Temporal and genotype-dependent transcriptomic responses to cold treatment and recovery in winter rapeseed (Brassica rapa L.).

Winter rapeseed (Brassica rapa L.) is exposed to recurrent low-temperature episodes, yet cultivar differences and reference-genome choice can complicate the interpretation of transcriptomic variation. Here, we reanalyzed 24 growth-point RNA-seq samples from the cold-tolerant cultivar 'Longyou-7' and the cold-sensitive cultivar 'Lenox' under control conditions (CK), after 3 and 24 h at 4 °C, and after 24 h of recovery at 22 °C. Transcriptome analysis was performed using the chromosome-level Longyou-7 reference genome and compared with results obtained using Chiifu v1.5. Functional analyses revealed stage-dependent transcriptional changes involving photosynthesis, lipid metabolism, hormone signaling, circadian regulation, sulfur and amino acid metabolism, and other stress-related processes. Glutathione metabolism was represented among the top-ranked pathways in several within-genotype contrasts, and genotype-specific co-expression networks linked glutathione-related transcription with antioxidant enzyme activities, soluble sugar, and ROS-associated traits. Biochemical measurements further showed stage-dependent changes in GSH and GSSG contents and GST activity, with 'Longyou-7' maintaining a higher GSH/GSSG ratio and higher GST activity than 'Lenox' during cold treatment. qRT-PCR analysis of 22 candidate genes showed overall concordance with RNA-seq (R² = 0.736), and 17 candidates contained conserved GST_N and GST_C domains, including nine Tau and eight Phi GSTs. Relative to Chiifu v1.5, the Longyou-7 reference increased uniquely mapped read rates by 1.28-1.69 percentage points in Longyou-7 samples, whereas differences in 'Lenox' ranged from -0.26 to 0.30 percentage points, while shared DEG responses remained highly concordant between the two reference backgrounds. Together, these results reveal stage-dependent transcriptional and biochemical adjustment during cold treatment and recovery and identify GSH/GST-related genes as candidates for further functional analysis in winter rapeseed.