The Experts below are selected from a list of 204 Experts worldwide ranked by ideXlab platform
Qingxi J Shen - One of the best experts on this subject based on the ideXlab platform.
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Transcriptomic analysis of rice Aleurone Cells identified a novel abscisic acid response element.
Plant cell & environment, 2017Co-Authors: Kenneth A. Watanabe, Arielle Homayouni, Kuan‐ying Huang, Qingxi J ShenAbstract:Seeds serve as a great model to study plant responses to drought stress, which is largely mediated by abscisic acid (ABA). The ABA responsive element (ABRE) is a key cis-regulatory element in ABA signaling. However, its consensus sequence, (ACGTG(G/T)C), is present in the promoters of only about 40% of ABA induced genes in rice Aleurone Cells, suggesting other ABREs may exist. To identify novel ABREs, RNA-sequencing was performed on Aleurone Cells of rice seeds treated with 20 μM ABA. Gibbs sampling was used to identify enriched elements and particle bombardment-mediated transient expression studies were performed to verify the function. Gene ontology analysis was performed to predict the roles of genes containing the novel ABREs. This study revealed 2,443 ABA-inducible genes and a novel ABRE, designated as ABREN, which was experimentally verified to mediate ABA signaling in rice Aleurone Cells. Many of the ABREN-containing genes are predicted to be involved in stress responses and transcription. Analysis of other species suggest that the ABREN may be monocot-specific. This study also revealed interesting expression patterns of genes involved in ABA metabolism and signaling. Collectively, this study advanced our understanding of diverse cis-regulatory sequences and the transcriptomes underlying ABA responses in rice Aleurone Cells.
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rna sequencing reveals previously unannotated protein and microrna coding genes expressed in Aleurone Cells of rice seeds
Genomics, 2014Co-Authors: Kenneth A. Watanabe, Patricia Ringler, Qingxi J ShenAbstract:The rice genome annotation has been greatly improved in recent years, largely due to the availability of full length cDNA sequences derived from many tissues. Among those yet to be studied is the Aleurone layer, which produces hydrolases for mobilization of seed storage reserves during seed germination and post germination growth. Herein, we report transcriptomes of Aleurone Cells treated with the hormones abscisic acid, gibberellic acid, or both. Using a comprehensive approach, we identified hundreds of novel genes. To minimize the number of false positives, only transcripts that did not overlap with existing annotations, had a high level of expression, and showed a high level of uniqueness within the rice genome were considered to be novel genes. This approach led to the identification of 553 novel genes that encode proteins and/or microRNAs. The transcriptome data reported here will help to further improve the annotation of the rice genome.
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a negative regulator encoded by a rice wrky gene represses both abscisic acid and gibberellins signaling in Aleurone Cells
Plant Molecular Biology, 2009Co-Authors: Xiaolu Zou, Zhonglin Zhang, Margaret Shin, Jianzhi Huang, Qingxi J ShenAbstract:Abscisic acid (ABA) and gibberellins (GAs) control several developmental processes including seed maturation, dormancy, and germination. The antagonism of these two hormones is well-documented. However, recent data from transcription profiling studies indicate that they can function as agonists in regulating the expression of many genes although the underlying mechanism is unclear. Here we report a rice WRKY gene, OsWRKY24, which encodes a protein that functions as a negative regulator of both GA and ABA signaling. Overexpression of OsWRKY24 via particle bombardment-mediated transient expression in Aleurone Cells represses the expression of two reporter constructs: the β-glucuronidase gene driven by the GA-inducible Amy32b α-amylase promoter (Amy32b-GUS) and the ABA-inducible HVA22 promoter (HVA22-GUS). OsWRKY24 is unlikely a general repressor because it has little effect on the expression of the luciferase reporter gene driven by a constitutive ubiquitin promoter (UBI-Luciferase). As to the GA signaling, OsWRKY24 differs from OsWRKY51 and −71, two negative regulators specifically function in the GA signaling pathway, in several ways. First, OsWRKY24 contains two WRKY domains while OsWRKY51 and −71 have only one; both WRKY domains are essential for the full repressing activity of OsWRKY24. Second, binding of OsWRKY24 to the Amy32b promoter appears to involve sequences in addition to the TGAC cores of the W-boxes. Third, unlike OsWRKY71, OsWRKY24 is stable upon GA treatment. Together, these data demonstrate that OsWRKY24 is a novel type of transcriptional repressor that inhibits both GA and ABA signaling.
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interactions of two transcriptional repressors and two transcriptional activators in modulating gibberellin signaling in Aleurone Cells
Plant Physiology, 2008Co-Authors: Xiaolu Zou, Dawn S Neuman, Qingxi J ShenAbstract:Gibberellins (GAs) regulate many aspects of plant development, such as germination, growth, and flowering. The barley (Hordeum vulgare) Amy32b α-amylase promoter contains at least five cis-acting elements that govern its GA-induced expression. Our previous studies indicate that a barley WRKY gene, HvWRKY38, and its rice (Oryza sativa) ortholog, OsWRKY71, block GA-induced expression of Amy32b-GUS. In this work, we investigated the functional and physical interactions of HvWRKY38 with another repressor and two activators in barley. HvWRKY38 blocks the inductive activities of SAD (a DOF protein) and HvGAMYB (a R2R3 MYB protein) when either of these proteins is present individually. However, SAD and HvGAMYB together overcome the inhibitory effect of HvWRKY38. Yet, the combination of HvWRKY38 and BPBF (another DOF protein) almost diminishes the synergistic effect of SAD and HvGAMYB transcriptional activators. Electrophoretic mobility shift assays indicate that HvWRKY38 blocks the GA-induced expression of Amy32b by interfering with the binding of HvGAMYB to the cis-acting elements in the α-amylase promoter. The physical interaction of HvWRKY38 and BPBF repressors is demonstrated via bimolecular fluorescence complementation assays. These data suggest that the expression of Amy32b is modulated by protein complexes that contain either activators (e.g. HvGAMYB and SAD) or repressors (e.g. HvWRKY38 and BPBF). The relative amounts of the repressor or activator complexes binding to the Amy32b promoter regulate its expression level in barley Aleurone Cells.
