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.

In vertebrates, the endocannabinoid signaling pathway is an important lipid regulatory pathway that modulates a variety of physiological and behavioral processes. N-Acylethanolamines (NAEs) comprise a group of fatty acid derivatives that function within this pathway, and their signaling activity is terminated by an enzyme called fatty acid amide hydrolase (FAAH), which hydrolyzes NAEs to ethanolamine and their corresponding free fatty acids. Bioinformatic approaches led to the identification of plant homologues of FAAH that are capable of hydrolyzing NAEs in vitro. To better understand the role of NAEs in plants, we identified T-DNA knockouts to Arabidopsis FAAH (AtFAAH; At5g64440) and generated plants overexpressing AtFAAH. Here we show that seeds of AtFAAH knockouts had elevated levels of endogenous NAEs, and seedling growth was hypersensitive to exogenously applied NAE. On the other hand, seeds and seedlings of AtFAAH overexpressors had lower endogenous NAE content, and seedlings were less sensitive to exogenous NAE. Moreover, AtFAAH overexpressors displayed enhanced seedling growth and increased cell size. AtFAAH expression and FAAH catalytic activity increased during seed germination and seedling growth, consistent with the timing of NAE depletion during seedling establishment. Collectively, our results show that AtFAAH is one, but not the only, modulator of endogenous NAE levels in plants, and that NAE depletion likely participates in the regulation of plant growth.endocannabinoids ͉ lipids ͉ seedling growth ͉ signaling

The green fluorescent protein (GFP) gene was fused to the potato virus X (PVX) TGBp2 gene, inserted into either the PVX infectious clone or pRTL2 plasmids, and used to study protein subcellular targeting. In protoplasts and plants inoculated with PVX-GFP:TGBp2 or transfected with pRTL2-GFP:TGBp2, fluorescence was mainly in vesicles and the endoplasmic reticulum (ER). During late stages of virus infection, fluorescence became increasingly cytosolic and nuclear. Protoplasts transfected with PVX-GFP:TGBp2 or pRTL2-GFP:TGBp2 were treated with cycloheximide and the decline of GFP fluorescence was greater in virus-infected protoplasts than in pRTL2-GFP:TGBp2-transfected protoplasts. Thus, protein instability is enhanced in virusinfected protoplasts, which may account for the cytosolic and nuclear fluorescence during late stages of infection. Immunogold labeling and electron microscopy were used to further characterize the GFP:TGBp2-induced vesicles. Label was associated with the ER and vesicles, but not the Golgi apparatus. The TGBp2-induced vesicles appeared to be ER derived. For comparison, plasmids expressing GFP fused to TGBp3 were transfected to protoplasts, bombarded to tobacco leaves, and studied in transgenic leaves. The GFP:TGBp3 proteins were associated mainly with the ER and did not cause obvious changes in the endomembrane architecture, suggesting that the vesicles reported in GFP:TGBp2 studies were induced by the PVX TGBp2 protein. In double-labeling studies using confocal microscopy, fluorescence was associated with actin filaments, but not with Golgi vesicles. We propose a model in which reorganization of the ER and increased protein degradation is linked to plasmodesmata gating.Plant viruses use the cellular endomembrane system to support virus replication (Schaad et al., 1997;Reichel and Beachy, 1998;Carette et al., 2002aCarette et al., , 2002bSchwartz et al., 2002). Among positive-strand RNA viruses, the viral replicase is typically anchored to membranes associated with the endoplasmic reticulum (ER), the endocytic pathway, or cellular organelles (Rubino and Russo, 1998;Mas and Beachy, 1999;Rubino et al., 2001;Schwartz et al., 2002Schwartz et al., , 2004. Many viral replicationassociated proteins cause reorganization of cellular membranes with the purpose of creating centers that protect the viral replication complexes (VRCs) from cellular nucleases and host defenses (Schwartz et al., 2002(Schwartz et al., , 2004Ding et al., 2004). Specifically, the replication complexes of plant viruses belonging to the genera Potyvirus, Comovirus, Dianthovirus, Pecluvirus, and Bromovirus cause proliferation and vesiculation of either the perinuclear or the cortical ER (Schaad et al., 1997;Dunoyer et al., 2002;Lee and Ahlquist, 2003). The replicases of cowpea mosaic virus and red clover necrotic mosaic virus induce ER proliferation (Carette et al., 2002a(Carette et al., , 2002bTurner et al., 2004). The best-studied example is the brome mosaic virus (BMV) 1a replicase, which localizes to the ER and induces membrane i...

Activation of the guard cell S-type anion channel SLAC1 is important for stomatal closure in response to diverse stimuli, including elevated CO The majority of known SLAC1 activation mechanisms depend on abscisic acid (ABA) signaling. Several lines of evidence point to a parallel ABA-independent mechanism of CO-induced stomatal regulation; however, molecular details of this pathway remain scarce. Here, we isolated a dominant mutation in the protein kinase HIGH LEAF TEMPERATURE1 (HT1), an essential regulator of stomatal CO responses, in an ozone sensitivity screen of Arabidopsis thaliana The mutation caused constitutively open stomata and impaired stomatal CO responses. We show that the mitogen-activated protein kinases (MPKs) MPK4 and MPK12 can inhibit HT1 activity in vitro and this inhibition is decreased for the dominant allele of HT1. We also show that HT1 inhibits the activation of the SLAC1 anion channel by the protein kinases OPEN STOMATA1 and GUARD CELL HYDROGEN PEROXIDE-RESISTANT1 (GHR1) in Xenopus laevis oocytes. Notably, MPK12 can restore SLAC1 activation in the presence of HT1, but not in the presence of the dominant allele of HT1. Based on these data, we propose a model for sequential roles of MPK12, HT1, and GHR1 in the ABA-independent regulation of SLAC1 during CO-induced stomatal closure.