Progress 05/09/06 to 09/30/10
Outputs
Progress Report Objectives (from AD-416) The long-term goal of this research is to define the molecular mechanisms by which the phytochrome (phy) family of photoreceptors perceive informational light signals from the environment and transduce them to photoresponsive nuclear genes, thereby controlling plant growth and development. Using genome-scale microarray-based expression profiling, we have begun to define the global transcriptional network regulated by these photoreceptors and have identified a set of rapidly light-induced or -repressed genes (primarily encoding various transcription factors) that are potential direct targets of the phytochrome signaling pathway. The specific objectives of this project plan are: 1. To identify which phy family members are responsible for signaling to each of these genes, by examining light-induced expression profiles in null mutants of each phytochrome. 2. To define the cis elements in the promoters of these genes responsible for coordinate light-regulated expression, using computational analysis coupled with targeted mutagenesis and transgenic expression of promoter::reporter fusion constructs. 3. To identify downstream targets of these genes in the light-regulated transcriptional network, using microarray-based expression profiling in knockout mutants null for each transcription factor, coupled with chromatin immunoprecipitation (ChIP) for target promoter identification and characterization. Approach (from AD-416) The specific objectives of this proposal are: (a) to identify which phys are responsible for signaling to these genes; (b) to define the cis elements in the promoters of these genes responsible for coordinate light- regulated expression; (c) to identify downstream targets of these genes in the light-regulated transcriptional network. The experimental approaches will include: (a) microarray-based expression profiling of mutants null for each phy, both to define the phy-regulated transcriptional networks and to identify rapidly light-responsive genes for targeted reverse-genetic mutagenesis; (b) computational analysis of the promoters of these genes, coupled with targeted mutagenesis and transgenic expression of promoter::reporter fusion constructs; (c) expression profiling in knockout mutants null for each transcription factor, coupled with chromatin immunoprecipitation for identification of promoters that are direct targets of these factors. REPLACES 5335-21000- 023-00D (4/06). Definition of early transcriptional circuitry involved in light-induced reversal of PIF (phytochrome-interacting bHLH factor)-imposed repression of photomorphogenesis in young Arabidopsis seedlings. Light-signals perceived by the phytochrome (phy) photoreceptor family induce the transition from skotomorphogenic to photomorphogenic development (deetiolation) in young dark-germinated seedlings. Recent genetic evidence, showing that a quadruple mutant (pifq) lacking four phy- interacting bHLH transcription factors (PIF1, 3, 4 and 5) displays a constitutive photomorphogenic phenotype in the dark, establishes that these factors act partially redundantly to sustain the skotomorphogenic state in dark-grown seedlings. Together with evidence that the photoactivated phy molecule binds to and induces rapid intranuclear degradation of the PIF proteins, these data indicate that the photoreceptor acts to reverse the constitutive activity of these factors upon light exposure, thereby initiating the switch to photomorphogenic development. To delineate the scope of the cellular, subcellular and gene- expression pathways controlled by these PIF proteins in directing this visible morphogenic transition, and to define the mode of transcriptional regulation of responsive genes exerted by the PIFs, we examined the deetiolating tissues cytologically, and have performed expression profiling of the dark-grown pifq mutant compared to the light-treated wild type. Dark-grown mutant seedlings display cellular and subcellular development that extensively phenocopies that of wild-type seedlings grown continuously in red light, including cell expansion, vacuolization, oil-body abundance and distribution, and initiation of chloroplast development. Similarly, 80 per cent of the gene-expression changes elicited by the genetic removal of the PIFs in dark-grown pifq seedlings are normally induced by light in establishing the deetiolated state in wild-type seedlings over the two-day growth period used here. By comparing the genes that respond rapidly (within 1 h) to initial red- light exposure in wild-type seedlings with those responding in the pifq mutant in the dark, we have identified a small subset, enriched in transcription-factor-encoding genes, that are potential direct targets of PIF transcriptional regulation. Collectively, these data suggest that the transcriptional response elicited by the light-induced proteolytic removal of the PIF proteins comprises a major component of the mechanism by which the phys exert pleiotropic regulation of multiple facets of the deetiolation process, and that at least some of the rapidly light- responsive genes identified may comprise a primary transcriptional network directly targeted by the PIF proteins. Accomplishments 01 Conversion of seedlings into plants. A critical factor in successful seedling establishment in crop plants is a developmental strategy that first enables postgerminative seedlings emerging from buried seed to gro vigorously upward in the subterranean darkness toward the soil surface, and then, upon initial exposure to light, undergo a process, termed deetiolation, involving conversion to the fully-photosynthetically activ state of green plants. The underlying molecular processes that control this strategy were not well defined. ARS scientists in Albany, CA have discovered that a central component of the mechanism underlying this strategy is the repression, by a family of proteins called PIFs (phytochrome-interacting factors), in darkness, of the gene-expression network normally induced by light. The experiments show that light triggers this process by activating the phy photoreceptor molecules whic then bind to and induce rapid degradation of the PIF proteins in the nucleus, thereby releasing the the normal gene expression pattern observ in green seedlings. This research provides basic information for understanding a key aspect of crop seedling establishment.
