The evolutionary development of grass stomata appears to have been a gradual progression

Molecular evolution of grass stomata

Chen Z.-H., Chen G., Dai F., Wang Y., Hills A., Ruan Y.-L., Zhang G., Franks P. J., Nevo E., Blatt M. R., (2017)

Trends Plant Sci 22: 124-139 – PMID:27776931 – https://doi.org/10.1016/j.tplants.2016.09.005 –

https://www.tandfonline.com/doi/full/10.1080/15592324.2017.1339858

Evolutionary trajectories of land plants have led to structurally complex and functionally active stomata for terrestrial life. A likely scenario for the emergence of active stomatal control is ‘evolutionary capture’ of key stomatal development, membrane transport, and abscisic acid signaling proteins in the divergence from liverworts to mosses.The unique morphology, development, and molecular regulation of grass stomata enable their rapid environmental response. Evolution of the molecular mechanism behind stomatal development and membrane transport has clearly drawn on conserved and sophisticated signaling networks common to stomata of all vascular plants and some mosses. Understanding this evolutionary trend will inform predictive modeling and functional manipulation of plant productivity and water use at all scales, and will benefit future efforts towards food security and ecological diversity.

Grasses began to diversify in the late Cretaceous Period and now dominate more than one third of global land area, including three-quarters of agricultural land. We hypothesize that their success is likely attributed to the evolution of highly responsive stomata capable of maximizing productivity in rapidly changing environments. Grass stomata harness the active turgor control mechanisms present in stomata of more ancient plant lineages, maximizing several morphological and developmental features to ensure rapid responses to environmental inputs. The evolutionary development of grass stomata appears to have been a gradual progression. Therefore, understanding the complex structures, developmental events, regulatory networks, and combinations of ion transporters necessary to drive rapid stomatal movement may inform future efforts towards breeding new crop varieties.

PYR/PYL/RCAR receptor coupling to the activation by ABA of plasma membrane Ca(2+) channels through ROS, affecting [Ca(2+)]i and its regulation of stomatal closure

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

by Wang Y., Chen Z. H., Zhang B., Hills A., Blatt M. R. ( 2013)

Laboratory of Plant Physiology and Biophysics, University of Glasgow, Glasgow G12 8QQ, United Kingdom.

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In Plant Physiol 163: 566– 577 – DOI: 10.1104/pp.113.219758 –

https://www.ncbi.nlm.nih.gov/pubmed/23899646?dopt=Abstract

Abstract

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.

The OnGuard model providing a framework for systems analysis of stomatal guard cells

 

 

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

by Chen Z. H., Hills A., Bätz U., Amtmann A., Lew V. L., Blatt M. R. (2012)

in Plant Physiol 159: 1235–1251 – doi:10.1104/pp.112.197350 –

CrossRef | CAS –

http://researchdirect.westernsydney.edu.au/islandora/object/uws:13280

Abstract

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.

Extracellular Ba2‡ and voltage interact in stomatal guard cells

 

Extracellular Ba2‡ and voltage interact to gate Ca2‡ channels at the plasma membrane of stomatal guard cells

by Hamilton D. W. A.,  Hills A., Blatt M. R. (2001)

in FEBS Letters 491 (2001) 99-103 – 

http://onlinelibrary.wiley.com/store/10.1016/S0014-5793(01)02176-7/asset/feb2s0014579301021767.pdf?v=1&t=ip5xzwp2&s=16a1dd5e6e60dc5a7e36dea9e88360ec36821ea3

Abstract

Ca2+ channels at the plasma membrane of stomatal guard cells contribute to increases in cytosolic free [Ca2+] ([Ca2+]i) that regulate K+ and Cl3 channels for stomatal closure in higher-plant leaves.

Under voltage clamp, the initial rate of increase in [Ca2+]i in guard cells is sensitive to the extracellular divalent concentration, suggesting a close interaction between the permeant ion and channel gating.

To test this idea, we recorded single-channel currents across the Vicia guard cell plasma membrane using Ba2+ as a charge carrying ion. Unlike other Ca2+ channels characterised to date, these channels activate at hyperpolarising voltages.

