Biosynthesis of monoterpene alcohols, derivatives and conjugates in plants : roles in resistance to western flower thrips

Western flower thrips (WFT), <em>Frankliniella occidentalis</em>, is one of the most serious pests in several vegetable and flower crops worldwide. It is a highly polyphagous insect and a vector of several plant viruses of which the Tomato Spotted Wilt Virus and the Impatiens Necrotic Spot Virus are the most important. Feeding by WFT causes light coloured patches on leaves, petals and fruits, stunted plant growth, and flower and fruit deformation. Synthetic pesticides has been widely used to control WFT. However, the frequent use of these pesticides leads to rapid resistance in WFT, and they are a threat to the environment. Therefore, it is desirable to identify natural sources of resistance effective against WFT to allow breeders to improve resistance in crop species. Monoterpenes, as constituents of floral scents and plant resins, play an important role in pollinator attraction and in direct and indirect plant defence against pest insects and pathogens. For example, linalool is a common floral scent constituent and found to be emitted from the leaves by many plant species after herbivore attack. In earlier work, linalool-overexpressing Arabidopsis has been tested for resistance to the pest aphid, <em>Myzus persicae</em>, in dual-choice assays, and transgenic plants significantly repelled or deterred the aphids. A linalool synthase (LIS) was overexpressed in chrysanthemum plants and studied the effect of transgenic plants on WFT (Chapter 2). The volatiles from leaves of transgenic plants were significantly attractive to WFT, however, WFT were significantly deterred by the content of leaf discs from transgenic plants. The headspace analysis showed that the volatiles of LIS chrysanthemum leaves were strongly dominated by linalool,but, they also emitted small amounts of the C₁₁-homoterpene, (3<em>E</em>)-4,8-dimethyl-1,3,7-nonatriene, a derivative of nerolidol. In addition, LC-MS analysis showed that several non-volatile linalool glycosides were significantly increased in the leaves of LIS chrysanthemum compared with leaves of wild-type plants. A geraniol synthase (GES) was overexpressed in maize to see whether WFT could be affected by geraniol or its derivatives (Chapter 3). However, geraniol produced in transgenic maize was all efficiently converted to non-volatile glycoside, geranoyl-6-<em>O</em>-malonyl-β-D-glucopyranoside, and GES maize had no effect on WFT behaviour. These studies demonstrate complex effects of terpene engineering on the metabolic changes in transgenic plants. These results suggest that the release/glycosylation of terpenes should be controlled to improve plant resistance against WFT upon metabolic engineering with terpene synthases. The research subsequently focused on a well-known natural pesticide—pyrethrins. Pyrethrins comprise a group of six closely related esters, derived from the monoterpene alcohol chrysanthemol. Pyrethrins are the economically most important natural insecticide with broad uses in homes, agriculture and stored products for more than 150 years. The effect of pyrethrins against WFT was evaluated on its survival, feeding behaviour, and reproduction both <em>in vitro</em> and <em>in planta</em> (infiltrated chrysanthemum leaves) (Chapter 4). Pyrethrins at 0.1% (w/v) and 1% (w/v) exhibited a significantly negative effect on feeding, and the effects of natural concentrations of pyrethrins in pyrethrum leaves can explain the observed high mortality of WFT feeding on pyrethrum leaves. After the finding of this strong effect of pyrethrins on WFT, the study on the biosynthetic pathway of pyrethrins was continued in order to introduce pyrethrin biosynthesis in transgenic plants. A second function of the published enzyme, chrysanthemyl diphosphate synthase (CDS) was identified (Chapter 5). CDS has been reported to catalyse the formation of chrysanthemyl diphosphate (CPP). However, CDS was demonstrated to also catalyse the next step of CPP into chrysanthemol both <em>in vitro</em> and <em>in vivo</em>. CDS was proposed to be renamed as a chrysanthemol synthase (CHS) using DMAPP as substrate. The gene involved in the next step converting chrysanthemol to chrysanthemic acid has also been characterized (Ramirez, 2013). A chrysanthemic acid:CoA ligase, which is involved in the final stage of pyrethrin biosynthesis was also studied (Chapter 6). The function of this enzyme was confirmed <em>in vitro</em> and the encoding gene showed a similar expression pattern as CHS in several different tissues and flower developmental stages. The gene responsible for making the final esters is a GDSL-lipase-like acyltransferase (Kikuta et al., 2012). We assume still three to four enzymes are required for the biosynthesis of the basic one of the six pyrethrin esters, jasmolin I, from the precursors DMAPP and jasmonic acid which are universal in plants. And four to five extra genes are required for the complete biosynthesis of all six pyrethrin esters. In this study, new insights were gained for the biosynthesis of monoterpenes and their derivatives and conjugates, as well as for plant resistance to WFT mediated by these compounds. The characterization of genes involved in pyrethrin biosynthesis paves the way for metabolic engineering of this natural pesticide in other crops.