Identification and regulation of TPS04/GES, an Arabidopsis geranyllinalool synthase catalyzing the first step in the formation of the insect-induced volatile C16-homoterpene TMTT

Abstract

Volatile secondary metabolites emitted by plants contribute to plant-plant, plant-fungus, and plant-insect interactions. The C(16)-homoterpene TMTT (for 4,8,12-trimethyltrideca-1,3,7,11-tetraene) is emitted after herbivore attack by a wide variety of plant species, including Arabidopsis thaliana, and is assumed to play a role in attracting predators or parasitoids of herbivores. TMTT has been suggested to be formed as a degradation product of the diterpene alcohol (E,E)-geranyllinalool. Here, we report the identification of Terpene Synthase 04 (TPS04; At1g61120) as a geranyllinalool synthase (GES). Recombinant TPS04/GES protein expressed in Escherichia coli catalyzes the formation of (E,E)-geranyllinalool from the substrate geranylgeranyl diphosphate. Transgenic Arabidopsis lines carrying T-DNA insertions in the TPS04 locus are deficient in (E,E)-geranyllinalool and TMTT synthesis, a phenotype that can be complemented by expressing the GES gene under the control of a heterologous promoter. GES transcription is upregulated under conditions that induce (E,E)-geranyllinalool and TMTT synthesis, including infestation of plants with larvae of the moth Plutella xylostella and treatment with the fungal peptide alamethicin or the octadecanoid mimic coronalon. Induction requires jasmonic acid but is independent from salicylic acid or ethylene. This study paves the ground to address the contribution of TMTT in ecological interactions and to elucidate the signaling network that regulates TMTT synthesis.

Figure 1.

Proposed Biosynthesis of the Volatile C₁₆-Homoterpene TMTT. GES is proposed to catalyze the formation of (E,E)-geranyllinalool from the substrate all-trans-GGPP, the central precursor in diterpene biosynthesis. (E,E)-Geranyllinalool is further converted into TMTT by uncharacterized sequential steps of oxidative degradation (Boland et al., 1998).

Figure 2.

Induced Emission of (E,E)-Geranyllinalool and TMTT from Arabidopsis Leaves in Response to Treatment with the Fungal Elicitor Alamethicin. Total ion GC-MS chromatograms of volatiles emitted from mock-treated (0.1% ethanol; top panel) and alamethicin-treated (in 0.1% ethanol; bottom panel) Arabidopsis leaves. Volatiles were collected from detached rosette leaves during 0 to 9 h and 21 to 30 h of treatment by a closed-loop stripping procedure. Results are shown for the second interval of volatile collection. Mock-treated leaves emitted trace amounts of TMTT but no MeSA, (E,E)-α-farnesene, or (E,E)-geranyllinalool (GL). S, nonyl acetate standard.

Figure 3.

Comparison of Volatile Emissions and Expression of At1g61120 in Arabidopsis Leaves upon Elicitor Treatment and Herbivore Challenge. (A) Quantitative analysis of the four major volatiles MeSA, (E,E)-α-farnesene, TMTT, and (E,E)-geranyllinalool (GL). As highest emission rates in response to most treatments were observed in the second light phase, only results obtained during this period (21 to 30 h of treatment) are reported. Alamethicin (5 μg/mL) was applied to detached leaves, and coronalon (100 μM) was added to the medium of hydroponically grown plants. Continuous insect-feeding experiments were conducted by the application of two P. xylostella larvae in the third to fourth instar on each medium-sized and fully expanded rosette leaf. The results represent means ± SE of three replicates. Experiments were repeated at least once with similar results. C, control; FW, fresh weight; T, treatment. (B) RNA gel blot analysis of At1g61120 transcription in Arabidopsis leaves after alamethicin treatment (5 μg/mL) applied through petioles of cut leaves, coronalon treatment (100 μM; applied to roots of intact plants for 31 h), and P. xylostella feeding for 31 h. Control RNA was collected from leaves treated with 0.1% ethanol through petioles (mock) or from intact hydroponically grown plants. The blot was rehybridized with a probe for Actin2 to document equal loading.

Figure 4.

GC-MS Analysis of Products Formed from GGPP by Recombinant GES Enzyme. GES was expressed in E. coli, extracted, purified, and incubated with the substrate all-trans-GGPP. The resulting terpene products were separated by GC-MS. (A) MS detector traces are shown for the products obtained from assays with the purified GES enzyme. An extract from E. coli carrying the empty expression vector was subjected to the same purification procedure and served as a negative control. The major peak was identified as (E,E)-geranyllinalool (1) via comparison with an authentic standard, as shown in the bottom chromatogram. (B) The catalytic activity of the partially purified enzyme was measured in the presence of the divalent metal ions Mg²⁺ and Mn²⁺ at different concentrations. Means ± SE of triplicate assays are shown.

