*3.3. Photosynthesis Was Not Boosted by LED Light Supplements*

*3.3. Photosynthesis Was Not Boosted by LED Light Supplements*  In mint plants, monoterpenes are biosynthesized from isopentenyl pyrophosphate and dimethylallyl pyrophosphate, which are provided by the plastidial methylerythritol phosphate (MEP) pathway [27]. The initial compound of the MEP pathway is pyruvic acid, which is supplied from sugars as a photosynthetic by-product. Next, we confirmed whether the photosynthetic activity was the source of monoterpene biosynthesis under light supplementation. We measured chlorophyll contents in the two-week light-treated mint leaves and compared them among the different light wavelengths. Similar to GT density results, BL and FR treatments significantly increased chlorophyll but not anthocyanin (Figure 6A). Anthocyanin biosynthesis was induced by BL treatment to plants. In *Arabidopsis thaliana*, anthocyanin biosynthesis was stimulated by 2.5 W/m2 of BL (approximately 11 µmol/m2/s) [28], which is higher than the intensity we used in this study (6.7 µmol/m2/s). We considered that the increase in the chlorophyll contents induced by either BL or FR supplements could help the biosynthesis of cyclic monoterpenes in GTs. To check the contribution of chlorophyll to photosynthesis, we quantified the quantum yield of In mint plants, monoterpenes are biosynthesized from isopentenyl pyrophosphate and dimethylallyl pyrophosphate, which are provided by the plastidial methylerythritol phosphate (MEP) pathway [27]. The initial compound of the MEP pathway is pyruvic acid, which is supplied from sugars as a photosynthetic by-product. Next, we confirmed whether the photosynthetic activity was the source of monoterpene biosynthesis under light supplementation. We measured chlorophyll contents in the two-week light-treated mint leaves and compared them among the different light wavelengths. Similar to GT density results, BL and FR treatments significantly increased chlorophyll but not anthocyanin (Figure 6A). Anthocyanin biosynthesis was induced by BL treatment to plants. In *Arabidopsis thaliana*, anthocyanin biosynthesis was stimulated by 2.5 W/m<sup>2</sup> of BL (approximately 11 µmol/m2/s) [28], which is higher than the intensity we used in this study (6.7 µmol/m2/s). We considered that the increase in the chlorophyll contents induced by either BL or FR supplements could help the biosynthesis of cyclic monoterpenes in GTs. To check the contribution of chlorophyll to photosynthesis, we quantified the quantum yield of photosystem II based on chlorophyll fluorescence.

.

photosystem II based on chlorophyll fluorescence.

**Figure 6.** Effects of light supplementation on leaf pigment and photosynthesis in two-week lightsupplemented Japanese mint leaves. (**A**) Total chlorophyll (*n* = 4) and anthocyanin contents (*n* = 4) were quantified using a spectrophotometer. (**B**) Photosynthetic parameters, Fv/Fm, and PSII quantum yield on leaves were monitored using a PAM chlorophyll fluorometer (*n* = 8–13). The data were obtained from independent experiments as biological replications. Error bars indicate standard deviations from the mean. Different alphabets indicate significant differences according to Tukey's **Figure 6.** Effects of light supplementation on leaf pigment and photosynthesis in two-week light-supplemented Japanese mint leaves. (**A**) Total chlorophyll (*n* = 4) and anthocyanin contents (*n* = 4) were quantified using a spectrophotometer. (**B**) Photosynthetic parameters, Fv/Fm, and PSII quantum yield on leaves were monitored using a PAM chlorophyll fluorometer (*n* = 8–13). The data were obtained from independent experiments as biological replications. Error bars indicate standard deviations from the mean. Different alphabets indicate significant differences according to Tukey's HSD test; *p* < 0.05.

Contrary to our expectations, both Fv/Fm and the quantum yield showed no change with any wavelength of light treatment (Figure 6B). This suggests that the increase in cyclic monoterpenes shown in Figure 3 was not due to the promotion of photosynthesis by the light supplements. Evans and Terashima reported no correlation between the chlorophyll content in spinach leaves and photosynthetic activity [29]. In *Arabidopsis*, red and far-red wavelengths supplemented with WL enhanced chlorophyll biosynthesis [30]. A BL supplement was also shown to increase the chlorophyll content in seven plant species [31]. In conclusion, chlorophyll generation observed in light-supplementation experiments might be the result of the transient activation of photoreceptors and does not influ-Contrary to our expectations, both Fv/Fm and the quantum yield showed no change with any wavelength of light treatment (Figure 6B). This suggests that the increase in cyclic monoterpenes shown in Figure 3 was not due to the promotion of photosynthesis by the light supplements. Evans and Terashima reported no correlation between the chlorophyll content in spinach leaves and photosynthetic activity [29]. In *Arabidopsis*, red and farred wavelengths supplemented with WL enhanced chlorophyll biosynthesis [30]. A BL supplement was also shown to increase the chlorophyll content in seven plant species [31]. In conclusion, chlorophyll generation observed in light-supplementation experiments might be the result of the transient activation of photoreceptors and does not influence photosynthesis and terpene biosynthesis.

### ence photosynthesis and terpene biosynthesis. **4. Conclusions**

HSD test; *p* < 0.05.

cells in GTs.

