Manipulation of flavour and aroma compound sequestration and release using a glycosyltransferase with specificity for terpene alcohols

Abstract

SUMMARYGlycosides are an important potential source of aroma and flavour compounds for release as volatiles in flowers and fruit. The production of glycosides is catalysed by UDP-glycosyltransferases (UGTs) that mediate the transfer of an activated nucleotide sugar to acceptor aglycones. A screen of UGTs expressed in kiwifruit (Actinidia deliciosa) identified the gene AdGT4 which was highly expressed in floral tissues and whose expression increased during fruit ripening. Recombinant AdGT4 enzyme glycosylated a range of terpenes and primary alcohols found as glycosides in ripe kiwifruit. Two of the enzyme's preferred alcohol aglycones, hexanol and (Z)-hex-3-enol, contribute strongly to the 'grassy-green' aroma notes of ripe kiwifruit and other fruit including tomato and olive. Transient over-expression of AdGT4 in tobacco leaves showed that enzyme was able to glycosylate geraniol and octan-3-ol in planta whilst transient expression of an RNAi construct in Actinidia eriantha fruit reduced accumulation of a range of terpene glycosides. Stable over-expression of AdGT4 in transgenic petunia resulted in increased sequestration of hexanol and other alcohols in the flowers. Transgenic tomato fruit stably over-expressing AdGT4 showed changes in both the sequestration and release of a range of alcohols including 3-methylbutanol, hexanol and geraniol. Sequestration occurred at all stages of fruit ripening. Ripe fruit sequestering high levels of glycosides were identified as having a less intense, earthier aroma in a sensory trial. These results demonstrate the importance of UGTs in sequestering key volatile compounds in planta and suggest a future approach to enhancing aromas and flavours in flowers and during fruit ripening.

Volatile Glycosylation in Tea Plants: Sequential Glycosylations for the Biosynthesis of Aroma β-Primeverosides Are Catalyzed by Two Camellia sinensis Glycosyltransferases

“…These data suggest that geraniol and linalool are separately biosynthesized in leaves and stems of tea plants, respectively, and that members of an unknown class of glucosyltransferases specifically catalyze the glucosylation of linalool and linalyl-glc. This notion is supported by recent reports that kiwi AdGT4 and grape VvGT14, which are UGT85-class glucosyltransferases for volatiles, also showed little enzyme activity toward linalool, in contrast to their preferred substrate, geraniol (Bönisch et al, 2014b;Yauk et al, 2014). Since CsGT1, AdGT4, and VvGT14 commonly exhibited substrate preference to the primary alcohols geraniol and (Z)-3-hexenol over the tertiary alcohol linalool, there might be a structural feature of substrate recognition that is shared by these enzymes.…”

“…S2 and S3; Bönisch et al, 2014a;Yauk et al, 2014). The estimated apparent K m of CsGT1 for geraniol (44 mM) was comparable to those of other volatile UGTs isolated from kiwi and grape [AdGT4 for (Z)-3-hexenol, 57 mM; VvGt14, VvGT15a, VvGT15b, VvGT15c, and VvGT16 for citronellol, 9, 29, 55, 20, and 108 mM, respectively].…”

“…2). CsGT1 belongs to the UGT85 family and shows similarities in structure and function to kiwi AdGT4 and grape VvGT14, VvGT16, VvGT17, and VvGT19 that were recently shown to catalyze the glucosylation of small terpenes and primary alcohols that are accumulated as glycosides in ripe kiwi and grape (Bönisch et al, 2014a(Bönisch et al, , 2014bYauk et al, 2014). It is noteworthy that AdGT4, VvGT14, and VvGT16 also have broad substrate specificities toward volatile sugar acceptors (Bönisch et al, 2014a;Yauk et al, 2014).…”

“…CsGT1 belongs to the UGT85 family and shows similarities in structure and function to kiwi AdGT4 and grape VvGT14, VvGT16, VvGT17, and VvGT19 that were recently shown to catalyze the glucosylation of small terpenes and primary alcohols that are accumulated as glycosides in ripe kiwi and grape (Bönisch et al, 2014a(Bönisch et al, , 2014bYauk et al, 2014). It is noteworthy that AdGT4, VvGT14, and VvGT16 also have broad substrate specificities toward volatile sugar acceptors (Bönisch et al, 2014a;Yauk et al, 2014). Taken together, our data and these studies support the notion that the machinery behind the glucosylation of monoterpenes and primary alcohols is fairly conserved among phylogenetically discrete various plant species (tea plant, kiwi, grapevine, Arabidopsis, snapdragon, and sweet potato; Supplemental…”

“…These UGTs were found to be highly expressed in young tea leaves, ripe kiwi, and grape, where aroma glucosides are dominantly accumulated. Furthermore, the concentrations of substrates, (Z)-3-hexenol in ripe kiwi, citronellol in grape, and geraniol in young tea leaves, were determined to be at least 0.8, 1.0, and 5.8 mM, respectively (Bönisch et al, 2014a;Yauk et al, 2014). Their relatively lower substrate specificity toward volatile sugar acceptors, compared with those of previously characterized nonvolatile UGTs, might reflect their promiscuous biochemical nature, recognizing structurally diverse substrates.…”