Source: UNIVERSITY OF CALIFORNIA, DAVIS submitted to
STRUCTURE FUNCTION STUDIES ON PHYTOCHROME
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
REVISED
Funding Source
Reporting Frequency
Annual
Accession No.
0084385
Grant No.
(N/A)
Project No.
CA-D-MCB-4126-H
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Oct 1, 2012
Project End Date
Sep 30, 2017
Grant Year
(N/A)
Project Director
Lagarias, J. C.
Recipient Organization
UNIVERSITY OF CALIFORNIA, DAVIS
410 MRAK HALL
DAVIS,CA 95616-8671
Performing Department
Molecular and Cellular Biology
Non Technical Summary
Our studies seek to provide insight into the origin of phytochromes in photosynthetic eukaryotes - the organisms responsible for conversion of light energy into chemical energy that sustains all animal life and fuels the machines we use daily. Over a billion years ago, a blue-green alga (cyanobacterium) was domesticated by an ancestral host cell that served as the progenitor of nearly all eukaryotic photosynthetic organisms on earth. This process required the host genome to capture algal genes needed for regulation of the photosynthetic organelle known as the chloroplast. In flowering plants, phytochromes function as such a master regulator. The presence of phytochrome-related genes in more primitive plants species indicates that the genesis of this light sensor was critical for the successful domestication of cyanobacteria. While the light-sensing region of phytochromes is clearly derived from this cyanobacterium, the origin of the regulatory domains involved in the translocation of light-activated phytochrome into the nucleus is unknown. Comparative analysis of phytochromes from extant distantly related species is expected to give us new knowledge into the novel regulatory domains harnessed by the cyanobacterial phytochrome photosensory domains and the processes that they regulate in these organisms. Such basic knowledge has already lead to development of new tools for light-dependent regulation of important cellular processes in plants, and also hold great potential for applications in agriculture, medicine and bioengineering. Novel phytochrome genes discovered in the previous grant period are being used to develop plant-derived, antibiotic-free genetic selection markers to produced new varieties of agronomically important plant species. These studies hold great potential for improving yield, flowering behavior and agronomic performance of food, fiber and energy crop plant species such as rice, alfalfa and canola. The human population is estimated to reach 10 billion before the end of this century while the amount of arable land is not expected to increase proportionately. These issues are further compounded by climate change that threatens agronomic performance of plant varieties suited to their local environment at rates faster than conventional breeding can compensate. Climate change is further exacerbated by the reliance on fossil fuels for energy and the concomitant release of greenhouse gases. The toll of rising costs of energy and increasing urbanization will elevate food prices that will fuel famine, disease and political unrest throughout the world. While there will be no easy fix for these problems, it is inevitable that agricultural biotechnology will play a key role in sustaining and increasing crop yield on the limited arable land. We therefore anticipate that basic knowledge gained by our studies and their application to agricultural improvement will play a significant role therein.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2062499100080%
2062499104020%
Goals / Objectives
Modern agriculture relies on crops that have been carefully tailored for environments by selective breeding. Phytochromes are attractive targets for crop improvement efforts aimed to enhance seed germination, seedling establishment, plant architecture and flowering time. The overall goal of this research is to understand the structural basis of both photosensory and regulatory functions of phytochromes from cyanobacteria to plants. Such basic knowledge will be leveraged for development of new strategies to alter the light responsiveness of agriculturally significant plant species. For example, our discovery of dominant photochemically uncoupled (punD) mutant alleles of phytochromes that are either constitutively active or constitutively inactive hold great promise for crop improvement. Such plant-derived reagents may overcome concerns raised by opponents of genetically modified organisms while addressing the pressing need for increased food production on a finite amount of arable land needed to nourish the growing world population. During the next five years, we will address both basic and applied questions. One major avenue of study focuses on gaining fundamental new knowledge about the diversity of the phytochrome family of protein light sensors. These investigations will address the hypothesis that the fundamental mechanism of light sensing has remained conserved for over one billion years since endosymbiotic capture of a cyanobacterium by a eukaryotic host progenitor of nearly all oxygenic photosynthetic eukaryotes on earth. The three specific aims of this project focus on the conservation of photoconversion and protein-chromophore interactions in plant and cyanobacterial phytochromes, the evolutionary genesis of plant phytochrome, and phytochrome photoconversion and signaling activity in land plants. Phytochromes from the glaucophyte Cyanophora paradoxa, the chlorophyte Micromonas pusilla, and the land plants Arabidopsis thaliana and Triticum aestivum (wheat), will be used to examine the hypothesis that a light-regulated conformational change triggers phytochrome translocation to the nucleus to alter transcription while also altering cytosolic functions in all eukaryotic phytochromes. In the second line of investigation, we plan to pursue potential translational applications of pun alleles as antibiotic-free selectable markers, as regulators of flowering time and as modulators of shade avoidance in alfalfa, wheat and representative Brassica species. Expected outcomes of this research include: 1) dissemination of research findings in scientific journals, at national and international scientific meetings and with local collaborators within the College of Agriculture and Environmental Sciences and the private sector, 2) training of the next generation of scientists who will address issues relevant to agricultural, environmental and societal issues, 3) development of novel tools, new alfalfa, wheat and Brassica sp. plant lines and technologies for crop plant improvement, and 4) contribute to the economic development, health and welfare of the general public.
Project Methods
Basic Knowledge Studies. Our research plan leverages recent published findings and technologies previously developed in my laboratory. Studies under Aim 1 address whether the photoconversion mechanism and light-triggered protein-chromophore interactions are conserved in extant phytochromes from cyanobacteria to plants. This will be done by examining the role of conserved residues within the photosensory core for formation of the signaling active Pfr state of phytochromes using both deletion and site-directed mutagenesis, and by testing the role of the bilin chromophore methine-bridges for light signaling using unnatural chromophore precursors. Aim 2 addresses the evolution of phytochromes via biochemical, spectroscopic and reverse-genetic functional analysis of phytochromes from the chlorophyte Micromonas pusilla CCMP1545 (MicroPhy) and the family of phytochromes from the glaucophyte, Cyanophora paradoxa (CyPhys, with at least five examples). Knowledge to be gained by these studies will fill in a large gap in our understanding of phytochrome structure and function following capture of a cyanobacterium by a eukaryotic host, while also laying the foundation for long-term photobiological studies of these interesting organisms with our collaborators, Drs. Alex Worden and Debashish Bhattacharya. Studies under Aim 3 address the functional significance of photoconversion and protein-chromophore interactions identified in Aim 1 by examining the cellular photobiology of wild-type and mutants of these various phytochrome species in transgenic phytochrome-deficient Arabidopsis plants. Translational Studies. We will utilize synthetic full-length, codon-optimized punD cDNAs and two truncated constructs each possessing all three or the two core photosensory domains fused with a small dimerization domain and a nuclear localization signal peptide to develop a plant-derived, antibiotic-free genetic selection marker in Medicago and Brassica species. These will first be applied to Brassica sp. that can be directly transformed in planta, e.g. Brassica napus - the third leading source for vegetable oil and second leading source of protein meal in the world. Selection will be based on the ability of punD transformants to germinate in darkness following treatment with far red light. For transformation of alfalfa, a species that presently requires selection in tissue culture followed by regeneration, we will exploit the expertise of David Tricoli of the UCD Plant Transformation Facility. We anticipate that shoots regenerating in darkness from punD-transformed callus will have a visually distinct non-etiolated phenotype, compared to chlorotic, etiolated shoots regenerating from non-transgenic callus - a result that will avoid the need for antibiotic selection. These studies also will leverage the non-dormant Highline alfalfa cultivar of Dr. Larry Teuber AE&S Plant Sciences Department that will permit evaluation of pun alleles to regulate various phytochrome-mediated responses, e.g. flowering, leaf senescence and shade avoidance, for an agronomically important forage crop species under natural field conditions.

