Stomatal pores, formed by two surrounding guard cells in the epidermis of plant leaves, allow influx of atmospheric carbon dioxide in exchange for transpirational water loss. Stomata also restrict the entry of ozone-an important air pollutant that has an increasingly negative impact on crop yields, and thus global carbon fixation 1 and climate change 2 . The aperture of stomatal pores is regulated by the transport of osmotically active ions and metabolites across guard cell membranes 3,4 . Despite the vital role of guard cells in controlling plant water loss 3,4 , ozone sensitivity 1,2 and CO 2 supply 2,5-7 , the genes encoding some of the main regulators of stomatal movements remain unknown. It has been proposed that guard cell anion channels function as important regulators of stomatal closure and are essential in mediating stomatal responses to physiological and stress stimuli 3,4,8 . However, the genes encoding membrane proteins that mediate guard cell anion efflux have not yet been identified. Here we report the mapping and characterization of an ozone-sensitive Arabidopsis thaliana mutant, slac1. We show that SLAC1 (SLOW ANION CHANNEL-ASSOCIATED 1) is preferentially expressed in guard cells and encodes a distant homologue of fungal and bacterial dicarboxylate/malic acid transport proteins. The plasma membrane protein SLAC1 is essential for stomatal closure in response to CO 2 , ©2008 Nature Publishing GroupCorrespondence and requests for materials should be addressed to J.K. (jaakko.kangasjarvi@helsinki.fi). † Present address: Division of Biology, Imperial College London, London SW7 2AZ, UK. * These authors contributed equally to this work Fig. 3d and Supplementary Fig. 6a. N.N. performed experiments in Fig. 3a and Supplementary Fig. 7. Y.-F.W. performed experiments in Fig. 4 and Supplementary Figs 8 and 9. J.K. and J.I.S. wrote the paper. All the authors discussed the results, and commented on and edited the manuscript.The primary microarray data reported has been deposited with the ArrayExpress database under accession number E-MEXP-1388.Reprints and permissions information is available at www.nature.com/reprints. 8,11 by mediating anion efflux and causing membrane depolarization, which controls K + efflux through K + channels. So far, none of the candidates for plant anion channels -the plant homologues to the animal CLC chloride channels -has been localized to the plasma membrane 10 , and the first plant CLC channel that was functionally characterized encodes a central vacuolar proton/nitrate exchanger 12 , rather than an anion channel. Thus, despite their proposed importance in several physiological and stress responses in plants 8,10,11 , the molecular identity of the guard cell plasma membrane proteins that mediate anion channel activity has remained unknown. NIH Public AccessIn a mutant screen for O 3 sensitivity, a series of Arabidopsis ethyl methanesulphonate (EMS) mutants called radical-induced cell death (rcd) was identified 13,14 . One of them, a recessive mutant originally referred ...

Abscisic acid (ABA) is a key hormone for plant growth, development, and stress adaptation. Perception of ABA through four types of receptors has been reported. We show here that impairment of ABA perception through the PYRABACTIN RESISTANCE1 (PYR1)/PYR1-LIKE (PYL)/REGULATORY COMPONENTS OF ABA RECEPTORS (RCAR) branch reduces vegetative growth and seed production and leads to a severe open stomata and ABA-insensitive phenotype, even though other branches for ABA perception remain functional. An Arabidopsis thaliana sextuple mutant impaired in six PYR/PYL receptors, namely PYR1, PYL1, PYL2, PYL4, PYL5, and PYL8, was able to germinate and grow even on 100 mM ABA. Wholerosette stomatal conductance (Gst) measurements revealed that leaf transpiration in the sextuple pyr/pyl mutant was higher than in the ABA-deficient aba3-1 or ABA-insensitive snrk2.6 mutants. The gradually increasing Gst values of plants lacking three, four, five, and six PYR/PYLs indicate quantitative regulation of stomatal aperture by this family of receptors. The sextuple mutant lacked ABA-mediated activation of SnRK2s, and ABA-responsive gene expression was dramatically impaired as was reported in snrk2.2/2.3/2.6. In summary, these results show that ABA perception by PYR/PYLs plays a major role in regulation of seed germination and establishment, basal ABA signaling required for vegetative and reproductive growth, stomatal aperture, and transcriptional response to the hormone.

Experiments with

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SUMMARYThe air pollutant ozone can be used as a tool to unravel in planta processes induced by reactive oxygen species (ROS). Here, we have utilized ozone to study ROS-dependent stomatal signaling. We show that the ozonetriggered rapid transient decrease (RTD) in stomatal conductance coincided with a burst of ROS in guard cells. RTD was present in 11 different Arabidopsis ecotypes, suggesting that it is a genetically robust response. To study which signaling components or ion channels were involved in RTD, we tested 44 mutants deficient in various aspects of stomatal function. This revealed that the SLAC1 protein, essential for guard cell plasma membrane S-type anion channel function, and the protein kinase OST1 were required for the ROS-induced fast stomatal closure. We showed a physical interaction between OST1 and SLAC1, and provide evidence that SLAC1 is phosphorylated by OST1. Phosphoproteomic experiments indicated that OST1 phosphorylated multiple amino acids in the N terminus of SLAC1. Using TILLING we identified three new slac1 alleles where predicted phosphosites were mutated. The lack of RTD in two of them, slac1-7 (S120F) and slac1-8 (S146F), suggested that these serine residues were important for the activation of SLAC1. Mass-spectrometry analysis combined with site-directed mutagenesis and phosphorylation assays, however, showed that only S120 was a specific phosphorylation site for OST1. The absence of the RTD in the dominant-negative mutants abi1-1 and abi2-1 also suggested a regulatory role for the protein phosphatases ABI1 and ABI2 in the ROS-induced activation of the S-type anion channel.