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interactions of two abscisic acid induced wrky genes in repressing gibberellin signaling in Aleurone Cells
Plant Journal, 2006Co-Authors: Zhen Xie, Zhonglin Zhang, Xiaolu Zou, Guangxiao Yang, Setsuko Komatsu, Qingxi J ShenAbstract:Gibberellins (GA) promote while abscisic acid (ABA) inhibits seed germination and post-germination growth. To address the cross-talk of GA and ABA signaling, we studied two rice WRKY genes (OsWRKY51 and OsWRKY71) that are ABA-inducible and GA-repressible in embryos and Aleurone Cells. Over-expression of these two genes in Aleurone Cells specifically and synergistically represses induction of the ABA-repressible and GA-inducible Amy32b alpha-amylase promoter reporter construct (Amy32b-GUS) by GA or the GA-inducible transcriptional activator, GAMYB. The physical interactions of OsWRKY71 proteins themselves and that of OsWRKY71 and OsWRKY51 are revealed in the nuclei of Aleurone Cells using bimolecular fluorescence complementation (BiFC) assays. Although OsWRKY51 itself does not bind to the Amy32b promoter in vitro, it interacts with OsWRKY71 and enhances the binding affinity of OsWRKY71 to W boxes in the Amy32b promoter. The binding activity of OsWRKY71 is abolished by deleting the C-terminus containing the WRKY domain or substituting the key amino acids in the WRKY motif and the zinc finger region. However, two of these non-DNA-binding mutants are still able to repress GA induction by enhancing the binding affinity of the wild-type DNA-binding OsWRKY71 repressors. In contrast, the third non-DNA-binding mutant enhances GA induction of Amy32b-GUS, by interfering with the binding of the wild-type OsWRKY71 or the OsWRKY71/OsWRKY51 repressing complex. These data demonstrate the synergistic interaction of ABA-inducible WRKY genes in regulating GAMYB-mediated GA signaling in Aleurone Cells, thereby establishing a novel mechanism for ABA and GA signaling cross-talk.
Zhonglin Zhang - One of the best experts on this subject based on the ideXlab platform.
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a negative regulator encoded by a rice wrky gene represses both abscisic acid and gibberellins signaling in Aleurone Cells
Plant Molecular Biology, 2009Co-Authors: Xiaolu Zou, Zhonglin Zhang, Margaret Shin, Jianzhi Huang, Qingxi J ShenAbstract:Abscisic acid (ABA) and gibberellins (GAs) control several developmental processes including seed maturation, dormancy, and germination. The antagonism of these two hormones is well-documented. However, recent data from transcription profiling studies indicate that they can function as agonists in regulating the expression of many genes although the underlying mechanism is unclear. Here we report a rice WRKY gene, OsWRKY24, which encodes a protein that functions as a negative regulator of both GA and ABA signaling. Overexpression of OsWRKY24 via particle bombardment-mediated transient expression in Aleurone Cells represses the expression of two reporter constructs: the β-glucuronidase gene driven by the GA-inducible Amy32b α-amylase promoter (Amy32b-GUS) and the ABA-inducible HVA22 promoter (HVA22-GUS). OsWRKY24 is unlikely a general repressor because it has little effect on the expression of the luciferase reporter gene driven by a constitutive ubiquitin promoter (UBI-Luciferase). As to the GA signaling, OsWRKY24 differs from OsWRKY51 and −71, two negative regulators specifically function in the GA signaling pathway, in several ways. First, OsWRKY24 contains two WRKY domains while OsWRKY51 and −71 have only one; both WRKY domains are essential for the full repressing activity of OsWRKY24. Second, binding of OsWRKY24 to the Amy32b promoter appears to involve sequences in addition to the TGAC cores of the W-boxes. Third, unlike OsWRKY71, OsWRKY24 is stable upon GA treatment. Together, these data demonstrate that OsWRKY24 is a novel type of transcriptional repressor that inhibits both GA and ABA signaling.