Progress 01/01/13 to 09/30/13

Outputs
Target Audience: The major target audience at UC Davis was undergraduates. In the past year, PI Lagarias presented general research presentations to students in MCB 191 entitled "Biliprotein Photoreceptors and Light Signaling" (March 13, 2013) and MCB Club Members entitled "Research in the Lagarias Lab (May 22, 2013). These lectures were designed to educate students on the role basic research plays in solving important problems in agriculture such as crop performance and yield in a changing environment. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? The Lagarias laboratory provides a rich environment for the interdisciplinary training of graduate students, postdoctoral fellows, undergraduates and project/visiting scientists. Current personnel include one postdoctoral fellow, two project scientists, one career technician (SRA II), two graduate students and one undergraduate lab assistant. Formal collaborations include those funded by the Department of Energy and National Science Foundation, with six UCD colleagues from MCB, Chemistry and Plant Sciences Departments. The PI also participates in two NIH training grant programs and is a member of three graduate programs, Plant Biology, Chemistry, and Biochemistry, Molecular, Cellular and Developmental Biology. How have the results been disseminated to communities of interest? PI Lagarias presented invited lectures at the following meetings: International Symposium on Plant Photobiology (ISPP), Edinburgh, Scotland UK (June 3-6, 2013, entitled "Sailing in deep waters: Algal phytochromes inform our understanding of phytochrome evolution and of photoreceptor adaptation for marine environments), the 10th International Phycological Congress "Algae and a Changing World", Orlando FL (August 4-10, 2013, entitled "Sailing in deep waters: Algal phytochromes inform our understanding of phytochrome evolution and of photoreceptor adaptation for marine environments", the11th International Conference on Tetrapyrrole Photoreceptors in Photosynthetic Organisms, Huazhong Agricultural University, Wuhan, China(September 11-15, 2013, entitled "Spectral tuning in cyanobacteriochromes" and DFG Round Table Discussion "Photorezeptoren: Novel systems and new methodologies", Schloss Ringberg, Kreuth-Tegersee, Germany, (September 22-25, 2013, entitled "Spectral engineering of the phytochrome superfamily". UCLA Biochemistry Seminar Series, "Spectral Tuning in the Phytochrome Superfamily" on February 1, 2013. PI Lagarias also presented invited lectures at the following research universities or institutes: Salk Institute, "Spectral Tuning in the Phytochrome Superfamily" on February 5, 2013, Halocarbon Corporation Lecture, Cornell University Department of Chemistry, "Chemical Basis of Spectral Tuning by Phytochromes" on April 25, 2013, Rutgers Energy Institute, NSF IGERT on Sustainable Fuels Seminar Series, "Anticipating the Dawn: A Critical Role for Bilins in Plants and Algae" on May 1, 2013, Cambridge University, UK, Department of Plant Sciences, "Anticipating the Dawn: A Critical Role for Bilins in Plants and Algae" on June 6, 2013 and Technical University of Berlin, Institut für Chemie Max-Volmer-Laboratorium, "Spectral Tuning in the Phytochrome Superfamily" on September 20, 2013. PI Lagarias also presented a poster at the Western Section of the American Society of Plant Biologists Meeting, Davis CA (April 12-13, 2013, entitled "Engineering a phytochrome-based, 'chemical-free,' selection marker system"). Wei Hu, and J. Clark Lagarias, poster What do you plan to do during the next reporting period to accomplish the goals? The potential identification of a signaling pathway in plants similar to the green algal pathway discovered by my lab in the past year will open new avenues for improving the photo-tolerance of crop plant species.

Impacts
What was accomplished under these goals? There were two major accomplishments of the past year. First, we discovered a new signaling pathway in green algae that allow them to anticipate light exposure every day at dawn by triggering gene expression that helps protect against photooxidative stress that accompanies photosynthesis. This pathway enables the chloroplast to signal to the algal cell nucleus, a process critical for algal survival when too much, and possibly too little light is available for optimal photosynthesis. We believe that this pathway is present in crop plant species as well, but has been hidden by the dominant role of phytochromes, not found in these green algal species. The second accomplishment was the isolation of an Arabidopsis mutant that lacks all five phytochrome photoreceptors. Our studies show that phytochromes are not essential for survival, as long as the light environment is carefully regulated. While such mutants would not survive competition with other plants in the natural world, they represent a powerful experimental tool for us to examine whether the signaling pathway found in green algae is present in a flowering plants. The potential identification of similar signaling pathway in plants will open new avenues for improving the photo-tolerance of crop plant species.

Publications

  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Hu, W., Franklin, K.A., Sharrock, R.A., Jones, M.A., Harmer, S. L. and Lagarias, J.C. (2013) Unanticipated regulatory roles for Arabidopsis phytochromes revealed by null mutant analysis. Proc. Natl. Acad. Sci. (USA) 110, 1542-1547.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Duanmu, D., Casero, D., Dent, R.M., Gallaher, S., Yang, W., Rockwell, N.C., Martin, S.S., Pellegrini, M., Niyogi, K.K., Merchant, S.S., Grossman, A.R. and Lagarias, J.C. (2013) Retrograde bilin signaling enables Chlamydomonas greening and phototrophic survival. Proc. Natl. Acad. Sci. (USA) 110, 3621-3626.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Hirose, Y., Rockwell, N.C., Martin, S.S., Narikawa, R., Inomata, K., Lagarias, J.C. and Ikeuchi, M. (2013) Green/red cyanobacteriochromes regulate complementary chromatic acclimation via a protochromic photocycle, Proc. Natl. Acad. Sci. (USA) 110, 4974-4979.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Kabasakal, B., Gae, D.D., Li, J., Lagarias, J.C., Koehl, P. and Fisher, A.J. (2013) Structure and dynamic analysis of PcyA variants of a conserved histidine residue. Archives of Biochemistry and Biophysics 537, 233-242.


Progress 01/01/12 to 12/31/12

Outputs
OUTPUTS: The major outputs of this work reflect our research on plant phytochromes and their distant cousins from cyanobacteria, the cyanobacteriochromes (CBCRs). We have successfully constructed and characterized a mutant in Arabidopsis thaliana that lacks all five phytochromes. By reintroduction of individual phytochromes, we are poised to understand the biological function and regulatory interplay among phytochrome species for the first time. Notable among these goals is the identification of the elusive functions of phyC for which conflicting data is known. In a second line of research, we have made great progress in our understanding of the extraordinary spectral diversity within the CBCR family. Third, our studies with phytochromes from lower eukaryote photosynthetic species lend strong support to the conclusion that phytochrome was present in the first eukaryote progenitor of all extant photosynthetic species. In addition to publications acknowledged below, PI Lagarias presented invited contributions at two Gordon Research Conferences, Photosensory Receptors & Signal Transduction, Galveston TX, 2012 January 22-27 and Chemistry and Biology of Tetrapyrroles, Salve Regina University, Newport RI, 2012 July 22-27, 10th International Congress on Plant Molecular Biology, Jeju, Korea October 21-26, 2012, and 10th International Conference on the Frontiers of Plant Biology: Development and Environment, Huangshan Anhui, China 2012 November 4-8, 2012. PI Lagarias also sent a representative to Plant Biology 2012. American Society of Plant Biology Annual Meeting, Austin, TX 2012 July 20-24. PI Lagarias was a panel member, NSF Structural Biochemistry Program, September 5-7, 2012. PARTICIPANTS: The PI's laboratory continues to provide a rich environment for the interdisciplinary training of graduate students, postdoctoral fellows, undergraduates and visiting scientists. Current personnel include one postdoctoral fellow, two Project Scientists, a career technician (SRA II) and one graduate student who are funded by three extramural grants and a fellowship. Formal collaborations include those funded by the Department of Energy and National Science Foundation, with six UCD colleagues from MCB, Chemistry and Plant Sciences. The PI also participates in three training grant programs (NIH and NSF) and is a member of three graduate programs, Plant Biology, Chemistry, and Biochemistry, Molecular, Cellular and Developmental Biology. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Research progress during the past year has heavily focused on the structural basis of the photochemical mechanism of phytochromes and CBCRs. We have completed the molecular description of the four major classes of CBCRs, which include those with violet/blue, violet/orange, blue/teal, blue/green, green/red and red/green photocycles (Kim et al, 2012a-d; Rockwell et al, 2012ab; Freer et al, 2012). This remarkable diversity arises from a single conserved, ultrafast primary photochemical process, i.e. 15Z-to-15E double bond isomerization, followed by proton release/uptake, thioether bond cleavage/formation or conformation twisting in/out of the plane of conjugation. We have begun to leverage these discoveries by introducing mutations into plant phytochromes to change their wavelength selectivity and test their effect on plant performance. Our recently isolated phytochrome null line (Hu et al, 2013) has proven exceedingly useful to test the biological consequences of these mutations on plant growth and development. The knowledge gained by these studies also provides the rationale for development of optogenetic light switches for regulation of gene expression of great interest to synthetic biological applications, eg. for potential biofuel, bioplastic and/or drug production in plant/algal/cyanobacterial hosts.

Publications

  • Kim, P.W., Freer, L.H. Rockwell, N.C., Martin, S.S., Lagarias, J.C. and Larsen, D.S. (2012a) Second-Chance Forward Isomerization Dynamics of the Red/Green Cyanobacteriochrome NpR6012g4 from Nostoc punctiforme. J. Am. Chem. Soc. 134, 130-133.
  • Kim, P.W., Freer, L.H. Rockwell, N.C., Martin, S.S., Lagarias, J.C. and Larsen, D.S. (2012b) Femtosecond Photodynamics of the Red/Green Cyanobacteriochrome NpR6012g4 from Nostoc punctiforme. 1. Forward Dynamics. Biochemistry 51, 608-618.
  • Kim, P.W., Freer, L.H. Rockwell, N.C., Martin, S.S., Lagarias, J.C. and Larsen, D.S. (2012c) Femtosecond Photodynamics of the Red/Green Absorbing NpR6012g4 Photoswitching Cyanobacteriochrome Domain from Nostoc punctiforme: 2. Reverse Dynamics. Biochemistry 51, 619-630.
  • Rockwell, N.C., Martin, S.S., Gulevich, A.G. and Lagarias, J.C. (2012a) Phycoviolobilin formation and spectral tuning in DXCF cyanobacteriochromes, Biochemistry, 51, 1449-1463.
  • Rockwell, N.C., Martin, S.S., and Lagarias, J.C. (2012b) Mechanistic Insight into the Photosensory Versatility of DXCF Cyanobacteriochromes, Biochemistry 51, 3576-3585.
  • Freer, L.H., Kim, P.W., Corley, S.C., Rockwell, N.C., Zhao, L., Thibert, A.J., Lagarias, J.C. and Larsen, D.S. (2012) Chemical Inhomogeneity in the Ultrafast Dynamics of the DXCF Cyanobacteriochrome Tlr0924, Journal of Physical Chemistry 116, 10571-81.
  • Park, E., Park, J., Kim, J., Nagatani, A., Lagarias, J.C. and Choi, G (2012) Phytochrome inhibits binding of Phytochrome-Interacting Factors to their target promoters. Plant Journal 72, 537-546.
  • Kim, P.W., Pan, J., Rockwell, N.C., Chang, C-W., Taylor, K.C., Lagarias, J.C. and Larsen, D.S. (2012d) Ultrafast E to Z Photoisomerization Dynamics of Phytochrome Cph1, Chemical Physics Letters 549, 86-92
  • Hu, W., Franklin, K.A., Sharrock, R.A., Jones, M.A., Harmer, S. L. and Lagarias, J.C. (2013) Unanticipated regulatory roles for Arabidopsis phytochromes revealed by null mutant analysis. Proc. Natl. Acad. Sci. (USA), in press doi: 10.1073/pnas.1221738110


Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: The major outputs of this work arise from exciting work on representatives of the phytochrome-related cyanobacteriochrome (CBCR) family of bilin photosensors from cyanobacteria that extend the wavelength sensing range of phytochromes into the near ultraviolet. These studies continue to surprise us with new photocycles and light sensing diversity by this family. In addition to publications acknowledged below, Principal Investigator Lagarias presented a Plenary Lectures at the 10th International Conference on Tetrapyrrole Photoreceptors in Photosynthetic Organisms, Berlin Germany, July 24-31, 2011 and sent representatives from the laboratory to the 22nd International Conference on Arabidopsis Research, Madison, WI, June 23-25, 2011 and to the DOE BES 2011 Photosynthetic Systems Research Meeting, Baltimore, MD, November 6-9, 2011 to present a poster and an invited lecture. Principal investigator Lagarias also was a participant on an NSF Committee of Visitors in 2011 to evaluate the NSF's Division of Molecular & Cellular Biosciences for the past five years. PARTICIPANTS: The PI's laboratory continues to provide a rich environment for the interdisciplinary training of graduate students, postdoctoral fellows, undergraduates and visiting scientists. Current personnel include two postdoctoral fellows, one Project Scientist, a career technician (SRA II) and one graduate student who are funded by three extramural grants and a fellowship. Formal collaborations include those funded by the Department of Energy (see above) and National Science Foundation, with five UCD colleagues from MCB, Chemistry and UCDMC. The PI also participates in three training grant programs and is a member of three graduate programs, Plant Biology, Chemistry, and Biochemistry, Molecular, Cellular and Developmental Biology. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Research progress during the past year has continued to focus on the photochemical mechanism of phytochromes. In 2009 with funding from the DOE, we initiated research to characterize the structure and function of the complete phytochrome/CBCR family in the cyanobacterium Nostoc punctiforme along with four other UCD investigators, Dr. Jack Meeks from Microbiology, Thomas Huser from CBST and Drs. Dave Britt and Delmar Larsen from Chemistry. Our studies have so far established that this cyanobacterial species harbors 22 phytochrome-related photosensors that possess 42 bilin-binding GAF domains. During the past years and a half, our laboratory have cloned, expressed and purified all 42 of these bilin-bound GAFs - studies that reveal that the expansion of the phytochrome superfamily in cyanobacteria reflects the unique light sensing specificity of each GAF domain. One major published study documents that two-cysteine photocycles, which are responsible for near UV/blue sensing, have independently arisen at least three times via insertion of a second cysteine at different positions within the GAF domain (Rockwell et al, 2011). In a collaborative investigation with Dr. Youn-il Park from Chungnam University, Korea, we showed that one of these two cysteine CBCRs functions as a near ultraviolet light sensor that regulates negative phototaxis in the cyanobacterial species Synechocystis sp. PCC 6803 (Song et al, 2011). These studies provided a molecular understanding of the light-regulated two component signaling system that controls expression of an integrator of high light, oxidative and chemical stress in Synechocystis. The knowledge gained by these studies now provides a springboard to develop optogenetic light switches for regulation of gene expression of great interest to synthetic biological applications, eg. for potential biofuel, bioplastic and/or drug production in plant/algal/cyanobacterial hosts.

Publications

  • Song, J-Y., Cho, H.S., Cho, J-I., Jeon, J.S., Lagarias, J.C. and Park, Y-I. (2011) A Near Ultraviolet Cyanobacteriochrome Signaling System Elicits Negative Phototaxis in the Cyanobacterium Synechocystis sp. PCC 6803 Proc. Natl. Acad. Sci. (USA) 108, 10780-10785.
  • Rockwell, N.C, Martin, S.S., Feoktistova, K. and Lagarias, J.C. (2011) Diverse two-cysteine photocycles in phytochromes and cyanobacteriochromes Proc. Natl. Acad. Sci. (USA) 108, 11854-11859.


Progress 01/01/10 to 12/31/10

Outputs
OUTPUTS: The major outputs of this work arise from ongoing research to leverage constitutively active mutant alleles of phytochrome to regulate plant development and to understand the basic mechanism of light signaling by phytochromes. In addition to publications acknowledged below, Principal Investigator Lagarias presented three Plenary Lectures at the Biochemistry and Molecular Biology Department, Pennsylvania State University, University Park, PA "One photochemistry to rule them all: Spectral diversity of the phytochrome family of bilin-GAF photosensors" (Pollard Memorial Lecture), at the Gordon Research Conference on the Chemistry and Biology of Tetrapyrroles, Salve Regina University, Newport RI, entitled "Lord of the Broken Rings: From Near UV to Near IR And Back Again." and at the Annual Meeting of the Korean Society for Molecular and Cellular Biology, COEX Center, Seoul, Korea entitled "Molecular Mechanisms of Light Perception by Phytochromes". Lectures entitled "Molecular Mechanisms of Light Perception by Phytochromes: Exploiting Constitutive Alleles to Regulate Plant Architecture" were contributed by Principal Investigator Lagarias at Pohang University (Pohang, Korea), Jeju National University (Jeju, Korea), and KAIST (Daejeon, Korea). Principal investigator Lagarias also chaired an NSF Committee of Visitors to evaluate the NSF's Plant Genome Program for the past five years. PARTICIPANTS: The PI's laboratory continues to provide a rich environment for the interdisciplinary training of graduate students, postdoctoral fellows, undergraduates and visiting scientists. The PI participates in three training grant programs and is a member of three graduate programs, Plant Biology, Biochemistry Molecular Cellular and Developmental Biology and Chemistry. This past year, our laboratory again hosted a visiting student from the Nara Institute of Science and Technology for two months. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Research progress during the past year has continued to focus on the photochemical mechanism of phytochromes. Using comparative biochemical and spectroscopic analysis of plant, cyanobacterial and bacterial phytochromes, our previous studies reveal that the primary pathway of photosensing (i.e. 15Z to 15E isomerization) has been preserved throughout evolution of this photoreceptor family. The research in the field has exploded in the past few years, owing to the growing studies on the widely distributed cyanobacteriochrome (CBCR) family - all of which share the same primary photochemistry as phytochromes (see Rockwell & Lagarias, 2010). Ongoing research has shown that the spectral diversity of CBCRs which extends the wavelength range sensed by phytochromes from the near ultraviolet through the near infrared arises from introduction of cysteine residues at critical positions within the bilin-binding GAF scaffold (Rockwell et al, to be submitted). However, research with chromophore analogs published during the past year supports the hypothesis of our earlier study that the direction of light-driven rotation of the chromophore D-ring differs amongst key classes of phytochromes (Shang et al, 2010). These studies suggest that the output signaling cascades will also differ amongst the extended phytochrome photosensor family. Research addressing the mechanism of plant phytochrome signaling exploits novel gain-of-function mutants of phytochrome previously discovered by our laboratory. We have demonstrated that these gain-of-function responses can be seen in important crop plant species - both dicots (tomato, alfalfa) and monocots (rice). These studies implicate many potential agricultural applications, with particular emphasis on improving seed germination, mitigating yield losses from shade avoidance responses and altering flowering time in agriculturally important plant species. In addition, a US Patent on these novel alleles issued in September underscores the potential applications of phytochromes as in vivo fluorescent probes of cellular function (Lagarias & Fischer, 2010).

Publications

  • Rockwell, N.C. and Lagarias, J.C. (2010) A brief history of phytochromes. ChemPhysChem 11, 1172-1180.
  • Shang, L., Rockwell, N.C., Martin S.S. and Lagarias, J.C. (2010) Biliverdin amides reveal roles for propionate side chains in bilin reductase recognition and in holophytochrome assembly and photoconversion. Biochemistry 49, 6070-6082.
  • John Clark Lagarias, Amanda J. Fischer, A Red and Near Infrared Fluorescent Phytochrome. US Patent No. 7,795,397, Issued on September 14, 2010.


