Abscisic acid (ABA) is a phytohormone critical for plant growth, development, and adaptation to various stress conditions. Plants have to adjust ABA levels constantly to respond to changing physiological and environmental conditions. To date, the mechanisms for fine-tuning ABA levels remain elusive. Here we report that AtBG1, a beta-glucosidase, hydrolyzes glucose-conjugated, biologically inactive ABA to produce active ABA. Loss of AtBG1 causes defective stomatal movement, early germination, abiotic stress-sensitive phenotypes, and lower ABA levels, whereas plants with ectopic AtBG1 accumulate higher ABA levels and display enhanced tolerance to abiotic stress. Dehydration rapidly induces polymerization of AtBG1, resulting in a 4-fold increase in enzymatic activity. Furthermore, diurnal increases in ABA levels are attributable to polymerization-mediated AtBG1 activation. We propose that the activation of inactive ABA pools by polymerized AtBG1 is a mechanism by which plants rapidly adjust ABA levels and respond to changing environmental cues.

The actin-associated protein Sla1p, through its SHD1 domain, acts as an adaptor for the NPFX (1,2) D endocytic targeting signal in yeast. Here we report that Wsc1p, a cell wall stress sensor, depends on this signal-adaptor pair for endocytosis. Mutation of NPFDD in Wsc1p or expression of Sla1p lacking SHD1 blocked Wsc1p internalization. By live cell imaging, endocytically defective Wsc1p was not concentrated at sites of endocytosis. Polarized distribution of Wsc1p to regions of cell growth was lost in the absence of endocytosis. Mutations in genes necessary for endosome to Golgi traffic caused redistribution of Wsc1p from the cell surface to internal compartments, indicative of recycling. Inhibition of Wsc1p endocytosis caused defects in polarized deposition of the cell wall and increased sensitivity to perturbation of cell wall synthesis. Our results reveal that the NPFX (1,2) D-Sla1p system is responsible for directing Wsc1p into an endocytosis and recycling pathway necessary to maintain yeast cell wall polarity. The dynamic localization of Wsc1p, a sensor of the extracellular wall in yeast, resembles polarized distribution of certain extracellular matrix-sensing integrins through endocytic recycling. INTRODUCTIONEndocytosis plays a fundamental role in regulating the dynamic organization of the plasma membrane in eukaryotic cells (Conner and Schmid, 2003). A well-characterized endocytic entry route involves formation of clathrin-coated vesicles (CCV). Proteins destined for uptake via CCV generally harbor endocytic targeting signals that direct incorporation into emergent vesicles (Traub, 2005). Such signals are recognized by adaptors that link the cargo to core components of the clathrin coat. Identifying endocytic targeting signals and their partner adaptors, and defining the roles of signal/adaptor pairs in cell physiology are key issues in understanding the endocytic process.Several types of endocytic targeting signals for CCV have been identified in mammalian cells including YXX⌽, FXNPXY (where X is any amino acid and ⌽ is a bulky hydrophobic amino acid), and ubiquitin (Hicke and Dunn, 2003;Traub, 2005). The YXX⌽ signal, present in many endocytic cargo proteins, is recognized by the AP-2 adaptor complex, a core structural component of CCV that plays important roles in clathrin coat assembly. In contrast, the less common FXNPXY and ubiquitin signals are recognized by alternative adaptors known as CLASPs (clathrin-associated sorting proteins), that appear to act more specifically in cargo collection (Traub, 2005).In the yeast Saccharomyces cerevisiae, two distinct classes of endocytic targeting signals have been defined: ubiquitin, which is added post-translationally to lysine residues in endocytic cargo and the peptide signal NPFX (1,2) D (Hicke and Riezman, 1996;Tan et al., 1996). Monoubiquitylation is sufficient to direct internalization and this signal is likely recognized by components of the endocytic machinery such as the epsins Ent1p and Ent2p and the Eps15 homologue Ede1p, all of which contain ub...

