Downstream signaling mechanisms of phytochrome dimers

• Applicant: Dr. Maximilian Ulbrich
• Subject Area: Plant Biochemistry and Biophysics ; Cell Biology
• Term: since 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 439422115

Plant development is regulated by a range of light-sensitive proteins. Proteins of the phytochrome family perceive red and far-red light. The fact that phytochromes dimerize has been largely neglected in the past. In this project we aim to elucidate the impact of the phytochromes' dimerization on their wavelength-specificity, function and physiological responses using single-molecule microscopy and classical physiological approaches.Type II phytochromes phyB–E become active upon red and inactive upon far-red light exposure. In contrast, the type I phytochrome phyA shows the highest physiological response in the range between red and far-red, an effect that is not trivial since the light sensing chromophore is the same for all phytochromes. One factor that likely contributes to this so-called far-red shift of phyA's response is the dimeric state that all phytochromes assume. In the dimer, the state with one monomer in the active and one in the inactive form is most prevalent in the range between red and far-red light. Several observations we made suggest that this intermediate state of the phyA dimer is predominant in binding to downstream signaling factors.Single-molecule imaging of fluorescently labeled proteins is a method that is well suited to accurately determine the constituents of a protein complex. In the proposed project, we will determine the properties of phytochrome dimers, and the interaction partners they bind. In particular, we will decipher in how far the different states of the phyA dimer differ in the profile of their interaction partners.To investigate the physiological properties of phyA dimers, we plan to express constitutively active or inactive mutants of phyA in plants, or a dimer that is permanently trapped in the intermediate state with one active and one inactive monomer. The characterization of the mutant plants will include the measurement of plant development under different light conditions, biochemical assessment of several proteins and metabolites, quantification of expression of light-response marker genes, and fluorescence microscopy of plant tissue.We are also interested in the type II phytochromes phyB–E. Heterodimer formation of type II phytochromes has previously been suggested, but not thoroughly investigated on a quantitative level. We intend to use single-molecule imaging to determine the heterotypic interactions of the type II phytochromes, and the interaction partners that bind to the different forms. Eventually, we plan to use chimeric proteins of phyA and phyB–E to assess which domains of the proteins are responsible for their characteristic behaviors.

• DFG Programme: Research Grants
• Co-Investigator: Professor Dr. Andreas Hiltbrunner