Functional Genomics Reveals That a Compact Terpene Synthase Gene Family Can Account for Terpene Volatile Production in Apple  

Abstract

Terpenes are specialized plant metabolites that act as attractants to pollinators and as defensive compounds against pathogens and herbivores, but they also play an important role in determining the quality of horticultural food products. We show that the genome of cultivated apple (Malus domestica) contains 55 putative terpene synthase (TPS) genes, of which only 10 are predicted to be functional. This low number of predicted functional TPS genes compared with other plant species was supported by the identification of only eight potentially functional TPS enzymes in apple 'Royal Gala' expressed sequence tag databases, including the previously characterized apple (E,E)-a-farnesene synthase. In planta functional characterization of these TPS enzymes showed that they could account for the majority of terpene volatiles produced in cv Royal Gala, including the sesquiterpenes germacrene-D and (E)-b-caryophyllene, the monoterpenes linalool and a-pinene, and the homoterpene (E)-4,8-dimethyl-1,3,7-nonatriene. Relative expression analysis of the TPS genes indicated that floral and vegetative tissues were the primary sites of terpene production in cv Royal Gala. However, production of cv Royal Gala floral-specific terpenes and TPS genes was observed in the fruit of some heritage apple cultivars. Our results suggest that the apple TPS gene family has been shaped by a combination of ancestral and more recent genome-wide duplication events. The relatively small number of functional enzymes suggests that the remaining terpenes produced in floral and vegetative and fruit tissues are maintained under a positive selective pressure, while the small number of terpenes found in the fruit of modern cultivars may be related to commercial breeding strategies.

Natural Variation in Monoterpene Synthesis in Kiwifruit: Transcriptional Regulation of Terpene Synthases by NAC and ETHYLENE-INSENSITIVE3-Like Transcription Factors

“…Primers and vectors used are given in Supplemental Table S2D. Recombinant N-terminal His-tagged proteins were expressed by autoinduction in Escherichia coli BL21 Star (DE3; Invitrogen) and purified by Ni 2+ affinity and size exclusion chromatography as described previously (Nieuwenhuizen et al, 2013). AcTPS1 purified recombinant protein (500 mg) was used to raise a polyclonal antibody in rabbit (AgResearch).…”

“…Volatile terpenes were solvent extracted from eating ripe A. arguta 'Hortgem Tahi' fruit (a3/a4) according to Matich et al (2003). Enantioselective analysis of terpene products was performed as described in Nieuwenhuizen et al (2013).…”

“…Fruit volatile terpenoids have been well characterized in Citrus spp. (Moufida and Marzouk, 2003), mango (Mangifera indica;MacLeod and Pieris, 1984), apple (Malus 3 domestica; Nieuwenhuizen et al, 2013), tomato (Solanum lycopersicum; Buttery et al, 1988), and grape (Vitis vinifera; Battilana et al, 2009;Duchêne et al, 2009). In kiwifruit (Actinidia spp.…”

Biochemistry of apple aroma: A review

“…The catalytic conversion of the terpene precursors is carried out by terpene synthase enzymes. Nieuwenhuizen et al (154) reported that genome of cultivated apple (Malus domestica) contains 55 putative terpene synthase genes, ten of which appear to be functional.…”

“…The acyclic branched sesquiterpene (E,E)-α--farnesene, synthesized predominantly in epidermal and hypodermal cell layers of the fruit (155), is the most associated with ripe apple fruit (154,156,157). However, α-farnesene has also been associated with superfi cial scald in the skin of several apple cultivars (155).…”