Variation of herbivore-induced volatile terpenes among Arabidopsis ecotypes depends on allelic differences and subcellular targeting of two terpene synthases, TPS02 and TPS03

Abstract

When attacked by insects, plants release mixtures of volatile compounds that are beneficial for direct or indirect defense. Natural variation of volatile emissions frequently occurs between and within plant species, but knowledge of the underlying molecular mechanisms is limited. We investigated intraspecific differences of volatile emissions induced from rosette leaves of 27 accessions of Arabidopsis (Arabidopsis thaliana) upon treatment with coronalon, a jasmonate mimic eliciting responses similar to those caused by insect feeding. Quantitative variation was found for the emission of the monoterpene (E)-beta-ocimene, the sesquiterpene (E,E)-alpha-farnesene, the irregular homoterpene 4,8,12-trimethyltridecatetra-1,3,7,11-ene, and the benzenoid compound methyl salicylate. Differences in the relative emissions of (E)-beta-ocimene and (E,E)-alpha-farnesene from accession Wassilewskija (Ws), a high-(E)-beta-ocimene emitter, and accession Columbia (Col-0), a trace-(E)-beta-ocimene emitter, were attributed to allelic variation of two closely related, tandem-duplicated terpene synthase genes, TPS02 and TPS03. The Ws genome contains a functional allele of TPS02 but not of TPS03, while the opposite is the case for Col-0. Recombinant proteins of the functional Ws TPS02 and Col-0 TPS03 genes both showed (E)-beta-ocimene and (E,E)-alpha-farnesene synthase activities. However, differential subcellular compartmentalization of the two enzymes in plastids and the cytosol was found to be responsible for the ecotype-specific differences in (E)-beta-ocimene/(E,E)-alpha-farnesene emission. Expression of the functional TPS02 and TPS03 alleles is induced in leaves by elicitor and insect treatment and occurs constitutively in floral tissues. Our studies show that both pseudogenization in the TPS family and subcellular segregation of functional TPS enzymes control the variation and plasticity of induced volatile emissions in wild plant species.

Figure 1.

Correlation of (E)-β-ocimene and (E,E)-α-farnesene emission from coronalon-treated leaves of 27 Arabidopsis accessions. Treatment with coronalon and volatile collection were conducted as described in “Materials and Methods.” Emissions are in ng g⁻¹ fresh weight h⁻¹. Numbers indicate individual accessions according to Table I. Each value represents the mean ± se of three replicates.

Figure 2.

Molecular nature of the TPS02 and TPS03 alleles in accessions Col-0 and Ws. A, Schematic representation of the structures of TPS02 and TPS03. Exons are represented by the gray boxes, and flanking regions and introns are represented by the lines between boxes. B, Alignment of nucleotide and amino acid sequence regions of the TPS02 (left) and TPS03 (right) alleles from accessions Col-0 and Ws indicating frame shift mutations caused by base pair insertions in the Col-0 TPS02 and Ws TPS03 genes. The white box marks premature stop codons. The gene-specific positions of the sequences are indicated. C, Amino acid sequence alignment of the full-length and truncated proteins of the Col-0 and Ws TPS02 and TPS03 alleles. Amino acids shaded in black are conserved in all sequences, and gray shading indicates amino acids conserved in two or three sequences. Dashes indicate gaps inserted for optimal alignment. Horizontal lines mark the highly conserved DDXXD, RXR, and RRX₈W motifs. A motif similar to the H-α1 loop region of apple MdAFS1 is marked by a thick dashed line. The asterisk indicates the putative cleavage site for a 25-amino acid plastidial transit peptide of the TPS02 protein.

Figure 3.

Semiquantitative RT-PCR analysis of TPS02 and TPS03 transcript levels in Col-0 and Ws tissues. Actin8 transcripts were analyzed as a control. Results are representative for at least three independent experiments. A, TPS02 and TPS03 transcript analysis from rosette leaves of accession Col-0 treated with coronalon (coro; top panel), with P. xylostella larvae (middle panel), and after mechanical wounding (bottom panel). The two amplicons obtained for TPS02 represent splice variants (Supplemental Fig. S2). No TPS02 transcript was detected upon insect feeding and wounding. Treatments were conducted as described in “Materials and Methods.” B, Transcript levels of TPS02 in leaves of accession Ws in response to treatments as described for A. No mRNA of TPS03 was detected. C, Analysis of TPS02 and TPS03 transcripts in flowers of Col-0 and Ws.

Figure 4.

