Adrian Hills

Nitric oxide regulates K+ and Cl- channels in guard cells through a subset of abscisic acid-evoked signaling pathways

Abscisic acid (ABA) triggers a complex sequence of signaling events that lead to concerted modulation of ion channels at the plasma membrane of guard cells and solute efflux to drive stomatal closure in plant leaves. Recent work has indicated that nitric oxide (NO) and its synthesis are a prerequisite for ABA signal transduction in Arabidopsis and Vicia guard cells. Its mechanism(s) of action is not well defined in guard cells and, generally, in higher plants. Here we show directly that NO selectively regulates Ca2+-sensitive ion channels of Vicia guard cells by promoting Ca2+ release from intracellular stores to raise cytosolic-free [Ca2+]. NO-sensitive Ca2+ release was blocked by antagonists of guanylate cyclase and cyclic ADP ribose-dependent endomembrane Ca2+ channels, implying an action mediated via a cGMP-dependent cascade. NO did not recapitulate ABA-evoked control of plasma membrane Ca2+ channels and Ca2+-insensitive K+ channels, and NO scavengers failed to block the activation of these K+ channels evoked by ABA. These results place NO action firmly within one branch of the Ca2+-signaling pathways engaged by ABA and define the boundaries of parallel signaling events in the control of guard cell movements.

EZ-Rhizo: integrated software for the fast and accurate measurement of root system architecture

The root system is essential for the growth and development of plants. In addition to anchoring the plant in the ground, it is the site of uptake of water and minerals from the soil. Plant root systems show an astonishing plasticity in their architecture, which allows for optimal exploitation of diverse soil structures and conditions. The signalling pathways that enable plants to sense and respond to changes in soil conditions, in particular nutrient supply, are a topic of intensive research, and root system architecture (RSA) is an important and obvious phenotypic output. At present, the quantitative description of RSA is labour intensive and time consuming, even using the currently available software, and the lack of a fast RSA measuring tool hampers forward and quantitative genetics studies. Here, we describe EZ-Rhizo: a Windows-integrated and semi-automated computer program designed to detect and quantify multiple RSA parameters from plants growing on a solid support medium. The method is non-invasive, enabling the user to follow RSA development over time. We have successfully applied EZ-Rhizo to evaluate natural variation in RSA across 23 Arabidopsis thaliana accessions, and have identified new RSA determinants as a basis for future quantitative trait locus (QTL) analysis.

Control of Guard Cell Ion Channels by Hydrogen Peroxide and Abscisic Acid Indicates Their Action through Alternate Signaling Pathways

Recent evidence has implicated the action of reactive oxygen species (ROS), notably hydrogen peroxide (H2O2), in abscisic acid (ABA) signaling of guard cells. ABA is known to evoke increases in cytosolic-free [Ca2+] ([Ca2+]i), dependent on flux through Ca2+ channels in the plasma membrane and

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 Ca2+ 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 Ca2+ 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.

Dynamic regulation of guard cell anion channels by cytosolic free Ca2+ concentration and protein phosphorylation

In guard cells, activation of anion channels (I(anion)) is an early event leading to stomatal closure. Activation of I(anion) has been associated with abscisic acid (ABA) and its elevation of the cytosolic free Ca(2+) concentration ([Ca(2+)](i)). However, the dynamics of the action of [Ca(2+)](i) on I(anion) has never been established, despite its importance for understanding the mechanics of stomatal adaptation to stress. We have quantified the [Ca(2+)](i) dynamics of I(anion) in Vicia faba guard cells, measuring channel current under a voltage clamp while manipulating and recording [Ca(2+)](i) using Fura-2 fluorescence imaging. We found that I(anion) rises with [Ca(2+)](i) only at concentrations substantially above the mean resting value of 125 +/- 13 nm, yielding an apparent K(d) of 720 +/- 65 nm and a Hill coefficient consistent with the binding of three to four Ca(2+) ions to activate the channels. Approximately 30% of guard cells exhibited a baseline of I(anion) activity, but without a dependence of the current on [Ca(2+)](i). The protein phosphatase antagonist okadaic acid increased this current baseline over twofold. Additionally, okadaic acid altered the [Ca(2+)](i) sensitivity of I(anion), displacing the apparent K(d) for [Ca(2+)](i) to 573 +/- 38 nm. These findings support previous evidence for different modes of regulation for I(anion), only one of which depends on [Ca(2+)](i), and they underscore an independence of [Ca(2+)](i) from protein (de-)phosphorylation in controlling I(anion). Most importantly, our results demonstrate a significant displacement of I(anion) sensitivity to higher [Ca(2+)](i) compared with that of the guard cell K(+) channels, implying a capacity for variable dynamics between net osmotic solute uptake and loss.

A Novel Motif Essential for SNARE Interaction with the K+ Channel KC1 and Channel Gating in Arabidopsis

The SNARE protein of Arabidopsis, SYP121, contributes to vesicle traffic and also controls the gating of K+ channels for K+ uptake by binding to the KC1 channel subunit. The identity of the KC1 binding site on the SNARE protein, described in this study, points to a novel role for the channel subunit in coordinating vesicle traffic. 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.

A minimal cysteine motif required to activate the SKOR K+ channel of Arabidopsis by the reactive oxygen species H2O2

Reactive oxygen species (ROS) are essential for development and stress signaling in plants. They contribute to plant defense against pathogens, regulate stomatal transpiration, and influence nutrient uptake and partitioning. Although both Ca2+ and K+ channels of plants are known to be affected, virtually nothing is known of the targets for ROS at a molecular level. Here we report that a single cysteine (Cys) residue within the Kv-like SKOR K+ channel of Arabidopsis thaliana is essential for channel sensitivity to the ROS H2O2. We show that H2O2 rapidly enhanced current amplitude and activation kinetics of heterologously expressed SKOR, and the effects were reversed by the reducing agent dithiothreitol (DTT). Both H2O2 and DTT were active at the outer face of the membrane and current enhancement was strongly dependent on membrane depolarization, consistent with a H2O2-sensitive site on the SKOR protein that is exposed to the outside when the channel is in the open conformation. Cys substitutions identified a single residue, Cys168 located within the S3 α-helix of the voltage sensor complex, to be essential for sensitivity to H2O2. The same Cys residue was a primary determinant for current block by covalent Cys S-methioylation with aqueous methanethiosulfonates. These, and additional data identify Cys168 as a critical target for H2O2, and implicate ROS-mediated control of the K+ channel in regulating mineral nutrient partitioning within the plant.

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.

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

A new strategy for recording Ca2+ channels in intact guard cells demonstrates a central role for Ca2+ channel activation and elevated cytosol-free Ca2+ concentrations in signaling mediated by the PYR/PYL/RCAR family of abscisic acid receptors. 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 Ca2+ 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 Ca2+ channels and Ca2+ release from endomembrane stores coordinately elevate cytosolic free Ca2+ concentration ([Ca2+]i) in guard cells, and both processes are facilitated by ABA. Here, we describe a method for recording Ca2+ 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 Ca2+ channel activity at the plasma membrane in the pyr1/pyl1/pyl2/pyl4 mutant and show the consequent suppression of [Ca2+]i increases in vivo. The basal activity of Ca2+ channels was not affected in the mutant; raising the concentration of Ca2+ outside was sufficient to promote Ca2+ 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 [Ca2+]i elevations. However, the ABA-dependent increase in reactive oxygen species (ROS) was impaired. Adding the ROS hydrogen peroxide was sufficient to activate the Ca2+ 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 Ca2+ channels through ROS, thus affecting [Ca2+]i and its regulation of stomatal closure.