Time-Resolved Transcriptomic and Widely Targeted Metabolomic Profiling of Tomato Seedlings During Short-Term Chilling

(1) Background: Chilling is a major environmental constraint on tomato growth and development, yet the temporal coordination of physiological, transcriptional, and metabolic responses remains insufficiently characterized. (2) Methods: Tomato seedlings were subjected to 4 °C chilling treatment for 0, 1, 3, 6, 12, and 24 h. We measured antioxidant enzyme activities and malondialdehyde (MDA) content, and performed transcriptomic and widely targeted metabolomic analyses to dissect time-course responses. qRT-PCR was used to validate gene-expression profiles. (3) Results: Superoxide dismutase (SOD) and catalase (CAT) activities, together with MDA content, were rapidly altered at early chilling stages, while peroxidase (POD) activity markedly accumulated at 12 and 24 h, showing divergent physiological response patterns. In contrast to the 0 h control, 4819, 4471, 4442, 5742, and 8701 differentially expressed genes (DEGs) were detected at 1, 3, 6, 12, and 24 h, among which 945 DEGs were common across all time-point comparisons. The pronounced rise in DEG count at 24 h reflected large-scale late-stage transcriptional reprogramming. Correspondingly, 262, 173, 170, 247, and 195 differentially abundant metabolites (DAMs) were identified, with merely 26 shared DAMs across all five time points and the maximum DAM number occurring at 1 h, demonstrating fast but non-monotonic metabolic shifts. A pathway-level comparison pinpointed carbon metabolism, phenylpropanoid biosynthesis, and tyrosine metabolism as key perturbed pathways. qRT-PCR results generally validated the RNA-seq expression trends of eight selected genes and showed a significant positive correlation with RNA-Seq data (r = 0.852, p < 0.01). (4) Conclusions: This study demonstrates stage-dependent and partially asynchronous physiological, transcriptional, and metabolic responses upon short-term chilling in tomato seedlings. Candidate pathways, genes, and metabolites uncovered herein provide valuable resources for further functional characterization of tomato cold responses.