*4.1. Plant Materials, Growth Condition, and LED Treatment*

The barley (*Hordeum vulgare* L. variety "Keunalbori No.1") and wheat (*Triticum asetivum* L. variety "Baekchalmil") seeds used by this study were sourced from the National Institute of Crop Science (Korea). The seeds at full maturity were separated by soaking thrice in tap water. Then, visually healthy seeds were once again soaked separately in distilled water for one day. For germination, seeds were sown on 16 plastic sprout cultivating trays (90 cm by 30 cm; ~180 g per tray) and maintained in greenhouse conditions (25 °C, 16 h photoperiod) for one day. Four trays with sprouting seeds (per treatment) were moved to individual growth chambers (Mokmin Co., Ltd, Suwon, South Korea) equipped with FL or different spectra LED light irradiations (470 nm for blue, 380 nm for white, and 660 nm for red) over 9 days (Figure 5). Sprouts were watered at regular intervals. The photosynthetic photon flux density (PPFD) of FL (96 µmol m−<sup>2</sup> s −1 ), blue (100 µmol m−<sup>2</sup> s −1 ), white (100 µmol m−<sup>2</sup> s −1 ), and red (93 µmol m−<sup>2</sup> s −1 ) lights was measured at plant level by using a quantum meter (Apogee Instruments, Logan, USA). Fresh leaves and coleoptiles of each treatment were harvested simultaneously at 3, 5, 7, and 9 days after irradiation (DAI). All samples were frozen in liquid nitrogen and stored at −80 °C for RNA extraction and metabolomic analyses. All sprouting seeds throughout the treatments were maintained in a growth chamber with 16 h photoperiods, 60–80% humidity, and 22–25 °C. *Plants* **2020**, *9*, x FOR PEER REVIEW 11 of 15 m−2s−1), white (100 μmol m−2s−1), and red (93 μmol m−2s−1) lights was measured at plant level by using a quantum meter (Apogee Instruments, Logan, USA). Fresh leaves and coleoptiles of each treatment were harvested simultaneously at 3, 5, 7, and 9 days after irradiation (DAI). All samples were frozen in liquid nitrogen and stored at −80 ℃ for RNA extraction and metabolomic analyses. All sprouting seeds throughout the treatments were maintained in a growth chamber with 16 h photoperiods, 60%– 80% humidity, and 22–25 ℃.

**Figure 5.** Schematic representation of experimental design used for LED treatment on barley and **Figure 5.** Schematic representation of experimental design used for LED treatment on barley and wheat seedlings. The diagram illustrates the germination method, growth period, and light quality and intensity.

### wheat seedlings. The diagram illustrates the germination method, growth period, and light quality and intensity. *4.2. Extraction of C-glycosylated Flavones and Measurement*

*4.3. Extraction and Quantification of Policosanols from Barley and Wheat Seedlings* 

input for the statistical analyses.

*4.2. .Extraction of C-glycosylated Flavones and Measurement*  The extraction method of Lee et al. [11] was used for the preparation of crude extracts from dried barley and wheat seedlings. An ultra-high-performance liquid chromatograph equipped with UV detectors (Dionex Ultimate 3000; Thermo Scientific, Waltham, MA, USA) and reversed-phase

The extraction method of Lee et al. [11] was used for the preparation of crude extracts from dried

analyses [8]. One gram of freeze-dried and chopped seedlings from each treatment was extracted by treating with either 50% ethanol (barley) or 100% methanol (wheat) on a shaker for 24 h at room temperature. The ethanolic or methanolic extracts were then filtered through 0.2 μm syringe filters. Following evaporation under vacuum, the extracts were dissolved in 10% dimethyl sulfoxide (DMSO) containing 50% ethanol and 1.3 μL was injected into the column for separation and detection at 325 nm. The mobile phase was comprised of trifluoroacetic acid (TFA) (0.1%) in water (A) and acetonitrile (B) with a flow rate of 0.5 mL/min, which was applied for separation of the analyte. Saponarin, isoorientin, and isoschaftoside molecules were identified by comparing the retention times to those of standards (obtained from Extrasynthese, Lyon, France and NICS, Jeonju, Korea). A standard calibration curve was prepared by plotting the peak area (y) of the chromatogram and the respective concentrations (31.25, 62.5, 125, 250, 500, and 1000 μg/mL) (x). The equations of the calibration curves for saponarin, isoorientin, and isoschaftoside were y = 0.0876x + 0.3514 (r2 = 0.999), y = 0.0917x −0.5536 (r2 = 0.998), and y = 0.0.0547x + 0.22935 (r2 = 0.999), respectively. The metabolite content (mg/g DW) from three technical replicates of biologically independent samples were used as

The preparation of barley and wheat crude extracts, policosanol standards, GC/MS parameters,

and the quantification method of GC/MS as described elsewhere [17,36], was used in this study. Briefly, 1 g of freeze-dried and chopped samples collected from all treatments was extracted separately into 10 mL of hexane on a shaker for 24 h at room temperature. The supernatant of the mixture was collected by centrifugation at 3000 g for 3 min, filtered through a syringe filter with a pore size of 0.45 μm (Whatman Inc., Maidstone, UK), and kept under vacuum conditions until the hexane was completely removed. To the final extract, 250 μL of N-Methyl-N-

HPLC column (ACQUITY UPLC BEH C18, 2.1 mm by 100 mm) was utilized for separation and quantitative analyses [8]. One gram of freeze-dried and chopped seedlings from each treatment was extracted by treating with either 50% ethanol (barley) or 100% methanol (wheat) on a shaker for 24 h at room temperature. The ethanolic or methanolic extracts were then filtered through 0.2 µm syringe filters. Following evaporation under vacuum, the extracts were dissolved in 10% dimethyl sulfoxide (DMSO) containing 50% ethanol and 1.3 µL was injected into the column for separation and detection at 325 nm. The mobile phase was comprised of trifluoroacetic acid (TFA) (0.1%) in water (A) and acetonitrile (B) with a flow rate of 0.5 mL/min, which was applied for separation of the analyte. Saponarin, isoorientin, and isoschaftoside molecules were identified by comparing the retention times to those of standards (obtained from Extrasynthese, Lyon, France and NICS, Jeonju, Korea). A standard calibration curve was prepared by plotting the peak area (y) of the chromatogram and the respective concentrations (31.25, 62.5, 125, 250, 500, and 1000 µg/mL) (x). The equations of the calibration curves for saponarin, isoorientin, and isoschaftoside were y = 0.0876x + 0.3514 (r<sup>2</sup> = 0.999), <sup>y</sup> <sup>=</sup> 0.0917x <sup>−</sup>0.5536 (r<sup>2</sup> <sup>=</sup> 0.998), and y <sup>=</sup> 0.0.0547x <sup>+</sup> 0.22935 (r<sup>2</sup> <sup>=</sup> 0.999), respectively. The metabolite content (mg/g DW) from three technical replicates of biologically independent samples were used as input for the statistical analyses.