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interactions of two abscisic acid induced wrky genes in repressing gibberellin signaling in Aleurone Cells
Plant Journal, 2006Co-Authors: Zhen Xie, Zhonglin Zhang, Xiaolu Zou, Guangxiao Yang, Setsuko Komatsu, Qingxi J ShenAbstract:Gibberellins (GA) promote while abscisic acid (ABA) inhibits seed germination and post-germination growth. To address the cross-talk of GA and ABA signaling, we studied two rice WRKY genes (OsWRKY51 and OsWRKY71) that are ABA-inducible and GA-repressible in embryos and Aleurone Cells. Over-expression of these two genes in Aleurone Cells specifically and synergistically represses induction of the ABA-repressible and GA-inducible Amy32b alpha-amylase promoter reporter construct (Amy32b-GUS) by GA or the GA-inducible transcriptional activator, GAMYB. The physical interactions of OsWRKY71 proteins themselves and that of OsWRKY71 and OsWRKY51 are revealed in the nuclei of Aleurone Cells using bimolecular fluorescence complementation (BiFC) assays. Although OsWRKY51 itself does not bind to the Amy32b promoter in vitro, it interacts with OsWRKY71 and enhances the binding affinity of OsWRKY71 to W boxes in the Amy32b promoter. The binding activity of OsWRKY71 is abolished by deleting the C-terminus containing the WRKY domain or substituting the key amino acids in the WRKY motif and the zinc finger region. However, two of these non-DNA-binding mutants are still able to repress GA induction by enhancing the binding affinity of the wild-type DNA-binding OsWRKY71 repressors. In contrast, the third non-DNA-binding mutant enhances GA induction of Amy32b-GUS, by interfering with the binding of the wild-type OsWRKY71 or the OsWRKY71/OsWRKY51 repressing complex. These data demonstrate the synergistic interaction of ABA-inducible WRKY genes in regulating GAMYB-mediated GA signaling in Aleurone Cells, thereby establishing a novel mechanism for ABA and GA signaling cross-talk.
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Interactions of two abscisic‐acid induced WRKY genes in repressing gibberellin signaling in Aleurone Cells
The Plant journal : for cell and molecular biology, 2006Co-Authors: Zhen Xie, Zhonglin Zhang, Xiaolu Zou, Guangxiao Yang, Setsuko Komatsu, Qingxi J ShenAbstract:Gibberellins (GA) promote while abscisic acid (ABA) inhibits seed germination and post-germination growth. To address the cross-talk of GA and ABA signaling, we studied two rice WRKY genes (OsWRKY51 and OsWRKY71) that are ABA-inducible and GA-repressible in embryos and Aleurone Cells. Over-expression of these two genes in Aleurone Cells specifically and synergistically represses induction of the ABA-repressible and GA-inducible Amy32b alpha-amylase promoter reporter construct (Amy32b-GUS) by GA or the GA-inducible transcriptional activator, GAMYB. The physical interactions of OsWRKY71 proteins themselves and that of OsWRKY71 and OsWRKY51 are revealed in the nuclei of Aleurone Cells using bimolecular fluorescence complementation (BiFC) assays. Although OsWRKY51 itself does not bind to the Amy32b promoter in vitro, it interacts with OsWRKY71 and enhances the binding affinity of OsWRKY71 to W boxes in the Amy32b promoter. The binding activity of OsWRKY71 is abolished by deleting the C-terminus containing the WRKY domain or substituting the key amino acids in the WRKY motif and the zinc finger region. However, two of these non-DNA-binding mutants are still able to repress GA induction by enhancing the binding affinity of the wild-type DNA-binding OsWRKY71 repressors. In contrast, the third non-DNA-binding mutant enhances GA induction of Amy32b-GUS, by interfering with the binding of the wild-type OsWRKY71 or the OsWRKY71/OsWRKY51 repressing complex. These data demonstrate the synergistic interaction of ABA-inducible WRKY genes in regulating GAMYB-mediated GA signaling in Aleurone Cells, thereby establishing a novel mechanism for ABA and GA signaling cross-talk.
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annotations and functional analyses of the rice wrky gene superfamily reveal positive and negative regulators of abscisic acid signaling in Aleurone Cells
Plant Physiology, 2005Co-Authors: Zhen Xie, Zhonglin Zhang, Xiaolu Zou, Jie Huang, Paul Ruas, Daniel B Thompson, Qingxi J ShenAbstract:The WRKY proteins are a superfamily of regulators that control diverse developmental and physiological processes. This family was believed to be plant specific until the recent identification of WRKY genes in nonphotosynthetic eukaryotes. We have undertaken a comprehensive computational analysis of the rice (Oryza sativa) genomic sequences and predicted the structures of 81 OsWRKY genes, 48 of which are supported by full-length cDNA sequences. Eleven OsWRKY proteins contain two conserved WRKY domains, while the rest have only one. Phylogenetic analyses of the WRKY domain sequences provide support for the hypothesis that gene duplication of single- and two-domain WRKY genes, and loss of the WRKY domain, occurred in the evolutionary history of this gene family in rice. The phylogeny deduced from the WRKY domain peptide sequences is further supported by the position and phase of the intron in the regions encoding the WRKY domains. Analyses for chromosomal distributions reveal that 26% of the predicted OsWRKY genes are located on chromosome 1. Among the dozen genes tested, OsWRKY24, -51, -71, and -72 are induced by abscisic acid (ABA) in Aleurone Cells. Using a transient expression system, we have demonstrated that OsWRKY24 and -45 repress ABA induction of the HVA22 promoter-β-glucuronidase construct, while OsWRKY72 and -77 synergistically interact with ABA to activate this reporter construct. This study provides a solid base for functional genomics studies of this important superfamily of regulatory genes in monocotyledonous plants and reveals a novel function for WRKY genes, i.e. mediating plant responses to ABA.
