The relationship between guard cell water potential and the aperture of stomata in Populus

Abstract Previous work with clones of Populus trichocarpa demonstrated that the water vapour conductance of leaves from well-watered cuttings of this species does not decline with loss of turgor from the bulk leaf. In the present study, stomatal responses to water potential in Populus were examined with detached epidermal strips. Stomata in epidermal strips from well-watered plants of P. trichocarpa did not close at low water potentials which led to plasmolysis of the guard cells. In contrast, stomata of P. deltoides and a P. trichocarpa×deltoides hybrid closed when the guard cells lost turgor. A period of water stress preconditioning resulted in modified stomatal responses in P. trichocarpa such that stomata of stressed and re-watered plants nearly closed when guard cell turgor was lost.     

Involvement of sphingosine kinase in plant cell signalling

In mammalian cells sphingosine-1-phosphate (S1P) is a well-established messenger molecule that participates in a wide range of signalling pathways. The objective of the work reported here was to investigate the extent to which phosphorylated long-chain sphingoid bases, such as sphingosine-1-phosphate and phytosphingosine-1-phosphate (phytoS1P) are used in plant cell signalling. To do this, we manipulated Arabidopsis genes capable of metabolizing these messenger molecules. We show that Sphingosine kinase1 (SPHK1) encodes an enzyme that phosphorylates sphingosine, phytosphingosine and other sphingoid long-chain bases. The stomata of SPHK1-KD Arabidopsis plants were less sensitive, whereas the stomata of SPHK1-OE plants were more sensitive, than wild type to ABA. The rate of germination of SPHK1-KD was enhanced, whereas the converse was true for SPHK1-OE seed. Reducing expression of either the putative Arabidopsis S1P phosphatase (SPPASE) or the DPL1 gene, which encodes an enzyme with S1P lyase activity, individually, had no effect on guard-cell ABA signalling; however, stomatal responses to ABA in SPPASEDPL1 RNAi plants were compromised. Reducing the expression of DPL1 had no effect on germination; however, germination of SPPASE RNAi seeds was more sensitive to applied ABA. We also found evidence that expression of SPHK1 and SPPASE were coordinately regulated, and discuss how this might contribute to robustness in guard-cell signalling. In summary, our data establish SPHK1 as a component in two separate plant signalling systems, opening the possibility that phosphorylated long-chain sphingoid bases such as S1P and phytoS1P are ubiquitous messengers in plants.     

The role of calcium in ABA-induced gene expression and stomatal movements

There is much interest in the transduction pathways by which abscisic acid (ABA) regulates stomatal movements (ABA-turgor signalling) and by which this phytohormone regulates the pattern of gene expression in plant cells (ABA-nuclear signalling). A number of second messengers have been identified in both the ABA-turgor and ABA-nuclear signalling pathways. A major challenge is to understand the architecture of ABA-signalling pathways and to determine how the ABA signal is coupled to the appropriate response. We have investigated whether separate Ca2+-dependent and -independent ABA-signalling pathways are present in guard cells. Our data suggest that increases in [Ca2+]i are a common component of the guard cell ABA-turgor and ABA-nuclear signalling pathways. The effects of Ca2+ antagonists on ABA-induced stomatal closure and the ABA-responsive CDeT6-19 gene promoter suggest that Ca2+ is involved in both ABA-turgor signalling and ABA-nuclear signalling in guard cells. However, the sensitivity of these pathways to alterations in the external calcium concentration differ, suggesting that the ABA-nuclear and ABA-turgor signalling pathways are not completely convergent. Our data suggest that whilst Ca2+-independent signalling elements are present in the guard cell, they do not form a completely separate Ca2+-independent ABA-signalling pathway.     

