Response Factor Considerations for the Quantitative Analysis of Western Redcedar (Thuja plicata) Foliar Monoterpenes

Abstract

A method is described for quantitative analysis of monoterpenes in western redcedar (Thuja plicata) foliage by gas chromatography with flame ionization detection. Response factors for monoterpenes identified in redcedar are evaluated to determine similarities among monoterpene responses. Evaluation demonstrates that redcedar monoterpenes yield detector responses that fall into two groups. One monoterpene from each group is used as a standard for quantitative analysis. Redcedar monoterpenes are quantitated by comparing analyte response with the response factor of one of the standards in single-point calibrations. Homogenized foliage samples are extracted with ethyl acetate and the extracts passed through a solid phase extraction column of graphitized carbon to remove plant pigments. Method bias and repeatability are evaluated by fortifying foliage samples with (1S)-(+)-carvone and (1S)-(+)-2-carene and subjecting the samples to the extraction and analysis procedures. Detection limits are also assessed from fortified samples. Excellent recovery (> 95.0%) and precision (< 5%) are obtained from the analysis of 2-carene from fortified samples. Carvone recovery is approximately 80% with excellent precision (< 4%). The method limits of detection obtained from 2-carene and carvone fortified samples are 4.7 and 13.5 microg/g, respectively.

Identification of Genes in Thuja plicata Foliar Terpenoid Defenses

“…Thus, the entire profile of monoterpenoids in wild-type foliage as detected here (Fig. 6B) and described prior (Kimball et al, 2005) can be explained by the activity of TpSAB and the predicted conversion of sabinene to thujone. TpSAB and its protein may be considered a key gene and enzyme, respectively, for monoterpene-based defenses against herbivore feeding in the T. plicata foliage.…”

“…1C). The terpenoid profile obtained from glands is qualitatively the same as that published for whole foliage (Kimball et al, 2005). Thus, it appears that the majority of stored foliar terpenoids are confined to resin glands.…”

“…While it is known that T. plicata foliage contains sabinene and thujone (Kimball et al, 2005), the internal site or sites of production and storage are unknown. The limited literature available on T. plicata foliage anatomy describes the presence of resin glands in the scale-like leaves (hereafter referred to as scales; Suzuki, 1979), suggesting that these compounds could be synthesized and possibly stored in these glands, just as terpenoids are stored in needle resin ducts in Pinaceae species (Wooding and Northcote, 1965;Manninen et al, 2002).…”

“…Ungulates selectively feed on individuals with low levels of foliar monoterpenoids, while avoiding plantlets with higher levels (Vourc'h et al, 2002b). The two most important monoterpenoids for deterring ungulate feeding are a-and b-thujone (Vourc'h et al, 2002a), which are typically the most abundant monoterpenoids in T. plicata foliage (Kimball et al, 2005). T. plicata foliage consists of scale-like needles, where each scale has a prominent central resin gland.…”

The Gymnosperm Cytochrome P450 CYP750B1 Catalyzes Stereospecific Monoterpene Hydroxylation of (+)-Sabinene in Thujone Biosynthesis in Western Redcedar

“…The proposed pathway was redrawn based on Karp and Croteau (1982). of terpenoids in WRC essential oil (Von Rudloff et al, 1988;Kimball et al, 2005;Tsiri et al, 2009;Tisserand and Young, 2014). These three monoterpenes are also found in other Cupressaceae species, such as the genera Juniperus spp.…”