molecules Article Oxidosqualene Cyclase Knock-Down in Latex of Taraxacum koksaghyz Reduces Triterpenes in Roots and Separated Natural Rubber Nicole van Deenen 1, Kristina Unland 2, Dirk Prüfer 1,2 and Christian Schulze Gronover 2,* 1 Institute of Plant Biology and Biotechnology, University of Muenster, Schlossplatz 8, 48143 Muenster, Germany 2 Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Schlossplatz 8, 48143 Muenster, Germany * Correspondence: [email protected]; Tel.: +49(0)-251-83-24998 Received: 19 June 2019; Accepted: 24 July 2019; Published: 25 July 2019 Abstract: In addition to natural rubber (NR), several triterpenes are synthesized in laticifers of the Russian dandelion (Taraxacum koksaghyz). Detailed analysis of NR and resin contents revealed different concentrations of various pentacyclic triterpenes such as α-, β-amyrin and taraxasterol, which strongly affect the mechanical properties of the resulting rubber material. Therefore, the reduction of triterpene content would certainly improve the industrial applications of dandelion NR. We developed T. koksaghyz plants with reduced triterpene contents by tissue-specific downregulation of major laticifer-specific oxidosqualene cyclases (OSCs) by RNA interference, resulting in an almost 67% reduction in the triterpene content of NR. Plants of the T1 generation showed no obvious phenotypic changes and the rubber yield also remained unaffected. Hence, this study will provide a solid basis for subsequent modern breeding programs to develop Russian dandelion plants with low and stable triterpene levels. Keywords: pentacyclic triterpenes; polyisoprenes; Taraxacum koksaghyz; RNA interference; oxidosqualene cyclases 1. Introduction In Russian dandelion (Taraxacum koksaghyz), high concentrations of secondary metabolites such as natural rubber (NR) are found mainly in the latex of root laticifers [1,2]. NR is an important industrial biopolymer that is used for the production of more than 40,000 consumables, including tires and medical devices [3,4]. To date, the main source for high-quality NR is the rubber tree Hevea brasiliensis, but increased raw material demand has led to the development of Russian dandelion as an alternative and sustainable rubber source. T. koksaghyz is a self-incompatible, sexually reproductive plant. Consequently, the resulting offspring is heterogenic [4,5], leading to trait divergence among individual plants. One such example is the highly variable triterpene content of dandelion-derived NK, which negatively affects NR properties [6,7]. For this reason, genetic engineering of the corresponding triterpene biosynthesis could support the development of more uniform accessions with low and stable triterpene contents in their roots. The central isopentenyl diphosphate (IPP) building block for triterpenes as well as for poly(cis-1,4-isoprenes) such as dolichols or the long-chain NR-forming poly(cis-1,4-isoprene) is synthesized by the mevalonate (MVA) pathway in the latex of laticifers (Figure1)[8,9]. Molecules 2019, 24, 2703; doi:10.3390/molecules24152703 www.mdpi.com/journal/molecules Molecules 2019, 24, 2703 2 of 12 Molecules 2019, 24, x FOR PEER REVIEW 2 of 12 . Figure 1. IsoprenoidFigure 1. biosynthesis Isoprenoid biosynthesis pathway pathway in T. koksaghyz in T. koksaghyzlatex. latex.T. koksaghyz T. koksaghyzoxidosqualene oxidosqualenecyclase cyclase 1 1 (TkOSC1) is a multifunctional(TkOSC1) is a multifunctional OSC that catalyses OSC that catalyses the formation the formation of several of several highly highly abundant abundant pentacyclic pentacyclic triterpenes suchtriterpenes as taraxasterol, such as taraxasterol,α-, β-amyrin α-, β-amyrin and lup-19(21)-en-3-ol and lup-19(21)-en-3-ol [9]. [9]. Dashed Dashed arrows arrows indicateindicate multiple enzymemultiple steps. enzyme MVA, steps. mevalonate; MVA, mevalonate; DMAPP, DMAPP, dimethylallyl dimethylallyl diphosphate; diphosphate; IPP, IPP, isopentenyl isopentenyl diphosphate; FPP, farnesyl diphosphate. diphosphate; FPP, farnesyl diphosphate. Oxidosqualene cyclases (OSCs; E.C. 5.4.99) catalyse the cyclisation of 2,3-oxidosqualene [10,11], Oxidosqualenewhich leads cyclases to the (OSCs; production E.C. of 5.4.99) triterpenes, catalyse comp theounds cyclisation of 30 carbon of 2,3-oxidosqualeneatoms, that comprise [nearly10,11 ], which leads to100 the different production scaffolds of in triterpenes, plants [12]. Triterpenes compounds can of be 30 further carbon modified atoms, by that tailoring comprise enzymes nearly via 100 different scaacetylation,ffolds in oxidation plants [or12 by]. Triterpenesadding sugar canchains, be resulting further in modified a huge diversity by tailoring of more enzymes than 20,000 via acetylation, oxidationknown triterpenoids or by adding [11,12]. sugar Due chains,to the cyclisation resulting reaction in a huge type, diversitytwo groups of ofmore triterpenes than can 20,000 be known triterpenoidsformed: [The11, 12first]. Duegroup to is the characterised cyclisation by reaction a chair–boat–chair type, two groupsconformation of triterpenes resulting in can the be production of cycloartenol, which is a phytosterol intermediate [10,11]. Phytosterols function, for formed: The first group is characterised by a chair–boat–chair conformation resulting in the production example, as membrane compounds or signalling molecules, making them important for plant of cycloartenol,development which is and a phytosterol growth [13]. In intermediate the second group, [10, 11a chair–chair–chair]. Phytosterols conformation function, forresults example, in the as membraneformation compounds of pentacyclic or signalling triterpenes molecules, such as lupeol, making β-amyrin them importantand taraxasterol for plantamongdevelopment others [11]. A and growth [13large]. In number the second of pentacyclic group, triterpenes a chair–chair–chair show antimicrobial conformation and antifungal results characteristics in the formation and play a of pentacyclic triterpenesrole in plant such defence. as lupeol, Due toβ -amyrinthese characteristic and taraxasterols, they are among interesting others for [ 11applications]. A large in number food, of pentacycliccosmetics triterpenes and showpharmaceutical antimicrobial industries and [14–16]. antifungal characteristics and play a role in plant Recently, we provided a comprehensive analysis of the triterpene composition in NR extracts defence. Due to these characteristics, they are interesting for applications in food, cosmetics and from T. koksaghyz and identified 13 triterpenes and triterpenoids, also including the so far unknown pharmaceuticalpentacyclic industries compound [14–16]. lup-19(21)-en-3-ol [17]. Furthermore, we isolated and functionally Recently,characterized we provided seven a comprehensive different T. koksaghyz analysis OSCs of ( theTkOSC1-6 triterpene and TkLUP) composition and could in NR demonstrate extracts fromthat T. koksaghyz andTkOSC1 identified is highly 13triterpenes expressed in and laticifers triterpenoids, and represents also a including multifunctional the so OSC far capable unknown of synthesizing pentacyclic compound lup-19(21)-en-3-olat least four of the [17 latex-predominant]. Furthermore, we pentacyclic isolated andtriterpenes functionally (taraxasterol, characterized α-, β-amyrin seven and different lup- T. koksaghyz OSCs19(21)-en-3-ol) (TkOSC1-6 [17,18]and (Figure TkLUP) 1). and could demonstrate that TkOSC1 is highly expressed in In this study, we developed transgenic T. koksaghyz plants with reduced levels of pentacyclic laticifers and representstriterpenes a by multifunctional laticifer-specific OSC downregulation capable of synthesizing of TkOSC1 expression at least four via ofRNA the interference. latex-predominant Lower pentacyclic triterpenesamounts of (taraxasterol, triterpenes wereα-, β also-amyrin evident and in lup-19(21)-en-3-ol)extracted rubber, which [17 ,certainly18] (Figure will1 ).contribute to a In this study,significant we developedimprovement transgenic of the resultingT. koksaghyz material properties.plants with reduced levels of pentacyclic triterpenes by laticifer-specific downregulation of TkOSC1 expression via RNA interference. Lower amounts of triterpenes2. Results were also evident in extracted rubber, which certainly will contribute to a significant improvement2.1. Generation of of theT. koksaghyz resulting OSC material RNAi Plants properties. 2. Results 2.1. Generation of T. koksaghyz OSC RNAi Plants In order to generate T. koksaghyz plants with reduced triterpene levels in the latex, two strategies were applied. For TkOSC1 knock-down, a RNAi construct directed against a 125 bp TkOSC1-specific region was developed and designated as a TkOSC1-RNAi vector (Figure2A). In contrast, a parallel Molecules 2019, Molecules24, 2703 2019, 24, x FOR PEER REVIEW 3 of 12 3 of 12 In order to generate T. koksaghyz plants with reduced triterpene levels in the latex, two strategies were applied. For TkOSC1 knock-down, a RNAi construct directed against a 125 bp TkOSC1-specific knockdownregion of several was developedTkOSCs andshould designated be as achieved a TkOSC1-RNAi by a vector RNAi (Figure vector 2A). containing In contrast, a parallel a 445 bp sequence displaying aknockdown more conserved of several TkOSC regions should of be OSCs achieved (designated by a RNAi vector as containingTkOSC -RNAia 445 bp sequence vector, Figure2a). The expressiondisplaying of the a resulting more conserved constructs region of was OSCs controlled (designated by as theTkOSC predominant-RNAi