Impacts
(N/A)
Publications
- Quail, P.H. 2008. Harry Smith � recipient of the 2008 Molecular Ecology Prize. Molecular Ecology. 18(1):21-22.
- Franklin, K.A., Quail, P.H. 2009. Phytochrome functions in Arabidopsis development. Journal of Experimental Botany. 61(1)11-24; doi:10. 1093/jxb/erp304.
- Leivar, P., Teppermann, J.M., Monte, E., Calderon, R.H., Liu, T.L., Quail, P.H. 2009. Definition of Early Transcriptional Circuitry Involved in Light- Induced Reversal of PIF-Imposed Repression of Photomorphogenesis in Young Arabidopsis Seedlings. The Plant Cell. 21:3535-3553.
- Quail, P.H. 2010. Phytochromes. Current Biology. 20(12):R504-R507.
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Progress 10/01/08 to 09/30/09
Outputs
Progress Report Objectives (from AD-416) The long-term goal of this research is to define the molecular mechanisms by which the phytochrome (phy) family of photoreceptors perceive informational light signals from the environment and transduce them to photoresponsive nuclear genes, thereby controlling plant growth and development. Using genome-scale microarray-based expression profiling, we have begun to define the global transcriptional network regulated by these photoreceptors and have identified a set of rapidly light-induced or -repressed genes (primarily encoding various transcription factors) that are potential direct targets of the phytochrome signaling pathway. The specific objectives of this project plan are: 1. To identify which phy family members are responsible for signaling to each of these genes, by examining light-induced expression profiles in null mutants of each phytochrome. 2. To define the cis elements in the promoters of these genes responsible for coordinate light-regulated expression, using computational analysis coupled with targeted mutagenesis and transgenic expression of promoter::reporter fusion constructs. 3. To identify downstream targets of these genes in the light-regulated transcriptional network, using microarray-based expression profiling in knockout mutants null for each transcription factor, coupled with chromatin immunoprecipitation (ChIP) for target promoter identification and characterization. Approach (from AD-416) The specific objectives of this proposal are: (a) to identify which phys are responsible for signaling to these genes; (b) to define the cis elements in the promoters of these genes responsible for coordinate light- regulated expression; (c) to identify downstream targets of these genes in the light-regulated transcriptional network. The experimental approaches will include: (a) microarray-based expression profiling of mutants null for each phy, both to define the phy-regulated transcriptional networks and to identify rapidly light-responsive genes for targeted reverse-genetic mutagenesis; (b) computational analysis of the promoters of these genes, coupled with targeted mutagenesis and transgenic expression of promoter::reporter fusion constructs; (c) expression profiling in knockout mutants null for each transcription factor, coupled with chromatin immunoprecipitation for identification of promoters that are direct targets of these factors. Significant Activities that Support Special Target Populations Residues Clustered in the Light-Sensing Knot of Phytochrome B Are Necessary for Conformer-Specific Binding to Signaling Partner PIF3 Plants monitor their environment for informational light signals that are used to direct adaptive morphogenic responses. The phytochrome (phy) family of photoreceptors are central to this process. Upon photoperception, phy molecules rapidly translocate to the nucleus where they interact with basic helix-loop-helix (bHLH) transcription factors, termed PIFs (phy-Interacting Factors), and induce gene-expression changes that control morphogenic responses. The molecular determinants in the phy protein responsible for direct intermolecular signal transfer from the activated photoreceptor to transduction partners have been until now undefined. The bHLH factor, PIF3, interacts specifically with the photoactivated, Pfr, form of Arabidopsis phytochrome B (phyB). This interaction induces PIF3 