Genetic interactions between BEN1- and cytochrome P450-mediated brassinosteroid inactivation

Authors: TAYENGWA, REUBEN - University Of Maryland; WESTENSKOW, SHELBY - Washington State University; Peng, Hao; HULBERT, ANNA - Washington State University; NEFF, MICHAEL - Washington State University

Submitted to: Physiologia Plantarum
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/5/2023
Publication Date: 1/2/2024
Citation: Tayengwa, R., Westenskow, S.R., Peng, H., Hulbert, A.K., Neff, M.M. 2024. Genetic interactions between BEN1- and cytochrome P450-mediated brassinosteroid inactivation. Physiologia Plantarum. 176(1). Article e14141. https://doi.org/10.1111/ppl.14141.
DOI: https://doi.org/10.1111/ppl.14141

Interpretive Summary: Plant steroid hormones (BRs) regulate seed germination, flowering, growth and development, disease resistance, and abiotic (drought, salinity, heat, cold, flooding, etc.) stress tolerance. Unlike other plant hormones, BRs do not undergo long-distance transport. Thus, their biosynthesis and metabolism pathways are tightly regulated at the cell- and tissue-specific levels. We call this mechanism BR homeostasis. In the model plant Arabidopsis thaliana,multiple enzymes are known to inactivate BRs with distinctive biochemical activities. They are also largely conserved across plant species. In this research, we used available Arabidopsis T-DNA insertional mutants and the prevalent CRISPR-Cas9 genome editing system to generate the triple-null mutant of three BR-inactivating genes BAS1, SOB7, and BEN1. This triple-null mutant bas1-2 sob7-1 ben1-3 showed a long-hypocotyl phenotype with additive genetic contributions from the three genes. In addition, BEN1 regulates seedling cotyledon petiole elongation, which can only be disclosed in the absence of both BAS1 and SOB7. Unlike BAS1 or SOB7, BEN1 does not contribute to plant early flowering. Overall, our work reveal the overlapping and independent roles of BR inactivating genes BAS1, SOB7, and BEN1.

Technical Abstract: Brassinosteroids (BRs) are essential plant growth-promoting hormones 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. Generating higher-order null mutants in these genes has been hampered by epigenetic interactions between T-DNA insertion alleles and genetic linkage. For example, 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 no longer confer a null-allele. We used the CRISPR-Cas9 genome editing system to alleviate this problem by generating the bas1-2 sob7-1 ben1-3 triple-null mutant. This triple-null mutant resulted in an additive seedling long-hypocotyl phenotype. In contrast, the triple-null mutant 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 that BAS1 and SOB7 redundantly control. Our results show that BAS1, BEN1, and SOB7 have overlapping, and independent roles based on differential spatio-temporal tissue expression patterns.