Phosphorylation of Arabidopsis UVR8 photoreceptor modulates protein interactions and responses to UV-B radiation.

Exposure of plants to ultraviolet-B (UV-B) radiation initiates transcriptional responses that modify metabolism, physiology and development to enhance viability in sunlight. Many of these regulatory responses to UV-B radiation are mediated by the photoreceptor UV RESISTANCE LOCUS 8 (UVR8). Following photoreception, UVR8 interacts directly with multiple proteins to regulate gene expression, but the mechanisms that control differential protein binding to initiate distinct responses are unknown. Here we show that UVR8 is phosphorylated at several sites and that UV-B stimulates phosphorylation at Serine 402. Site-directed mutagenesis to mimic Serine 402 phosphorylation promotes binding of UVR8 to REPRESSOR OF UV-B PHOTOMORPHOGENESIS (RUP) proteins, which negatively regulate UVR8 action. Complementation of the uvr8 mutant with phosphonull or phosphomimetic variants suggests that phosphorylation of Serine 402 modifies UVR8 activity and promotes flavonoid biosynthesis, a key UV-B-stimulated response that enhances plant protection and crop nutritional quality. This research provides a basis to understand how UVR8 interacts differentially with effector proteins to regulate plant responses to UV-B radiation.

Cysteines have a role in conformation of the UVR8 photoreceptor.

The UV RESISTANCE LOCUS 8 (UVR8) photoreceptor mediates plant responses to Ultraviolet-B (UV-B) wavelengths. The UVR8 dimer dissociates into monomers following UV-B photoreception, a process accompanied by conformational changes that facilitate interaction of UVR8 with proteins that initiate responses. However, the importance of particular amino acids in maintaining UVR8 conformation and modulating protein interactions is poorly understood. Here we examine the roles of cysteine amino acids C231 and C335 in UVR8 structure and function. UVR8C231S,C335S mutant protein forms dimers and monomerizes similarly to wild-type UVR8. UVR8C231S,C335S interacts with CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1) in plants to initiate photomorphogenic responses to UV-B, although the interaction is weaker when examined in yeast two-hybrid assays. Similarly, the interaction of UVR8C231S,C335S with REPRESSOR OF UV-B PHOTOMORPHOGENESIS (RUP) proteins is weaker in both plants and yeast compared with wild-type UVR8. Re-dimerization of UVR8 in plants, which is mediated by RUP proteins, occurs with reduced efficiency in UVR8C231S,C335S . Fluorescence resonance energy transfer analysis indicates that UVR8C231S,C335S has an altered conformation in plants, in that the N- and C-termini appear closer together, which may explain the altered protein interactions.

The photoreceptor UVR8 mediates the perception of both UV-B and UV-A wavelengths up to 350 nm of sunlight with responsivity moderated by cryptochromes.

The photoreceptors UV RESISTANCE LOCUS 8 (UVR8) and CRYPTOCHROMES 1 and 2 (CRYs) play major roles in the perception of UV-B (280-315 nm) and UV-A/blue radiation (315-500 nm), respectively. However, it is poorly understood how they function in sunlight. The roles of UVR8 and CRYs were assessed in a factorial experiment with Arabidopsis thaliana wild-type and photoreceptor mutants exposed to sunlight for 6 or 12 hr under five types of filters with cut-offs in UV and blue-light regions. Transcriptome-wide responses triggered by UV-B and UV-A wavelengths shorter than 350 nm (UV-Asw ) required UVR8 whereas those induced by blue and UV-A wavelengths longer than 350 nm (UV-Alw ) required CRYs. UVR8 modulated gene expression in response to blue light while lack of CRYs drastically enhanced gene expression in response to UV-B and UV-Asw . These results agree with our estimates of photons absorbed by these photoreceptors in sunlight and with in vitro monomerization of UVR8 by wavelengths up to 335 nm. Motif enrichment analysis predicted complex signaling downstream of UVR8 and CRYs. Our results highlight that it is important to use UV waveband definitions specific to plants' photomorphogenesis as is routinely done in the visible region.

A FRET method for investigating dimer/monomer status and conformation of the UVR8 photoreceptor.

The photoreceptor UVR8 has a pivotal role in mediating plant responses to UV-B wavelengths. Dimeric UVR8 dissociates into monomers following UV-B photoreception, and there is evidence that this process is accompanied by conformational changes that may facilitate interaction of UVR8 with other proteins to initiate signaling. Hence monitoring UVR8 dimer/monomer status and conformation is key to understanding UVR8 action. Here we have used Fluorescence Resonance Energy Transfer (FRET) to study these processes in both wild-type and mutant UVR8 proteins in vivo. UVR8 was fused to GFP and mCherry at the C- and N-termini, respectively and both the FRET efficiency and loss of GFP fluorescence after photobleaching were measured. In addition, measurements were made for UVR8 fused to either GFP or mCherry to eliminate intra-molecular FRET signals. The results indicate that dissociation of UVR8 dimer to monomer principally accounts for the loss of FRET signal for wild-type UVR8 and there is little evidence of a contribution from conformational change in vivo. Examination of plants expressing UVR8W285F and UVR8D96N,D107N are consistent with these mutant proteins being constitutively dimeric and monomeric, respectively. The methods employed here will be valuable for monitoring UVR8 dimer/monomer status in vivo in relation to signaling, and will facilitate characterization of dimer/monomer status and conformation of further UVR8 mutants.

