Stomata are pores at the leaf surface that enable gas exchange and transpiration. The signaling pathways that regulate the differentiation of stomatal guard cells and the mechanisms of stomatal pore formation have been characterized in Arabidopsis thaliana. However, the process by which stomatal complexes develop after pore formation into fully mature complexes is poorly understood. We tracked the morphogenesis of young stomatal complexes over time to establish characteristic geometric milestones along the path of stomatal maturation. Using 3D-nanoindentation coupled with finite element modeling of young and mature stomata, we found that despite having thicker cell walls than young guard cells, mature guard cells are more energy efficient with respect to stomatal opening, potentially attributable to the increased mechanical anisotropy of their cell walls and smaller changes in turgor pressure between the closed and open states. Comparing geometric changes in young and mature guard cells of wild-type and cellulose-deficient plants revealed that although cellulose is required for normal stomatal maturation, mechanical anisotropy appears to be achieved by the collective influence of cellulose and additional wall components. Together, these data elucidate the dynamic geometric and biomechanical mechanisms underlying the development process of stomatal maturation.

Background Like all plant cells, the guard cells of stomatal complexes are encased in cell walls that are composed of diverse, interacting networks of polysaccharide polymers. The properties of these cell walls underpin the dynamic deformations that occur in guard cells as they expand and contract to drive the opening and closing of the stomatal pore, the regulation of which is crucial for photosynthesis and water transport in plants.Scope Our understanding of how cell wall mechanics are influenced by the nanoscale assembly of cell wall polymers in guard cell walls, how this architecture changes over stomatal development, maturation and ageing and how the cell walls of stomatal guard cells might be tuned to optimize stomatal responses to dynamic environmental stimuli is still in its infancy.Conclusion In this review, we discuss advances in our ability to probe experimentally and to model the structure and dynamics of guard cell walls quantitatively across a range of plant species, highlighting new ideas and exciting opportunities for further research into these actively moving plant cells.

The function of plant stomata is regulated by nanoscale cellular components and turgor pressure in flanking guard cells that control stomatal pore size to facilitate gas exchange and photosynthesis. Cell wall nanostructure plays a critical role in stomatal behavior. Stomatal aperture is regulated by tuning the mechanical properties of guard cell walls and by water uptake or release by the cells. However, the specific mechanical roles of different wall components, especially in newly maturing guard cells, are unclear. Here, we used a combination of molecular genetics, nanoindentation in normal and lateral directions, computational modeling, and microscopy to investigate the influences of wall components and maturation on wall mechanics and turgor pressure in stomatal guard cells. Lateral indentation measurements allow the unique anisotropic properties of each guard cell to be quantified with respect to age and genotype, including mutants with lower cellulose content and with lower or higher pectin molecular weight. Our results highlight the significance of cellulose in modulating the dynamics of guard cells by affecting wall anisotropy and emphasize the role of pectin modification for tuning stomatal responses to environmental stimuli. Our nanoindentation measurements when combined with finite element modeling allow the wall modulus, anisotropy, and turgor pressure to be measured. We also found that both cell wall mechanics and turgor pressure undergo significant changes during stomatal maturation. The role of cell wall architecture in determining anisotropic wall mechanics is also discussed. Together, these results provide new insights of wall mechanics and their effect on stomatal function and growth in plants.

Stomatal function in plants is regulated by the nanoscale architecture of the cell wall and turgor pressure, which together control stomatal pore size to facilitate gas exchange and photosynthesis. The mechanical properties of the cell wall and cell geometry are critical determinants of stomatal dynamics. However, the specific biomechanical functions of wall constituents, for example, cellulose and pectins, and their impact on the work required to open or close the stomatal pore are unclear. Here, we use nanoindentation in normal and lateral directions, computational modeling, and microscopic imaging of cells from the model plant Arabidopsis thaliana to investigate the precise influences of wall architecture and turgor pressure on stomatal biomechanics. This approach allows us to quantify and compare the unique anisotropic properties of guard cells with normal composition, lower cellulose content, or alterations in pectin molecular weight. Using these data to calculate the work required to open the stomata reveals that the wild type, with a circumferential-to-longitudinal modulus ratio of 3:1, is the most energy-efficient of those studied. In addition, the tested genotypes displayed similar changes in their pore size despite large differences in wall thickness and biomechanical properties. These findings imply that homeostasis in stomatal function is maintained in the face of varying wall compositions and biomechanics by tuning wall thickness.

Automating the three-dimensional (3D) segmentation of stomatal guard cells and other confocal microscopy data is extremely challenging due to hardware limitations, hard-to-localize regions, and limited optical resolution. We present a memory-efficient, attention-based, one-stage segmentation neural network for 3D images of stomatal guard cells. Our model is trained end to end and achieved expert-level accuracy while leveraging only eight human-labeled volume images. As a proof of concept, we applied our model to 3D confocal data from a cell ablation experiment that tests the "polar stiffening" model of stomatal biomechanics. The resulting data allow us to refine this polar stiffening model. This work presents a comprehensive, automated, computer-based volumetric analysis of fluorescent guard cell images. We anticipate that our model will allow biologists to rapidly test cell mechanics and dynamics and help them identify plants that more efficiently use water, a major limiting factor in global agricultural production and an area of critical concern during climate change.