Ubiquitin Ligase HcPUB30 Targets HcWRKY1 to Regulate Monoterpenoids Synthesis in Hedychium coronarium

Hedychium coronarium , a perennial herb belonging to the genus Hedychium Koenig within the family Zingiberaceae, is renowned for its pleasant fragrance. The volatile compounds of flowers are primarily terpenoids, which are catalyzed by terpenoid synthase (TPS). Earlier studies have shown that HcWRKY1 transcription factor can bind to the promoters of HcTPS1 , regulating the metabolism of terpenoids. To further investigate the upstream molecular mechanism regulating volatile release in Hedychium , we focused on a crucial U-box type of E3 ubiquitin ligase involved in regulating transcription factors. This study utilized genomic data to identify HcPUB gene family. Seven candidate HcPUB genes were identified and cloned with subsequent functional analysis and transcriptome data integration. Yeast two-hybrid demonstrated that HcPUB30 was the sole interactor of HcWRKY1 among the seven HcPUB candidates. In vivo and in vitro ubiquitination assays demonstrated that HcPUB30 ubiquitinates and promotes the degradation of HcWRKY1 via the 26S proteasome pathway. Multi-alignment analysis revealed that HcPUB30 possesses a conserved U-box domain and ARM motif, which are implicated in plant growth and development. Subcellular location studies indicated that HcPUB30 is localized in both the nucleus and cytoplasm. Quantitative real-time PCR analysis revealed elevated HcPUB30 expression in H. coronarium petals, particularly during senescence. Virus-induced gene silencing of HcPUB30 in Hedychium petals resulted in a significant decrease in monoterpenoid content, accompanied by a significant reduction in the relative expression levels of HcWRKY1 and HcTPS1 . These findings indicate that HcPUB30 participates in the regulation of monoterpenoid biosynthesis by mediating HcWRKY1 in Hedychium petals.