Subcellular localisation and functional analysis of UVR8, a UV-B specific signalling component in Arabidopsis

UV-B is an integral component of the daylight spectrum that regulates plant gene expression and development, but very little is known about how plants perceive UV-B. Although UV-B-induced damage and repair have been extensively investigated, the mechanisms by which UV-B is perceived as a signal, which mediates physiological and protective responses is not yet clearly understood neither in mammals, nor in higher plants. Low fluence rates of UV-B induce the expression of genes involved in UV-protective responses such as flavonoid biosynthesis and promote plant survival in UV-B. The aim of this study is to contribute to the elucidation of the signal transduction events that lead to the acclimation of plants in response to non-damaging levels of UV-B (< 3.5 μmol m-2 s-1). In particular, the characterisation of UVR8 (UV-RESISTANCE LOCUS 8), a UV-B specific signalling component, is carried out at the protein level. The function of UVR8 involves the orchestration of the expression of a range of genes mediating vital UV-protective responses, including those encoding light-regulated transcription factors HY5 and HYH, enzymes involved in the phenylpropanoid pathway, antioxidant and stress proteins (Brown et al., 2005). UVR8 shows 30% sequence identity to the human regulator of chromatin condensation (RCC1) but differs both in activity and function. The phenotype of uvr8 mutant plants is characterised by an increased susceptibility to UV-B and the lack of the UV-B-specific induction of genes involved in UV-protection, such as CHS (encoding the flavonoid biosynthetic enzyme chalcone synthase) and the transcription factor HY5. The UVR8-mediated regulation of transcription in response to UV-B seems to occur via the association of UVR8 with chromatin via histones in the promoter region of HY5 (Brown et al., 2005) and other genes involved in light signalling. In this study, further investigation of the mechanism by which UVR8 acts as a UV-B specific signalling component is performed by employing a number of approaches including: spatial, temporal protein analysis, subcellular localisation studies, structure-function analyses, and the yeast-two-hybrid assay for the identification of UVR8 interacting proteins. To study spatial, temporal and wavelength specific UVR8 protein abundance anti-UVR8 peptide antibodies were generated. Western blot analyses showed that UVR8 is ubiquitously expressed in all plant tissues from the very early stages of development and at every light treatment tested (dark, white light, UV-B). The subcellular localisation of UVR8 analysed by confocal fluorescence microscopy revealed that a fusion of UVR8 with green fluorescent protein (GFP) is localised in the cytoplasm and the nucleus of various plant tissues (leaf, hypocotyl, root, flower) and under various light fluence rates and qualities (white, red, UV-A, UV-B). Interestingly, a treatment of low fluence rates of UV-B led to an increase of GFP-UVR8 protein accumulation in the nucleus, which was confirmed by western blot analysis based on protein fractionation studies in wild-type plants. The wavelength specificity, the kinetics and the fluence-rate sensitivity of GFP-UVR8 nuclear accumulation suggest that this response is UV-B specific, rapid (10 min UV-B) and very sensitive to very low fluence rates of UV-B (0.1 μmol m-2 s-1). Protein synthesis does not seem to be involved in this process, as there is no change in the protein levels before and after a UV-B irradiation. To assess the importance of the presence of UVR8 in the nucleus and the cytoplasm of the plant cell, uvr8-1 transgenic plants were produced expressing either constitutively nuclear localised GFP-UVR8 fused to a nuclear localisation signal (NLS), or cytosolically retained GFP-UVR8 fused to a nuclear export signal (NES). Nuclear exclusion of NES-GFP-UVR8 fusion protein was sustained under most light conditions apart from UV-B, which induced nuclear import of the protein. This indicates that the mechanism involved in the nuclear accumulation of UVR8 can overcome an export signal either by masking it or by simply superseding it. Furthermore, the NES-GFP-UVR8 construct was functional after UV-B treatment, since it rescued the mutant uvr8 phenotype. None of the inhibitor treatments tested (staurosporine, cycloheximide, cantharidin) was successful in blocking the UV-B induced nuclear import of NES-GFP-UVR8, although they impaired the UVR8 regulated induction of CHS expression. Thus, no evidence is presented for a specific protein modification, which could control this response. Constitutive nuclear localisation of NLS-GFP-UVR8 had no effect on the function of the protein according to complementation analyses. Furthermore, no change in localisation, fluorescence intensity or protein abundance was observed in response to white light or after a UV-B irradiation. These results indicate that the constitutive nuclear localisation of UVR8 is not sufficient for constitutive activation of UVR8 regulated gene expression and that a UV-B stimulus is still necessary to trigger these responses. Unfortunately, based on the current data it cannot be concluded whether the UV-B signal perception occurs in the nucleus or in the cytosol of the plant cell. To investigate the structure-function relationship within the UVR8 protein, deletion analyses followed by complementation studies in transgenic plants were performed. More specifically, deletion of the first 23 amino acids at the N-terminus of UVR8 impaired its nuclear accumulation in response to UV-B. Deletion of a 27 amino acid region near the C-terminus had no effect on the UV-B dependent re-localisation of the protein, but abolished UVR8 regulated gene expression. In addition, a highly basic sequence at the extreme C-terminal of UVR8, resembling a putative monopartite nuclear localisation signal, was deleted. Subcellular localisation and complementation analyses suggest that this sequence does not serve as a nuclear localisation signal, it is not involved in the UV-B induced nuclear accumulation and its absence does not affect UVR8 protein function. Chromatin immunoprecipitation assays show that none of the regions deleted is required for chromatin association and none of the deletions affects subcellular localisation in white light. In order to identify interacting partners for UVR8, the yeast-two hybrid system was used. Unfortunately no interacting proteins have been identified, neither from a screen, nor by directed-interaction studies. A different approach could be employed in the future involving size exclusion chromatography of protein extracts from plants in order to establish whether UVR8 functions as part of a complex in vivo

