Herbivore-induced defense response in a model legume. Two-spotted spider mites induce emission of (E)-beta-ocimene and transcript accumulation of (E)-beta-ocimene synthase in Lotus japonicus

Abstract

Indirect defense of plants against herbivores often involves the induced emission of volatile infochemicals including terpenoids that attract natural enemies of the herbivores. We report the isolation and characterization of a terpene synthase cDNA (LjEbetaOS) from a model legume, Lotus japonicus. Recombinant LjEbetaOS enzyme produced (E)-beta-ocimene (98%) and its Z-isomer (2%). Transcripts of LjEbetaOS were induced in L. japonicus plants infested with two-spotted spider mites (Tetranychus urticae), coinciding with increasing emissions of (E)-beta-ocimene as well as other volatiles, (Z)-3-hexenyl acetate and (E)-4,8-dimethyl-1,3,7-nonatriene, by the infested plants. We suggest that LjEbetaOS is involved in the herbivore-induced indirect defense response of spider mite-infested L. japonicus via de novo formation and emission (E)-beta-ocimene. Mechanical wounding of the leaves or application of alamethicin (ALA), a potent fungal elicitor of plant volatile emission, also induced transiently increased levels of LjEbetaOS transcripts in L. japonicus. However, wounding or ALA did not result in elevated release of (E)-beta-ocimene. Differences in volatile emissions after herbivory, mechanical wounding, or treatment with ALA suggest that neither a single mechanical wounding event nor ALA simulate the effect of herbivore activity and indicate that herbivore-induced emission of (E)-beta-ocimene in L. japonicus involves control mechanisms in addition to up-regulation of LjEbetaOS transcripts.

Figure 1.

Amino acid sequence alignment of LjEβOS (LjEBOS), Arabidopsis (E)-β-ocimene synthase (AtTPS03, GenBank accession no. AY151086), snapdragon (E)-β-ocimene synthase (ama0a23, AY195607), and Populus alba × Populus tremula isoprene synthase (IspS, AJ294819). Amino acid residues that are identical among at least three sequences are indicated by white letters on black background. Other conserved residues are shaded in gray. Dashes indicate sequence gaps introduced to optimize the alignment. Conserved motifs R(R/K/G)x₈W and DDxxD are indicated.

Figure 2.

Expression in E. coli of recombinant LjEβOS protein and GC-MS analysis of monoterpene products formed by recombinant LjEβOS enzyme activity in vitro. Total ion chromatogram and mass spectra are of LjEβOS in vitro enzyme activity products with GPP as substrate. The mass spectrum of the most abundant product (Peak II) and a minor compound (Peak I), identified as (E)- and (Z)-β-ocimene, respectively, are shown together with that of the respective of authentic standards.

Figure 3.

GC profiles of volatiles emitted from L. japonicus plants after 24 h of spider mite infestation or mechanical wounding compared with undamaged plants. Identification of compounds: 1, (Z)-3-hexenyl acetate; 2, (E)-β-ocimene; 3, (E)-4,8-dimethyl-1,3,7-nonatriene (DMNT); internal standard (IS), n-tridecane.

Figure 4.

LjEβOS transcripts and (E)-β-ocimene emissions in L. japonicus plants in response to spider mite infestation or mechanical wounding. A, Northern analysis of transcripts in the L. japonicus shoots exposed to spider mites or wounded mechanically for 0, 1, 6, and 24 h. B, Volatiles were collected over 2 h at time points, 1 to 3 h, 6 to 8 h, and 24 to 26 h from the headspace of L. japonicus exposed to spider mites or wounded mechanically and subjected to GC-MS. Data represents the mean ± se (n = 4–9, ion intensity of (E)-β-ocimene per gram fresh weight). Untreated plants served as control. A bar with asterisk is significantly different from the untreated control within each sampling interval (Dunnet's test at the 0.05 level).

Figure 5.

Effects of ALA treatment on transcripts accumulation and emission of (E)-β-ocimene in L. japonicus. A, Northern hybridization signals for LjEβOS and LjSqS in the L. japonicus shoots treated with water or ALA solution. B, RT-PCR analysis using the gene-specific primers of LjEβOS or LjTPS1 followed the same time courses shown in A. C, (E)-β-Ocimene emissions from L. japonicus plants treated with ALA solution. Data represent the mean ± se (n = 3–6, ion intensity of (E)-β-ocimene per gram fresh weight).