Gethyn J. Allen

Calcium channels activated by hydrogen peroxide mediate abscisic acid signalling in guard cells.

Drought is a major threat to agricultural production. Plants synthesize the hormone abscisic acid (ABA) in response to drought, triggering a signalling cascade in guard cells that results in stomatal closure, thus reducing water loss. ABA triggers an increase in cytosolic calcium in guard cells ([Ca2+]cyt) that has been proposed to include Ca2+ influx across the plasma membrane. However, direct recordings of Ca 2+ currents have been limited and the upstream activation mechanisms of plasma membrane Ca2+ channels remain unknown. Here we report activation of Ca2+-permeable channels in the plasma membrane of Arabidopsis guard cells by hydrogen peroxide. The H2O2-activated Ca2+ channels mediate both influx of Ca2+ in protoplasts and increases in [Ca 2+]cyt in intact guard cells. ABA induces the production of H2O2 in guard cells. If H2O2 production is blocked, ABA-induced closure of stomata is inhibited. Moreover, activation of Ca2+ channels by H2O2 and ABA- and H2O2-induced stomatal closing are disrupted in the recessive ABA-insensitive mutant gca2. These data indicate that ABA-induced H2O2 production and the H2O 2-activated Ca2+ channels are important mechanisms for ABA-induced stomatal closing.

Drought- and salt-tolerant plants result from overexpression of the AVP1 H+-pump

Transgenic plants overexpressing the vacuolar H+-pyrophosphatase are much more resistant to high concentrations of NaCl and to water deprivation than the isogenic wild-type strains. These transgenic plants accumulate more Na+ and K+ in their leaf tissue than the wild type. Moreover, direct measurements on isolated vacuolar membrane vesicles derived from the AVP1 transgenic plants and from wild type demonstrate that the vesicles from the transgenic plants have enhanced cation uptake. The phenotypes of the AVP1 transgenic plants suggest that increasing the vacuolar proton gradient results in increased solute accumulation and water retention. Presumably, sequestration of cations in the vacuole reduces their toxic effects. Genetically engineered drought- and salt-tolerant plants could provide an avenue to the reclamation of farmlands lost to agriculture because of salinity and a lack of rainfall.

Guard cell abscisic acid signalling and engineering drought hardiness in plants

Guard cells are located in the epidermis of plant leaves, and in pairs surround stomatal pores. These control both the influx of CO2 as a raw material for photosynthesis and water loss from plants through transpiration to the atmosphere. Guard cells have become a highly developed system for dissecting early signal transduction mechanisms in plants. In response to drought, plants synthesize the hormone abscisic acid, which triggers closing of stomata, thus reducing water loss. Recently, central regulators of guard cell abscisic acid signalling have been discovered. The molecular understanding of the guard cell signal transduction network opens possibilities for engineering stomatal responses to control CO2 intake and plant water loss.

A defined range of guard cell calcium oscillation parameters encodes stomatal movements

Oscillations in cytosolic calcium concentration ([Ca2+]cyt) are central regulators of signal transduction cascades, although the roles of individual [Ca2+]cyt oscillation parameters in regulating downstream physiological responses remain largely unknown. In plants, guard cells integrate environmental and endogenous signals to regulate the aperture of stomatal pores and [Ca2+]cyt oscillations are a fundamental component of stomatal closure. Here we systematically vary [Ca2+]cyt oscillation parameters in Arabidopsis guard cells using a ‘calcium clamp’ and show that [Ca2+]cyt controls stomatal closure by two mechanisms. Short-term ‘calcium-reactive’ closure occurred rapidly when [Ca2+]cyt was elevated, whereas the degree of long-term steady-state closure was ‘calcium programmed’ by [Ca2+]cyt oscillations within a defined range of frequency, transient number, duration and amplitude. Furthermore, in guard cells of the gca2 mutant, [Ca2+]cyt oscillations induced by abscisic acid and extracellular calcium had increased frequencies and reduced transient duration, and steady-state stomatal closure was abolished. Experimentally imposing [Ca2+]cyt oscillations with parameters that elicited closure in the wild type restored long-term closure in gca2 stomata. These data show that a defined window of guard cell [Ca2+]cyt oscillation parameters programs changes in steady-state stomatal aperture.

