Joost Lücker

Increased and Altered Fragrance of Tobacco Plants after Metabolic Engineering Using Three Monoterpene Synthases from Lemon

Wild-type tobacco (Nicotiana tabacum) plants emit low levels of terpenoids, particularly from the flowers. By genetic modification of tobacco cv Petit Havana SR1 using three different monoterpene synthases from lemon (Citrus limon L. Burm. f.) and the subsequent combination of these three into one plant by crossings, we show that it is possible to increase the amount and alter the composition of the blend of monoterpenoids produced in tobacco plants. The transgenic tobacco plant line with the three introduced monoterpene synthases is emitting β-pinene, limonene, and γ-terpinene and a number of side products of the introduced monoterpene synthases, from its leaves and flowers, in addition to the terpenoids emitted by wild-type plants. The results show that there is a sufficiently high level of substrate accessible for the introduced enzymes.

Functional genomic analysis of potato tuber life-cycle

SummaryPotato tuber life-cycle is composed of many individual developmental stages including tuber formation, tuber development, dormancy and sprouting. We have used cDNA-AFLP fingerprinting to analyse gene expression in 24 individual stages of development, over the period from stolon formation through sprouting. In addition to these developmental stages, different tissues were analysed to assess tissue specificity and various controls were incorporated to determine process specificity. In total around 18000 transcript derived cDNA fragments (TDFs) were visualised from which circa 2600 were included in a statistical analysis allowing general conclusions about gene expression during development. More than 200 process specific TDFs were isolated and sequenced throughout the potato tuber life-cycle. The sequence similarities of these TDFs to known genes give an insight into the kinds of processes occurring during tuberisation, dormancy and sprouting.

Domain swapping of Citrus limon monoterpene synthases: impact on enzymatic activity and product specificity

Monoterpene cyclases are the key enzymes in the monoterpene biosynthetic pathway, as they catalyze the cyclization of the ubiquitous geranyl diphosphate (GDP) to the specific monoterpene skeletons. From Citrus limon, four monoterpene synthase-encoding cDNAs for a beta-pinene synthase named Cl(-)betaPINS, a gamma-terpinene synthase named ClgammaTS, and two limonene synthases named Cl(+)LIMS1 and Cl(+)LIMS2 were recently isolated [J. Lücker et al., Eur. J. Biochem. 269 (2002) 3160]. The aim of our work in this study was to identify domains within these monoterpene synthase enzymes determining the product specificity. Domain swapping experiments between Cl(-)betaPINS and ClgammaTS and between Cl(+)LIMS2 and ClgammaTS were conducted. We found that within the C-terminal domain of these monoterpene synthases, a region comprising 200 amino acids, of which 41 are different between Cl(-)betaPINS and ClgammaTS, determines the specificity for the formation of beta-pinene or gamma-terpinene, respectively, while another region localized further downstream is required for a chimeric enzyme to yield products in the same ratio as in the wild-type ClgammaTS. For Cl(+)LIMS2, the two domains together appear to be sufficient for its enzyme specificity, but many chimeras were inactive probably due to the low homology with ClgammaTS. Molecular modeling was used to further pinpoint the amino acids responsible for the differences in product specificity of ClgammaTS and Cl(-)betaPINS.