Photomorphogenic responses to ultraviolet-B light

Exposure to UV-B light regulates numerous aspects of plant metabolism, morphology and physiology through the differential expression of hundreds of genes. Photomorphogenic responses to UV-B are mediated by the photoreceptor UV RESISTANCE LOCUS8 (UVR8). Considerable progress has been made in understanding UVR8 action: the structural basis of photoreceptor function, how interaction with CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1) initiates signaling and how REPRESSOR OF UV-B PHOTOMORPHOGENESIS (RUP) proteins negatively regulate UVR8 action. In addition, recent research shows that UVR8 mediates several responses through interaction with other signaling pathways, in particular auxin signaling. Nevertheless, many aspects of UVR8 action remain poorly understood. Most research to date has been undertaken with Arabidopsis, and it is important to explore the functions and regulation of UVR8 in diverse plant species. Furthermore, it is essential to understand how UVR8, and UV-B signaling in general, regulates processes under natural growth conditions. UV-B regulates the expression of many genes through UVR8-independent pathways, but the activity and importance of these pathways in plants growing in sunlight are poorly understood

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

Regulation of transcription by the Arabidopsis UVR8 photoreceptor involves a specific histone modification

The photoreceptor UV RESISTANCE LOCUS 8 (UVR8) specifically mediates photomorphogenic responses to UV-B wavelengths. UVR8 acts by regulating transcription of a set of genes, but the underlying mechanisms are unknown. Previous research indicated that UVR8 can associate with chromatin, but the specificity and functional significance of this interaction are not clear. Here we show, by chromatin immunoprecipitation, that UV-B exposure of Arabidopsis increases acetylation of lysines K9 and/or K14 of histone H3 at UVR8-regulated gene loci in a UVR8-dependent manner. The transcription factors HY5 and/or HYH, which mediate UVR8-regulated transcription, are also required for this chromatin modification, at least for the ELIP1 gene. Furthermore, sequencing of the immunoprecipitated DNA revealed that all UV-B-induced enrichments in H3K9,14diacetylation across the genome are UVR8-dependent, and approximately 40 % of the enriched loci contain known UVR8-regulated genes. In addition, inhibition of histone acetylation by anacardic acid reduces the UV-B induced, UVR8 mediated expression of ELIP1 and CHS. No evidence was obtained in yeast 2-hybrid assays for a direct interaction between either UVR8 or HY5 and several proteins involved in light-regulated histone modification, nor for the involvement of these proteins in UVR8-mediated responses in plants, although functional redundancy between proteins could influence the results. In summary, this study shows that UVR8 regulates a specific chromatin modification associated with transcriptional regulation of a set of UVR8-target genes

The Photomorphogenic Signal: An Essential Component of Photoautotrophic Life

In this chapter, we focus on the role of the photomorphogenic signal to trigger the synthesis of photosynthetic genes and pigments during the greening process and later on, during photosynthetic plant development, with emphasis on the regulation of gene expression.Fil: Iñigo, Sabrina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Barber, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Sanchez Lamas, Maximiliano. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Iglesias, Francisco Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Cerdan, Pablo Diego. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentin

UV-B exposure, ROS, and stress: inseparable companions or loosely linked associates?

Ultraviolet-B (UV-B) radiation has long been perceived as a stressor. However, a conceptual U-turn has taken place, and UV-B damage is now considered rare. We question whether UV-stress and UV-B-induced reactive oxygen species (ROS) are still relevant concepts, and if ROS-mediated signaling contributes to UV-B acclimation. Measurements of antioxidants and of antioxidant genes show that both low and high UV-B doses alter ROS metabolism. Yet, there is no evidence that ROS control gene expression under low UV-B. Instead, expression of antioxidant genes is linked to the UV RESISTANCE LOCUS 8 pathway. We hypothesize that low UVB doses cause ‘eustress’ (good stress) and that stimulispecific signaling pathways pre-dispose plants to a state of low alert that includes activation of antioxidant defenses.Funding agencies are:COST Action  FA0906UV4Growth  Faculty of Business, Science, and Technology at Örebro University  Science Foundation Ireland (SFI)  11/RFP.1/EOB/3303 Hungarian Scientific Research Fund  OTKA NN-85349 UV-B-fotobiologi: mekanismer för perception och cellulära response

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

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

On the mechanism of photoinduced dimer dissociation in the plant UVR8 photoreceptor

