Poster: Metabolic Engineering

Abs # 1003: Functional characterization of a tobacco, glandular- trichome-expressed CBT-ol cyclase, and the novel trichome exudation chemistry of CBT-ol cyclase suppressed plants

Presenter:	Wang, Erming , ewang2@uky.edu
Authors	Wang, Erming  (A)   Wagner, George J (A)
Affiliations:	(A): Agronomy Department, University of Kentucky

Geranylgeranyl pyrophosphate (GGPP) is the universal substrate for diterpene cyclases. Enzymatic and genetic studies indicate that there are perhaps three major and some minor diterpene cyclases in the glandular trichomes of Nicotiana tabacum, cultivar T.I. 1068. The three major cyclases; cembratriene (CBT)-ol cyclase, cis-abienol cyclase, and labdenediol cyclase, are responsible for conversion of GGPP to (CBT)-ols, cis-abienol and labdenediol, respectively. CBT-ols (plus their hydroxylated products the CBT-diols), cis-abienol and labdenediol account for ~62%, ~10% and ~0.8% of total trichome exudate weight in T.I. 1068. We identified a partial cDNA clone of a terpene cyclase-like gene, and the full-length cDNA (named CYC-2) was isolated by RACE-PCR. CYC-2 mRNAs were only accumulated in trichomes, and reached the maximum level just before flowering. The expression of CYC-2 gene was down-regulated by drought stress. The mature protein of CYC-2 was expressed in E. coli. Preliminary enzyme assays showed that this heterologously expressed protein was capable of catalyzing the formation of CBT-ols from GGPP, indicating that the CYC-2 gene encodes a CBT-ol cyclase. RNAi suppression of CYC-2 further showed that CYC-2 is a CBT-ol cyclase gene. In the trichomes of CYC-2 suppressed tobacco plants, CBT-ols (and CBT-diols) were almost absent while cis-abienol and labdenediol increased substantially. In addition, several novel diterpene-like compounds were produced. Our results demonstrate that surplus GGPP, resulting from suppression of a major diterpene cyclase, can be diverted to diterpene products of normally minor branch pathways.