OnGuard3e: A predictive, ecophysiology-ready tool for gas exchange and photosynthesis research.

Gas exchange across the stomatal pores of leaves is a focal point in studies of plant-environmental relations. Stomata regulate atmospheric exchange with the inner air spaces of the leaf. They open to allow CO2 entry for photosynthesis and close to minimize water loss. Models that focus on the phenomenology of stomatal conductance generally omit the mechanics of the guard cells that regulate the pore aperture. The OnGuard platform fills this gap and offers a truly mechanistic approach with which to analyse stomatal gas exchange, whole-plant carbon assimilation and water-use efficiency. Previously, OnGuard required specialist knowledge of membrane transport, signalling and metabolism. Here we introduce OnGuard3e, a software package accessible to ecophysiologists and membrane biologists alike. We provide a brief guide to its use and illustrate how the package can be applied to explore and analyse stomatal conductance, assimilation and water use efficiencies, addressing a range of experimental questions with truly predictive outputs.

Guard Cell Starch Degradation Yields Glucose for Rapid Stomatal Opening in Arabidopsis.

Starch in Arabidopsis (Arabidopsis thaliana) guard cells is rapidly degraded at the start of the day by the glucan hydrolases α-AMYLASE3 (AMY3) and ß-AMYLASE1 (BAM1) to promote stomatal opening. This process is activated via phototropin-mediated blue light signaling downstream of the plasma membrane H+-ATPase. It remains unknown how guard cell starch degradation integrates with light-regulated membrane transport processes in the fine control of stomatal opening kinetics. We report that H+, K+, and Cl- transport across the guard cell plasma membrane is unaltered in the amy3 bam1 mutant, suggesting that starch degradation products do not directly affect the capacity to transport ions. Enzymatic quantification revealed that after 30 min of blue light illumination, amy3 bam1 guard cells had similar malate levels as the wild type, but had dramatically altered sugar homeostasis, with almost undetectable amounts of Glc. Thus, Glc, not malate, is the major starch-derived metabolite in Arabidopsis guard cells. We further show that impaired starch degradation in the amy3 bam1 mutant resulted in an increase in the time constant for opening of 40 min. We conclude that rapid starch degradation at dawn is required to maintain the cytoplasmic sugar pool, clearly needed for fast stomatal opening. The conversion and exchange of metabolites between subcellular compartments therefore coordinates the energetic and metabolic status of the cell with membrane ion transport.

Evolution of chloroplast retrograde signaling facilitates green plant adaptation to land.

Chloroplast retrograde signaling networks are vital for chloroplast biogenesis, operation, and signaling, including excess light and drought stress signaling. To date, retrograde signaling has been considered in the context of land plant adaptation, but not regarding the origin and evolution of signaling cascades linking chloroplast function to stomatal regulation. We show that key elements of the chloroplast retrograde signaling process, the nucleotide phosphatase (SAL1) and 3'-phosphoadenosine-5'-phosphate (PAP) metabolism, evolved in streptophyte algae-the algal ancestors of land plants. We discover an early evolution of SAL1-PAP chloroplast retrograde signaling in stomatal regulation based on conserved gene and protein structure, function, and enzyme activity and transit peptides of SAL1s in species including flowering plants, the fern Ceratopteris richardii, and the moss Physcomitrella patens Moreover, we demonstrate that PAP regulates stomatal closure via secondary messengers and ion transport in guard cells of these diverse lineages. The origin of stomata facilitated gas exchange in the earliest land plants. Our findings suggest that the conquest of land by plants was enabled by rapid response to drought stress through the deployment of an ancestral SAL1-PAP signaling pathway, intersecting with the core abscisic acid signaling in stomatal guard cells.

Predicting the unexpected in stomatal gas exchange: not just an open-and-shut case.

Plant membrane transport, like transport across all eukaryotic membranes, is highly non-linear and leads to interactions with characteristics so complex that they defy intuitive understanding. The physiological behaviour of stomatal guard cells is a case in point in which, for example, mutations expected to influence stomatal closing have profound effects on stomatal opening and manipulating transport across the vacuolar membrane affects the plasma membrane. Quantitative mathematical modelling is an essential tool in these circumstances, both to integrate the knowledge of each transport process and to understand the consequences of their manipulation in vivo. Here, we outline the OnGuard modelling environment and its use as a guide to predicting the emergent properties arising from the interactions between non-linear transport processes. We summarise some of the recent insights arising from OnGuard, demonstrate its utility in interpreting stomatal behaviour, and suggest ways in which the OnGuard environment may facilitate 'reverse-engineering' of stomata to improve water use efficiency and carbon assimilation.