Progress 01/01/09 to 12/31/09

Outputs
OUTPUTS: The major outputs of this work arise from ongoing research to leverage constitutively active mutant alleles of phytochrome to regulate plant development and to understand the basic mechanism of light signaling by phytochromes. In addition to publications acknowledged below and one newly accepted patent entitled "A Red and Near Infrared Fluorescent Phytochrome", we have presented public lectures on our studies at the Plant Molecular Biology Consortium and the North Carolina Biotechnology Center (Durham, NC), at Plant Biology 2009. American Society of Plant Biology Annual Meeting (Honolulu, HI), at the 9th International Conference on Tetrapyrrole Photoreceptors in Photosynthetic Organisms, and at the International Symposium of the Collaborative Research Center Sfb-498 Protein-Cofactor Interactions in Biological Processes (Berlin, Germany). We are also cultivating a relationship with Arcadia Biosciences to pursue collaborative research arising from participation in a joint ARPA-E proposal with plant biology colleagues and we continue to work with PIPRA (and CalWest Seeds) to exploit constitutive phytochrome alleles for agronomic benefits particularly in transgenic alfalfa. PARTICIPANTS: AE&S support has been leveraged to secure support from the Department of Energy Basic Energy Biosciences Division. This support enabled the PI to fund stipends for a postdoctoral fellow and technical assistants (one staff research associate). The PI's laboratory provides a rich environment for the interdisciplinary training of graduate students, postdoctoral fellows and visiting scientists. The PI participates in three training grant programs and is a member of three graduate programs, Plant Biology, Biochemistry Molecular Cellular and Developmental Biology and Chemistry. Last year, our laboratory hosted a visiting student from the Nara Institute of Science and Technology for two months. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Research progress during the past year has primarily focused on the photochemical mechanism of phytochromes. Using comparative biochemical and spectroscopic analysis of plant, cyanobacterial and bacterial phytochromes, our studies have revealed that the primary pathway of photosensing has strikingly diverged throughout evolution of this photoreceptor family. Specifically, we have shown that two classes of photochemistries have evolved - one leading to the cyanobacterial/plant phytobilin-type phytochromes and the other to the bacterial biliverdin-type phytochromes. Our studies shown that the two pathways correspond to different "rotation" directions of double bond isomerization (Rockwell et al, 2009). This not only leads to a profound difference in the structure of the activated Pfr form for the two classes of phytochrome, but likely to differences in the signaling output mechanisms. In collaborative studies with the Mathies lab at UC Berkeley, spectroscopic measurements of the cyanobacterial phytochrome 1 (Cph1) reveal that double bond isomerization occurs within the excited state (DasGupta et al, 2009) and that the bifurcated excited state decays arises from a homogeneous ground state population (Spillane et al, 2009). Research addressing the mechanism of plant phytochrome signaling that exploit novel gain-of-function mutants of phytochrome previously discovered by our laboratory is ongoing (although unpublished). A screen for mutants that suppress the gain-of-function activity of our mutant phytochrome alleles has been undertaken and has identified both intragenic and extragenic mutations that are presently under study. The discovery that the phytochrome chromophore is required for the gain-of-function activity also has lead to the development of a chemical inducible system to regulate phytochrome activity. This will enable us to study rapid protein-mediated and slower transcriptional-dependent pathways with or without the activation of other light-dependent processes (eg. photosynthesis and other light sensory systems). Our studies also impact potential agricultural applications, with particular emphasis on improving seed germination, mitigating yield losses from shade avoidance responses and altering flowering time in agriculturally important plant species. Ongoing experiments in rice and tobacco species are underway.

Publications

  • Rockwell, N.C, Shang, L., Martin, S.S. and Lagarias, J.C. (2009) J.C. Distinct classes of red/far-red photochemistry within the phytochrome superfamily. Proc. Natl. Acad. Sci. (USA) 106, 6123-6127.
  • Dasgupta, J., Frontiera, R.R., Taylor, K.C., Lagarias, J.C. and Mathies, R. A. (2009) Ultrafast Excited state isomerization in phytochrome revealed by femtosecond stimulated Raman spectroscopy. Proc. Natl. Acad. Sci. (USA) 106, 1784-1789
  • Spillane, K.M., Dasgupta, J., Lagarias, J.C. and Richard A. Mathies (2009) Homogeneity of phytochrome Cph1 vibronic absorption revealed by resonance Raman intensity analysis. J. Am. Chem. Soc. 131, 13946-13948.


Progress 01/01/08 to 12/31/08

Outputs
OUTPUTS: The major output of this work relates to the discovery and applications of a novel phytochrome gene that can be used to regulate light-mediated growth and development. In addition to publication and pending patents on these findings, we have publically reported on this work at national/international conferences (GRC Photosensory Receptors & Signal Transduction, !st International Congress of Biophotonics, 25th Annual Plant Biochemistry Symposium at University of Missouri, 34th Meeting of the American Society of Photobiology, GRC Chemistry and Biology of Tetrapyrroles and the 236th Annual ACS Meeting), at college/university/research institutes (University of Texas, Austin, University of Chicago, Salk Institute) and at private industry (Arcadia Biosciences, Pioneer). We are also cultivating a relationship with Arcadia Biosciences to pursue development of a research agreement and PIPRA plans to market/license this technology for agricultural applications in the third world. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Research during the past year has mainly focused on Objectives 1 and 3. Objective 1 addresses the photochemical mechanism of phytochromes. Using comparative biochemical and spectroscopic analysis of plant, cyanobacterial and bacterial phytochromes, our studies under Objective 1 have revealed that the primary pathway of photosensing has unexpectedly diverged throughout evolution of this photoreceptor family. These studies thus impact our fundamental understanding of evolutionary adaptations developed by photosynthetic organisms to adjust to their rapidly changing light environment while also providing insight for new approaches to improve photosynthetic efficiency. Our studies clearly indicate that rampant molecular evolution of the phytochrome family in cyanobacteria has yielded new families of photosensors that perceive light throughout the entire visible light spectrum. One such cyanobacteriochrome from a thermophilic cyanobacterium was studied in detail during the past year (Rockwell et al 2008). The major take home message of these studies is that the wavelength sensitivity can be tuned by new residues that have by introduced into the phytochrome protein during evolution. Despite this novel spectral sensitivity, the structural basis of the light sensing has been preserved in these blue-green sensing molecules. Objective 3 addresses the mechanism of plant phytochrome signaling by focusing on a novel gain-of-function mutation. Using molecular genetic, genomic and cell biological approaches, we have further documented that these mutants show light-independent signaling at the global level (Hu et al, 2009). Our studies thus impact potential agricultural applications, with particular emphasis on improving seed germination, mitigating yield losses from shade avoidance responses and altering flowering time in agriculturally important plant species. Ongoing experiments in rice and tobacco species are underway.

Publications

  • Rockwell, N.C., Njuguna, S.L., Dwojak, S., Castillo, E., Roberts, L., Parson, V.L, Lagarias, J.C, and Spiller, S.S. (2008) A second conserved GAF domain cysteine is required for the blue/green photoreversibility of cyanobacteriochrome Tlr0924 from Thermosynechococcus elongatus. Biochemistry, 47, 7304-7316.Enter publications in this block.
  • Hu, W., Su, Y-S. and Lagarias, J.C. (2009) A light-independent allele of phytochrome B faithfully recapitulates photomorphogenic transcriptional networks. Molecular Plant, 2, 166-182; doi:10.1093/mp/ssn086


Progress 01/01/07 to 12/31/07

Outputs
We have developed a new software tool for mapping homology onto protein structure, aiding in interpretation of structure and homology modeling projects (Rockwell and Lagarias, 2007). This tool is broadly applicable to any protein family for which a 3D structure of one member has been solved. We have used this new tool to predict and assess the functional significance of conserved residues within the chromophore binding pocket of phytochromes. The research published in Plant Cell (Su & Lagarias, 2007) represents one of the most significant discoveries of my career. These studies document the discovery of a new family of constitutively active mutant alleles of plant phytochromes, that encode a single tyrosine-to-histidine (YH) mutation within the pigment binding pocket of phytochromes. Dominant, gain-of-function alleles of phytochromes have long been sought, but have proven elusive targets of genetic screens by many labs. Transgenic plants expressing YH phytochrome alleles develop in darkness -as if they were grown in light- and also exhibit light-independent photomorphogenesis. The isolation of constitutively active alleles of phytochromes represents a significant breakthrough for many reasons. First, their 'signaling-on' activity of YH mutants should permit biochemical and biophysical characterization of a stable 'photoactivated' phytochrome. Second, our observation that bilin biosynthesis is required for YHB activation can be used to initiate signal transduction, permitting both visualization of nuclear translocation by fluorescence microscopy and elucidation of the transcriptional networks initiated by phytochromes without activation of other photoreceptor systems. Third, suppressor mutant screens, now in progress, are also expected to identify new phytochrome-specific signaling pathway components. Since YH plants show reduced shade avoidance, enhanced seed germination, and other light-insensitive phenotypes of agronomic significance, we ultimately envisage a bright future for applications of this unexpected discovery to tailor the development of food, fiber and energy crops to particular light environments.

