Turgor pressure change in stomatal guard cells arises from interactions between water influx and mechanical responses of their cell walls.

The ability of plants to absorb CO2 for photosynthesis and transport water from root to shoot depends on the reversible swelling of guard cells that open stomatal pores in the epidermis. Despite decades of experimental and theoretical work, the biomechanical drivers of stomatal opening and closure are still not clearly defined. We combined mechanical principles with a growing body of knowledge concerning water flux across the plant cell membrane and the biomechanical properties of plant cell walls to quantitatively test the long-standing hypothesis that increasing turgor pressure resulting from water uptake drives guard cell expansion during stomatal opening. To test the alternative hypothesis that water influx is the main motive force underlying guard cell expansion, we developed a system dynamics model accounting for water influx. This approach connects stomatal kinetics to whole plant physiology by including values for water flux arising from water status in the plant .

The stomatal flexoskeleton: how the biomechanics of guard cell walls animate an elastic pressure vessel.

In plants, stomatal guard cells are one of the most dynamic cell types, rapidly changing their shape and size in response to environmental and intrinsic signals to control gas exchange at the plant surface. Quantitative and systematic knowledge of the biomechanical underpinnings of stomatal dynamics will enable strategies to optimize stomatal responsiveness and improve plant productivity by enhancing the efficiency of photosynthesis and water use. Recent developments in microscopy, mechanical measurements, and computational modeling have revealed new insights into the biomechanics of stomatal regulation and the genetic, biochemical, and structural origins of how plants achieve rapid and reliable stomatal function by tuning the mechanical properties of their guard cell walls. This review compares historical and recent experimental and modeling studies of the biomechanics of stomatal complexes, highlighting commonalities and contrasts between older and newer studies. Key gaps in our understanding of stomatal functionality are also presented, along with assessments of potential methods that could bridge those gaps.

Synergistic Pectin Degradation and Guard Cell Pressurization Underlie Stomatal Pore Formation.

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.

Mechanical Effects of Cellulose, Xyloglucan, and Pectins on Stomatal Guard Cells of Arabidopsis thaliana.

Stomata function as osmotically tunable pores that facilitate gas exchange at the surface of plants. Stomatal opening and closure are regulated by turgor changes in guard cells that result in mechanically regulated deformations of guard cell walls. However, how the molecular, architectural, and mechanical heterogeneities that exist in guard cell walls affect stomatal dynamics is unclear. In this work, stomata of wild type Arabidopsis thaliana plants or of mutants lacking normal cellulose, hemicellulose, or pectins were experimentally induced to close or open. Three-dimensional images of these stomatal complexes were collected using confocal microscopy, images were landmarked, and three-dimensional finite element models (FEMs) were constructed for each complex. Stomatal opening was simulated with a 5 MPa turgor increase. By comparing experimentally measured and computationally modeled changes in stomatal geometry across genotypes, anisotropic mechanical properties of guard cell walls were determined and mapped to cell wall components. Deficiencies in cellulose or hemicellulose were both predicted to stiffen guard cell walls, but differentially affected stomatal pore area and the degree of stomatal opening. Additionally, reducing pectin molecular mass altered the anisotropy of calculated shear moduli in guard cell walls and enhanced stomatal opening. Based on the unique architecture of guard cell walls and our modeled changes in their mechanical properties in cell wall mutants, we discuss how each polysaccharide class contributes to wall architecture and mechanics in guard cells. This study provides new insights into how the walls of guard cells are constructed to meet the mechanical requirements of stomatal dynamics.

Balancing Strength and Flexibility: How the Synthesis, Organization, and Modification of Guard Cell Walls Govern Stomatal Development and Dynamics.