Figure 5.

Coronalon-Induced Volatile Emission and GES Expression in Leaves of Arabidopsis Wild-Type and GES T-DNA Insertion Lines. (A) Positions of T-DNA insertions in At1g61120 (GES). The exons are represented by the gray boxes, and flanking regions and introns are represented by the black line. Black boxes symbolize untranslated regions (UTR) of the first and last exons. The two independent T-DNA insertions are indicated as boxes A and B. (B) RNA gel blot analysis of GES transcript levels in leaves of wild-type and T-DNA insertion lines after application of 100 μM coronalon for 30 h to roots of hydroponically grown plants. The blot was rehybridized with a probe for Actin2 to document equal loading. (C) Quantitative analysis of volatiles emitted between 21 and 30 h after the beginning of coronalon treatment of single hydroponically grown wild-type and mutant plants. The results represent means + se of three replicates. FW, fresh weight; tr, trace.

Figure 6.

Complementation Analysis of the GES Insertion Line salk_039864. (A) Schematic drawing of the chimeric gene used to complement the Arabidopsis insertion line salk_039864. The transgene (ProAlcA:GES) consists of a 6-kb genomic fragment starting with the presumed transcriptional start site (as inferred from the cDNA) under the control of the alcohol-inducible AlcA promoter. The exons are represented by the gray boxes, and flanking regions and introns are represented by the black line. UTR, untranslated region. (B) GC-MS analysis of volatiles emitted from leaves of untransformed salk_039864 and of ProAlcA:GES transformants. Twenty 3-week-old plants grown on soil under long-day conditions were sprayed with 4.7% ethanol prior to continuous volatile collection for 31 h (control plants were sprayed with water only). Chromatograms selected for 69 m/z are shown. GL, (E,E)-geranyllinalool; S, nonyl acetate standard. (C) GES transcript analysis in leaves of mutant lines. Twenty 3-week-old plants grown on soil under long-day conditions were sprayed with 4.7% ethanol (EtOH; control plants were sprayed with water only). Material for RNA analysis was harvested after 31 h. The blot was rehybridized with a probe for Actin2 to document equal loading.

Figure 7.

Phenotype and Volatile Analysis of Seedlings with Constitutive GES Expression. (A) Schematic drawing of the chimeric gene used to complement the Arabidopsis insertion line salk_039864. The transgene consists of a 6-kb genomic fragment starting with the presumed transcriptional start site (as inferred from the cDNA) under the control of the constitutive CaMV 35S promoter. The exons are represented by the gray boxes, and flanking regions and introns are represented by the black line. UTR, untranslated region. (B) RNA gel blot analysis of GES transcripts in leaves of two Pro35S:GES lines (1 and 2) compared with the wild type and the knockout line salk_039864. Leaf material was harvested from 20 3-week-old untreated plants grown under long-day conditions. The blot was rehybridized with a probe for Actin2 to document equal loading. (C) GC-MS analysis of volatiles emitted from leaves of salk_039864 transformed with the Pro35S:GES gene. Volatiles were collected continuously for 31 h from 20 plants grown as described in (B). Total ion chromatograms are shown. (D) Top row, phenotype of 3-week-old plants grown under long-day conditions. Bottom row, phenotype of 10-d-old seedlings. The phenotype is representative for at least six independent lines showing expression of the GES transcript. (E) Emission rates of MeSA, (E,E)-α-farnesene, TMTT, and (E,E)-geranyllinalool (GL) from leaves of wild-type, salk_039864, and Pro35S:GES lines. Plants were first grown for 3 weeks under long-day conditions, which allows the selection of strong expressors (deduced from the phenotype; see [D]). Subsequently, they were cultivated for 4 weeks under short-day conditions. Volatiles were collected from 16 to 20 detached leaves during 0 to 7 h of treatment in 20 mL of tap water (white bars), 0.1% ethanol (mock; gray bars), and alamethicin (5 μg/mL 0.1% ethanol; black bars). Volatile collection and analysis were performed as described in Methods. The results represent means ± SE of six replicates (wild type and Pro35S:GES) or four replicates (salk_039864; n = 3 for mock treatment). Statistical analysis (one-way analysis of variance) was performed for each compound on log-transformed data. Letters above each bar indicate significant differences among each set of volatiles after Tukey's test (P < 0.05). For statistical values, see Supplemental Table 1 online. FW, fresh weight.

