Model-based inference of a plant-specific dual role for HOPS in regulating guard cell vacuole fusion

Abstract

Stomata are the pores on a leaf surface that regulate gas exchange. Each stoma consists of two guard cells whose movements regulate pore opening and thereby control CO2 fixation and water loss. Guard cell movements depend in part on the remodeling of vacuoles, which have been observed to change from a highly fragmented state to a fused morphology during stomata opening. This change in morphology requires a membrane fusion mechanism that responds rapidly to environmental signals, allowing plants to respond to diurnal and stress cues. With guard cell vacuoles being both large and responsive to external signals, stomata represent a unique system in which to delineate mechanisms of membrane fusion. Fusion of vacuole membranes is a highly conserved process in eukaryotes, with key roles played by two multi-subunit complexes: HOPS (homotypic fusion and vacuolar protein sorting) and SNARE (soluble NSF attachment protein receptor). HOPS is a vacuole tethering factor that is thought to chaperone SNAREs from apposing vacuole membranes into a fusion-competent complex capable of rearranging membranes. To resolve a counter-intuitive observation regarding the role of HOPS in regulating plant vacuole morphology, we derived a quantitative model of vacuole fusion dynamics and used it to generate testable predictions about HOPS-SNARE interactions. We derived our model by applying simulation-based inference to integrate prior knowledge about molecular interactions with limited, qualitative observations of emergent vacuole phenotypes. By constraining the model parameters to yield the emergent outcomes observed for stoma opening - as induced by two distinct chemical treatments - we predicted a dual role for HOPS and identified a stalled form of the SNARE complex that differs from phenomena reported in yeast. We predict that HOPS has contradictory actions at different points in the fusion signaling pathway, promoting the formation of SNARE complexes, but limiting their activity. Author summaryPlants "breathe" through pores in their leaves where each pore is formed by two specialized cells called guard cells. To open these pores, guard cells change in volume. This volume change is controlled by water-filled organelles called vacuoles that morph from multiple small entities to a few large ones capable of taking up more water to reshape the cell. Specialized proteins in vacuole membranes make this change happen by pulling vacuoles together until they fuse. Some of these proteins reside in membranes, but others must be drawn to the membrane from the cells cytoplasm. Specific lipid molecules in the membrane play an important role in recruiting those proteins to the vacuole membrane. We previously made an unexpected finding that removing this lipid induces plant vacuole fusion. To make sense of this observation, we used a mathematical model to piece together our knowledge of the proteins involved in this process and what we know about the chemical treatments that cause vacuoles to morph. Using computer simulations, we uncovered new rules about how molecules interact in membranes to accomplish the task of vacuole fusion in plants. We think the rules uncovered through mathematical modeling allow plants to respond quickly to environmental cues.