The Arabidopsis abscisic acid (ABA)-insensitive abi5 mutants have pleiotropic defects in ABA response, including decreased sensitivity to ABA inhibition of germination and altered expression of some ABA-regulated genes. We isolated the ABI5 gene by using a positional cloning approach and found that it encodes a member of the basic leucine zipper transcription factor family. The previously characterized abi5-1 allele encodes a protein that lacks the DNA binding and dimerization domains required for ABI5 function. Analyses of ABI5 expression provide evidence for ABA regulation, cross-regulation by other ABI genes, and possibly autoregulation. Comparison of seed and ABA-inducible vegetative gene expression in wild-type and abi5-1 plants indicates that ABI5 regulates a subset of late embryogenesis-abundant genes during both developmental stages. INTRODUCTIONAbscisic acid (ABA) regulates many agronomically important aspects of seed development, including synthesis of storage proteins and lipids (Finkelstein and Somerville, 1988;Rock and Quatrano, 1995) and acquisition of desiccation tolerance and dormancy (Black, 1983;Karssen et al., 1983;Koornneef et al., 1989). In addition, vegetative responses to ABA include induction of stomatal closure and tolerance of drought, salt, and cold stresses (reviewed in Leung and Giraudat, 1998). Molecular studies have identified many ABA-regulated genes and an array of corresponding transcriptional regulators (reviewed in Busk and Pages, 1998). Genetic studies, especially those with Arabidopsis plants, have identified a large number of loci involved in responses to ABA. Mutants with defects at these loci are being characterized physiologically, and as the affected genes are cloned, their products are being characterized biochemically.To date, six genes required for wild-type ABA response have been reported cloned. These genes represent four classes of protein: two orthologous transcriptional regulators (VIVIPAROUS1 [VP1] of maize and ABA INSENSITIVE3 [ABI3] of Arabidopsis) (McCarty et al., 1991; Giraudat et al., 1992), two highly homologous members of the protein phosphatase 2C family (ABI1 and ABI2 of Arabidopsis) (Leung et al., , 1997Meyer et al., 1994), a member of the APETALA2 domain family (ABI4 of Arabidopsis) (Finkelstein et al., 1998), and a farnesyl transferase (ENHANCED RE-SPONSE TO ABA1 [ERA1] of Arabidopsis) (Cutler et al., 1996). Two additional genes demonstrated to interact with ABI3 in regulating seed maturation, FUSCA3 ( FUS3 ) and LEAFY COTYLEDON1 ( LEC1 ), have been found to encode presumed transcription factors (Lotan et al., 1998;Luerssen et al., 1998). To fully describe the molecular events during ABA signaling, we need to identify the biochemical functions of many more of the genes that are required for ABA response.The Arabidopsis abi5 mutants, like many of the other ABA-insensitive mutants, were selected on the basis of ABA-resistant germination (Finkelstein, 1994). Initial physiologic and genetic analyses suggested that ABI5 represented a new element of a...

The identification of genetic causes for Mendelian disorders has been based on the collection of multi-incident families, linkage analysis, and sequencing of genes in candidate intervals. This study describes the application of next-generation sequencing technologies to a Swiss kindred presenting with autosomal-dominant, late-onset Parkinson disease (PD). The family has tremor-predominant dopa-responsive parkinsonism with a mean onset of 50.6 ± 7.3 years. Exome analysis suggests that an aspartic-acid-to-asparagine mutation within vacuolar protein sorting 35 (VPS35 c.1858G>A; p.Asp620Asn) is the genetic determinant of disease. VPS35 is a central component of the retromer cargo-recognition complex, is critical for endosome-trans-golgi trafficking and membrane-protein recycling, and is evolutionarily highly conserved. VPS35 c.1858G>A was found in all affected members of the Swiss kindred and in three more families and one patient with sporadic PD, but it was not observed in 3,309 controls. Further sequencing of familial affected probands revealed only one other missense variant, VPS35 c.946C>T; (p.Pro316Ser), in a pedigree with one unaffected and two affected carriers, and thus the pathogenicity of this mutation remains uncertain. Retromer-mediated sorting and transport is best characterized for acid hydrolase receptors. However, the complex has many types of cargo and is involved in a diverse array of biologic pathways from developmental Wnt signaling to lysosome biogenesis. Our study implicates disruption of VPS35 and retromer-mediated trans-membrane protein sorting, rescue, and recycling in the neurodegenerative process leading to PD.

The Arabidopsis abscisic acid (ABA)-insensitive abi5 mutants have pleiotropic defects in ABA response, including decreased sensitivity to ABA inhibition of germination and altered expression of some ABA-regulated genes. We isolated the ABI5 gene by using a positional cloning approach and found that it encodes a member of the basic leucine zipper transcription factor family. The previously characterized abi5-1 allele encodes a protein that lacks the DNA binding and dimerization domains required for ABI5 function. Analyses of ABI5 expression provide evidence for ABA regulation, cross-regulation by other ABI genes, and possibly autoregulation. Comparison of seed and ABA-inducible vegetative gene expression in wild-type and abi5-1 plants indicates that ABI5 regulates a subset of late embryogenesis-abundant genes during both developmental stages.

