Anion channel sensitivity to cytosolic organic acids implicates a central role for oxaloacetate in integrating ion flux with metabolism in stomatal guard cells

Stomatal guard cells play a key role in gas exchange for photosynthesis and in minimizing transpirational water loss from plants by opening and closing the stomatal pore. The bulk of the osmotic content driving stomatal movements depends on ionic fluxes across both the plasma membrane and tonoplast, the metabolism of organic acids, primarily Mal (Imitate), and its accumulation and loss. Anion channels at the plasma membrane are thought to comprise a major pathway for Mal efflux during stomatal closure, implicating their key role in linking solute flux with metabolism. Nonetheless, little is known of the regulation of anion channel current (I(Cl)) by cytosolic Mal or its immediate metabolite OAA (oxaloacetate). In the present study, we have examined the impact of Mal, OAA and of the monocarboxylic acid anion acetate in guard cells of Vicia faba L. and report that all three organic acids affect I(Cl), but with markedly different characteristics and sidedness to their activities. Most prominent was a suppression of I(Cl) by OAA within the physiological range of concentrations found in vivo. These findings indicate a capacity for OAA to co-ordinate organic acid metabolism with I(Cl), through the direct effect of organic acid pool size. The findings of the present study also add perspective to in vivo recordings using acetate-based electrolytes

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<sup>2+</sup> signals evoked by ABA has proven more difficult to resolve, although it has been implicated by studies of the pyrbactin-insensitive <i>pyr1/pyl1/pyl2/pyl4</i> quadruple mutant. One difficulty is that flux through plasma membrane Ca<sup>2+</sup> channels and Ca<sup>2+</sup> release from endomembrane stores coordinately elevate cytosolic free Ca<sup>2+</sup> concentration ([Ca<sup>2+</sup>]<sub>i</sub>) in guard cells, and both processes are facilitated by ABA. Here, we describe a method for recording Ca<sup>2+</sup> 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<sup>2+</sup> channel activity at the plasma membrane in the <i>pyr1/pyl1/pyl2/pyl4</i> mutant and show the consequent suppression of [Ca<sup>2+</sup>]<sub>i</sub> increases in vivo. The basal activity of Ca<sup>2+</sup> channels was not affected in the mutant; raising the concentration of Ca<sup>2+</sup> outside was sufficient to promote Ca<sup>2+</sup> entry, to inactivate current carried by inward-rectifying K<sup>+</sup> channels and to activate current carried by the anion channels, both of which are sensitive to [Ca<sup>2+</sup>]<sub>i</sub> elevations. However, the ABA-dependent increase in reactive oxygen species (ROS) was impaired. Adding the ROS hydrogen peroxide was sufficient to activate the Ca<sup>2+</sup> 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<sup>2+</sup> channels through ROS, thus affecting [Ca<sup>2+</sup>]<sub>i</sub> and its regulation of stomatal closure

Exploring emergent properties in cellular homeostasis using OnGuard to model K+ and other ion transport in guard cells

It is widely recognized that the nature and characteristics of transport across eukaryotic membranes are so complex as to defy intuitive understanding. In these circumstances, quantitative mathematical modeling is an essential tool, both to integrate detailed knowledge of individual transporters and to extract the properties emergent from their interactions. As the first, fully integrated and quantitative modeling environment for the study of ion transport dynamics in a plant cell, OnGuard offers a unique tool for exploring homeostatic properties emerging from the interactions of ion transport, both at the plasma membrane and tonoplast in the guard cell. OnGuard has already yielded detail sufficient to guide phenotypic and mutational studies, and it represents a key step toward ‘reverse engineering’ of stomatal guard cell physiology, based on rational design and testing in simulation, to improve water use efficiency and carbon assimilation. Its construction from the HoTSig libraries enables translation of the software to other cell types, including growing root hairs and pollen. The problems inherent to transport are nonetheless challenging, and are compounded for those unfamiliar with conceptual ‘mindset’ of the modeler. Here we set out guidelines for the use of OnGuard and outline a standardized approach that will enable users to advance quickly to its application both in the classroom and laboratory. We also highlight the uncanny and emergent property of OnGuard models to reproduce the ‘communication’ evident between the plasma membrane and tonoplast of the guard cell

