Calcium-dependent protein kinases (CDPKs) are specific to plants and some protists. Their activation by calcium makes them important switches for the transduction of intracellular calcium signals. Here, we identify the subcellular targeting potentials for nine CDPK isoforms from Arabidopsis, as determined by expression of green fluorescent protein (GFP) fusions in transgenic plants. Subcellular locations were determined by fluorescence microscopy in cells near the root tip. Isoforms AtCPK3-GFP and AtCPK4-GFP showed a nuclear and cytosolic distribution similar to that of free GFP. Membrane fractionation experiments confirmed that these isoforms were primarily soluble. A membrane association was observed for AtCPKs 1, 7, 8, 9, 16, 21, and 28, based on imaging and membrane fractionation experiments. This correlates with the presence of potential N-terminal acylation sites, consistent with acylation as an important factor in membrane association. All but one of the membrane-associated isoforms targeted exclusively to the plasma membrane. The exception was AtCPK1-GFP, which targeted to peroxisomes, as determined by covisualization with a peroxisome marker. Peroxisome targeting of AtCPK1-GFP was disrupted by a deletion of two potential N-terminal acylation sites. The observation of a peroxisome-located CDPK suggests a mechanism for calcium regulation of peroxisomal functions involved in oxidative stress and lipid metabolism.

The United States Environmental Protection Agency is committed to developing new recreational water quality criteria for coastal waters by 2012 to provide increased protection to swimmers.We review the uncertainties and shortcomings of the current recreational water quality criteria, describe critical research needs for the development of new criteria, as well as recommend a path forward for new criteria development. We believe that among the most needed research needs are the completion of epidemiology studies in tropical waters and in waters adversely impacted by urban runoff and animal feces, as well as studies aimed to validate the use of models for indicator and pathogen concentration and health risk predictions.

Living organisms maintain a cellular solute composition very different from that of the externa1 environment. This implicitly requires the transport of solutes across the cell membrane, and ion channels are integral membrane proteins that play indispensable roles in such transport. In the past dozen years, radical advances have aided in our understanding of ion channel function and regulation in higher plants. Nowhere are these advances more striking than with respect to K+ channels, where the synergistic application of electrophysiological, cell biological, physiological, and molecular techniques has demonstrated an array of channel types playing diverse but defined roles in plant physiology.The major function of K+ channels in animal cells is that of membrane voltage control and short-term repolarization of the membrane. Although K+ channels in plants share similar roles in the regulation of the membrane voltage, early research on guard cells led to the model that shows that plant K+ channels in addition provide important pathways for long-term physiological K+ uptake and release. An extensive range of recent studies suggests diverse longterm transport functions of plant K+ channels, including participation in osmotically driven movements, solute loading into the xylem, cation nutrition, and, by virtue of the presence of K+ channels at endomembranes, intracellular solute redistribution and cytosolic volume control. Most plant K+ channels remain activated for long periods of time, which is critica1 for this proposed long-term transport function of K+ channels in plants. Because higher plant K+ channels are proposed to play a role in regulating both the influx and the efflux of K+ from cells, activity of these channels may impinge upon aspects of turgor and water relations of a11 plant cells. In this Update we focus on important principles of plant K+ channel function and on the proposed physiological roles of specific plant K + channel types in the plasma membrane and tonoplast (Fig. 1A) of different plant cells.