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

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.

EZ-Root-VIS: A Software Pipeline for the Rapid Analysis and Visual Reconstruction of Root System Architecture.

If we want to understand how the environment has shaped the appearance and behavior of living creatures, we need to compare groups of individuals that differ in genetic makeup and environment experience. For complex phenotypic features, such as body posture or facial expression in humans, comparison is not straightforward because some of the contributing factors cannot easily be quantified or averaged across individuals. Therefore, computational methods are used to reconstruct representative prototypes using a range of algorithms for filling in missing information and calculating means. The same problem applies to the root system architecture (RSA) of plants. Several computer programs are available for extracting numerical data from root images, but they usually do not offer customized data analysis or visual reconstruction of RSA. We developed Root-VIS, a free software tool that facilitates the determination of means and variance of many different RSA features across user-selected sets of root images. Furthermore, Root-VIS offers several options to generate visual reconstructions of root systems from the averaged data to enable screening and modeling. We confirmed the suitability of Root-VIS, combined with a new version of EZ-Rhizo, for the rapid characterization of genotype-environment interactions and gene discovery through genome-wide association studies in Arabidopsis (Arabidopsis thaliana).

A constraint-relaxation-recovery mechanism for stomatal dynamics.

Models of guard cell dynamics, built on the OnGuard platform, have provided quantitative insights into stomatal function, demonstrating substantial predictive power. However, the kinetics of stomatal opening predicted by OnGuard models were threefold to fivefold slower than observed in vivo. No manipulations of parameters within physiological ranges yielded model kinetics substantially closer to these data, thus highlighting a missing component in model construction. One well-documented process influencing stomata is the constraining effect of the surrounding epidermal cells on guard cell volume and stomatal aperture. Here, we introduce a mechanism to describe this effect in OnGuard2 constructed around solute release and a decline in turgor of the surrounding cells and its subsequent recovery during stomatal opening. The results show that this constraint-relaxation-recovery mechanism in OnGuard2 yields dynamics that are consistent with experimental observations in wild-type Arabidopsis, and it predicts the altered opening kinetics of ost2 H+ -ATPase and slac1 Cl- channel mutants. Thus, incorporating solute flux of the surrounding cells implicitly through their constraint on guard cell expansion provides a satisfactory representation of stomatal kinetics, and it predicts a substantial and dynamic role for solute flux across the apoplastic space between the guard cells and surrounding cells in accelerating stomatal kinetics.

Global Sensitivity Analysis of OnGuard Models Identifies Key Hubs for Transport Interaction in Stomatal Dynamics.

The physical requirement for charge to balance across biological membranes means that the transmembrane transport of each ionic species is interrelated, and manipulating solute flux through any one transporter will affect other transporters at the same membrane, often with unforeseen consequences. The OnGuard systems modeling platform has helped to resolve the mechanics of stomatal movements, uncovering previously unexpected behaviors of stomata. To date, however, the manual approach to exploring model parameter space has captured little formal information about the emergent connections between parameters that define the most interesting properties of the system as a whole. Here, we introduce global sensitivity analysis to identify interacting parameters affecting a number of outputs commonly accessed in experiments in Arabidopsis (Arabidopsis thaliana). The analysis highlights synergies between transporters affecting the balance between Ca2+ sequestration and Ca2+ release pathways, notably those associated with internal Ca2+ stores and their turnover. Other, unexpected synergies appear, including with the plasma membrane anion channels and H+-ATPase and with the tonoplast TPK K+ channel. These emergent synergies, and the core hubs of interaction that they define, identify subsets of transporters associated with free cytosolic Ca2+ concentration that represent key targets to enhance plant performance in the future. They also highlight the importance of interactions between the voltage regulation of the plasma membrane and tonoplast in coordinating transport between the different cellular compartments.

Evolutionary Conservation of ABA Signaling for Stomatal Closure.

