The signaling pathway of the phytohormone abscisic acid (ABA) regulates responses towards abiotic stress such as drought and high osmotic conditions. The multitude of functionally redundant components involved in ABA signaling, poses a major challenge for elucidating the largely unresolved response selectivity. We decided rebuilding single linear ABA signaling pathways in yeast for combinatoric permutation of ABA receptors and coreceptors, as well as the response-mediating SnRK2 protein kinases and their targeted transcription factors to drive luciferase expression in a heterologous host. We show that SnRK2s differ in the regulation by ABA receptor complexes, affect ABA responsiveness of the pathway, and differ in their transactivation activity but have similar preferences for ABA-responsive transcription factors. SnRK2s thought to act ABA-independently and known to be activated under osmotic stress in plants were regulated by ABA receptor complexes in yeast and competed with ABA receptor components in an ABA-dependent manner in plant tissue. The study reveals the suitability of the yeast system for analysis of ABA signaling factors and allowing the future dissection of ligand-receptor specificities in a functional response pathway. The analysis provides new insights into SnRK2 regulation indicating that four SnRK2 members of the osmotic stress response are tightly embedded into the ABA signaling pathway.

To quantitatively monitor phosphorylation sites in the activation loop of SnRK2 kinases, we set up a parallel reaction monitoring assay, using MS/MS spectra identified in a previous DDA experiment to construct a phosphopeptide-specific spectral library. For the PRM analysis, raw files were imported into Skyline (version 4.1.0.11796). Transition selection was manually refined to include site determining ions for each phosphorylation site. The library ion match tolerance was set to 0.02 m/z and transitions were extracted using the centroided product mass analyzer with 10 ppm mass accuracy. Peaks were integrated using the automatic peak finding function followed by the manual curation of all peak boundaries and transitions. The summed area under the fragment ion traces data for every transition was exported for data visualization in Microsoft Excel. Phosphopeptide intensities were normalized to the unmodified counterpart peptide to correct for unequal SnRK2 protein abundance and peptide measurability.

Yeast cells expressing 3xFLAG:SnRK2 were grown in galactose-containing SD-minimal medium. Crude protein extracts were incubated with ANTI-FLAG® M2 Affinity Gel (Sigma-Aldrich) and bound proteins were eluted by boiling the samples in LDS-sample buffer. SnRK2 protein gel bands were excised from Coomassie-stained SDS-PAGE gels, reduced (DTT), alkylated (CAA) and in-gel digested with Trypsin. PROCAL peptide standard was added prior to LC-MS/MS analysis.