Functional, Biotinylproteomic and Bioinformatic Analysis of Both Cytoskeletal and Plastoskeletal Proteins in Plant Mechanoresponse
To investigate the early signaling components of skeletal proteins in mediating Arabidopsis thigmomorphogenesis, both microscopic and proximity labeling (PL)-based quantitative biotinylproteomics were applied to investigate the subcellular location and putative interactors of a touch-responsive WPRa4 protein. These experiments have demonstrated that the cytoskeletal protein WPRa4 is localized nearby the plastid. Several cytosolic Plastid Movement-Impaired (PMI) proteins and a member of the plastidic translocon were identified as putative interactors of WPRa4, suggesting an integrated network of skeletal proteins linking the cytoskeleton with the plastid membrane. Further bioinformatic analysis of both Proximity Labeling- and XL-MS-based proteomic results suggested that Plastid Movement-Impaired 4 (PMI4) protein may serve as a candidate in mediating the plant touch response. The loss-of-function pmi4 mutant showed neither the touch-induced bolting delay nor the rosette size reduction upon repetitive touches, suggesting that pmi4 is a unique type of mutant of Arabidopsis thigmomorphogenesis. Moreover, the null mutant pmi4 displayed a severe defect in the touch-induced Ca2+ oscillation. Further transcriptomic analysis performed on both the wild-type Arabidopsis and pmi4 mutant indicated that the mutated pmi4 gene suppressed the expression of a number of touch rapidly induced transcripts and a JA-responsive gene, LOX2. These findings led us to propose a revised touch force-sensing theory, in which the interconnected cytosolic and plastidic skeletal proteins serve as the early mechano-sensing components mediating Arabidopsis thigmomorphogenesis and the retrograde calcium signaling in response to touch.