Impacts
The ability to selectively modify phytochrome function in agronomically important crop plants has profound implications for agriculture, and is therefore particularly relevant to "the mission of the UC Davis Agricultural Experiment Station". For example, it is desirable to alter the phytochrome-mediated shade avoidance response of crop plants whereby growth is adversely affected by neighboring vegetation. This could enable us to increase the density of crop plants in the field without yield losses associated with shade avoidance. Compounds that alter the synthesis of phytochrome's linear tetrapyrrole (bilin) chromophore could improve the germination of crop plant species, prevent the germination of undesireable weed species and/or modify the length of time needed to flower.

Publications

  • Rockwell, N. C., and Lagarias, J.C. 2007 HOMOLMAPPER: Visualizing homology on a known protein structure. BMC Bioinformatics, 8:123, doi:10.1186-1471-2105-8-123
  • Su, Y-S. and Lagarias, J.C. 2007 Light independent phytochrome signaling mediated by dominant GAF-domain tyrosine mutants of Arabidopsis phytochromes in transgenic plants. Plant Cell, USA 19, 2124-2139


Progress 01/01/06 to 12/31/06

Outputs
A crystal structure of the BV-bound PAS-GAF domain of the bacteriophytochrome DrBphP from the bacterium Deinococcus radiodurans was recently reported. We were invited to contribute a perspective on this significant contribution to the field of phytochrome signaling for The Plant Cell (Rockwell & Lagarias, 2006). In a more comprehensive review published in the Annual Review of Plant Biology, we presented a new 'counterion switch' model for signal initiation by phytochromes that forms the basis of ongoing experiments (Rockwell et al, 2006). In collaboration with Thomas Huser and colleagues, single molecule diffusion measurements using the technique of fluorescence correlation spectroscopy (FCS) showed that the red-emitting phytofluor, i.e. the YH mutant of cyanobacterial phytochrome Cph1, has excellent fluorescence properties at the single molecule level. These studies not only presage the utility of phytofluor tags for probing single molecule dynamics in living cells), but also establish the feasibility of single molecule analyses of phytochrome biochemistry in living cells.

Impacts
The ability to genetically label other proteins tagged with phytofluors will impact the fields of functional genomics and cell biology. Since these mutations affect the primary mechanism of light activation, it is conceivable that the YH mutant alleles of plant phytochromes will be locked into 'active' and 'inactive' states which should have novel biological activity in plants. Expression of hyperactive phytochromes in agronomically important crop species should effectively counteract shade avoidance responses that result in yield losses from high-density plantings.

Publications

  • Miller, A.E., Fischer, A.J., Laurence, T., Hollars, C., Saykally, R., Lagarias, J.C. and Huser,T. (2006) Single molecule dynamics of phytochrome-bound fluorophores probed by fluorescence correlation spectroscopy. Proc. Natl. Acad. Sci (USA) 103, 11136-11141.
  • Rockwell, N. C., and Lagarias, J.C. (2006) The structure of phytochrome. A picture is worth a thousand spectra. Plant Cell 18, 4-14.
  • Rockwell, N. C., Su, Y-S., and Lagarias, J.C. (2006) Phytochrome structure and signaling mechanisms, Annual Review of Plant Biology 57, 837-858.


Progress 01/01/05 to 12/31/05

Outputs
We previously showed that the efficiency of this photochemical process is profoundly altered by mutation of a conserved tyrosine residue (Tyr176) within the bilin binding GAF domain of the cyanobacterial phytochrome Cph1. In the present study, we show that the equivalent mutation in plant phytochromes behaves similarly, indicating that the function of this tyrosine in the primary photochemical mechanism is conserved (FISCHER et al, 2005). Saturation mutagenesis of Tyr176 in Cph1 established that no other residue can support comparably efficient photoisomerization. The spectroscopic consequences of Tyr176 mutations also reveal that Tyr176 regulates the conversion of the porphyrin-like conformation of the bilin precursor to a more extended conformation. The porphyrin binding ability of the Tyr176Arg mutant protein indicates that Tyr176 also regulates the ligand binding specificity of apophytochrome. Based on the H-bonding ability of Tyr176 substitutions that support the non-photochemical C15-Z,syn to C15-Z,anti interconversion, we propose that Tyr176 orients the carboxyl side chain of a conserved acidic residue to stabilize protonation of the bilin chromophore. A homology model of the GAF domain of Cph1 predicts a C5-Z,syn, C10-Z,syn, C15-Z,anti configuration for the chromophore and implicates Glu189 as the proposed acidic residue stabilizing the extended conformation - an interpretation consistent with site-directed mutagenesis of this conserved acidic residue.

Impacts
The identification of a conserved residue in phytochrome that directly participates in its primary photochemistry led to the production of an intensely red fluorescent biliprotein or phytofluor. The ability to genetically label other proteins with phytofluors will impact the fields of functional genomics and cell biology. Since these mutations affect the primary mechanism of light activation, it is conceivabe that the Tyr mutant alleles of plant phytochromes will be locked into 'active' and 'inactive' states which should have novel biological activity in plants. Expression of hyperactive phytochromes in agronomically important crop species should effectively counteract shade avoidance responses that result in yield losses from high-density plantings.

Publications

  • FISCHER, A.J., ROCKWELL, N.C., YAP, A., ERNST, L., WAGGONER, A., DUAN, Y., LEI, H-X. AND LAGARIAS, J.C. (2005) Multiple Roles of a Conserved GAF Domain Tyrosine Residue in Cyanobacterial and Plant Phytochromes, Biochemistry 44, 15203-15215.


Progress 01/01/04 to 12/31/04

Outputs
Utilizing the technique of directed evolution, we identified a tyrosine residue in a cyanobacterial phytochrome that is essential for the primary step of phytochrome signaling, i.e. Z-to-E photoisomerization of its bilin chromophore (FISCHER & LAGARIAS, 2004). Mutagenesis of this tyrosine residue to histidine strongly inhibits the Pr to Pfr photoconversion, greatly enhancing the fluorescence emission yield. We propose a photochemical gating mechanism involving movement of this tyrosine which enables the electronically excited bilin chromophore to photoisomerize. Since this tyrosine residue is conserved is all phytochromes, we hypothesize that a similar mechanism occurs for plant phytochromes. Using an optical based screen, mutations that alter the absorption spectrum of phytochrome were also localized to a small region of the protein adjacent to the chromophore (FISCHER et al, 2004; FISCHER & LAGARIAS, 2004).

Impacts
The overall objective of this project is to provide information regarding both phytochrome structure and the primary signal transduction processes initiated by its photoactivation. The identification of a conserved residue in phytochrome that directly participates in its primary photochemistry led to the production of an intensely red fluorescent biliprotein or phytofluor. The ability to genetically label other proteins with phytofluors will impact the fields of functional genomic s and cell biology. Identification of the primary molecular mechanism of phytochrome signaling should help guide the design altered phytochrome genes that can be used to improve the productivity of agronomically important crop plants in their natural light environment.

Publications

  • FISCHER, A.J., COLEMAN, W.J., YANG, M.M., AND LAGARIAS, J.C. (2004) Engineering Phytochromes: Biliproteins that switch and glow. SPIE Proceedings Vol 5329, 33-43.
  • FISCHER, A.J. AND LAGARIAS, J.C. (2004) Harnessing Phytochrome's Glowing Potential. Proc. Natl. Acad. Sci (USA), published 17 November 2004, 10.1073/pnas.0407645101.


Progress 01/01/03 to 12/31/03

Outputs
The widespread distribution of phytochrome-related genes in nature has stimulated collaborative investigations to study the structure and function of some of these interesting molecules. The rcaE locus from the cyanobacterium Fremyella diplosiphon encodes a phytochrome-related protein that has been implicated to be a light sensor which enables these organisms to modify their photosynthetic apparatus to optimize light harvesting. In a collaborative study (TERAUCHI et al, in press), we present compelling genetic and biochemical evidence that RcaE is a major photoreceptor involved in this process. These studies provide the impetus for comparative biochemical studies with plant phytochromes to elucidate those features which are shared and which differ between the two photoreceptor families. Such basic knowledge not only should provide new avenues for regulation of light responsiveness in plants, but also growth and devlopment of cyanobacteria, which are both beneficial for their role in nitrogen fixation and noxious due to their production of toxic alkaloids.

Impacts
The overall objective of this project is to provide information regarding both phytochrome structure and the primary signal transduction processes initiated by its photoactivation. Through reconstruction of phytochrome signaling in yeast and bacteria, the power of molecular genetics can be brought to bear on this important plant signaling system. Identification of the primary molecular mechanism of phytochrome signaling should help guide the design of novel approaches to improve the productivity of agronomically important crop plants in their natural light environment.

Publications

  • TERAUCHI, K., MONTGOMERY, B.L., GROSSMAN, A.R., LAGARIAS, J.C., and KEHOE, D.M. 2003. RcaE is a complementary chromatic adaptation photoreceptor required for green and red light responsiveness. Molecular Microbiology. In Press.


