Progress 10/01/09 to 09/30/13
Outputs
Target Audience: This project aims to develop new strategies to manipulate plant architecture, growth, and harvestable yield to the benefit of agriculture, especially related to improved yield under high density cropping systems. Its particular focus in on engineering desirable signaling traits into the phytochrome family of photoreceptors that control many aspects of plant growth and development, including seed germination, stem growth and shade avoidance, photosynthetic efficiency, flowering time, and senescence. The target audiences of this project are ag biotech companies and plant breeders that could exploit this technology. The project also aims to develop future scientists at the graduate and undergraduate levels that are experienced in modern plant biology research and will become the next leaders in this field. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? This worked enabled the training of two graduate students that resulted in two Ph.D thesis projects by Dr. Junrui Zhang and Dr. Robert Stankey. Both are now working at postdoctoral fellows in other research laboratories. In addition, a collection of undergraduates also participated, which provided practical training in experimental design and methods to these beginning investigators. How have the results been disseminated to communities of interest? The work generated by this project was presented at several national and international meetings focused on photobiology and plant physiology. In addition, six research papers and two invited reviews were published. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The goal of this project was to develop structural information related to the molecular mechanisms that underpin signaling by phytochromes, and then to use this knowledge to begin to rationally redesign light perception in plants for agricultural benefit. Our approach was to exploit bacterial version of plant phytochromes for structural studies, using their ability to be highly expressed in bacterial recombinant systems and thus generate ample amounts of chromoproteins needed for these approaches. We then wanted to understand how phytochromes signal by biochemical and biophysical analyses of these photoreceptors combined with site-directed mutagenesis of key amino acids that contact the chromophore. With knowledge of how these amino acids impact phytochrome photochemistry and structure, we finally proposed to generate plant phytochromes with novel signaling properties by expressing these altered chromoproteins in transgenic Arabidopsis. Ultimately, we hoped to generate a toolbox of phytochrome variants that can be used to modify the growth and reproduction of individual food and biofuel crops to better suit specific cropping systems and/or environments. Taken together, this project fit well into the mission of the Hatch research program through its use of basic molecular information to solve real-life agricultural problems. Starting from our first three-dimentional structure of the photosenory module of a bacterial phytochrome (Deinococcus radiodurans BphP) generated by Wagner et al. (2006 and 2007), we began to exploit a collection of other prokaryotic versions with potentially novel or insightful properties photodynamics for X-ray crystallographic or solution two-dimensional NMR studies. With the cyanobacteriochrome (CBCR) from Thermosynechococcus elongatus, we used NMR spectroscopy to determine the three-dimensional structure of the GAF domain bound with chromophore. Prior work showed that this GAF domain works alone in driving photoconversion, thus making it small enough for NMR studies, and revealed that it employs a second cysteine to bind the chromophore (Ulijasz et al., 2009). Surprisingly, this second cysteine thioether bond breaks during photoconversion from its blue-light absorbing Pb state to its green-light-absorbing Pg state. A crystallographic structure of the Pg state then revealed that during photoconversion the bilin isomerizes from a ZZZssa to ZZEssa conformation, slides with the GAF domain pocket, and ultimately impinges on the helical spine lining the sister GAF domains dimerization contacts, thus identifying a potential mechanism for how phytochromes translate light energy into a conformational signal (Burgie et al., 2013). We also determined the three-dimensional structure of the GAF domain from Synechcoccus OSB' Cph1 in both its red light-absorbing Pr and far-red light-absorbing Pfr states (Cornilescu et al., 2010). Comparisons indicated that this distinctive phytochrome actually rotates the A and not the D pyrrole ring, thus revealing a novel