Gain and Loss of Fruit Flavor Compounds Produced by Wild and Cultivated Strawberry Species

Abstract

The blends of flavor compounds produced by fruits serve as biological perfumes used to attract living creatures, including humans. They include hundreds of metabolites and vary in their characteristic fruit flavor composition. The molecular mechanisms by which fruit flavor and aroma compounds are gained and lost during evolution and domestication are largely unknown. Here, we report on processes that may have been responsible for the evolution of diversity in strawberry (Fragaria spp) fruit flavor components. Whereas the terpenoid profile of cultivated strawberry species is dominated by the monoterpene linalool and the sesquiterpene nerolidol, fruit of wild strawberry species emit mainly olefinic monoterpenes and myrtenyl acetate, which are not found in the cultivated species. We used cDNA microarray analysis to identify the F. ananassa Nerolidol Synthase1 (FaNES1) gene in cultivated strawberry and showed that the recombinant FaNES1 enzyme produced in Escherichia coli cells is capable of generating both linalool and nerolidol when supplied with geranyl diphosphate (GPP) or farnesyl diphosphate (FPP), respectively. Characterization of additional genes that are very similar to FaNES1 from both the wild and cultivated strawberry species (FaNES2 and F. vesca NES1) showed that only FaNES1 is exclusively present and highly expressed in the fruit of cultivated (octaploid) varieties. It encodes a protein truncated at its N terminus. Green fluorescent protein localization experiments suggest that a change in subcellular localization led to the FaNES1 enzyme encountering both GPP and FPP, allowing it to produce linalool and nerolidol. Conversely, an insertional mutation affected the expression of a terpene synthase gene that differs from that in the cultivated species (termed F. ananassa Pinene Synthase). It encodes an enzyme capable of catalyzing the biosynthesis of the typical wild species monoterpenes, such as a-pinene and b-myrcene, and caused the loss of these compounds in the cultivated strawberries. The loss of a-pinene also further influenced the fruit flavor profile because it was no longer available as a substrate for the production of the downstream compounds myrtenol and myrtenyl acetate. This phenomenon was demonstrated by cloning and characterizing a cytochrome P450 gene (Pinene Hydroxylase) that encodes the enzyme catalyzing the C10 hydroxylation of a-pinene to myrtenol. The findings shed light on the molecular evolutionary mechanisms resulting in different flavor profiles that are eventually selected for in domesticated species.

Variation of Herbivore-Induced Volatile Terpenes among Arabidopsis Ecotypes Depends on Allelic Differences and Subcellular Targeting of Two Terpene Synthases, TPS02 and TPS03

“…Subcellular segregation of homologous bifunctional TPSs in plastids and the cytosol has also been documented in cultivated plants, such as for two linalool/ nerolidol synthases in snapdragon (Nagegowda et al, 2008) and in cultivated strawberry (Aharoni et al, 2004). Our results on enzymes from a noncultivated species expand these findings by showing that differential subcellular targeting of dual-function TPSs is a molecular mechanism of general importance in the natural evolution of intraspecific volatile terpene diversity.…”

Rational Conversion of Substrate and Product Specificity in a Salvia Monoterpene Synthase: Structural Insights into the Evolution of Terpene Synthase Function

“…Although no immediate evolutionary advantage may result from an enzyme of such dual functionality because of the likely absence or inefficiency of sesquiterpene production in the plastids, a subsequent event resulting in the loss of the monoterpenespecific transit peptide could make this gene product a functional sesquiterpene synthase. Evidence that such an event can take place in nature is reported by Aharoni et al (2004), who identified a bifunctional linalool/nerolidol synthase from strawberry (Fragaria spp) (Fa-NES1) that has acquired cytosolic localization due to translation initiation from a downstream ATG after insertion of a stop codon in its transit peptide region.…”

“…With very few exceptions, monoterpene synthases use GPP as a substrate, and only a few have been reported to be able to also use the five-carbon-longer FPP, thus catalyzing the formation of sesquiterpenes (Schnee et al, 2002;Aharoni et al, 2004). Sf-CinS1 does not make sesquiterpenes when presented with FPP as substrate.…”

FaQR, Required for the Biosynthesis of the Strawberry Flavor Compound 4-Hydroxy-2,5-Dimethyl-3(2H)-Furanone, Encodes an Enone Oxidoreductase

“…Only recently, it was shown that the SAAT (for strawberry alcohol acylCoA transferase) and FaNES (for Fragaria 3 ananassa nerolidol synthase) genes are involved in strawberry fruit ester and terpene formation, respectively (Aharoni et al, 2000(Aharoni et al, , 2004 and that FaOMT (for Fragaria 3 ananassa O-methyltransferase) encodes an O-methyltransferase responsible for DMMF biosynthesis Wein et al, 2002). The first indications for the enzymatic formation of HDMF in strawberry fruit were provided by studies demonstrating the correlation of fruit ripening stage and HDMF concentration (Sanz et al, 1995).…”