Atherosclerosis and calcium signalling in endothelial cells

The link between atherosclerosis and regions of disturbed flow and low wall shear stress is now firmly established, but the causal mechanisms underlying the link are not yet understood. It is now recognised that the endothelium is not simply a passive barrier between the blood and the vessel wall, but plays an active role in maintaining vascular homeostasis and participates in the onset of atherosclerosis. Calcium signalling is one of the principal intracellular signalling mechanisms by which endothelial cells (EC) respond to external stimuli, such as fluid shear stress and ligand binding. Previous studies have separately modelled mass transport of chemical species in the bloodstream and calcium dynamics in EC via the inositol trisphosphate (IP(3)) signalling pathway. We review existing models of these two phenomena, before going on to integrate the two components to provide an inclusive new model for the calcium response of the endothelium in an arbitrary vessel geometry. This enables the combined effects of fluid flow and biochemical stimulation on EC to be investigated and is the first time spatially varying, physiological fluid flow-related environmental factors have been combined with intracellular signalling in a mathematical model. Model results show that low endothelial calcium levels in the area of disturbed flow at an arterial widening may be one contributing factor to the onset of vascular disease.     

Sustained calcium signalling and caspase-3 activation involve NMDA receptors in thymocytes in contact with dendritic cells

L-glutamate, the major excitatory neurotransmitter, also has a role in non-neuronal tissues and modulates immune responses. Whether NMDA receptor (NMDAR) signalling is involved in T-cell development is unknown. In this study, we show that mouse thymocytes expressed an array of glutamate receptors, including NMDARs subunits. Sustained calcium (Ca²⁺) signals and caspase-3 activation in thymocytes were induced by interaction with antigen-pulsed dendritic cells (DCs) and were inhibited by NMDAR antagonists MK801 and memantine. NMDARs were transiently activated, triggered the sustained Ca²⁺ signal and were corecruited with the PDZ-domain adaptor postsynaptic density (PSD)-95 to thymocyte-DC contact zones. Although T-cell receptor (TCR) activation was sufficient for relocalization of NMDAR and PSD-95 at the contact zone, NMDAR could be activated only in a synaptic context. In these T-DC contacts, thymocyte activation occurred in the absence of exogenous glutamate, indicating that DCs could be a physiological source of glutamate. DCs expressed glutamate, glutamate-specific vesicular glutamate transporters and were capable of fast glutamate release through a Ca²⁺-dependent mechanism. We suggest that glutamate released by DCs could elicit focal responses through NMDAR-signalling in T cells undergoing apoptosis. Thus, synapses between T and DCs could provide a functional platform for coupling TCR activation and NMDAR signalling, which might reflect on T-cell development and modulation of the immune response.     

Abscisic acid and CO2 signalling via calcium sensitivity priming in guard cells, new CDPK mutant phenotypes and a method for improved resolution of stomatal stimulus-response analyses

Background

Stomatal guard cells are the regulators of gas exchange between plants and the atmosphere. Ca²⁺-dependent and Ca²⁺-independent mechanisms function in these responses. Key stomatal regulation mechanisms, including plasma membrane and vacuolar ion channels have been identified and are regulated by the free cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt).

Scope

Here we show that CO₂-induced stomatal closing is strongly impaired under conditions that prevent intracellular Ca²⁺ elevations. Moreover, Ca²⁺ oscillation-induced stomatal closing is partially impaired in knock-out mutations in several guard cell-expressed Ca²⁺-dependent protein kinases (CDPKs) here, including the cpk4cpk11 double and cpk10 mutants; however, abscisic acid-regulated stomatal movements remain relatively intact in the cpk4cpk11 and cpk10 mutants. We further discuss diverse studies of Ca²⁺ signalling in guard cells, discuss apparent peculiarities, and pose novel open questions. The recently proposed Ca²⁺ sensitivity priming model could account for many of the findings in the field. Recent research shows that the stomatal closing stimuli abscisic acid and CO₂ enhance the sensitivity of stomatal closing mechanisms to intracellular Ca²⁺, which has been termed ‘calcium sensitivity priming’. The genome of the reference plant Arabidopsis thaliana encodes for over 250 Ca²⁺-sensing proteins, giving rise to the question, how can specificity in Ca²⁺ responses be achieved? Calcium sensitivity priming could provide a key mechanism contributing to specificity in eukaryotic Ca²⁺ signal transduction, a topic of central interest in cell signalling research. In this article we further propose an individual stomatal tracking method for improved analyses of stimulus-regulated stomatal movements in Arabidopsis guard cells that reduces noise and increases fidelity in stimulus-regulated stomatal aperture responses ( Box 1). This method is recommended for stomatal response research, in parallel to previously adopted blind analyses, due to the relatively small and diverse sizes of stomatal apertures in the reference plant Arabidopsis thaliana.

