## 3 Discussion  
While low temperatures improving the plant growth and Hyp accumulation by stimulating related gene expressions has been observed in \(H\). _perforatum_[20; 22; 27; 28], the mechanism responsible for temperature-dependent growth and Hyp biosynthesis has not been dissected. Here, it is shown that there is a greater plant biomass and Hyp accumulation, more leaf dark glands, stomatal density and rounded stomatal shape at 15 compared to 22 \({}^{\circ}\)C. By treating seedlings at 15 and 22 \({}^{\circ}\)C, ca. 1600 genes were differentially expressed, with over 56% of the 749 characterized genes being UR at 15 \({}^{\circ}\)C compared to 22 \({}^{\circ}\)C (Figure 4). By grouping genes based on biological functions, 150 genes were associated with Hyp biosynthesis, plant growth and stress responses, including photosynthesis, carbohydrate metabolism, fatty acid metabolism, CYPs, morphophysiological traits, HSPs, CRPs and TFs (Figure 5). Experimental evidence shows that \(H\). _perforatum_ plants can adapt to low temperatures by increasing the biomass and morphogenesis (e.g., leaf area, number of stem and its internode and root), and low temperatures can improve Hyp biosynthesis by increasing the number of dark glands and related gene expressions [10; 12; 20; 22; 27; 28]. The current study exhibited greater biomass by increasing the stomatal density and opening percentages, as well as a more rounded stomatal shape with the ratio of length/width and greater Hyp content via the overexpression of 61 genes involved in Hyp biosynthesis (Figures 2 and 3 and Table 1). As is shown in Figure 1, the Hyp biosynthetic pathway depends on photosynthesis and is involved in carbohydrate metabolism and fatty acid metabolism in the upstream steps, as well as PKS, TER, POCR and CYPs. For the photosynthesis, the CABs are the most abundant protein complex on the thylakoid membrane and are light receptors that capture and deliver excitation energy to photosystems [31]. The UR of CAB genes that are involved in photosynthesis provides energy and fixation and a reduction of CO\({}_{2}\) into carbohydrate [32]. For the carbohydrate metabolism, the carbonic anhydrases (CAs) catalyze the reversible interconversion between carbon dioxide and bicarbonate [33]. The L-lactate dehydrogenase A (LDHA) synthesizes lactate from pyruvate in glycolysis [34]. The phosphoglycerate mutase-like protein (PGAM) catalyzes the reversible interconversion between 3-phosphoglycerate and 2-phosphoglycerate [35]. The beta-glucosidase (BGLU11) hydrolyzes the terminal, nonreducing beta-d-glucosyl residues to generate beta-d-glucose [36]. The glutamate decarboxylase (GAD) catalyzes the conversion of glutamate to CO\({}_{2}\) and g-aminobutyrate [37]. The glyceraldehyde-3-phosphate dehydrogenase (GAPC) catalyzes D-glyceraldehyde 3-phosphate to 3-phospho-d-glyceroyl phosphate [38]. The UR of genes that are involved in carbohydrate metabolism enhances the accumulation of pyruvic acid by glycolysis pathways, which provide substrates for the biosynthesis of acetyl-CoA. For the fatty acid metabolism, for example, the phospholipase D zeta 1 (PLDZETA1) is involved in the lipid catabolic process by the hydrolysis of glycerol-phospholipids to produce phosphatidic acids [39]. The acetyl-CoA-benzyl alcohol acetyltransferase (BEAT) is involved in the biosynthesis of benzyl acetate [40]. The 2,4,6-trihydroxybenzophenone synthase (BPS) is involved in the malonyl-CoA metabolic process [41]. 3-ketoacyl-CoA thiolase 2 (PED1) is involved in long chain fatty acid beta-oxidation [42]. The fatty acid metabolic process will provide abundant acetyl-CoA, which can combine with its product malonyl-CoA to produce an octa-\(\beta\)-ketoacyl chain. Extensive studies have demonstrated that CYPs can catalyze several reactions, such as hydroxylation, dealkylation and oxidation reactions. Studies have demonstrated that the CYPs are a large and diverse family involved in secondary metabolism [43; 44; 45; 46; 47].