Progress 01/01/04 to 12/31/04
Outputs
Due to personnel turnover and repeated difficulties to obtain J-1 visa for qualified scientist, progress has been slow and I have asked for a 1-year no-cost extension. I was able to hire a well-qualified postdoctoral researcher in August who is continuing with the biochemical characterization of Aux/IAA proteins and the molecular genetic characterization of transgenic plants overexpressing Aux/IAA proteins. In addition, he has been developing the experimental tools to test the hypothesis that Aux/IAA proteins may directly interact with histidine phosphotransfer (HPT) proteins that are regulatory intermediates of the two-component signaling system in Arabidopsis. The idea for these experiments is the observation that Aux/IAA and HPT proteins share a conserved amphipathic helix that is known to mediate home- and heterodimerization of Aux/IAA and ARF proteins in auxin signaling. The prospect arises that the same helical fold may mediate regulatory interactions between
the two classes of auxin transcription factors and HPT proteins.
Impacts
Physiological data indicate that light and hormone signaling are inertwined. Demonstration of in vitro phosphorylation of Aux/IAA proteins by phytochrome is an original and significant observation. The prospect arises that Aux/IAA proteins play an essential integrative role at the nexus of auxin and light signaling, both of which are known to profoundly affect plant growth and crop performance.
Publications
- No publications reported this period
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Progress 09/01/01 to 08/31/04
Outputs
The plant hormone auxin (IAA) is a key regulator of plant development. To dissect auxin signaling, several classes of early genes and response mutants have been identified. Aux/IAA genes are primary response genes encoding short-lived nuclear proteins with properties reminiscent of transcription factors. In addition, the evidence suggests that Aux/IAA proteins are positive regulators of photo morphogenesis. Gain-of-function mutations that stabilize select Aux/IAA proteins cause partial photo morphogenesis in dark-grown seedlings, whereas loss-of-function mutations result in longer hypocotyls in light-grown plants. We have previously shown that various recombinant Aux/IAA proteins are phosphorylated in vitro by recombinant oat phytochrome A, and that IAA3 is a phosphoprotein in vivo. If select Aux/IAA proteins are substrates of phytochrome in vivo, we hypothesize that phytochrome-dependent phosphorylation increases their activity or nuclear abundance. To test whether
select Aux/IAA proteins are phosphorylated by phytochrome in vivo, we have generated plants that are transgenic for constructs encoding T7-epitope-tagged IAA3 and IAA17 under the control of the constitutive CaMV 35S promoter. For each construct, we isolated several independent lines in the Columbia wild-type, phyA, and phyB genetic backgrounds. Analysis of visual phenotypes revealed increased or reduced apical dominance and severely reduced plant height for some of the transgenic lines, which is consistent with over expression of the IAA transgenes. For several lines, we observed a long hypocotyl phenotype reminiscent of phyB loss-of-function mutants. We are investigating whether this phenotype is caused by co-suppression of endogenous Aux/IAA genes or by extreme over expression of IAA proteins. If T7-epitope tagged IAA3 and IAA17 can be detected in planta, either by western blot analysis or in vivo labeling, these transgenic lines will be used to monitor the phosphorylation status of
both proteins in response to various light conditions. Since Aux/IAA proteins contain a putative DNA binding domain and previous experiments have suggested auto regulation of IAA3, we tested whether IAA3 binds to its own promoter. We performed electrophoretic mobility shift assays with purified recombinant IAA3 protein and various promoter fragments of the IAA3 gene. Our data showed cooperative binding of IAA3 to its own promoter, however, in a nonspecific fashion. Since Aux/IAA proteins interact with auxin response factors (ARFs), our data suggest that IAA proteins increase the DNA binding specificity of ARFs to auxin-responsive promoter elements when present in ARF-Aux/IAA transcriptional complexes. As Aux/IAA and histidine phosphotransfer (HPT) proteins share a conserved amphipathic helix, we tested the hypothesis that Aux/IAA proteins may directly interact with HPT proteins, which are regulatory intermediates of the two-component signaling system in Arabidopsis. We produced
several recombinant HPT proteins and carried out co-sedimentation assays with epitope-tagged recombinant Aux/IAA proteins. Although we observed in vitro binding, we were unable to demonstrate specificity for this interaction.
Impacts
Physiological data indicate that light and hormone signaling are inertwined. Demonstration of in vitro phosphorylation of Aux/IAA proteins by phytochrome is an original and significant observation. The prospect arises that Aux/IAA proteins play an essential integrative role at the nexus of auxin and light signaling, both of which are known to profoundly affect plant growth and crop performance.