A unique subfamily of calmodulin-dependent Ca 2؉ -ATPases was recently identified in plants. In contrast to the most closely related pumps in animals, plasma membrane-type Ca 2؉ -ATPases, members of this new subfamily are distinguished by a calmodulin-regulated autoinhibitor located at the N-terminal instead of a C-terminal end. In addition, at least some isoforms appear to reside in non-plasma membrane locations. To begin delineating their functions, we investigated the subcellular localization of isoform ACA2p (Arabidopsis Ca 2؉ -ATPase, isoform 2 protein) in Arabidopsis. Here we provide evidence that ACA2p resides in the endoplasmic reticulum (ER). In buoyant density sucrose gradients performed with and without Mg 2؉ , ACA2p cofractionated with an ER membrane marker and a typical "ER-type" Ca 2؉ -ATPase, ACA3p/ECA1p. To visualize its subcellular localization, ACA2p was tagged with a green fluorescence protein at its C terminus (ACA2-GFPp) and expressed in transgenic Arabidopsis. We collected fluorescence images from live root cells using confocal and computational optical-sectioning microscopy. ACA2-GFPp appeared as a fluorescent reticulum, consistent with an ER location. In addition, we observed strong fluorescence around the nuclei of mature epidermal cells, which is consistent with the hypothesis that ACA2p may also function in the nuclear envelope. An ER location makes ACA2p distinct from all other calmodulin-regulated pumps identified in plants or animals. Ca2ϩ is thought to function as an important second messenger in all eukaryotes (Bootman and Berridge, 1995;Clapham, 1995). In addition, Ca 2ϩ is required for the stability and activity of many proteins and appears to play a critical role in protein processing in the secretory pathway (Rudolph et al., 1989; Gill et al., 1996) Type IIA and IIB pumps include the "ER-type" and the "PM-type" Ca 2ϩ pumps, respectively, first characterized in animal cells. Previously, homologs of ER-or PM-type pumps were distinguished by three criteria: (a) localization to either the ER or PM, respectively, (b) differential sensitivity to inhibitors (e.g. ER-type inhibition by cyclopiazonic acid and thapsigargin), and (c) direct activation of PM-type pumps by calmodulin. However, not all plant homologs conform to these criteria (Bush, 1995;Evans and Williams, 1998).In plants several genes encoding type IIA pumps (ERtype homologs) have been cloned, including LCA1 from tomato (Wimmers et al., 1992), OsCA from rice (Chen et al., 1997), and ACA3/ECA1 (Arabidopsis Ca 2ϩ -ATPase, isoform 3/ER-Ca 2ϩ -ATPase isoform 1) from Arabidopsis (Liang et al., 1997). Consistent with the criteria for a typical ER-type pump, ACA3p (ACA isoform 3 protein) appears to reside in the ER (Liang et al., 1997). However, non-ER locations have been suggested for other isoforms. For example, Ferrol and Bennett (1996) obtained evidence for tonoplast and PM isoforms from membrane fractionation and immunodetection of pumps cross-reacting with an anti-LCA1 antibody.Three plant genes encoding type IIB pumps (PM...

K+ channels play diverse roles in mediating K+ transport and in modulating the membrane potential in higher plant cells during growth and development. Some of the diversity in K+ channel functions may arise from the regulated expression of multiple genes encoding different K+ channel polypeptides. Here we report the isolation of a nove1 Arabidopsis fhaliana cDNA (AKTZ) that is highly homologous t o the two previously identified K+ channel genes, KATl and AKT7. This cDNA mapped t o the center of chromosome 4 by restriction fragment length polymorphism analysis and was highly expressed i n leaves, whereas AKT7 was mainly expressed in roots. I n addition, we show that diversity in K+ channel function may be attributable t o differences i n expression levels. lncreasing KAT7 expression i n Xenopus oocytes by polyadenylation of the KATl mRNA increased the current amplitude and led t o higher levels of KATl protein, as assayed i n western blots. The increase in KATl expression in oocytes produced shifts in the threshold potentia1 for activation to more positive membrane potentials and decreased half-activation times. These results suggest that different levels of expression and tissue-specific expression of different K+ channel isoforms can contribute to the functional diversity of plant K+ channels. The identification of a highly expressed, leaf-specific K+ channel homolog in plants should allow further molecular characterization of K+ channel functions for physiological K+ transport processes in leaves.

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Inward-rectifying K+ (K+in) channels in the guard cell plasma membrane have been suggested to function as a major pathway for K+ influx into guard cells during stomatal opening. When K+in channels were blocked with external Cs+ in wild-type Arabidopsis guard cells, light-induced stomatal opening was reduced. Transgenic Arabidopsis plants were generated that expressed a mutant of the guard cell K+in channel, KAT1, which shows enhanced resistance to the Cs+ block. Stomata in these transgenic lines opened in the presence of external Cs+. Patch-clamp experiments with transgenic guard cells showed that inward K+(in) currents were blocked less by Cs+ than were K+ currents in controls. These data provide direct evidence that KAT1 functions as a plasma membrane K+ channel in vivo and that K+in channels constitute an important mechanism for light-induced stomatal opening. In addition, biophysical properties of K+in channels in guard cells indicate that components in addition to KAT1 may contribute to the formation of K+in channels in vivo.