Abscisic acid (ABA) is important for land plants to survive in terrestrial conditions. Protein phosphorylation/dephosphorylation has a central role in ABA signaling, mediated by protein phosphatase 2C (PP2C) and SNF1-related protein kinase 2 (SnRK2). Recently, we are taking a phosphoproteomic approach to elucidate the ABA signaling network in multi-species of land plants, such as Arabidopsis1), Physcomitrella2) and barley3).
For example, Physcomitrella patens is a model species of basal land plants. Our phosphoproteomic analysis detected 4,630 phosphopeptides from P. patens wild-type and two ABA-responsive mutants, such as a PP2C disruptant (ppabi1a/b), and an ARK-defective mutant (AR7), identified as an upstream regulator of SnRK2. Our data revealed: 1) the entire ABA-responsive phosphoproteome in P. patens was quite diverse, 2) P. patens PP2C regulated multiple pathways in addition to the ABA response, and 3) ARK is mainly involved in ABA signaling.
We also obtained a dataset of phosphoproteomic profiles in barley seeds. Seed dormancy is an important trait for agricultural production, and it changes significantly during after-ripening process. We treated ABA to fresh seeds and after-ripened seeds of barley (cv. Golden Promise), and compared their phosphoproteomic profiles. The data suggested that ABA response in fresh seeds could be more active than that in after-ripened seeds at signaling level, suggesting that ABA signaling is somehow impaired by after-ripening process.
On the other hand, recently we identified Raf36 from a protein-protein interaction screening for SnRK2. Biochemical analysis revealed that SnRK2 phosphorylates N-terminal region of Raf36, and reverse-genetic analysis showed that Raf36 negatively regulates ABA response. Furthermore, we found Raf22 functions redundantly with Raf36. Now we are trying to identify substrates of Raf36 or Raf22 by phosphoproteomic analysis.