How does the plant UV-B photoreceptor UVR8 initiate signalling?

Lead Research Organisation: University of Glasgow
Department Name: College of Medical, Veterinary, Life Sci

Abstract

Ultraviolet-B (UV-B) radiation is a minor but very energetic component of sunlight. Exposure to UV-B wavelengths (280-315 nm) has numerous effects on plants, including changes in metabolism and development. Importantly, UV-B stimulates responses in plants that protect them against the potentially damaging consequences of UV-B exposure. The effects of UV-B are due to its ability to regulate the expression of numerous plant genes, including those involved in UV-protection, biosynthesis and chloroplast function. It is therefore important to understand how UV-B is perceived by plants and how it initiates responses. A key component involved in these processes has been identified, a protein called UVR8. UVR8 acts as a photoreceptor to detect UV-B light. UVR8 physically interacts with another protein, COP1, to initiate responses to UV-B in plants. The aim of this project is to enhance understanding of how UVR8 interacts with COP1 to initiate plant responses to UV-B. Establishing the molecular mechanism of this interaction will help us to understand how UV-B regulates aspects of plant growth and development and how plants survive in sunlight.

Technical Summary

UV-B wavelengths (280-315 nm) induce a range of physiological responses in plants, but the mechanisms by which these responses are initiated are poorly understood. Our research has shown that the Arabidopsis protein UV RESISTANCE LOCUS8 (UVR8) is a UV-B-specific photoreceptor that regulates the expression of a set of genes involved in processes that protect the plant against damage by UV-B, including secondary metabolism, anti-oxidant defence and DNA repair. UVR8 associates with chromatin to regulate the expression of target genes. UV-B absorption converts the UVR8 protein from a dimer to a monomer and induces its direct interaction with the plant protein COP1. Recently we reported the crystal structure of UVR8 and showed how it acts to absorb UV-B and how photoreception causes monomer formation. However, we do not know how photoreception enables UVR8 to interact with COP1. The aim of this project is to gain a molecular understanding of how UV-B absorption changes UVR8 so that it is able to interact physically with COP1 to initiate signal transduction.

Planned Impact

Beneficiaries The beneficiaries and users of the research will include: organisations in the commercial sector interested in novel strategies to improve crop productivity; agencies and policy makers interested in the effects of UV-B on organisms and ecosystems in relation to depletion of the ozone layer and effects of increasing UV-B radiation (including the relevant United Nations Environmental Programme - UNEP - panel); individuals (including text book authors) and organisations (e.g. Glasgow Science Centre) involved in science communication to schools and the wider public. The general public, in so far as they are interested in the effects of UV-B on organisms and ecosystems.
Benefits The impact of the research to the beneficiaries derives both from (i) its potential relevance to crop plant improvement and agricultural practice and (ii) the relevance to understanding the impact of UV-B on the biosphere. (i) UV-B impacts on agricultural as well as natural ecosystems and therefore has direct relevance to crop plants. UV-B signalling pathways regulate biosynthetic activities (and hence plant biochemical composition), prime defence responses (e.g. UV-B exposure reduces damage by herbivorous insects in a range of species) and regulate aspects of morphogenesis and development of relevance to crops (e.g. leaf expansion, extension growth and branching). Furthermore there is evidence that UV-B interacts with a number of signalling pathways to modify responses to a variety of abiotic factors (e.g. drought, low temperature and various mineral nutrients). Research to understand UV-B perception, signalling and response therefore has the potential to generate novel strategies for crop plant improvement that could benefit farmers, consumers and the environment and contribute to the economic competitiveness of the UK. In addition, there are examples where manipulation of the UV-B environment is being used in agriculture to help control pests. Thus it is important to understand how altering the UV-B environment may affect plant processes and investigation of the mechanisms of UV-B perception and signalling is key to this. (ii) Plants are key components of natural ecosystems and UV-B has broad impacts on ecosystem function. Concern over depletion of the stratospheric ozone layer by human activities has promoted an interest in understanding how plants perceive UV-B. This information is being used to inform policy makers who are concerned with maintaining human health and the quality of life. The general public will benefit in a cultural sense from the increase in knowledge and understanding of the effects of UV-B on plants. The public can relate to effects of UV-B such as sunburn and skin cancer and so the idea that plants manage to avoid damage by UV-B through perceiving and responding to UV-B is accessible. The above impacts of the research will be realised over the short to medium term. Staff working on the project will obtain knowledge and expertise that can be applied in related research or more widely in the commercial or public sectors.