phosphorylation and degradation in vivo and modulates phyB-mediated seedling deetiolation in response to red light. To identify missense mutations in the phyB N-terminal domain that disrupt this interaction, we developed a yeast reverse-hybrid screen. Fifteen individual mutations identified in this screen, or in previous genetic screens for Arabidopsis mutants showing reduced sensitivity to red light, were shown to also disrupt light-induced binding of phyB to PIF3 in in vitro co-immunoprecipitation assays. These phyB missense mutants fall into two general classes: Class I (eleven mutants) containing those defective in light signal perception, due to aberrant chromophore attachment or photoconversion, and Class II (four mutants) containing those normal in signal perception, but defective in the capacity to transduce this signal to PIF3. By generating a homology model for the three-dimensional structure of the Arabidopsis phyB chromophore-binding region, based on the crystal structure of Deinococcus radiodurans phytochrome, we predict that three of the four Class II mutated phyB residues are solvent exposed in a cleft between the presumptive PAS and GAF domains. This deduction suggests that these residues could be directly required for the physical interaction of phyB with PIF3. Because these three residues are also necessary for phyB-imposed inhibition of hypocotyl elongation in response to red light, they are functionally necessary for signal transfer from photoactivated phyB, not only to PIF3 and other related bHLH transcription factors tested here, but also to other downstream signaling components involved in regulating seedling deetiolation.
Impacts
(N/A)
Publications
- Kikis, E., Oka, Y., Hudson, M.E., Nagatani, A., Quail, P.H. 2009. Residues Clustered in the Light-Sensing Knot of Phytochrome B Are Necessary for Conformer-Specific Binding to Signaling Partner PIF3. PLoS Genetics 5(1): e1000352. doi:10.1371/journal.pgen.1000352.
- Oka, Y., Matsushita, T., Mochizuki, N., Quail, P.H., Nagatani, A. 2008. Mutant Screen Distinguishes between Residues Necessary for Light-Signal Perception and Signal Transfer by Phytochrome B. PLoS Genet 4(8): e1000158. doi:10.1371/journal.pgen.1000158.
- Leivar, P., Monte, E., Oka, Y., Liu, T., Carle, C., Castillon, A., Huq, E., Quail, P.H. 2008. Multiple Phytochrome-Interacting bHLH Transcription Factors Repress Premature Seedling Photomorphogenesis in Darkness. Current Biology 18(23):1815-23.
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Progress 10/01/07 to 09/30/08
Outputs
Progress Report Objectives (from AD-416) The long-term goal of this research is to define the molecular mechanisms by which the phytochrome (phy) family of photoreceptors perceive informational light signals from the environment and transduce them to photoresponsive nuclear genes, thereby controlling plant growth and development. Using genome-scale microarray-based expression profiling, we have begun to define the global transcriptional network regulated by these photoreceptors and have identified a set of rapidly light-induced or -repressed genes (primarily encoding various transcription factors) that are potential direct targets of the phytochrome signaling pathway. The specific objectives of this project plan are: 1. To identify which phy family members are responsible for signaling to each of these genes, by examining light-induced expression profiles in null mutants of each phytochrome. 2. To define the cis elements in the promoters of these genes responsible for coordinate light-regulated expression, using computational analysis coupled with targeted mutagenesis and transgenic expression of promoter::reporter fusion constructs. 