Ultraviolet rays light up transcriptional networks regulating plant growth

Light and hormones tightly regulate plant growth and development by both synergistic and antagonistic actions. In the current issue of Developmental Cell, Liang et al. (2018) uncover how the UV-B photoreceptor UVR8 mediates inhibition of plant growth via direct interactions with key transcriptional regulators of brassinosteroid signaling

Light and temperature shape nuclear architecture and gene expression

Environmental stimuli play a major role in modulating growth and development throughout the life-cycle of a plant. Quantitative and qualitative variations in light and temperature trigger changes in gene expression that ultimately shape plant morphology for adaptation and survival. Although the phenotypic and transcriptomic basis of plant responses to the constantly changing environment have been examined for decades, the relationship between global changes in nuclear architecture and adaption to environmental stimuli is just being uncovered. This review presents recent discoveries investigating how changes in light and temperature trigger changes in chromatin structure and nuclear organization with a focus on the role of gene repositioning and chromatin accessibility in regulating gene expression

Let it bloom: crosstalk between light and flowering signalling in Arabidopsis

The terrestrial environment is complex, with many parameters fluctuating on daily and seasonal basis. Plants in particular, have developed complex sensory and signalling networks to extract and integrate information about their surroundings, in order to maximise their fitness and mitigate some of the detrimental effects of their sessile lifestyles. Light and temperature each provide crucial insight on the surrounding environment and in combination allow plants to appropriately develop, grow and adapt. Crosstalk between light and temperature signalling cascades allow plants to time key developmental decisions accordingly to ensure they are “in sync” with their environment. In this review, we discuss the major players that regulate light and temperature signalling, and the cross‐talk between them, in reference to a crucial developmental decision faced by plants: to bloom or not to bloom

The impact of light and temperature on chromatin organisation and plant adaptation

Light and temperature shape the developmental trajectory and morphology of plants. Changes in chromatin organisation and nuclear architecture can modulate gene expression and lead to short and long-term plant adaptation to the environment. Here, we review recent reports investigating how changes in chromatin composition, structure and topology modulate gene expression in response to fluctuating light and temperature conditions resulting in developmental and physiological responses. Furthermore, the potential application of novel revolutionary techniques such as RNA and padlock fluorescence in situ hybridization (FISH), and Hi-C to study the impact of environmental stimuli such as light and temperature on nuclear compartmentalisation in plants is discussed

The diverse and unanticipated roles of Histone deacetylase 9 in coordinating plant development and environmental acclimation

Plants tightly control gene transcription to adapt to environmental conditions and steer growth and development. Different types of epigenetic modifications are instrumental in this. In recent years, an important role for the chromatin modifying RPD3/HDA1 class I HDAC HISTONE DEACETYLASE 9 (HDA9) emerged in the regulation of a multitude of plant traits and responses. HDACs are widely considered transcriptional repressors and are typically part of multiprotein complexes containing co-repressors, DNA and histone binding proteins. By catalyzing the removal of acetyl groups from lysine residues of histone protein tails, HDA9 indeed negatively controls gene expression in many cases, in concert with interacting proteins such as POWERDRESS (PWR), HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENES 15 (HOS15), WRKY53, ELONGATED HYPOCOTYL 5 (HY5), ABA INSENSITIVE 4 (ABI4) and EARLY FLOWERING 3 (ELF3). However, HDA9 activity has also been directly linked to transcriptional activation. In addition, following the recent breakthrough discovery of mutual negative feedback regulation between HDA9 and its interacting WRKY-domain transcription factor WRKY53, swift progress in gaining understanding of HDA9 biology is expected. In this review, we summarize knowledge on this intriguing versatile – and long underrated – protein and propose novel leads to further unravel HDA9-governed molecular networks underlying plant development and environmental biology