Box 1. Improved resolution of stimulus-induced stomatal movements in guard cells by tracking of individual stomatal apertures

Arabidopsis guard cells have become a prime model system for analysing signal transduction, since early research combining genetic and ion channel analyses in this system (Ichida et al., 1997; Pei et al., 1997, 1998; Roelfsema and Prins, 1997). Arabidopsis stomata are small relative to other stomatal model systems and stomatal apertures of various plant types including Arabidopsis are known to show variability in the size of individual stomatal complexes and also variability in the opening apertures of stomata of similar size in a given leaf (Gorton et al., 1988; Mott and Buckley, 2000; Mott and Peak, 2007). Thus stomatal aperture measurements are expected to show a clear degree of statistical variation. Use of blind experiments, in which the genotype and, when possible, the stimulus being applied to guard cells is unknown to the experimenter (Murata et al., 2001) has been employed by several laboratories, has become a standard in the field and has aided in addressing the above limitations of the range of stomatal aperture sizes found under any given condition.Research in our laboratory has shown that a major additional improvement in experiments can be made, by adding imaging of the same individual stomatal apertures over time (Allen et al., 2001; Mori et al., 2006; Vahisalu et al., 2008; Siegel et al., 2009), while performing blind experiments. In such ‘stomatal tracking’ experiments the lower side of a leaf is attached to a glass coverslip in an extracellular incubation medium (Webb et al., 2001; Young et al., 2006). The mesophyll and upper leaf epidermis are removed surgically for better optical resolution of stomatal apertures in the intact lower leaf epidermis (Young et al., 2006). For stimulus-induced stomatal closing analyses, a field of well-opened stomata is located and images are captured (e.g. using Scion Image software) for later analyses and data storage. The bottom (dry side) of coverslips can be marked with colour marker pens to label grids in the regions where apertures where imaged, for finding these same stomata subsequently if needed. Images of the same stomatal apertures are taken over time and can be stored for later analyses of individual stomatal apertures and for deposition of image files. While this approach has been used as a standard for imposed Ca²⁺ oscillation studies (Allen et al., 2001; Mori et al., 2006; Vahisalu et al., 2008; Fig. 4), we have found that this method also substantially improves stomatal movement response analyses to any given stimulus (Siegel et al., 2009; see Figs 1 and 4 and, Box Fig. 1). For example, while individual stomata are known to have diverse apertures (e.g. Box Fig. 1C), the relative responses of wide open stomata and smaller stomatal apertures to ABA or to CO₂ were comparable (Fig. 1 and Box Fig. 1; Siegel et al., 2009). Note that this method has previously been proposed and used in Vicia faba (Gorton et al., 1988), for which stomata exhibit relatively weak ABA and CO₂ responses, compared with, for example, Arabidopsis. We propose that this simple image-capturing approach, together with blind analyses, be used as a standard for stomatal response research in arabidopsis. Our research experience with this method shows that this approach will aid in greatly improving resolution and robustness and in defining the functions of individual Ca²⁺-independent and Ca²⁺-dependent components and mechanisms in stomatal response analyses. Open in a separate windowBox Fig. 1.ABA-induced stomatal closing of individually tracked stomatal apertures. (A) Average individually tracked stomatal apertures in the presence of 50 µm Ca²⁺ (open triangles) and in the presence of 200 nm free Ca²⁺ (open squares) in the bath solution from three experiments are shown and were normalized to the stomatal apertures at time = 0. (B, C) ABA-induced stomatal closing in the presence of 50 µm Ca²⁺ in five individually tracked stomatal apertures. In (A; open triangles) normalized stomatal apertures of the same stomata depicted in (B) and (C) are shown. Methods used in these experiments tracking individual stomatal apertures are described in Siegel et al. (2009). ABA-induced stomatal closing experiments are reproduced from Siegel et al. (2009) with permission of the publisher.     

