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

• Published in: Physiologia plantarum Vol. 176; no. 1
• Main Authors: Tayengwa, Reuben, Westenskow, Shelby R., Peng, Hao, Hulbert, Anna K., Neff, Michael M.
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Publishing Ltd 01-01-2024; Wiley Subscription Services, Inc
• Subjects: Alleles; Biosynthesis; Brassinosteroids; CRISPR; Cytochrome P450; Cytochromes P450; Deactivation; Deoxyribonucleic acid; DNA; Epigenetics; Flowering; Genetic analysis; Genomes; Germination; Hormones; Inactivation; Insertion; Linkage analysis; Mutants; Phenotypes; Plant growth; Seed germination; Seedlings
• DOI: 10.1111/ppl.14141
• EISSN: 1399-3054
• ISSN: 0031-9317

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