Biosynthesis and Transport of Terpenes
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Biosynthesis and transport of terpenes Hieng-Ming Ting Thesis committee Promotor Prof. Dr H.J. Bouwmeester Professor of Plant Physiology Wageningen University Co-promotor Dr A.R. van der Krol Associate professor, Laboratory of Plant Physiology Wageningen University Other members Prof. Dr A.H.J. Bisseling, Wageningen University Prof. Dr M. Boutry, University of Louvain, Belgium Dr M.A. Jongsma, Wageningen University Dr M.H.A.J. Joosten, Wageningen University This research was conducted under the auspices of the Graduate School of Experimental Plant Sciences. Biosynthesis and transport of terpenes Hieng-Ming Ting Thesis submitted in fulfilment of the requirements for the degree of doctor at Wageningen University by the authority of the Rector Magnificus Prof. Dr M.J. Kropff, in the presence of the Thesis Committee appointed by the Academic Board to be defended in public on Monday 24 March 2014 at 11 a.m. in the Aula. Hieng-Ming Ting Biosynthesis and transport of terpenes 184 pages. PhD thesis, Wageningen University, Wageningen, NL (2014) With references, with summaries in English and Dutch ISBN 978-90-6173-892-9 To my beloved wife Ya-Fen my lovely son Teck-Yew my family in Malaysia Contents Chapter 1 General introduction 9 Chapter 2 The metabolite chemotype of Nicotiana benthamiana transiently 23 expressing artemisinin biosynthetic pathway genes is a function of CYP71AV1 type and relative gene dosage Chapter 3 Experiments to address the potential role of LTPs in sesquiterpene 65 emission Chapter 4 Characterisation of aberrant pollen and ovule phenotypes associated 91 with chromosomal translocations in two T-DNA insertion mutants of Arabidopsis Chapter 5 Inhibition of vesicle transport during terpene biosynthesis causes 105 proteasome malfunction Chapter 6 General discussion 145 References 161 Summary 173 Samenvatting 175 Acknowledgements 177 Curriculum Vitae 180 Publications 181 Education statement 182 Chapter 1 General introduction Chapter 1 Biological function of terpenes Terpenes or terpenoids are a large and structurally diverse family of primary and secondary metabolites in plants (Trapp&Croteau, 2001). Some terpenes are only produced in minute amounts to function as plant phytohormones, such as gibberellic acid, abscisic acid, brassinosteroid, cytokinins and the strigolactones. Terpenes can also be produced in bulk amounts in plastids where they form (part of) the pigments functioning in photosynthesis, such as chlorophylls, plastoquinones and carotenoids. In addition, plants contain the structurally diverse monoterpenes, sesquiterpenes, diterpenes and triterpenes that function as secondary metabolites with important ecological functions in the interaction of plants with other organisms (Aharoni et al., 2005, Pichersky&Gershenzon, 2002, Staniek et al., 2013, Trapp&Croteau, 2001). Many of the terpenoids have biological activity in humans - as medicine or flavour and fragrance compounds - and therefore there are many efforts to elucidate and engineer the different steps in the biosynthesis of these compounds in plants (Staniek et al., 2013). Figure 1. Compartmentation of terpene biosynthesis in the plant cell. 10 General introduction Terpene biosynthesis Terpenes can be divided into hemiterpenes (C5), monoterpenes (C10), sesquiterpenes (C15), diterpenes (C20), sesterterpenes (C25) and triterpenes (C30), and all are synthesized from the condensation of the five-carbon isoprenoid precursors, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). IPP and DMAPP are synthesized by two independent pathways in different subcellular compartment, the cytosolic mevalonic acid (MVA) pathway and the plastidial methylerythritol phosphate (MEP) pathway (Fig. 1). The isoprenoid precursors are derived from acetyl-CoA in the MVA pathway, and from pyruvate and glyceraldehyde-3-phosphate in the MEP pathway. IPP and DMAPP are condensed by geranyl diphosphate synthase to form geranyl diphosphate (GPP, C10) in the plastids, to form the direct precursor for monoterpene biosynthesis. Geranylgeranyl diphosphate synthase also catalyses a condensation reaction with IPP and DMAPP in the plastid to form geranylgeranly diphosphate (GGPP, C20), which is the precursor for diterpenes and carotenoids. In the cytosol, IPP and DMAPP are condensed by farnesyl diphosphate synthase to form farnesyl diphosphate (FPP, C15) in the cytosol, the direct precursor for sesquiterpenes, triterpenes and sterol biosynthesis (Aharoni et al., 2005, Bohlmann&Keeling, 2008, Nagegowda, 2010, Staniek et al., 2013). In this thesis, I have mainly worked with two sesquiterpene biosynthesis pathways: the multi-enzyme pathway for artemisinin biosynthesis in Artemisia annua and the single enzymatic pathway of caryophyllene