool, 9-OH-linalool, and 8-COOH-linalool, the only linalool-derived metabolites detected in the transformed leaves and for which the accumulation was modified depending on the enzymes coexpressed. Data are mean ± se of three biological replicates. Statistically significant differences relative to empty vector are indicated (Student’s t test: *P ≤ 0.05; **P > 0.01; ***P < 0.001).

Figure 8.

Behavior of Flower Pollinators and Floral Antagonists on CYP76C1 Mutant Lines and Pure Compounds. (A) Thrips dual-choice test between flowers from the wild type and cyp76c1-1 mutant. Results represent the average number of thrips (±se) on the flowers from five biological replicates and for three independent tests. Statistically significant differences relative to the wild type are indicated (Wilcoxon test: n = 5, *P ≤ 0.05). (B) Thrips dual-choice test between volatiles from Col-0, cyp76c1, and 35S:CYP76C1 flowers performed in a Y-shaped arena of an olfactometer. Three headspace combinations were tested: Col-0 versus cyp76c1-1, Col-0 versus 35S:CYP76C1, and cyp76c1-1 versus 35S:CYP76C1. Results represent the average thrips’ choice + se from five independent replicates, each using different individual plants and for which choice of 30 to 40 individual thrips was scored. Statistically significant differences are indicated (paired t test: n = 5, **P < 0.01; ***P < 0.001). (C) Thrips dual-choice test between pure compounds performed in Y-shaped arena of an olfactometer. Results represent the average thrips’ choice + se from six independent replicates and for which the choice of 30 individual thrips was scored. One hundred micrograms of each compound alone or 100 µg of both lilac compounds for the mix were used for each assay. Statistically significant differences are indicated (paired t test: n = 6, **P < 0.01). (D) Hoverfly dual-choice test between pure compounds performed in a star-shaped arena of an olfactometer. Results represent the average percentage of time + se spent in substance or control fields for 30 individual hoverflies. One hundred micrograms of each compound alone or in mix were used for each assay. Statistically significant differences are indicated (paired t test: n = 25, **P < 0.05).

Figure 9.

Behavior of Florivores on Flowers of CYP76C1 Mutant Lines and Pure Compounds. (A) Feeding preferences of larvae of P. xylostella and S. littoralis and adults of P. cochleariae for flowers from the wild type and cyp76c1-1 mutant in dual-choice test. Response index R represents the mean proportion consumed from cyp76c1-1 flowers minus the mean proportion of Col-0 flowers consumed in each case (±se) for 30 individuals per insect species. Negative values indicate a preference for the mutants. Statistically significant differences relative to the wild type are indicated (Student’s t test: n = 3, **P < 0.01; ***P < 0.001). (B) Dual-choice test with adults of P. cochleariae feeding on cabbage leaves treated with pure 8-OH and 8-COOH-linalool. One gram of cabbage leave was treated with 10, 100, or 1000 ng of pure compounds dissolved in methanol or only with methanol (Control). Results represent the percentage mean choice of 30 individuals between treated or control leaves. Statistically significant differences relative to control are indicated (paired t test: n = 30, *P < 0.05).