Role of tryptophans in UVR8 photoreceptor function in arabidopsis

Plants are exposed to a variety of environmental cues that can affect their development. Of these, light is the most important as apart from being their energy source, it also serves as an informational cue that will allow them to control a range of photomorphogenic responses throughout their life cycle. Due to their sessile nature, plants are inevitably exposed to the highest energy photons of the solar radiation that reach the Earth’s surface, Ultraviolet-B (UV-B) light. Although being a potentially harmful and damaging agent, UV-B also acts as a key environmental signal that will allow regulation of several aspects of plant metabolism, morphology and physiology via differential regulation of gene expression. These photomorphogenic responses UV-B light such as gene expression, inhibition of hypocotyl elongation, leaf expansion or increased biosynthesis of UV-B absorbing compounds like flavonoids, are activated by the photoreceptor UV RESISTANCE LOCUS 8 (UVR8) after exposure to low intensity ultraviolet-B (UV-B) (Jenkins, 2017a). ELONGATED HYPOCOTYL 5 (HY5) is one of the genes regulated by UVR8 which has a key-role in UVR8-mediated responses. Its transcription activates rapidly after exposure to UV-B and it has been found to regulate approximately one half of the genes regulated by UVR8. Additionally, the hyper sensitivity of hy5 mutant to low UV-B levels has further confirmed its important role in UVR8-mediated signalling (Brown et al., 2005; Brown & Jenkins, 2008). In the absence of UV-B, UVR8 is present as a homodimer in the cytosol and nucleus. The most upstream event of the UVR8-signalling pathway is its monomerisation followed by perception of UV-B. This will lead to conformational changes that will allow interaction to CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1), subsequent nuclear accumulation and initiation of the photomorphogenic responses (Jenkins, 2014b). Recent in vitro and in vivo studies have shown that UVR8 monomer can undergo UV-B photoreception as a constitutively monomeric UVR8 mutant can activate photomorphogenic responses after UV-B perception (Heilmann et al., 2016). Whereas the absorption spectrum of UVR8 peaks at 280 nm (Christie et al., 2012), the action spectra of several photomorphogenic UV responses show maximal photon effectiveness at 290-300 nm (Díaz-Ramos et al., 2018). To investigate this discrepancy, the wavelength effectiveness of two UV responses were measured: UVR8 monomerisation and the accumulation of HY5 transcripts. When exposing intact plants, monomerisation peaked at 290 nm, whereas the accumulation of HY5 transcripts, measured in the same plant tissue samples, was maximal at 300 nm. This inconsistency was thought to be due to photoreception of UV-B made by UVR8 dimer and monomer would shift the peak of HY5 transcript accumulation to the longer wavelengths. Therefore, the constitutively monomeric mutant was also tested for wavelength effectiveness of HY5 transcript accumulation. Results showed that the UVR8 dimer was more efficient in responding to UV-B than UVR8 monomer. The monomer had a major peak at 295 nm and a more reciprocate response to the longer wavelengths (300 and 305 nm) than the shorter ones (285 and 290 nm). An important characteristic of UVR8, which makes it different from other plant photoreceptors is that it does not employ an external co-factor as a chromophore. UV-B perception by UVR8 is done via tryptophans located in the dimer interface (Christie et al., 2012; Wu et al., 2012). UVR8 is rich in aromatic residues it contains six Trps in the core of the protein, seven in the dimer interface and one in the C-terminal. UVR8 has a conserved and repeated motif GWRHT that generates a triad of closely packed tryptophan residues (W233, W285 and W337). These together with W94 from the other monomer form a Trp pyramid of excitonically coupled orbitals in the dimer interface (Christie et al., 2012; Wu et al., 2012). In vitro studies have shown that proton-coupled electron transfer constitutes the photoactivation mechanism of UVR8 (Mathes et al., 2015). Furthermore, W285 and W233 have been shown to be the key players in UV-B photoreception (Christie et al., 2012; Huang et al., 2014; O'Hara & Jenkins, 2012; Rizzini et al., 2011; Wu et al., 2012). The role of the rest of the UVR8 Trp in UV-B photoreception in vivo has been analysed previously with results showing the Trps located in the core are not important for UVR8 function and some of them have a role in maintaining UVR8 structure (O'Hara & Jenkins, 2012). Moreover, apart from W285 and W233 Trps in the dimer interface are not essential for structure or function (Huang et al., 2014; O'Hara & Jenkins, 2012). These studies analysed mutation of this Trps under saturating UV-B conditions so a potential role under non-saturating conditions has not been defined yet. In this research, conserved mutations of the six core Trps to Tyrs produced a very unstable UVR8 protein that was able to form the homodimer in the absence of UV-B and monomerise in response to it, further confirming their role in UVR8 structure. Additionally, the contribution of the other 5 tryptophans in the dimer interface to UV-B photoreception was analysed under non-saturating UV-B. Mutations of tryptophans W94F, W337F and W198/250/302F were found to weaken the interaction between the monomers in the dimer interface at different degrees and decrease their efficiency in generating a response (HY5 transcript accumulation), when plants were exposed to different doses of UV-B. These mutants were also tested for wavelength effectiveness experiments with results showing a major peak at 295 nm, consistent with the monomeric UVR8 mutant and WT GFP-UVR8. Furthermore, the possibility of these Trps acting as light harvesters of UV-B was confirmed