Abscisic acid (ABA)-driven stomatal regulation reportedly evolved after the divergence of ferns, during the early evolution of seed plants approximately 360 million years ago. This hypothesis is based on the observation that the stomata of certain fern species are unresponsive to ABA, but exhibit passive hydraulic control. However, ABA-induced stomatal closure was detected in some mosses and lycophytes. Here, we observed that a number of ABA signaling and membrane transporter protein families diversified over the evolutionary history of land plants. The aquatic ferns Azolla filiculoides and Salvinia cucullata have representatives of 23 families of proteins orthologous to those of Arabidopsis (Arabidopsis thaliana) and all other land plant species studied. Phylogenetic analysis of the key ABA signaling proteins indicates an evolutionarily conserved stomatal response to ABA. Moreover, comparative transcriptomic analysis has identified a suite of ABA-responsive genes that differentially expressed in a terrestrial fern species, Polystichum proliferum These genes encode proteins associated with ABA biosynthesis, transport, reception, transcription, signaling, and ion and sugar transport, which fit the general ABA signaling pathway constructed from Arabidopsis and Hordeum vulgare The retention of these key ABA-responsive genes could have had a profound effect on the adaptation of ferns to dry conditions. Furthermore, stomatal assays have shown the primary evidence for ABA-induced closure of stomata in two terrestrial fern species Pproliferum and Nephrolepis exaltata In summary, we report, to our knowledge, new molecular and physiological evidence for the presence of active stomatal control in ferns.

An Optimal Frequency in Ca2+ Oscillations for Stomatal Closure Is an Emergent Property of Ion Transport in Guard Cells.

Oscillations in cytosolic-free Ca(2+) concentration ([Ca(2+)]i) have been proposed to encode information that controls stomatal closure. [Ca(2+)]i oscillations with a period near 10 min were previously shown to be optimal for stomatal closure in Arabidopsis (Arabidopsis thaliana), but the studies offered no insight into their origins or mechanisms of encoding to validate a role in signaling. We have used a proven systems modeling platform to investigate these [Ca(2+)]i oscillations and analyze their origins in guard cell homeostasis and membrane transport. The model faithfully reproduced differences in stomatal closure as a function of oscillation frequency with an optimum period near 10 min under standard conditions. Analysis showed that this optimum was one of a range of frequencies that accelerated closure, each arising from a balance of transport and the prevailing ion gradients across the plasma membrane and tonoplast. These interactions emerge from the experimentally derived kinetics encoded in the model for each of the relevant transporters, without the need of any additional signaling component. The resulting frequencies are of sufficient duration to permit substantial changes in [Ca(2+)]i and, with the accompanying oscillations in voltage, drive the K(+) and anion efflux for stomatal closure. Thus, the frequency optima arise from emergent interactions of transport across the membrane system of the guard cell. Rather than encoding information for ion flux, these oscillations are a by-product of the transport activities that determine stomatal aperture.

Nitrate reductase mutation alters potassium nutrition as well as nitric oxide-mediated control of guard cell ion channels in Arabidopsis.

Maintaining potassium (K(+) ) nutrition and a robust guard cell K(+) inward channel activity is considered critical for plants' adaptation to fluctuating and challenging growth environment. ABA induces stomatal closure through hydrogen peroxide and nitric oxide (NO) along with subsequent ion channel-mediated loss of K(+) and anions. However, the interactions of NO synthesis and signalling with K(+) nutrition and guard cell K(+) channel activities have not been fully explored in Arabidopsis. Physiological and molecular techniques were employed to dissect the interaction of nitrogen and potassium nutrition in regulating stomatal opening, CO2 assimilation and ion channel activity. These data, gene expression and ABA signalling transduction were compared in wild-type Columbia-0 (Col-0) and the nitrate reductase mutant nia1nia2. Growth and K(+) nutrition were impaired along with stomatal behaviour, membrane transport, and expression of genes associated with ABA signalling in the nia1nia2 mutant. ABA-inhibited K(+) in current and ABA-enhanced slow anion current were absent in nia1nia2. Exogenous NO restored regulation of these channels for complete stomatal closure in nia1nia2. While NO is an important signalling component in ABA-induced stomatal closure in Arabidopsis, our findings demonstrate a more complex interaction associating potassium nutrition and nitrogen metabolism in the nia1nia2 mutant that affects stomatal function.

Systems analysis of guard cell membrane transport for enhanced stomatal dynamics and water use efficiency.

