Quantitative phosphoproteomics identifies SnRK2 protein kinase substrates and reveals the effectors of abscisic acid action - PubMed (original) (raw)

Quantitative phosphoproteomics identifies SnRK2 protein kinase substrates and reveals the effectors of abscisic acid action

Pengcheng Wang et al. Proc Natl Acad Sci U S A. 2013.

Abstract

Sucrose nonfermenting 1 (SNF1)-related protein kinase 2s (SnRK2s) are central components of abscisic acid (ABA) signaling pathways. The snrk2.2/2.3/2.6 triple-mutant plants are nearly completely insensitive to ABA, suggesting that most of the molecular actions of ABA are triggered by the SnRK2s-mediated phosphorylation of substrate proteins. Only a few substrate proteins of the SnRK2s are known. To identify additional substrate proteins of the SnRK2s and provide insight into the molecular actions of ABA, we used quantitative phosphoproteomics to compare the global changes in phosphopeptides in WT and snrk2.2/2.3/2.6 triple mutant seedlings in response to ABA treatment. Among the 5,386 unique phosphorylated peptides identified in this study, we found that ABA can increase the phosphorylation of 166 peptides and decrease the phosphorylation of 117 peptides in WT seedlings. In the snrk2.2/2.3/2.6 triple mutant, 84 of the 166 peptides, representing 58 proteins, could not be phosphorylated, or phosphorylation was not increased under ABA treatment. In vitro kinase assays suggest that most of the 58 proteins can serve as substrates of the SnRK2s. The SnRK2 substrates include proteins involved in flowering time regulation, RNA and DNA binding, miRNA and epigenetic regulation, signal transduction, chloroplast function, and many other cellular processes. Consistent with the SnRK2 phosphorylation of flowering time regulators, the snrk2.2/2.3/2.6 triple mutant flowered significantly earlier than WT. These results shed new light on the role of the SnRK2 protein kinases and on the downstream effectors of ABA action, and improve our understanding of plant responses to adverse environments.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

(A) Venn diagram illustrating the overlap of up-regulated (Left) and down-regulated (Right) phosphopeptides identified in WT and snrk2.2/2.3/2.6 triple mutant. (B) GO catalogs of the 58 putative SnRK2 substrates. (C) In vitro phosphorylation of putative substrates by recombinant SnRK2.6. (D) Sequence logos (generated using Motif-X) of the phosphorylation sites seen only in ABA up-regulated phosphopeptides.

Fig. 2.

Fig. 2.

Role of SnRK2.2/2.3/2.6 and ABA up-regulated phosphoproteins in flowering time regulation. (A) Early flowering phenotype of the snrk2.2/2.3/2.6 triple mutant (snrk2) under short-day (Upper; 35-d-old plants) and long-day (Lower; 24-d-old plants) photoperiods. (Scale bars: 10 mm). (B and C) Number of days (B) and vegetative leaf number (C) at bolting of WT and the snrk2.2/2.3/2.6 triple mutant under long-day and short-day photoperiods. Error bars depict SE (n > 25). Asterisks denote significant differences (P < 0.05) between triple-mutant and WT plants. (D) Expression of the flowering time-related genes analyzed by qPCR in leaves of 21-d-old plants in WT and snrk2.2/2.3/2.6 triple mutant under long-day conditions. The experiment was repeated at least twice, with similar results obtained each time. (E) Model showing the function of flowering time-related phosphoproteins identified in this study. The phosphoproteins up-regulated and those down-regulated by ABA treatment are shown in red and green, respectively. Bold letters indicate putative SnRK2 substrates.

Fig. 3.

Fig. 3.

Model showing the regulation and functional grouping of putative SnRK2 substrates in ABA response pathways. The phosphoproteins up-regulated or down-regulated by ABA treatment are shown in red and green, respectively. Bold letters indicate putative SnRK2 substrates.

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