• Broccoli HSPs were identified by RNA-sequencing and de novo assembly. • Broccoli HSP expression at basal state and during storage was investigated. • Treatment of 17-AAG induced accumulation of HSP in broccoli. • Treatment of 10 nM 17-AAG alleviates senescence of broccoli. Heat shock proteins (HSPs) are chaperones that elicit stress tolerance in plants. Treatments that induce HSPs accumulation have been widely used as a mean to improve stress tolerance and extend shelf life of postharvest produces. Broccoli is a vegetable appreciated worldwide for its high nutrition value. However, the quality of harvested broccoli deteriorates quickly due to rapid senescence. Here, we examined the expression of broccoli HSP and investigated the effect of 17-(allylamino)-17-demethoxygeldanamycin (17-AAG), an HSP inducer, on broccoli senescence. Through RNA-sequencing and de novo assembly of the broccoli transcriptome, 11 HSP and related genes were identified. The expression of ClpB1 , HSC70 , GRP78 , HSP18.1 , and HSP17.6CII were upregulated in broccoli during 20 °C storage. Treatment of 1–100 nM 17-AAG dose-dependently induced HSP101 and HSP70 proteins accumulation, while treatment of 10 nM 17-AAG induced the expression of all 11 HSP and related genes and substantially alleviated yellowing of broccoli. In addition, 10 nM 17-AAG treatment significantly suppressed the expression of chlorophyll catabolic genes NYC1 , CLH1 , and PAO. Taken together, we provide the first report of the broccoli HSP and demonstrate the potential of using HSP inducers such as 17-AAG to tune up HSP level and enhance stress tolerance in postharvest crops. [ABSTRACT FROM AUTHOR]