photochemistry for this phytochrome type. With our extensive structural information on microbial phytochromes in hand, our next goal was to develop a suite of plant phytochrome mutants that can be useful to agriculture (Zhang et al., Plant Physiology 2013). In brief, mutations found to generate potentially useful photochemical defects during our studies on microbial phytochromes were introduced at comparable positions into Arabidopsis PhyB, the main phytochrome isoform in light grown plants. Altered signaling mutants of PhyB are especially interesting given the dominant role of this isoform in controlling plant seed germination, height and shade avoidance, axillary branching, and flowering time. Those PhyB mutants with novel photochemical properties in vitro were introduced into an Arabidopsis background missing the corresponding isoform (phyB-9). The transgenic plants were then examined for altered photomorphogenesis, using a set of well-understood photoresponses, such as seed germination, de-etiolation, hypocotyl elongation, leaf epinasty, and flowering time. Interestingly, we found several PhyB mutants substantially modified Arabidopsis growth and development, including one (Tyr361-Phe) variant that generated plants with a >50 fold increase in sensitivity to light (Zhang et al., Plant Physiology 2013). This mutant was so novel, that a patent was submitted by WARF based on its potential commercial value. We are now attempting to introduce these potentially useful PhyB alleles into maize (Zea mays), both wild-type and a mutant missing its two PhyB isoforms, to test their effects in this crop under greenhouse conditions that replicate field environments. In an attempt to improve our predictions for how mutations would impact plant phytochrome photochemistry, we also generated the first three-dimensional structure of the photosensory module of a plant phytochrome by X-ray crystallography (Burgie et al., PNAS 2014). For this groundbreaking work, we used a fragment of Arabidopsis PhyB encompassing the photosensory module with the bound bilin chromophore that was assembled recombinantly with its native bilin phytochromobilin and crystallized it in its dark-adapted Pr state. This structure can now be used as a more reliable scaffold to build the next generation of PhyB mutants. Taken together, the project has provided a concrete example where highly basic biological information (i.e., three-dimensional structures of microbial relatives) can be used to rationally manipulate crop yield for human benefit.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2009
Citation:
Ulijasz, A.T., G. Cornilescu, D. von Stetten, C. Cornilescu, F. Velazquez, J. Zhang, R.J. Stankey, M. Rivera, P. Hildebrandt, and R.D. Vierstra (2009) Cyanochromes: blue-green light photoreversible phytochromes defined by a stable double cysteine linkage to a phycoviolobilin-type chromophore. J. Biol. Chem. 284: 29757-29772.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Zhang, J., R.J. Stankey, and R.D. Vierstra (2013) Structure-guided engineering of phytochrome B with altered photochemistry and light signaling. Plant Physiol. 161: 1445-1457.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Burgie, E.S., A.N. Bussell, K. Dubiel, J.M. Walker, and R.D. Vierstra (2014) Crystal structure of the photosensing module from a red/far-red light-absorbing plant phytochrome. Proc. Natl. Acad. Sci. USA 111: 10179-10184.
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Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: Understanding the mechanisms used by plants to sense their environment and regulate their physiology, growth and development have important ramifications on our ability to manipulate crop plants for agricultural benefit. One such sensing system involves the phytochrome (Phy) family of photoreceptors, which sense red and far-red light and then initiate a set of photomorphogenic processes which allow plants to better shape their growth and development to the ambient light environment. The goals of this project are to define at the atomic level how Phys sense light and then exploit these structures to rationally redesign Phy signaling to enhance agricultural yield. In particular, we will exploit Phys in both Arabidopsis and a number of prokaryotes as models. Goals of this project are to (i) define the structures of the chromophore-binding domain of a Phy in both the Pr and Pfr forms, (ii) construct the structure of the entire Phy dimer by X-ray crystallographic and single particle electron microscopic (EM) techniques, (iii) determine the structures of a new set of Phys called cyanobacteriochromes (CBCRs) that