**3. Discussion**

Phytochromes and cryptochromes are the specialized photoreceptors of plants that sense the spectral quality and quantity, transducing the light signal to regulate genes responsible for secondary metabolite production [30]. Therefore, it is possible to determine the metabolic composition or to enhance the nutritional functionality of the target crops through selective application of light resources and photoperiodism. The application of LEDs for special metabolite production is considered promising, where it has been shown that the metabolic profiles also depend on several other factors, including crop genetics [18,26,31]. The increasing application of LED irradiation sources for the development of designer foods/functional foods may revolutionize the food industry. In this study, we attempted to enhance the *C*-glycosylated flavones/flavonoids and policosanol contents in barley and wheat sprouts using varying light qualities.

*C*-glycosylated flavones constitute the major portion of flavonoids found in barley seedlings [32]. Saponarin (isovitexin-7-O-glucoside) is a major *C*-glycosylated flavone, which is naturally present in young barley seedlings [11]. Among cellular organelles, saponarin is efficiently stored in vacuoles [33] and high accumulation is typically observed in primary leaves [4]. Similarly, isoorientin and isoschaftoside are the major *C*-glycosylated flavones often reported in wheat seedlings [29]. These *C*-glycosylflavones have potential roles as beneficial flavonoids in the human diet [4,8,34]. In this study, we found that the *C*-glycosylflavone (saponarin, isoorientin, and isoschaftoside) content was high in the early growth stages of seedlings, where the maximum accumulation was induced by blue LED light irradiation. We found an inverse relationship between *C*-glycosylated flavone content and growth times, indicating that the *C*-glycosylflavone content remains high in young sprouts. In a previous study investigating barley sprouts (13–56 days post-sprouting), it was stated that the saponarin content continued to decline with increasing growth periods [9]. Now, it is clear that the saponarin content in barley sprouts starts declining just three days post-sprouting. In terms of light quality, blue LED light had a positive impact on saponarin content. Blue LED light showed the highest accumulation (57.7% and 68.68% than that in the control, respectively) in barley and wheat sprouts. On the other hand, the impact of red LED light irradiation on saponarin content in barley, and isoorientin and isoschaftoside in wheat sprouts, seemed to differ. In barley, the saponarin content was reduced, while the levels of isoorientin and isoschaftoside were increased in wheat sprouts, suggesting that the effect of red LED light is specific to metabolites and/or crops.

Policosanol (PC) is another beneficial metabolite in the human diet, which is frequently found in cuticular waxes in primary leaves of young cereal sprouts. It represents a mixture of long-chain fatty alcohols (20–36 carbon) mostly comprised of docosanol (C22), tetracosanol (C24), hexacosanol (C26), octacosanol (C28), and triacontanol (C30) [35,36]. Octacosanol and policosanol (long-chain saturated fatty alcohols) are useful in preventing high-fat diet-induced obesity [37]. Owing to its importance in lowering blood cholesterol and protection from platelet aggregation, it has been commercialized in the health industry for a long time [35]. Among the PCs, hexacosanol (C26) and octacosanol (C28) are often observed in barley and wheat sprouts [36,38]. LED light irradiation showed a differential influence on the policosanol content in barley and wheat sprouts, suggesting that the LED response is likely specific to either metabolites or crops. Compared to FL conditions, LED light irradiation reduced the hexacosanol content in barley, while a similar condition in wheat sprouts showed an irregular pattern of octacosanol accumulation; suggesting that factors other than light quality also influence its content in wheat sprouts. Interestingly, in most cases, the policosanol content in barley and wheat sprouts gradually increased with the extension of growth time. Statistical analyses confirmed that the saponarin content under LED treatment was negatively correlated with barley growth periods. A similar negative relationship between content and growth period was also evident in wheat sprouts, albeit restricted to white and/or red LED light treatments. Unlike *C*-glycosylflavone, the hexacosanol content in sprouts appeared to have a positive correlation with sprout growth periods, suggesting the importance of growth level in the determination of policosanol content in barley. Our study corroborates previous findings which have reported a positive correspondence between hexacosanol

and growth periods in barley [36]. However, a similar correlation pattern was not observed between octacosanol content and growth periods in FL, white, and blue LED irradiated wheat sprouts, possibly suggesting that this relationship might be specific to crop genetics. Altogether, our results show that light qualities and growth periods are two crucial factors in determining the C-glycosylflavone and policosanol contents in barley and wheat sprouts. In addition, light conditions are important parameters in determining the photomorphogenesis of plants [39]. Herein, we found that red LED light irradiation mostly reduced the root growth of sprouts, while white and blue LED light mediated root growth inconsistently across growth periods, indicating the role of other factors regulating growth parameters. The LED light responses of leaves of young seedlings also indicated that LED irradiation does not induce a regular growth pattern for leaves in barley and wheat sprouts. At this stage, it is difficult to conclude the growth impact of LED light irradiation, as this study utilized low intensity LED spectra. Further studies concerning the selection of optimum light intensity and LED spectra for enhancing sprout growth are, therefore, essential.

