Brassinosteroids regulate petal spur length in Aquilegia by controlling cell elongation

Background and Aims Aquilegia produce elongated, three-dimensional petal spurs that fill with nectar to attract pollinators. Previous studies have shown that the diversity of spur length across the Aquilegia genus is a key innovation that is tightly linked with its recent and rapid diversification into new ranges, and that evolution of increased spur lengths are achieved via anisotropic cell elongation. Previous work identified a brassinosteroid response transcription factor as being enriched in the early developing spur cup. Brassinosteroids (BRs) are known to be important for cell elongation, suggesting that brassinosteroid-mediated response may be an important regulator of spur elongation and potentially a driver of spur length diversity in Aquilegia. In this study, we investigated the role of brassinosteroids in the development of the Aquilegia coerulea petal spur. Methods We exogenously applied the biologically active BR brassinolide to developing petals spurs to investigate spur growth under high hormone conditions. We used virus induced gene silencing and gene expression experiments to understand the function of brassinosteroid-related transcription factors in Aquilegia coerulea petal spurs. Key Results We identified a total of three Aquilegia homologs of the BES1/BZR1 protein family and found that these genes are ubiquitously expressed in all floral tissues during development, yet consistent with the previous RNAseq study, we found that two of these paralogs are enriched in early developing petals. Exogenously applied brassinosteroid increased petal spur length due to increased anisotropic cell elongation as well as cell division. We found that targeting of the AqBEH genes with VIGS resulted in shortened petals, a phenotype caused in part by a loss of cell anisotropy. Conclusions Collectively, our results support a role for brassinosteroids in anisotropic cell expansion in Aquilegia petal spurs and highlight the BR pathway as a potential player in the diversification of petal spur length in Aquilegia.