Temporal phosphate gradients reveal diverse acclimation responses in phytoplankton phosphate uptake

Phytoplankton face environmental nutrient variations that occur in the dynamic upper layers of the ocean. Phytoplankton cells are able to rapidly acclimate to nutrient fluctuations by adjusting their nutrient-uptake system and metabolism. Disentangling these acclimation responses is a critical step in bridging the gap between phytoplankton cellular physiology and community ecology. Here, we analyzed the dynamics of phosphate (P) uptake acclimation responses along different P temporal gradients by using batch cultures of the diatom Phaeodactylum tricornutum. We employed a multidisciplinary approach that combined nutrient-uptake bioassays, transcriptomic analysis, and mathematical models. Our results indicated that cells increase their maximum nutrient-uptake rate (Vmax) both in response to P pulses and strong phosphorus limitation. The upregulation of three genes coding for different P transporters in cells experiencing low intracellular phosphorus levels supported some of the observed Vmax variations. In addition, our mathematical model reproduced the empirical Vmax patterns by including two types of P transporters upregulated at medium-high environmental and low intracellular phosphorus levels, respectively. Our results highlight the existence of a sequence of acclimation stages along the phosphate continuum that can be understood as a succession of acclimation responses. We provide a novel conceptual framework that can contribute to integrating and understanding the dynamics and wide diversity of acclimation responses developed by phytoplankton

ZINC-FINGER interactions mediate transcriptional regulation of hypocotyl growth in Arabidopsis

Integration of environmental signals and interactions among photoreceptors and transcriptional regulators is key in shaping plant development. TANDEM ZINC-FINGER PLUS3 (TZP) is an integrator of light and photoperiodic signaling that promotes flowering in Arabidopsis thaliana. Here we elucidate the molecular role of TZP as a positive regulator of hypocotyl elongation. We identify an interacting partner for TZP, the transcription factor ZINC-FINGER HOMEODOMAIN 10 (ZFHD10), and characterize its function in coregulating the expression of blue-light–dependent transcriptional regulators and growth-promoting genes. By employing a genome-wide approach, we reveal that ZFHD10 and TZP coassociate with promoter targets enriched in light-regulated elements. Furthermore, using a targeted approach, we show that ZFHD10 recruits TZP to the promoters of key coregulated genes. Our findings not only unveil the mechanism of TZP action in promoting hypocotyl elongation at the transcriptional level but also assign a function to an uncharacterized member of the ZFHD transcription factor family in promoting plant growth

Wavelength-dependent effects of artificial light at night on phytoplankton growth and community structure

Artificial light at night (ALAN) is a disruptive form of pollution, impacting physiological and behavioural processes that may scale up to population and community levels. Evidence from terrestrial habitats show that the severity and type of impact depend on the wavelength and intensity of ALAN; however, research on marine organisms is still limited. Here, we experimentally investigated the effect of different ALAN colours on marine primary producers. We tested the effect of green (525 nm), red (624 nm) and broad-spectrum white LED ALAN, compared to a dark control, on the green microalgae Tetraselmis suesica and a diatom assemblage. We show that green ALAN boosted chlorophyll production and abundance in T. suesica. All ALAN wavelengths affected assemblage biomass and diversity, with red and green ALAN having the strongest effects, leading to higher overall abundance and selective dominance of specific diatom species, some known to cause harmful algal blooms. Our findings show that green and red ALAN should be used with caution as alternative LED colours in coastal areas, where there might be a need to strike a balance between the effects of green and red light on marine primary producers with the benefit they appear to bring to other organisms

Interaction specificity of Arabidopsis 14-3-3 proteins with phototropin receptor kinases

Phototropin receptor kinases play an important roles in optimising plant growth in response to blue light. Much is known regarding their photochemical reactivity, yet little progress has been made to identify downstream signalling components. Here, we isolated several interacting proteins for Arabidopsis phototropin 1 (phot1) by yeast two-hybrid screening. These include members of the NPH3/RPT2 (NRL) protein family, proteins associated with vesicle trafficking, and the 14-3-3 lambda (?) isoform from Arabidopsis . 14-3-3? and phot1 were found to colocalise and interact in vivo. Moreover, 14-3-3 binding to phot1 was limited to non-epsilon 14-3-3 isoforms and was dependent on key sites of receptor autophosphorylation. No 14-3-3 binding was detected for Arabidopsis phot2, suggesting that 14-3-3 proteins represent specific mode of phot1 signalling