## 2 Results and Discussion
### PathVisio Pathway Analysis  
To interpret the findings of multivariate statistical analyses described in Sections 2.1 and 2.2 in the context of metabolic pathways, PathVisio was used to visualize metabolic changes in 110 metabolites in pathway diagrams (18 metabolites are not shown in these diagrams) (Figure 4) [29]. The fold change (FC) was calculated by dividing the average  
Figure 3: Correlation matrix and cluster analysis of data for 110 metabolites of black, red, and non-pigmented (white) rice cultivars (**A**). Detailed correlation matrix of each compound cluster (**B**). Each square indicates a Pearson’s correlation coefficient for a pair of compounds. The value for the correlation coefficient is represented by the intensity of the blue or red color, as indicated on the color scale. Hierarchical clusters are presented as a cluster tree. C20-ol, Eicosanol; C21-ol, Heneicosanol; C22-ol, Docosanol; C24-ol, Tetracosanol; C26-ol, Hexacosanol; C27-ol, Heptacosanol; C28-ol, Octacosanol; C30-ol, Triacontanol; C16:0, Palmitic acid; C18:0, Stearic acid; C18:1, Oleic acid; C18:2, Linoleic acid; C18:3, alpha-Linolenic acid; C20:0, Arachidic acid. values for pigmented rice by the average values for non-pigmented rice (WR), 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 by PathVisio. Figure 4: 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 pigmented rice than in non-pigmented rice (WR: white rice). If log\({}_{2}\)FC value is less than zero (indicated in green), metabolite content is lower in pigmented rice than in non-pigmented rice. If log\({}_{2}\)FC value is zero (indicated in white), metabolite content is identical in pigmented and non-pigmented rice. (BR: Black Rice, RR: Red Rice). Concerning the phenylpropanol pathway, BR contained more cyanidin-3-glucose and peonidin-3-glucoside than WR (Figure 4A). On the contrary, RR contained more catechin and epicatechin than WR. These metabolites determine the color of pigmented rice [3,4]. Hence, these observations indicated that the synthesis of color metabolites in pigmented rice proceeds via a different route in the same phenylpropanol pathway. These findings were in agreement with the HCA and Pearson's correlation analysis, which indicated that catechin and epicatechin are negatively correlated with anthocyanins (Figure 3). In addition, in PLS-DA and OPLS-DA, catechin and epicatechin were significant contributing metabolites in RR, whereas anthocyanins were important metabolites in BR (Figures 1 and 2). Shikimic acid is the starting point of the phenylpropanol pathway, which then proceeds to phenylalanine, _trans_-cinnamic acid, and _p_-coumaric acid. These metabolites are important precursors of flavonoids and phenolics, and were more abundant in pigmented rice than in WR, except for phenylalanine (lower levels in BR than in WR) and _trans_-cinnamic acid (not detected). Although BR contained less phenylalanine than WR, _p_-coumaric acid was notably more abundant in BR than in RR and WR. These observations suggested that the high level of _p_-coumaric acid in BR might arise from the biotransformation of phenylalanine, stimulated by upregulation of phenylalanine ammonia-lyase (PAL) activity. PAL is a key enzyme that acts at the first step of the phenylpropanol pathway, and converts phenylalanine to _trans_-cinnamic acid and ammonia. According to a previous study, PAL activity impacts anthocyanin levels in the flavonoid pathway [30]. In addition, PAL regulates anthocyanin accumulation [31]. A further, genomics, study is needed to address this issue.