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a rice wrky gene encodes a transcriptional repressor of the gibberellin signaling pathway in Aleurone Cells
Plant Physiology, 2004Co-Authors: Zhonglin Zhang, Jose A Casaretto, Tuanhua David Ho, Qingxi J ShenAbstract:The molecular mechanism by which GA regulates plant growth and development has been a subject of active research. Analyses of the rice (Oryza sativa) genomic sequences identified 77 WRKY genes, among which OsWRKY71 is highly expressed in Aleurone Cells. Transient expression of OsWRKY71 by particle bombardment specifically represses GA-induced Amy32b α-amylase promoter but not abscisic acid-induced HVA22 or HVA1 promoter activity in Aleurone Cells. Moreover, OsWRKY71 blocks the activation of the Amy32b promoter by the GA-inducible transcriptional activator OsGAMYB. Consistent with its role as a transcriptional repressor, OsWRKY71 is localized to nuclei of Aleurone Cells and binds specifically to functionally defined TGAC-containing W boxes of the Amy32b promoter in vitro. Mutation of the two W boxes prevents the binding of OsWRKY71 to the mutated promoter, and releases the suppression of the OsGAMYB-activated Amy32b expression by OsWRKY71, suggesting that OsWRKY71 blocks GA signaling by functionally interfering with OsGAMYB. Exogenous GA treatment decreases the steady-state mRNA level of OsWRKY71 and destabilizes the GFP:OsWRKY71 fusion protein. These findings suggest that OsWRKY71 encodes a transcriptional repressor of GA signaling in Aleurone Cells.
Frank Gubler - One of the best experts on this subject based on the ideXlab platform.
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Increased expression of HvGAMYB in transgenic barley increases hydrolytic enzyme production by Aleurone Cells in response to gibberellin
Journal of Cereal Science, 2006Co-Authors: F. Murray, John V. Jacobsen, Peter R. Matthews, Frank GublerAbstract:HvGAMYB is a transcription factor protein, which is part of the gibberellin (GA) signal transduction pathway in barley Aleurone Cells. It activates GA-regulated genes to cause synthesis of alpha-amylase and other hydrolytic enzymes. In Aleurone Cells, HvGAMYB is up-regulated by GA prior to hydrolytic enzyme gene expression. In this study we tested the hypothesis that over-expression of HvGAMYB by inserting additional gene copies might increase production of the GA-induced Aleurone enzymes. Transgenic lines of barley cv.Golden Promise with extra copies of the HvGAMYB gene were previously created and shown to produce increased amounts of HvGAMYB. In this study grains which were null and homozygous for the transgene were compared, and it was found that increased expression of HvGAMYB resulted in increased amounts of two GA-induced hydrolytic enzymes in both germinating grains and in GA-treated de-embryonated grains. Enzyme production by malting barley is an important quality parameter and these results indicate that it might be possible to increase the production of all GA-induced enzymes in Aleurone by increasing the production of HvGAMYB. Such an outcome would be a valuable adjunct to conventional breeding programs.
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cprg hcl a potential h cl symporter prevents acidification of storage vacuoles in Aleurone Cells and inhibits ga dependent hydrolysis of storage protein and phytate
Plant Journal, 2003Co-Authors: Yong-sic Hwang, Paul C. Bethke, Frank Gubler, Russell L. JonesAbstract:Summary The putative H+/Cl− symporter cycloprodigiosin-HCl (cPrG-HCl) was used to investigate the role of vacuole acidification in cereal Aleurone cell function. The protein storage vacuole (PSV) becomes acidified rapidly when Aleurone Cells are treated with gibberellic acid (GA) but not abscisic acid (ABA). We show that cPrG prevents PSV acidification in Aleurone layers and prevents synthesis of secretory proteins such as α-amylase. Our data support the hypothesis that decreased hydrolase synthesis is a consequence of decreased hydrolysis of storage proteins in PSV. Support for this hypothesis comes from experiments showing that breakdown of barley 7S globulins and phytate is inhibited by cPrG in GA-treated Aleurone layers. Decreased mobilization of PSV reserves is accompanied by reductions in the free amino acid pool size and in the amount of ions released from the Aleurone layer. Vacuolation of the Aleurone cell is a diagnostic feature of the response to GA, and vacuolation is also inhibited by cPrG. Evidence that cPrG acts as a potential H+/Cl− symporter in Aleurone is presented. We show that cPrG does not inhibit the synthesis and secretion of α-amylase when Cl− ions are omitted from the incubation medium. Although cPrG blocks many GA-induced responses of Aleurone layers, it does not affect early steps in GA signaling. The SLN1 protein, a negative regulator of GA signaling, is turned over in GA-treated Cells in the presence and absence of cPrG. Similarly, synthesis of the transcriptional activator GAMYB is unaffected by the presence of cPrG in GA-treated Cells.
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cPrG-HCl a potential H+/Cl- symporter prevents acidification of storage vacuoles in Aleurone Cells and inhibits GA-dependent hydrolysis of storage protein and phytate.
