Endocytosis is an essential process by which eukaryotic cells internalize exogenous material or regulate signaling at the cell surface [1]. Different endocytic pathways are well established in yeast and animals; prominent among them is clathrin-dependent endocytosis [2, 3]. In plants, endocytosis is poorly defined, and no molecular mechanism for cargo internalization has been demonstrated so far [4, 5], although the internalization of receptor-ligand complexes at the plant plasma membrane has recently been shown [6]. Here we demonstrate by means of a green-to-red photoconvertible fluorescent reporter, EosFP [7], the constitutive endocytosis of PIN auxin efflux carriers [8] and their recycling to the plasma membrane. Using a plant clathrin-specific antibody, we show the presence of clathrin at different stages of coated-vesicle formation at the plasma membrane in Arabidopsis. Genetic interference with clathrin function inhibits PIN internalization and endocytosis in general. Furthermore, pharmacological interference with cargo recruitment into the clathrin pathway blocks internalization of PINs and other plasma-membrane proteins. Our data demonstrate that clathrin-dependent endocytosis is operational in plants and constitutes the predominant pathway for the internalization of numerous plasma-membrane-resident proteins including PIN auxin efflux carriers.

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 tapetum, the innermost of four sporophytic layers in the anther wall, comes in direct contact with the developing male gametophyte and is thought to play a crucial role in the development and maturation of microspores. Here, we report the identification of rice (Oryza sativa) Undeveloped Tapetum1 (Udt1), which is required for the differentiation of secondary parietal cells to mature tapetal cells. T-DNA or retrotransposon Tos17 insertions in the Udt1 gene caused male sterility. The anther walls and meiocytes of the mutants were normal during the early premeiosis stage, but their tapeta failed to differentiate and became vacuolated during the meiotic stage. In addition, meiocytes did not develop to microspores, and middle layer degeneration was inhibited. Consequently, the anther locules contained no pollen. The UDT1:green fluorescent protein fusion protein was localized to the nucleus. This, together with its homology with other basic helix-loop-helix proteins, suggests that UDT1 is a transcription factor. DNA microarray analysis identified 958 downregulated and 267 upregulated genes in the udt1-1 anthers, suggesting that Udt1 plays a major role in maintaining tapetum development, starting in early meiosis.

The phytohormone abscisic acid (ABA) regulates stress-responsive gene expression during vegetative growth. The ABA regulation of many genes is mediated by a subfamily of basic leucine zipper class transcription factors referred to as ABFs (i.e. ABF1-ABF4), whose transcriptional activity is induced by ABA. Here we show that a calcium-dependent protein kinase is involved in the ABA-dependent activation process. We carried out yeast two-hybrid screens to identify regulatory components of ABF4 function and isolated AtCPK32 as an ABF4-interacting protein. AtCPK32 has autophosphorylation activity and can phosphorylate ABF4 in vitro. Mutational analysis indicated that serine-110 of ABF4, which is highly conserved among ABF family members, may be phosphorylated by AtCPK32. The serine-110 residue is essential for ABF4-AtCPK32 interaction, and transient expression assay revealed that it is also required for the normal transcriptional function of ABF4. The expression patterns and subcellular localization of AtCPK32 are similar to those of ABF4. Furthermore, its overexpression affects both ABA sensitivity and the expression of a number of ABF4-regulated genes. Together, our data demonstrate that AtCPK32 is an ABA signaling component that regulates the ABA-responsive gene expression via ABF4.

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