Quantitation of Cell Type-Specific Responses to Brassinosteroid by Deep Sequencing of Polysome-Associated Polyadenylated RNA.

Hormonal signaling pathways control almost every aspect of plant physiology and development. Extensive analysis of hormonal signaling output, i.e., gene expression, has therefore been the focus of many studies. These analyses have been primarily conducted on total extracts derived from a mixture of tissues and cell types, consequentially limiting delineation of precise models. In this chapter, methods for tissue-specific functional genomics are overviewed, in which hormonal responses are analyzed at the transcriptional and the translational levels. Deep sequencing of polysome-associated polyadenylated RNA is employed for cell type-specific quantitation of translatome responses to brassinosteroids. Polysomes are purified by the previously established Translating Ribosome Affinity Purification (TRAP) method, in which the expression of a tagged ribosomal protein is targeted to the tissue of interest, allowing tissue-specific immunopurification of the polysome complexes. The methods presented assess establishment and selection of suitable transgenic lines. A protocol for hormonal treatment of the Arabidopsis thaliana root as a case study, TRAP and linear amplification of the purified polysome-associated polyadenylated RNA are described. Finally, a step-by-step presentation is included of the analysis of the RNA deep-sequencing data and Rscript for plotting hierarchically clustered heatmap of the expressed genes.

Translatome analyses capture of opposing tissue-specific brassinosteroid signals orchestrating root meristem differentiation.

The mechanisms ensuring balanced growth remain a critical question in developmental biology. In plants, this balance relies on spatiotemporal integration of hormonal signaling pathways, but the understanding of the precise contribution of each hormone is just beginning to take form. Brassinosteroid (BR) hormone is shown here to have opposing effects on root meristem size, depending on its site of action. BR is demonstrated to both delay and promote onset of stem cell daughter differentiation, when acting in the outer tissue of the root meristem, the epidermis, and the innermost tissue, the stele, respectively. To understand the molecular basis of this phenomenon, a comprehensive spatiotemporal translatome mapping of Arabidopsis roots was performed. Analyses of wild type and mutants featuring different distributions of BR revealed autonomous, tissue-specific gene responses to BR, implying its contrasting tissue-dependent impact on growth. BR-induced genes were primarily detected in epidermal cells of the basal meristem zone and were enriched by auxin-related genes. In contrast, repressed BR genes prevailed in the stele of the apical meristem zone. Furthermore, auxin was found to mediate the growth-promoting impact of BR signaling originating in the epidermis, whereas BR signaling in the stele buffered this effect. We propose that context-specific BR activity and responses are oppositely interpreted at the organ level, ensuring coherent growth.

Root growth is modulated by differential hormonal sensitivity in neighboring cells.

Coherent plant growth requires spatial integration of hormonal pathways and cell wall remodeling activities. However, the mechanisms governing sensitivity to hormones and how cell wall structure integrates with hormonal effects are poorly understood. We found that coordination between two types of epidermal root cells, hair and nonhair cells, establishes root sensitivity to the plant hormones brassinosteroids (BRs). While expression of the BR receptor BRASSINOSTEROID-INSENSITIVE1 (BRI1) in hair cells promotes cell elongation in all tissues, its high relative expression in nonhair cells is inhibitory. Elevated ethylene and deposition of crystalline cellulose underlie the inhibitory effect of BRI1. We propose that the relative spatial distribution of BRI1, and not its absolute level, fine-tunes growth.