The Plant journal : for cell and molecular biology, 2003Co-Authors: Yong-sic Hwang, Paul C. Bethke, Frank Gubler, Russell L. JonesAbstract:Summary The putative H+/Cl− symporter cycloprodigiosin-HCl (cPrG-HCl) was used to investigate the role of vacuole acidification in cereal Aleurone cell function. The protein storage vacuole (PSV) becomes acidified rapidly when Aleurone Cells are treated with gibberellic acid (GA) but not abscisic acid (ABA). We show that cPrG prevents PSV acidification in Aleurone layers and prevents synthesis of secretory proteins such as α-amylase. Our data support the hypothesis that decreased hydrolase synthesis is a consequence of decreased hydrolysis of storage proteins in PSV. Support for this hypothesis comes from experiments showing that breakdown of barley 7S globulins and phytate is inhibited by cPrG in GA-treated Aleurone layers. Decreased mobilization of PSV reserves is accompanied by reductions in the free amino acid pool size and in the amount of ions released from the Aleurone layer. Vacuolation of the Aleurone cell is a diagnostic feature of the response to GA, and vacuolation is also inhibited by cPrG. Evidence that cPrG acts as a potential H+/Cl− symporter in Aleurone is presented. We show that cPrG does not inhibit the synthesis and secretion of α-amylase when Cl− ions are omitted from the incubation medium. Although cPrG blocks many GA-induced responses of Aleurone layers, it does not affect early steps in GA signaling. The SLN1 protein, a negative regulator of GA signaling, is turned over in GA-treated Cells in the presence and absence of cPrG. Similarly, synthesis of the transcriptional activator GAMYB is unaffected by the presence of cPrG in GA-treated Cells.
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Gibberellin Signaling in Barley Aleurone Cells. Control of SLN1 and GAMYB Expression
Plant physiology, 2002Co-Authors: Frank Gubler, Peter M. Chandler, Rosemary G. White, Danny J. Llewellyn, John V. JacobsenAbstract:We have previously identified GAMYB, a gibberellin (GA)-regulated transcriptional activator of α-amylase gene expression, in Aleurone Cells of barley ( Hordeum vulgare ). To examine the regulation of GAMYB expression, we describe the use of nuclear run-on experiments to show that GA causes a 2-fold increase in the rate of GAMYB transcription and that the effect of GA can be blocked by abscisic acid (ABA). To identify GA-signaling components that regulate GAMYB expression, we examined the role of SLN1, a negative regulator of GA signaling in barley. SLN1, which is the product of the Sln1 ( Slender1 ) locus, is necessary for repression of GAMYB in barley Aleurone Cells. The activity of SLN1 in Aleurone Cells is regulated posttranslationally. SLN1 protein levels decline rapidly in response to GA before any increase in GAMYB levels. Green fluorescent protein-SLN1 fusion protein was targeted to the nucleus of Aleurone protoplasts and disappeared in response to GA. Evidence from a dominant dwarf mutant at Sln1 , and from the gse1 mutant (that affects GA “sensitivity”), indicates that GA acts by regulating SLN1 degradation and not translation. Mutation of the DELLA region of SLN1 results in increased protein stability in GA-treated layers, indicating that the DELLA region plays an important role in GA-induced degradation of SLN1. Unlike GA, ABA had no effect on SLN1 stability, confirming that ABA acts downstream of SLN1 to block GA signaling.
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Gibberellin-regulated expression of a myb gene in barley Aleurone Cells: evidence for Myb transactivation of a high-pI alpha-amylase gene promoter.
The Plant cell, 1995Co-Authors: Frank Gubler, Roger Kalla, James K. Roberts, John V. JacobsenAbstract:Functional analysis of a barley high-pI alpha-amylase gene promoter has identified a gibberellin (GA) response complex in the region between -174 and -108. The sequence of the central element, TAACAAA, is very similar to the c-Myb and v-Myb consensus binding site. We investigated the possibility that a GA-regulated Myb transactivates alpha-amylase gene expression in barley Aleurone Cells. A cDNA clone, GAmyb, which encodes a novel Myb, was isolated from a barley Aleurone cDNA library. RNA blot analysis revealed that GAmyb expression in isolated barley Aleurone layers is up-regulated by GA. The kinetics of GAmyb expression indicates that it is an early event in GA-regulated gene expression and precedes alpha-amylase gene expression. Cycloheximide blocked alpha-amylase gene expression but failed to block GAmyb gene expression, indicating that protein synthesis is not required for GAmyb gene expression. Gel mobility shift experiments with recombinant GAMyb showed that GAMyb binds specifically to the TAACAAA box in vitro. We demonstrated in transient expression experiments that GAMyb activates transcription of a high-pI alpha-amylase promoter fused to a beta-glucuronidase reporter gene in the absence of GA. Our results indicate that the GAMyb is the sole GA-regulated transcription factor required for transcriptional activation of the high-pI alpha-amylase promoter. We therefore postulate that GAMyb is a part of the GA-response pathway leading to alpha-amylase gene expression in Aleurone Cells.
Xiaolu Zou - One of the best experts on this subject based on the ideXlab platform.
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a negative regulator encoded by a rice wrky gene represses both abscisic acid and gibberellins signaling in Aleurone Cells
Plant Molecular Biology, 2009Co-Authors: Xiaolu Zou, Zhonglin Zhang, Margaret Shin, Jianzhi Huang, Qingxi J ShenAbstract:Abscisic acid (ABA) and gibberellins (GAs) control several developmental processes including seed maturation, dormancy, and germination. The antagonism of these two hormones is well-documented. However, recent data from transcription profiling studies indicate that they can function as agonists in regulating the expression of many genes although the underlying mechanism is unclear. Here we report a rice WRKY gene, OsWRKY24, which encodes a protein that functions as a negative regulator of both GA and ABA signaling. Overexpression of OsWRKY24 via particle bombardment-mediated transient expression in Aleurone Cells represses the expression of two reporter constructs: the β-glucuronidase gene driven by the GA-inducible Amy32b α-amylase promoter (Amy32b-GUS) and the ABA-inducible HVA22 promoter (HVA22-GUS). OsWRKY24 is unlikely a general repressor because it has little effect on the expression of the luciferase reporter gene driven by a constitutive ubiquitin promoter (UBI-Luciferase). As to the GA signaling, OsWRKY24 differs from OsWRKY51 and −71, two negative regulators specifically function in the GA signaling pathway, in several ways. First, OsWRKY24 contains two WRKY domains while OsWRKY51 and −71 have only one; both WRKY domains are essential for the full repressing activity of OsWRKY24. Second, binding of OsWRKY24 to the Amy32b promoter appears to involve sequences in addition to the TGAC cores of the W-boxes. Third, unlike OsWRKY71, OsWRKY24 is stable upon GA treatment. Together, these data demonstrate that OsWRKY24 is a novel type of transcriptional repressor that inhibits both GA and ABA signaling.
