Postharvest broccoli is prone to yellowing during storage, which is the key factor leading to a reduction in value. To explore appropriate control methods, it is important to understand the mechanisms of yellowing. We analyzed the genes related to the metabolism of chlorophyll, carotenoids, and flavonoids and the transcription factors (TFs) involved in broccoli yellowing using transcriptome sequencing profiling. Broccoli stored at 10 °C showed slight yellowing on postharvest day 5 and serious symptoms on day 12. There were significant changes in chlorophyll fluorescence kinetics, mainly manifesting as a decrease in the Fv/Fm value and an increase in nonphotochemical quenching, during the yellowing process. Transcriptome sequencing profiles from samples of fresh broccoli and broccoli with slight and severe yellowing revealed 6, 5, and 4 differentially expressed genes involved in chlorophyll metabolism, carotenoid biosynthesis, and flavonoid biosynthesis, respectively. The transcription factor gene ontology categories showed that the MYB, bHLH, and bZip gene families were involved in chlorophyll metabolism. In addition, the transcription factor families included NACs and ethylene response factors (ERFs) that regulated carotenoid biosynthesis. Reverse transcription polymerase chain reaction further confirmed that bHLH66, PIF4, LOB13, NAC92, and APL were vital transcription factors that potentially regulated the CAO and HYD genes and were involved in chlorophyll metabolism and the carotenoid biosynthetic process. The flavonoid biosynthetic pathway was mainly regulated by MYBs, NACs, WRKYs, MADSs, and bZips. The results of the differentially expressed gene (DEG) and pigment content analyses indicated that the transcriptome data were accurately and positively associated with broccoli yellowing.

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