Awakening buds: navigating tomato axillary bud dormancy from the BRC1 hub

Samenvatting

Plants exhibit remarkable plasticity, continuously growing and regenerating to cope with various challenges. This capacity is facilitated by meristematic cells, serving as reservoirs, allowing, when activated, the addition of new organs and axes both above and below ground. Specifically, axillary meristems support continuous lateral growth. The precise timing of their activation is critical as prioritizing the growth of the primary shoot ensures reproductive success and survival. Plants have evolved a complex and robust system that responds to and regulates both external and internal cues, ensuring optimal timing for bud outgrowth. Central to this regulatory network is the TEOSINTE BRANCHED 1/CYCLOIDEA/PCF1 (TCP) transcription factor (TF) BRANCHED1/TEOSINTE BRANCHED 1 (BRC1/TB1), which plays a sophisticated role in inhibiting bud outgrowth, thereby ensuring favorable conditions for growth activation. This thesis explores the mechanism of dormancy regulation through BRC1 in tomato axillary buds. Chapter 2 aims to dissect cis-acting elements and their associated trans-regulatory factors in the BRC1 promoter. I explore the evolutionary trajectories of sequences upstream and downstream of BRC1-like genes in Solanaceae and other angiosperms. This chapter reveals four highly conserved regions (CRs) in the upstream sequences of BRC1 genes of tomato. Subsequently, we employ Yeast one-hybrid (Y1H) screens between CRs and an axillary bud-specific cDNA library to identify putative regulators. The results highlight members of MYB, zinc finger, NAC, and MADS transcription factor families as putative regulators. Moreover, Arabidopsis homologs of these TFs are involved in light and ABA-mediated signals, suggesting that these TFs could integrate these signals at the BRC1 promoter. Chapter 3 explores the in vivo function of identified CRs in BRC1-mediated bud dormancy. I employed CRISPR/Cas9-mutagenesis on individual CRs to create a promoter mutant library. The detailed bud growth and development analysis in these mutants reveals that disruption of CR sequences led to decreased bud outgrowth. This observation suggests that the binding sites of repressor TFs were effectively removed. Comparative analysis of mutant and wild-type CR sequences indicates the elimination of consensus binding sites for MYB, zinc finger, SBP, and TCP TF families, potentially leading to the removal of their repressive activity on SlBRC1B. Mutations in two regions, CR2 and CR4, directly affected SlBRC1B expression levels. In other CR mutants with significant effects on axillary bud outgrowth, no effect on SlBRC1B expression could be detected by RT-qPCR, suggesting altered spatiotemporal expression rather than absolute expression levels. These findings underscore the critical role of CRs in finetuning SlBRC1B activity, which is essential for precise shoot branching regulation through these putative regulatory sequences. Chapter 4 scrutinizes the downstream targets and pathways acting parallel to BRC1-mediated dormancy regulation to investigate this molecular network. I studied the transcriptome of dormant and activated buds following decapitation, focusing on genes explicitly expressed in the buds. These results highlight a highly conserved network including cytokinin-, ABA-, cell wall-, nitrogen- and GA- associated signals. Bud outgrowth phenotypes of knockout and knockdown mutants for selected candidates underline the involvement of multiple genes and signals in regulating dormancy, confirming the complexity of the network.