Guard cells are pairs of epidermal cells that control gas diffusion by regulating the opening and closure of stomatal pores. Guard cells, like other types of plant cells, are surrounded by a three-dimensional, extracellular network of polysaccharide-based wall polymers. In contrast to the walls of diffusely growing cells, guard cell walls have been hypothesized to be uniquely strong and elastic to meet the functional requirements of withstanding high turgor and allowing for reversible stomatal movements. Although the walls of guard cells were long underexplored as compared to extensive studies of stomatal development and guard cell signaling, recent research has provided new genetic, cytological, and physiological data demonstrating that guard cell walls function centrally in stomatal development and dynamics. In this review, we highlight and discuss the latest evidence for how wall polysaccharides are synthesized, deposited, reorganized, modified, and degraded in guard cells, and how these processes influence stomatal form and function. We also raise open questions and provide a perspective on experimental approaches that could be used in the future to shed light on the composition and architecture of guard cell walls.

POLYGALACTURONASE INVOLVED IN EXPANSION3 Functions in Seedling Development, Rosette Growth, and Stomatal Dynamics in Arabidopsis thaliana.

Plant cell separation and expansion require pectin degradation by endogenous pectinases such as polygalacturonases, few of which have been functionally characterized. Stomata are a unique system to study both processes because stomatal maturation involves limited separation between sister guard cells and stomatal responses require reversible guard cell elongation and contraction. However, the molecular mechanisms for how stomatal pores form and how guard cell walls facilitate dynamic stomatal responses remain poorly understood. We characterized POLYGALACTURONASE INVOLVED IN EXPANSION3 (PGX3), which is expressed in expanding tissues and guard cells. PGX3-GFP localizes to the cell wall and is enriched at sites of stomatal pore initiation in cotyledons. In seedlings, ablating or overexpressing PGX3 affects both cotyledon shape and the spacing and pore dimensions of developing stomata. In adult plants, PGX3 affects rosette size. Although stomata in true leaves display normal density and morphology when PGX3 expression is altered, loss of PGX3 prevents smooth stomatal closure, and overexpression of PGX3 accelerates stomatal opening. These phenotypes correspond with changes in pectin molecular mass and abundance that can affect wall mechanics. Together, these results demonstrate that PGX3-mediated pectin degradation affects stomatal development in cotyledons, promotes rosette expansion, and modulates guard cell mechanics in adult plants.

Activation tagging of Arabidopsis POLYGALACTURONASE INVOLVED IN EXPANSION2 promotes hypocotyl elongation, leaf expansion, stem lignification, mechanical stiffening, and lodging.

Pectin is the most abundant component of primary cell walls in eudicot plants. The modification and degradation of pectin affects multiple processes during plant development, including cell expansion, organ initiation, and cell separation. However, the extent to which pectin degradation by polygalacturonases affects stem development and secondary wall formation remains unclear. Using an activation tag screen, we identified a transgenic Arabidopsis thaliana line with longer etiolated hypocotyls, which overexpresses a gene encoding a polygalacturonase. We designated this gene as POLYGALACTURONASE INVOLVED IN EXPANSION2 (PGX2), and the corresponding activation tagged line as PGX2AT . PGX2 is widely expressed in young seedlings and in roots, stems, leaves, flowers, and siliques of adult plants. PGX2-GFP localizes to the cell wall, and PGX2AT plants show higher total polygalacturonase activity and smaller pectin molecular masses than wild-type controls, supporting a function for this protein in apoplastic pectin degradation. A heterologously expressed, truncated version of PGX2 also displays polygalacturonase activity in vitro. Like previously identified PGX1AT plants, PGX2AT plants have longer hypocotyls and larger rosette leaves, but they also uniquely display early flowering, earlier stem lignification, and lodging stems with enhanced mechanical stiffness that is possibly due to decreased stem thickness. Together, these results indicate that PGX2 both functions in cell expansion and influences secondary wall formation, providing a possible link between these two developmental processes.

Integrating cell biology, image analysis, and computational mechanical modeling to analyze the contributions of cellulose and xyloglucan to stomatal function.

