Gesell Andreas, Blaukopf Markus, Madilao Lina, Yuen Macaire M S, Withers Stephen G, Mattsson Jim, Russell John H, Bohlmann Jörg
Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z4 (A.G., L.M., M.M.S.Y., J.B.);Department of Chemistry, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z1 (M.B., S.G.W.);Department of Biological Sciences, Simon Fraser University, Burnaby, British Columbia, Canada V5A 1S6 (J.M.); andBritish Columbia Ministry of Forests, Lands and Natural Resource Operations, Cowichan Lake Research Station, Mesachie Lake, British Columbia, Canada V0R 2N0 (J.H.R.).
Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z4 (A.G., L.M., M.M.S.Y., J.B.);Department of Chemistry, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z1 (M.B., S.G.W.);Department of Biological Sciences, Simon Fraser University, Burnaby, British Columbia, Canada V5A 1S6 (J.M.); andBritish Columbia Ministry of Forests, Lands and Natural Resource Operations, Cowichan Lake Research Station, Mesachie Lake, British Columbia, Canada V0R 2N0 (J.H.R.)
Plant Physiol. 2015 May;168(1):94-106. doi: 10.1104/pp.15.00315. Epub 2015 Mar 31.
Western redcedar (WRC; Thuja plicata) produces high amounts of oxygenated thujone monoterpenoids associated with resistance against herbivore feeding, particularly ungulate browsing. Thujones and other monoterpenoids accumulate in glandular structures in the foliage of WRC. Thujones are produced from (+)-sabinene by sabinol and sabinone. Using metabolite analysis, enzyme assays with WRC tissue extracts, cloning, and functional characterization of cytochrome P450 monooxygenases, we established that trans-sabin-3-ol but not cis-sabin-3-ol is the intermediate in thujone biosynthesis in WRC. Based on transcriptome analysis, full-length complementary DNA cloning, and characterization of expressed P450 proteins, we identified CYP750B1 and CYP76AA25 as the enzymes that catalyze the hydroxylation of (+)-sabinene to trans-sabin-3-ol. Gene-specific transcript analysis in contrasting WRC genotypes producing high and low amounts of monoterpenoids, including a glandless low-terpenoid clone, as well as assays for substrate specificity supported a biological role of CYP750B1 in α- and β-thujone biosynthesis. This P450 belongs to the apparently gymnosperm-specific CYP750 family and is, to our knowledge, the first member of this family to be functionally characterized. In contrast, CYP76AA25 has a broader substrate spectrum, also converting the sesquiterpene farnesene and the herbicide isoproturon, and its transcript profiles are not well correlated with thujone accumulation.
西部红雪松（WRC；北美乔柏）会产生大量与抵御食草动物取食相关的氧化侧柏酮单萜类化合物，尤其是有蹄类动物的啃食。侧柏酮和其他单萜类化合物在西部红雪松叶片的腺状结构中积累。侧柏酮由（+）-桧烯通过桧醇和桧酮生成。通过代谢物分析、对西部红雪松组织提取物进行酶活性测定、克隆以及细胞色素P450单加氧酶的功能表征，我们确定反式桧-3-醇而非顺式桧-3-醇是西部红雪松侧柏酮生物合成的中间体。基于转录组分析、全长互补DNA克隆以及对表达的P450蛋白的表征，我们鉴定出CYP750B1和CYP76AA25为催化（+）-桧烯羟基化为反式桧-3-醇的酶。在产生高含量和低含量单萜类化合物的不同西部红雪松基因型中进行基因特异性转录分析，包括一个无腺体低萜类克隆，以及底物特异性测定，支持了CYP750B1在α-和β-侧柏酮生物合成中的生物学作用。这种P450属于明显为裸子植物特有的CYP750家族，据我们所知，这是该家族中首个进行功能表征的成员。相比之下，CYP76AA25具有更广泛的底物谱，还能转化倍半萜金合欢烯和除草剂异丙隆，并且其转录谱与侧柏酮积累没有很好的相关性。
