## 3 Results
### Identification and Functional Annotation of Differentially Expressed Metabolites
\begin{table}
\begin{tabular}{l c c c c} \hline \hline \multicolumn{1}{c}{**Metabolite**} & **Class** & **VIP 1** & **Fold Change (FC)** & **log\({}_{2}\)FC 2** \\ \hline
1-Phenylethanol & Phenolic acids & 1.44 & 269,754.80 & 18.04 \\ Dicaffeoylshikimic acid & Phenolic acids & 1.44 & 86,594.42 & 16.40 \\
2-Methoxycinnamic acid & Phenolic acids & 1.44 & 10,264.53 & 13.33 \\
1,3-O-Dicaffeoylquinic Acid (Cynarin) & Phenolic acids & 1.42 & 266.71 & 8.06 \\
4-O-(4\({}^{\prime}\)-O-alpha-D-Glucopyranosyl)caffeoylquinic acid & Phenolic acids & 1.43 & 182.93 & 7.52 \\ Chlorogenic acid methyl ester & Phenolic acids & 1.44 & 103.94 & 6.70 \\ Cyanidin-3-O-(6\({}^{\prime\prime}\)-O-caffeoyl-2\({}^{\prime\prime}\)-O-xylosyl)glucose & Flavonoids & 1.43 & 82.45 & 6.37 \\ Tri-O-galloyl Methyl gallate & Phenolic acids & 1.41 & 82.20 & 6.36 \\ Luteolin-7-O-Sophoroside-5-O-arabinoside & Flavonoids & 1.43 & 74.07 & 6.21 \\
5-O-Feruloyl quinic acid glucoside & Phenolic acids & 1.37 & 73.70 & 6.20 \\ \hline
3,6-Di-O-caffeoyl glucose & Phenolic acids & 1.44 & \(9.18\times 10^{-6}\) & \(-16.73\) \\ Rosmarinic acid methyl ester & Phenolic acids & 1.44 & \(2.37\times 10^{-5}\) & \(-15.36\) \\
2,3-Dimethoxybenzaldehyde & Phenolic acids & 1.44 & \(3.2\times 10^{-5}\) & \(-14.93\) \\ Quercetin-3-O-(2\({}^{\prime\prime}\)-O-Hammosyl)rutinoside-7-O-glucose & Flavonoids & 1.44 & \(2.5\times 10^{-4}\) & \(-11.96\) \\ Quercetin-3-O-(6\({}^{\prime\prime}\)-O-acetyl)glucoseJ-(1\(\to\)3)-Galactoside & Flavonoids & 1.41 & 0.016 & \(-6.01\) \\ P-Counaroylaginate & Alkaloids & 1.43 & 0.020 & \(-5.64\) \\ Quercetin-7-O-(6\({}^{\prime\prime}\)-malonyl)glucosyl-5-O-glucose & Flavonoids & 1.42 & 0.023 & \(-5.43\) \\ Quercetin-3-O-rutinoside-7-O-Hammoside & Flavonoids & 1.40 & 0.0251 & \(-5.31\) \\ Quercetin-3-O-xylosyl(1\(\to\)2)glucoseJ (1\(\to\)2)glucoseJ & Flavonoids & 1.40 & 0.0253 & \(-5.30\) \\ Quercetin-3-O-(6\({}^{\prime\prime}\)-O-malonyl)glucoseJ-5-O-glucose & Flavonoids & 1.38 & 0.031 & \(-5.00\) \\ \hline \hline \end{tabular}  
* VIP—Variable importance in projection value. 2 Positive and negative values for log\({}_{2}\)FC indicate upregulation and downregulation, respectively. Screening results were based on a comparison where ’NuMex LotaLutein’ was used as the control. Upregulation signifies that the relative content of the metabolite is higher in ’NuMex Pumpin Spice’ than in ’NuMex LotaLutein’ and vice versa.  
\end{table} 
Table 1: Top 10 most highly upregulated and downregulated metabolites. Figure 4: Pearson correlation coefficients among the biological replicates for the cultivars. Numbers 1, 2, and 3 correspond to the first, second, and third biological replications, respectively. Correlation coefficients were significant at \(p<0.0001\). The KEGG database was used in order to understand the metabolite content as a whole network in the form of metabolic pathways. A total of 89 differentially expressed metabolites had annotation from the KEGG database, where 36 metabolites were upregulated and 53 were downregulated (Table S2). The top 10 upregulated metabolites comprised mostly phenolic acids, whereas the top 10 downregulated metabolites with annotation consisted mostly of flavonoids (Table 2). Classification based on annotation from the KEGG database revealed that of the differentially expressed metabolites, 70 (78.65%) were annotated to be involved in metabolic pathways. A total of 42 (47.19%) and 17 (19.1%) metabolites were associated with the biosynthesis of secondary metabolites and of cofactors, respectively (Figure 5). The upregulated metabolites with KEGG annotation were classified into seven different groups that comprised organic acids, nucleotides and derivatives, and flavonoids, among others. Similarly, downregulated metabolites comprised eight different groups, including amino acids and derivatives, organic acids, alkaloids, and phenolic acids. Flavonoids, phenolic acids, organic acids, and nucleotides and derivatives comprised the top 10 differentially expressed upregulated metabolites with KEGG database map annotation.