biosynthesis of Arabidopsis by caryophyllene synthase (CST) (Fig. 1). Artemisinin is the sesquiterpene lactone endoperoxide produced by the plant A. annua L (Fig. 1). The biosynthesis of artemisinin starts with the cyclization of FPP to amorpha- 4,11-diene by amorphadiene synthase (ADS) (Bouwmeester et al., 1999, Mercke et al., 2000). Amorpha-4,11-diene is subsequently oxidized by amorphadiene oxidase (AMO/CYP71AV1), a P450 enzyme, to artemisinic alcohol, artemisinic aldehyde and artemisinic acid (Ro et al., 2006, Teoh et al., 2006). In the low artermisinin production chemotype, artemisinic acid is likely spontaneously converted to arteanniun B. However, in the high artermisinin production chemotype, artemisinic aldehyde is reduced by artemisinic aldehyde reductase (DBR2), to the intermediate dihydroartemisinic aldehyde which is converted by aldehyde dehydrogenase 1 (ALDH1) to the final intermediate before artemisinin, dihydroartemisinic acid (Bertea et al., 2005, Zhang et al., 2008). The last step, conversion of dihydroartemisinic acid to artemisinin, is likely non-enzymatic and occurs spontaneously through photo-oxidation (Sy&Brown, 2002, Wallaart et al., 1999). Recently, it was shown that dihydroartemisinic aldehyde can also be 11 Chapter 1 converted to dihydroartemisinic alcohol by artemisinic aldehyde reductase (RED1) (Rydén et al., 2010) (Fig. 1). A. annua has two types of trichomes: non-glandular and glandular, secretory, trichomes. It has been shown that the artemisinin biosynthesis genes (ADS, CYP71AV1, DBR2 and ALDH1) are expressed in both apical and sub-apical cells of the glandular secretory trichomes (Olofsson et al., 2012). Hence, dihydroartemisinic acid is produced in the apical and sub-apical cells, while artemisinin is excreted into and stored in the subcuticular space of the glandular secretory trichomes (Duke et al., 1994). Caryophyllene is a common sesquiterpene floral volatile, which is a constituent of more than 50% of the angiosperm families’ floral odor (Knudsen et al., 2006). In Arabidopsis, for example, more than 40% of the total floral volatiles emitted by the flowers is represented by caryophyllene (Chen et al., 2003). The biosynthesis of caryophyllene consists of a single step, catalysed by caryophyllene synthase (CST), which cyclizes FPP to caryophyllene (Fig. 1). CST-promoter:GUS analysis showed that Arabidopsis CST is mainly expressed in the stigma of the flower suggesting that caryophyllene is exclusively released from the stigmas (Chen et al., 2003, Tholl et al., 2005). Metabolic engineering of terpene biosynthesis Because of their commercial and ecological importance, there is strong interest in the possibilities to enhance or chance the production of terpenoids in plants. The assumption was that with some understanding of the biosynthesis pathways terpene biosynthesis can be engineered in the host plant itself or in heterologous expression systems. Engineering of plants Because many of the medicinal plants that contain commercially interesting terpenes often have low yields and are difficult to cultivate it may be of benefit to engineer the pathway of an interesting terpene in an alternative plant host, which is easy to grow and propagate. Tobacco is a fast-growing and high-biomass producing crop species that seems to be a suitable heterologous host for terpene engineering. For instance, monoterpene synthases from lemon (Citrus limon L. Burm. f.) (Lücker et al., 2004); patchoulol synthases from Pogostemon cablin , ADS from A. annua and limonene synthase from lemon (Wu et al., 2006); geraniol synthases from Valeriana officinalis and Lippia dulcis (Dong et al., 2013) have been successfully transformed into tobacco. Furthermore, multiple attempts are under 12 General introduction way to engineer the artemisinin biosynthesis pathway in tobacco (Farhi et al., 2011, Zhang et al., 2011). Artemisinin was indeed detected in N. tabacum after transformation with multiple genes (five genes involved in the mevalonate and artemisinin pathways: HMGR, ADS, DBR2, CYP71AV1 and CPR) combined into a single transformation vector. All of these multiple genes were separated and driven by different promoters and terminators. To increase the availability of precursor for artemisinin production, these multiple genes were targeted to different subcellular compartments, such as the mitochondria for amorphadiene synthase (Farhi et al., 2011). This was the first heterologous host plant overexpressing the artemisinin biosynthetic genes, which can produce artemisinin. However, the amount of the artemisinin produced, 7 μg/g dry weight, is still much lower than A. annua (0.01~1g / g dry weight) and the results have not been confirmed by a peer reviewed publication. Hence, further studies are needed to improve and optimize artemisinin production