Isolated plant nuclei as mechanical and thermal sensors involved in calcium signalling

Calcium signals in the nucleus elicit downstream effects that are distinct from those of cytosolic calcium signals. In the present work, we have evaluated the ability of plant nuclei to sense stimuli directly and to convert them into calcium changes. We show that individual mechanical stimulation of isolated nuclei elicits a single calcium transient at acidic pHs, whereas a series of stimulations leads to oscillations whose frequency reflects that of the stimuli. Conversely, at alkaline pHs, nuclei respond to temperature but not to stretch. The stretch- and the temperature-activated processes differ by their sensitivity to pharmacological drugs known to affect ion channel activities in animal cells. Our data demonstrate that isolated nuclei are able to gauge physical parameters of their environment. This might have a profound influence on the functioning of calcium-dependent processes known to control a large array of molecular events in the nucleus.     

Two guard cell‐preferential MAPKs,MPK9 and MPK12, regulate YEL signalling in Arabidopsis guard cells

We report that two mitogen‐activated protein kinases (MAPKs), MPK9 and MPK12, positively regulate abscisic acid (ABA)‐induced stomatal closure in Arabidopsis thaliana. Yeast elicitor (YEL) induced stomatal closure accompanied by intracellular reactive oxygen species (ROS) accumulation and cytosolic free calcium concentration ([Ca²⁺]_cyt) oscillation. In this study, we examined whether these two MAP kinases are involved in YEL‐induced stomatal closure using MAPKK inhibitors, PD98059 and U0126, and MAPK mutants, mpk9, mpk12 and mpk9 mpk12. Both PD98059 and U0126 inhibited YEL‐induced stomatal closure. YEL induced stomatal closure in the mpk9 and mpk12 mutants but not in the mpk9 mpk12 mutant, suggesting that a MAPK cascade involving MPK9 and MPK12 functions in guard cell YEL signalling. However, YEL induced extracellular ROS production, intracellular ROS accumulation and cytosolic alkalisation in the mpk9, mpk12 and mpk9 mpk12 mutants. YEL induced [Ca²⁺]_cyt oscillations in both wild type and mpk9 mpk12 mutant. These results suggest that MPK9 and MPK12 function redundantly downstream of extracellular ROS production, intracellular ROS accumulation, cytosolic alkalisation and [Ca²⁺]_cyt oscillation in YEL‐induced stomatal closure in Arabidopsis guard cells and are shared with ABA signalling.     

Early events in signalling high-temperature stress in tobacco BY2 cells involve alterations in membrane fluidity and enhanced hydrogen peroxide production

Alterations in membrane fluidity are among the early events in plants that detect changes in ambient temperature. However, signal transduction downstream of the membrane-associated processes is still not well understood. We have focused here on the role of hydrogen peroxide (H(2)O(2)) in high-temperature signalling in relation to changes in membrane fluidity in cells of tobacco (Nicotiana tabacum L.) cv. Bright Yellow 2 (BY2). As final indicators of the heat-signalling cascade, we have monitored the synthesis of small heat-shock proteins (sHSPs). Elevation of temperature between 32 and 38 degrees C resulted in a fast, transient stimulation of H(2)O(2) production in the tobacco cells. A similar H(2)O(2) burst could be induced at lower temperatures (28-32 degrees C) by membrane fluidization using benzyl alcohol (BA). Diphenylene iodonium (DPI), a NADPH oxidase inhibitor, prevented both the heat- and BA-triggered H(2)O(2) rise. The synthesis of sHSPs (14.5 and 16 kDa) was shifted to lower temperatures by BA application and was suppressed by DPI treatment in the same way. The results indicate that H(2)O(2) is an early component of the heat-signalling pathway, which responds rapidly to changes in membrane fluidity and is required for the activation of sHSP synthesis.     