absorb blue and green light, (iv) understand the photochemistry of Phys using various biophysical methods, and (v) use the information gained to alter plant Phys by structure-based mutagenesis. PARTICIPANTS: Dr. Richard D. Vierstra - Principal investigator. Joseph Walker - Laboratory Manager: oversaw experimental protocols and laboratory organization. Dr. Sethe Burgie- Research Associate: structure of cyanobacterial Phys. Junrui Zhang - Research Assistant: structure and function of bacterial Phys and reengineering of plant Phys. Robert Stankey - Research Assistant: structure and turnover mechanism of plant phytochromes. Collaborators: Dr John Markley, Gabriel Cornilescu, and Claudia Cornilescu: Department of Biochemistry, University of Wisconsin; Dr Huilin Li, Brookhaven National Laboratory. TARGET AUDIENCES: Target audience includes plant biologists and agricultural biotech companies aimed at improving plant development and physiology in attempts to enhance crop productivity. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
In the past year, we made great strides on several key objectives. Most importantly, we determined the first x-ray crystallographic structure of the ground state of the photosensing module from the CBCR PixJ from Thermosynechococcus elongatus (Te), a novel Phy variant that photoconverts between blue- and green-light absorbing forms Pb and Pg, respectively. CBCRs are unique to cyanobacteria and are likely used to help them measure their ambient light environments and move appropriately within the colonial mats prevalent within aquatic ecosystems. The Pb structure revealed the molecular basis behind their unusual photochemistry. Instead of coupling the bilin chromophore to the chromophore-binding GAF domain through a single cysteine thioesther linkage, Te PixJ uses two cysteines, one that links to the A pyrrole ring of the bilin and another that links to the methine bridge between the B and C pyrrole rings, thus forming a less conjugated and shorter wavelength-absorbing chromophore. Presumably, this second bridge is broken upon photoconversion to Pg, thus extending the π-conjugation system and concomitantly shifting the absorption of Te PixJ toward green light. Work is now underway to determine the solution structures of Pb and Pg using 2-D NMR methods. Using our catalog of microbial Phy 3-D structures generated by X-ray crystallography, NMR, and single particle EM as templates, we have started to rationally re-engineer plant Phys in an effort to manipulate photomorphogenesis for agricultural benefit. One application would be to control plant reproduction and architecture to better fit specific environments and to dramatically increase crop densities, the latter of which will require the redesign of plants that perform well in more competitive environments. In the past year, we generated a suite of site-directed mutants in Arabidopsis PhyB, introduced these mutants into Arabidopsis plants missing this Phy isoform, and then tested for the ability of the mutants to restore proper photomorphogenesis. The research identified several PhyB mutants with interesting photochemical and signaling behaviors. One mutant, which was created by a single amino acid substitution of a tyrosine (Tyr361-Phe in the PhyB isoform), was discovered to dramatically alter the light sensitivity of Arabidopsis plants. Altered photoresponses include an improved germination efficiency of seeds in low light, a dramatic hypersensitivity to white and red light with respect to hypocotyl and stem growth, larger leaf surface areas in white light, and dampened leaf and petiole epinasty. We propose that the Tyr361-to-Phe mutation in PhyB could generate plants with smaller sizes and that are more tolerant to the low light condition experienced in crowded field conditions, and therefore plants with this mutant PhyB would require much less space in the agricultural field. Given that this substituted tyrosine is conserved in all plant phytochromes, analogous substitutions should elicit a similar hypersensitivity in most, if not all, crop plants when the host PhyB is engineered and expressed, thus providing a facile way to alter crop reproduction and architecture.
Publications
- Burgie, E.S., J.M. Walker, G.N. Phillips Jr, and R.D. Vierstra (2013) A photo-labile thioether linkage to phycoviolobilin provides the foundation for the unique blue/green photocycles in DXCF cyanobacterio-chromes. Structure 21: 88-97.
- Zhang, J., R.J. Stankey, and R.D. Vierstra (2013) Structure-guided engineering of phytochrome B with altered photochemistry and light signaling. Plant Physiol. (in press, doi:10.1104/pp.112.208892).