**4. Conclusions**  We propose a model based on the results as shown in Figure 7. The background WL illumination was still important for photosynthesis to provide sugars for terpene biosynthesis in GTs. We then speculate that the increase in cyclic monoterpenes in the GTs was probably due to the direct activation of processes of terpene biosynthesis. Studies have revealed that light modulated the biosynthesis of terpenes in many plant species. For example, a RL receptor, phytochrome, was shown to promote monoterpene production in thyme seedlings [32]. In cannabis plants, the content of a meroterpenoid, cannabinoid, was increased by BL treatment with a short photoperiod (12 h light/12 h dark) [33]. This indicates that photoreceptors or related factors, such as transcription factors, might directly or indirectly facilitate the enzymatic processes of terpene biosynthesis in secretory We propose a model based on the results as shown in Figure 7. The background WL illumination was still important for photosynthesis to provide sugars for terpene biosynthesis in GTs. We then speculate that the increase in cyclic monoterpenes in the GTs was probably due to the direct activation of processes of terpene biosynthesis. Studies have revealed that light modulated the biosynthesis of terpenes in many plant species. For example, a RL receptor, phytochrome, was shown to promote monoterpene production in thyme seedlings [32]. In cannabis plants, the content of a meroterpenoid, cannabinoid, was increased by BL treatment with a short photoperiod (12 h light/12 h dark) [33]. This indicates that photoreceptors or related factors, such as transcription factors, might directly or indirectly facilitate the enzymatic processes of terpene biosynthesis in secretory cells in GTs.

**Figure 7.** Schematic of glandular trichomes on Japanese mint leaves. Background WL provides energy for photosynthesis and provides a sugar source, which is necessary for terpene biosynthesis in glandular trichomes. LED light supplementation may promote the terpene biosynthetic pathway in glandular trichome cells through photoreceptor activation. S: secretory cells; ST: stalk cells; SC: storage cavity. **Figure 7.** Schematic of glandular trichomes on Japanese mint leaves. Background WL provides energy for photosynthesis and provides a sugar source, which is necessary for terpene biosynthesis in glandular trichomes. LED light supplementation may promote the terpene biosynthetic pathway in glandular trichome cells through photoreceptor activation. S: secretory cells; ST: stalk cells; SC: storage cavity.

Recently, GT research is spotlighted because certain secondary metabolites produced by plants have high pharmaceutical value [4,5]. A lot of chemical compounds for producing medicines still rely on plant-derived starting materials. For example, *Artemisia annua* is recognized as a precious plant resource of artemisinin, an antimalarial agent, stored in GTs. Since a net synthesis of artemisinin has great difficulty and costly, the cultivation of *Artemisia* plants is necessary to obtain the compound. In addition to the plant, many other medicinal plants store valuable second metabolites, including terpenes in GTs. It is known that the contents of the compounds in GTs are dependent on the growing environment [8]. Therefore, a considerable effort is being paid to cultivating medicinal plants in an artificially modified environment to maximize the contents of desired compounds in GTs. Results obtained from Japanese mint as a model of GTs metabolism showed that light, which is one of the critical environmental factors, promotes monoterpene synthesis, and it will be helpful for GT research. Although we first speculated that the BL affects specific terpene biosynthesis, all four monoterpenes (pulegone, menthofuran, menthone, and menthol) were significantly increased. It suggests that the BL might stimulate the biosynthesis and accumulation of terpene precursors such as DMAPP or IPP in GTs (Figures 1 and 7). These compounds are the necessary starting materials for all higher plant monoterpene biosynthesis, including *Artemisia* plants, as mentioned above. Thus, the information on supplement lighting obtained from the Japanese mint study will be helpful to boost the contents of valuable terpene compounds in medicinal plants for industrial cultivating conditions. As no genomic information on the Japanese mint used in the study was provided, genetic verification was not conducted in this study. Therefore, in future research, further details on physiological regulation by the light perception in the Japanese mint need clarification. Recently, GT research is spotlighted because certain secondary metabolites produced by plants have high pharmaceutical value [4,5]. A lot of chemical compounds for producing medicines still rely on plant-derived starting materials. For example, *Artemisia annua* is recognized as a precious plant resource of artemisinin, an antimalarial agent, stored in GTs. Since a net synthesis of artemisinin has great difficulty and costly, the cultivation of *Artemisia* plants is necessary to obtain the compound. In addition to the plant, many othermedicinal plants store valuable second metabolites, including terpenes in GTs. It is known that the contents of the compounds in GTs are dependent on the growing environment [8]. Therefore, a considerable effort is being paid to cultivating medicinal plants in an artificially modified environment to maximize the contents of desired compounds in GTs. Results obtained from Japanese mint as a model of GTs metabolism showed that light, which is one of the critical environmental factors, promotes monoterpene synthesis, and it will be helpful for GT research. Although we first speculated that the BL affects specific terpene biosynthesis, all four monoterpenes (pulegone, menthofuran, menthone, and menthol) were significantly increased. It suggests that the BL might stimulate the biosynthesis and accumulation of terpene precursors such as DMAPP or IPP in GTs (Figures 1 and 7). These compounds are the necessary starting materials for all higher plant monoterpene biosynthesis, including *Artemisia* plants, as mentioned above. Thus, the information on supplement lighting obtained from the Japanese mint study will be helpful to boost the contents of valuable terpene compounds in medicinal plants for industrial cultivating conditions. As no genomic information on the Japanese mint used in the study was provided, genetic verification was not conducted in this study. Therefore, in future research, further details on physiological regulation by the light perception in the Japanese mint need clarification.

**Author Contributions:** Conceptualization, K.Y. and M.M.; methodology, K.Y.; formal analysis, K.Y.; investigation, T.U.; writing and editing manuscript, K.Y., M.M. and T.U. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Lotte Research Promotion Grant (K.Y.) and in part by the Leading Initiative for Excellent Young Researchers of MEXT, Japan (K.Y.).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data that support the findings of this study are available from the corresponding author, K.Y., upon reasonable request.

**Acknowledgments:** We thank research collaboration between Kitami Hakka Tsusho and HakkaLab, Kitami Institute of Technology.

**Conflicts of Interest:** The authors declare no conflict of interest.