Experiments with several Arabidopsis thaliana mutants have revealed a web of interactions between hormonal signaling. Here, we show that the Arabidopsis mutant radical-induced cell death1 (rcd1), although hypersensitive to apoplastic superoxide and ozone, is more resistant to chloroplastic superoxide formation, exhibits reduced sensitivity to abscisic acid, ethylene, and methyl jasmonate, and has altered expression of several hormonally regulated genes. Furthermore, rcd1 has higher stomatal conductance than the wild type. The rcd1-1 mutation was mapped to the gene At1g32230 where it disrupts an intron splice site resulting in a truncated protein. RCD1 belongs to the (ADP-ribosyl)transferase domain-containing subfamily of the WWE protein-protein interaction domain protein family. The results suggest that RCD1 could act as an integrative node in hormonal signaling and in the regulation of several stress-responsive genes.

Rapid stomatal closure induced by changes in the environment, such as elevation of CO2, reduction of air humidity, darkness, and pulses of the air pollutant ozone (O3), involves the SLOW ANION CHANNEL1 (SLAC1). SLAC1 is activated by OPEN STOMATA1 (OST1) and Ca2+-dependent protein kinases. OST1 activation is controlled through abscisic acid (ABA)-induced inhibition of type 2 protein phosphatases (PP2C) by PYRABACTIN RESISTANCE/REGULATORY COMPONENTS OF ABA RECEPTOR (PYR/RCAR) receptor proteins. To address the role of signaling through PYR/RCARs for whole-plant steady-state stomatal conductance and stomatal closure induced by environmental factors, we used a set of Arabidopsis (Arabidopsis thaliana) mutants defective in ABA metabolism/signaling. The stomatal conductance values varied severalfold among the studied mutants, indicating that basal ABA signaling through PYR/RCAR receptors plays a fundamental role in controlling whole-plant water loss through stomata. PYR/RCAR-dependent inhibition of PP2Cs was clearly required for rapid stomatal regulation in response to darkness, reduced air humidity, and O3. Furthermore, PYR/RCAR proteins seem to function in a dose-dependent manner, and there is a functional diversity among them. Although a rapid stomatal response to elevated CO2 was evident in all but slac1 and ost1 mutants, the bicarbonate-induced activation of S-type anion channels was reduced in the dominant active PP2C mutants abi1-1 and abi2-1. Further experiments with a wider range of CO2 concentrations and analyses of stomatal response kinetics suggested that the ABA signalosome partially affects the CO2-induced stomatal response. Thus, we show that PYR/RCAR receptors play an important role for the whole-plant stomatal adjustments and responses to low humidity, darkness, and O3 and are involved in responses to elevated CO2.

Plants grow and reproduce within a highly dynamic environment that can see abrupt changes in conditions, such as light intensity, temperature, humidity, or interactions with biotic agents. Recent studies revealed that plants can respond within seconds to some of these conditions, engaging many different metabolic and molecular networks, as well as rapidly altering their stomatal aperture. Some of these rapid responses were further shown to propagate throughout the entire plant via waves of reactive oxygen species (ROS) and Ca 2+ that are possibly mediated through the plant vascular system. Here, we propose that the integration of these signals is mediated through pulses of gene expression that are coordinated throughout the plant in a systemic manner by the ROS/Ca +2 waves. The Dynamic Environment of PlantsWhile growing within their natural habitat, or in a manmade field environment, plants are subjected to many different changes in their physical and biological surroundings. These can be gradual, such as the slow decrease in soil water content over time during summer, or rapid, such as changes in light intensity occurring as a result of sun flecks during a cloudy day [1-9] (Figure 1, Key Figure). Some of these rapid changes can occur simultaneously, or on the background of other more persistent stress conditions, such as a prolonged drought or a heat wave, essentially representing a state of stress combination [4,5,10]. In addition, due to the placement of the plant within its environment, for example its position within a row of plants or plot in the field, or its position in nature in close proximity to a tree cover, not all parts or tissues of the plant may experience the rapid change in environmental conditions simultaneously [4,11]. Under such conditions, the rapid responses (see Glossary) at the affected tissue could trigger systemic signaling pathways, such as the ROS [12] and calcium waves [13], electric signals [14], and/or hydraulic waves [15]. The occurrence of rapid changes in the physical and/ or biological environment of the plant, coupled with the discovery of rapid systemic signaling pathways activated by many of these conditions [12][13][14][15], and the discovery of rapid transcriptional and metabolic responses to stress [16,17], support a hypothesis that plants evolved sensing and acclimation mechanisms that may function [ 3 9 0 _ T D $ D I F F ] within the seconds to minute timescale and are important for the overall fitness of the plant and its ability to survive rapid changes within its environment (Figure 1). Here, we examine the molecular networks, and physiological and metabolic changes that occur in plants during rapid responses to stress, and propose that the integration of these responses is mediated through pulses of gene expression that are coordinated throughout the plant in a systemic manner by [ 3 9 1 _ T D $ D I F F ] the ROS/Ca +2 waves. HighlightsRecent studies reveal that plants respond within the seconds to minutes time-scale to different biotic and/or abiotic stimuli.The ra...