Publications

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Jenkins GI (2014) Structure and function of the UV-B photoreceptor UVR8. in Current opinion in structural biology

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Heilmann M (2015) Photoinduced transformation of UVR8 monitored by vibrational and fluorescence spectroscopy. in Photochemical & photobiological sciences : Official journal of the European Photochemistry Association and the European Society for Photobiology

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Heilmann M (2016) Dimer/monomer status and in vivo function of salt-bridge mutants of the plant UV-B photoreceptor UVR8. in The Plant journal : for cell and molecular biology

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Liao X (2019) A FRET method for investigating dimer/monomer status and conformation of the UVR8 photoreceptor. in Photochemical & photobiological sciences : Official journal of the European Photochemistry Association and the European Society for Photobiology

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Camacho IS (2019) Native mass spectrometry reveals the conformational diversity of the UVR8 photoreceptor. in Proceedings of the National Academy of Sciences of the United States of America

 
Description NMR We have used nuclear magnetic resonance (NMR) spectroscopy to examine the location of the C-terminus and conformational changes induced by photoreception. One of the findings of the NMR study is that the C-terminal region of UVR8 does not have an ordered structure. It has been suggested recently that the N- and C-termini may be locked together through electrostatic interactions to form a separate domain containing a 4-stranded ß-sheet in the inactive state to prevent the C27 region interacting with COP1 and RUP proteins, and that UV-B induced dimer dissociation enables unlocking the termini. However, our data do not support this model. Both the N- and C-termini adopt random coil conformations and there is no sign of regular secondary structure. We are presently completing the NMR experiments and data interpretation in preparation for publication.
Fluorescence anisotropy As a complementary approach to NMR to probe the relative mobility of UVR8's C-terminal region, we have used fluorescence polarisation anisotropy spectroscopy. We have undertaken the proposed mutagenesis of the exposed C-terminal C317 to serine in UVR8. In the C317S background we made a series of mutant proteins with cysteine mutations introduced at different positions into the C-terminus to enable labelling with a fluorophore. We fluorescently labelled each mutant protein and made measurements of fluorescence anisotropy before and after UV-B illumination of the protein. The results show increasing mobility of more distal residues in the C-terminus. In addition, there is no indication that UV-B exposure alters mobility of the C-terminus. So our data do not indicate that the C-terminus is in some way fixed to the core of the protein prior to UV-B exposure and becomes released to interact with other proteins following photoreception.
As an additional approach to examine the C-terminus we used FTIR spectroscopy in collaboration with Drs J. Kennis and T. Mathes (Amsterdam). These data (Heilmann et al. 2014) also support the concept that there are no substantial conformational changes in the C-terminus. This collaboration also provided new information on the mechanism of UVR8 photoreception (Mathes et al. 2015).

Which amino acids in the C27 region are required for COP1 binding? We used the yeast 2-hybrid assay to test binding of mutants of UVR8 to COP1 and RUP1 and RUP2. Mutants were made for each residue in the C27 region and the assays provided information on the importance of individual amino acids in the interactions. In addition, we produced transgenic plants to test the functional significance of particular mutations. These experiments are being completed in advance of publication.
In summary, we substantially completed our original objectives, and used some additional approaches. Two papers have been published and at least one more is planned.
Exploitation Route The findings provide a basis for further studies of the relationship between UVR8 structure and function. We are pursuing several aspects of this research. Since UV-B light has a wide ranging impact on plant development, metabolism, defence and responses to abiotic stimuli, knowledge of how UVR8 functions has potential applications in the agricultural sector. In addition, fundamental knowledge of how UVR8 protein functions has application in providing ontogenetic tools for biotechnological applications.
Sectors Agriculture

Food and Drink