Deciphering the Protein Phosphorylation Dynamics Triggered by Seconds of Force Stimulation

• High resolution phosphoproteomics was performed on both 20s multiple inversion force-treated and 30s gravity force-treated aerial organs of Arabidopsis and have identified 2,735 and 2,885 phosphoproteins, respectively. • SILIA-based phosphoproteomic quantitation identified 34 and 52 significantly regulated phosphoprotein groups from Inversion and Gravistimulation, respectively. • The 20-sec rotation force-specific phosphoproteins, corresponding to the initial calcium code of gravitropism, might participate both in transducing calcium signals by CPK1 and mediating the phosphor-relays through RAF15 and MKK1/2 kinases. • The 30-sec gravity force-specific phosphoproteins, associated with the secondary calcium code of gravitropism, are mainly involved in membrane vesicle trafficking and lipid signaling, supporting PIN protein/auxin molecule transport and orchestrating gravity force response. • GRavity force Enhanced PHosphoprotein 1 ( GREPH1 ) regulates both inversion- and gravistimulation-related protein phosphorylation of TREPH2, ATEH2 and PALT3. GREPH1 gene plays a role in shoot gravitropism. High resolution phosphoproteomics was performed on both 20s multiple inversion force-treated and 30s gravity force-treated aerial organs of Arabidopsis and have identified 2,735 and 2,885 phosphoproteins, respectively. SILIA-based phosphoproteomic quantitation identified 34 and 52 significantly regulated phosphoprotein groups from Inversion and Gravistimulation, respectively. The 20-sec rotation force-specific phosphoproteins, corresponding to the initial calcium code of gravitropism, might participate both in transducing calcium signals by CPK1 and mediating the phosphor-relays through RAF15 and MKK1/2 kinases. The 30-sec gravity force-specific phosphoproteins, associated with the secondary calcium code of gravitropism, are mainly involved in membrane vesicle trafficking and lipid signaling, supporting PIN protein/auxin molecule transport and orchestrating gravity force response. GRavity force Enhanced PHosphoprotein 1 ( GREPH1 ) regulates both inversion- and gravistimulation-related protein phosphorylation of TREPH2, ATEH2 and PALT3. GREPH1 gene plays a role in shoot gravitropism. 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 Ca 2+ 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.