# Transcriptional Regulatory Networks Oscillate Seasonal Plasticity of Fruit Metabolism in Melon
## 3 Results  
### Metabolomic Analysis of Melons Grown in Spring and Autumn  
Natural gradual changes in temperature and illumination were observed and recorded for melons grown in spring and autumn. The temperature of melons grown in spring showed an overall upward trend as time went by after pollination, while the opposite was observed in autumn (Figure 1a). Meanwhile, the difference in illumination between spring and autumn gradually increased (Figure 1b). This may lead to differences in phenology between the two seasons of melon fruits. We examined the metabolome of melon fruits at 40 DAP (Figure 1c) using a widely targeted metabolic analysis. Correlation analysis and principal component analysis of metabolites in three biological replicates showed reliable sampling of melon fruits, with strong intra-group repeat correlation during the same period (Figure S1a,b). The differentially accumulated metabolites (DAMs) of the melon fruits between the two seasons were determined based on the criteria of VIP \(\geq\) 1 and 1 Log2(Fold Change)\(\mid\)\(\geq\) 1. In total, 184 significant DAMs with 87 up-regulated and 97 down-regulated metabolites (Figure S1c) were detected between spring and autumn. These DAMs were classified into 10 categories (Figure 1d; Table S1), including phenolic acids (46, 25.0%), flavonoids (32, 17.3%), lipids (20, 10.8%), alkaloids (19, 10.3%), organic acids (19, 10.3%), amino acids and derivatives (18, 9.7%), nucleotides and derivatives (13, 7.0%), lignans and coumarins (6, 3.2%), terpenids (2, 1.0%), and others (9, 4.8%). Phenolic acids and flavonoids, both belong to phenylpropanoids, accounting for almost half (42.3%) of all DAMs. Summarized and sorted, all DAMs according to the 1 log2 (FoldChange)\(\mid\) value (Table S1), the content changes, and up-down information of the top30 DAMs can be visually observed using radar map and OPLS-DA S-plot (Figure S1d,e). Among them, mws0576 (organic acids, 3-hydroxybutyric acid), pme1975 (organic acids, malonic acid), mws1075 (lignans and coumarins, 7-methoxycoumarin), pme2122 (alkaloids, histamine), Zmdp000976 (amino acids and derivatives, S-(2-Carboxopropyl)cysteine), Zmgn002106 (amino acids and derivatives, N-Acetyl-L-phenylalanine), Jmwn002494 (phenolic acids, (2E)-3-[4-(\(\beta\)-D-glucopyranoside)-phenylacrylic]-acid), pmb3075 (phenolic acids, 3-O-p-Cumaroylshikimic acid), mws0954 (lipids, 5-Hydroxy-6,8,11,14-eicosaetraenoic acid), mws0145 (phenolic acids, O-Anisic acid) were only present in autumn melon fruits and were not detected in spring. On the contrary, Cmbn004127 (alkaloids, (S)-2-amino-5-((R)-1-carboxy-2-((E)-3-(4-hydroxy-3-methoxyphenyl)allylthio)ethyl-amino)-5-oxopentanoic acid), MWSmce355 (phenolic acids, methyl Syngnate), pmn001695 (phenolic acids, trihydroxycinnamoyl quinic acid), Lmln001195 (phenolic acids, picein) and two flavonoid compounds pme0321 (flavonols, kaempfer-7-O-Hammoside), and Lmxn007456 (flavones, acerosin) were only found in spring melon fruits (Table S1). In addition, the majority of flavonoid DAMs (29/32, 90.63%) showed up-regulation in spring, while out of all 87 DAMs up-regulated, there were 29 flavonoid metabolites and 28 phenolic acid metabolites. We speculate that differences in the composition and content of phenolic acids and flavonoids in spring fruits may be one of the factors leading to differences in the quality of spring and autumn melons. KEGG pathway enrichment analysis demonstrated that the DAMs were significantly enriched mainly in glutathione metabolism, histidine metabolism, glycerophospholipid metabolism, flavone and flavanol biosynthesis, and phenylpropanoid biosynthesis, and overall expression tended to be up-regulated in spring (Figure 1e; Table S2). Correspondingly, phenylpropanoid biosynthesis, flavone and flavonoid biosynthesis, and flavonoid biosynthesis were also highly represented pathways for up-regulated DAMs (Figure S1f).