Stomatal transpiration is at the center of a crisis in water availability and crop production that is expected to unfold over the next 20 to 30 years. Global water usage has increased 6-fold in the past 100 years, twice as fast as the human population, and is expected to double again before 2030, driven mainly by irrigation and agriculture. Guard cell membrane transport is integral to controlling stomatal aperture and offers important targets for genetic manipulation to improve crop performance. However, its complexity presents a formidable barrier to exploring such possibilities. With few exceptions, mutations that increase water use efficiency commonly have been found to do so with substantial costs to the rate of carbon assimilation, reflecting the trade-off in CO2 availability with suppressed stomatal transpiration. One approach yet to be explored in detail relies on quantitative systems analysis of the guard cell. Our deep knowledge of transport and homeostasis in these cells gives real substance to the prospect for reverse engineering of stomatal responses, using in silico design in directing genetic manipulation for improved water use and crop yields. Here we address this problem with a focus on stomatal kinetics, taking advantage of the OnGuard software and models of the stomatal guard cell recently developed for exploring stomatal physiology. Our analysis suggests that manipulations of single transporter populations are likely to have unforeseen consequences. Channel gating, especially of the dominant K⁺ channels, appears the most favorable target for experimental manipulation.

PYR/PYL/RCAR abscisic acid receptors regulate K+ and Cl- channels through reactive oxygen species-mediated activation of Ca2+ channels at the plasma membrane of intact Arabidopsis guard cells.

The discovery of the START family of abscisic acid (ABA) receptors places these proteins at the front of a protein kinase/phosphatase signal cascade that promotes stomatal closure. The connection of these receptors to Ca(2+) signals evoked by ABA has proven more difficult to resolve, although it has been implicated by studies of the pyrbactin-insensitive pyr1/pyl1/pyl2/pyl4 quadruple mutant. One difficulty is that flux through plasma membrane Ca(2+) channels and Ca(2+) release from endomembrane stores coordinately elevate cytosolic free Ca(2+) concentration ([Ca(2+)]i) in guard cells, and both processes are facilitated by ABA. Here, we describe a method for recording Ca(2+) channels at the plasma membrane of intact guard cells of Arabidopsis (Arabidopsis thaliana). We have used this method to resolve the loss of ABA-evoked Ca(2+) channel activity at the plasma membrane in the pyr1/pyl1/pyl2/pyl4 mutant and show the consequent suppression of [Ca(2+)]i increases in vivo. The basal activity of Ca(2+) channels was not affected in the mutant; raising the concentration of Ca(2+) outside was sufficient to promote Ca(2+) entry, to inactivate current carried by inward-rectifying K(+) channels and to activate current carried by the anion channels, both of which are sensitive to [Ca(2+)]i elevations. However, the ABA-dependent increase in reactive oxygen species (ROS) was impaired. Adding the ROS hydrogen peroxide was sufficient to activate the Ca(2+) channels and trigger stomatal closure in the mutant. These results offer direct evidence of PYR/PYL/RCAR receptor coupling to the activation by ABA of plasma membrane Ca(2+) channels through ROS, thus affecting [Ca(2+)]i and its regulation of stomatal closure.

Systems dynamic modeling of the stomatal guard cell predicts emergent behaviors in transport, signaling, and volume control.

The dynamics of stomatal movements and their consequences for photosynthesis and transpirational water loss have long been incorporated into mathematical models, but none have been developed from the bottom up that are widely applicable in predicting stomatal behavior at a cellular level. We previously established a systems dynamic model incorporating explicitly the wealth of biophysical and kinetic knowledge available for guard cell transport, signaling, and homeostasis. Here we describe the behavior of the model in response to experimentally documented changes in primary pump activities and malate (Mal) synthesis imposed over a diurnal cycle. We show that the model successfully recapitulates the cyclic variations in H⁺, K⁺, Cl⁻, and Mal concentrations in the cytosol and vacuole known for guard cells. It also yields a number of unexpected and counterintuitive outputs. Among these, we report a diurnal elevation in cytosolic-free Ca²âº concentration and an exchange of vacuolar Cl⁻ with Mal, both of which find substantiation in the literature but had previously been suggested to require additional and complex levels of regulation. These findings highlight the true predictive power of the OnGuard model in providing a framework for systems analysis of stomatal guard cells, and they demonstrate the utility of the OnGuard software and HoTSig library in exploring fundamental problems in cellular physiology and homeostasis.

OnGuard, a computational platform for quantitative kinetic modeling of guard cell physiology.