Progress 01/01/02 to 12/31/02

Outputs
The widespread distribution of phytochrome-related genes in nature has led us to develop new hypotheses for the ancestry and function of this rapidly expanding class of signaling molecules (MONTGOMERY and LAGARIAS). Ongoing work has also sought to develop a yeast cell system for reconstitution of photoactive holophytochromes. Engineering yeast cells to produce bilin pigments has proven more difficult than that devised for bacteria in the previous grant period. Presently, we are exploring chimeric genes between heme oxygenases, bilin reductases and cytochrome P450 reductases to produce an enzymatically active fusion protein that can function within yeast cells. In a collaborative study with Dr. Joseph Lakowicz's laboratory, the two-photon photophysical properties of a Cph1-derived phytofluor was described (GRYCZYNSKI et al).

Impacts
The overall objective of this project is to provide information regarding both phytochrome structure and the primary signal transduction processes initiated by its photoactivation. Through reconstruction of phytochrome signaling in yeast and bacteria, the power of molecular genetics can be brought to bear on this important plant signaling system. Identification of the primary molecular mechanism of phytochrome signaling should help guide the design of novel approaches to improve the productivity of agronomically important crop plants in their natural light environment.

Publications

  • MONTGOMERY, B.L. and LAGARIAS, J.C. (2002) Phytochrome ancestry: Sensors of bilins and light. Trends in Plant Science 7, 357-366.
  • GRYCZYNSKI, I., G. PISZCZEK, G., J. LAKOWICZ and J.C. LAGARIAS (2002) Two photon excitation of a phytofluor protein. Photochemistry and Photobiology, in press.


Progress 01/01/01 to 12/31/01

Outputs
The bilin prosthetic groups of the phytochrome photoreceptors and the light-harvesting phycobiliprotein antennae arise from the oxygen-dependent ring opening of heme. Two ferredoxin-dependent enzymes contribute to this conversion, a heme oxygenase and a bilin reductase with discrete double bond specificity. Using a dual plasmid system - one expressing a truncated cyanobacterial apophytochrome 1 and the other expressing a two gene operon consisting of a heme oxygenase and a bilin reductase, these studies establish the feasibility to produce photoactive phytochromes in any heme-containing cell. Heterologous expression systems for phytochromes will not only facilitate genetic analysis of their assembly, spectrophotometric activity and biological function, but might also afford the means to regulate gene expression by light in non-plant cells.

Impacts
The overall objective of this project is to provide information regarding both phytochrome structure and the primary signal transduction processes initiated by its photoactivation. Through purification and molecular characterization of phytochromes from evolutionarily diverged organisms, structural features which may confer common and distinct functions can be determined. Identification of targets of phytochrome action should enable us to design novel approaches to improve the productivity of agronomically important crop plants.

Publications

  • GAMBETTA, G.A. and J.C. LAGARIAS (2001) Genetic engineering of phytochrome biosynthesis in bacteria. Proc. Natl. Acad. Sci (USA) 98, 10566-10571.


Progress 01/01/00 to 12/31/00

Outputs
Through pattern searches of genomic databases, new members of the growing family of phytochrome-related genes were identified. The deduced protein products of these genes were used to construct a small amino acid motif that delimits the bilin lyase domain, a subdomain of the extended phytochrome family that is sufficient for covalent attachment of linear tetrapyrroles (bilins). Spectroscopic analysis of bilin-adducts of a 24 kDa fragment of a new phytochrome, Cph2, definitively established that the motif identified by pattern searches represents a bonafide bilin lyase domain. Site-directed mutagenesis of highly conserved charged residues within bilin lyase domains of nearly all members of the extended phytochrome superfamily has identified a glutamate residue critical for bilin binding. Other conserved residues in the bilin lyase domains have no apparent role in bilin binding however may be essential for transduction of the signal generated by (bilin) ligand binding and/or light activation of new members of the phytochrome superfamily.

Impacts
The overall objective of this project is to provide information regarding both phytochrome structure and the primary signal transduction processes initiated by its photoactivation. Through purification and molecular characterization of phytochromes from evolutionarily diverged organisms, structural features which may confer common and distinct functions can be determined. Identification of targets of phytochrome action sahould enable us to design novel approaches to improve the productivity of agronomically important crop plants.

Publications

  • Wu, S-H. and Lagarias, J.C. (2000) Defining the bilin lyase domain: Lessons from the extended phytochrome superfamily, Biochemistry 39, 13487-13495.


Progress 01/01/99 to 12/31/99

Outputs
Research progress during the past year has primarily involved two collaborative projects. In a collaboration with Joanne Chory's lab (FANKHAUSER ET AL, 1999), we documented that the phytochrome-interacting protein PKS1 was a good substrate for phytochrome's kinase activity, thus provoiding a link between protein phosphorylation and the mode of phytochrome action. To more clearly elucidate the chromophore structures in Pr and Pfr, Raman spectra of full-length 124-kDa recombinant oat phytochromes reconstituted with phytochromobilin, the phycobilin analogs phycocyanobilin and phycoerythrobilin and a novel C5,10,15-d-3-deuterated phytochromobilin were obtained in a collaborative study with Rich Mathies (ANDEL ET AL, 2000). In this study, we have assigned modes of vibration for the Pr and Pfr chromophores by normal mode analyses and by comparison to the Raman spectra of native and Pr and Pfr phytochromes. The study of these analogs reveals that the modes of Pr and Pfr at approx. 800 cm-1 are C-H out-of-plane vibrations and that the modes at approx. 650 cm-1 are likely candidates for C=C torsional modes. A model for the mechanism of phytochrome phototransformation was proposed.

Impacts
This research should lead to the development of new tools to improve plant growth and development.

Publications

  • Fankhauser, C., K.-C. Yeh, K.C., Lagarias, J.C., Zhang, H., Elich, T.D. and Chory, J. 1999. PKS1, a substrate phosphorylated by phytochrome that modulates light signaling in Arabidopsis.Science 284, 1539-154.
  • Andel, F. III, Murphy, J.T., J. A. Haas, J.A.. McDowell, M.T., van der Hoef, I. Lugtenburg, J., Lagarias, J.C. and Mathies, R.A. 2000. Probing the photoreaction mechanism of phytochrome through analysis of resonance Raman vibrational spectra of recombinant analogs. Biochemistry, in press.


Progress 01/01/98 to 12/01/98

Outputs
Research progress during the past year has confirmed the hypothesis that phytochrome from plants is an light-regulated enzyme. In one study (YEH ET AL 1998), we demonstrate that purified recombinant phytochromes from a higher plant and a green alga exhibit serine/threonine kinase activity similar to that of phytochrome isolated from dark grown seedlings. We document that phosphorylation of recombinant oat phytochrome is a light- and chromophore-regulated intramolecular process. Based on comparative protein sequence alignments and biochemical crosstalk experiments with the response regulator substrate of the cyanobacterial phytochrome Cphl, we propose that eukaryotic phytochromes are histidine kinase paralogs with serine/threonine specificity whose enzymatic activity diverged from that of a prokaryotic ancestor after duplication of the transmitter module. In a collaborative study (NOZUE ET AL, 1998), we describe the discovery of a novel photoreceptor gene from the fern Adiantum that encodes a protein with features of both phytochrome and NPH1, the putative blue light receptor for second-positive phototropism in seed plants. The fusion of a functional photosensory domain of phytochrome with a nearly full length NPH1 homolog suggests that this polypeptide could mediate both red/far-red and blue light responses in Adiantum normally ascribed to distinct photoreceptors.

Impacts
(N/A)

Publications

  • YEH, K-C. and LAGARIAS, J. C. 1998. Eukaryotic phytochromes: Light-regulated serine/threonine protein kinases with histidine kinase ancestry. Proc. Natl. Acad. Sci (USA). 95: 13976-13981.
  • NOZUE, K., KANEGAE, T., IMAIZUMI, T., FUKUDA, S., OKAMOTO, H., YEH, K-C., LAGARIAS, J.C. and WADA, M. 1998. A novel phytochrome from the fern Adiatum with features of NPH1. Proc. Natl. Acad. Sci (USA). 95:


Progress 01/01/97 to 12/01/97

Outputs
Research progress during the past year has confirmed the hypothesis that phytochrome from cyanobacteria is an light-regulated enzyme. Utilizing affinity tagged versions of the cyanobacterial phytochrome Cphl and its response regulator Rcpl encoded by the adjacent open reading frame, we have established that Cphl is a light-regulated histidine kinase. The proposed experiments to test the affects of Rcpl and Cphl expression in transgenic Arabidopsis have so far failed to reveal significant functional cross-talk between the cyanobacterial phytochrome components and higher plant phytochrome signalling pathways. An initial manuscript on the application of recombinant phytochromes as a new family of in vivo fluorescent protein probe has been published. This work was jointly supported by the NSF and USDA. In August of 1997, a patent application was filed on behalf of the University of California.

Impacts
(N/A)

Publications

  • YEH, K.-C., WU, S.-H., MURPHY, J.T. and LAGARIAS, J.C. 1997. A cyanobacterial phytochrome two-component light sensory system.
  • MURPHY, J.T. and LAGARIAS, J.C. 1997. Phytochromes: A new family of fluorescent protein probes. Current Biology 7:870-876.