Cell walls are likely to be essential determinants of the amazing strength and flexibility of the guard cells that surround each stomatal pore in plants, but surprisingly little is known about cell wall composition, organization, and dynamics in guard cells. Recent analyses of cell wall organization and stomatal function in the guard cells of Arabidopsis thaliana mutants with defects in cellulose and xyloglucan have allowed for the development of new hypotheses about the relative contributions of these components to guard cell function. Advanced image analysis methods can allow for the automated detection of key structures, such as microtubules (MTs) and Cellulose Synthesis Complexes (CSCs), in guard cells, to help determine their contributions to stomatal function. A major challenge in the mechanical modeling of dynamic biological structures, such as guard cell walls, is to connect nanoscale features (e.g., wall polymers and their molecular interactions) with cell-scale mechanics; this challenge can be addressed by applying multiscale computational modeling that spans multiple spatial scales and physical attributes for cell walls.

Multiscale stress-strain characterization of onion outer epidermal tissue in wet and dry states.

UNLABELLED: • PREMISE OF THE STUDY: Quantitative measurements of water's effects on the tension response of plant tissue will assist in understanding the regulatory mechanism underlying expansive growth. Such measurements should be multiscale in nature to account for plants' hierarchical structure.• METHODS: Outer onion epidermal tissues were cut and bonded to uniaxial displacement-controlled mechanical loading devices to apply and measure the force on the sample. Fluorescent polystyrene beads (500 nm in diameter) were dispersed on the sample surface under various levels of tensile load conditions to obtain displacement maps with a confocal fluorescent microscope. The resulting strain was measured using a digital image correlation technique by tracking individual bead displacements. The applied forces were obtained by measuring the displacement of the calibrated force-sensing device. Tissue- and cell-scale mechanical properties were quantified by calculating the applied stress and the corresponding global and local strains.• KEY RESULTS: The Young's modulus values of individual cell walls of dehydrated and rehydrated samples were 3.0 ± 1.0 GPa and 0.4 ± 0.2 GPa, respectively, and are different from the Young's modulus values of the global tissue-scale dehydrated and rehydrated samples, which were 1.9 ± 0.3 GPa and 0.08 ± 0.02 GPa, respectively. Poisson's ratio increased more than 3-fold due to hydration.• CONCLUSION: The results on global, cell-to-cell, and point-to-point mechanical property variations suggest the importance of the mechanical contribution of extracellular features including the middle lamella, cell shape, and dimension. This study shows that a multiscale investigation is essential for fundamental insights into the hierarchical deformation of biological systems.

Mechanical characterization of outer epidermal middle lamella of onion under tensile loading.

UNLABELLED: • PREMISE OF THE STUDY: The cells in plant tissue are joined together by a distinct layer called the middle lamella (ML). Understanding the mechanical properties of the ML is crucial in studying how tissue-level mechanical properties emerge from the subcellular-level mechanical properties. However, the nanoscale size of the ML presents formidable challenges to its characterization as a separate layer. Consequently, the mechanical properties of the ML under tensile loading are as yet unknown.• METHODS: Here, we characterize the ML from a subcellular sample excised from two adjacent cells and composed of two wall fragments and a single line of ML in between. Two techniques, cryotome sectioning and milling with a focused ion beam, were used to prepare ML samples, and tensile experiments were performed using microelectromechanical system (MEMS) tensile testing devices.• KEY RESULTS: Our test results showed that even at a subcellular scale, the ML appears to be stronger than the wall fragments. There was also evidence that the ML attached at the corner of cells more strongly than at the rest of the contact area. The contribution of the additional ML contact area was estimated to be 40.6 MPa. Wall fragment samples containing an ML layer were also significantly stronger (p < 0.05) than the wall fragments without an ML layer.• CONCLUSIONS: The tensile properties of the ML might not have a major impact on the tissue-scale mechanical properties. This conclusion calls for further study of the ML, including characterization under shear loading conditions and elucidation of the contributions of other extracellular parameters, such as cell size and shape, to the overall tissue-level mechanical response.

Contributions of the mechanical properties of major structural polysaccharides to the stiffness of a cell wall network model.

