**1. Introduction**

Secondary metabolites are chemical products enzymatically converted from primary metabolites in plants. Plants use these compounds to adapt to their environment, for example, defense against pathogens or insect attacks, or other stresses. In human history, secondary metabolites have also been an essential source of medicines [1]. Although the development of modern chemistry enables us to synthesize a broad range of chemical compounds, many plant secondary metabolites, especially terpenes, are still considered important pharmaceutical materials. This is because it is convenient to use plant-derived compounds as synthetic starting materials to obtain the desired molecules. Thus, significant efforts have been made to acquire valuable secondary metabolites [2,3].

A glandular trichome (GT) is a plant-specific storage organ distributed in the aerial part of the plant body. In mint plants, GTs play an essential role as a tiny cell factory to synthesize and accumulate secondary metabolites. In terms of biotechnological or pharmacological interests, the number of studies on plant GTs has increased recently [4,5]. Transcriptomic analysis was conducted to elucidate the regulation of GT-specific terpene biosynthesis, for example, in spearmint [6] and *Artemisia* plants [7]. An essential oil generated catalytically in GTs is susceptible to environmental fluctuations or stresses [8]. Particularly, an increase in the terpene contents after various abiotic stress treatments in medicinal plants was confirmed [9].

**Citation:** Ueda, T.; Murata, M.; Yokawa, K. Single Wavelengths of LED Light Supplement Promote the Biosynthesis of Major Cyclic Monoterpenes in Japanese Mint. *Plants* **2021**, *10*, 1420. https:// doi.org/10.3390/plants10071420

Academic Editors: Valeria Cavallaro and Rosario Muleo

Received: 21 May 2021 Accepted: 6 July 2021 Published: 12 July 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Mint plants belonging to the *Mentha* genus are the most famous herbs that have been used in the past. A major terpene produced by the plant is menthol. Menthol causes a sensation of coolness through the direct reaction with the transient receptor potential melastatin 8 (TRPM8) channel [10]. Menthol-containing essential oil is used extensively for many purposes. Menthol has many biological actions, and its antifungal activity is a well-known function [11]. In addition to menthol, mint plants produce cyclic monoterpenes, for example, pulegone, menthone (the intermediates in menthol biosynthesis), and menthofuran (Figure 1). The balance of these contents features an odor of the mint leaves, and the growth environment influences the biosynthesis of the compounds. used in the past. A major terpene produced by the plant is menthol. Menthol causes a sensation of coolness through the direct reaction with the transient receptor potential melastatin 8 (TRPM8) channel [10]. Menthol-containing essential oil is used extensively for many purposes. Menthol has many biological actions, and its antifungal activity is a wellknown function [11]. In addition to menthol, mint plants produce cyclic monoterpenes, for example, pulegone, menthone (the intermediates in menthol biosynthesis), and menthofuran (Figure 1). The balance of these contents features an odor of the mint leaves, and the growth environment influences the biosynthesis of the compounds.

erated catalytically in GTs is susceptible to environmental fluctuations or stresses [8]. Particularly, an increase in the terpene contents after various abiotic stress treatments in me-

Mint plants belonging to the *Mentha* genus are the most famous herbs that have been

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dicinal plants was confirmed [9].

**Figure 1.** Cyclic monoterpene 12 synthesized in glandular trichomes in Japanese mint. These four molecules are the major components of Japanese mint flavor. PR: pulegone reductase; MR: (−)-menthol reductase; MFS: menthofuran synthase. **Figure 1.** Cyclic monoterpene 12 synthesized in glandular trichomes in Japanese mint. These four molecules are the major components of Japanese mint flavor. PR: pulegone reductase; MR: (−)-menthol reductase; MFS: menthofuran synthase.

> Light is one of the physical factors necessary for plant growth, development, and metabolism. Many studies have focused on light and changes in secondary metabolites, such as anthocyanins, carotenoids, and flavanols, under the control of photoreceptors [12]. The light effect on monoterpene metabolism of mint plants has long been studied [13]. It Light is one of the physical factors necessary for plant growth, development, and metabolism. Many studies have focused on light and changes in secondary metabolites, such as anthocyanins, carotenoids, and flavanols, under the control of photoreceptors [12]. The light effect on monoterpene metabolism of mint plants has long been studied [13]. It was assessed that a short-day photoperiod treatment for three mint species significantly increased the oil content [14]. In a controlled light environment using light-emitting diodes (LED), red LED was highly effective in increasing the oil content in *M. piperita*; blue and white LEDs were also effective [15]. However, it is still unclear which wavelength of light affects both the contents and composition of monoterpenes produced by mint plants.