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interactions of two transcriptional repressors and two transcriptional activators in modulating gibberellin signaling in Aleurone Cells
Plant Physiology, 2008Co-Authors: Xiaolu Zou, Dawn S Neuman, Qingxi J ShenAbstract:Gibberellins (GAs) regulate many aspects of plant development, such as germination, growth, and flowering. The barley (Hordeum vulgare) Amy32b α-amylase promoter contains at least five cis-acting elements that govern its GA-induced expression. Our previous studies indicate that a barley WRKY gene, HvWRKY38, and its rice (Oryza sativa) ortholog, OsWRKY71, block GA-induced expression of Amy32b-GUS. In this work, we investigated the functional and physical interactions of HvWRKY38 with another repressor and two activators in barley. HvWRKY38 blocks the inductive activities of SAD (a DOF protein) and HvGAMYB (a R2R3 MYB protein) when either of these proteins is present individually. However, SAD and HvGAMYB together overcome the inhibitory effect of HvWRKY38. Yet, the combination of HvWRKY38 and BPBF (another DOF protein) almost diminishes the synergistic effect of SAD and HvGAMYB transcriptional activators. Electrophoretic mobility shift assays indicate that HvWRKY38 blocks the GA-induced expression of Amy32b by interfering with the binding of HvGAMYB to the cis-acting elements in the α-amylase promoter. The physical interaction of HvWRKY38 and BPBF repressors is demonstrated via bimolecular fluorescence complementation assays. These data suggest that the expression of Amy32b is modulated by protein complexes that contain either activators (e.g. HvGAMYB and SAD) or repressors (e.g. HvWRKY38 and BPBF). The relative amounts of the repressor or activator complexes binding to the Amy32b promoter regulate its expression level in barley Aleurone Cells.
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interactions of two abscisic acid induced wrky genes in repressing gibberellin signaling in Aleurone Cells
Plant Journal, 2006Co-Authors: Zhen Xie, Zhonglin Zhang, Xiaolu Zou, Guangxiao Yang, Setsuko Komatsu, Qingxi J ShenAbstract:Gibberellins (GA) promote while abscisic acid (ABA) inhibits seed germination and post-germination growth. To address the cross-talk of GA and ABA signaling, we studied two rice WRKY genes (OsWRKY51 and OsWRKY71) that are ABA-inducible and GA-repressible in embryos and Aleurone Cells. Over-expression of these two genes in Aleurone Cells specifically and synergistically represses induction of the ABA-repressible and GA-inducible Amy32b alpha-amylase promoter reporter construct (Amy32b-GUS) by GA or the GA-inducible transcriptional activator, GAMYB. The physical interactions of OsWRKY71 proteins themselves and that of OsWRKY71 and OsWRKY51 are revealed in the nuclei of Aleurone Cells using bimolecular fluorescence complementation (BiFC) assays. Although OsWRKY51 itself does not bind to the Amy32b promoter in vitro, it interacts with OsWRKY71 and enhances the binding affinity of OsWRKY71 to W boxes in the Amy32b promoter. The binding activity of OsWRKY71 is abolished by deleting the C-terminus containing the WRKY domain or substituting the key amino acids in the WRKY motif and the zinc finger region. However, two of these non-DNA-binding mutants are still able to repress GA induction by enhancing the binding affinity of the wild-type DNA-binding OsWRKY71 repressors. In contrast, the third non-DNA-binding mutant enhances GA induction of Amy32b-GUS, by interfering with the binding of the wild-type OsWRKY71 or the OsWRKY71/OsWRKY51 repressing complex. These data demonstrate the synergistic interaction of ABA-inducible WRKY genes in regulating GAMYB-mediated GA signaling in Aleurone Cells, thereby establishing a novel mechanism for ABA and GA signaling cross-talk.