Coronalon-induced expression of TPS03 and volatile emission in detached leaves of Col-0 wild-type plants and the TPS03 T-DNA insertion line SALK_132694. A, Position of the T-DNA insertion in the TPS03 gene. Gray boxes represent exons, and flanking regions and introns are shown by the black line. B, Semiquantitative RT-PCR analysis of transcripts of TPS03 in comparison with genes TPS02 and TPS10. Transcript levels of Actin8 were analyzed as a control. coro, Coronalon. C, Emission of MeSA, TMTT, and (E,E)-α-farnesene as measured between 21 and 30 h of coronalon treatment of Col-0 wild-type plants and the T-DNA insertion line. Normalized peak areas are shown for each compound as analyzed by GC-MS (see “Materials and Methods”). No (E)-β-ocimene could be detected from wild-type or mutant plants under these conditions. Results are average values ± se (n = 3). None of the volatiles was detected in mock controls.

Figure 5.

Coronalon-induced expression of TPS02 and volatile emission in detached leaves of Ws wild-type plants and the TPS02 T-DNA insertion line FLAG_406A04. A, Schematic presentation of the position of the T-DNA insertion in the TPS02 gene. Exons are represented by gray boxes, and flanking regions and introns are shown by the black line. B, Semiquantitative RT-PCR analysis of transcripts of TPS02. Transcript levels of Actin8 were analyzed as a control. No full-length transcripts were found for TPS03 and TPS10 in wild-type and mutant plants. coro, Coronalon. C, Emission of MeSA, TMTT, (E,E)-α-farnesene, and (E)-β-ocimene between 21 and 30 h of coronalon treatment of Ws wild-type plants and the T-DNA insertion line. Normalized peak areas are shown for each compound as analyzed by GC-MS (see “Materials and Methods”). Results represent mean values ± se (n = 3). None of the volatiles was detected from mock control leaves.

Figure 6.

GC-MS analysis of monoterpene and sesquiterpene products of recombinant Ws TPS02 and Col-0 TPS03 enzymes. Recombinant proteins were expressed in E. coli, extracted, partially purified, and applied for TPS assays using the substrates GPP and FPP. A, Total ion GC-MS chromatograms of monoterpenes (top) and sesquiterpenes (bottom) produced by recombinant Ws TPS02 protein from GPP and FPP, respectively. The molecular structures of (E)-β-ocimene and (E,E)-α-farnesene are shown. B, Monoterpene products (top) and sesquiterpene products (bottom) of recombinant Col-0 TPS03 enzyme detected in assays with GPP and FPP, respectively. Terpene products were identified by comparison with authentic standards or by library suggestion [for (Z,E)-α-farnesene]. No products were found in purified extracts from E. coli carrying the empty expression vector.

Figure 7.

Confocal laser scanning microscopy of stably expressed Ws TPS02 and Col-0 TPS03 peptide-GFP fusion proteins. Microscopic images were taken from the hypocotyls of 2-week old seedlings. The first column (A, E, and I) shows light microscopic images of hypocotyl cells. Chlorophyll autofluorescence, detected in the red channel, is shown in the second column (B, F, and J). The third column (C, G, and K) shows GFP fluorescence, detected in the green channel. The fourth column (D, H, and L) shows merged green and red channel images. A 35-amino acid N-terminal TPS02 peptide (containing a putative 25-amino acid plastidial transit peptide) fused to GFP localizes to chloroplasts (A–D). No plastidial localization was detected for a fusion protein containing a 50-amino acid N-terminal peptide of Col-0 TPS03 (E–H). Ferredoxin N-reductase-eGFP carrying a plastidial target peptide was used as a chloroplast marker (I–L). Bars = 20 μm.

Figure 8.

Effects of the MEP pathway inhibitor fosmidomycin (fos) and the mevalonate pathway inhibitor lovastatin (lov) on emission of (E)-β-ocimene and (E,E)-α-farnesene from leaves of accessions Ws and Col-0. Volatiles were collected for 8 h from detached rosette leaves treated with coronalon in the presence of a single inhibitor. Relative peak areas of compounds are shown. Peak areas from controls without the addition of inhibitors were arbitrarily set to 1.0. Results represent mean values ± se (n = 3).

Figure 9.

GUS activity in Col-0 ProTPS03:GUS and Ws ProTPS02:GUS plants. The results are representative for at least three independent lines. A, Histochemical GUS staining of an inflorescence (1), pollen grains (2, arrow), a silique (3), and a P. xylostella-damaged mature leaf from a Col-0 ProTPS03:GUS plant (4). In panel 4, GUS activity is induced locally around the sites of feeding damage. B, Induced GUS activity in a rosette leaf of a Ws ProTPS02:GUS plant treated for 24 h with coronalon through the petiole. The results are representative for at least three independent lines.