The importance of specific tryptophans to UVR8 function: an intrinsic chromophore for a UV-B photoreceptor

[摘要] Although sessile organisms, unable to run away from danger, plants are well adapted to the potential harmful effects of sunlight’s high energy photons within the UV-B wavelength range (280-315 nm). For instance they are able to, among other things; produce their own sunscreen to counter any damage to their proteins, lipids and DNA. Plants of course depend on light as a source of energy for photosynthesis but also use specific wavelengths within the electromagnetic spectrum in a number of ways to act as an informational signal, including UV-B wavelengths, which can induce photomorphogenic responses that allow adaptation and survival for plants in the ever-changing environmental conditions they inhabit. It is now well established in plants that there are more than two pathways operating in response to different wavelengths and fluence rates of UV-B. In response to high, potentially damaging UV-B levels plants utilize a non-specific pathway which overlaps with other stress pathways such as pathogen attack and wounding by, for example, herbivores. And in response to low non-damaging UV-B levels plants utilize the UV-B specific photomorphogenic pathways which bring about acclimation, preparing the plant for potential higher doses and actively promoting plant survival (Jenkins and Brown, 2007). A number of photoreceptors have been identified in plants which act throughout the electromagnetic spectrum, but only in the last year has one been discovered operating at UV-B wavelengths. In fact until then no UV-B- specific photoreceptor had been found in any organism and it was not known how plants perceive UV-B light to initiate photomorphogenic responses. Over the last decade evidence was mounting in favour of the most upstream component of the UV-B photomorphogenic pathway and the only UV-B specific component, UVR8 (UV-RESISTANCE LOCUS 8) as being a UV-B photoreceptor. Now it has been demonstrated in plants to be a bona fide UV-B photoreceptor and to perceive UV-B by a novel mechanism (Rizzini et al., 2011, Christie et al., 2012, Wu et al., 2012). It has been demonstrated upon UV-B irradiation that UVR8 can dissociate from a homodimer to a monomer in vivo and in vitro. And unlike other conventional photoreceptors, which use a chromophore to detect specific wavelengths of light, UVR8 uses tryptophan residues found within its protein structure to carry out photoperception. When UV-B is detected via specific tryptophan residues found within the dimeric UVR8 protein, the energy is captured and used to cause disruption and breakage of several salt bridges between adjacent homodimers causing monomerization and subsequently leading to interaction with COP1 (CONSTITUTIVELY PHOTOMORPHOGENIC 1), nuclear accumulation and signal transduction (Christie et al., 2012; Wu et al., 2012; Favory et al 2009; Kaiserli and Jenkins 2007; Brown et al., 2005). Once UVR8 is in its active form it can then regulate the transcription of a number of UV-B responsive photomorphogenic genes allowing the plant to acclimate to counteract any future potential damage, which in turn promotes the plant’s survival and reproduction (Brown et al., 2005; Oravecz et al., 2006; Favory et al., 2009). When I first started my studies UVR8 was implicated in UV-B responses but it was unknown if it functioned as a photoreceptor. The purpose of my Ph.D was to determine if UVR8 was a UV-B photoreceptor and if so how it perceives UV-B. And more specifically, to address the question: can tryptophan residues within its structure act as an intrinsic chromophore?To investigate this aim I firstly used site directed mutagenesis to mutate specific and multiple tryptophan residues of the 14 found within UVR8’s structure to alanine, phenylalanine and tyrosine. Then I carried out transient expression studies in Nicotiana benthamiana to determine if the mutant protein tagged to GFP was stable and to determine if its subcellular localisation was affected. These UVR8 Trp mutant variants were further analyzed using yeast 2-hybrid assays (Y2H) to test for interaction with COP1, RUP1/RUP2 (REPRESSOR OF UV-B PHOTOMORPHOGENESIS) and also homodimerization. This allowed me to identify Trp mutant candidates to introduce transgenically into Arabidopsis and test further for their ability to complement the null mutant uvr8-1. The mutants were tested using a number of assays to check for monomer/dimer status, subcellular localisation, protein stability, COP1 interaction, photomorphogenic gene expression, hypocotyl inhibition and chromatin binding. Herein I present in vivo data in yeast and plants which shows, as reported by Rizzini et al. (2011), Christie et al. (2012) and Wu et al. (2012), that specific Trps, mainly W285 and W233 within the triad W233, W285, W337 have key roles in photoreception. W337 has a lesser role. These triad Trps, which are all in the conserved motif GWRHT, have now been shown in the UVR8 crystal structure to be brought into close proximity (Christie et al., 2012, Wu et al., 2012). The W285A mutant did not complement uvr8-1 and the W233A mutant only partially complemented, whereas W337A substantially complemented uvr8-1. And although all three Trp mutants constitutively interact with COP1 in planta before and after UV-B irradiation, this is not sufficient to rescue the uvr8-1 mutant for W285A and W233A, suggesting that although COP1 interaction is required for UV-B specific photomorphogenic responses it is not sufficient to mediate a response. Furthermore, for each of the triad mutants their dimer/monomer status is affected, and W285A is constitutively monomeric without being functional. Therefore, similar to COP1 interaction, monomerization on its own is not sufficient for UVR8 activation. In addition, I show that of the remaining 11 trps left of the 14 in total found within UVR8, some (W39, W144, W352) are important for structure and hence function, and the others (W92, 94, 196, 198, 250, 300, 302, 400) are not essential for function and/or structure. To further support the intrinsic Trp chromophore model of UVR8 I also present an action spectrum for dimer to monomer conversion for pure UVR8 protein in vitro from samples expressed and purified from E.coli. The spectrum closely resembles the absorption spectra of UVR8 and Trp in solution, with a maximum response at 280 nm. Moreover, the action spectrum partially resembles the in vivo UVR8 dependent HY5 (ELONGATED HYPOCOTYL 5) expression action spectrum published previously (Brown et al., 2009), although the in vivo HY5 study shows a substantial response at 300 nm, which this in vitro study lacks. Overall I show the importance of specific Trps to the UV-B photoreceptor UVR8 in yeast and in planta and demonstrate that W285 and W233 in particular are important in allowing UVR8 to function as a photoreceptor by acting as intrinsic chromophores.

[发布机构] University:University of Glasgow;Department:Institute of Molecular Cell and Systems Biology

[关键词] UVR8, tryptophans, intrinsic chromophore