De Novo Terpenes Emitted from Juvenile Leaves of Eucalyptus globulus Labill. subsp. globulus

Abstract

The contributions of de novo synthesis to terpene emissions from Eucalyptus globulus subsp. globulus were determined by fumigating branchlets with ¹³CO₂ in a gas exchange system. Of more than thirty-four terpenes emitted by this species, only four, i.e., isoprene, iso-valeraldehyde, cis-ocimene, and trans-caryophyllene, incorporated ¹³C into the terpene carbon skeleton during the ~5-6 h experiment. ¹³C incorporation into isoprene and iso-valeraldehyde reached a maximum of ca. 82% of the carbon skeleton, similar to cis-ocimene, with a maximum of 77% ¹³C incorporation after ~2.5 h exposure to ¹³CO₂. Only ca. 20% of carbon was labelled in trans-caryophyllene after 5-6 h. the incorporation of ¹³C was observed only in compounds emitted from leaves, and was not detected in either individual oil glands or in bulk leaf tissue. The results suggest the de novo synthesis of some terpenes (isoprene, cis-ocimene, trans-caryophyllene, and iso-valeraldehyde) and their emission is independent of emissions of terpenes stored in oil glands.

Keywords: 13C incorporation; BVOC; Eucalyptus globulus; GC-MS; PTR-MS; de novo synthesis; terpene emissions.

Figure 1

Typical GC-MS chromatogram of leaf oils of E. globulus. See Table 1 for the identity of the compounds, indicated in bold numbers, eluting under the respective peak.

Figure 2

PTR-MS (SIM) monitoring of the time course of the ion intensities of ¹³C incorporation into isoprene emitted from the leaves of E. globulus. Ions were identified as follows: m/z 69 = ¹²C isoprene; m/z 70 = isoprene with one ¹³C atom; m/z 71 = isoprene with two ¹³C atoms, etc. The vertical dotted lines indicate switching between 1.1% and 99.5% ¹³CO₂. Leaves were exposed at 28 °C under 500 μmol m⁻² s⁻¹ PAR for the duration of the experiment. The data represent a typical isotope profile representative of four replicates.

Figure 3

PTR-MS (SIM) monitoring the time course of the ion intensities of ¹³C incorporation for the m/z range 85–92. Ions at m/z 87 and m/z 85 declined in response to fumigation with ¹³CO₂. Transient increases and decreases in the abundances of m/z 86, 88, and 89 are evident, as are sustained increases in m/z 91and m/z 92, indicating the incorporation of ¹³C. Evidence suggests that ¹³C is incorporated into a five-carbon BVOC, later identified as iso-valeraldehyde (C₅H₁₀O). Vertical dotted lines indicate switching between 1.1% and 99.5% ¹³CO₂. Experimental conditions were identical to those in Figure 1. The data represent a typical isotope profile representative of four replicates.

Figure 4

PTR-MS mass spectra of (A) iso-valeraldehyde and (B) 2-methyl-3-buten-2-ol (1 μL), diluted into individual 1 L Tedlar bags and filled with ultrapure N₂. Spectra represent the background-subtracted average of 10–15 cycles of the PTR-MS in scan mode.

Figure 5

EI mass spectra from GC-MS analysis of (A) cis-ocimene and (B) trans-caryophyllene, emitted from leaves of E. globulus, showing a shift in fragmentation resulting from the incorporation of ¹³C. The ¹³C-labelled spectra (red) were captured 6 h after the onset of labelling and are compared with the unlabelled spectra (black) showing the natural isotopic abundance.

Figure 6

Time course of ¹³C incorporation into (A) cis-ocimene and (B) trans-caryophyllene emitted from leaves of E. globulus. The relative incorporation of ¹³C was calculated as [m/z (labelled)/(m/z unlabelled + m/z labelled)] × 100 using ions from EI mass spectra averaged across the total ion chromatogram for a given compound at each time point. The duration of ¹³CO₂ fumigation (0.5 to 6.5 h) is indicated on each graph by the vertical dotted lines. Data are from a single tree but are typical of the pattern observed in four trees.

Figure 7

In vitro synthesis of cis-ocimene in response to incrementally higher incubation temperatures during the monoterpene synthase assay for leaves of E. globulus (squares) and E. viminalis (circles). Each symbol is the mean of two assays at the temperature indicated on the x-axis.