Progress 01/01/96 to 12/30/96

Outputs
The overall objective of this project is to provide new information regarding both phytochrome structure and the primary signal transduction process(es) initiated by phytochrome photoactivation in both a higher plant (AVENA SATIVA) and a green alga (MESOTAENIUM CALDARIORUM). We have recently constructed and expressed a full length cDNA clone for one of the green algal phytochromes in the yeast PICHIA PASTORIS. In that study, we made the unexpected discovery that Pichia pastoris possesses the ability to synthesize the bilin pigment precursor of the phytochrome chromophore - an observation that has important biochemical and evolutionary implications. In another study, we have engineered an Avena phytochrome cDNA clone for expression of the phytochrome apoprotein, its reconstitution with bilin pigments and purification to homogeneity. This has enabled us to characterize the properties of the recombinant photoreceptor which possess both natural and phycobilin prosthetic groups. In a collaborative study, the structural changes which occur in the primary step of phytochrome phototransformation were deduced by resonance Raman spectroscopy. These studies have led to insight into pigment-protein interactions which are necessary for light-activation of this photoreceptor.

Impacts
(N/A)

Publications

  • ANDEL, F. III, LAGARIAS, J. C., and MATHIES, R.A. 1996. Resonance Raman analysis of the Lumi-R photoproduct of phytochrome. Biochemistry 35:15997-16008.
  • MURPHY, J.T. and LAGARIAS, J.C. 1997. Purification and biochemical characterization of Strep-tagged recombinant oat phytochrome A expressed in yeast. Photochemistry and Photobiology, in press.
  • WU, S-H. and LAGARIAS, J.C. 1996. The methylotrophic yeast Pichia pastoris synthesize a functionally active chromophore precursor of the plant photoreceptor phytochrome. Proc. Natl. Acad. Sci (USA) 93:8989-8994.


Progress 01/01/95 to 12/30/95

Outputs
The overall objective of this project is to provide new information regarding both phytochrome structure and the primary signal transduction processes initiated by phytochrome photoactivation in both a higher plant (AVENA SATIVA) and a green alga (MESOTAENIUM CALDARIORUM). A long term goal of these studies is to define three dimensional structural relationships between amino acid residues within the phytochrome polypeptide. Recently we showed that the phytochrome photoreceptor in the green alga MESOTAENIUM CALDARIORUM is encoded by a small family of highly related genes. Two of these genes have been cloned and shown to encode polypeptides which differ by one one amino acid. The deduced amino acid sequence of one of these genes reveals that the algal photoreceptor has an overall protein structure similar to other known phytochromes. Phylogenetic analyses indicate that the algal phytochrome falls into a distinct subfamily with other lower plant phytochromes. These studies also led to the discovery of a novel protein sequence motif which provides a new clue to the molecular mechanism of phytochrome function.

Impacts
(N/A)

Publications


    Progress 01/01/94 to 12/30/94

    Outputs
    The overall objective of this project is to provide new information regarding both phytochrome structure and the primary signal transduction process(es) initiated by phytochrome photoactivation in both a higher plant (Avena sativa) and a green alga (Mesotaenium caldariorum). A long term goal of these studies is to define three dimensional structural relationships between amino acid residues within the phytochrome polypeptide. Recent progress in the expression and assembly of recombinant phytochrome in my laboratory provides a powerful new direction for structural analysis on phytochrome. With regard to the primary function of phytochrome in plant cells, the hypothesis that phytochrome is a protein kinase is under intensive scrutiny at the present time. In addition, recent studies have shown that phytochrome can be expressed and assembled in living yeast cells. These studies suggest that it may be possible to reconstruct a phytochrome-mediated signal transduction pathway in yeast. Molecular dissection of the phytochrome system in the green alga Mesotaenium caldariorum is also in progress. To date, we have identified, clone and partially sequenced four genes for phytochrome in this alga. DNA sequence analysis of these clones is already providing new information regarding the structure and evolution of the phytochrome molecule.

    Impacts
    (N/A)

    Publications


      Progress 01/01/93 to 12/30/93

      Outputs
      The overall objective of this project is to provide new information regarding both phytochrome structure and the primary signal transduction process(es) initiated by phytochrome photoactivation in both a higher plant (Avena sativa) and a green alga (Mesotaenium caldariorum). A long term goal of these studies is to define three dimensional structural relationships between amino acid residues within the phytochrome polypeptide. Recent progress in the expression and assembly of recombinant phytochrome in my laboratory provides a powerful new direction for structural analysis on phytochrome. With regard to the primary function of phytochrome in plant cells, the hypothesis that phytochrome is a protein kinase is under intensive scutiny at the present time. In addition, recent studies have shown that phytochrome can be expressed and assembled in living yeast cells (Li. L. and Lagarias, J.C., manuscript in preparation). These studies suggest that it may be possible to reconstruct a phytochrome-mediated signal transduction pathway in yeast. Molecular dissection of the phytochrome system in the green alga Mesotaenium caldariorum is also in progress. To date, we have identified, clone and partially sequenced four genes for phytochrome in this alga. DNA sequence analysis of these clones is already providing new infromation regarding the structure and evolution of the phytochrome molecule.

      Impacts
      (N/A)

      Publications


        Progress 01/01/92 to 12/30/92

        Outputs
        The overall objective of this project is to provide new information regarding both phytochrome structure and the primary signal transduction process(es) initiated by phytochrome photoactivation in both a higher plant (AVENA SATIVA) and a green alga (MESOTAENIUM CALDARIORUM). A long term goal of these studies is to define three dimensional structural relationships between amino acid residues within the phytochrome polypeptide. Recent progress in the expression and assembly of recombinant phytochrome in my laboratory provides a powerful new direction for structural analysis on phytochrome. With regard to the primary function of phytochrome in plant cells, the hypothesis that phytochrome is a protein kinase is under intensive scrutiny at the present time. Molecular dissection of the phytochrome system in the green alga MESOTAENIUM CALDARIORUM is also in progress. In one recent study (Morand et al., 1993), we showed that the algal phytochrome levels are light regulated. This interesting phenomenon suggests that the regulation of phytochrome in this alga is similar to that in higher plants. Thus, this alga appears to be a good model system for the analysis of higher plant phytochrome-mediated phenomena. Experiments to clone the algal phytochrome gene(s) and cDNAs are also in progress.

        Impacts
        (N/A)

        Publications


          Progress 01/01/86 to 12/30/91

          Outputs
          The overall objective of this project is to provide new information regarding both phytochrome structure and the primary signal transduction process(es) initiated by phytochrome photoactivation in both a higher plant (AVENA SATIVA) and a green alga (MESOTAENIUM CALDARIORUM). A long term goal of these studies is to define three dimensional structural relationships between amino acid residues within the phytochrome polypeptide. Recent progress in the expression and assembly of recombinant phytochrome in my laboratory provides a powerful new direction for structural analysis on phytochrome. With regard to the primary function of phytochrome in plant cells, the hypothesis that phytochrome is a protein kinase (or is functionally associated with a protein kinase) has been implicated by ongoing investigations in my laboratory. Molecular dissection of the phytochrome system in the green alga MESOTAENIUM CALDARIORUM also is well underway. In this regard, the algal phytochrome has been purified to homogeneity and molecular probes (i.e. antibodies and cDNA clones) are now being developed. Methods for fractionation for membranes and organelles from this alga have also been developed. Hence we are now in a good position to biochemically characterize the components of the phytochrome signal transduction pathway in this organism.

          Impacts
          (N/A)

          Publications


            Progress 01/01/90 to 12/30/90

            Outputs
            A major goal of our program is provide new information at the molecular level ofthe protein components and second messengers which participate in the light-initiated phytochrome-mediated intracellular signal cascade. To achieve this goal, one line of research has focussed on the development of a unicellular green algal model system (MESOTAENIUM CALDARIORUM) in which phytochrome regulates chloroplast orientation. Based on the hypothesis that phytochrome photoactivation effects a change in the cytosolic levels of calcium, we set out to fractionate microsomal membranes from M. CALDARIORUM and to study the role of light in the regulation of calcium transport in these membranes. As a result of our studies, subcellular localization and initial biochemical characterization of calcium transport systems in this organism have been accomplished (ref 1). As outlined in ref 2, the development of an IN VITRO assay system for light-regulated transmembrane Ca+2 transport in microsomal membranes from M. CALDARIORUM has so far proven unsuccessful. In ref. 3, we critically examine published and unpublished data pertaining to the hypothesis that phytochrome is itself a protein kinase. We conclude that this hypothesis is still viable. Resonance Raman spectroscopy of phytochrome is described in ref. 4. These studies demonstrate the feasibility of this technique for elucidation of the structural basis for phytochrome photoreversibility.