PREMISE OF THE STUDY: The molecular mechanisms regulating the expansive growth of the plant cell wall have yet to be fully understood. The recent development of a computational cell wall model allows quantitative examinations of hypothesized cell wall loosening mechanisms. METHODS: Computational cell wall network (CWN) models were generated using cellulose microfibrils (CMFs), hemicelluloses (HCs), and their interactions (CMF-HC). For each component, a range of stiffness values, representing various situations hypothesized as potential cell-wall-loosening mechanisms, were used in the calculation of the overall stiffness of the computational CWN model. Thus, a critical mechanism of the loosening of the primary cell wall was investigated using a computational approach by modeling the molecular structure. KEY RESULTS: The increase in the stiffness equivalent of the CMF-HC interaction results in an increase in the Young's modulus of the CWN. In the major growth direction, the CWN stiffness is most sensitive to the CMF-HC interaction (75%). HC stiffness contributes moderately (24%) to the change in the CWN stiffness, whereas the CMF contribution is marginal (1%). Minor growth direction exhibited a similar trend except that the contributions of CMFs and HCs are higher than for the major growth direction. CONCLUSIONS: The stiffness of the CMF-HC interaction is the most critical mechanical component in altering stiffness of the CWN model, which supports the hypothesized mechanism of expansin's role in efficient loosening of the plant cell wall by disrupting HC binding to CMFs. The comparison to experiments suggests additional load-bearing mechanisms in CMF-HC interactions.

Characterizing microscale biological samples under tensile loading: stress-strain behavior of cell wall fragment of onion outer epidermis.

PREMISE OF THE STUDY: The results of published studies investigating the tissue-scale mechanical properties of plant cell walls are confounded by the unknown contributions of the middle lamella and the shape and size of each cell. However, due to their microscale size, cell walls have not yet been characterized at the wall fragment level under tensile loading. It is imperative to understand the stress-strain behavior of cell wall fragments to relate the wall's mechanical properties to its architecture. • METHODS: This study reports a novel method used to characterize wall fragments under tensile loading. Cell wall fragments from onion outer epidermal peels were cut to the desired size (15 × 5 µm) using the focused ion beam milling technique, and these fragments were manipulated onto a microelectromechanical system (MEMS) tensile testing device. The stress-strain behavior of the wall fragments both in the major and minor growth directions were characterized in vacuo. • KEY RESULTS: The measured mean modulus, fracture strength, and fracture strain in the major growth direction were 3.7 ± 0.8 GPa, 95.5 ± 24.1 MPa, and 3.0 ± 0.5%, respectively. The corresponding properties along the minor growth direction were 4.9 ± 1.2 GPa, 159 ± 48.4 MPa, and 3.8 ± 0.5%, respectively. • CONCLUSIONS: The fracture strength and fracture strain were significantly different along the major and minor growth directions, the wall fragment level modulus of elasticity anisotropy for a dehydrated cell wall was 1.23, suggesting a limited anisotropy of the cell wall itself compared with tissue-scale results.

Architecture-based multiscale computational modeling of plant cell wall mechanics to examine the hydrogen-bonding hypothesis of the cell wall network structure model.

A primary plant cell wall network was computationally modeled using the finite element approach to study the hypothesis of hemicellulose (HC) tethering with the cellulose microfibrils (CMFs) as one of the major load-bearing mechanisms of the growing cell wall. A computational primary cell wall network fragment (10 × 10 µm) comprising typical compositions and properties of CMFs and HC was modeled with well-aligned CMFs. The tethering of HC to CMFs is modeled in accordance with the strength of the hydrogen bonding by implementing a specific load-bearing connection (i.e. the joint element). The introduction of the CMF-HC interaction to the computational cell wall network model is a key to the quantitative examination of the mechanical consequences of cell wall structure models, including the tethering HC model. When the cell wall network models with and without joint elements were compared, the hydrogen bond exhibited a significant contribution to the overall stiffness of the cell wall network fragment. When the cell wall network model was stretched 1% in the transverse direction, the tethering of CMF-HC via hydrogen bonds was not strong enough to maintain its integrity. When the cell wall network model was stretched 1% in the longitudinal direction, the tethering provided comparable strength to maintain its integrity. This substantial anisotropy suggests that the HC tethering with hydrogen bonds alone does not manifest sufficient energy to maintain the integrity of the cell wall during its growth (i.e. other mechanisms are present to ensure the cell wall shape).