Progress 07/12/01 to 06/30/06
Outputs
Progress Report 1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? This project is aligned to NP 302, Plant Biological and Molecular Processes. The major problem being addressed is the need to produce more food of enhanced nutritional value in an ecofriendly manner, without increasing acreage, in order to feed the expanding world population. Toward this end we are investigating the fundamental molecular mechanisms by which plants regulate their growth and development in response to the prevailing light environment. Our overall research goal is to define the molecular mechanisms by which the phytochrome family of photoreceptors perceives, interprets and transduces informational light signals from the environment to photoresponsive genes. Our specific objectives are to identify, molecularly clone, and functionally define the biological and biochemical
activities of the components involved in this process using genetic, molecular and biochemical strategies. Many plant responses to light that have evolved to provide competitive advantage and survival under natural conditions are agronomically undesirable. These include the shade-avoidance response and photoperiodic control of such processes as flowering and leaf senescence. Identification of the molecular components and understanding of the biochemical and cellular mechanisms involved in light signaling to nuclear genes will provide potential targets for biotechnologically based improvement of crop plants. 2. List by year the currently approved milestones (indicators of research progress) This specific cooperative agreement directly supports approved milestones identified in the reports for Plant Gene Expression Center projects 5335- 21000-015-00D, 5335-21000-016-00D, 5335-21000-017-00D, 5335-21000-018-00D, 5335-21000-020-00D, 5335-21430-005-00D, and 5335-22000-006-00D. Key ongoing
milestones include: YEAR 1 (FY 2006/07) 1. Continue analysis of monogenic phyA and PhyB mutants. 2. Generate promoter-deletion constructs for selected genes; initiate transgenic plant production. 3. Identify early-response transcription factor genes; obtain insertional mutants for selected genes; obtain cDNAs for selected genes YEAR 2 (FY 2007/08) 1. Microarray-based expression proviling of phyAphyB double mutants. 2. Continue production of homozygous transgenic production; begin expression analysis. 3. Produce homozygous knockout mutant lines for selected genes; intiate microarray analysis produce GR-fusion expression transgenic lines. YEAR 3 (FY 2008/09) 1. Microarray-based expression profiling of monogenic phyC and phyD mutants. 2. Continue expression analysis of promoter-deletion lines; make targeted motif mutant constructions; initiate transgenic production. 3. Continue microarray analysis in knockout mutants; initiate ChIP experiments with GR-fusion lines; produce recombinant
protein for antibody production. YEAR 4 (FY 2009/10) 1. Microarray-based expression profiling of higher order phy mutants. 2. Continue production of homozygous motif-mutant lines; performa expression analysis. 3. Continue microarray analysis; continue ChIP experiments. YEAR 5 (FY 2010/11) 1. Microarry-absed expression provigin of higher order phy mutants. 2. Initiate gain-of-funciton construct and transgenic production; screen for motif- binding proteins. 3. Continue microarray analysis; continue ChIP experiments. 4a List the single most significant research accomplishment during FY 2006. Research under this project is relevant to NP 302, Component 1, "Functional Utilization of Plant Genomes." Identification of genes encoding phytochrome signaling components. All aspects of plant biology hinge on the plant's ability to perceive light. Because phytochrome is the major light receptor for plants, understanding how it functions is crucial for crop improvement. We identified a bHLH
transcription factor, designated PIF1 (PHYTOCHROME (phy) -INTERACTING FACTOR 1). We showed that this factor negatively regulates chlorophyll biosynthesis. PIF1 interacts specifically with the photoactivated conformer of both phytochromes A and B. This observation suggests a signaling pathway by which chlorophyll biosynthetic rates are tightly controlled during the critical initial emergence of seedlings from subterranean darkness into sunlight (Huq et al. (2004) Science 305: 1937-1941). These findings demonstrate that PIF1 has a critical function in the agronomically important process of seedling establishment. 4d Progress report. This report serves to document research conducted under a Specfic Cooperative Agreement between ARS and UC Berkeley (Agreement #58-5335-1- 0446). Additional details of research can be found in the report for the parent project (5335-21000-027-00D). 5. Describe the major accomplishments to date and their predicted or actual impact. Research under this
project is relevant to NP 302, Component 1, "Functional Utilization of Plant Genomes." The identification and cloning of the phytochrome gene family in Arabidopsis, and homologs in such crop plants as rice and corn. The identification of functionally active promoter cis elements in the phyA genes of oat, rice and corn, and the cloning of transcription factors that interact with these elements. The overexpression of phytochrome in transgenic plants leading to modified architecture due to suppression of the shade-avoidance response. Discovery of the differential functional roles of individual phytochrome family members through creation of null mutants in the photoreceptors. Genetic and molecular identification of phytochrome signaling components and definition of phytochrome-regulated transcriptional networks.