Evolutionary conservation of structure and function of the UVR8 photoreceptor from the liverwort Marchantia polymorpha and the moss Physcomitrella patens.

The ultraviolet-B (UV-B) photoreceptor UV RESISTANCE LOCUS 8 (UVR8) mediates photomorphogenic responses to UV-B in Arabidopsis through differential gene expression, but little is known about UVR8 in other species. Bryophyte lineages were the earliest diverging embryophytes, thus being the first plants facing the UV-B regime typical of land. We therefore examined whether liverwort and moss species have functional UVR8 proteins and whether they are regulated similarly to Arabidopsis UVR8. We examined the expression, dimer/monomer status, cellular localisation and function of Marchantia polymorpha and Physcomitrella patens UVR8 in experiments with bryophyte tissue and expression of green fluorescent protein (GFP)-UVR8 fusions in Nicotiana leaves and transgenic Arabidopsis. P. patens expresses two UVR8 genes that encode functional proteins, whereas the single M. polymorpha UVR8 gene expresses two transcripts by alternative splicing that encode functional UVR8 variants. P. patens UVR8 proteins form dimers that monomerise and accumulate in the nucleus following UV-B exposure, similar to Arabidopsis UVR8, but M. polymorpha UVR8 has weaker dimers and the proteins appear more constitutively nuclear. We conclude that liverwort and moss species produce functional UVR8 proteins. Although there are differences in expression and regulation of P. patens and M. polymorpha UVR8, the mechanism of UVR8 action is strongly conserved in evolution.

Q&A: How do plants sense and respond to UV-B radiation?

Plants are able to sense UV-B through the UV-B photoreceptor UVR8. UV-B photon absorption by a UVR8 homodimer leads to UVR8 monomerization and interaction with the downstream signaling factor COP1. This then initiates changes in gene expression, which lead to several metabolic and morphological alterations. A major response is the activation of mechanisms associated with UV-B acclimation and UV-B tolerance, including biosynthesis of sunscreen metabolites, antioxidants and DNA repair enzymes. To balance the response, UVR8 is inactivated by regulated re-dimerization. Apart from their importance for plants, UVR8 and its interacting protein COP1 have already proved useful for the optogenetic toolkit used to engineer synthetic light-dependent responses.

Multiple roles for UV RESISTANCE LOCUS8 in regulating gene expression and metabolite accumulation in Arabidopsis under solar ultraviolet radiation.

Photomorphogenic responses triggered by low fluence rates of ultraviolet B radiation (UV-B; 280-315 nm) are mediated by the UV-B photoreceptor UV RESISTANCE LOCUS8 (UVR8). Beyond our understanding of the molecular mechanisms of UV-B perception by UVR8, there is still limited information on how the UVR8 pathway functions under natural sunlight. Here, wild-type Arabidopsis (Arabidopsis thaliana) and the uvr8-2 mutant were used in an experiment outdoors where UV-A (315-400 nm) and UV-B irradiances were attenuated using plastic films. Gene expression, PYRIDOXINE BIOSYNTHESIS1 (PDX1) accumulation, and leaf metabolite signatures were analyzed. The results show that UVR8 is required for transcript accumulation of genes involved in UV protection, oxidative stress, hormone signal transduction, and defense against herbivores under solar UV. Under natural UV-A irradiance, UVR8 is likely to interact with UV-A/blue light signaling pathways to moderate UV-B-driven transcript and PDX1 accumulation. UVR8 both positively and negatively affects UV-A-regulated gene expression and metabolite accumulation but is required for the UV-B induction of phenolics. Moreover, UVR8-dependent UV-B acclimation during the early stages of plant development may enhance normal growth under long-term exposure to solar UV.

The UV-B photoreceptor UVR8 promotes photosynthetic efficiency in Arabidopsis thaliana exposed to elevated levels of UV-B.

