Plants control CO2 uptake and water loss by modulating the aperture of stomata located in the epidermis. Stomatal opening is initiated by the activation of H+-ATPases in the guard-cell plasma membrane. In contrast to regulation of H+-ATPase activity, little is known about the translocation of the guard cell H+-ATPase to the plasma membrane. Here we describe the isolation of an Arabidopsis gene, PATROL1, that controls the translocation of a major H+-ATPase, AHA1, to the plasma membrane. PATROL1 encodes a protein with a MUN domain, known to mediate synaptic priming in neuronal exocytosis in animals. Environmental stimuli change the localization of plasma membrane-associated PATROL1 to an intracellular compartment. Plasma membrane localization of AHA1 and stomatal opening require the association of PATROL1 with AHA1. Increased stomatal opening responses in plants overexpressing PATROL1 enhance the CO2 assimilation rate, promoting plant growth.

Pavement cells in cotyledons and true leaves exhibit a jigsaw puzzle-like morphology in most dicotyledonous plants. Among the molecular mechanisms mediating cell morphogenesis, two antagonistic Rho-like GTPases regulate local cell outgrowth via cytoskeletal rearrangements. Analyses of several cell wall-related mutants suggest the importance of cell wall mechanics in the formation of interdigitated patterns. However, how these factors are integrated is unknown. In this study, we observed that the application of exogenous cellulase to hydroponically grown Arabidopsis thaliana cotyledons switched the interdigitation of pavement cells to the production of smoothly elongated cells. The cellulase-induced inhibition of cell interdigitation was not observed in a RIC1 knockout mutant. This gene encodes a Rho-like GTPase-interacting protein important for localized cell growth suppression via microtubule bundling on concave cell interfaces. Additionally, to characterize pavement cell morphologies, we developed a mathematical model that considers the balance between cell and cell wall growth, restricted global cell growth orientation, and regulation of local cell outgrowth mediated by a Rho-like GTPase-cytoskeleton system. Our computational simulations fully support our experimental observations, and suggest that interdigitated patterns form because of mechanical buckling in the absence of Rho-like GTPase-dependent regulation of local cell outgrowth. Our model clarifies the cell wall mechanics influencing pavement cell morphogenesis.

The Arabidopsis stomatal complex is composed of a pair of guard cells and surrounding anisocytic subsidiary cells. Subsidiary cells are thought to function as a supplier and receiver of bulk water and ions, and to assist turgor-driven stomatal movement, but the molecular mechanisms are largely unknown. In this work, we studied the dynamic behavior and environmental responses of PATROL1, which has been identified as a translocation factor of the plasma membrane proton pump ATPase (PM H(+)-ATPase) AHA1 in guard cells and subsidiary cells in Arabidopsis thaliana. Variable-angle epifluorescence microscopic observation revealed that green fluorescent protein (GFP)-PATROL1 localized on dot-like compartments that resided on plasma membranes for several seconds. The GFP-PATROL1-labeled dots were sensitive to phosphatidylinositol 4-kinase inhibitors but not to a phosphatidylinositol 3-kinase inhibitor. GFP-PATROL1 and red fluorescent protein (RFP)-AHA1 co-localized in hyperosmotic conditions, and a mutation of PATROL1 resulted in an increase in GFP-AHA1 internalization, suggesting a role in the translocation of PM H(+)-ATPase in subsidiary cells. Interestingly, subsidiary cells showed changes in localization of GFP-PATROL1 in response to environmental stimuli that were opposite to those in guard cells. Our observations suggested that PATROL1 may contribute to stomatal movement by translocations of PM H(+)-ATPase in subsidiary cells.

The evaluation of cytoskeletal bundling is a fundamental experimental method in the field of cell biology. Although the skewness of the pixel intensity distribution derived from fluorescently-labeled cytoskeletons has been widely used as a metric to evaluate the degree of bundling in digital microscopy images, its versatility has not been fully validated. Here, we applied the coefficient of variation (CV) of intensity values as an alternative metric, and compared its performance with skewness. In synthetic images representing extremely bundled conditions, the CV successfully detected degrees of bundling that could not be distinguished by skewness. On actual microscopy images, CV was better than skewness, especially on variable-angle epifluorescence microscopic images or stimulated emission depletion and confocal microscopy images of very small areas of around 1 μm2. When blur or noise was added to synthetic images, CV was found to be robust to blur but deleteriously affected by noise, whereas skewness was robust to noise but deleteriously affected by blur. For confocal images, CV and skewness showed similar sensitivity to noise, possibly because optical blurring is often present in microscopy images. Therefore, in practical use with actual microscopy images, CV may be more appropriate than skewness, unless the image is extremely noisy.

To comprehensively grasp cell biological events in plant stomatal movement, we have captured microscopic images of guard cells with various organelles markers. The 28,530 serial optical sections of 930 pairs of Arabidopsis guard cells have been released as a new image database, named Live Images of Plant Stomata (LIPS). We visualized the average organellar distributions in guard cells using probabilistic mapping and image clustering techniques. The results indicated that actin microfilaments and endoplasmic reticulum (ER) are mainly localized to the dorsal side and connection regions of guard cells. Subtractive images of open and closed stomata showed distribution changes in intracellular structures, including the ER, during stomatal movement. Time-lapse imaging showed that similar ER distribution changes occurred during stomatal opening induced by light irradiation or femtosecond laser shots on neighboring epidermal cells, indicating that our image analysis approach has identified a novel ER relocation in stomatal opening.