A Quick Method to Analyze Peptide-Regulated Anthocyanin Biosynthesis.

Post-translationally modified peptides are now recognized as important regulators of plant stress responses. We recently identified the sulfated CLE-LIKE6 (CLEL6) peptide as a negative regulator of anthocyanin biosynthesis in dark-grown and in light-stressed Arabidopsis seedlings. The function of CLEL6 depends on proteolytic processing by subtilisin-like serine proteinase SBT6.1, and on tyrosine sulfation by tyrosylprotein sulfotransferase (TPST), and CLEL6 signaling relies on the ROOT MERISTEM GROWTH FACTOR 1 INSENSITIVE (RGI) receptor family. In this chapter, we describe in detail how to quantify peptide-regulated and stress-induced anthocyanin biosynthesis. We include protocols for peptide treatment of Arabidopsis seedlings and growth under different stress conditions, for the extraction and quantification of anthocyanins, and for the expression analysis of anthocyanin biosynthetic genes.