The non-osmotic volume of Commelina guard cells

Abstract. The non-osmotic volume (NOV) of Connnelina communis L. guard cells was estimated by observing the volumes of guard cell protoplasts incubated in mannitol solutions of different solute potential, and applying the Boyle-van't Hoff relation to the results. NOV values of between 517 and 1782 μm³ were obtained for different batches of protoplasts. There was a negative correlation between NOV and apparent protoplast solute contents, and the NOV and solute content were observed to alter when pretreatments affecting stomatal aperture were given. H is hypothesized that changes in guard cell chloroplast starch levels could account for variation in NOV and solute content.
For closed stomata, it is calculated that the NOV could reduce the proportion of the total guard cell volume which is osmotically active by over 40%. Serious inaccuracy may thus result if the NOV is not taken into account in the estimation of guard cell solute potential or solute concentration from measurements of solute levels per cell. The error is maximal at low stomatal apertures.     

ATP-induced calcium signalling in acinar cells of the submandibular salivary gland

To study changes in the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) and the total amount of calcium in cells, we used, respectively, the fluorescent dye fura 2/AM and the metallochrome dye arsenazo III. The total amount of calcium in acinar cells after their incubation in calcium-free ATP-containing extracellular solution decreased. The action of ATP induced a dose-dependent increase in the [Ca²⁺]_i; the EC₅₀ was, on average, 130 ± ± 36 μM. Calcium transients induced by ATP demonstrated no desensitization. Against the background of a blocker of ionotropic P2X receptors, pyridoxal phosphate-6-azophenyl-2′,4′-disulfonic acid, we observed a decrease in the ATP-induced calcium transients by 72%. In addition, these transients were reduced by 65% in the calcium-free milieu, while after thapsigargin-induced exhaustion of the endoplasmic reticulum store they disappeared. This is indicative of the involvement of metabotropic P2Y receptors in the formation of the above calcium transients. Therefore, P2X and P2Y receptors participate in ATP-induced calcium signalling in acinar cells of the submandibular salivary gland; activation of these channels results in a rise in the [Ca²⁺]_i. The P2X receptors to a higher extent contribute to the formation of calcium signals; the P2Y-determined increase in the [Ca²⁺]_i is smaller (equal to about 35%). Therefore, the functionally active ligand-operated ionotropic P2Y receptors and metabotropic G protein-related P2Y receptors do exist in acinar cells of the submandibular salivary gland and play an important role in the control of functioning of this gland. Neirofiziologiya/Neurophysiology, Vol. 37, Nos. 5/6, pp. 395–402, September–December, 2005.     

Stochastic dynamics of actin filaments in guard cells regulating chloroplast localization during stomatal movement

Actin filaments and chloroplasts in guard cells play roles in stomatal function. However, detailed actin dynamics vary, and the roles that they play in chloroplast localization during stomatal movement remain to be determined. We examined the dynamics of actin filaments and chloroplast localization in transgenic tobacco expressing green fluorescent protein (GFP)-mouse talin in guard cells by time-lapse imaging. Actin filaments showed sliding, bundling and branching dynamics in moving guard cells. During stomatal movement, long filaments can be severed into small fragments, which can form longer filaments by end-joining activities. With chloroplast movement, actin filaments near chloroplasts showed severing and elongation activity in guard cells during stomatal movement. Cytochalasin B treatment abolished elongation, bundling and branching activities of actin filaments in guard cells, and these changes of actin filaments, and as a result, more chloroplasts were localized at the centre of guard cells. However, chloroplast turning to avoid high light, and sliding of actin fragments near the chloroplast, was unaffected following cytochalasin B treatment in guard cells. We suggest that the sliding dynamics of actin may play roles in chloroplast turning in guard cells. Our results indicate that the stochastic dynamics of actin filaments in guard cells regulate chloroplast localization during stomatal movement.