Maria Papanatsiou

Systems Dynamic Modeling of a Guard Cell Cl− Channel Mutant Uncovers an Emergent Homeostatic Network Regulating Stomatal Transpiration

Stomata account for much of the 70% of global water usage associated with agriculture and have a profound impact on the water and carbon cycles of the world. Stomata have long been modeled mathematically, but until now, no systems analysis of a plant cell has yielded detail sufficient to guide phenotypic and mutational analysis. Here, we demonstrate the predictive power of a systems dynamic model in Arabidopsis (Arabidopsis thaliana) to explain the paradoxical suppression of channels that facilitate K+ uptake, slowing stomatal opening, by mutation of the SLAC1 anion channel, which mediates solute loss for closure. The model showed how anion accumulation in the mutant suppressed the H+ load on the cytosol and promoted Ca2+ influx to elevate cytosolic pH (pHi) and free cytosolic Ca2+ concentration ([Ca2+]i), in turn regulating the K+ channels. We have confirmed these predictions, measuring pHi and [Ca2+]i in vivo, and report that experimental manipulation of pHi and [Ca2+]i is sufficient to recover K+ channel activities and accelerate stomatal opening in the slac1 mutant. Thus, we uncover a previously unrecognized signaling network that ameliorates the effects of the slac1 mutant on transpiration by regulating the K+ channels. Additionally, these findings underscore the importance of H+-coupled anion transport for pHi homeostasis.

Hydrogen Sulfide Regulates Inward-Rectifying K+ Channels in Conjunction with Stomatal Closure

Hydrogen sulfide affects inward-rectifying K+ channels in guard cells and implicates an additional, but as yet unidentified, signaling pathway in stomatal closure. Hydrogen sulfide (H2S) is the third biological gasotransmitter, and in animals, it affects many physiological processes by modulating ion channels. H2S has been reported to protect plants from oxidative stress in diverse physiological responses. H2S closes stomata, but the underlying mechanism remains elusive. Here, we report the selective inactivation of current carried by inward-rectifying K+ channels of tobacco (Nicotiana tabacum) guard cells and show its close parallel with stomatal closure evoked by submicromolar concentrations of H2S. Experiments to scavenge H2S suggested an effect that is separable from that of abscisic acid, which is associated with water stress. Thus, H2S seems to define a unique and unresolved signaling pathway that selectively targets inward-rectifying K+ channels.

Clustering of the K+ channel GORK of Arabidopsis parallels its gating by extracellular K+.

GORK is the only outward-rectifying Kv-like K+ channel expressed in guard cells. Its activity is tightly regulated to facilitate K+ efflux for stomatal closure and is elevated in ABA in parallel with suppression of the activity of the inward-rectifying K+ channel KAT1. Whereas the population of KAT1 is subject to regulated traffic to and from the plasma membrane, nothing is known about GORK, its distribution and traffic in vivo. We have used transformations with fluorescently-tagged GORK to explore its characteristics in tobacco epidermis and Arabidopsis guard cells. These studies showed that GORK assembles in puncta that reversibly dissociated as a function of the external K+ concentration. Puncta dissociation parallelled the gating dependence of GORK, the speed of response consistent with the rapidity of channel gating response to changes in the external ionic conditions. Dissociation was also suppressed by the K+ channel blocker Ba2+. By contrast, confocal and protein biochemical analysis failed to uncover substantial exo- and endocytotic traffic of the channel. Gating of GORK is displaced to more positive voltages with external K+, a characteristic that ensures the channel facilitates only K+ efflux regardless of the external cation concentration. GORK conductance is also enhanced by external K+ above 1 mm. We suggest that GORK clustering in puncta is related to its gating and conductance, and reflects associated conformational changes and (de)stabilisation of the channel protein, possibly as a platform for transmission and coordination of channel gating in response to external K+.

Functional characterization of Ostreococcus tauri phototropin

Phototropins (phots) regulate a range of adaptive processes in plants that serve to optimize photosynthetic efficiency and promote growth. Light sensing by Arabidopsis thaliana phots is predominantly mediated by the Light, Oxygen and Voltage sensing 2 (LOV2) flavin-binding motif located within the N-terminus of the photoreceptor. Here we characterize the photochemical and biochemical properties of phot from the marine picoalga Ostreococcus tauri phototropin (Otphot) and examine its ability to replace phot-mediated function in Arabidopsis. Photochemical properties of Otphot rely on both LOV1 and LOV2. Yet, biochemical analysis indicates that light-dependent receptor autophosphorylation is primarily dependent on LOV2. As found for Arabidopsis phots, Otphot associates with the plasma membrane and partially internalizes, albeit to a limited extent, in response to blue-light irradiation. Otphot is able to elicit a number of phot-regulated processes in Arabidopsis, including petiole positioning, leaf expansion, stomatal opening and chloroplast accumulation movement. However, Otphot is unable to restore phototropism and chloroplast avoidance movement. Consistent with its lack of phototropic function in Arabidopsis, Otphot does not associate with or trigger dephosphorylation of the phototropic signalling component Non-Phototropic Hypocotyl 3 (NPH3). Taken together, these findings indicate that the mechanism of action of plant and evolutionarily distant algal phots is less well conserved than previously thought.

