*3.2. Alteration of GT Density*

We next observed whether the light treatment promoted the emergence of GTs. After two weeks of LED light treatment, the second and third leaves from the top of mint cuttings were harvested. The number of GTs in three regions of the leaf (apex, middle and basal regions) was counted separately and averaged. Overall, the density of GTs in the abaxial side of the leaves was slightly higher than that on the adaxial side (Figure 4A,B and Figure 5A,B). On both sides of the second leaf, except the apex region of the abaxial side, the GT density was decreased by the BL treatment (Figure 4A,B). There was a tendency for an increase in GT density by the FR light treatment. On the third leaf, a BL-dependent GT increase was similarly observed, whereas FR showed no promotion

of GT emergence (Figure 5A,B). We observed three regions of the second and third leaves to determine whether GT was newly generated as the leaf expands (apex region) or light directly regulated GT density regardless of the leaf regions. Here, we confirmed that environmental factors, including light conditions, could flexibly control GT density. A previous study showed that two light conditions (full solar radiation vs. shade) had no impact on GT density in a medicinal plant, *Ocimum campechianum* [22]. In cultivated tomato plants, a density of type VI leaf GTs increased under high light conditions (approximately 300 µmol/m2/s) [23]. Thus, light supplementation is likely to affect the density of GTs. The emergence of GTs is under the regulation of the well-known MYB transcription factor. Overexpression of AaMYB17 in sweet wormwood (*Artemisia annua* L.) increased the density of GTs [24]. Homeodomain-leucine zipper IV transcription factor was also shown to be involved in the control of GT density [25,26]. Our results show that the modulation of GT density by BL and FR is possible because of the regulation of transcription factors by light perception. However, the reduction of GT density with BL treatment was inconsistent with the increase of terpene contents, as shown in Figure 3. This suggests that biological processes of terpene biosynthesis were promoted directly by single wavelengths of LED light supplements. increase was similarly observed, whereas FR showed no promotion of GT emergence (Figure 5A,B). We observed three regions of the second and third leaves to determine whether GT was newly generated as the leaf expands (apex region) or light directly regulated GT density regardless of the leaf regions. Here, we confirmed that environmental factors, including light conditions, could flexibly control GT density. A previous study showed that two light conditions (full solar radiation vs. shade) had no impact on GT density in a medicinal plant, *Ocimum campechianum* [22]. In cultivated tomato plants, a density of type VI leaf GTs increased under high light conditions (approximately 300 µmol/m2/s) [23]. Thus, light supplementation is likely to affect the density of GTs. The emergence of GTs is under the regulation of the well-known MYB transcription factor. Overexpression of AaMYB17 in sweet wormwood (*Artemisia annua* L.) increased the density of GTs [24]. Homeodomain-leucine zipper IV transcription factor was also shown to be involved in the control of GT density [25,26]. Our results show that the modulation of GT density by BL and FR is possible because of the regulation of transcription factors by light perception. However, the reduction of GT density with BL treatment was inconsistent with the increase of terpene contents, as shown in Figure 3. This suggests that biological processes of terpene biosynthesis were promoted directly by single wavelengths of LED light supplements.

basal regions) was counted separately and averaged. Overall, the density of GTs in the abaxial side of the leaves was slightly higher than that on the adaxial side (Figures 4A,B and 5A,B). On both sides of the second leaf, except the apex region of the abaxial side, the GT density was decreased by the BL treatment (Figure 4A,B). There was a tendency for an increase in GT density by the FR light treatment. On the third leaf, a BL-dependent GT

*Plants* **2021**, *10*, x FOR PEER REVIEW 7 of 12

**Figure 4.** The number of glandular trichomes generated on the second leaf under different light treatments for two weeks. (**A**) Quantification on the adaxial side in the three leaf regions (*n* = 16). (**B**) Quantification on the abaxial side in the three leaf regions (*n* = 16). The data were obtained from four plants in four 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. **Figure 4.** The number of glandular trichomes generated on the second leaf under different light treatments for two weeks. (**A**) Quantification on the adaxial side in the three leaf regions (*n* = 16). (**B**) Quantification on the abaxial side in the three leaf regions (*n* = 16). The data were obtained from four plants in four independent experiments as biological replications. Error bars indicate standard deviations from the mean. Different alphabets indicate significant differences according toTukey's HSD test; *<sup>p</sup>* < 0.05.

Tukey's HSD test; *p* < 0.05.

**Figure 5.** The number of glandular trichomes generated on the third leaf under different light treatments for two weeks. (**A**) Quantification on the adaxial side in the three leaf regions (*n* = 16). (**B**) Quantification on the abaxial side in the three leaf regions (*n* = 16). The data were obtained from four plants in four independent experiments as biological replications. Error bars indicate standard deviations from the mean. Different alphabets indicate significant differences according to **Figure 5.** The number of glandular trichomes generated on the third leaf under different light treatments for two weeks. (**A**) Quantification on the adaxial side in the three leaf regions (*n* = 16). (**B**) Quantification on the abaxial side in the three leaf regions (*n* = 16). The data were obtained from four plants in four 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.
