Identification and Functional Characterization of Two Cytochrome P450 Reductases in Scutellaria barbata

Scutellaria barbata is a traditional Chinese medicinal herb rich in bioactive clerodane diterpenoid alkaloids with diverse pharmaceutical activities, yet their biosynthetic pathways remain largely unelucidated. Cytochrome P450 plays important roles in these pathways, and cytochrome P450 reductase (CPR) serves as an indispensable redox partner of P450s. Here we identified two CPRs, SbarCPR1 and SbarCPR2, from S. barbata. Phylogenetic analysis showed both CPRs belong to class II CPRs and are from two separate clades adjacent to the major clade containing typical class II CPRs. Functional characterization in yeast and plant systems demonstrated that both CPRs are active redox partners, supporting the catalytic activity of an 11-hydroxyferruginol synthase and producing comparable amounts of product. qRT-PCR analysis revealed distinct tissue-specific expression modes between the two CPRs: SbarCPR1 was uniformly expressed at relatively high levels across tissues, whereas SbarCPR2 was preferentially expressed in reproductive organs such as flowers and fruits. Upon MeJA treatment, SbarCPR2 responded much more rapidly than SbarCPR1, reaching its maximum expression at 6 h, compared to 24 h for SbarCPR1. Genome-wide co-expression analysis revealed that the two CPRs were co-expressed with distinct sets of P450 genes, which were enriched in both primary and secondary metabolisms. Further co-expression analysis with diterpenoid biosynthetic genes showed that SbarCPR1 was mainly co-expressed with abietane-type diTPS and P450 genes, whereas SbarCPR2 showed preferential co-expression with clerodane-type diTPS genes. Together, these results provide insights into the functional specialization of class II CPRs at the physiological level and highlight the association of SbarCPR2 with clerodane diterpenoid biosynthesis in S. barbata.