Deciphering the Protein Phosphorylation Dynamics Triggered by Seconds of Force Stimulation

Plants perceive mechanical forces through phosphosignaling networks, but their relationship with gravity signaling remains elusive. To dissect gravity force signaling components, we performed SILIA-based phosphoproteomics on Arabidopsis aerial organs subjected to 20-s inversion or 30-s gravistimulation, identifying 2,733 and 2,878 phosphoproteins, respectively. Quantitative analysis revealed 34 significantly regulated phosphoproteins specific to inversion and 52 specific to gravistimulation. Inversion-specific phosphoproteins, associated with the initial calcium code, likely mediate calcium signals through EF-hand proteins, CPK1, and calmodulin-interacting proteins, potentially intersecting with receptor-like kinase-initiated MAPK cascades via RAF15 and MKK1/2 to induce gravitropic responses. Gravistimulation-specific phosphoproteins, linked to the secondary calcium code, function in calcium signaling/homeostasis (ACA8, ZAC, IQD2, ANNAT1), membrane vesicle trafficking (ABCG36, ARF-GAP8), and lipid signaling (PIP5K8/9), supporting auxin transport and stress signal transduction. Immunoblot validation confirmed treatment-associated phosphosites pS108-PATL3 and pS107-TREPH2, along with inversion-specific pS1145-ATEH2, exhibiting stem-specific phosphorylation enhancement and force-discriminatory responses. Functional analysis identified the integrin-like protein GREPH1 as a key gravitropism regulator, with greph1 mutants displaying reduced inflorescence stem gravicurvature. Notably, hyperphosphorylation of pS107-TREPH2 and pS1145-ATEH2 peaked at 20 to 50 s in greph1 mutants but persisted from 20 s to 2 h in WT plants. These findings establish a stem-enriched phosphorylation code for gravity force discrimination, with GREPH1 modulating spatiotemporal phosphoprotein dynamics and shoot gravicurvature, potentially functioning as a receptor reminiscent of sedimenting plastids.