3. To identify downstream targets of these genes in the light-regulated transcriptional network, using microarray-based expression profiling in knockout mutants null for each transcription factor, coupled with chromatin immunoprecipitation (ChIP) for target promoter identification and characterization. Approach (from AD-416) The specific objectives of this proposal are: (a) to identify which phys are responsible for signaling to these genes; (b) to define the cis elements in the promoters of these genes responsible for coordinate light- regulated expression; (c) to identify downstream targets of these genes in the light-regulated transcriptional network. The experimental approaches will include: (a) microarray-based expression profiling of mutants null for each phy, both to define the phy-regulated transcriptional networks and to identify rapidly light-responsive genes for targeted reverse-genetic mutagenesis; (b) computational analysis of the promoters of these genes, coupled with targeted mutagenesis and transgenic expression of promoter::reporter fusion constructs; (c) expression profiling in knockout mutants null for each transcription factor, coupled with chromatin immunoprecipitation for identification of promoters that are direct targets of these factors. REPLACES 5335-21000- 023-00D (4/06). Significant Activities that Support Special Target Populations Duality of transcription factor activity in mediating phytochrome-induced seedling photomorphogenesis The phytochrome (phy) family of sensory photoreceptors (phyA-E in Arabidopsis) elicit changes in gene expression following light-induced migration to the nucleus, where they interact with bHLH transcription factors, such as PIF3. The mechanism by which PIF3 relays phy signals, both early following initial light exposure and later during long-term irradiation, is not understood. Using transgenically-expressed PIF3 variants, carrying site-specific amino-acid substitutions that block the protein from binding either to DNA, phyA and/or phyB, we examined the involvement of PIF3 in early, phy-induced marker-gene expression, and in modulating long-term, phy-imposed inhibition of hypocotyl cell elongation under prolonged, continuous irradiation. We discovered an unanticipated dual mechanism of PIF3 action that involves the temporal uncoupling of its two most central molecular functions. We found that, in early signaling, PIF3 acts positively as a transcription factor, exclusively requiring its DNA-binding capacity. Contrary to previous proposals, PIF3 functions as a constitutive coactivator in this process, without the need for phy binding and subsequent phy-induced modifications. This implies that another factor(s) is conditionally activated by phy, and functions in concert with PIF3, to induce target-gene transcription. In contrast, during long-term irradiations, PIF3 acts exclusively through its phyB- interacting capacity to control hypocotyl cell elongation, independently of its ability to bind DNA. Unexpectedly, PIF3 uses this capacity to regulate phyB protein abundance (and thereby global photosensory sensitivity) to modulate this long-term response, rather than participating directly in the transduction chain as a signaling intermediate. This relates to NP 302, Component 1.
Impacts
(N/A)
Publications
- Leivar, P., Monte, E., Al-Sady, B., Carle, C., Storer, A., Alonso, J.M., Ecker, J.R., Quail, P.H. 2008. The Arabidopsis Phytochrome-Interacting Factor PIF7, Together with PIF3 and PIF4, Regulates Responses to Prolonged Red Light by Modulating phyB Levels. The Plant Cell. 20:337-352.
- Al-Sady, B., Kikis, E.A., Monte, E., Quail, P.H. 2008. Mechanistic duality of transcription factor function in phytochrome signaling. Proceedings of the National Academy of Sciences. 105(6):2232-2237.
- Hwang, Y.S., Quail, P.H. 2008. Phytochrome-Regulated PIL1 Derepression is Developmentally Modulated. Plant Cell Physiology. 49(4):501-511.
- Shen, Y., Khanna, R., Carle, C.M., Quail, P.M. 2007. Phytochrome Induces Rapid PIF5 Phosphorylation and Degradation in Response to Red-Light Activation. Plant Physiology. 145:1043-1051.
- Khanna, R., Shen, Y., Marion, C.M., Tsuchisaka, A., Theologis, A., Schafer, E., Quail, P.M. 2007. The Basic Helix-Loop-Helix Transcription Factor PIF5 Acts on Ethylene Biosynthesis and Phytochrome Signaling by Distinct Mechanisms. The Plant Cell. Dec 19(12):3915-29. Epub 2007 Dec 7.
- Monte, E., Al-Sady, B., Leivar, P., Quail, P. 2007. Out of the dark: how the PIFs are unmasking a dual temporal mechanism of phytochrome signalling. Journal of Experimental Biology. 3(58):3125-3133.