Publications
- ABEL, S., WISELEY, J., COLON-CARMONA, A. and D.L. CHEN (2002) Light modulation of Aux/IAA gene expression. Keystone Symposium on Specificity and Crosstalk in Plant Signal Transduction. Tahoe City, CA, January 22-27, Poster Number 101
- BRUENTRUP, I., WISELEY, J. and S. ABEL (2002) Light modulation of IAA3/SHY2 gene expression. Plant Biology 2002, Denver, CO, August 3-7, Poster number 501
- LASKOWSKI, M.J., DREHER, K.A., GEHRING, M., ABEL, S., GENSLER, A., and I.M. SUSSEX (2002) FQR1, a novel primary auxin-response gene, encodes an FMN-binding quinone reductase. Plant Physiol. 128:578-590.
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Progress 01/01/03 to 12/31/03
Outputs
The plant hormone indole-3-acetic acid (IAA or auxin) is a key regulator of plant growth and development. To dissect auxin signaling, several classes of early genes and response mutants have been isolated by molecular and genetic approaches, respectively. Aux/IAA genes are primary response genes encoding short-lived nuclear proteins with properties reminiscent of transcription factors. In addition, genetic and molecular evidence suggests that Aux/IAA proteins are positive regulators of photomorphogenesis. Gain-of-function mutations that stabilize select Aux/IAA proteins cause partial photomorphogenesis in dark-grown seedlings, whereas loss-of-function mutations result in longer hypocotyls in light-grown plants (partial skotomorphogenesis). We have previously shown that various recombinant Aux/IAA proteins are phosphorylated in vitro by recombinant oat phytochrome A, and that IAA3 is a phosphoprotein in vivo. If select Aux/IAA proteins are substrates of phytochrome in
vivo, we hypothesize that phytochrome-dependent phosphorylation increases their activity or nuclear abundance. To test whether select Aux/IAA proteins are phosphorylated by phytochrome in vivo, we have generated plants that are transgenic for constructs encoding T7-epitope-tagged IAA3 and IAA17 under the control of the constitutive CaMV 35S promoter. For each construct, we have isolated several independent, homozygous transgenic lines in the Columbia wild-type, phyA, and phyB genetic backgrounds. Analysis of visual phenotypes revealed significantly increased or reduced apical dominance and severely reduced plant height for some of the transgenic lines, which is consistent with overexspression of the IAA transgenes. For several of the lines, we observed a long hypocotyls phenotype reminiscent of phyB loss-of-function mutants. We are currently investigating whether this phenotype is caused by co-suppression of endogenous Aux/IAA genes or by extreme overexpression of IAA proteins. If
T7-epitope tagged IAA3 and IAA17 can be detected in planta, either by western blot analysis or in vivo labeling, these transgenic lines will be used to monitor the phosphorylation status of both proteins in response to various light conditions. Since Aux/IAA proteins contain a putative DNA binding domain and previous experiments have suggested autoregulation of IAA3, we tested whether IAA3 binds to its own promoter. We performed electrophoretic mobility shift assays with purified recombinant IAA3 protein and various promoter fragments of the IAA3 gene. Our data clearly showed highly cooperative binding of IAA3 to its own promoter, however, in a nonspecific fashion. Since Aux/IAA proteins interact with auxin response factors (ARFs), our data suggest that IAA proteins increase the DNA binding specificity of ARFs to auxin-responsive promoter elements when present in ARF-Aux/IAA transcriptional complexes.
Impacts
Physiological data indicate that light and hormone signaling are inertwined. Demonstration of in vitro phosphorylation of Aux/IAA proteins by phytochrome is an original and significant observation. The prospect arises that Aux/IAA proteins play an essential integrative role at the nexus of auxin and light signaling, both of which are known to profoundly affect plant growth and crop performance.
