*3.1. Promotion of Terpene Biosynthesis by Light Supplements*

After two weeks of light supplementation, the increase of the cyclic monoterpene content in the treated leaves was observed. Especially, BL increased all four monoterpenes (Figure 3): pulegone (362% increase compared to the WL control), menthofuran (285%), menthone (223%), and menthol (389%). RL slightly promoted pulegone (256%), menthofuran (178%), and menthol (197%). These results are consistent with a previous study that showed an increase in fresh weight and essential oils in several mint species (*Mentha piperita*, *Mentha spicata*, etc.) cultivated in a red-blue LED incubator [15]. Another previous report also observed the increase of terpene contents in *Cannabis* plants under RL-BL subcanopy LED lighting [21]. The precursors of all higher plant monoterpene biosynthesis, isopentenyl diphosphate (IPP) or dimethylallyl pyrophosphate (DMAPP), were proposed to be increased by the RL-BL supplement [21]. It is likely to explain our result showing the increase of cyclic monoterpene with either BL or RL supplement in Japanese mint (Figure 3). Interestingly, the accumulation of menthone (229%) or menthofuran (339%) was observed with FR light treatment. Our result is the first report that showed that FR light treatment increased terpenes in mint plants. In natural conditions, long-day treatment to

mint plants showed the accumulation of menthofuran [14]. It indicates that the content of menthofuran is associated with the life cycle of the mint plant. Our result showed that the FR treatment disturbed the plant response to the photoperiod, affecting the metabolism of menthofuran. Furthermore, the reaction to FR indicates that the biosynthesis of these compounds is possibly under the control of phytochromes. In our study, no significant promotion of plant growth was observed during two weeks of LED light supplementation at the fluence rate used. Also, the contents of cyclic monoterpenes shown in Figure 3 were normalized with the weight of fresh leaves. This suggests that the increase of cyclic monoterpenes was not due to the leaf area's expansion. In many *Lamiaceae* plants, GTs in the aerial part of the plant body influence the biosynthesis of secondary metabolites, including terpenes, and store them in a cavity space surrounded by a cuticle. Here, we hypothesized why the terpenes increased after LED treatments as follows: (1) BL treatment increased the density of GTs, and (2) light stimulated the processes of terpene biosynthesis. the life cycle of the mint plant. Our result showed that the FR treatment disturbed the plant response to the photoperiod, affecting the metabolism of menthofuran. Furthermore, the reaction to FR indicates that the biosynthesis of these compounds is possibly under the control of phytochromes. In our study, no significant promotion of plant growth was observed during two weeks of LED light supplementation at the fluence rate used. Also, the contents of cyclic monoterpenes shown in **Figure 3** were normalized with the weight of fresh leaves. This suggests that the increase of cyclic monoterpenes was not due to the leaf area's expansion. In many *Lamiaceae* plants, GTs in the aerial part of the plant body influence the biosynthesis of secondary metabolites, including terpenes, and store them in a cavity space surrounded by a cuticle. Here, we hypothesized why the terpenes increased after LED treatments as follows: (1) BL treatment increased the density of GTs, and (2) light stimulated the processes of terpene biosynthesis.

*Plants* **2021**, *10*, x FOR PEER REVIEW 7 of 15

After two weeks of light supplementation, the increase of the cyclic monoterpene content in the treated leaves was observed. Especially, BL increased all four monoterpenes (**Figure 3**): pulegone (362% increase compared to the WL control), menthofuran (285%), menthone (223%), and menthol (389%). RL slightly promoted pulegone

(256%), menthofuran (178%), and menthol (197%). These results are consistent with a previous study that showed

cultivated in a red-blue LED incubator **[15]**. Another previous report also observed the increase of terpene contents in *Cannabis* plants under RL-BL sub-canopy LED lighting **[21]**. The precursors of all higher plant monoterpene biosynthesis, isopentenyl diphosphate (IPP) or dimethylallyl pyrophosphate (DMAPP), were proposed to be increased by the RL-BL supplement **[21]**. It is likely to explain our result showing the increase of cyclic

an increase in fresh weight and essential oils in several mint species (*Mentha piperita*, *Mentha spicata*, etc.)

monoterpene with either BL or RL supplement in Japanese mint (**Figure 3**). Interestingly, the accumulation of

showed that FR light treatment increased terpenes in mint plants. In natural conditions, long-day treatment to mint plants showed the accumulation of menthofuran **[14]**. It indicates that the content of menthofuran is associated with

**Figure 3.** Modulation of the contents of cyclic monoterpenes in two-week light-supplemented Japanese mint leaves. The contents of pulegone, menthone, menthofuran, and menthol are presented. The values in the graphs indicate the increasing rates compared to the WL control. The measurements were conducted independently six times for biological replicates. Error bars indicate standard deviations from the mean. \* Asterisks represent a significant difference from the WL control in the Mann–Whitney *U* test (*p* < 0.05).
