Brassinosteroids Regulate Plant Growth through Distinct Signaling Pathways in Selaginella and Arabidopsis

Brassinosteroids (BRs) are growth-promoting steroid hormones that regulate diverse physiological processes in plants. Most BR biosynthetic enzymes belong to the cytochrome P450 (CYP) family. The gene encoding the ultimate step of BR biosynthesis in Arabidopsis likely evolved by gene duplication followed by functional specialization in a dicotyledonous plant-specific manner. To gain insight into the evolution of BRs, we performed a genomic reconstitution of Arabidopsis BR biosynthetic genes in an ancestral vascular plant, the lycophyte Selaginella moellendorffii. Selaginella contains four members of the CYP90 family that cluster together in the CYP85 clan. Similar to known BR biosynthetic genes, the Selaginella CYP90s exhibit eight or ten exons and Selaginella produces a putative BR biosynthetic intermediate. Therefore, we hypothesized that Selaginella CYP90 genes encode BR biosynthetic enzymes. In contrast to typical CYPs in Arabidopsis, Selaginella CYP90E2 and CYP90F1 do not possess amino-terminal signal peptides, suggesting that they do not localize to the endoplasmic reticulum. In addition, one of the three putative CYP reductases (CPRs) that is required for CYP enzyme function co-localized with CYP90E2 and CYP90F1. Treatments with a BR biosynthetic inhibitor, propiconazole, and epi-brassinolide resulted in greatly retarded and increased growth, respectively. This suggests that BRs promote growth in Selaginella, as they do in Arabidopsis. However, BR signaling occurs through different pathways than in Arabidopsis. A sequence homologous to the Arabidopsis BR receptor BRI1 was absent in Selaginella, but downstream components, including BIN2, BSU1, and BZR1, were present. Thus, the mechanism that initiates BR signaling in Selaginella seems to differ from that in Arabidopsis. Our findings suggest that the basic physiological roles of BRs as growth-promoting hormones are conserved in both lycophytes and Arabidopsis; however, different BR molecules and BRI1-based membrane receptor complexes evolved in these plants.