Quantitative phosphoproteomics reveals the role of wild soybean GsSnRK1 as a metabolic regulator under drought and alkali stresses.

Drought and alkali stresses cause detrimental effects on plant growth and development. SnRK1 protein kinases act as key energy and stress sensors by phosphorylation-mediated signaling in the regulation of plant defense reactions against adverse environments. To understand SnRK1-dependent phosphorylation events in signaling pathways triggered by abiotic factors, we employed quantitative phosphoproteomics to compare the global changes in phosphopeptides and phosphoproteins in 2kinm mutant Arabidopsis (SnRK1.1 T-DNA knockout and SnRK1.2 knockdown by β-estradiol-induced RNAi) complemented with wild soybean GsSnRK1(wt) or dominant negative mutant GsSnRK1(K49M) in response to drought and alkali stresses. Among 4014 phosphopeptides (representing 2380 phosphoproteins) identified in this study, we finalized 74 phosphopeptides (representing 61 phosphoproteins), and 75 phosphopeptides (representing 57 phosphoproteins) showing significant changes in phosphorylation levels under drought and alkali treatments respectively. Function enrichment and protein-protein interaction analyses indicated that the differentially-expressed phosphoproteins (DPs) under drought and alkali stresses were mainly involved in signaling transduction, stress response, carbohydrate and energy metabolism, transport and membrane trafficking, RNA splicing and processing, DNA binding and gene expression, and protein synthesis/folding/degradation. These results provide assistance to identify bona fide and novel SnRK1 phosphorylation substrates and shed new light on the biological functions of SnRK1 kinase in responses to abiotic stresses. SIGNIFICANCE: These results provide assistance to identify novel SnRK1 phosphorylation substrates and regulatory proteins, and shed new light on investigating the potential roles of reversible phosphorylation in plant responses to abiotic stresses.