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

Abstract

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.