UV-B absorption by the photoreceptor UV resistance locus 8 (UVR8) consisting of two identical protein units triggers a signal chain used by plants in connection with protection and repair of UV-B induced damage. X-ray structural analysis of the purified protein [Christie JM, et al. (2012) Science 335(6075):1492–1496] [Wu D, et al. (2012) Nature 484(7393): 214–220] has revealed that the dimer is held together by arginine–aspartate salt bridges. In this paper we address the initial processes in the signal chain. On the basis of high-level quantum-chemical calculations, we propose a mechanism for the photodissociation of UVR8 that consists of three steps: (i) In each monomer, multiple tryptophans form an extended light-harvesting system in which the L_a excited state of Trp233 experiences strong electrostatic stabilization by the protein environment. The strong stabilization singles out this tryptophan to be an efficient exciton acceptor that accumulates the excitation energy from the entire protein subunit. (ii) A fast decay of the locally excited state by charge separation generates the radical ion pair Trp285(+)-Trp233(−) with a dipole moment of ∼18 D. (iii) Key to the proposed mechanism is that this large dipole moment drives the breaking of the salt bridges between the two monomer subunits. The suggested mechanism for the UV-B–driven dissociation of the dimer that rests on the prominent players Trp233 and Trp285 explains the experimental results obtained from mutagenesis of UVR8

INVESTIGATION INTO THE ROLE OF UVR8 IN BALANCING GROWTH AND ACCLIMATION TO UV-B RADIATION IN NATURAL AND TRANSGENIC POPULUS VARIANTS

Research on woody plants offers promise for the development of next-generation biofuel feedstocks with reduced lignin recalcitrance and enhanced saccharification for ethanol production. Natural variants of Populus trichocarpa with diverse lignin content and saccharification differences, and transgenic Populus deltoides constructed for reduced lignin levels for improved cellulose extraction, offer clues to enhance biofuel production but with a tradeoff to overall fitness and biomass. One concern of engineering lignin relates to the protection of plants against environmental stress such as UV-B radiation. Secondary metabolite biosynthesis initiated by UV-B, particularly phenylpropanoids (lignin precursors) and flavonoids, plays an important role in managing and protection of UV stress. Genetic modifications affecting the production of these compounds may have significant physiological consequences. Thus, the goal of this research was to develop a model for biosynthetic compensation of low-lignin Populus to UV-B stress. The effect of UV-B on Populus was evaluated by spectroscopic and metabolomic measurements on leaves. UV-B promoted shifts in physiological and metabolomic responses of natural and transgenic Populus with varying levels of lignin were complex, reflecting compensation from variety of biosynthetic alterations. Therefore, the impact of modulating the expression of the photoreceptor, UVR8, in regulating the response of Populus to UV-B was pursued. Modulation of UVR8 expression in Populus hybrid was achieved by constructing transgenic plants using CRISPR and RNAi, in wild-type, and an RNAi-constructed cinnamyl alcohol dehydrogenase knockdown line. UV-B response of UVR8 modulated Populus indicated that flavonoids were upregulated in UVR8 overexpression lines, and that in a CAD knockdown background, these effects were slightly enhanced. Salicylates were upregulated in UVR8 knockout poplars, suggesting metabolic flux in the pathway, but little difference was seen relative to wild-type plants in CAD lines, and UV-B treatment had little effect. An interesting and unexpected finding was that UVR8 modulated Populus exhibited more rapid growth than wild-type plants. The findings underscore the key role of UVR8 in synchronizing protective metabolic responses to UV-B and suggest an additional function of the photoreceptor in regulating growth and development of Populus through shifts in the chemical equilibria of UVR8 monomers and dimers and interactions with other regulatory factors

Regulation of transcription by Ultraviolet-B radiation in Arabidopsis thaliana

Plants are sessile photo-autotrophic organisms and need to adapt constantly to a dynamic environment. Light is of utmost importance for plants to be able to monitor their surroundings. Ultraviolet-B radiation (UV-B; 280-315 nm) is an intrinsic part of sunlight and, depending on the wavelength and the fluence rate, it may be a stressful signal or an “informational” one. The so called photomorphogenic responses of plants to UV-B are largely mediated by the UV-B specific photoreceptor UV RESISTANCE LOCUS 8 (UVR8), which “senses” UV-B via a tryptophan based mechanism. UVR8 is localised in the cytoplasm and the nucleus mainly as a homodimer. Upon UV-B irradiation it splits to its monomers and accumulates in the nucleus where it has been found to interact with the E3 Ubiquitin ligase COP1. In the nucleus UVR8 has been shown to associate with chromatin on loci of UV-B responsive genes, including that encoding for the bZIP transcription factor (TF) ELONGATED HYPOCOTYL 5 (HY5), a key effector of UVR8-dependent signalling pathways. The binding of UVR8 to chromatin appears to take place via interaction with histones (H2B in particular) rather than DNA itself. However, this association with chromatin seems not to be UV-B specific. The above data suggest a mechanistic basis for an assumed function of UVR8 in the regulation transcription. It seems likely that UVR8 interacts with other proteins associated with chromatin to promote remodelling and/or recruits/activates TFs which in turn stimulate transcription of its target genes. The main objective of this study was to address the above working hypothesis