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Stomatal pores are vital for the diffusion of gasses into and out of land plants and are, therefore, gatekeepers for photosynthesis and transpiration. Although much published literature has described the intercellular signaling and transcriptional regulators involved in early stomatal development, little is known about the cellular details of the local separation between sister guard cells that give rise to the stomatal pore or how formation of this pore is achieved. Using three-dimensional (3D) time-lapse imaging, we found that stomatal pore formation in Arabidopsis (Arabidopsis thaliana) is a highly dynamic process involving pore initiation and enlargement and traverses a set of morphological milestones in 3D. Confocal imaging data revealed an enrichment of exocytic machinery, de-methyl-esterified pectic homogalacturonan (HG), and an HG-degrading enzyme at future pore sites, suggesting that both localized HG deposition and degradation might function in pore formation. By manipulating HG modification via enzymatic, chemical, and genetic perturbations in seedling cotyledons, we found that augmenting HG modification promotes pore formation, whereas preventing HG de-methyl-esterification delays pore initiation and inhibits pore enlargement. Through mechanical modeling and experimentation, we tested whether pore formation is an outcome of sister guard cells being pulled away from each other upon turgor increase. Osmotic treatment to reduce turgor pressure did not prevent pore initiation but did lessen pore enlargement. Together, these data provide evidence that HG delivery and modification, and guard cell pressurization, make functional contributions to stomatal pore initiation and enlargement. [ABSTRACT FROM AUTHOR]