Impacts
(N/A)
Publications
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Progress 10/01/04 to 09/30/05
Outputs
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? The major problem being addressed is the need to produce more food of enhanced nutritional value in an ecofriendly manner, without increasing acreage, in order to feed the expanding world population. Toward this end we are investigating the fundamental molecular mechanisms by which plants regulate their growth and development in response to the prevailing light environment. Our overall research goal is to define the molecular mechanisms by which the phytochrome family of photoreceptors perceives, interprets and transduces informational light signals from the environment to photoresponsive genes. Our specific objectives are to identify, molecularly clone, and functionally define the biological and biochemical activities of the components involved in this process using genetic, molecular and biochemical
strategies. Many plant responses to light that have evolved to provide competitive advantage and survival under natural conditions are agronomically undesirable. These include the shade-avoidance response and photoperiodic control of such processes as flowering and leaf senescence. Identification of the molecular components and understanding of the biochemical and cellular mechanisms involved in light signaling to nuclear genes will provide potential targets for biotechnologically based improvement of crop plants. 2. List the milestones (indicators of progress) from your Project Plan. a. Identify phytochrome signaling intermediates (i) Identify phytochrome-interacting factors (PIFS) (ii) Perform forward genetic screens for signaling intermediates b. Define phytochrome-regulated transcription networks (i) Perform microarray-based expression profiling (ii) Characterize the basic helix-loop-helix (bHLH) transcription factor family 3a List the milestones that were scheduled to be
addressed in FY 2005. For each milestone, indicate the status: fully met, substantially met, or not met. If not met, why. 1. We will continue to screen for phytochrome-interacting factors and assess their functional relevance to phytochrome signaling using reverse-genetic disruption of the identified loci. Milestone Fully Met 2. We will continue to perform forward genetic screens for additional signaling intermediates in the phytochrome pathway by screening for abnormalities in the seedling de-etiolation process. Genes will be isolated and characterized and their protein products assessed for potential biochemical function. Milestone Fully Met 3. We will continue to map the phytochrome-regulated transcriptional networks by performing microarray expression profiling on wild-type and mutant seedlings. This will permit placement of the activity of the mutated locus in the network hierarchy. Milestone Fully Met 4. We will continue a systematic assessment of the functional role of
members of the bHLH family in phytochrome signaling by reverse-genetic disruption of targets related to the phytochrome-interacting members of the family. Milestone Fully Met 3b List the milestones that you expect to address over the next 3 years (FY 2006, 2007, and 2008). What do you expect to accomplish, year by year, over the next 3 years under each milestone? FY 2006, 2007, 2008 A. Identify phytochrome signaling intermediates (i) Identify phytochrome-interacting factors (PIFS). We will continue to screen for phytochrome-interacting factors and assess their functional relevance to phytochrome signaling using reverse-genetic disruption of the identified loci. (ii) Perform forward genetic screens for signaling intermediates. We will continue to perform forward genetic screens for additional signaling intermediates in the phytochrome pathway by screening for abnormalities in the seedling de-etiolation process. Genes will be isolated and characterized and their protein products
assessed for potential biochemical function. B. Define phytochrome-regulated transcription networks (i) Perform microarray-based expression profiling. We will continue to map the phytochrome-regulated transcriptional networks by performing microarray expression profiling on wild-type and mutant seedlings. This will permit placement of the activity of the mutated locus in the network hierarchy. (ii) Characterize the basic helix-loop-helix (bHLH) transcription factor family. We will continue a systematic assessment of the functional role of members of the bHLH family in phytochrome signaling by reverse- genetic disruption of targets related to the phytochrome-interacting members of the family. Year by year we expect to identify increasing numbers of signaling intermediates and phytochrome target genes and determine their biological and biochemical functions. 4a What was the single most significant accomplishment this past year? Identification of genes encoding phytochrome signaling
components. The phytochrome (phy) family of sensory photoreceptors transduce informational light signals to selected nuclear genes. These signals induce agriculturally important plant growth and developmental responses to the prevailing light environment and, therefore, it is important to define the molecular mechanisms involved in this signaling process. The Plant Gene Expression Center in collaboration with U. C. Berkeley, identified new mutants of the centrally important transcription factor, PHYTOCHROME INTERACTING FACTOR 3 (PIF3) which show that PIF3 is necessary for early chloroplast greening and for rapid phy-induced expression of nuclear genes encoding chloroplast components, upon first exposure of seedlings to light (Monte et al. (2004) Proc. Natl. Acad. Sci. 101: 16091- 16098). These data demonstrate that PIF3 functions to transduce phy signals to genes involved in the generation of a functional photosynthetic apparatus. This is an agronomically critical facet of the early