Publications
- No publications reported this period
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Progress 01/01/02 to 12/31/02
Outputs
The plant hormone indole-3-acetic acid (IAA or auxin) is a key regulator of plant growth and development. To dissect auxin signaling, several classes of early genes and response mutants have been isolated by molecular and genetic approaches, respectively. Aux/IAA genes are primary response genes encoding short-lived nuclear proteins with properties reminiscent of transcription factors. In addition, genetic and molecular evidence suggests that Aux/IAA proteins are positive regulators of photomorphogenesis. Gain-of-function mutations that stabilize select Aux/IAA proteins cause partial photomorphogenesis in dark-grown seedlings, whereas loss-of-function mutations result in longer hypocotyls in light-grown plants (partial skotomorphogenesis). We have previously shown that various recombinant Aux/IAA proteins are phosphorylated in vitro by recombinant oat phytochrome A, and that IAA3 is a phosphoprotein in vivo. If select Aux/IAA proteins are substrates of phytochrome in
vivo, we hypothesize that phytochrome-dependent phosphorylation increases their activity or nuclear abundance. To test whether select Aux/IAA proteins are phosphorylated by phytochrome in vivo, we have generated plants that are transgenic for constructs encoding T7-epitope-tagged IAA3 and IAA17 under the control of the constitutive CaMV 35S promoter. For each construct, we have isolated several independent, homozygous transgenic lines in the Columbia wild-type, phyA, and phyB genetic backgrounds. Analysis of visual phenotypes revealed significantly increased or reduced apical dominance and severely reduced plant height for some of the transgenic lines, which is consistent with overexspression of the IAA transgenes. For several of the lines, we observed a long hypocotyls phenotype reminiscent of phyB loss-of-function mutants. We are currently investigating whether this phenotype is caused by co-suppression of endogenous Aux/IAA genes or by extreme overexpression of IAA proteins. If
T7-epitope tagged IAA3 and IAA17 can be detected in planta, either by western blot analysis or in vivo labeling, these transgenic lines will be used to monitor the phosphorylation status of both proteins in response to various light conditions. Since Aux/IAA proteins contain a putative DNA binding domain and previous experiments have suggested autoregulation of IAA3, we tested whether IAA3 binds to its own promoter. We performed electrophoretic mobility shift assays with purified recombinant IAA3 protein and various promoter fragments of the IAA3 gene. Our data clearly showed highly cooperative binding of IAA3 to its own promoter, however, in a nonspecific fashion. Since Aux/IAA proteins interact with auxin response factors (ARFs), our data suggest that IAA proteins increase the DNA binding specificity of ARFs to auxin-responsive promoter elements when present in ARF-Aux/IAA transcriptional complexes.
Impacts
Physiological data indicate that light and hormone signaling are inertwined. Demonstration of in vitro phosphorylation of Aux/IAA proteins by phytochrome is an original and significant observation. The prospect arises that Aux/IAA proteins play an essential integrative role at the nexus of auxin and light signaling, both of which are known to profoundly affect plant growth and crop performance.
Publications
- LASKOWSKI, M.J., DREHER, K.A., GEHRING, M., ABEL, S., GENSLER, A., and I.M. SUSSEX (2002) FQR1, a novel primary auxin-response gene, encodes an FMN-binding quinone reductase. Plant Physiol. 128:578-590.
- ABEL, S., WISELEY, J., COLON-CARMONA, A. and D.L. CHEN (2002) Light modulation of Aux/IAA gene expression. Keystone Symposium on Specificity and Crosstalk in Plant Signal Transduction. Tahoe City, CA, January 22-27, Poster #101
- BRUENTRUP, I., WISELEY, J. and S. ABEL (2002) Light modulation of IAA3/SHY2 gene expression. Plant Biology 2002, Denver, CO, August 3-7, Poster #501
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Progress 01/01/01 to 12/31/01
Outputs
The plant hormone indole-3-acetic acid (IAA or auxin) is a key regulator of plant growth and development. To dissect auxin signaling, several classes of early genes and response mutants have been isolated by molecular and genetic approaches, respectively. Aux/IAA genes are primary response genes encoding short-lived nuclear proteins with properties reminiscent of transcription factors. In addition, genetic and molecular evidence suggests that Aux/IAA proteins are positive regulators of photomorphogenesis. Gain-of-function mutations that stabilize select Aux/IAA proteins cause partial photomorphogenesis in dark-grown seedlings, whereas loss-of-function mutations result in longer hypocotyls in light-grown plants (partial skotomorphogenesis). We have previously shown that various recombinant Aux/IAA proteins are phosphorylated in vitro by recombinant oat phytochrome A, and that IAA3 is a phosphoprotein in vivo. If select Aux/IAA proteins are substrates of phytochrome in
vivo, we hypothesize that phytochrome-dependent phosphorylation increases their activity or nuclear abundance. To test whether select Aux/IAA proteins are phosphorylated by phytochrome in vivo, we have begun to generate transgenic plants that constitutively overexpress T7-epitope-tagged IAA3 and IAA17 in wild-type plants as well as in various phytochrome mutant backgrounds. It is hoped that these lines will support higher gene expression and specific detection of IAA3 and IAA17 proteins in planta. If that is the case, these transgenic lines will be used to monitor the phosphorylation status of both proteins in response to various light conditions. To specifically detect the IAA3 protein in wild-type plants, we have raised an epitope-specific antibody. This antibody has been characterized and will be used during in vivo labeling/co-immunoprecipitation experiments to follow steady-state protein levels and to determine metabolic turnover rates in response to various treatments.
Impacts
Physiological data indicate that light and hormone signaling are inertwined. Demonstration of in vitro phosphorylation of Aux/IAA proteins by phytochrome is an original and significant observation. The prospect arises that Aux/IAA proteins play an essential integrative role at the nexus of auxin and light signaling, both of which are known to profoundly affect plant growth and crop performance.
Publications
- No publications reported this period
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