Molecular regulation of plant monoterpene biosynthesis in relation to fragrance

Citrus belongs to an important economical group of crops. Fruits have a distinguished and pleasant taste partly due to the presence of terpenes. Furthermore, these terpenes potentially play a role in the resistance against pathogens such as insects and fungi. The aim of this thesis was therefore to study the regulation of terpene biosynthesis in Citrus limon L. Burm. Therefore, monoterpene synthase cDNAs from Citrus limon L. Burm. peel were isolated by a cDNA library-screening approach and to identify specific domains, and possibly amino acids, involved in product specificity within the active site of these enzymes. In addition, we aimed at investigating factors that might positively contribute to the regeneration of transgenic Citrus sinensis (L.) Osbeck cv. Valencia Late plants. Because transforming Citrus was not practical, we aimed at studying the odor effects of transforming a model plant, Nicotiana tabacum , with the isolated Citrus limon monoterpene synthases. In chapter 2, the isolation, expression and characterization of four new cDNAs belonging to the terpene synthase b sub-family ( tpsb ) family (Bohlmann et al., 1998) and coding for monoterpene synthases in Citrus limon L. Burm . has been described in detail. A random sequencing approach was applied to a cDNA library from mRNA isolated from the peel of a young developing fruit. These monoterpene synthase cDNAs were isolated and all four cDNAs could be functionally expressed in E. coli after removal of their plastid targeting signals. The main products of the enzymes in assays with geranyl diphosphate (GDP) as substrate were (+)-limonene (two cDNAs), (-)-β-pinene andγ-terpinene and these enzymes were named Cl(+)LIMS1, Cl(+)LIMS2, Cl(-)βPINS and ClγTS, respectively. In chapter 3, the functional differences which were present within the domains of the monoterpene synthases and leading to product specificity in the enzymatic catalysis, have been studied. Domain swapping experiments were conducted as well between Cl(-)βPINS and ClγTS as between Cl(+)LIMS2 and ClγTS . We showed that within the same putative C-terminal structural domain of monoterpene synthases, a region comprising 200 amino acids, of which 41 are different between Cl(-)βPINS and ClγTS, determines the specificity for the formation ofβ-pinene orγ- terpinene, respectively, while another region localized further downstream is required for a chimeric enzyme to yield products with the same ratio as in the wild type ClγTS. For Cl(+)LIMS2, both domains together appear to be sufficient for its enzyme specificity, but probably due to the low homology with ClγTS, many chimeric enzymes were inactive. In order to find factors that might improve the transformation efficiency of Citrus sinensis (L.) Osbeck cv. Valencia Late, the interactive effects within the regeneration of Citrus sinensis have been studied (Chapter 4). The latter was focused on the influence of the initial germination medium, of the explant type (source) and of the regeneration medium on shoot regeneration. Results show that the germination on MS30 (Murashige & Skoog, 1962) and the regeneration on NNIII (Nitsch & Nitsch, 1969) containing a 20:1 cytokinin: auxin ratio supplied with the riboside form of the benzyl amino purine cytokinin, are essential for an efficient induction of shoot regeneration. In addition, hypocotyl-derived shoot regenerants incubated on NNRtVIII (Nitsch & Nitsch based medium) showed better rooting than epicotyl-derived shoot regenerants and incubation on other media tested. Finally, with the help of a human panel, the floral fragrance effect of transforming Nicotiana tabacum either together with the Cl(+)LIMS1, Cl(-)βPINS and ClγTS genes, or singly with the Cl(-)βPINS gene has been investigated (Chapter 5). In a triangle experimental design, the panel was able to discriminate significantly between the smell of a transgenic plant and a wild type plant. However, a descriptive panel had difficulties to generate accurate sensory descriptors for smells, and to reach consensus on the precise meaning of chosen attributes in a predetermined small number of panel sessions. Nevertheless, this thesis illustrates an example where the combination of molecular biology, cell biology and biochemistry, and sensory analysis offers an integrated approach as a first step in trying to unravel the world of plant fragrance emission and perception.