Biosynthesis of the monoterpenes limonene and carvone in the fruit of caraway. I. Demonstration Of enzyme activities and their changes with development

Abstract

The biosynthesis of the monoterpenes limonene and carvone in the fruit of caraway (Carum carvi L.) proceeds from geranyl diphosphate via a three-step pathway. First, geranyl diphosphate is cyclized to (+)-limonene by a monoterpene synthase. Second, this intermediate is stored in the essential oil ducts without further metabolism or is converted by limonene-6-hydroxylase to (+)-trans-carveol. Third, (+)-trans-carveol is oxidized by a dehydrogenase to (+)-carvone. To investigate the regulation of monoterpene formation in caraway, we measured the time course of limonene and carvone accumulation during fruit development and compared it with monoterpene biosynthesis from [U-14C]Suc and the changes in the activities of the three enzymes. The activities of the enzymes explain the profiles of monoterpene accumulation quite well, with limonene-6-hydroxylase playing a pivotal role in controlling the nature of the end product. In the youngest stages, when limonene-6-hydroxylase is undetectable, only limonene was accumulating in appreciable levels. The appearance of limonene-6-hydroxylase correlates closely with the onset of carvone accumulation. At later stages of fruit development, the activities of all three enzymes declined to low levels. Although this correlates closely with a decrease in monoterpene accumulation, the latter may also be the result of competition with other pathways for substrate.

Figure 1

Biosynthetic pathway of (+)-limonene and (+)-carvone in fruits of caraway.

Figure 2

GC-MS analysis (in selected ion-monitoring mode) of products of limonene synthase assay using octakis-(6-O-methyl-2,3-di-O-pentyl)-γ-cyclodextrin as the chiral stationary phase. Ions monitored: for limonene, m/z 68, 93, and 136; for carvone, m/z 82, 108, and 150; for trans-carveol, m/z 84, 109, and 152; and for cis-carveol, m/z 84, 109, and 134. A, Reference compounds. B, Product of partially purified limonene synthase. For further details, see Methods.

Figure 3

GC-MS analyses (in selected ion-monitoring mode) of products of carveol dehydrogenase assays using octakis-(6-O-methyl-2,3-di-O-pentyl)-γ-cyclodextrin as the chiral stationary phase. Ions monitored: for limonene, m/z 68, 93, and 136; for carvone, m/z 82, 108, and 150; for trans-carveol, m/z 84, 109, and 152; for cis-carveol, m/z 84, 109, and 134. A, Reference compounds. B through E, Products of carveol dehydrogenase activity with a (+)/(−)-trans-carveol mixture (B), a (+)/(−)-cis-carveol mixture (C), (−)-trans-carveol (D), and (−)-cis-carveol (E) as the substrates. For further details, see Methods.

Figure 4

Accumulation of limonene (○), carvone (▵), and fatty acids (□) (A), and changes in activities of limonene synthase (○), limonene-6-hydroxylase (▵), and trans-carveol dehydrogenase (□) (B) during development of fruits of annual caraway. Dotted lines in A indicate sigmoidal curves of the equation: y = a + b/(1 + exp[−(x − c)/d]). r² values are 0.52 for limonene, 0.86 for carvone, and 0.99 for fatty acids. The dotted line in B indicates the carvone accumulation rate, which was calculated by taking the 1st-order derivative of the sigmoidal-curve fit to the carvone content data as shown in A. Data for A were obtained from pooled samples of 0.5 g of fruits of each developmental stage containing 40 to 130 individual fruits. Data for B were obtained from pooled samples of 1.0 g of fruits. Enzyme assays were carried out in triplicate (limonene synthase) or in duplicate (other activities) under linear conditions. Error bars indicate se.