The UV-B photoreceptor UVR8 regulates expression of genes in response to UV-B, some encoding chloroplast proteins, but the importance of UVR8 in maintaining photosynthetic competence is unknown. The maximum quantum yield of PSII (F (v)/F(m)) and the operating efficiency of PSII (Φ(PSII)) were measured in wild-type and uvr8 mutant Arabidopsis thaliana. The importance of specific UVR8-regulated genes in maintaining photosynthetic competence was examined using mutants. Both F (v)/F(m) and Φ(PSII) decreased when plants were exposed to elevated UV-B, in general more so in uvr8 mutant plants than wild-type. UV-B increased the level of psbD-BLRP (blue light responsive promoter) transcripts, encoding the PSII D2 protein. This increase was mediated by the UVR8-regulated chloroplast RNA polymerase sigma factor SIG5, but SIG5 was not required to maintain photosynthetic efficiency at elevated UV-B. Levels of the D1 protein of PSII decreased markedly when plants were exposed to elevated UV-B, but there was no significant difference between wild-type and uvr8 under conditions where the mutant showed increased photoinhibition. The results show that UVR8 promotes photosynthetic efficiency at elevated levels of UV-B. Loss of the DI polypeptide is probably important in causing photoinhibition, but does not entirely explain the reduced photosynthetic efficiency of the uvr8 mutant compared to wild-type.

UVR8 in Arabidopsis thaliana regulates multiple aspects of cellular differentiation during leaf development in response to ultraviolet B radiation.

Responses specific to ultraviolet B (UV-B) wavelengths are still poorly understood, both in terms of initial signalling and effects on morphogenesis. Arabidopsis thaliana UV RESISTANCE LOCUS8 (UVR8) is the only known UV-B specific signalling component, but the role of UVR8 in leaf morphogenesis is unknown. The regulatory effects of UVR8 on leaf morphogenesis at a range of supplementary UV-B doses were characterized, revealing both UVR8-dependent and independent responses to UV irradiation. Inhibition of epidermal cell division in response to UV-B is largely independent of UVR8. However, overall leaf growth under UV-B irradiation in wild-type plants is enhanced compared with a uvr8 mutant because of a UVR8-dependent compensatory increase of cell area in wild-type plants. UVR8 was also required for the regulation of endopolyploidy in response to UV-B, and the uvr8 mutant also has a lower density of stomata than the wild type in the presence of UV-B, indicating that UVR8 has a regulatory role in other developmental events. Our findings show that, in addition to regulating UV-protective gene expression responses, UVR8 is involved in controlling aspects of leaf growth and morphogenesis. This work extends our understanding of how UV-B response is orchestrated at the whole-plant level.

Signal transduction in responses to UV-B radiation.

UV-B radiation is a key environmental signal that initiates diverse responses in plants that affect metabolism, development, and viability. Many effects of UV-B involve the differential regulation of gene expression. The response to UV-B depends on the nature of the UV-B treatment, the extent of adaptation and acclimation to UV-B, and interaction with other environmental factors. Responses to UV-B are mediated by both nonspecific signaling pathways, involving DNA damage, reactive oxygen species, and wound/defense signaling molecules, and UV-B-specific pathways that mediate photomorphogenic responses to low levels of UV-B. Importantly, photomorphogenic signaling stimulates the expression of genes involved in UV-protection and hence promotes plant survival in UV-B. Photomorphogenic UV-B signaling is mediated by the UV-B-specific component UV RESISTANCE LOCUS8 (UVR8). Both UVR8 and CONSTITUTIVE PHOTOMORPHOGENESIS1 (COP1) are required for UV-B-induced expression of the ELONGATED HYPOCOTYL5 (HY5) transcription factor, which plays a central role in the regulation of genes involved in photomorphogenic UV-B responses.

Members of the c1/pl1 regulatory gene family mediate the response of maize aleurone and mesocotyl to different light qualities and cytokinins.

We investigated the role of transcription factors (R, SN, C1, and PL) in the regulation of anthocyanin biosynthesis by different light qualities (white, red, blue, and ultraviolet) and by cytokinin in maize (Zea mays). We analyzed anthocyanin accumulation, structural gene expression, and regulatory gene expression in the seed aleurone and the seedling mesocotyl. In the mesocotyl, white, blue, and ultraviolet-B light strongly induced anthocyanin accumulation and expression of two key structural genes. In contrast, red light had little effect. Cytokinin enhanced the response to light but was not sufficient to induce anthocyanin accumulation in darkness. Plants with the pl-bol3 allele showed high levels of anthocyanin accumulation in response to light, whereas those with the pl-W22 allele did not, demonstrating the importance of pl1 in the light response. The expression of the pl-bol3 gene, encoding an MYB-related transcription factor, was induced by light and enhanced by cytokinin in a very similar manner to the structural genes and anthocyanin accumulation. Expression of the bHLH (basic helix-loop-helix) Sn1-bol3 gene was stimulated by several light qualities, but not enhanced by cytokinin, and was less well correlated with the induction of anthocyanin biosynthesis. In the aleurone, white, red, and blue light were effective in stimulating anthocyanin accumulation and expression of the MYB-related gene C1. The bHLH R gene was constitutively expressed. We conclude that specific members of the MYB-related c1/pl1 gene family play important roles in the regulation of anthocyanin synthesis in maize in response to different light qualities and cytokinin.