            Impacts
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            Publications


              Progress 01/01/89 to 12/30/89

              Outputs
              The overall objective of this project is to provide new information regarding both phytochrome structure and the primary signal transduction process(es) initiated by phytochrome photoactivation in both a higher plant (Avena sativa) and green alga (Mesotaenium caldariorum). With regard to the primary function of phytochrome in plant cells, the hypothesis that phytochrome is a protein kinase (or is functionally associated with a protein kinase) suggested by recent studies in our laboratory, is under active investigation. The properties of the phytochrome kinase which is present in purified Avena phytochrome preparations are described in ref 1. In ref 2., phosphopeptide mapping of Avena phytochrome phosphorylated by protein kinases, including the phytochrome kinase, is described. These studies have defined specific regions of light-dependent conformational change on the phytochrome molecule. The purification of phytochrome from the green alga Mesotaenium caldariorum is described in ref 3. These studies show that this unicellular alga is amenable to biochemical analyses which should facilitate elucidation of the components of a phytochrome-mediated signal transduction pathway.

              Impacts
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              Publications


                Progress 01/01/88 to 12/30/88

                Outputs
                Results from two collaborative projects were communicated in the last year. In acollaborative study with Drs. Rich Mathies and Steve Fodor (UCB), the first resonance Raman spectra of phytochrome were recently obtained (1). This technique is expected to lead to a complete molecular description of photochemical interconversions which occur in the phytochrome chromophore. In the second collaboration with Drs. Lee H. Pratt (University of Georgia) and Marie-Michele Cordonnier (CIBA GEIGY), localization of epitopes of a large number of monoclonal antibodies on the Avena phytochrome subunit was reported (2). The results of these studies confirmed and extended the model of the light-dependent changes in the Avena phytochrome protein structure proposed in an earlier study. We reported previously that highly purified Avena phytochrome preparations exhibit ATP-dependent polycation-stimulated protein kinase activity. Based on analysis of over 30 different Avena phytochrome preparations, a more detailed description of the characteristic properties of this unusual protein kinase has emerged (Wong, McMichael, and Lagarias, in preparation). To test the possibility that phytochrome itself is the protein kinase we explored the ATP binding properties of Avena phytochrome using two ATP-affinity analogs, 8-azido ATP and 5'-p-fluorosulfonylbenzoyl adenosine (3). These studies provided evidence for the presence of a nucleoside triphosphate binding site on phytochrome which depends on the presence of polycations.

                Impacts
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                Publications


                  Progress 01/01/87 to 12/30/87

                  Outputs
                  Extensive structural studies performed in my laboratory have led to a detailed picture of the phytochrome subunit structure as well as a more thorough description of the changes in protein structure which accompany phytochrome's photoactivation. Using a methodology we devised earlier for the quantitation of the efficiency of photoconversion be compared the photochemical properties of purified rye and oat phytochromes. These studies revealed differences in photoconversion efficiency which may indicate that the photoconversion can be regulated by a biochemical modification. Our earlier discovery of a co-purifying (or endogenous) protein kinase activity in Avena phytochrome preparations has raised the possibility that phytochrome is itself a protein kinase. The properties of this polycation-stimulated protein kinase have been extensively characterized (manuscript in preparation). By contrast to these studies with dark grown Avena seedlings, the molecular properties of phytochrome from the green alga Mesotaenium are poorly defined at present. We have devised a protocol for obtaining soluble extracts enriched in phytochrome from Mesotaenium which has entailed the preparation of a algal protoplasts followed by gentle cell lysis (manuscript in preparation). Work is in progress to purify and fully charcterize the algal phytochrome protein as well as to clone the gene.

                  Impacts
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                  Publications


                    Progress 01/01/86 to 12/30/86

                    Outputs
                    Our research can be summarized as follows: Protein kinases have proven particularly useful probes of light induced conformational changes in purified Avena phytochrome. Three mammalian protein kinases showed preferential modification of a region near the N-terminus of phytochrome while a region distant from the N-terminus became accessible for phosphorylation by the cAMP-dependent protein kinase. The discovery of an endogenous protein kinase activity in purified phytochrome preparation raised the exciting possibility that phytochrome itself is a protein kinase. Inhibition of the biosynthesis of the linear tetrapyrrolic prosthetic group of Avena phytochrome was accomplished with the transaminase inhibitor gabaculine. This inhibition was overcome by administration of gabaculine-treated seedlings with 5-aminolevulinic acid and biliverdin-demonstrating the intermediacy of these compounds in the chromophore biosynthesis. Comparative absorption and photochemical measurements of purified 124 kDa oat and rye phytochromes revealed differences which have been ascribed to structural differences. Recent structure-function studies on Avena phytochrome in our laboratory is summarized in reference 4.

                    Impacts
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                    Publications


                      Progress 01/01/85 to 12/30/85

                      Outputs
                      Light absorption by phytochrome leads to a significant conformational change in the protein as observed by steric exclusion chromatography and limited proteolysis (ref 1). Peptide mapping studies have provided a detailed picture of specific regions of light induced conformational change (ref. 1). The quantum yields for both phytochrome photoconversions have been determined using a new experimental approach (ref. 2). These have allowed predictions of the photostationary equilibrium produced under monochromatic irradiation throughout the ultraviolet and visible spectrum (ref. 2). A new method for visualization of linear tetrapyrrole containing peptides in polyacrylamide gels has been devised (ref. 4). Antibodies prepared to the chromophore binding site on Avena phytochrome show cross reactivity with phytochrome from other monocots and dicot species as well (ref. 5). This allows that the chromophore binding site is conserved evolutionarily.

                      Impacts
                      (N/A)

                      Publications


                        Progress 01/01/84 to 12/30/84

                        Outputs
                        Our research progress on this project has been in two areas - the identificationand structural characterization of light-induced conformational changes in 124 kDa Avena phytochrome in vitro and the determination of the quantum yields for phytochrome phototransformation in vitro. In the first study (Lagarias and Mercurio) we have shown a light-mediated shape change of Avena phytochrome by steric exclusion chromatography and have identified specific regions in the 124 kDa subunit which are involved in light-induced structural changes by limited proteolysis. We have also prepared site-directed antibodies using a peptide immunization approach. This has permitted the assignment of specific polypeptides obtained via limited proteolysis of phytochrome to predetermined structural regions of the native subunit. In the second study (Kelly and Lagarias), we have developed a new experimental approach to determine the quantum yields of the two phytochrome phototransformations and the photoequilibrium mixture of Pr and Pfr forms established under continuous red illumination. This method involves 1) the use of potassium ferrioxalate actinometry to calibrate a photodiode-array spectrophotometer for absolute radiometry and 2) the determination of the photochemical cross sections of the transformations for use in calculating the quantum yields. The quantum yields and equilibrium mole fraction of Pfr under constant illumination have been determined for a number of different buffer conditions.

                        Impacts
                        (N/A)

                        Publications


                          Progress 01/01/83 to 12/30/83

                          Outputs
                          We have recently developed a new purification protocol for the undegraded, 124,000 dalton phytochrome species from etiolated oat seedlings. Based upon spectral observations, we believe that our phytochrome preparations are indeed native and therefore are representative of the functionally active chromoprotein. A sensitive photoassay has been devised which we will use to study the effect of protein modifying reagents on overall protein structure. Site specific antibodies are being used as probes of protein structure. These studies are aimed at providing a detailed picture of the molecular events which occur during photomorphogenetic development in higher plants.

                          Impacts
                          (N/A)

                          Publications


                            Progress 01/01/82 to 12/30/82

                            Outputs
                            With the successful isolation and purification of phytochrome from etiolated oats seedlings, which is enriched in the native species with monomer M(r) of 124,000 on SDS PAGE, we have begun to investigate the molecular properties of this unusual chromoprotein. Spectroscopic comparison of our phytochrome preparation with phytochrome preparations described by others has been accomplished. Several significant differences were observed, which we have ascribed to differences in the monomer M(r)'s between samples. These include: a significantly reduced amount of dark reversion, a shift of absorption maxima, a larger P(fr)/P(r) photoequilibrium ratio, a new CD band at 430 nm. and significant difference in the luminescence properties of the chromoprotein prepared in our laboratory. These spectroscopic properties, especially (1)-(3), more closely correspond to those observed in vivo. The significance of the proteolytically unstable region of the native phytochrome molecule with regard to the biophysical changes in the molecule which occur upon light absorption are currently under study.

                            Impacts
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                            Publications


                              Progress 01/01/81 to 12/30/81

                              Outputs
                              Our first goals, that of isolating and purifying large quantities of phytocrome from oats, Arena sativa, cv. Garry, has been realized. Utilizing the protocol pioneered by G. Bolton and further refined by J.C. Litts, a postdoctoral research associate presently in this laboratory, the routine isolation of 20 mg of phytochrome in >90% purity from 2 kg freshly-harvested oats has been accomplished. This phytochrome has been characterized spectrophotometrically and exhibits spectral characteristics (i.e. photoequilibria, photor eversibility and peak position P(FR)) which are virtually identical with those observed in vivo. SDS PAGE has revealed that this phytochrome has partially proteolyzed and a mixture of difference118-125 kD biliprotein fragments are obtained. Methods are being developed to minimize these presurmed, artifictual cleavages of the holoprotein. Presently we are developing peptide mapping techniques to address several structural questions: Chromophore number, phosphate attachment site, and differences in protein surfaces P(R) versus P(FR).

                              Impacts
                              (N/A)

                              Publications