The phytohormone abscisic acid (ABA) is one of the major determinant of seed dormancy in plants. Seed dormancy is gradually broken during dry storage in what is known as “after-ripening”, and this decay in dormancy is correlated with a decay in ABA sensitivity and content in seeds after imbibition. In this study we aim to investigate the effect of after-ripening on ABA signaling in our cereal model species, barley. Phosphosignaling networks in barley grains were investigated by a large-scale analysis of phosphoproteins to examine potential changes in response pathways to after-ripening. We used freshly harvested (FH) and after-ripened (AR) barley grains which showed different ABA sensitivity. In total 1,730 phosphopeptides were identified in barley embryos isolated from half-cut grains. Comparative analysis revealed that 329 and 235 phosphopeptides were up- or down-regulated respectively after ABA treatment, and phosphoproteomic profiles were quite different between FH and AR grains. These results were supported by peptide motif analysis which suggested that different sets of protein kinases are active in FH and AR grains. Furthermore, in vitro phosphorylation assays confirmed that some phosphopeptides were phosphorylated by SnRK2s, which are major protein kinases involved in ABA signaling. Taken together, our results revealed very distinctive phosphosignaling networks in FH and AR grains of barley, and suggested that the after-ripening of barley grains is associated with differential regulation of phosphosignaling pathways leading to a decay of ABA signaling.

Seed embryos were dissected from barley to remove storage protein in grain. Protein was extracted from the embryo and digested by trypsin. Phosphopeptides were enriched by hydroxy acid-modified metal oxide chromatography (HAMMOC) and analyzed by LC-MS system (AB-SCIEX TripleTOF 5600).

Two barley grains which have different dormancy were used for this analysis. Freshly harvested (FH) seed maintains strong dormancy, while dormancy is impaired in after-ripened (AR) seed.