seedling deetiolation process, an important process to seedling establishment. 4d Progress report. This report serves to document research conducted under a Specific Cooperative Agreement between ARS and UC Berkeley. Additional details of research can be found in the report for the parent project (5335-21000- 017-00D). Different Arabidopsis phytochrome (phy) family members (phyA through phyE) display differential photosensory and/or physiological functions in regulating growth and developmental responses to light signals. To identify those genes regulated by phyB in response to continuous red light (Rc) during the induction of seedling deetiolation, we have performed time-course, microarray-based expression profiling of wild-type and phyB null mutants. Comparison of the observed expression patterns with those induced by continuous far-red light (FRc) (perceived exclusively by phyA) in wild-type and phyA null mutant seedlings suggests early convergence of the FRc and Rc photosensory
pathways to control a largely common transcriptional network. phyB mutant seedlings retain a surprisingly high level of responsiveness to Rc for the majority of Rc- regulated genes on the microarray, indicating that one or more other phys has a major role in regulating their expression. Combined with the robust visible morphogenic phenotype of the phyB mutant in Rc, these data provide evidence that different members of the phy family act in organ- specific fashion in regulating seedling deetiolation. Specifically, phyB appears to be the dominant, if not exclusive, photoreceptor in regulating a minority population of genes involved in suppression of hypocotyl cell elongation in response to Rc light signals. By contrast, this sensory function is apparently shared by one or more other phytochromes in regulating the majority, Rc-responsive gene-set involved in other important facets of the deetiolation process in the apical region, such as cotyledon cell expansion (Tepperman et al. (2004)
Plant J. 38: 725-739) . 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. The identification and cloning of the phytochrome gene family in Arabidopsis, and homologs in such crop plants as rice and corn. The identification of functionally active promoter cis elements in the phyA genes of oat, rice and corn, and the cloning of transcription factors that interact with these elements. The overexpression of phytochrome in transgenic plants leading to modified architecture due to suppression of the shade-avoidance response. Discovery of the differential functional roles of individual phytochrome family members through creation of null mutants in the photoreceptors. Genetic and molecular identification of phytochrome signaling components and definition of phytochrome-regulated transcriptional networks. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become
available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? The information is transferred to the research community and general public through peer-reviewed publications, presentations at international meetings and at diverse universities, and in communications aimed at the lay person. On an ongoing basis, we receive and respond to large numbers of requests for clones, mutant lines and antibodies from the scientific community throughout the U.S. and internationally.
Impacts
(N/A)
Publications
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Progress 10/01/03 to 09/30/04
Outputs
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? The major problem being addressed is the need to produce more food of enhanced nutritional value in an ecofriendly manner, without increasing acreage, in order to feed the expanding world population. Toward this end we are investigating the fundamental molecular mechanisms by which plants regulate their growth and development in response to the prevailing light environment. Our overall research goal is to define the molecular mechanisms by which the phytochrome family of photoreceptors perceives, interprets and transduces informational light signals from the environment to photoresponsive genes. Our specific objectives are to identify, molecularly clone, and functionally define the biological and biochemical activities of the components involved in this process using genetic, molecular and biochemical
strategies. Many plant responses to light that have evolved to provide competitive advantage and survival under natural conditions are agronomically undesirable. These include the shade-avoidance response and photoperiodic control of such processes as flowering and leaf senescence. Identification of the molecular components and understanding of the biochemical and cellular mechanisms involved in light signaling to nuclear genes will provide potential targets for biotechnologically based improvement of crop plants. 2. List the milestones (indicators of progress) from your Project Plan. a. Identify phytochrome signaling intermediates (i) Identify phytochrome-interacting factors (PIFS) (ii) Perform forward genetic screens for signaling intermediates b. Define phytochrome-regulated transcription networks (i) Perform microarray-based expression profiling (ii) Characterize the basic helix-loop-helix (bHLH) transcription factor family 3. Milestones: A. FY 2004 a. Identify phytochrome
signaling intermediates (i) Identify phytochrome interacting factors. Using yeast two-hybrid screens and pull-down assays, we have identified three additional PIFs, designated, PIF1, PIF5 and PIF6, which are transcription factors related to the previously identified PIF3 and PIF4 factors. (ii) Perform forward genetic screens for signaling intermediates. We have identified and characterized two additional genes involved in phytochrome signaling, using both forward- and reverse-genetic screens, and have published this work in peer-reviewed journals. These two genes are designated PRR7, a pseudoresponse regulator (Kaczorowski and Quail, 2003), and ELF4, a novel gene that is rapidly induced by light (Khanna, et al., 2003). b. Define phytochrome-regulated transcriptional networks (i) Perform microarray-based expression profiling. Using Affymetrix oliognucleotide microarrays, we have defined the expression profiles induced by red light, and have identified those genes regulated by phyB,
