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Subcellular localisation and functional analysis of UVR8, a UV-B specific signalling component in Arabidopsis

Kaiserli, Eirini (2008) Subcellular localisation and functional analysis of UVR8, a UV-B specific signalling component in Arabidopsis. PhD thesis, University of Glasgow.

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Abstract

UV-B is an integral component of the daylight spectrum that regulates plant gene expression and development, but very little is known about how plants perceive UV-B. Although UV-B-induced damage and repair have been extensively investigated, the mechanisms by which UV-B is perceived as a signal, which mediates physiological and protective responses is not yet clearly understood neither in mammals, nor in higher plants. Low fluence rates of UV-B induce the expression of genes involved in UV-protective responses such as flavonoid biosynthesis and promote plant survival in UV-B. The aim of this study is to contribute to the elucidation of the signal transduction events that lead to the acclimation of plants in response to non-damaging levels of UV-B (< 3.5 μmol m-2 s-1). In particular, the characterisation of UVR8 (UV-RESISTANCE LOCUS 8), a UV-B specific signalling component, is carried out at the protein level. The function of UVR8 involves the orchestration of the expression of a range of genes mediating vital UV-protective responses, including those encoding light-regulated transcription factors HY5 and HYH, enzymes involved in the phenylpropanoid pathway, antioxidant and stress proteins (Brown et al., 2005). UVR8 shows 30% sequence identity to the human regulator of chromatin condensation (RCC1) but differs both in activity and function. The phenotype of uvr8 mutant plants is characterised by an increased susceptibility to UV-B and the lack of the UV-B-specific induction of genes involved in UV-protection, such as CHS (encoding the flavonoid biosynthetic enzyme chalcone synthase) and the transcription factor HY5. The UVR8-mediated regulation of transcription in response to UV-B seems to occur via the association of UVR8 with chromatin via histones in the promoter region of HY5 (Brown et al., 2005) and other genes involved in light signalling. In this study, further investigation of the mechanism by which UVR8 acts as a UV-B specific signalling component is performed by employing a number of approaches including: spatial, temporal protein analysis, subcellular localisation studies, structure-function analyses, and the yeast-two-hybrid assay for the identification of UVR8 interacting proteins. To study spatial, temporal and wavelength specific UVR8 protein abundance anti-UVR8 peptide antibodies were generated. Western blot analyses showed that UVR8 is ubiquitously expressed in all plant tissues from the very early stages of development and at every light treatment tested (dark, white light, UV-B). The subcellular localisation of UVR8 analysed by confocal fluorescence microscopy revealed that a fusion of UVR8 with green fluorescent protein (GFP) is localised in the cytoplasm and the nucleus of various plant tissues (leaf, hypocotyl, root, flower) and under various light fluence rates and qualities (white, red, UV-A, UV-B). Interestingly, a treatment of low fluence rates of UV-B led to an increase of GFP-UVR8 protein accumulation in the nucleus, which was confirmed by western blot analysis based on protein fractionation studies in wild-type plants. The wavelength specificity, the kinetics and the fluence-rate sensitivity of GFP-UVR8 nuclear accumulation suggest that this response is UV-B specific, rapid (10 min UV-B) and very sensitive to very low fluence rates of UV-B (0.1 μmol m-2 s-1). Protein synthesis does not seem to be involved in this process, as there is no change in the protein levels before and after a UV-B irradiation. To assess the importance of the presence of UVR8 in the nucleus and the cytoplasm of the plant cell, uvr8-1 transgenic plants were produced expressing either constitutively nuclear localised GFP-UVR8 fused to a nuclear localisation signal (NLS), or cytosolically retained GFP-UVR8 fused to a nuclear export signal (NES). Nuclear exclusion of NES-GFP-UVR8 fusion protein was sustained under most light conditions apart from UV-B, which induced nuclear import of the protein. This indicates that the mechanism involved in the nuclear accumulation of UVR8 can overcome an export signal either by masking it or by simply superseding it. Furthermore, the NES-GFP-UVR8 construct was functional after UV-B treatment, since it rescued the mutant uvr8 phenotype. None of the inhibitor treatments tested (staurosporine, cycloheximide, cantharidin) was successful in blocking the UV-B induced nuclear import of NES-GFP-UVR8, although they impaired the UVR8 regulated induction of CHS expression. Thus, no evidence is presented for a specific protein modification, which could control this response. Constitutive nuclear localisation of NLS-GFP-UVR8 had no effect on the function of the protein according to complementation analyses. Furthermore, no change in localisation, fluorescence intensity or protein abundance was observed in response to white light or after a UV-B irradiation. These results indicate that the constitutive nuclear localisation of UVR8 is not sufficient for constitutive activation of UVR8 regulated gene expression and that a UV-B stimulus is still necessary to trigger these responses. Unfortunately, based on the current data it cannot be concluded whether the UV-B signal perception occurs in the nucleus or in the cytosol of the plant cell. To investigate the structure-function relationship within the UVR8 protein, deletion analyses followed by complementation studies in transgenic plants were performed. More specifically, deletion of the first 23 amino acids at the N-terminus of UVR8 impaired its nuclear accumulation in response to UV-B. Deletion of a 27 amino acid region near the C-terminus had no effect on the UV-B dependent re-localisation of the protein, but abolished UVR8 regulated gene expression. In addition, a highly basic sequence at the extreme C-terminal of UVR8, resembling a putative monopartite nuclear localisation signal, was deleted. Subcellular localisation and complementation analyses suggest that this sequence does not serve as a nuclear localisation signal, it is not involved in the UV-B induced nuclear accumulation and its absence does not affect UVR8 protein function. Chromatin immunoprecipitation assays show that none of the regions deleted is required for chromatin association and none of the deletions affects subcellular localisation in white light. In order to identify interacting partners for UVR8, the yeast-two hybrid system was used. Unfortunately no interacting proteins have been identified, neither from a screen, nor by directed-interaction studies. A different approach could be employed in the future involving size exclusion chromatography of protein extracts from plants in order to establish whether UVR8 functions as part of a complex in vivo.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: UV-B, light, nuclear translocation
Subjects: Q Science > QK Botany
Q Science > Q Science (General)
Colleges/Schools: College of Medical Veterinary and Life Sciences > Institute of Molecular Cell and Systems Biology
Supervisor's Name: Jenkins, Professor Gareth I
Date of Award: 2008
Depositing User: Miss Eirini Kaiserli
Unique ID: glathesis:2008-57
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 06 Nov 2008
Last Modified: 10 Dec 2012 13:15
URI: http://theses.gla.ac.uk/id/eprint/57

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