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Progress 10/01/06 to 09/30/07
Outputs
Progress Report Objectives (from AD-416) The long-term goal of this research is to define the molecular mechanisms by which the phytochrome (phy) family of photoreceptors perceive informational light signals from the environment and transduce them to photoresponsive nuclear genes, thereby controlling plant growth and development. Using genome-scale microarray-based expression profiling, we have begun to define the global transcriptional network regulated by these photoreceptors and have identified a set of rapidly light-induced or -repressed genes (primarily encoding various transcription factors) that are potential direct targets of the phytochrome signaling pathway. The specific objectives of this project plan are: 1. To identify which phy family members are responsible for signaling to each of these genes, by examining light-induced expression profiles in null mutants of each phytochrome. 2. To define the cis elements in the promoters of these genes responsible for coordinate light-regulated expression, using computational analysis coupled with targeted mutagenesis and transgenic expression of promoter::reporter fusion constructs. 3. To identify downstream targets of these genes in the light-regulated transcriptional network, using microarray-based expression profiling in knockout mutants null for each transcription factor, coupled with chromatin immunoprecipitation (ChIP) for target promoter identification and characterization. Approach (from AD-416) The specific objectives of this proposal are: (a) to identify which phys are responsible for signaling to these genes; (b) to define the cis elements in the promoters of these genes responsible for coordinate light- regulated expression; (c) to identify downstream targets of these genes in the light-regulated transcriptional network. The experimental approaches will include: (a) microarray-based expression profiling of mutants null for each phy, both to define the phy-regulated transcriptional networks and to identify rapidly light-responsive genes for targeted reverse-genetic mutagenesis; (b) computational analysis of the promoters of these genes, coupled with targeted mutagenesis and transgenic expression of promoter::reporter fusion constructs; (c) expression profiling in knockout mutants null for each transcription factor, coupled with chromatin immunoprecipitation for identification of promoters that are direct targets of these factors. REPLACES 5335-21000- 023-00D (4/06). BSL 1; 7/1/05. Accomplishments Phytochrome A Dominates in Transduction of Red-light Signals to Rapidly- responding Genes at the Initiation of Seedling Deetiolation All aspects of plant biology hinge on the plant's ability to perceive light. Because the phytochrome (phy) family (phyA to phyE) are the major light receptors for plants, understanding how they function in regulating gene expression, is crucial for crop improvement. At the PGEC, using microarray-based gene-expression profiling, we found, contrary to expectation based on previous data, that phyA is the dominant photoreceptor regulating rapidly-induced genes in response to red-light signals (Tepperman et al., 2006). This finding represents a major shift in thinking about the molecular basis of phy-regulated gene expression and opens up new opportunities for manipulating this system for agronomic improvement of crop species. Accomplishments fall under NP 302 Problem Statement 1B (Applying Genomics to Crop Improvement) of Component 1 (Functional Utilization of Plant Genomes: Translating Plant Genomics into Crop Improvement). Technology Transfer Number of New CRADAS and MTAS: 20 Number of Web Sites managed: 1 Number of Non-Peer Reviewed Presentations and Proceedings: 4
Impacts
(N/A)
Publications
- Quail, P.H. 2007. Phytochrome-regulated Gene Expression. Journal of Integrative Plant Biology 49(1):11-20.
- Quail, P.C. 2007. Phytochrome Interacting Factors. Annual Plant Reviews, Vol 30. In: Whitelam, G., Halliday, K., editors. Light and Plant Development. Oxford, UK: Blackwell Publishing. Ch. 4, p. 81-105.
- Tepperman, J.M., Hwang, Y., Quail, P.H. 2006. phyA dominates in transduction of red-light signals to rapidly responding genes at the initiation of Arabidopsis seedling de-etiolation. Plant Journal 48(5) :728�742.
- Khanna, R., Shen, Y., Toledo-Ortiz, G., Kikis, E.A., Johannesson, H., Hwang, Y., Quail, P.H. 2006. Functional Profiling Reveals that Only a Small Number of Phytochrome-Regulated Early-Response Genes in Arabidopsis Are Necessary for Optimal Deetiolation. The Plant Cell 18:2157-2171.
- Al-Sady, B., Ni, W., Kircher, S., Schafer, E., Quail, P.H. 2006. Photoactivated Phytochrome Induces Rapid PIF3 Phosphorylation Prior to Proteasome-Mediated Degradation. Molecular Cell 23(3):439-446.
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