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: Understanding the mechanisms used by plants to sense their environment and regulate their physiology, growth and development have important ramifications on our ability to manipulate crop plants for agricultural benefit. One such sensing system involves the phytochrome (Phy) family of photoreceptors, which sense red and far-red light and then initiate a set of photomorphogenic processes which allow plants to better shape their growth and development to the ambient light environment. The goals of this project are to define at the atomic level how Phys sense light and then use this information to rationally redesign Phy signaling to enhance agricultural yield. In particular, we will exploit Phys in both Arabidopsis and a number of prokaryotes as models. Goals of this project are to: (i) define the structures of the chromophore-binding domain of a Phy in both the Pr and Pfr forms, (ii) constructed the structure of the entire Phy dimer by x-ray crystallographic and single particle electron microscopic (EM) techniques, (iii) determine the structures of a new set of Phys called cyanochromes, that absorb blue and green light, (iv) understand the photochemistry of Phys using various biophysical methods, and (v) use the information gained to alter plant Phys by structure-based mutagenesis. PARTICIPANTS: Dr. Richard D. Vierstra - Principal investigator. Joseph Walker - Laboratory Manager: oversaw experimental protocols and laboratory organization. Dr. Sethe Burgie- Research Associate: structure of cyanobacterial Phys. Junrui Zhang - Research Assistant: structure and function of bacterial Phys and reengineering of plant Phys. Robert Stankey - Research Assistant: structure and turnover mechanism of plant phytochromes. Collaborators: Dr John Markley, Gabriel Cornilescu, and Claudia Cornilescu: Department of Biochemistry, University of Wisconsin; Dr Huilin Li, Brookhaven National Laboratory TARGET AUDIENCES: These studies are designed to understand how plants detect and respond to light. Understanding thus signaling processes will aid in the design of strategies and reagents to enhance crop productivity. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
In the past year, we made great strides on several key objectives. Most importantly, we determined the first x-ray crystallographic structure of the Pb form of the photosensing module from the PixJ cyanochrome from Thermosynechococcus elongatus, a unique Phy variant that photoconverts between a blue and green-light absorbing forms Pb and Pg, respectively. These cyanochromes are unique to cyanobacteria and likely used to help them measure their ambient light environments and move appropriately within the colonial mats prevalent within aquatic ecosystems. The structure revealed the molecular basis behind their unusual photochemistry. Instead of coupling the bilin chromophore to the chromophore-binding GAF domain through a single cysteine thioesther linkage, this cyanochrome uses two cysteines, one that links to the A pyrrole ring of the bilin and another that links to the methine bridge between the B and C pyrrole rings, thus forming a less conjugated and shorter wavelength-absorbing chromophore. Work is now underway to determine the solution structures of Pb and Pg using 2D NMR methods. Using our catalog of microbial Phy 3D structures generated by x-ray crystallography, NMR, and single particle EM as templates, we have started to rationally reengineer plant Phys in an effort to manipulate photomorphogenesis for agricultural benefit. One application would be to control plant reproduction and architecture to better fit specific environments and to dramatically increase crop densities, the latter of which will require the redesign of plants that perform well in more competitive environments. In the past year, we generated a suite of site-directed mutants in Arabidopsis PhyB, introduced these mutants into Arabidopsis plants missing this Phy isoform, and then tested for the ability of the mutants to restore proper photomorphogenesis. One such mutant, which was created by a single amino acid substitution of a tyrosine (Tyr361-Phe in the PhyB isoform), was discovered to dramatically alter the light sensitivity of Arabidopsis plants. Altered photoresponses include: an improved germination efficiency of seeds in low light, a dramatic hypersensitivity to white and red light with respect to hypocotyl and stem growth, and larger leaf surface areas in white light. We propose that the Tyr361-to-Phe mutation in PhyB could generate plants with smaller sizes and more tolerant to the low light condition experienced in crowded field conditions, and therefore plants with this mutant PhyB would require much less space in the agricultural field. Given that this substituted tyrosine is conserved in all plant phytochromes, analogous substitutions should elicit a similar hypersensitivity when the host PhyB is engineered and expressed in most, if not all, crop plants, thus providing a facile way to alter crop reproduction and architecture.
Publications
- Vierstra, R.D., and Zhang, J. (2011) Phytochrome signaling, solving the Gordian knot with microbial relatives. Trends Plant Sci. 16: 417-426.
- Ulijasz, A.T., and R.D. Vierstra (2011) Phytochrome structure and photochemistry: recent advances toward a complete molecular picture. Curr. Opin. Plant Biol. 14: 498-506.
- Zhang, J., and R.D. Vierstra (2012) Structure-guided engineering of plant phytochromes with alter light signaling. Curr. Biol. (in preparation).
