Integrative Network Analysis of the Signaling Cascades in Seedling Leaves of Bread Wheat by Large-Scale Phosphoproteomic Profiling

Here,
we conducted the first large-scale leaf phosphoproteome analysis
of two bread wheat cultivars by liquid chromatography-tandem mass
spectrometry. Altogether, 1802 unambiguous phosphorylation sites representing
1175 phosphoproteins implicated in various molecular functions and
cellular processes were identified by gene ontology enrichment analysis.
Among the 1175 phosphoproteins, 141 contained 3–10 phosphorylation
sites. The phosphorylation sites were located more frequently in the
N- and C-terminal regions than in internal regions, and ∼70%
were located outside the conserved regions. Conservation analysis
showed that 90.5% of the phosphoproteins had phosphorylated orthologs
in other plant species. Eighteen significantly enriched phosphorylation
motifs, of which six were new wheat phosphorylation motifs, were identified.
In particular, 52 phosphorylated transcription factors (TFs), 85 protein
kinases (PKs), and 16 protein phosphatases (PPs) were classified and
analyzed in depth. All the Tyr phosphorylation sites were in PKs such
as mitogen-activated PKs (MAPKs) and SHAGGY-like kinases. A complicated
cross-talk phosphorylation regulatory network based on PKs such as
Snf1-related kinases (SnRKs), calcium-dependent PKs (CDPKs), and glycogen
synthase kinase 3 (GSK3) and PPs including PP2C, PP2A, and BRI1 suppressor
1 (BSU1)-like protein (BSL) was constructed and was found to be potentially
involved in rapid leaf growth. Our results provide a series of phosphoproteins
and phosphorylation sites in addition to a potential network of phosphorylation
signaling cascades in wheat seedling leaves.