Characterisation of a UV-B-specific signalling pathway

Lead Research Organisation: University of Glasgow
Department Name: Institute of Biomedical & Life Sciences

Abstract

UV-B wavelengths (280-320 nm) are an integral component of the daylight spectrum. UV-B induces physiological responses in plants, including changes in morphology and development and the regulation of various genes involved in secondary metabolism, chloroplast function and protection of the plant from damage by UV-B. Remarkably very little is known about how UV-B is perceived by plants and how it initiates responses. However, the first component involved in these processes has recently been identified, a protein called UVR8. The aim of this project is to characterise the UVR8 pathway, its interaction with other photoregulatory pathways and components, to understand how UV-B regulates UVR8 and to determine how UVR8 regulates transcription of target genes. Establishing how UVR8 is regulated and how it functions to control gene expresion 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 (280-320 nm) induce physiological responses in plants, but very little is known about the processes of UV-B perception and signal transduction. We have recently discovered that the Arabidopsis protein UVR8 is a UV-B-specific signalling component that mediates a range of gene expression responses involved in secondary metabolism, anti-oxidant defence and UV-protection and have shown that UVR8 associates with chromatin to regulate the expression of a key target gene, encoding the transcription factor HY5. The initial aim of this project is to characterise the UVR8 pathway, determine its interaction with other light signalling pathways and examine whether it regulates UV-B responses other than gene expression. We will then investigate the UV-B regulation of the cellular localisation, abundance and phosphorylation of UVR8 and how this regulation is influenced by specific components. Finally we will examine whether UV-B regulates the association of UVR8 with chromatin and how UVR8 acts to control HY5 transcription and protein accumulation.
 
Description Characterisation of the UVR8 pathway
Photobiological characterisation We defined conditions to produce quantitative dose response data at different UV-B wavelengths for HY5 transcript levels and completed an action spectrum for HY5 transcript accumulation that showed maximum action at 280 nm and a smaller peak at 300 nm (published in Photochem. Photobiol.).
Integration with other light signalling pathways We have defined a number of genes that are either UVR8-dependent or UVR8-independent, examined their fluence-response profiles and showed that the known photoreceptors (phy, cry, phot) are not involved in their expression. In addition, we have demonstrated that the UVR8 pathway is mediated by HY5 acting to some extent redundantly with HYH. We have evidence that all the UVR8 target genes are regulated in this way and, moreover, that HY5/HYH do not appear to regulate UV-B stimulated, UVR8-independent genes. This work was published in Plant Physiology.
Role of UVR8 in other responses. We have examined the involvement of UVR8 in morphogenic responses in collaboration with Dr J. Wargent and Prof. N. Paul (University of Lancaster) and Dr R. Ulm (University of Freiburg). The uvr8 mutant lacks suppression of hypocotyl extension in low fluence rates of UV-B. UV-B reduces leaf area and this response includes two components, a reduction in epidermal cell number and an increase in epidermal cell size at low fluence rates of UV-B. The uvr8 mutant is unaltered in the change in cell number but lacks the stimulation of cell size and therefore has smaller leaves than wild-type in UV-B. These findings are published in two joint papers (Favory et al., 2009; Wargent et al., 2009).

Regulation of UVR8 by UV-B
Regulation of UVR8 localisation UV-B illumination promotes nuclear accumulation of GFP-UVR8 in plants grown in light lacking UV-B (published in Plant Cell). However, movement out of the nucleus in darkness is very slow (about 10-20% reduction after 24 hours) and hence there is little change over the diurnal cycle.
Regulation of UVR8 abundance We found that the levels of native UVR8 and of GFP-UVR8 expressed from the UVR8 promoter are essentially constant over the diurnal cycle and in response to various light qualities (published in Plant Cell). In addition, we monitored the abundance of UVR8 in plants treated with either MG132 or cycloheximide and found there was little change over a 12 hour period, indicating that UVR8 is not subject to rapid turnover.
Is UVR8 subject to phospohorylation? We have not obtained any reproducible data to indicate that UVR8 is modified post-translationally resulting in a change of mobility following electrophoresis.
Do other components modulate UVR8 localisation, abundance or activity? We have found that the cop1-4 mutant is not altered in the accumulation of UVR8.

Regulation of HY5 by UVR8
We found that UV-B is not required for the association of UVR8 with chromatin containing the HY5 gene (published in Molecular Plant). The association of UVR8 with chromatin is not altered in the cop1-4 mutant and this observation has been published in Favory et al. (2009).
Exploitation Route The findings provide a basis for further studies of the physiological function, UVR8 structure/function relationships and regulation of UVR8. 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.
Sectors Agriculture

Food and Drink