The development and growth of the plant body are regulated by aninterplay between plant hormone signaling pathways that both interpret anddetermine their accumulation and distribution. Brassinosteroids (BRs) are oneof these hormones. Although the BR signaling pathway is well known, there arestill gaps in our knowledge regarding the site of BR production, the extent ofBR movement, and how its signaling pathway is decoded at the tissue andcellular levels. Using the Arabidopsis root as a developmental system, recentstudies showed that BR biosynthesis genes are expressed in different tissues,suggesting that the BR hormone might move radially between tissues. Inaddition, BR signaling has been shown to have different developmental effectson the root meristem, depending on the tissue in which it is perceived,suggesting that BR signaling is decoded in a tissue-specific manner. Thisthesis was conducted with the following open questions in mind: do activehormones or their precursors move between tissues in the root, istissue-specific BR signaling essential for meristem development, and howcellular BR activity can be quantified in the growing root.
To address these questions, I applied established approaches and alsoevaluated new strategies. These were designed to cover the three main steps ofthe hormone action: i) biosynthesis, ii) signaling input (i.e., receptoractivity), and iii) signaling output (i.e., regulation of gene expression). Forthe first part, I took a tissue-specific-complementation approach for BRproduction to evaluate the extent of BR movement in the root meristem. This wasperformed alongside the characterization of the root system architecture andits dependency on BR biosynthesis. For studies involving BR signaling input, Iapplied a novel approach for a tissue-specific knockout using the CRISPR-CAS9technology. This tool aimed to target the BR receptor BRI1 in select tissues asa means to infer the essentiality of BRI1’s activity in a particular tissue inthe meristem. Finally, I started to develop a spatiotemporal transcriptionalreadout for BR signaling output as part of a collaborative effort. Thisstrategy is based on designing a promoter sequence that includes conservedmotifs from promoters activated as a response to BR presence. This promoter isfused to nuclear localization fragment and fluorophore to detect BR responsesites in the meristem different tissues of the root. The results of theseapproaches will be presented and discussed.