# Transcriptional Regulatory Networks Oscillate Seasonal Plasticity of Fruit Metabolism in Melon
## 3 Results
### Transcriptomic Analysis of Melons Grown in Spring and Autumn  
To investigate the mechanism of transcriptional plasticity caused by seasonal variation on the formation of differences in metabolites of melon fruits, transcriptomic analysis was performed in melon fruits at five stages (0, 10, 20, 30, and 40 DAP) in spring and autumn. Transcriptome sequencing yielded a total of 240.13 Gb of clean data after filtration. More than 91.19% of the clean reads were sequentially mapped to the _C. melo_ genome DHL 92 genome (Table S3). The percentages of Q30 and GC content in each library exceeded 96.43% and 43.20%, respectively (Table S4), indicating the high-quality transcriptome data that can be used for further DEG analysis. PCA of the FPKM values for all expressed genes revealed the relationships between samples from different seasons and stages (Figure S2a). A total of 16,525 DEGs were identified in melon fruits at different developmental stages between spring and autumn. The Venn diagram showed the DEGs detected at each stage (Figure S2b). We constructed protein-protein interaction (PPI) networks using Arabidopsis genes blasted by melon DEGs, and identified potential core networks containing central hub genes using k-means cluster analysis. At 0 DAP, a total of 3005 DEGs were used in the analysis, resulting in 220 genes forming the putative hub cluster. Similarly, 2200, 853, 1772, and 1626 DEGs were imported into 10, 20, 30, and 40 DAP analyses, respectively, and yielded a main cluster consisting of 242, 66, 210, and 122 nodes. Functional enrichment analysis (FDR < 0.05) on gene clusters was performed using the KEGG database and GO-biological process to reveal a PPI network comprising potential hub genes and their associated functional networks (Tables S5-S9). By comparing different seasons at the same stage, distinct enrichment pathways were projected to correspond to diverse phenotypic traits. Five-stage gene sets all showed (Figures S3-S6 and 2b) that cellular process (GO: 0009987) and metabolic process (GO: 0008152) were the pathways enriched with the highest number of DEGs, and they were most significantly enriched at 0, 10, and 30 DAP (Tables S5-S9), suggesting that these pathways were representative in the development process of fruit ripening affected by seasonal changes and were dynamically regulated at the transcriptional level. Differences in genes related to photosynthesis (GO: 0015979) began to appear from the 0 DAP stage and continued until the ripening stage, 40 DAP. There were also pigment biosynthesis/metabolic processes (GO: 0046148/GO: 0042440), which mainly consist of two types of pigments, chlorophyll and carotenoid biosynthesis/metabolic processes, and showed different significance in the biosynthesis or metabolism of different pigments according to different periods. Carbon metabolism (ath01200) was also a functional pathway that cannot be ignored during the developmental process, and its inclusion of carbon fixation in photosynthetic organizations (ath00710) suggested changes in the flow of carbon source metabolism caused by environmental signals. These same processes were reflected in the concentration of DEGs at different stages, indicating that these functions always played a role during development. In addition to the pathways commonly present in various stages of the network mentioned above, genes related to cell division (GO: 0051301) and mitotic cell cycle (GO: 0051301) were significantly enriched in the core network of 0 DAP and up-regulated in spring. Moreover, the enrichment pathways detected include the hormone-mediated signaling pathway (GO: 0009755), MAPK signaling pathway (ath04016), and phenylpropanol biosynthetic process (ath00280), as well as precursor substances of melon, and volatiles such as valine, leucine, and isoleucine degradation (GO: 0009699) [23]. It can be seen that the key metabolic pathway, the phenylpropanolid pathway, detected in the metabolome data was transcriptionally regulated at this stage.