Stomatal guard cells play a key role in gas exchange for photosynthesis while minimizing transpirational water loss from plants by opening and closing the stomatal pore. Foliar gas exchange has long been incorporated into mathematical models, several of which are robust enough to recapitulate transpirational characteristics at the whole-plant and community levels. Few models of stomata have been developed from the bottom up, however, and none are sufficiently generalized to be widely applicable in predicting stomatal behavior at a cellular level. We describe here the construction of computational models for the guard cell, building on the wealth of biophysical and kinetic knowledge available for guard cell transport, signaling, and homeostasis. The OnGuard software was constructed with the HoTSig library to incorporate explicitly all of the fundamental properties for transporters at the plasma membrane and tonoplast, the salient features of osmolite metabolism, and the major controls of cytosolic-free Ca²âº concentration and pH. The library engenders a structured approach to tier and interrelate computational elements, and the OnGuard software allows ready access to parameters and equations 'on the fly' while enabling the network of components within each model to interact computationally. We show that an OnGuard model readily achieves stability in a set of physiologically sensible baseline or Reference States; we also show the robustness of these Reference States in adjusting to changes in environmental parameters and the activities of major groups of transporters both at the tonoplast and plasma membrane. The following article addresses the predictive power of the OnGuard model to generate unexpected and counterintuitive outputs.

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 Ca(2+) influx to elevate cytosolic pH (pH(i)) and free cytosolic Ca(2+) concentration ([Ca(2+)](i)), in turn regulating the K(+) channels. We have confirmed these predictions, measuring pH(i) and [Ca(2+)](i) in vivo, and report that experimental manipulation of pH(i) and [Ca(2+)](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 pH(i) homeostasis.

A novel motif essential for SNARE interaction with the K(+) channel KC1 and channel gating in Arabidopsis.

The SNARE (for soluble N-ethylmaleimide-sensitive factor protein attachment protein receptor) protein SYP121 (=SYR1/PEN1) of Arabidopsis thaliana facilitates vesicle traffic, delivering ion channels and other cargo to the plasma membrane, and contributing to plant cell expansion and defense. Recently, we reported that SYP121 also interacts directly with the K(+) channel subunit KC1 and forms a tripartite complex with a second K(+) channel subunit, AKT1, to control channel gating and K(+) transport. Here, we report isolating a minimal sequence motif of SYP121 prerequisite for its interaction with KC1. We made use of yeast mating-based split-ubiquitin and in vivo bimolecular fluorescence complementation assays for protein-protein interaction and of expression and electrophysiological analysis. The results show that interaction of SYP121 with KC1 is associated with a novel FxRF motif uniquely situated within the first 12 residues of the SNARE sequence, that this motif is the minimal requirement for SNARE-dependent alterations in K(+) channel gating when heterologously expressed, and that rescue of KC1-associated K(+) current of the root epidermis in syp121 mutant Arabidopsis plants depends on expression of SNARE constructs incorporating this motif. These results establish the FxRF sequence as a previously unidentified motif required for SNARE-ion channel interactions and lead us to suggest a mechanistic framework for understanding the coordination of vesicle traffic with transmembrane ion transport.

What can mechanistic models tell us about guard cells, photosynthesis, and water use efficiency?

Stomatal pores facilitate gaseous exchange between the inner air spaces of the leaf and the atmosphere. The pores open to enable CO2 entry for photosynthesis and close to reduce transpirational water loss. How stomata respond to the environment has long attracted interest in modeling as a tool to understand the consequences for the plant and for the ecosystem. Models that focus on stomatal conductance for gas exchange make intuitive sense, but such models need also to connect with the mechanics of the guard cells that regulate pore aperture if we are to understand the 'decisions made' by stomata, their impacts on the plant and on the global environment.

Engineering a K+ channel 'sensory antenna' enhances stomatal kinetics, water use efficiency and photosynthesis.

Stomata of plant leaves open to enable CO2 entry for photosynthesis and close to reduce water loss via transpiration. Compared with photosynthesis, stomata respond slowly to fluctuating light, reducing assimilation and water use efficiency. Efficiency gains are possible without a cost to photosynthesis if stomatal kinetics can be accelerated. Here we show that clustering of the GORK channel, which mediates K+ efflux for stomatal closure in the model plant Arabidopsis, arises from binding between the channel voltage sensors, creating an extended 'sensory antenna' for channel gating. Mutants altered in clustering affect channel gating to facilitate K+ flux, accelerate stomatal movements and reduce water use without a loss in biomass. Our findings identify the mechanism coupling channel clustering with gating, and they demonstrate the potential for engineering of ion channels native to the guard cell to enhance stomatal kinetics and improve water use efficiency without a cost in carbon fixation.