Guard cell-enriched phosphoproteome reveals phosphorylation of endomembrane proteins in closed stomata

Control of the stomatal aperture is multifaceted, involving a complex interplay of environmental cues and intracellular signaling pathways. It is well established that changes in ion gradients drive water movement into and out of the guard cell, thereby altering cell volume and modulating the opening or closing of the stomatal pore. These rapid responses are often regulated by phosphorylation cascades to efficiently transmit environmental status and either reduce water loss or enhance carbon assimilation. The role of endomembrane trafficking networks in stomatal dynamics is not well characterized. Here, we investigated the regulation of stomatal opening and closing by generating a proteome and phosphoproteome of guard cell-enriched tissue. This deep proteome captured a protein profile that was similar to previously characterized guard cell proteomes. The guard cell-enriched tissue with closed stomata showed greater levels of phosphorylation of proteins related to endomembrane trafficking and vacuoles when compared to both whole leaf tissue with closed stomata and guard cell-enriched tissue with open stomata. These results support the hypothesis that phosphorylation of endomembrane proteins may contribute to the regulation of stomatal movements. SIGNIFICANCE: Stomatal movements are tightly regulated and are critical for gas exchange and water retention in plants. Stomatal movements are regulated by light, temperature, CO₂, humidity and hormones, and are driven by changes in cellular turgor and metabolism. Stomatal opening is the result of a complex signaling pathway involving the activity of blue light photoreceptors, plasma membrane H⁺ ATPases, and inward-rectifying K⁺ channels. Stomatal closing is induced by the stress hormone ABA, and involves the activation of specific anion channels that lead to K⁺ efflux. Phosphorylation cascades are key signaling cues for this pathway, and many kinases and phosphatases have been implicated in regulating stomatal movements. This work describes the first phosphoproteome of guard cell-enriched tissue. Isolation of intact guard cells, followed by treatment with opening or closing conditions, allowed the isolation of proteins in their open or closed-responsive state. Quantitative phosphoproteomics enabled quantitative detection of changes in phosphorylated peptide abundance between open and closed states. GO enrichment analysis supports the hypothesis that phosphorylation cascades induced by stomatal closing may target endomembrane proteins, which could be important for the endomembrane remodeling that follows decreases in cellular volume. Overall, this dataset serves as a key resource for hypothesis generation regarding plant stomatal function.