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, 161 (4), 1993-2004

Identification of Genes in Thuja Plicata Foliar Terpenoid Defenses

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Identification of Genes in Thuja Plicata Foliar Terpenoid Defenses

Adam J Foster et al. Plant Physiol.

Abstract

Thuja plicata (western redcedar) is a long-lived conifer species whose foliage is rarely affected by disease or insect pests, but can be severely damaged by ungulate browsing. Deterrence to browsing correlates with high foliar levels of terpenoids, in particular the monoterpenoid α-thujone. Here, we set out to identify genes whose products may be involved in the production of α-thujone and other terpenoids in this species. First, we generated a foliar transcriptome database from which to draw candidate genes. Second, we mapped the storage of thujones and other terpenoids to foliar glands. Third, we used global expression profiling to identify more than 600 genes that are expressed at high levels in foliage with glands, but can either not be detected or are expressed at low levels in a natural variant lacking foliar glands. Fourth, we used in situ RNA hybridization to map the expression of a putative monoterpene synthase to the epithelium of glands and used enzyme assays with recombinant protein of the same gene to show that it produces sabinene, the monoterpene precursor of α-thujone. Finally, we identified candidate genes with predicted enzymatic functions for the conversion of sabinene to α-thujone. Taken together, this approach generated both general resources and detailed functional characterization in the identification of genes of foliar terpenoid biosynthesis in T. plicata.

Figures

Figure 1.
Figure 1.
A, Comparison of monoterpene content in glands (arows I, II) and areas without glands (arrow III). Terpenoids were collected from tissue samples by piercing with sterile needles. B, No monoterpenes were detected by GC from tissue extracted from areas surrounding the resin glands. C, Monoterpenes were detected in resin glands by GC. IS, Internal standard (isobutyl benzene); 1, thujene; 2, α-pinene; 3, sabinene; 4, myrcene; 5, α-thujone; 6, β-thujone. [See online article for color version of this figure.]
Figure 2.
Figure 2.
Anatomical and chemical differences between wild-type and variant T. plicata genotypes. A, Wild-type genotype foliage with a visible resin gland. B, Variant genotype foliage without visible resin glands. C and D, Total ion chromatogram (TIC; Varian Saturn 2000 Ion Trap gas chromatograph-mass spectrometer) of monoterpenes extracted from the foliage of 2-year-old trees. C, Wild-type foliage. D, Variant foliage. Variant TIC displays 103 rather than 106 counts to show that no traces of monoterpenes were detected. RG, Resin gland; IS, internal standard (n-nonyl acetate); 1, thujene; 2, α-pinene; 3, sabinene; 4, myrcene; 5, α-thujone; 6, β-thujone. [See online article for color version of this figure.]
Figure 3.
Figure 3.
Distribution of contigs with greater than 10-fold up-regulation in the wild-type genotype compared with the variant genotype.
Figure 4.
Figure 4.
Quantitative PCR results for genes expressed in the leaves of wild-type and variant T. plicata trees. A, Terpene synthases. B, Cytochrome P450 monooxygenases. C, Dehydrogenases. D, Reductases. RTA, Relative transcript abundance between wild-type and variant genotypes. Error bars indicate se. Single asterisk indicates statistically significant data at P < 0.05 using Student’s t test; double asterisk indicates absence of detectable levels of transcript in the variant genotype after 35 quantitative PCR cycles and that RTA values are based on an artificial CT = 35 in the variant.
Figure 5.
Figure 5.
Contig788 expression is localized to the epithelial cells of foliar resin glands. A, Cross section of leaf hybridized with sense strand. B, Cross section of leaf hybridized with antisense strand. C, Longitudinal section of scale primordia hybridized with antisense strand. D, Longitudinal section of foliar shoot apex hybridized with antisense strand. Red arrows point at glands.
Figure 6.
Figure 6.
TICs (Agilent 6890A gas chromatograph with a 5973N mass spectrometer) of products formed in vitro by recombinant contig788 protein. A, Contig788 protein produced (+)-sabinene as the major product. B, TIC of monoterpenes found in T. plicata foliage. C, TIC of (+)-sabinene analytical standard (Chromadex). TIC was used to determine the identity of each peak by comparison to authentic standards. 1, α-thujene; 2, (+)-α-pinene; 3, myrcene; 4, (+)-sabinene; 5, (+)-β-pinene; 6, internal standard (isobutyl benzene); 7, (+)-limonene; 8, γ-terpinene; 9, terpinolene; 10, α-thujone.
Figure 7.
Figure 7.
Candidate genes in thujone biosynthesis pathway. A, Thujone biosynthesis pathway, including enzymatic activities proposed by Croteau (1996). B, Genes identified in this study whose products are candidates for the enzymatic conversions of geranyl diphosphate (GPP) to α-thujone.

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