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Interactions of two abscisic‐acid induced WRKY genes in repressing gibberellin signaling in Aleurone Cells
The Plant journal : for cell and molecular biology, 2006Co-Authors: Zhen Xie, Zhonglin Zhang, Xiaolu Zou, Guangxiao Yang, Setsuko Komatsu, Qingxi J ShenAbstract:Gibberellins (GA) promote while abscisic acid (ABA) inhibits seed germination and post-germination growth. To address the cross-talk of GA and ABA signaling, we studied two rice WRKY genes (OsWRKY51 and OsWRKY71) that are ABA-inducible and GA-repressible in embryos and Aleurone Cells. Over-expression of these two genes in Aleurone Cells specifically and synergistically represses induction of the ABA-repressible and GA-inducible Amy32b alpha-amylase promoter reporter construct (Amy32b-GUS) by GA or the GA-inducible transcriptional activator, GAMYB. The physical interactions of OsWRKY71 proteins themselves and that of OsWRKY71 and OsWRKY51 are revealed in the nuclei of Aleurone Cells using bimolecular fluorescence complementation (BiFC) assays. Although OsWRKY51 itself does not bind to the Amy32b promoter in vitro, it interacts with OsWRKY71 and enhances the binding affinity of OsWRKY71 to W boxes in the Amy32b promoter. The binding activity of OsWRKY71 is abolished by deleting the C-terminus containing the WRKY domain or substituting the key amino acids in the WRKY motif and the zinc finger region. However, two of these non-DNA-binding mutants are still able to repress GA induction by enhancing the binding affinity of the wild-type DNA-binding OsWRKY71 repressors. In contrast, the third non-DNA-binding mutant enhances GA induction of Amy32b-GUS, by interfering with the binding of the wild-type OsWRKY71 or the OsWRKY71/OsWRKY51 repressing complex. These data demonstrate the synergistic interaction of ABA-inducible WRKY genes in regulating GAMYB-mediated GA signaling in Aleurone Cells, thereby establishing a novel mechanism for ABA and GA signaling cross-talk.
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annotations and functional analyses of the rice wrky gene superfamily reveal positive and negative regulators of abscisic acid signaling in Aleurone Cells
Plant Physiology, 2005Co-Authors: Zhen Xie, Zhonglin Zhang, Xiaolu Zou, Jie Huang, Paul Ruas, Daniel B Thompson, Qingxi J ShenAbstract:The WRKY proteins are a superfamily of regulators that control diverse developmental and physiological processes. This family was believed to be plant specific until the recent identification of WRKY genes in nonphotosynthetic eukaryotes. We have undertaken a comprehensive computational analysis of the rice (Oryza sativa) genomic sequences and predicted the structures of 81 OsWRKY genes, 48 of which are supported by full-length cDNA sequences. Eleven OsWRKY proteins contain two conserved WRKY domains, while the rest have only one. Phylogenetic analyses of the WRKY domain sequences provide support for the hypothesis that gene duplication of single- and two-domain WRKY genes, and loss of the WRKY domain, occurred in the evolutionary history of this gene family in rice. The phylogeny deduced from the WRKY domain peptide sequences is further supported by the position and phase of the intron in the regions encoding the WRKY domains. Analyses for chromosomal distributions reveal that 26% of the predicted OsWRKY genes are located on chromosome 1. Among the dozen genes tested, OsWRKY24, -51, -71, and -72 are induced by abscisic acid (ABA) in Aleurone Cells. Using a transient expression system, we have demonstrated that OsWRKY24 and -45 repress ABA induction of the HVA22 promoter-β-glucuronidase construct, while OsWRKY72 and -77 synergistically interact with ABA to activate this reporter construct. This study provides a solid base for functional genomics studies of this important superfamily of regulatory genes in monocotyledonous plants and reveals a novel function for WRKY genes, i.e. mediating plant responses to ABA.
Russell L. Jones - One of the best experts on this subject based on the ideXlab platform.
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cprg hcl a potential h cl symporter prevents acidification of storage vacuoles in Aleurone Cells and inhibits ga dependent hydrolysis of storage protein and phytate
Plant Journal, 2003Co-Authors: Yong-sic Hwang, Paul C. Bethke, Frank Gubler, Russell L. JonesAbstract:Summary The putative H+/Cl− symporter cycloprodigiosin-HCl (cPrG-HCl) was used to investigate the role of vacuole acidification in cereal Aleurone cell function. The protein storage vacuole (PSV) becomes acidified rapidly when Aleurone Cells are treated with gibberellic acid (GA) but not abscisic acid (ABA). We show that cPrG prevents PSV acidification in Aleurone layers and prevents synthesis of secretory proteins such as α-amylase. Our data support the hypothesis that decreased hydrolase synthesis is a consequence of decreased hydrolysis of storage proteins in PSV. Support for this hypothesis comes from experiments showing that breakdown of barley 7S globulins and phytate is inhibited by cPrG in GA-treated Aleurone layers. Decreased mobilization of PSV reserves is accompanied by reductions in the free amino acid pool size and in the amount of ions released from the Aleurone layer. Vacuolation of the Aleurone cell is a diagnostic feature of the response to GA, and vacuolation is also inhibited by cPrG. Evidence that cPrG acts as a potential H+/Cl− symporter in Aleurone is presented. We show that cPrG does not inhibit the synthesis and secretion of α-amylase when Cl− ions are omitted from the incubation medium. Although cPrG blocks many GA-induced responses of Aleurone layers, it does not affect early steps in GA signaling. The SLN1 protein, a negative regulator of GA signaling, is turned over in GA-treated Cells in the presence and absence of cPrG. Similarly, synthesis of the transcriptional activator GAMYB is unaffected by the presence of cPrG in GA-treated Cells.
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cPrG-HCl a potential H+/Cl- symporter prevents acidification of storage vacuoles in Aleurone Cells and inhibits GA-dependent hydrolysis of storage protein and phytate.
