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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.

SummaryCdk specificity is determined by the intrinsic selectivity of the active site and by substrate docking sites on the cyclin subunit. There is a long-standing debate about the relative importance of these factors in the timing of Cdk1 substrate phosphorylation. We analyzed major budding yeast cyclins (the G1/S-cyclin Cln2, S-cyclin Clb5, G2/M-cyclin Clb3, and M-cyclin Clb2) and found that the activity of Cdk1 toward the consensus motif increased gradually in the sequence Cln2-Clb5-Clb3-Clb2, in parallel with cell cycle progression. Further, we identified a docking element that compensates for the weak intrinsic specificity of Cln2 toward G1-specific targets. In addition, Cln2-Cdk1 showed distinct consensus site specificity, suggesting that cyclins do not merely activate Cdk1 but also modulate its active-site specificity. Finally, we identified several Cln2-, Clb3-, and Clb2-specific Cdk1 targets. We propose that robust timing and ordering of cell cycle events depend on gradual changes in the substrate specificity of Cdk1.

Studies on multisite phosphorylation networks of cyclin-dependent kinase (CDK) targets have opened a new level of signaling complexity by revealing signal processing routes encoded into disordered proteins. A model target, the CDK inhibitor Sic1, contains linear phosphorylation motifs, docking sites, and phosphodegrons to empower an N-to-C terminally directed phosphorylation process. Here, we uncover a signal processing mechanism involving multi-step competition between mutually diversional phosphorylation routes within the S-CDK-Sic1 inhibitory complex. Intracomplex phosphorylation plays a direct role in controlling Sic1 degradation, and provides a mechanism to sequentially integrate both the G1-and S-CDK activities while keeping S-CDK inhibited towards other targets. The competing phosphorylation routes prevent premature Sic1 degradation and demonstrate how integration of MAPK from the pheromone pathway allows one to tune the competition of alternative phosphorylation paths. The mutually diversional phosphorylation circuits may be a general way for processing multiple kinase signals to coordinate cellular decisions in eukaryotes.

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DSP-4 is a neurotoxin highly selective for the noradrenergic nerve terminals of the locus coeruleus projections. Data on the effect of DSP-4 treatment on amphetamine-induced hyperlocomotion are contradictory. In this study, DSP-4 (50 mg/kg) caused reduction of noradrenaline levels by 70% in the cerebral cortex and by 79% in the cerebellum. This treatment resulted in upregulation of dopamine D2 receptors in the striatum as evidenced by [3H]-raclopride binding. In an open field test, DSP-4 reduced locomotor activity. D-Amphetamine (1.5 mg/kg) caused a similar increase in locomotor activity in control and DSP-4-pretreated animals not familiar to the apparatus. However, when the rats were habituated to the test apparatus, the effect of amphetamine on horizontal activity was significantly larger in the DSP-4-pretreated animals. These data suggest that supersensitivity of D2 receptors develops after locus coeruleus denervation, but that the enhanced efficacy of amphetamine in DSP-4-treated rats is masked by neophobia.

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DSP-4 is a neurotoxin highly selective for the noradrenergic nerve terminals of the locus coeruleus projections. Data on the effect of DSP-4 treatment on amphetamine-induced hyperlocomotion are contradictory. In this study, DSP-4 (50 mg/kg) caused reduction of noradrenaline levels by 70% in the cerebral cortex and by 79% in the cerebellum. This treatment resulted in upregulation of dopamine D 2 receptors in the striatum as evidenced by [ In an open field test, DSP-4 reduced locomotor activity. D-Amphetamine (1.5 mg/kg) caused a similar increase in locomotor activity in control and DSP-4-pretreated animals not familiar to the apparatus. However, when the rats were habituated to the test apparatus, the effect of amphetamine on horizontal activity was significantly larger in the DSP-4-pretreated animals. These data suggest that supersensitivity of D 2 receptors develops after locus coeruleus denervation, but that the enhanced efficacy of amphetamine in DSP-4-treated rats is masked by neophobia.After the first report on N(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) (Ross 1976), this neurotoxin has been widely used for selective destruction of the nerve terminals originating from the locus coeruleus (Jonsson et al. 1981;Fritschy & Grzanna 1989). Peripheral administration of DSP-4 depletes noradrenaline (NA) in the brain regions which are innervated by the locus coeruleus and it has no direct effect on serotonin and dopamine (DA) terminals in the rat (Ö gren et al. 1980;Chrobak et al. 1985;Harro et al. 1992;Somboonthum et al. 1997;Al-Zahrani et al. 1998;Hughes & Stanford 1998), even though some investigators have found small but significant reductions in 5-HT levels (Jonsson et al. 1982;Ohno et al. 1996).Previous studies have shown that DSP-4-induced lesions cause no gross changes in spontaneous behaviour (Jonsson et al. 1982), nor does treatment with the drug interfere with simple attentional tasks (Ruotsalainen et al. 1997). However, in more complex learning tasks, the locus coeruleusdenervated rats are less successful and the animals fail to engage in active behaviour necessary to cope with environmental changes (Archer 1983;Archer et al. 1984), possibly due to an increase in neophobia and some reduction in their motivation to explore (Harro et al. 1995).Contradictory data exist about the effect of locus coeruleus denervation by DSP-4 treatment on locomotor activity Author for correspondence: Jaanus Harro, Department of Psychology, University of Tartu, Tiigi 78, EE-50410 Tartu, Estonia (fax π372 7 375900, e-mail jharro/ut.ee).and amphetamine-induced hyperlocomotion. In early studies using 6-hydroxydopamine lesions of the NA-containing pathways, it was found that large NA depletions in several forebrain regions did not modify the locomotor stimulant effect of amphetamine (Hollister et al. 1974;Roberts et al. 1975). However, since the study of Ö gren et al. (1983), it is generally believed that the hyperactivity induced by amphetamine is significantly reduced after selective locus coeruleus denervatio...