Transcriptome-wide Characterization of Novel WRKY Family Genes Engaged in Crocin Biosynthesis in Crocus sativus

Crocins comprise a family of hydrophilic carotenoids with pharmacological properties that are produced in significant quantities in stigma of Crocus sativus . Although the biosynthesis pathway of crocins has been sufficiently elucidated, there is a paucity of information regarding how transcription factors (TFs) regulate crocin biosynthesis in various stigma developmental stages. WRKY TFs play a role in modulating carotenoid/apocarotenoid metabolism. To provide an overview of the WRKY family in Crocus sativus (CsWRKY) and characterize candidate CsWRKY TFs involved in the biosynthesis of crocins, CsWRKY genes were identified from RNA-sequenced stigma at different developmental stages. A phylogenetic analysis was performed to characterize their evolutionary interrelatedness. A coexpression analysis of CsWRKY genes and crocin biosynthesis-related genes was performed. A quantitative real-time polymerase chain reaction was used to corroborate the expression level of CsWRKY TFs in various tissue and at different developmental stages. A total of 34 CsWRKY TFs were identified from the stigma of C. sativus . The CsWRKY TFs, together with their orthologs from Arabidopsis , were clustered into group I, II, or III following phylogenetic analysis. A correlation analysis revealed that the expressions of the TFs CsWRKY1, CsWRKY2, CsWRKY8, CsWRKY10, CsWRKY15, and CsWRKY28 were strongly related to the expression of crocin biosynthesis-related genes CsBCH , CsCCD2L , CsALDH , and CsUGT . CsWRKY2, CsWRKY15, and CsWRKY28 exhibited identical motifs and were stratified into group IId. Transcript levels of candidate CsWRKY genes were higher in stigma than in other tissues and were proportional to the crocin content.