The Plant journal : for cell and molecular biology, 2003Co-Authors: Yong-sic Hwang, Paul C. Bethke, Frank Gubler, Russell L. JonesAbstract:Summary The putative H+/Cl− symporter cycloprodigiosin-HCl (cPrG-HCl) was used to investigate the role of vacuole acidification in cereal Aleurone cell function. The protein storage vacuole (PSV) becomes acidified rapidly when Aleurone Cells are treated with gibberellic acid (GA) but not abscisic acid (ABA). We show that cPrG prevents PSV acidification in Aleurone layers and prevents synthesis of secretory proteins such as α-amylase. Our data support the hypothesis that decreased hydrolase synthesis is a consequence of decreased hydrolysis of storage proteins in PSV. Support for this hypothesis comes from experiments showing that breakdown of barley 7S globulins and phytate is inhibited by cPrG in GA-treated Aleurone layers. Decreased mobilization of PSV reserves is accompanied by reductions in the free amino acid pool size and in the amount of ions released from the Aleurone layer. Vacuolation of the Aleurone cell is a diagnostic feature of the response to GA, and vacuolation is also inhibited by cPrG. Evidence that cPrG acts as a potential H+/Cl− symporter in Aleurone is presented. We show that cPrG does not inhibit the synthesis and secretion of α-amylase when Cl− ions are omitted from the incubation medium. Although cPrG blocks many GA-induced responses of Aleurone layers, it does not affect early steps in GA signaling. The SLN1 protein, a negative regulator of GA signaling, is turned over in GA-treated Cells in the presence and absence of cPrG. Similarly, synthesis of the transcriptional activator GAMYB is unaffected by the presence of cPrG in GA-treated Cells.
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Active oxygen and cell death in cereal Aleurone Cells.
Journal of experimental botany, 2002Co-Authors: Angelika Fath, Paul C. Bethke, Veronica Beligni, Russell L. JonesAbstract:The cereal Aleurone layer is a secretory tissue whose function is regulated by gibberellic acid (GA) and abscisic acid (ABA). Aleurone Cells lack functional chloroplasts, thus excluding photosynthesis as a source of active oxygen species (AOS) in cell death. Incubation of barley Aleurone layers or protoplasts in GA initiated the cell death programme, but incubation in ABA delays programmed cell death (PCD). Light, especially blue and UV-A light, and H 2 O 2 accelerate PCD of GA-treated Aleurone Cells, but ABA-treated Aleurone Cells are refractory to light and H 2 O 2 and are not killed. It was shown that light elevated intracellular H 2 O 2 , and that the rise in H 2 O 2 was greater in GA-treated Cells compared to Cells in ABA. Experiments with antioxidants show that PCD in Aleurone is probably regulated by AOS. The sensitivity of GA-treated Aleurone to light and H 2 O 2 is a result of lowered amounts of enzymes that metabolize AOS. mRNAs encoding catalase, ascorbate peroxidase and superoxide dismutase are all reduced during 6-18 h of incubation in GA, but these mRNAs were present in higher amounts in Cells incubated in ABA. The amounts of protein and enzyme activities encoded by these mRNAs were also dramatically reduced in GA-treated Cells. Aleurone Cells store and metabolize neutral lipids via the glyoxylate cycle in response to GA, and glyoxysomes are one potential source of AOS in the GA-treated Cells. Mitochondria are another potential source of AOS in GA-treated Cells. AOS generated by these organelles bring about membrane rupture and cell death.
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Hormonally regulated programmed cell death in barley Aleurone Cells
The Plant cell, 1999Co-Authors: Paul C. Bethke, Angelika Fath, Jennifer E. Lonsdale, Russell L. JonesAbstract:Cell death was studied in barley (cv Himalaya) Aleurone Cells treated with abscisic acid and gibberellin. Aleurone protoplasts incubated in abscisic acid remained viable in culture for at least 3 weeks, but exposure to gibberellin initiated a series of events that resulted in death. Between 4 and 8 days after incubation in gibberellin, >70% of all protoplasts died. Death, which occurred after Cells became highly vacuolated, was manifest by an abrupt loss of plasma membrane integrity followed by rapid shrinkage of the cell corpse. Hydrolysis of DNA began before death and occurred as protoplasts ceased production of α-amylase. DNA degradation did not result in the accumulation of discrete low molecular weight fragments. DNA degradation and cell death were prevented by LY83583, an inhibitor of gibberellin signaling in barley Aleurone. We conclude that cell death in Aleurone Cells is hormonally regulated and is the final step of a developmental program that promotes successful seedling establishment.
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Barley Aleurone cell death is not apoptotic: characterization of nuclease activities and DNA degradation
The Plant journal : for cell and molecular biology, 1999Co-Authors: Angelika Fath, Paul C. Bethke, Russell L. JonesAbstract:Barley Aleurone Cells undergo programmed cell death (PCD) when exposed to gibberellic acid (GA), but incubation in abscisic acid (ABA) prevents PCD. We tested the hypothesis that PCD in Aleurone Cells occurs by apoptosis, and show that the hallmarks of apoptosis, namely DNA cleavage into 180 bp fragments, plasma membrane blebbing, and the formation of apoptotic bodies do not occur when Aleurone Cells die. We show that endogenous barley Aleurone nucleases and nucleases present in enzymes used for protoplast preparation degrade Aleurone DNA and that DNA degradation by these nucleases is rapid and can result in the formation of 180 bp DNA ladders. Methods are described that prevent DNA degradation during isolation from Aleurone layers or protoplasts. Barley Aleurone Cells contain three nucleases whose activities are regulated by GA and ABA. GA induction and ABA repression of nuclease activities correlate with PCD in Aleurone Cells. Cells incubated in ABA remain alive and do not degrade their DNA, but living Aleurone Cells treated with GA accumulate nucleases and hydrolyze their nuclear DNA. We propose that barley nucleases play a role in DNA cleavage during Aleurone PCD.