SummaryGSK3/shaggy-like protein kinases have been shown to play diverse roles in development and signal transduction pathways in various organisms. An Arabidopsis homologue of GSK3/shaggy-like kinase, AtGSK1, has been shown to be involved in NaCl stress responses. In order to further clarify the role of AtGSK1 in NaCl stress responses in plants, we generated transgenic Arabidopsis plants that overexpressed AtGSK1 mRNA. These plants showed enhanced resistance to NaCl stress when assayed either as whole plants or by measurement of root growth on NaCl plates. In addition, AtGSK1 transgenic plants in the absence of NaCl stress showed phenotypic changes, such as accumulation of anthocyanin, that were similar to those observed in wild-type plants under NaCl stress. Transgenic plants accumulated 30±50% more Na + than did wild-type plants when subjected to NaCl stress, and Ca 2+ content was increased by 15±30% in the transgenic plants regardless of the NaCl stress level. Northern blotting revealed that AtGSK1 over-expression induced expression of the NaCl stress-responsive genes AtCP1, RD29A and CHS1 in the absence of NaCl stress. In addition, AtCBL1 and AtCP1 were superinduced in the NaCl-stressed transgenic plants. Taken together, these results suggest that AtGSK1 is involved in the signal transduction pathway(s) of NaCl stress responses in Arabidopsis.

GSK3/shaggy-like genes encode kinases that are involved in a variety of biological processes. By functional complementation of the yeast calcineurin mutant strain DHT22-1a with a NaCl stresssensitive phenotype, we isolated the Arabidopsis cDNA AtGSK1, which encodes a GSK3/shaggy-like protein kinase. AtGSK1 rescued the yeast calcineurin mutant cells from the effects of high NaCl. Also, the AtGSK1 gene turned on the transcription of the NaCl stress-inducible PMR2A gene in the calcineurin mutant cells under NaCl stress. To further define the role of AtGSK1 in the yeast cells we introduced a deletion mutation at the MCK1 gene, a yeast homolog of GSK3, and examined the phenotype of the mutant. The mck1 mutant exhibited a NaCl stress-sensitive phenotype that was rescued by AtGSK1. Also, constitutive expression of MCK1 complemented the NaCl-sensitive phenotype of the calcineurin mutants. Therefore, these results suggest that Mck1p is involved in the NaCl stress signaling in yeast and that AtGSK1 may functionally replace Mck1p in the NaCl stress response in the calcineurin mutant. To investigate the biological function of AtGSK1 in Arabidopsis we examined the expression of AtGSK1. Northern-blot analysis revealed that the expression is differentially regulated in various tissues with a high level expression in flower tissues. In addition, the AtGSK1 expression was induced by NaCl and exogenously applied ABA but not by KCl. Taken together, these results suggest that AtGSK1 is involved in the osmotic stress response in Arabidopsis.

Dynamin, a CTP-binding protein, is involved in endocytosis in animal cells. We found that a dynamin-like protein, ADLl, is present in multiple forms in Arabidopsis leaf tissue. Subcellular fractionation experiments, together with gel-filtration and nondenaturing-gel electrophoresis revealed that most of ADLl is present as a high-molecular-mass complex of 400 to 600 kD in the membrane or pellet fraction, whereas ADLl is present in the soluble fraction as a monomer. l h e subcellular distribution of ADLl is affected by various agents such as Ca2+, cyclosporin A, CTP, and ATP. Ca2+ increases the amount of ADLl present in the membrane fraction, whereas cyclosporin A inhibits the membrane association. Furthermore, CaZ+ and CTP change the migration pattern of ADLl in nondenaturing polyacrylamide gels, indicating that these chemicals influence either the complex formation and/or the conformation of the ADLl complex. Our results demonstrate that ADLl has characteristics that are similar to Dynamin I, which is found in animal cells. Therefore, it is possible that ADLl is also involved in biological processes that require vesicle formation.