- Copyright © 2001 American Society of Plant Physiologists
The guard cells that flank the stomatal pores of leaves and stems must integrate and respond appropriately to a multitude of instantaneously varying stimuli. In addition to a background circadian rhythmicity, the principle physiological determinants of stomatal aperture are the respective levels of blue light, CO2, and the stress-induced hormone abscisic acid (ABA). The 1990s witnessed a blossoming in our understanding of how guard cells work, largely because of the advent of patch-clamp recording techniques, new ways to measure cytoplasmic free Ca2+([Ca2+]cyt), and the adoption of Arabidopsis as a model organism. This month's The Hot and the Classic presents a brief synopsis of the most cited guard cell research contribution for each year of the 1990s.
1990: Ca2+ Increase Precedes ABA-Induced Closure
Although there had been much indirect evidence that Ca2+ fluxes might be involved in regulating the responses of guard cells to ABA, McAinsh et al. (1990) were able to demonstrate this conclusively by application of fura-2, a fluorescent Ca2+ indicator to Commelina communisguard cells. Physiological concentrations of ABA caused a 2- to 10-fold increase in [Ca2+]cyt.
1991: Multiple Stretch-Activated Channels
Mechanosensitive ion channels in the plasma membrane of fava bean (Vicia faba) guard cell protoplasts were studied by the patch clamp technique (Cosgrove and Hedrich, 1991). Stretch-activated (SA) channels in outside-out patches were analyzed for channel conductance, kinetics, and ion selectivity. Three distinct SA channels were found that were permeable to Cl−, K+, and Ca2+. These SA channels may mediate ion transport across the plasma membrane directly, as well as influence the activity of non-SA channels via effects on membrane voltage and [Ca2+]cyt.
1992: Foreign Expression of Plant K+Channel Gene
KAT1 had previously been cloned from Arabidopsis by complementation of Saccharomyces cerevisiae mutants deficient in K+ uptake. Schachtman et al. (1992)report that a single mRNA transcript from the ArabidopsisKAT1 cDNA confers the functional expression of a hyperpolarization-activated K+ channel inXenopus laevis oocytes. The channel encoded byKAT1 is highly selective for K+ over other monovalent cations and is blocked by tetraethylammonium and Ba2+. These characteristics demonstrate thatKAT1 encodes an inward-rectifying K+channel.
1993: Control of Outward K+ Channel by Cytoplasmic pH
The activation by ABA of outward-rectifying K+ channels and its dependence on cytoplasmic pH were examined in stomatal guard cells of V. faba (Blatt and Armstrong, 1993). ABA caused a cytoplasmic alkalinization and a parallel rise in the outward-rectifying K+channel current. Acid loads, imposed with external butyrate, abolished the ABA-evoked K+ current. These results establish a causal link between cytoplasmic alkalinization and the activation of the outward K+ current by ABA and thus affirm a role for H+ in signaling and transport control in plants.
1994: Vacuolar K+ Channel
More than 90% of the K+ released from guard cells during stomatal closure originates from the guard cell vacuole.Ward & Schroeder (1994) report upon a novel type of K+ channel in the vacuolar membrane of V. faba guard cells that is activated by physiological increases in [Ca2+]cyt. The Ca2+, voltage, and pH dependences, high selectivity for K+, and high density of the K+ channels in the vacuolar membrane suggest a central role for these channels in vacuolar K+ release. The authors also presented a model of a possible mechanism of Ca2+-induced Ca2+ release involving the vacuolar K+ channel and a previously described slow vacuolar channel.
1995: Protein Phosphatase Regulation of K+Channels
Disruption of ABA sensitivity in wilty abi1-1 mutants of Arabidopsis and evidence that this gene encodes a protein phosphatase suggest that protein (de-)phosphorylation contributes to stomatal control by ABA. Armstrong et al. (1995)stably introduced the abi1-1 mutant allele intoNicotiana benthamiana, and monitored its influence on ion channel activity in guard cells under voltage clamp. Expression of theabi1-1 gene was associated with 2- to 6-fold reductions in an outward K+ current and the desensitization of both inward and outward K+ currents to ABA. In guard cells from the abi1-1 transformants, the protein kinase antagonists H7 or staurosporine restored the normal responses of both types of K+ channels and stomatal aperture to ABA. These results implicate ABI1 as part of a phosphatase/kinase pathway that modulates the sensitivity of guard-cell K+ channels to ABA-evoked signal cascades.
1996: Ca2+ and CO2-Induced Closure
Webb et al. (1996) used fura-2 fluorescence to measure increases in guard cell [Ca2+]cyt in stomatal guard cells of C. communisin response to increased CO2. Removal of extracellular Ca2+ both prevented the CO2-induced increase in [Ca2+]cyt and inhibited the associated reduction in stomatal aperture. These data suggest that an influx of Ca2+ is required for stomatal response to CO2.
1997: Activation of an Anion Channel by ABA
ABA strongly activates slow anion channels in wild-type Arabidopsis guard cells (Pei et al., 1997). Protein phosphatase inhibitors suppressed ABA-induced anion channel activation and stomatal closing. ABA activation of slow anion channels and ABA-induced stomatal closing were abolished in wilty abi1 andabi2 mutant guard cells. These impairments in ABA signaling were partially rescued by kinase inhibitors in abi1 but not in abi2 guard cells. These data provide evidence that theabi2 locus disrupts early ABA signaling, thatabi1 and abi2 affect ABA signaling at different steps in the cascade, and that protein kinases act as negative regulators of ABA signaling in Arabidopsis.
1998: Farnesyltransferase and ABA-Induced Closure
Protein farnesylation, a posttranslational modification process, mediates the COOH-terminal lipidation of specific cellular proteins such as Ras and G-proteins. Pei et al. (1998) report that deletion of the Arabidopsis farnesyltransferase geneERA1 or application of farnesyltransferase inhibitors resulted in ABA hypersensitivity of guard cell anion-channel activation and of stomatal closing (Pei et al., 1998). Double-mutant analyses ofera1 with the ABA-insensitive mutants abi1 andabi2 showed that era1 suppresses the ABA-insensitive phenotypes. Moreover, era1 plants exhibited a reduction in transpirational water loss during drought treatment.
1999: Phospholipase C and Ca Oscillations
ABA induces oscillations in C. communis guard cell [Ca2+]cyt(Staxen et al., 1999). The pattern of the oscillations depended on the ABA concentration and is correlated with the final stomatal aperture. U-71322, an inhibitor of phosphoinositide-specific phospholipase, inhibited both ABA-induced oscillations in [Ca2+]cyt and stomatal closure. An inactive analog of U-71322 was without effect. These findings suggest a role for phosphoinositide-specific phospholipase in the generation of ABA-induced oscillations in [Ca2+]cyt and suggest the involvement of oscillations in [Ca2+]cyt in the maintenance of stomatal aperture by ABA.
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- Article
- 1990: Ca2+ Increase Precedes ABA-Induced Closure
- 1991: Multiple Stretch-Activated Channels
- 1992: Foreign Expression of Plant K+Channel Gene
- 1993: Control of Outward K+ Channel by Cytoplasmic pH
- 1994: Vacuolar K+ Channel
- 1995: Protein Phosphatase Regulation of K+Channels
- 1996: Ca2+ and CO2-Induced Closure
- 1997: Activation of an Anion Channel by ABA
- 1998: Farnesyltransferase and ABA-Induced Closure
- 1999: Phospholipase C and Ca Oscillations
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