We found that the open probability (Po) increased strongly with external Ba2+ concentration, consistent with a 4-fold cooperative action of Ba2+ in which its binding promoted channel opening in the steady state.

Dwell time analyses indicated the presence of a single open state and at least three closed states of the channel, and showed that both hyperpolarising voltage and external Ba2+ concentration prolonged channel residence in the open state.

Remarkably, increasing Ba2+ concentration also enhanced the sensitivity of the open channel to membrane voltage.

We propose that Ba2+ binds at external sites distinct from the permeation pathway and that divalent binding directly influences the voltage gate.

ABA, Ca2+ and stomatal guard cells

Photo credit: NCBI

P_o is suppressed by micromolar [Ca²⁺]_i. Means ± SE of P_o from mean open times of 100-s recordings at −120 mV (n = 3). Ca²⁺ added on the cytosolic side (inside) during inside-out recordings against a background of 30 mM Ba²⁺ and with 10 mM Ba²⁺ outside. (Insets) Segments of traces at each [Ca²⁺]_i. Data from one patch. Scale: vertical, 1 pA; horizontal, 1 s.

Ca²⁺ channels at the plasma membrane of stomatal guard cells are activated by hyperpolarization and abscisic acid.

by Hamilton D. W. A.,  Hills A., Kohler B., Blatt M. R. (2000)

in Proc. Natl Acad. Sci. USA, 97, 4967–4972. –

CrossRef | PubMed | CAS | ADS

http://www.ncbi.nlm.nih.gov/pubmed/10781106

Abstract

In stomatal guard cells of higher-plant leaves, abscisic acid (ABA) evokes increases in cytosolic free Ca(2+) concentration ([Ca(2+)](i)) by means of Ca(2+) entry from outside and release from intracellular stores. The mechanism(s) for Ca(2+) flux across the plasma membrane is poorly understood.

Because [Ca(2+)](i) increases are voltage-sensitive, we suspected a Ca(2+) channel at the guard cell plasma membrane that activates on hyperpolarization and is regulated by ABA.

We recorded single-channel currents across the Vicia guard cell plasma membrane using Ba(2+) as a charge-carrying ion. Both cell-attached and excised-patch measurements uncovered single-channel events with a maximum conductance of 12.8 +/- 0.4 pS and a high selectivity for Ba(2+) (and Ca(2+)) over K(+) and Cl(-).

Unlike other Ca(2+) channels characterized to date, these channels rectified strongly toward negative voltages with an open probability (P(o)) that increased with [Ba(2+)] outside and decreased roughly 10-fold when [Ca(2+)](i) was raised from 200 nM to 2 microM. Adding 20 microM ABA increased P(o), initially by 63- to 260-fold; in both cell-attached and excised patches, it shifted the voltage sensitivity for channel activation, and evoked damped oscillations in P(o) with periods near 50 s. A similar, but delayed response was observed in 0.1 microM ABA.

These results identify a Ca(2+)-selective channel that can account for Ca(2+) influx and increases in [Ca(2+)](i) triggered by voltage and ABA, and they imply a close physical coupling at the plasma membrane between ABA perception and Ca(2+) channel control.

NO, K and Cl in stomatal pathways

 

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

by Garcia-Mata C.,

 Gay R.,

 Sokolovski S.,

 Hills A.,

Lamattina L.,

Blatt M. R.  (2003)

in Proc. Natl Acad. Sci. USA, 100, 11116–11121. –

CrossRef | PubMed | CAS | ADS

Abstract

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.

OnGuard and stomatal guard cell physiology

 

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

by Blatt M. R.,

Wang Y.,

Leonhardt N.,

Hills A.

(2014)

in Journal of Plant Physiology, 2014, 171, 9, 770-778 – doi:10.1016/j.jplph.2013.09.014 –

http://www.sciencedirect.com/science/article/pii/S017616171300401X

Abstract

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.

Guard cell anion channels, Ca2+ and proteins

 

 

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

by Chen Z.-H.,

Hills A., Lim C. K.,

Blatt M. R.

