## 4 Discussion
### Flavonoid Biosynthesis Up-Regulated by Low-Temperature Stress in Sandrice  
A total of 14 flavonoids were detected in sandrice (Table S2), with 12 of them showing an increased number of flavonoids after exposure to low-temperature stress. (Figure 2A). This observation indicated that sandrice responded to low-temperature stress by accumulating a large number of flavonoids. This phenomenon was supported by extensive research that indicated low-temperature stress can induce the accumulation of flavonoids in plants [24; 25; 27; 48]. It was also found that five flavonoids, namely rutin, isoquercetin, astragalain, quercetin, and isorhamnetin, displayed relatively high content in sandrice, collectively accounting for over 99% of the total flavonoid content. These flavonoids showed increased accumulation under cold stress. When subjected to external stressors, plants can accumulate substantial flavonoids and other substances to mitigate the damage caused by the reactive oxygen species [49; 50; 51]. In addition, these flavonoid metabolites are known to possess various medicinal values, including antioxidant, hypoglycemic, antiviral, anti-inflammatory, anti-tumor, cardiovascular, and cerebrovascular protective properties [52; 53; 54; 55; 56; 57]. This discovery underscores the rich flavonoid content in the above-ground tissue of sandrice, highlighting its potential as a valuable medicinal resource for development and application. Furthermore, in practical production, sandrice can be harvested after undergoing appropriate cold stress to ensure a high flavonoid content.  
To screen the elite genetic resource for flavonoids industry, the molecular regulation mechanism of the flavonoid biosynthesis pathway responding to cold stress was explored through a joint analysis of DAs and DEGs. The common DAs among the three cotypes were naringenin and naringenin chalcone under low-temperature stress, which serve as precursor substances for the other 12 flavonoid metabolites (Figure 7). A correlation network analysis revealed that cold stress significantly up-regulated at least three upstream DEGs, including _AsqAEX006535\(-\)CHS_, _AsqAEX016074\(-\)C_H_, and _AsqAEX004011\(-\)4 CL_. These genes were positively regulated by _AsqAEX015868\(-\)hHLH62_ and _AsqAEX001711\(-\)MYB12_, while also being negatively regulated by _AsqAEX002220\(-\)MYB1R1_, promoting the biosynthesis of naringenin in sandrice (Figure 6 and Table S5). Interestingly, _MYBs_ and _hHLHs_, known to regulate flavonoid synthesis, are widely conserved across various plants species [38; 58; 59; 60; 61]. For example, in _L. barbarrum LrMYB1_ activates _LbCHS_ to accumulate flavonoids [62; 63], while some other MYBs, such as two R2R3-MYB proteins of poplar [64] and GbMYBF2 of _Ginkgo biloba_[65], inhibit flavonoid synthesis. In _Erigeron breiscaps_, _EbbHLH80_ regulates the expression of some of the structural genes involved in flavonoid biosynthesis, influencing both flavonoid accumulation and plant development [66]. In this study, we identified both positive and negative regulators of flavonoid synthesis in response to cold stress in sandrice. However, further functional analyses are necessary to fully elucidate these mechanisms.