Genome-wide analysis of WRKY gene family in high-CBD hemp (Cannabis sativa L.) and identification of the WRKY genes involved in abiotic stress responses and regulation cannabinoid accumulation

Cannabinoids are vital metabolites produced by Cannabis sativa. The growing market demand for cannabinoids has made C. sativa a prominent research focus. The biosynthetic pathway of cannabinoids has been extensively investigated, but the information on the transcription factors (TFs) involved in cannabinoids biosynthesis remains limited. In this study, a total of 48 CsWRKYS were identified in high-cannabidiol (CBD) hemp using a combination of two-way blast, plantTFDB prediction, and CDD batching methods. These CsWRKYs exhibited uneven distribution across ten chromosomes, and encoded 162-728 amino acids. Phylogenetic analysis revealed that CsWRKY genes were categorized into three groups, with group II further divided into five subgroups. Members within each subfamily exhibited similar conserved motifs and exon-intron structures. Furthermore, gene expression analysis in various tissues, accompanied by cis-regulatory elements analysis, and alternative splicing analysis, provided insight into the potential roles of CsWRKYs in hemp growth and development. Notably, the expression of group III CsWRKY genes (CsWRKY1/2/20/26/28) was induced by salt, drought, and cold stress treatments. Metabolic analysis revealed an increase in the contents of cannabinoids under these treatments. Furthermore, CsWRKY20 and CsWRKY28 were proved to be positive regulatory factors of cannabinoids biosynthesis pathway. Altogether, the results of this study significantly contributes to understand the function of WRKY genes in response to abiotic stress, and their involvement in the transcriptional regulation of cannabinoid biosynthesis in high-CBD hemp.