Conserved regions upstream of BRC1B regulate bud dormancy in tomato

Plant architecture is majorly influenced by shoot branching through the development of axillary meristems in the leaf axils. These meristems develop into axillary buds, which are kept dormant until endogenous or exogenous cues allow their activation. The key TCP class II transcription factor (TF) BRANCHED1 (BRC1) regulates the early stages of bud outgrowth. <i>BRC1</i> expression is highly specific to axillary buds, where it inhibits outgrowth by integrating hormonal, nutrient, and environmental signals. While the function of <i>BRC1/TB1</i> genes is highly conserved in numerous species, the <i>cis</i>- and <i>trans</i>-regulation of <i>BRC1</i> expression remains poorly understood. In this study, we explored how modifications in the <i>SlBRC1B</i> promoter affect bud outgrowth in tomato. We identified four highly conserved regions (<i>CR1 to CR4</i>) in the sequence upstream of the <i>SlBRC1B</i> translation start site by performing a phylogenetic footprinting. A collection of promoter mutants was generated by separately targeting each <i>CR</i> using CRISPR/Cas9. These <i>CR</i> mutants were employed for a detailed bud outgrowth characterization to investigate the effect of mutations on SlBRC1B-mediated bud dormancy. Most <i>CR</i> mutants consistently showed decreased bud outgrowth, suggesting that <i>SlBRC1B</i> is under tight control of various transcriptional repressors. Screening <i>CR4</i> with an axillary bud-specific cDNA library in a Yeast one-hybrid assay identified MYB, GRF, NAC, MADS, and zinc finger TF family members. Based on our findings, we concluded that the identified <i>CRs</i> play a crucial role in regulating <i>SlBRC1B</i> expression, and that they could be strategically targeted to achieve a desired level of shoot branching.