We have characterized developmental, environmental, and genetic regulation of abscisic acid-insensitive (ABI)4 gene expression in Arabidopsis. Although expressed most strongly in seeds, ABI4 transcripts are also present at low levels in vegetative tissue; vegetative expression is not induced by abscisic acid (ABA) or stress treatments. Comparison of transcript levels in mature seeds of ABA-insensitive, ABA-hypersensitive, ABA-deficient, or heterochronic mutants indicates that ABI4 expression is altered in only two of the backgrounds, the ABA-insensitive mutants abi1-1 and abi3-1. To determine whether ABI4 is necessary and/or sufficient for ABA response, we assayed the effects of loss of ABI4 function and ectopic ABI4 expression on growth and gene expression. We examined genetic interactions among three ABA response loci, ABI3, ABI4, and ABI5, by comparing phenotypes of mutants, ectopic expression lines, mutants carrying an ectopically expressed transgene, and the corresponding wild-type lines. Our results indicate some cross-regulation of expression among ABI3, ABI4, and ABI5 and suggest that they function in a combinatorial network, rather than a regulatory hierarchy, controlling seed development and ABA response.

Arabidopsis abscisic acid (ABA)-insensitive abi4 mutants have pleiotropic defects in seed development, including decreased sensitivity to ABA inhibition of germination and altered seed-specific gene expression. This phenotype is consistent with a role for ABI4 in regulating seed responses to ABA and/or seed-specific signals. We isolated the ABI4 gene by positional cloning and confirmed its identity by complementation analysis. The predicted protein product shows homology to a plant-specific family of transcriptional regulators characterized by a conserved DNA binding domain, the APETALA 2 domain. The single mutant allele identified has a single base pair deletion, resulting in a frameshift that should disrupt the C-terminal half of the protein but leave the presumed DNA binding domain intact. Expression analyses showed that despite the seed-specific nature of the mutant phenotype, ABI4 expression is not seed specific.

Abscisic acid (ABA) and stress response from late embryonic growth through early seedling development is regulated by a signaling network that includes the Arabidopsis ABA-insensitive (ABI)5 gene, which encodes a basic leucine zipper transcription factor. We have characterized genetic, developmental, and environmental regulation of ABI5 expression. Although expressed most strongly in seeds, the ABI5 promoter is also active in vegetative and floral tissue. Vegetative expression is strongly induced by ABA, and weakly by stress treatments during a limited developmental window up to approximately 2 d post-stratification, but ABA and some stresses can induce expression in specific tissues at later stages. ABI5 expression is autoregulated in transgenic plants and yeast (Saccharomyces cerevisiae), and stress response appears to involve ABI5-dependent and -independent mechanisms. To determine whether ABI5 is necessary and/or sufficient for ABA or stress response, we assayed the effects of increased ABI5 expression on growth and gene expression. Although overexpression of ABI5 confers hypersensitivity to ABA and sugar, as previously described for ABI4 and ABI3 overexpression lines, it has relatively limited effects on enhancing ABA-responsive gene expression. Comparison of expression of eight ABI5-homologous genes shows overlapping regulation by ABI3, ABI4, and ABI5, suggestive of a combinatorial network involving positive and negative regulatory interactions.The phytohormone abscisic acid (ABA) regulates many agronomically important aspects of plant growth and development, including seed maturation, dormancy, stress tolerances, and water relations (for review, see Leung and Giraudat, 1998; Rock, 2000;. All of these processes are regulated by additional signals, including other phytohormones, stage-specific regulators, and abiotic stresses. Studies of ABA-deficient mutants have shown that ABA is an essential mediator in triggering some plant responses to abiotic stresses, including drought, salinity, and cold (Rock, 2000; Shinozaki and Yamaguchi-Shinozaki, 2000; Xiong and Zhu, 2001). Dehydration and low temperatures result in elevated levels of ABA, which, in turn, trigger the synthesis of some proteins responsible for drought or freezing tolerance. However, although most of the drought-and salt-induced genes studied to date can be induced by ABA, many aspects of stress response are also mediated by ABA-independent mechanisms (Shinozaki and Yamaguchi-Shinozaki, 2000). These results indicate that ABA participates in only part of a network of stress-signaling mechanisms. Furthermore, even the ABA-dependent portions of this network rely on both independent and partially redundant signaling components, some of which are produced only in specific developmental stages or tissues of the plant.Components of this signaling network have been identified by a combination of biochemical, cell biological, and forward and reverse genetic approaches. To date, nearly 50 loci in Arabidopsis alone have been demonstrated to function in v...

). ² These authors contributed equally to this work. SummaryGenetic and physiological studies have shown that the Arabidopsis thaliana abscisic acid-insensitive (ABI) loci interact to regulate seed-speci®c and/or ABA-inducible gene expression. We have used the yeast two-hybrid assay to determine whether any of these genetic interactions re¯ect direct physical interactions. By this criterion, only ABI3 and ABI5 physically interact with each other, and ABI5 can form homodimers. The B1 domain of ABI3 is essential for this interaction; this is the ®rst speci®c function ascribed to this domain of the ABI3/VP1 family. The ABI5 domains required for interaction with ABI3 include two conserved charged domains in the amino-terminal half of the protein. An additional conserved charged domain appears to have intrinsic transcription activation function in this assay. Yeast one-hybrid assays with a lacZ reporter gene under control of the late embryogenesis-abundant AtEm6 promoter show that only ABI5 binds directly to this promoter fragment.

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