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

GORK is the only outward-rectifying Kv-like K<sup>+</sup> channel expressed in guard cells. Its activity is tightly regulated to facilitate K<sup>+</sup> efflux for stomatal closure and is elevated in ABA in parallel with suppression of the activity of the inward-rectifying K<sup>+</sup> 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<sup>+</sup> 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<sup>+</sup> channel blocker Ba<sup>2+</sup>. 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<sup>+</sup>, a characteristic that ensures the channel facilitates only K<sup>+</sup> efflux regardless of the external cation concentration. GORK conductance is also enhanced by external K<sup>+</sup> 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<sup>+</sup>

CDPKs CPK6 and CPK3 Function in ABA Regulation of Guard Cell S-Type Anion- and Ca(2+)- Permeable Channels and Stomatal Closure

Abscisic acid (ABA) signal transduction has been proposed to utilize cytosolic Ca(2+) in guard cell ion channel regulation. However, genetic mutants in Ca(2+) sensors that impair guard cell or plant ion channel signaling responses have not been identified, and whether Ca(2+)-independent ABA signaling mechanisms suffice for a full response remains unclear. Calcium-dependent protein kinases (CDPKs) have been proposed to contribute to central signal transduction responses in plants. However, no Arabidopsis CDPK gene disruption mutant phenotype has been reported to date, likely due to overlapping redundancies in CDPKs. Two Arabidopsis guard cell–expressed CDPK genes, CPK3 and CPK6, showed gene disruption phenotypes. ABA and Ca(2+) activation of slow-type anion channels and, interestingly, ABA activation of plasma membrane Ca(2+)-permeable channels were impaired in independent alleles of single and double cpk3cpk6 mutant guard cells. Furthermore, ABA- and Ca(2+)-induced stomatal closing were partially impaired in these cpk3cpk6 mutant alleles. However, rapid-type anion channel current activity was not affected, consistent with the partial stomatal closing response in double mutants via a proposed branched signaling network. Imposed Ca(2+) oscillation experiments revealed that Ca(2+)-reactive stomatal closure was reduced in CDPK double mutant plants. However, long-lasting Ca(2+)-programmed stomatal closure was not impaired, providing genetic evidence for a functional separation of these two modes of Ca(2+)-induced stomatal closing. Our findings show important functions of the CPK6 and CPK3 CDPKs in guard cell ion channel regulation and provide genetic evidence for calcium sensors that transduce stomatal ABA signaling

Systems Analysis of Guard Cell Membrane Transport for Enhanced Stomatal Dynamics and Water Use Efficiency

Stomatal transpiration is at the centre of a crisis in water availability and crop production that is expected to unfold over the next 20-30 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 any 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 (www.psrg.org.uk) 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

A chloroplast retrograde signal, 3’phosphoadenosine 5’-phosphate, acts as a secondary messenger in abscisic acid signaling in stomatal closure and germination

Organelle-nuclear retrograde signaling regulates gene expression, but its roles in specialized cells and integration with hormonal signaling remain enigmatic. Here we show that the SAL1-PAP (3'-phosphoadenosine 5'- phosphate) retrograde pathway interacts with abscisic acid (ABA) signaling to regulate stomatal closure and seed germination in Arabidopsis. Genetically or exogenously manipulating PAP bypasses the canonical signaling components ABA Insensitive 1 (ABI1) and Open Stomata 1 (OST1); priming an alternative pathway that restores ABA-responsive gene expression, ROS bursts, ion channel function, stomatal closure and drought tolerance in ost1-2. PAP also inhibits wild type and abi1-1 seed germination by enhancing ABA sensitivity. PAP-XRN signaling interacts with ABA, ROS and Ca2+; up-regulating multiple ABA signaling components, including lowly-expressed Calcium Dependent Protein Kinases (CDPKs) capable of activating the anion channel SLAC1. Thus, PAP exhibits many secondary messenger attributes and exemplifies how retrograde signals can have broader roles in hormone signaling, allowing chloroplasts to fine-tune physiological responses.Wannarat Pornsiriwong, Gonzalo M Estavillo, Kai Xun Chan, Estee E Tee, Diep Ganguly, Peter A Crisp, Su Yin Phua, Chenchen Zhao, Jiaen Qiu, Jiyoung Park, Miing Tiem Yong, Nazia Nisar, Arun Kumar Yadav, Benjamin Schwessinger, John Rathjen, Christopher I Cazzonelli, Philippa B Wilson, Matthew Gilliham, Zhong-Hua Chen, Barry J Pogso