as well as those regulated by one or more other members of the phytochrome family. This work has been recently published (Tepperman, et al., 2004). (ii) Characterize the basic helix-loop-helix (bHLH) transcription factor family. As part of our interest in determining whether members of the Arabidopsis family, related to phytochrome-interacting factor 3 (PIF3) , might be involved in phy signaling and transcriptional regulation, we undertook a comprehensive computational analysis of this family. This was necessitated because the existing preliminary analysis based on the published Arabidopsis genome sequence was incomplete and the annotation was frequently incorrect. (see publications, Toledo-Ortiz et al., 2003; Bailey et al., 2003). Our initial analysis identified 147 members of the family (Toledo-Ortiz et al., 2003). However, subsequent further analysis in collaboration with another research group, led to the identification of additional members, such that now it appears that the
complete bHLH family consists of 162 members in Arabidopsis (Bailey et al., 2003). This is the first apparently complete delineation of this gene family in a plant, and indicates that this family is the second-largest transcription factor family in Arabidopsis. We have utilized the phylogenetic relationships between the genes generated by this analysis to identify targets for reverse-genetic analysis for potential involvement in photomorphogenesis. We are currently investigating knock-out mutants in several of the family members most closely related to PIF3. B. FY 2005, 2006, 2007 a (i) We will continue to screen for phytochrome-interacting factors and assess their functional relevance to phytochrome signaling using reverse-genetic disruption of the identified loci. a (ii) We will continue to perform forward genetic screens for additional signaling intermediates in the phytochrome pathway by screening for abnormalities in the seedling deetiolation process. Genes will be isolated and
characterized and their protein products assessed for potential biochemical function. b (i) We will continue to map the phytochrome-regulated transcriptional networks by performing microarray expression profiling on wild-type and mutant seedlings. This will permit placement of the activity of the mutated locus in the network hierarchy. b (ii) We will continue a systematic assessment of the functional role of members of the bHLH family in phytochrome signaling by reverse-genetic disruption of targets related to the phytochrome-interacting members of the family. 4. What were the most significant accomplishments this past year? A. Phytochrome is the major light receptor for plants and thus understanding how it functions is crucial for crop improvement. Using a series of reverse-genetic screens, Scientists in the Quail lab at the Plant Gene Expression Center, Albany, CA, isolated mutants in Phytochrome C, the only member of the Arabidopsis phytochrome family that had not yet been
characterized at the genetic and molecular level, and succeeded in identifying several mutant alleles of phyC and analyzing the monogenic phyC mutants plus extended the work to include double and triple phy mutants. The data suggest that phyC is involved in regulating photomorphogenesis throughout the life cycle, with a photosensory specificity similar to the phyB, D and E and that it interacts with phyA and phyB in the control of multiple developmental processes. Research provides fundamental understanding of molecular mechanism to achieve enhanced crop productivity and improve efficiency. B. Other Significant Accomplishment(s), if any: NONE. C. Significant Accomplishments/Activities that Support Target Populations: NONE. D. Progress Report: This report serves to document research conducted under a Specific Cooperative Agreement between ARS and UC Berkeley. Additional details of research can be found in the report for the parent project (5335-21000- 017-00D). Necessity of nuclear
translocation of phyB for biological function. Although evidence for light-induced nuclear translocation of phy molecules is well established, direct evidence of the functional relevance of this translocation to phy signaling in vivo had been lacking. We used a glucocorticoid-receptor-based fusion system to demonstrate that nuclear translocation of phyB is indeed necessary for its biological function in the living cell, and combined with photoconversion to the active Pfr form, is sufficient for this activity. These data provide strong support for the conclusion that primary phy signaling events occur in the nucleus and, conversely suggest the absence of a cytosolic signaling pathway. (see publication, Huq et al., 2003). 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. The identification and cloning of the phytochrome gene family in Arabidopsis, and homologs in such crop plants as rice and corn. The identification of
functionally active promoter cis elements in the phyA genes of oat, rice and corn, and the cloning of transcription factors that interact with these elements. The overexpression of phytochrome in transgenic plants leading to modified architecture due to suppression of the shade-avoidance response. Discovery of the differential functional roles of individual phytochrome family members through creation of null mutants in the photoreceptors. Genetic and molecular identification of phytochrome signaling components and definition of phytochrome-regulated transcriptional networks. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? The information is transferred to the research community and general public through peer-reviewed publications, presentations at
international meetings and at diverse universities, and in communications aimed at the lay person. On an ongoing basis, we receive and respond to large numbers of requests for clones, mutant lines and antibodies from the scientific community throughout the U.S. and internationally.
Impacts
(N/A)
Publications
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