Time-Series Transcriptomics Reveals Photoperiod-Responsive Regulatory Networks in Soybean Cotyledons

Abstract Photoperiod is a major environmental cue that regulates flowering, maturity, and ecological adaptation in soybean. Although soybean photoperiodic regulation has been extensively studied in true leaves and whole plants, the contribution of cotyledons, the first light-exposed organs after seedling emergence, remains poorly understood. In this study, we used cotyledons of the photoperiod-sensitive soybean cultivar ‘Zigongdongdou’ to investigate early transcriptional responses to day length. Seedlings were grown under short-day conditions, 12 h light/12 h dark, or long-day conditions, 16 h light/8 h dark, and cotyledons were collected every 4 h during the first 72 h after transfer from darkness to photoperiodic conditions, generating a 19-time-point RNA-seq dataset. A total of 663 differentially expressed genes were identified between short-day and long-day conditions, including 340 upregulated and 323 downregulated genes. Functional enrichment analysis showed that these genes were mainly associated with photoperiodic flowering, circadian regulation, hormone signaling, oxidative stress responses, glutathione metabolism, and primary metabolism. Core photoperiod-related genes, including photoreceptors, circadian clock genes, CONSTANS-like genes, E1-family genes, and FT genes, displayed clear photoperiod-dependent expression patterns in cotyledons. Weighted gene co-expression network analysis combined with predicted regulatory network analysis further identified C2H2, WRKY, ERF, NAC, Dof, MYB, and TCP transcription factors as candidate regulators associated with photoperiod-responsive modules. In particular, LATE and several GmNAC genes were connected with flowering- and hormone-related modules, suggesting possible links between transcription factor regulation and the COL–FT pathway. These results demonstrate that soybean cotyledons establish an active photoperiod-responsive transcriptional network shortly after emergence. Cotyledon expression profiling captures major regulatory features of the soybean photoperiod pathway and provides a compact, time-saving, and space-efficient system for investigating early photoperiodic regulation in soybean.