## 2 Results and Discussion
### PathVisio Pathway Analysis
Furthermore, fatty acid levels in pigmented rice were lower than those in WR. Malonyl-CoA is the precursor of both, fatty acid and flavonoid biosynthesis. Chalcone synthase (CHS) converts three molecules of malonyl-CoA and one molecule of 4-coumaroyl-CoA into naringenin chalcone [32,33]. Therefore, pigmented rice might consume more malonyl-CoA than WR to synthesize flavonoids for pigment formation, such as black and red. Consequently, pigmented rice might lack malonyl-CoA for fatty acid synthesis. On the contrary, WR does not synthesize flavonoids, and contained relatively more fatty acids than pigmented rice, as explained above. Similarly, wild beans, which contain high levels of flavonoids, have relative lower fatty acid levels than cultivated beans [34]. The above results were consistent with PLS-DA and OPLA-DA (Figures 1 and 2). As shown in Figure 4B, carotenoids and phytosterols, such as \(\beta\)-carotene, lutein, zexaxanthin, campesterol, \(\beta\)-sitosterol, cholesterol, and stigmasted, were more abundant in BR than in RR and WR, which was consistent with previous studies reporting the abundance of carotenoids and phytosterols in BR [3,20]. However, in the present study, pigmented rice contained less \(\beta\)-tocopherol, \(\gamma\)-tocopherol, and \(\gamma\)-tocotrienol than WR. In addition, these metabolites contributed to the separation of WR from other rice samples in PLS-DA and OPLS-DA, and they clustered together, separately from other terpenids. The major tocopherols in rice are \(\alpha\)-tocopherol, \(\alpha\)-tocotrienol, \(\gamma\)-tocopherol, and \(\gamma\)-tocotrienol [35]. According to a previous study, \(\alpha\)-tocopherol levels are highest in BR, [36]. In addition, most volatiles, such as monoterpenes, fatty acid-driven volatiles, and other volatiles, were most abundant in BR. Therefore, BR exhibited higher secondary metabolic activity, such as that involving terpenoid (monoterpene, triterpene, and tetraterpene) and phenylpropanol (anthocyanin) biosynthesis, than RR and WR. To the best of our knowledge, this is the first report on the differences in terpenoid metabolism in non-pigmented and pigmented rice associated with their color. To elucidate the metabolic differences between pigmented rice samples, FC was calculated by dividing the average data of BR by the average data of RR, and then log\({}_{2}\)-transforming (log\({}_{2}\)FC) (Table S11). The log\({}_{2}\)FC data (ranging from \(-1\) to \(1\)) were visualized on pathway diagrams in PathVisio (Figure 5). Most amino acids and organic acids were more abundant in RR than in BR, except for \(\gamma\)-aminobutyric acid, asparagine, proline, glutamine, tryptophan, glutamic acid, oxalic acid, and citric acid (Figure 5A). Furthermore, most organic acids and amino acids significantly contributed to the separation of RRfrom BR in the PLS-DA loading plot and OPLS-DA VIP plot (Figures 1 and 2). Among \"other\" metabolites, \(\gamma\)-aminobutyric acid and asparagine are reportedly highly abundant in BR [37; 38], which is consistent with the data presented herein. In addition, the \(t\)-test \(p\)-value for these metabolites comparing BR and RR was below 0.05, and these metabolites contributed to the separation of BR from RR in the PLS-DA loading plot and OPLS-DA VIP plot. Figure 5: Metabolic pathway diagrams visualized by PathVisio 3. Detailed metabolic pathway diagrams of amino acids, organic acids, sugars, phenylpropanoids, and fatty acids (**A**); terpenoids and volatiles (**B**). The expression data consist of log\({}_{2}\)-transformed fold change (FC) values (log\({}_{2}\)FC). A log\({}_{2}\)FC value range is \(-1<\) log\({}_{2}\)FC \(<1\). If log\({}_{2}\)FC value is higher than zero (indicated in red), metabolite content is higher in black rice than in red rice. If log\({}_{2}\)FC value is less than zero (indicated in green), metabolite content is higher in red rice than in black rice. If log\({}_{2}\)FC value is zero (indicated in white), metabolite content is identical in black and red rice. (BR: Black Rice, RR: Red Rice).