Differential incorporation of 1-deoxy-d-xylulose into (3S)-linalool and geraniol in grape berry exocarp and mesocarp

Abstract

In vivo feeding experiments with [5,5-²H₂]mevalonic acid lactone (MVL) and [5,5-²H₂]-1-deoxy-d-xylulose (DOX) indicate that the novel mevalonate-independent 1-deoxy- d-xylulose 5-phosphate/2C-methyl- d-erythritol 4-phosphate (DOXP/MEP) pathway is the dominant metabolic route for monoterpene biosynthesis in grape berry exocarp and mesocarp and in grape leaves. The highly uneven distribution of the monoterpene alcohols (3S)-linalool and geraniol between leaves, berry exocarp and berry mesocarp can be attributed to a compartmentation of monoterpene metabolism. In grape berries incorporation of [5,5-²H₂]-DOX into geraniol is mainly restricted to the exocarp, whereas (3S)-linalool biosynthesis can be detected in exocarp as well as in mesocarp tissue. The results demonstrate that grape berries exhibit an autonomic monoterpene biosynthesis via the novel DOXP/MEP route throughout the ripening process.

Introduction

Monoterpenes are biologically active molecules that belong to the structurally diverse group of isoprenoids. In higher plants, monoterpenes participate in a wide variety of functions and are synthesized and stored in specialized structures like glandular trichomes in several essential oil producing plants (Wise and Croteau, 1999). Numerous nonglandular plants, including many crop and ornamental species, produce at least trace levels of monoterpenes, some of which are components of herbivore or pathogen defense systems (Boland et al., 1998). In flowers volatile monoterpenes act as pollinating insect attractants (Raguso and Pichersky, 1999) and the odor impression of several edible fruits is determined by monoterpenes (Rouseff and Leahy, 1995). Particularly linalool has been shown to contribute to the characteristic varietal aroma of grapes of Vitis vinifera cultivars Muscat and White Riesling (Ebeler, 2001). In addition to the free odour-producing forms of monoterpenes, several glycosidically bound forms of monoterpenes have been described. Upon chemical or enzymatic hydrolysis these bound monoterpenes can contribute significantly to the characteristic floral aroma in Muscat and related aromatic grape varieties. These findings have been a major stimulus to research on free and bound monoterpenoids in grapes, especially on their structure elucidation, their distribution within the grape, and their changes in levels during grape maturation and vinification (Strauss et al., 1986). However, relatively little is known about the biosynthesis of monoterpenoids in V. vinifera. Free and bound monoterpenes were detected in blades and petioles of vine leaves as well as in the exocarp and mesocarp of berries (Gunata et al., 1985, Gunata et al., 1986). The principal biosynthetic capability and contribution of each of these organs or tissues to the total biosynthesis of monoterpenes in V. vinifera is still unclear and it has been assumed that the bulk of monoterpenes is synthesized in the leaf blade and transported to the berries in glycosidically bound form (Gunata et al., 1986). Feeding experiments with the cultivar Muscat of Alexandria with ¹⁴C-labelled mevalonic acid indicated that monoterpene glycosides were translocated from the leaves to the berries (Shoseyov, 1988). Nevertheless, it could be shown by approach grafting experiments that biosynthesis of monoterpene glycosides in the grape berry is independent of other parts of the vine and is determined by the genotype of the grape bunch (Gholami et al., 1995). In support of this finding is the demonstration of glucosidation activity in detached grape berries grown in vitro (Bravdo et al., 1990). This points to an autonomy of grape berries concerning terpenoid metabolism and, indeed, plastids isolated from cell suspension cultures of cv. Muscat de Frontignan berries are able to import isopentenyl pyrophosphate (IPP) which is incorporated into geranyl diphosphate by a geranyl-diphosphate synthase located within the plastids (Feron et al., 1990, Soler et al., 1993, Clastre et al., 1993). However, the cell suspension cultures used in these studies did not accumulate detectable amounts of monoterpenoids (as it is frequently observed in plant cell cultures of essential oil accumulating plants due to an increased activity of catabolic enzymes (Falk et al., 1990)) and the principal biosynthesis and origin of IPP in grapes remained unclear. Thus, the question of how monoterpene metabolism is compartmentalized between and within different tissues of complex structures such as fruit is largely unanswered. An immunohistochemical study of the compartmentation of sugar, organic acid and amino acid metabolism during the development of the flesh and seeds of grape berries has shown that the distribution of the corresponding enzymatic activities was extremely heterogeneous even in apparently structurally homogeneous tissues (Famiani et al., 2000).

Against this background it is important to note that recently a mevalonate-independent pathway has been discovered in bacteria, algae and plants that leads to the formation of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), the two basic precursors of monoterpenoids (for reviews see Lichtenthaler et al., 1997, Lichtenthaler, 1999, Lichtenthaler, 2000, Rohmer, 1999, Eisenreich et al., 2001). According to the key intermediates 1-deoxyxylulose-5-phosphate (DOXP) and 2C-methyl-d-erythritol-4-phosphate (MEP) this novel route is called the DOXP/MEP pathway and operates in the plastids of higher plants where hemiterpenes, monoterpenes, diterpenes and carotenoids are formed. Isoprenoids synthesized in the cytoplasma and mitochondria like sterols and sesquiterpenes are formed predominately via the classical mevalonate route (MVA). However, the compartmental separation of the two pathways is not absolute and the extent of this crosstalk depends on the species and the physiological conditions (Arigoni et al., 1997, Piel et al., 1998, Adam et al., 1999). It is still unclear to which extent the MVA and DOXP/MEP route are utilized for monoterpenoid biosynthesis in V. vinifera and to which extent the demonstrated uptake of IPP in isolated plastids in vitro contributes to the biosynthesis in vivo. Therefore, the biosynthesis of monoterpenes in V. vinifera was investigated by in vivo feeding experiments using different berry tissues and deuterium labelled DOX and MVL with subsequent enantioselective multidimensional GC/MS analysis of the formed metabolites. The results demonstrate that in grape berry exocarp and mesocarp as well as in grape leaves the novel DOXP/MEP route is almost exclusively utilized for monoterpene biosynthesis. This localization of monoterpene biosynthesis in grape berry is an essential prerequisite for the selection of suitable grape tissues for more detailed studies on isolation and characterization of enzymes involved in monoterpene metabolism.