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As the occurrence of record-breaking drought events continue to rise, understanding how plants regulate water loss becomes an increasingly important area of study for protecting and improving crop productivity. Tight control of stomatal opening and closing is essential for enabling CO2 uptake while mitigating water vapor loss, particularly under drought stress. Control of the stomatal aperture is multifaceted, involving a complex response of intracellular signaling pathways to environmental cues. It is well established that changes in ion gradients drive water influx or efflux, thereby altering guard cell volume and modulating the opening or closing of the stomatal pore. These rapid responses are often mediated by phosphorylation cascades. Additionally, the opening of stomata is regulated in part by homotypic vacuole fusion, which is mediated by conserved homotypic vacuole protein sorting (HOPS) and vacuolar SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptors) complexes. HOPS tethers apposing vacuole membranes and promotes the formation of trans-SNARE complexes to mediate fusion. While little is known about this process in plants, it is known that the HOPS-specific subunits VACUOLE PROTEIN SORTING39 (VPS39) and VPS41 are required for homotypic plant vacuole fusion. In this body of work, I investigated the role of phosphorylation in regulating endomembrane trafficking components, including HOPS subunits, during stomatal opening.
n the first chapter, I review the current understanding of the role of endomembrane trafficking proteins in regulating stomatal opening and closing.
In the second chapter, I investigated the role of VPS39 and its phosphorylation in stomatal opening and closing. I found that VPS39 is important for vacuole fusion during stomatal opening and that phosphorylation of VPS39 is essential for its function. Additionally, I detected increased levels of VPS39 phosphorylation in closed stomata when compared to the open state. These results indicate that vacuole fusion corresponds to a new target for stomatal regulation.
In the third chapter, I investigated whether additional endomembrane trafficking proteins are regulated by phosphorylation during stomatal opening and closing. I found supporting evidence for proteins related to endomembrane trafficking being more phosphorylated in the closed stomata of guard cell-enriched tissue. These results suggest an additional layer of stomatal regulation that involves phosphorylation of endomembrane proteins not yet explored. The proteome and phosphoproteome produced from this work can also serve as a resource for others investigating proteomic and phosphoproteomic changes in guard cells.
The fourth chapter is a summary of technical achievements of this body of work, and it highlights future directions.
The addendum consists of a mathematical modeling paper published in collaboration with the laboratory of Dr. Belinda Akpa. The model was generated to investigate the mechanisms that control vacuole membrane fusion in stomata. The model resulting from this work was pivotal to our hypothesis in chapter 2 that a post-translational modifications of HOPS is needed for stomatal vacuole fusion