Arabidopsis abi1-1 and abi2-1 phosphatase mutations reduce abscisic acid-induced cytoplasmic calcium rises in guard cells

Elevations in cytoplasmic calcium ([Ca2+]cyt) are an important component of early abscisic acid (ABA) signal transduction. To determine whether defined mutations in ABA signal transduction affect [Ca2+]cyt signaling, the Ca2+-sensitive fluorescent dye fura 2 was loaded into the cytoplasm of Arabidopsis guard cells. Oscillations in [Ca2+]cyt could be induced when the external calcium concentration was increased, showing viable Ca2+ homeostasis in these dye-loaded cells. ABA-induced [Ca2+]cyt elevations in wild-type stomata were either transient or sustained, with a mean increase of ∼300 nM. Interestingly, ABA-induced [Ca2+]cyt increases were significantly reduced but not abolished in guard cells of the ABA-insensitive protein phosphatase mutants abi1 and abi2. Plasma membrane slow anion currents were activated in wild-type, abi1, and abi2 guard cell protoplasts by increasing [Ca2+]cyt, demonstrating that the impairment in ABA activation of anion currents in the abi1 and abi2 mutants was bypassed by increasing [Ca2+]cyt. Furthermore, increases in external calcium alone (which elevate [Ca2+]cyt) resulted in stomatal closing to the same extent in the abi1 and abi2 mutants as in the wild type. Conversely, stomatal opening assays indicated different interactions of abi1 and abi2, with Ca2+-dependent signal transduction pathways controlling stomatal closing versus stomatal opening. Together, [Ca2+]cyt recordings, anion current activation, and stomatal closing assays demonstrate that the abi1 and abi2 mutations impair early ABA signaling events in guard cells upstream or close to ABA-induced [Ca2+]cyt elevations. These results further demonstrate that the mutations can be bypassed during anion channel activation and stomatal closing by experimental elevation of [Ca2+]cyt.

Convergence of Calcium Signaling Pathways of Pathogenic Elicitors and Abscisic Acid in Arabidopsis Guard Cells

A variety of stimuli, such as abscisic acid (ABA), reactive oxygen species (ROS), and elicitors of plant defense reactions, have been shown to induce stomatal closure. Our study addresses commonalities in the signaling pathways that these stimuli trigger. A recent report showed that both ABA and ROS stimulate an NADPH-dependent, hyperpolarization-activated Ca2+ influx current in Arabidopsis guard cells termed “ICa” (Z.M. Pei, Y. Murata, G. Benning, S. Thomine, B. Klüsener, G.J. Allen, E. Grill, J.I. Schroeder, Nature [2002] 406: 731–734). We found that yeast (Saccharomyces cerevisiae) elicitor and chitosan, both elicitors of plant defense responses, also activate this current and activation requires cytosolic NAD(P)H. These elicitors also induced elevations in the concentration of free cytosolic calcium ([Ca2+]cyt) and stomatal closure in guard cells. ABA and ROS elicited [Ca2+]cytoscillations in guard cells only when extracellular Ca2+was present. In a 5 mm KCl extracellular buffer, 45% of guard cells exhibited spontaneous [Ca2+]cytoscillations that differed in their kinetic properties from ABA-induced Ca2+ increases. These spontaneous [Ca2+]cyt oscillations also required the availability of extracellular Ca2+ and depended on the extracellular potassium concentration. Interestingly, when ABA was applied to spontaneously oscillating cells, ABA caused cessation of [Ca2+]cyt elevations in 62 of 101 cells, revealing a new mode of ABA signaling. These data show that fungal elicitors activate a shared branch with ABA in the stress signal transduction pathway in guard cells that activates plasma membrane ICa channels and support a requirement for extracellular Ca2+ for elicitor and ABA signaling, as well as for cellular [Ca2+]cyt oscillation maintenance.

