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UV-resistance locus 8 and UV-B specific signaling in Arabidopsis thaliana

Headland, Lauren R. (2010) UV-resistance locus 8 and UV-B specific signaling in Arabidopsis thaliana. PhD thesis, University of Glasgow.

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Abstract

UV-B is a natural component of the sunlight spectrum. As a result of the potentially harmful effects of this radiation, plants have evolved a highly effective suit of protective and repair mechanisms. However, the signalling pathways that control such responses are not yet well known. For example while the photoreceptors responsible for red and blue light responses are well characterised, no such UV-B photoreceptor has yet been identified. Despite this particularly large gap in our knowledge, previous work identified the first UV- B specific signalling component which, unlike the more general stress-associated pathways often seen at high doses, specifically regulates expression of genes in response to even very low fluence rates of UV-B. This protein, UV-RESISTANCE LOCUS 8 (UVR8) regulates the induction of a number of photoprotective genes mostly via the transcription factors ELONGATED HYPOCOTYL 5 (HY5) and HY5 HOMOLOGUE (HYH). The end result of this pathway is the production of photoprotective compounds such as the flavonoids which enhance a plants ability to withstand UV-B stress. Thus UVR8 promotes plant fitness under these conditions. While we know that UVR8 binds to chromatin in the promoter region of HY5 and that it accumulates in the nucleus under UV-B, many other questions about this particular protein remain unanswered. For example, we do not yet know if UVR8-mediated UV-B signalling involves other factors which interact with UVR8 nor do we understand the mechanism by which UVR8 localisation is mediated. In addition, although we are aware of the importance of UVR8 in UV-B acclimation, it is unclear what roles might be played by other genes and proteins acting independently of this pathway. Therefore, the aims of this study were to investigate low fluence UV-B pathways that may act independently of UVR8 and to further examine the UVR8 protein itself both in terms of its interactions with other proteins and also in the role of the N-terminal region in regulation of its localisation. To achieve the first of these aims, RNA samples derived from plants treated with low fluence UV-B were submitted for microarray analysis. It was initially determined that the total number of genes induced was roughly equal in both low fluence treated samples and also to that found in the previous microarray performed by Brown et al. (2005) at a comparatively higher fluence. Thus, as only 72 genes have currently been linked to UVR8, there do appear to be many low-fluence UV-B induced pathways besides that regulated by UVR8. Several genes were analysed further using RT-PCR and qPCR methods in order to confirm their independence from the UVR8 signalling pathway components as well as assess their dependence on other hypothesised UV-B sensory mechanisms. It was found that while some genes did seem to be expressed independently of known photoreceptors, DNA damage signals as well as UVR8, HY5, HYH and COP1; one gene was expressed in a COP1-dependent but UVR8 independent manner. It therefore appears that at least four classes of genes are induced by UV-B; low fluence UVR8/HY5/HYH independent COP1 dependent, low fluence UVR8/HY5/HYH/COP1 dependent, low fluence UVR8/HY5/HYH/COP1 independent and finally high fluence non- specific signalling. The second portion of this thesis examined the structure and function of UVR8 in greater detail. To assist in this analysis, the BLAST sequence homology tool was used to probe both the Arabidopsis genome and available green plant sequences. It was found that 23 UVR8-like sequences exist in Arabidopsis but none of these appear to have similar N or C-terminal sequences to UVR8. As these two regions have previously been shown to be of vital importance in UVR8 function (Kaiserli and Jenkins, 2008; Kaiserli unpublished data) it is unlikely that any are acting in a redundant fashion to UVR8. A number of similar proteins to UVR8 can be found in other plant species. These potential homologues however fall into two categories based on their closer similarity with either UVR8 or its close homologue in humans REGULATOR OF CHROMATIN CONDENSATION 1 (RCC1). The wide variety of plant species that did show UVR8-like proteins suggests that this particular means of UV-B acclimation may have arisen relatively early with the colonisation of land plants. Interestingly, many of these likely homologues had a conserved N terminal. The N-terminal of UVR8 has previously been show to have a role in UV-B dependent nuclear accumulation (Kaiserli and Jenkins, 2008). This was examined further in Chapter 4 through the generation of a number of deletion and addition constructs in both a stable Arabidopsis uvr8-1 background as well as transiently in tobacco. From analysis of localisation of these constructs via confocal microscopy it was determined that the first 12 amino acids are sufficient but not necessary for nuclear accumulation, while the first 20 appear to be both necessary and sufficient. Indeed, it was shown that the initial 32 amino acids also confer constitutive localisation of a GFP tag in the nucleus regardless of light condition and despite the presence of a nuclear exclusion signal (NES). It therefore appears that this region, which shows strong conservation with UVR8-like proteins in other plant species, is of vital importance to the nuclear accumulation seen under UV-B. Finally, in Chapter 5, the possibility that UVR8 may be acting as part of a complex was explored. This involved use of size exclusion chromatography to provide approximate sizes of the UVR8 protein complex. It was found that native UVR8 appears to exist in a complex of about 70-90 kDa in size. This suggests that at least one other protein interacts stably with UVR8. Other fusion constructs were also analysed in this way, however the results were more difficult to interpret due the apparent artificial dimerisation of the GFP tag. In summary, the work presented here has shown that although UVR8 dependent pathways are predominant, a variety of low fluence UV-B induced genes and pathways may exist. Homology searches and mutational analyses suggest that the N-terminal region of UVR8 plays a critical role in its function and localisation. Finally, size exclusion chromatography suggests that UVR8 forms a complex in vivo with as yet uncharacterised partner proteins. In total these results provide further insight into the mechanisms UVR8 action and highlight new avenues for both UVR8 dependent and independent UV-B signalling.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Arabidopsis, UV-B, UVR8, light, signal transduction
Subjects: Q Science > QK Botany
Colleges/Schools: College of Medical Veterinary and Life Sciences > Institute of Molecular Cell and Systems Biology
Supervisor's Name: Jenkins, Prof. Gareth I.
Date of Award: 2010
Depositing User: Miss Lauren R Headland
Unique ID: glathesis:2010-1757
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 25 May 2010
Last Modified: 25 Apr 2013 07:35
URI: http://theses.gla.ac.uk/id/eprint/1757

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