Regulation of monoterpene accumulation in leaves of peppermint

Abstract

Plants synthesize numerous classes of natural products that accumulate during development and are thought to function as constitutive defenses against herbivores and pathogens. However, little information is available about how the levels of such defenses are regulated. We measured the accumulation of monoterpenes, a model group of constitutive defenses, in peppermint (Mentha x piperita L.) leaves and investigated several physiological processes that could regulate their accumulation: the rate of biosynthesis, the rate of metabolic loss, and the rate of volatilization. Monoterpene accumulation was found to be restricted to leaves of 12 to 20 d of age, the period of maximal leaf expansion. The rate of monoterpene biosynthesis determined by (14)CO(2) incorporation was closely correlated with monoterpene accumulation, as determined by gas chromatographic analysis, and appeared to be the principal factor controlling the monoterpene level of peppermint leaves. No significant catabolic losses of monoterpenes were detected throughout leaf development, and monoterpene volatilization was found to occur at a very low rate, which, on a monthly basis, represented less than 1% of the total pool of stored monoterpenes. The composition of volatilized monoterpenes differed significantly from that of the total plant monoterpene pool, suggesting that these volatilized products may arise from a separate secretory system. With the demonstration that the rate of biosynthesis is the chief process that determines monoterpene accumulation in peppermint, efforts to improve production in this species can now focus on the genes, enzymes, and cell differentiation processes that regulate monoterpene biosynthesis.

Figure 1

Major monoterpene constituents of peppermint leaves.

Figure 2

Changes in monoterpene content (A), leaf weight (B), and rate of monoterpene biosynthesis (C) during peppermint leaf development. Monoterpenes were extracted with diethyl ether and analyzed by gas chromatography. To determine the rate of monoterpene biosynthesis, leaves of various ages were exposed to a 5-min pulse of ¹⁴CO₂, and the incorporation of ¹⁴C into monoterpenes was measured after a 6-h chase period. Each data point is the mean of three to six independent measurements. Bars indicate sd.

Figure 3

Lack of monoterpene turnover in peppermint leaves. Plants were exposed to a 5-min pulse of ¹⁴CO₂, and samples were harvested over the next 6 weeks for determination of monoterpene content. Incorporation of ¹⁴C into monoterpenes (A) did not change significantly (Tukey's studentized range test, P > 0.05) over the time course of the experiment, indicating the lack of detectable monoterpene loss. In contrast, total monoterpene content (B) and leaf weight (C) increased steadily over the time course of the experiment. Each data point represents the mean of at least three independently measured samples, each consisting of a pair of leaves from a single stem. Bars indicate sd.

Figure 4

Comparison of the composition of monoterpenes stored (black bars) and emitted (white bars) by peppermint shoots. Aerial parts of 6-week-old plants were examined. Stored monoterpenes were extracted with diethyl ether and analyzed by gas chromatography. Emitted monoterpenes were collected by headspace sorption from intact plants (see “Materials and Methods” for details). Each value is the mean of five determinations. Key to compounds (in order of elution on gas chromatography, which approximates the range from most to least volatile): 1, α-Pinene; 2, β-pinene; 3, sabinene; 4, myrcene; 5, limonene; 6, 1,8-cineole; 7, menthone; 8, menthofuran; 9, isomenthone; 10, linalool; 11, neomenthol; 12, menthol; 13, pulegone; 14, α-terpineol; and 15, piperitone.