An interesting observation is that comparison of the metabolite accumulation trends revealed the *C*-glycosylflavones and policosanol contents to have an inverse relationship in barley sprouts. It is unknown whether this is due to a balancing act of metabolic pathways or just an influencing act of sprout growth periods. It warrants further in-depth studies, in order to understand the underlying mechanisms of metabolite biosynthesis. The molecular basis for the specialized metabolite accumulation to a particular physiological condition is mainly due to changes in the expression pattern of one or more specialized biosynthetic genes. Lee et al. [11] suggested that the expression pattern of UDP-Glc: Isovitexin 7-O-glucosyltransferase (OGT) is likely responsible for saponarin biosynthesis in barley. *OGT* is responsible for the conversion of isovitexin to saponarin in barley [33]. There are two classes of OGT (*OGT1* and *OGT2*) found in the barley genome, where a change in the expression level of *OGT1* has been associated with saponarin concentration [11]. In the present study, we found that a positive correlation exists between the *HvOGT1* expression pattern and saponarin accumulation, suggesting the possibility of *OGT1* involvement in saponarin biosynthesis. Both metabolite accumulation and *HvOGT1* expression were found to be highest after 3 days of treatment. It is clear that blue LED light irradiation accumulated saponarin by upregulating *HvOGT1* expression in barley sprouts. The flavonoid biosynthesis pathway gene, *HvCHS1*, is not linked with saponarin accumulation, indicating the possibility of a specific pathway controlling saponarin biosynthesis in barley. Studies on other crops have shown that fatty acyl-coenzyme A reductases (FAR) are involved in long-chain primary alcohol biosynthesis, part of the cuticular waxes found in leaf surfaces [40]. Studies have also shown that the number of *FAR* genes and their functions may vary, according to species-specific genetics. In other crops, it is evident that *FAR2* and *FAR3* are responsible for the biosynthesis of C<sup>26</sup> and C<sup>28</sup> primary alcohols [41]. Another report has claimed that at least five *FARs* are responsible for primary alcohol (C<sup>16</sup> to C28) biosynthesis in *Aegilops tauschii* [42]. Until recently, there has been no information about *HvFARs* and their potential role in hexacosanol biosynthesis. Identification of the *FARs* responsible for policosanol biosynthesis is inevitable for tailoring metabolic pathways towards enhanced production. Hence, we used the homology-based gene identification method to predict the gene sequences of *HvFAR2, HvFAR3, HvFAR4, HvFAR5*, and *HvFAR6* from available barley genome information. We also measured their expression changes using quantitative PCR during differential light treatments and growth periods, in order to infer their relationship to hexacosanol biosynthesis. Of all the *HvFARs* analyzed in this study, the expression changes of *HvFAR3* were positively associated with hexacosanol accumulation, suggesting their involvement in hexacosanol biosynthesis. Other *HvFARs* did not have a positive correlation with hexacosanol content, suggesting that they may not be associated with its biosynthesis. In this study, we identified the potential candidate genes involved in saponarin and hexacosanol biosynthesis. These genes can be effectively used to enhance the metabolite concentration by means of genetic manipulation. We also provided an expression atlas of *HvFARs* during LED light irradiation treatment in sprouts, which may be useful in future studies associated with other policosanol biosynthesis routes. A system-wide

identification and characterization would add more information about the genetic factors controlling metabolite biosynthesis in barley sprouts.

To conclude, we showed that light qualities and growth times are crucial factors determining the contents of *C*-glycosylflavones and policosanols in barley and wheat sprouts. Blue LED light may be useful for increasing the *C*-glycosylflavone contents in cereal sprouts. Regardless of the light quality, management of growth time of sprouts is essential for policosanol content. This study will help to maximize the beneficial flavonoids and policosanol contents in cereal sprouts through LED applications in the future.