Hypersensitivity of Abscisic Acid–Induced Cytosolic Calcium Increases in the Arabidopsis Farnesyltransferase Mutant era1-2

Cytosolic calcium increases were analyzed in guard cells of the Arabidopsis farnesyltransferase deletion mutant era1-2 (enhanced response to abscisic acid). At low abscisic acid (ABA) concentrations (0.1 μM), increases of guard cell cytosolic calcium and stomatal closure were activated to a greater extent in the era1-2 mutant compared with the wild type. Patch clamping of era1-2 guard cells showed enhanced ABA sensitivity of plasma membrane calcium channel currents. These data indicate that the ERA1 farnesyltransferase targets a negative regulator of ABA signaling that acts between the points of ABA perception and the activation of plasma membrane calcium influx channels. Experimental increases of cytosolic calcium showed that the activation of S-type anion currents downstream of cytosolic calcium and extracellular calcium-induced stomatal closure were unaffected in era1-2, further supporting the positioning of era1-2 upstream of cytosolic calcium in the guard cell ABA signaling cascade. Moreover, the suppression of ABA-induced calcium increases in guard cells by the dominant protein phosphatase 2C mutant abi2-1 was rescued partially in era1-2 abi2-1 double mutant guard cells, further reinforcing the notion that ERA1 functions upstream of cytosolic calcium and indicating the genetic interaction of these two mutations upstream of ABA-induced calcium increases.

Combining Genetics and Cell Biology to Crack the Code of Plant Cell Calcium Signaling

Plant hormones, light receptors, pathogens, and abiotic signals trigger elevations in the cytosolic calcium concentration, which mediate physiological and developmental responses. Recent studies are reviewed here that reveal how specific genetic mutations impair or modify stimulus-induced calcium elevations in plant cells. These studies provide genetic evidence for the importance of calcium as a second messenger in plant signal transduction. A fundamental question arises: How can different stimuli use the same second messenger, calcium, to mediate different responses? Recent research and models are reviewed that suggest that several important mechanisms contribute to specificity in calcium signaling in plant cells. These mechanisms include (i) activation of different calcium channels in the plasma membrane and organellar membranes, (ii) stimulus-specific calcium oscillation parameters, (iii) cell type-specific responses, and (iv) intracellular localization of calcium gradients and calcium elevations in plant cells. Plant hormones and light signals enable plants to respond to changes in their environment. These stimuli are perceived by receptors that transmit responses to the inside of cells by inducing changes in cytoplasmic concentrations of chemical messengers, such as calcium. Many plant hormone and light signals cause elevations in the cytoplasmic calcium concentration. Recent studies are reviewed here that reveal how genetic mutations disrupt stimulus-induced calcium elevations in plant cells. These studies provide genetic evidence for the importance of calcium as a second messenger in plant signal transduction. A classical question has remained unanswered until recently, namely, how can different stimuli use the same second messenger, calcium, to mediate different responses in plants? Recent research and models are reviewed here that show several important mechanisms contributing to specificity in calcium signaling in plant cells. These mechanisms include (i) the activation of different calcium channels in the plasma membrane and organellar membranes of plant cells; (ii) the specific frequencies needed by calcium oscillations to mediate specific responses; (iii) the differential responses of different cell types to the same stimulus; and (iv) within each cell, the importance of intracellular localization of calcium gradients and calcium elevations to elicit the response. These advances show that plant cells contain a sophisticated apparatus that allows them to respond to many different environmental conditions through the second messenger, calcium.

Cyclic ADP-ribose and ABA signal transduction

A new assay system utilizing ABA-inducible gene expression in tomato hypocotyl cells has placed cADPR firmly in the signal transduction cascade of this hormone[1xAbscisic acid signaling through cyclic ADP-ribose in plants. Wu, Y. et al. Science. 1997; 278: 2126–2130Crossref | PubMed | Scopus (273)See all References][1]. Furthermore, similar transduction pathways utilizing cADPR appear to be activated regardless of whether the cellular response to ABA is gene expression or stomatal closure (Fig. 2Fig. 2). Striking similarities are evident between cADPR action in plant and animal cells.Fig. 2Schematic representation of ABA signal-transduction pathways that involve cyclic ADP-ribose (cADPR) and result in gene expression in tomato hypocotyl cells or loss of guard cell turgor. The upper part shows ABA perception and transduction in tomato hypocotyls, resulting in gene expression mediated by cADPR and elevation of cytosolic Ca2+ concentration[1xAbscisic acid signaling through cyclic ADP-ribose in plants. Wu, Y. et al. Science. 1997; 278: 2126–2130Crossref | PubMed | Scopus (273)See all References][1]. The lower part shows ABA perception and transduction in guard cells, resulting in loss of cell turgor mediated by cADPR and elevation of cytosolic Ca2+ concentration.View Large Image | Download PowerPoint Slide