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 promoter of HcTPS1, regulating the metabolism of terpenoids. To further investigate the upstream molecular mechanisms that regulate the release of volatile compounds 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. In combination with transcriptome data, seven candidate HcPUB genes were identified and cloned with subsequent functional analysis. Yeast two-hybrid assay 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 motifs, which are implicated in plant growth and development. Subcellular localization indicated that HcPUB30 is localized in both the nucleus and cytoplasm. Quantitative real-time PCR analysis revealed that HcPUB30 exhibited the highest expression in petal tissues, and its expression peaked during floral senescence stage. 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.