BackgroundTerpenoids are among the most important constituents of grape flavour and wine bouquet, and serve as useful metabolite markers in viticulture and enology. Based on the initial 8-fold sequencing of a nearly homozygous Pinot noir inbred line, 89 putative terpenoid synthase genes (VvTPS) were predicted by in silico analysis of the grapevine (Vitis vinifera) genome assembly [1]. The finding of this very large VvTPS family, combined with the importance of terpenoid metabolism for the organoleptic properties of grapevine berries and finished wines, prompted a detailed examination of this gene family at the genomic level as well as an investigation into VvTPS biochemical functions.ResultsWe present findings from the analysis of the up-dated 12-fold sequencing and assembly of the grapevine genome that place the number of predicted VvTPS genes at 69 putatively functional VvTPS, 20 partial VvTPS, and 63 VvTPS probable pseudogenes. Gene discovery and annotation included information about gene architecture and chromosomal location. A dense cluster of 45 VvTPS is localized on chromosome 18. Extensive FLcDNA cloning, gene synthesis, and protein expression enabled functional characterization of 39 VvTPS; this is the largest number of functionally characterized TPS for any species reported to date. Of these enzymes, 23 have unique functions and/or phylogenetic locations within the plant TPS gene family. Phylogenetic analyses of the TPS gene family showed that while most VvTPS form species-specific gene clusters, there are several examples of gene orthology with TPS of other plant species, representing perhaps more ancient VvTPS, which have maintained functions independent of speciation.ConclusionsThe highly expanded VvTPS gene family underpins the prominence of terpenoid metabolism in grapevine. We provide a detailed experimental functional annotation of 39 members of this important gene family in grapevine and comprehensive information about gene structure and phylogeny for the entire currently known VvTPS gene family.

Terpenoid volatiles are important information molecules that enable pollinators to locate flowers and may protect reproductive tissues against pathogens or herbivores. Inflorescences of grapevine (Vitis vinifera L.) are composed of tiny green flowers that produce an abundance of sesquiterpenoid volatiles. We demonstrate that male flower parts of grapevines are responsible for sesquiterpenoid floral scent formation. We describe temporal and spatial patterns of biosynthesis and release of floral volatiles throughout the blooming of V. vinifera L. cv. Cabernet Sauvignon. The biosynthesis of sesquiterpene volatiles, which are emitted with a light-dependent diurnal pattern early in the morning at prebloom and bloom, is localized to anthers and, more specifically, within the developing pollen grains. Valencene synthase (VvValCS) enzyme activity, which produces the major sesquiterpene volatiles of grapevine flowers, is present in anthers. VvValCS transcripts are most abundant in flowers at prebloom stages. Western blot analysis identified VvValCS protein in anthers, and in situ immunolabeling located VvValCS protein in pollen grains during bloom. Histochemical staining, as well as immunolabeling analysis by fluorescent microscopy and transmission electron microscopy, indicated that VvValCS localizes close to lipid bodies within the maturing microspore.anthers ͉ floral scent ͉ flower development ͉ sesquiterpenes ͉ terpenoid biosynthesis A lthough the release of volatiles, including aliphatics, terpenoids, and phenylpropanoids, from anthers and/or pollen has been reported (1), the molecular mechanisms and localization of pollen volatile formation remain uncertain. Pollen volatiles serve as attractants for pollinators and also may function to deter herbivores and defend against pathogens (2). They can be distinct from the scents of other floral organs and may decrease after pollination, thereby advertising the pollen of unvisited flowers to pollinators (1, 3). Pollen volatiles are thought to be localized to the pollenkitt, a waxy substance localized to the grooves in the exine, which provides protection against pathogens, desiccation, or UV light and assists in pollen-pistil interactions (1, 4). Various nonvolatile compounds of the pollenkitt and sporopollenin are deposited on the surface of microspores by tapetum cells (4). Although it has been postulated that the tapetum also produces volatiles (1, 5), this has not been established experimentally.Grapevines produce dense panicles of small flowers with tiny petals fused into a cap (Fig. 1). Most cultivated grapevines have perfect hermaphroditic flowers and are thought to be at least partially autogamous or cleistogamous (6), whereas their wild relatives are dioecious and have functionally pistillate or staminate flowers that require insect pollinators for fertilization (7,8). The composition of floral volatiles of several cultivated grapevine varieties has been described (9-11), but little else is known about the molecular biochemistry of grapevine floral scent. Research...

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