Genetic Interactions Between BEN1-and Cytochrome P450-Mediated Brassinosteroid Inactivation

Brassinosteroids (BRs) are essential plant growth-promoting hormones involved in many processes throughout plant development, from seed germination to flowering time. Since BRs do not undergo long-distance transport, cell- and tissue-specific regulation of hormone levels involves both biosynthesis and inactivation. To date, ten BR-inactivating enzymes, with at least five distinct biochemical activities, have been experimentally identified in the model plant Arabidopsis thaliana. Epigenetic interactions between T-DNA insertion alleles and genetic linkage have hindered analysis of higher-order null mutants in these genes. A previous study demonstrated that the bas1-2 sob7-1 ben1-1 triple-null mutant could not be characterized due to epigenetic interactions between the exonic T-DNA insertions in bas1-2 and sob7-1, causing the intronic T-DNA insertion of ben1-1 to revert to a partial loss-of-function allele. We used CRISPR-Cas9 genome editing to avoid this problem and generated the bas1-2 sob7-1 ben1-3 triple-null mutant. This triple-null mutant resulted in an additive seedling long-hypocotyl phenotype. We also uncovered a role for BEN1-mediated BR-inactivation in seedling cotyledon petiole elongation that was not observed in the single ben1-2 null mutant but only in the absence of both BAS1 and SOB7. In addition, genetic analysis demonstrated that BEN1 does not contribute to the early-flowering phenotype, which BAS1 and SOB7 redundantly regulate. Our results show that BAS1, BEN1, and SOB7 have overlapping and independent roles based on their differential spatiotemporal tissue expression patterns