Deciphering the Protein Phosphorylation Dynamics Triggered by Seconds of Force Stimulation

Plants perceive mechanical forces through specialized phosphosignaling networks, yet how they are correlated with gravity force signaling remains unclear. To unravel the components of gravity force signalling, SILIA-based phosphoproteomics was performed on both 20s multiple inversion-treated and 30s gravistimulated aerial organs of Arabidopsis and has identified 2,733 and 2,878 phosphoproteins, respectively. Phosphoproteomic quantitation identified 34 and 52 significantly regulated phosphoprotein groups from Inversion and Gravistimulation, respectively. The Inversion-specific phosphoproteins, corresponding to the initial calcium code triggered by gravistimulation, might collectively mediate calcium signals sensed by EF-hand proteins, transduced by CPK1, and mediated by calmodulin-interacting proteins, which probably intersect with the receptor-like kinase(s)-initiated MAPK cascades via RAF15 and MKK1/2 kinases to induce gravitropic response. The Gravistimulation-specific phosphoproteins, associated with the secondary calcium code induced by gravistimulation, have the theme functions in Ca2+ signaling/homeostasis (ACA8, ZAC, IQD2, ANNAT1), membrane vesicle trafficking (ABCG36/C14, ARF-GAP8) and lipid signaling (PIP5K8/9), supporting PIN protein/auxin molecule transport, and auxin/stress signal transduction (TPR1), orchestrating responses environmental cues like physical force signals. Spatiotemporal analysis using immunoblots validation confirmed both treatments-associated phosphosites, pS108-PATL3 and pS107-TREPH2, as well as the Inversion-specific pS1145-ATEH2, with stem-specific phosphorylation enhancement. Crucially, phosphorylation on these representative phosphosites exhibited force-discriminatory responses. Functional validation has demonstrated the integrin-like protein GREPH1​ as a key regulator of gravitropism, with its mutants showing reduced inflorescence stem gravicurvature. Accelerated hyperphosphorylation on both pS107-TREPH2 and pS1145-ATEH2 phosphosites in greph1 mutant peaked at 20s - 50s while in WT plant the hyperphosphorylation of these phosphosites lasted from 20s to 2hr. These results established a stem-enriched unique phosphorylation for gravity force discrimination, with GREPH1 modulating spatiotemporal dynamics of some phosphoproteins and shoot gravicurvature and being a reminiscent receptor to the sediment plastid.