Modulation of Phytochrome B Signalling by Phosphorylation

Plants are sessile organisms and therefore have to adapt their growth to changes in the environment. Among the abiotic and biotic environmental factors that regulate plant growth, light plays a distinguished role. Light is not only used to drive photosynthesis but it is also an important developmental clue to ensure optimal adaptation to the changing environment. To monitor variations in the wavelength, intensity, direction and duration of light, plants evolved a battery of photoreceptors. The photoreceptors active in red/far-red light are called phytochromes. Phytochromes are dimeric chromoproteins with one covalently linked tetrapyrrol chromophore per molecule, and they cycle between their biologically inactive (Pr = red absorbing) and active (Pfr = far-red absorbing) forms. It is the Pfr conformer which is imported into the nuclei and whose interaction with specific cellular factors is required to launch the signaling cascade. It follows that phytochrome signaling is quantitatively determined (i) by the number of Pfr molecules available and (ii) by the kinetics of protein-protein interactions between Pfr molecules and signal transducers.

Phytochromes are often also referred to as light-regulated enzymes as it was shown that phytochrome-A (phyA) can autophosphorylate and phosphorylate other proteins in vitro and is phosphorylated by unknown kinase(s) in planta. These data were interpreted to mean that autophosphorylation reduces the amount of phytochrome Pfr by increasing its degradation whereas phosphorylation by an unknown kinase at other amino acid residues decreases the capacity of the Pfr form to bind to its authentic signaling partners. Thereby it is generally accepted that phosphorylation negatively regulates phyA controlled signaling and physiological responses. However, the topic is surrounded by considerable controversy as fundamental biochemical evidences are still missing to validate this theory.

In this proposal, we outline a research program to elucidate how reversible phosphorylation regulates phytochrome-B (phyB) controlled responses at the molecular level. We show that phyB, the major photoreceptor regulating photomorphogenic responses in adult plants, is (i) phosphorylated at multiple sites in planta, (ii) this post-translational modification increases the rate of dark-reversion, a light independent conversion of the thermodynamically unstable Pfr to Pr form and thereby (iii) results in decreased responsiveness to red light. To test if phosphorylation also affects interaction of phyB with its downstream signalling partners, we will perform in vitro experiments to characterise binding of the mutated phyB proteins to Phytochrome Interacting Factor (PIF) proteins. We also intend to identify those phosphatases which dephosphorylate phyB. We will therefore express candidate phosphatase proteins in bacterial cells and treat phosphorylated phyB purified from plants with recombinant phosphatases purified from E.coli.

Finally we will determine whether phyB indeed functions as a kinase. To this end, we will define if phosphorylation detected in planta can be recapitulated at least partly in vitro by characterising autophosphorylation of phyB Pr and Pfr purified from insect cells. If phyB indeed autophosphorylates in vitro, we will use this approach to identify the catalytic domain and the ATP binding sites essential for kinase activity of the photoreceptor. Taken together, these experiments will help us deciphering whether phyB is phopshorylated by itself or other yet unknown kinases or if both of these mechanisms are involved in mediating post-translational modification of the photoreceptor.

The red/far-red light absorbing phytochromes regulate growth and development throughout the entire life cycle of plants. Many components and events of the molecular mechanism underlying phytochrome controlled signalling, culminating in light regulated responses, have been uncovered, yet we have very limited knowledge about how reversible phosphorylation of phytochrome-B (phyB) affects light induced signalling and photomorphogenic responses. It has been reported that mutants lacking specific phosphatases display hypersensitivity to red light, but up to now, no data are available to demonstrate that phyB is actually phosphorylated in planta. In this proposal we outlined a comprehensive research plan, based on a considerable amount of unpublished data, to define how this particular post-translational modification modulates phyB controlled signalling. Our goal is to determine the positions of all amino acid residues phosphorylated in planta and validate the importance of these sites by expressing mutant derivatives in transgenic lines. We will define, by using a broad array of photobiological and molecular assays, if phosphorylation of these amino acids exclusively regulates phyB signalling through modulating the rate of dark reversion or whether phosphorylation also alters interaction of phyB with its known signalling partners. We will test whether the phosphatases implicated in regulating phytochrome signalling are indeed capable of dephosphorylating phyB in vitro and perform a limited Y2H screen to isolate novel phophatases that could be involved in catalysing dephosphorylation of phyB. Finally, we will determine whether phyB autophosphorylates and/or phosphorylates other proteins in vitro and identify the catalytic and ATP-binding domains which are required for kinase activity. As these studies require large quantities of properly folded, photobiologically active phyB, we will express phyB in insect cells or in Pichia cells shown to be suitable to express oat phyA.

Plants are of vital importance for agriculture and also significantly contribute to mitigate climate changes. Plants are increasingly used as raw material for the production of fuel, fibre and many other economically important products. It follows that designing and breeding new varieties of crop plants that produce higher yields, exhibit optimal architecture suitable for specialised processing, as well as increasingly withstanding detrimental environmental stresses has become a globally important issue. Plant productivity is regulated by many environmental factors among which light plays a distinguished role as it regulates plant development from germination to seed setting. To optimize adaptation to changes in their light environment, plants have evolved a battery of photoreceptors. Our proposal outlines a research plan to assess the function of reversible phosphorylation of the photoreceptor phyB, the most important member of the phytochrome photoreceptor family in mature plants, which modulates light dependent plant growth and productivity.

Since the discovery that oat phyA autophosphorylates in vitro, the topic of reversible phosphorylation of phytochromes has been the subject of a considerable debate and controversy within the academic community. Despite a number of articles published in high profile, leading international journals, our knowledge is still rudimentary about how phytochromes function as enzymes. Our research is aimed at clarifying this topic by focusing on elucidating the key molecular events and components mediating reversible phosphorylation of phyB in the model plant Arabidopsis thaliana. It follows that the proposal is expected to have a significant academic impact as beside photobiologists, researchers interested in chronobiology, stress signalling, applied biotechnology and optogenetics will also benefit. The methodology we wish to develop will be suitable to isolate large quantities of the purified photoreceptor, a method that is badly missing from plant phytochrome research, thus it will be of help to many academic researchers worldwide. The proposed work is challenging for each of the participants, including the PI, Co-PI, and PDRA, as individually we all face new theoretical and methodical problems. Yet we believe that the chemistry of our team will enable us to overcome these hurdles and make further progress in our scientific careers.

The current proposal promises to give an insight into the molecular machinery that mediates this particular post-translational modification of phyB, and thereby light regulated photoreceptor controlled signalling that culminates in such agriculturally important responses as shade-avoidance, flowering time, seed germination and seedling establishment. As these photoreceptors are evolutionarily highly conserved, data obtained can be readily extended to crop plants and biotechnology and have the promise to open new avenues of research to understand how light and other environmentally-induced abiotic and biotic stress signalling are integrated. As both applicants are engaged in more targeted research related to the development of optogenetic tools, information obtained from the proposed work will also be beneficial in supporting these projects.

Plant photoreceptors and plant photobiology played a distinguished function in the birth of optogenetics and holds the promise for developing new tools for academic as well as commercial research. Basic research undertaken with Arabidopsis has proven to be useful to promote technology transfer to commercial enterprises, fertilising research on improving crop plants and playing an increasingly important role in developing new strategies for sustainable growth under the changing climate. Both applicants will utilize their existing links and develop new ones to communicate these important messages to policy makers as well as to the general public to increase awareness about the value of plant biology research.