*2.3. HS-SPME and GC–MS Analysis for Cyclic Monoterpenes*

After two weeks of light treatment, the second and third leaves (total four leaves) were harvested by cutting the petiole and weighed. The first leaves newly emerged during the 2-week LED treatment, and the size of the leaves was small and immature. To observe the effect of the LED supplements on expanded leaves, we decided to use the second and third leaves for further analyses. The analysis of a volatile compound with the head-space solid phase microextraction (HS-SPME) method was adapted from a previous report [17]. The leaves were then frozen with liquid nitrogen and ground finely with a pre-cooled pestle and mortar. The ground powder was put into 2 mL of 2 M CaCl2·H2O solution in a head-space 20-mL glass vial (Supelco/Sigma-Aldrich, Tokyo, Japan) to prevent unnecessary enzymatic reactions. The tightly sealed vials were incubated in a hot bath at a temperature of 40 ◦C for 5 min. An SPME fiber coated with 60-µm-thick PDMS/DVB (polydimethylsiloxane/divinylbenzene, Supleco) was inserted into the headspace through the vial septum, and the volatile released from the warmed solution was absorbed. The fiber was removed from the vial and inserted quickly into the inlet of a gas chromatography–mass spectroscopy (GC–MS) system (GC-17A, Shimadzu, Kyoto, Japan) with a polyethylene glycol column (60 m long, 0.25 mm internal diameter, 0.25 µm film thickness; GL Sciences, Tokyo, Japan). The temperature of the inlet was held at 180 ◦C with a 9:1 split. Helium was used as the carrier gas at a 2 mL/min flow rate, 245.9 kPa. The starting oven temperature was 100 ◦C, followed by an 8 ◦C/min ramp until 180 ◦C was reached and held for 2 min. The interface to the mass spectrometer was held at 250 ◦C. The mass spectrometer was run in the scan mode with electron impact ionization at 1 keV, from *m*/*z* 40 to *m*/*z* 300. Compounds were identified using the National Institute of Standard and Technology Mass Spectral Database (NIST12). The GC–MS analyses of terpenes were conducted independently six times in different LED lightning treatments.