(2010)

in The Plant Journal, 2010, 61, 5, 816 – DOI: 10.1111/j.1365-313X.2009.04108.x

Wiley Online Library

http://onlinelibrary.wiley.com/doi/10.1111/j.1365-313X.2009.04108.x/full

Summary

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 Ca²⁺ concentration ([Ca²⁺]_i). However, the dynamics of the action of [Ca²⁺]_i on I_anion has never been established, despite its importance for understanding the mechanics of stomatal adaptation to stress.

We have quantified the [Ca²⁺]_i dynamics of I_anion in Vicia faba guard cells, measuring channel current under a voltage clamp while manipulating and recording [Ca²⁺]_i using Fura-2 fluorescence imaging.

We found that I_anion rises with [Ca²⁺]_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 Ca²⁺ ions to activate the channels.

Approximately 30% of guard cells exhibited a baseline of I_anionactivity, but without a dependence of the current on [Ca²⁺]_i. The protein phosphatase antagonist okadaic acid increased this current baseline over twofold.

Additionally, okadaic acid altered the [Ca²⁺]_i sensitivity of I_anion, displacing the apparent K_d for [Ca²⁺]_i to 573 ± 38 nm.

These findings support previous evidence for different modes of regulation for I_anion, only one of which depends on [Ca²⁺]_i, and they underscore an independence of [Ca²⁺]_i from protein (de-)phosphorylation in controlling I_anion.

Most importantly, our results demonstrate a significant displacement of I_anion sensitivity to higher [Ca²⁺]_icompared with that of the guard cell K⁺ channels, implying a capacity for variable dynamics between net osmotic solute uptake and loss.

Stomata and nitric oxide

 

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

by Garcıa-Mata C., Gay R., Sokolovski S., Hills A., Lamattina L., Blatt M.R. (2003)

in Proc. Natl. Acad. Sci. USA 100:11116–11121.

CrossRef | CAS | PubMed |

Abstract

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.

See the text: PNAS

Read the full article: PNAS – Lens

K+ and other ion transport in guard cells

 

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

• by Blatt M. R. , Wang Y., Leonhardt N., Hills A. (2014)

in Journal of Plant Physiology, Volume 171, Issue 9, 15 May 2014, Pages 770–778

Conclusion and outlook

From the simulations illustrated here, two general observations can be drawn. The first – that transporters interact when operating at one membrane – is implicit in the nature of transport, which must occur in parallel across a common membrane, and was highlighted at the start of this article. It arises from the commonality of membrane voltage, and in many cases also the pools of ionic species on either side of the membrane, and it leads to both direct and indirect interactions between otherwise unrelated transporters. The example of slac1 is a case in point in which eliminating a Cl⁻ channel results in substantial changes in the intrinsic activities of two different K⁺ channels at the same membrane. The reader will discover similar patterns with the ost2 mutant simulations outlined here, and in many other situations. The second observation similarly arises from the commonality of the pools of ionic species that are transported across the serial barriers of the plasma membrane and tonoplast. In this case, the resulting coordination between fluxes of each ionic species at the two membranes leads to an uncanny sense of ‘communication’ between membranes. Yet this emergent behavior arises simply from the interconnections between [Ca²⁺]_i and pH as well as the shared ‘substrates’ and ‘products’ that comprise the various solutes transported across these membranes.

In hindsight, it should be obvious that the consequence of manipulating a single transporter at a membrane is rarely (if ever) restricted to this one process or solely to the distributions of the transported species. The difficulty is in anticipating the consequences of such manipulations, because they are generally beyond intuitive grasp. Clearly, these problems can only be addressed satisfactorily through quantitative mathematical modeling, such as the modeling illustrated here. We anticipate that the HoTSig libraries, on which the OnGuard software is built, will find applications in exploring many other cell systems for which there is kinetic detail of transport sufficient to develop truly predictive models. The modular construction of the HoTSig libraries (Hills et al., 2012) means that solute content, volume and turgor can be ‘bolted on’ to any phenomenological descriptors for virtually any plant cell type.

Read the full article: Science Direct