Here, we analyzed the contents of four major cyclic monoterpenes in a Japanese mint plant cultivated under single-wavelength supplementation of blue, red, or far-red LED light. The number of GTs on the growing mint leaves was counted and compared between the treatments. The possible physiological mechanism of light supplementation on terpene biosynthesis was also discussed. Here, we analyzed the contents of four major cyclic monoterpenes in a Japanese mint plant cultivated under single-wavelength supplementation of blue, red, or far-red LED light. The number of GTs on the growing mint leaves was counted and compared between the treatments. The possible physiological mechanism of light supplementation on terpene biosynthesis was also discussed.

was assessed that a short-day photoperiod treatment for three mint species significantly increased the oil content [14]. In a controlled light environment using light-emitting diodes (LED), red LED was highly effective in increasing the oil content in *M. piperita*; blue and white LEDs were also effective [15]. However, it is still unclear which wavelength of light affects both the contents and composition of monoterpenes produced by mint plants.

#### **2. Materials and Methods 2. Materials and Methods**

#### *2.1. Plant Preparation and Growth Conditions 2.1. Plant Preparation and Growth Conditions*

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A rootstock of the major Japanese mint cultivar "HOKKAI JM 23" (*Mentha canadensis* L.) was obtained from the National Agriculture and Food Research Organization Genebank, Tsukuba, Japan (collection ID: JP176265). The plants were first recovered from the rootstock and grown in commercial soil. Fourteen young shoots were harvested from a well-grown mint plant by stem cutting at the position below the fourth leaf. The cuttings were incubated until adventitious roots were generated in distilled water for nine days at room temperature under a 16 h/8 h light/dark cycle at 23 ◦C. The young plants were then placed in a commercially available hydroponic cultivating system, as shown in Figure 2A (Green Farm, U-ing, Osaka, Japan). The box-shaped system was equipped with programmable white LED lighting, a ventilator, and automatic water flow/aeration. For acclimation to the new environment, the plants were pre-grown for one week with distilled water. At the beginning of the experiment, the hydroponic water tank was filled with 4 L of standard Hoagland's cultivating solution (2.5 mM KNO3, 1.25 mM Ca(NO3)2·4H2O, 0.5 mM NH4NO3, 1 mM MgSo4·7H2O, 0.25 mM KH2PO4, 25 mM NaFe(III)EDTA, 23 mM H3BO3, 4.55 mM MnCl2·4H2O, 0.39 mM ZnSO4·7H2O, 0.1 mM CuSO4·5H2O, and 0.25 mM Na2MoO4·2H2O; FUJIFILM Wako Chemicals, Osaka, Japan), and the pH of the solution was measured daily using a portable pH meter (LAQUAtwin pH-33B, HORIBA, Kyoto, Japan). The pH was adjusted to 6.2 using 1 M 2-morpholinoethanesulfonic acid (Dojindo, Kumamoto, Japan) buffer, and 5 M NaOH was used every two days [16]. A rootstock of the major Japanese mint cultivar "HOKKAI JM 23" (*Mentha canadensis* L.) was obtained from the National Agriculture and Food Research Organization Genebank, Tsukuba, Japan (collection ID: JP176265). The plants were first recovered from the rootstock and grown in commercial soil. Fourteen young shoots were harvested from a well-grown mint plant by stem cutting at the position below the fourth leaf. The cuttings were incubated until adventitious roots were generated in distilled water for nine days at room temperature under a 16 h/8 h light/dark cycle at 23 °C. The young plants were then placed in a commercially available hydroponic cultivating system, as shown in Figure 2A (Green Farm, U-ing, Osaka, Japan). The box-shaped system was equipped with programmable white LED lighting, a ventilator, and automatic water flow/aeration. For acclimation to the new environment, the plants were pre-grown for one week with distilled water. At the beginning of the experiment, the hydroponic water tank was filled with 4 L of standard Hoagland's cultivating solution (2.5 mM KNO3, 1.25 mM Ca(NO3)2・4H2O, 0.5 mM NH4NO3, 1 mM MgSo4・7H2O, 0.25 mM KH2PO4, 25 mM NaFe(Ⅲ)EDTA, 23 mM H3BO3, 4.55 mM MnCl2・4H2O, 0.39 mM ZnSO4・7H2O, 0.1 mM CuSO4・5H2O, and 0.25 mM Na2MoO4・2H2O; FUJIFILM Wako Chemicals, Osaka, Japan), and the pH of the solution was measured daily using a portable pH meter (LAQUAtwin pH-33B, HORIBA, Kyoto, Japan). The pH was adjusted to 6.2 using 1 M 2-morpholinoethanesulfonic acid (Dojindo, Kumamoto, Japan) buffer, and 5 M NaOH was used every two days [16].

**Figure 2.** Hydroponic cultivation of Japanese mint and LED supplement. (**A**) A commercially available hydroponic system was modified for the light-supplementation experiment. The system is semi-closed and automatic. (**B**) Three light spectra were emitted from three LED sources used in the study: blue light (BL), red light (RL), and far-red (FR). **Figure 2.** Hydroponic cultivation of Japanese mint and LED supplement. (**A**) A commercially available hydroponic system was modified for the light-supplementation experiment. The system is semi-closed and automatic. (**B**) Three light spectra were emitted from three LED sources used in the study: blue light (BL), red light (RL), and far-red (FR).