Stomatal spacing safeguards stomatal dynamics by facilitating guard cell ion transport independent of the epidermal solute reservoir

Clustering of stomata suppresses guard cell ion transport independent of ion stores in neighboring epidermal cells. Stomata enable gaseous exchange between the interior of the leaf and the atmosphere through the stomatal pore. Control of the pore aperture depends on osmotic solute accumulation by, and its loss from the guard cells surrounding the pore. Stomata in most plants are separated by at least one epidermal cell, and this spacing is thought to enhance stomatal function, although there are several genera that exhibit stomata in clusters. We made use of Arabidopsis (Arabidopsis thaliana) stomatal patterning mutants to explore the impact of clustering on guard cell dynamics, gas exchange, and ion transport of guard cells. These studies showed that stomatal clustering in the Arabidopsis too many mouths (tmm1) mutant suppressed stomatal movements and affected CO2 assimilation and transpiration differentially between dark and light conditions and were associated with alterations in K+ channel gating. These changes were consistent with the impaired dynamics of tmm1 stomata and were accompanied by a reduced accumulation of K+ ions in the guard cells. Our findings underline the significance of spacing for stomatal dynamics. While stomatal spacing may be important as a reservoir for K+ and other ions to facilitate stomatal movements, the effects on channel gating, and by inference on K+ accumulation, cannot be explained on the basis of a reduced number of epidermal cells facilitating ion supply to the guard cells.

Speedy Grass Stomata: Emerging Molecular and Evolutionary Features.

Stomata in most land plants are formed by a pair of guard cells, controlling the water loss and the carbon dioxide uptake. The development, patterning, and density of stomata are fundamental traits for stomatal function, contributing to plant growth and productivity (Pillitteri and Torii, 2012). The stomata of most plant species consist of two kidney-shaped guard cells, while stomata of grass species are formed by two dumbbell-shaped guard cells flanked by two subsidiary cells (Figure 1A). The four-celled stomatal complex in grasses may facilitate a fast response to environmental cues for efficient photosynthesis and water use, possibly through the rapid transport of ions and osmolytes between guard cells and subsidiary cells (see Jezek and Blatt, 2017; Chen et al., 2017 and references therein).

Stomatal clustering in Begonia associates with the kinetics of leaf gaseous exchange and influences water use efficiency

Highlight Non-contiguous clustering of small stomata in Begonia favours rapid stomatal closure by comparison with larger, solitary stomata, and enhances WUE under low light conditions.

The conceptual approach to quantitative modeling of guard cells

Much of the 70% of global water usage associated with agriculture passes through stomatal pores of plant leaves. The guard cells, which regulate these pores, thus have a profound influence on photosynthetic carbon assimilation and water use efficiency of plants. We recently demonstrated how quantitative mathematical modeling of guard cells with the OnGuard modeling software yields detail sufficient to guide phenotypic and mutational analysis. This advance represents an all-important step toward applications in directing “reverse-engineering” of guard cell function for improved water use efficiency and carbon assimilation. OnGuard is nonetheless challenging for those unfamiliar with a modeler’s way of thinking. In practice, each model construct represents a hypothesis under test, to be discarded, validated or refined by comparisons between model predictions and experimental results. The few guidelines set out here summarize the standard and logical starting points for users of the OnGuard software.

Unexpected Connections between Humidity and Ion Transport Discovered Using a Model to Bridge Guard Cell-to-Leaf Scales

A model that connects guard cell solute and water transport with whole-plant water relations accurately predicts foliar transpiration and stomatal kinetics, and their dependence on relative humidity. Stomatal movements depend on the transport and metabolism of osmotic solutes that drive reversible changes in guard cell volume and turgor. These processes are defined by a deep knowledge of the identities of the key transporters and of their biophysical and regulatory properties, and have been modeled successfully with quantitative kinetic detail at the cellular level. Transpiration of the leaf and canopy, by contrast, is described by quasilinear, empirical relations for the inputs of atmospheric humidity, CO2, and light, but without connection to guard cell mechanics. Until now, no framework has been available to bridge this gap and provide an understanding of their connections. Here, we introduce OnGuard2, a quantitative systems platform that utilizes the molecular mechanics of ion transport, metabolism, and signaling of the guard cell to define the water relations and transpiration of the leaf. We show that OnGuard2 faithfully reproduces the kinetics of stomatal conductance in Arabidopsis thaliana and its dependence on vapor pressure difference (VPD) and on water feed to the leaf. OnGuard2 also predicted with VPD unexpected alterations in K+ channel activities and changes in stomatal conductance of the slac1 Cl− channel and ost2 H+-ATPase mutants, which we verified experimentally. OnGuard2 thus bridges the micro-macro divide, offering a powerful tool with which to explore the links between guard cell homeostasis, stomatal dynamics, and foliar transpiration.

Natural Variation Reveals Interplay between C4 Biology and Water Use Efficiency

The year 2016 marked a half-century since the discovery of C4 photosynthesis, yet we still seek to elucidate many of the mechanisms underpinning the C4 cycle. Although C4 and C3 plants share molecular units involved in photosynthesis ([Miyao, 2003][1]; [Kellogg, 2013][2]), C4 plants have unique