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.2000 Jan;122(1):215-24.
doi: 10.1104/pp.122.1.215.

Developmental regulation of monoterpene biosynthesis in the glandular trichomes of peppermint

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Developmental regulation of monoterpene biosynthesis in the glandular trichomes of peppermint

M E McConkey et al. Plant Physiol.2000 Jan.

Abstract

Monoterpene production in peppermint (Mentha x piperita L.) glandular trichomes is determined by the rate of biosynthesis, as determined by (14)CO(2) incorporation, and is restricted to leaves 12 to 20 d of age. Using oil glands isolated from peppermint leaves of different ages, in vitro assay of the eight sequential enzymes responsible for the biosynthesis of the principal monoterpene (-)-menthol indicated that all but one biosynthetic enzyme had a very similar developmental profile. Activities were highest in leaves 12 to 20 d of age, with a sharp peak centered at 15 d. The exception, (-)-menthone reductase, the last enzyme of the pathway, exhibited a later peak of activity, which was centered at approximately 21 d. The correlation between in vitro enzyme activity and the rate of biosynthesis measured in vivo suggests that monoterpene formation is controlled mainly by the coordinately regulated activity of the relevant biosynthetic enzymes. Developmental immunoblotting of limonene synthase, which catalyzes the committed step of the pathway, demonstrated a direct correlation between enzyme activity and enzyme protein, suggesting that the dynamic time course for the remaining pathway enzyme activities also reflects the corresponding protein levels. RNA-blot analyses indicated that the genes encoding enzymes of the early pathway steps are transcriptionally activated in a coordinated fashion, with a time course superimpossible with activity measurements and immunoblot data. These results demonstrating coincidental temporal changes in enzyme activities, enzyme protein level, and steady-state transcript abundances indicate that most of the monoterpene biosynthetic enzymes in peppermint are developmentally regulated at the level of gene expression.

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Figures

Figure 1
Figure 1
The principal pathway for monoterpene biosynthesis in peppermint. The responsible enzymes are: (1) geranyl diphosphate synthase, (2) (4S)-(−)-limonene synthase, (3) Cyt P450 (−)-limonene-3-hydroxylase, (4) (−)-trans-isopiperitenol dehydrogenase, (5) (−)-isopiperitenone reductase, (6) (+)-cis-isopulegone isomerase, (7) (+)-pulegone reductase, and (8) (−)-menthone reductase.
Figure 2
Figure 2
Changes in overall biosynthetic rate, biosynthetic enzyme activities, and monoterpene content of peppermint as a function of leaf development. A, Changes in monoterpene biosynthetic rate measured by14CO2 incorporation (♦), total leaf monoterpene content (●), and leaf weight (○) as a function of development (see Gershenzon et al., 1999). Peppermint leaves are fully expanded at 21 d. B, Changes in the activities of geranyl diphosphate synthase (▪), (−)-limonene synthase (○), and (−)-limonene-3-hydroxylase (×102) (▴). C, Changes in the activities of (−)-trans-isopiperitenol dehydrogenase (×10−1) (▪), (−)-isopiperitenone reductase (○), and (+)-cis-isopulegone isomerase (▴). D, Changes in the activities of (+)-pulegone reductase (▪) and (−)-menthone reductase (×102) (○). E, Changes in the essential oil content of (−)-menthone (○) and (−)-menthol (●) as a function of leaf development (see Gershenzon et al., 1999). (−)-Menthone is also converted to small amounts of (+)-neomenthol and menthyl esters; these data are not shown. Thesds are indicated for (+)-pulegone reductase activity data points (D); these error limits are typical for the other enzyme assays but are not plotted for the sake of clarity of the presentation. The complete data set, with error analysis, can be accessed at websitewww.wsu.edu/∼ibc/faculty/rc.html. Enzyme assays were standardized based on the number of oil glands and, by using normalized gland count data, were converted to a per-leaf basis.
Figure 3
Figure 3
Temporal regulation of (−)-limonene synthase in the glandular trichomes of peppermint leaves as a function of development. A, The absolute amounts of limonene synthase protein determined by immunoblotting of extracts from the secretory cells of peppermint leaves of different ages. B, In vitro limonene synthase activity measured in soluble protein extracts from the glandular trichomes of peppermint leaves of different ages (○), the corresponding relative amounts of limonene synthase protein (♦) (from the immunoblot data of A), and the corresponding relative steady-state levels of limonene synthase transcripts (▴) (from the northern-blot data of Fig. 4). Peppermint leaves are fully expanded at 21 d.
Figure 4
Figure 4
Temporal changes in steady-state mRNA levels for monoterpene biosynthetic enzymes in the glandular trichomes of peppermint leaves as a function of development. For each time point, total glandular RNA was isolated, separated on a denaturing agarose gel (5 μg/lane), blotted, and hybridized to radiolabeled DNA probes directed toward geranyl diphosphate synthase (B), (−)-limonene synthase (C), Cyt P450 limonene-3-hydroxylase (D), and NADPH-dependent Cyt P450 reductase (E). A, Ribosomal bands visualized with ethidium bromide that were used to verify loading of equal amounts of total RNA. F, Plot of the relative amounts of message determined by northern-blot analysis for geranyl diphosphate synthase (●), limonene synthase (○), limonene-3-hydroxylase (♦), and Cyt P450 reductase (⋄).
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