Figure 8.

Subcellular Localization of GES-YFP Fusion Proteins in Arabidopsis. (A) Schematic drawing of the GES-YFP fusion gene used to transform the Arabidopsis insertion line salk_039864. The transgene consists of a 6-kb genomic fragment starting with the presumed transcriptional start site (as inferred from the cDNA) under the control of the constitutive CaMV 35S promoter. The YFP tag was integrated upstream of the GES stop codon. The exons are represented by gray boxes, and flanking regions and introns are represented by the black line. UTR, untranslated region. (B) and (C) Epidermal peels from true leaves of 10-d-old plants of the salk_039864 line (B) and the same knockout line transformed with Pro35S:GES-YFP (C) were used for fluorescence microscopy.

Figure 9.

GES Transcript Analysis and Leaf Volatile Emission in Different Genotypes Impaired in SA, JA, and Ethylene Signaling/Biosynthesis after Induction with Alamethicin. (A) RNA gel blot analysis of GES transcript levels in detached leaves of wild-type plants and mutants deficient in SA, JA, and ethylene abundance or signal transduction. Alamethicin (ala; 5 μg/mL) was applied through petioles of cut leaves; for mock treatment, a 0.1% ethanol solution was used. RNA was harvested after 24 h. The blots were rehybridized with a probe for Actin2 to document equal loading. Induction of the coi1 mutant was done in intact hydroponically grown plants as described in Methods. The experiment was repeated twice with similar results. (B) Volatiles were collected during 21 to 30 h after the beginning of alamethicin treatment from detached leaves of wild-type plants and mutants deficient in SA, JA, and ethylene abundance or signal transduction. Treatments and volatile analysis were conducted as described in Methods. The results represent means ± SE of three replicates. Letters above each bar indicate significant differences for each set of volatiles after Tukey's test (P < 0.05). For statistical values, see Supplemental Table 2 online. FW, fresh weight.

Figure 10.

GUS Transcript Levels and GUS Activity in ProGES:GUS Plants. (A) Schematic drawing of the ProGES:GUS construct. The GES promoter (−1162 to +3) was cloned upstream of the GUS reporter gene using Gateway technology. The attB2 site is part of the 5′ untranslated region of the GUS gene. (B) RNA gel blot analysis of GUS and GES transcript levels in hydroponically grown transgenic ProGES:GUS plants after induction with alamethicin (5 μg/mL) or ethanol (0.1%) for 31 h. (C) Histochemical GUS staining of a silique (1), a flower (2), an inflorescence (3), and a wounded leaf (4) from ProGES:GUS plants. The results are representative for at least 10 independent transgenic lines. (D) GES expression in wild-type plants in response to wounding (1 to 12 h) and treatment with alamethicin (ala; 24 h). Six-week-old hydroponically grown plants were wounded with forceps. Leaf material within ∼3 mm adjacent to the wound marks was harvested. Material from five leaves was combined before extraction. Relative levels of GES and VSP2 (a wound-inducible control gene) were determined by real-time RT-PCR using SYBR Green I chemistry. Values were normalized to the expression of At1g13320 (protein phosphatase type 2). As a control, plants were treated with 5 μg/mL alamethicin through the roots, and RNA was harvested after 24 h. The results represent means + SE of three technical replicates. The experiment was repeated with similar results (see Supplemental Figure 5 online).

Figure 11.

Sequence Comparison of Arabidopsis GES with Additional Arabidopsis and Other Plant TPSs. Bayesian tree generated from an alignment of 14 TPS proteins, including Arabidopsis GES (At1g61120), C. breweri LIS, C. concinna LIS, and selected Arabidopsis and other angiosperm monoterpene and diterpene synthases of primary and secondary metabolism. Arabidopsis genes used for the analysis are as follows: GES (At1g61120), LIS (At1g61680), myrcene/(E)-β-ocimene synthase (At3g25810), 1,8-cineole synthase (At3g25820), CPPS (At4g02780), and KS (At1g79460). Other monoterpene and diterpene synthase genes come from Nicotiana tabacum, Rhizinus communis, Mentha citrata, Solanum lycopersicum, Lactuca sativa, and Oryza sativa. Numbers above nodes represent Bayesian posterior probabilities.