- Burgie, E.S., G.N. Phillips, and R.D. Vierstra (2012) Three-dimensional structure of the photosensing module of a cyanochrome in the blue-light absorbing Pb form. Proc. Natl. Acad. Sci. USA (in preparation).
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: Understanding the mechanisms used by plants to sense their environment and regulate their physiology, growth and development have important ramifications on our ability to manipulate crop plants for agricultural benefit. One such sensing system involves the phytochrome family of photoreceptors, which sense red and far-red light and then initiate a set of photomorphogenic processes which allow plants to better shape their growth and development to the ambient light environment. The goals of this project are to define at the atomic level how phytochromes sense light and then use this information to rationally redesign phytochrome signaling for agricultural benefit. In particular, we will exploit phytochromes in both Arabidopsis and a number of prokaryotes as models. Goals of this project are to: (i) define the structures of the chromophore-binding domain of a phytochrome in both the Pr and Pfr forms, (ii) constructed the structure of the entire phytochrome dimer by x-ray crystallographic and single particle electron microscopic techniques, (iii) determine the structures of a new set of phytochromes called cyanochromes, that absorb blue and green light, (iv) understand the photochemistry of phytochromes using various biophysical methods, and (v) use the information gained to alter plant phytochromes by structure-based mutagenesis. PARTICIPANTS: Dr. Richard D. Vierstra - Principal investigator. Joseph Walker - Laboratory Manager: oversaw experimental protocols and laboratory organization. Dr. Sethe Burgie- Research Associate: structure of cyanobacterial phytochromes. Junrui Zhang - Research Assistant: structure and function of bacterial phytochromes. Robert Stankey - Research Assistant: structure and turnover mechanism of plant phytochromes. Collaborators: Dr John Markley, Gabriel Cornilescu, and Claudia Cornilescu: Department of Biochemistry, University of Wisconsin; Dr Huilin Li, Brookhaven National Laboratory TARGET AUDIENCES: Target audience includes plant biologists aimed at improving plant development and physiology in attempts to enhance crop productivity. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
In the past year, we made great strides on several key objectives. Most importantly, we determined the first paired solution structures of the chromophore-binding domain of a phytochrome in both the ground Pr and photoactivated Pfr states using NMR. These structures showed that while the chromophore and surrounding pocket is remarkably rigid, a number of surface residues are highly mobile, which may be important for Pr to Pfr photoconversion. During photoconversion, the A pyrrole ring of the chromophore rotates approximately 90 degrees, when then induces a number of conformational changes within the phytochrome dimer to initiate signaling. To help confirm these conformation changes occur in other phytochrome-type photoreceptors, we began to exploit several cyanochromes to generate pair Pb and Pg structures of these chromoproteins. Using a full-length phytochrome from the eubacterium Deinococcus radiodurans, we developed the first full structure of a phytochrome dimer by single particle EM. Contrary to the long-standing view that the two monomers are held together solely via their C-terminal kinase domains, our structure provided unambiguous evidence that the N-terminal bilin-binding region is the primary dimerization contact in this phytochrome with the C-terminal kinase domain appearing as a more flexible appendage. The BphP monomers dimerize in parallel with the polypeptides intimately twisting around each other in a right-handed fashion. Based on this EM picture, we proposed that the light-driven conformational changes transmitted from the chromophore to the kinase domain along this extensive dimer interface is the central feature underpinning phytochrome signaling. With this information, we have now begun to manipulate plant phytochromes by site-directed mutagenesis base on our atomic appreciations, and used these mutant phytochromes to replace the wild-type forms. Analysis of these plants should reveal how plant phytochromes work mechanistically and provide new variants that can be used to manipulate photomorphogenesis.
Publications
- Ulijasz, A.T., G. Cornilescu, C. Cornilescu. J. Zhang, M. Rivera, J.L. Markley and R.D. Vierstra (2010) Structural basis for the photoconversion of a phytochrome to the activated Pfr form. Nature 463: 250-254.
- Li, H., J. Zhang, R.D. Vierstra, and H. Li (2010) Quaternary organization of a phytochrome dimer as revealed by cryo-electron microscopy. Proc. Natl. Acad. Sci. U.S.A. 107: 10872-10877.
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