In planta and in silico characterization of five sesquiterpene synthases from Vitis vinifera (cv. Shiraz) berries
- 193 Downloads
Five Vitis vinifera sesquiterpene synthases were characterized, two was previously uncharacterized, one being a caryophyllene/cubebene synthase and the other a cadinene synthase. Residue differences with other Vitis sesquiterpene synthases are described.
The biochemical composition of grape berries at harvest can have a profound effect on the varietal character of the wine produced. Sesquiterpenes are an important class of volatile compounds produced in grapes that contribute to the flavor and aroma of wine, making the elucidation of their biosynthetic origin an important field of research. Five cDNAs corresponding to sesquiterpene synthase genes (TPSs) were isolated from Shiraz berries and expressed in planta in Nicotiana benthamiana followed by chemical characterization by GC–MS. Three of the TPS cDNAs were isolated from immature berries and two were isolated from ripe Shiraz berries. Two of the investigated enzymes, TPS26 and TPS27, have been previously investigated by expression in E. coli, and the in planta products generally correspond to these previous studies. The enzyme TPS07 differed by eight amino acids (none of which are in the active site) from germacrene B and D synthase isolated from Gewürztraminer grapes and characterized in vitro. Here in planta characterization of VvShirazTPS07 yielded ylangene, germacrene D and several minor products. Two of the enzymes isolated from immature berries were previously uncharacterized enzymes. VvShirazTPS-Y1 produced cadinene as a major product and at least 17 minor sesquiterpenoid skeletons. The second, VvShirazTPS-Y2, was characterized as a caryophyllene/cubebene synthase, a combination of products not previously reported from a single enzyme. Using in silico methods, we identified residues that could play key roles regarding differences in product formation of these enzymes. The first ring closure that is either a 1,10- or 1,11-ring closure is likely controlled by three neighboring amino acids in helices G1, H2, and J. As for many other investigated TPS enzymes, we also observe that only a few residues can account for radical changes in product formation.
KeywordsSesquiterpene synthase Shiraz Isoprenoid Sesquiterpenes Wine aroma
This research was supported by the European Union Seventh Framework Programme (FP7/2007-2013) under Grant agreement 275422, which supported a Marie Curie International Outgoing Fellowship (DPD). The Danish Strategic Research Council Grant “SPOTLight” supported the work of HTS.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
- Bach SS, Bassard J-É, Andersen-Ranberg J et al (2014) High throughput testing of terpenoid biosynthesis candidate genes using transient expression in Nicotiana benthamiana. In: Rodríguez-Concepción M (ed) Plant isoprenoids, 1153rd edn. Springer, New YorkGoogle Scholar
- Davis EM, Croteau R (2000) Cyclization enzymes in the biosynthesis of monoterpenes, sesquiterpenes, and diterpenes. In: Leeper FJ, Vedera JC (eds) Biosynthesis. Topics in Current Chemistry, vol 209. Springer, Berlin, Heidelberg, pp 53–95Google Scholar
- Drew DP, Rasmussen SK, Avato P, Simonsen HT (2012) A comparison of headspace solid-phase microextraction and classic hydrodistillation for the identification of volatile constituents from Thapsia spp. provides insights into guaianolide biosynthesis in Apiaceae. Phytochem Anal 23:44–51. https://doi.org/10.1002/pca.1323 CrossRefGoogle Scholar
- Köllner TG, Schnee C, Gershenzon J, Degenhardt J (2004) The variability of sesquiterpenes emitted from two Zea mays cultivars is controlled by allelic variation of two terpene synthase genes encoding stereoselective multiple product enzymes. Plant Cell 16:1115–1131. https://doi.org/10.1105/tpc.019877.tive CrossRefPubMedCentralGoogle Scholar
- Martin DM, Aubourg S, Schouwey MB et al (2010) Functional annotation, genome organization and phylogeny of the grapevine (Vitis vinifera) terpene synthase gene family based on genome assembly, FLcDNA cloning, and enzyme assays. BMC Plant Biol 10:226. https://doi.org/10.1186/1471-2229-10-226 CrossRefPubMedCentralGoogle Scholar
- Martin DM, Chiang A, Jo STL et al (2012) Biosynthesis of wine aroma: transcript profiles of hydroxymethylbutenyl diphosphate reductase, geranyl diphosphate synthase, and linalool/nerolidol synthase parallel monoterpenol glycoside accumulation in Gewürztraminer grapes. Planta 236:919–929. https://doi.org/10.1007/s00425-012-1704-0 CrossRefGoogle Scholar