RESUMEN
Protein S-nitrosylation, which is defined by the covalent attachment of nitric oxide (NO) to the thiol group of cysteine residues, is known to play critical roles in plant development and stress responses. NO promotes seedling photomorphogenesis and NO emission is enhanced by light. However, the function of protein S-nitrosylation in plant photomorphogenesis is largely unknown. E3 ligase CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1) and transcription factor ELONGATED HYPOCOTYL 5 (HY5) antagonistically regulate seedling photomorphogenesis. COP1 inhibits plant photomorphogenesis by targeting photomorphogenic promoters like HY5 for 26S proteasome degradation. Here, we report that COP1 is S-nitrosylated in vitro. Mass spectrometry analyses revealed that two evolutionarily well conserved residues, cysteine 425 and cysteine 607, in the WD40 domain of COP1 are S-nitrosylated. S-nitrosylated glutathione (GSNO) is an important physiological NO donor for protein S-nitrosylation. The Arabidopsis (Arabidopsis thaliana) gsnor1-3 mutant, which accumulates higher level of GSNO, accumulated higher HY5 levels than wildtype (WT), indicating that COP1 activity is inhibited. Protein S-nitrosylation can be reversed by Thioredoxin-h5 (TRXh5) in plants. Indeed, COP1 interacts directly with TRXh5 and its close homolog TRXh3. Moreover, catalase 3 (CAT3) acts as a transnitrosylase that transfers NO to its target proteins like GSNO reductase (GSNOR). We found that CAT3 interacts with COP1 in plants. Taken together, our data indicate that the activity of COP1 is likely inhibited by NO via S-nitrosylation to promote the accumulation of HY5 and photomorphogenesis.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico , Óxido Nítrico , Ubiquitina-Proteína Ligasas , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/crecimiento & desarrollo , Arabidopsis/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Óxido Nítrico/metabolismo , Luz , Cisteína/metabolismo , Plantones/metabolismo , Plantones/crecimiento & desarrollo , Plantones/genética , Aldehído Oxidorreductasas/metabolismo , Aldehído Oxidorreductasas/genética , Regulación de la Expresión Génica de las PlantasRESUMEN
COP1 is a critical repressor of plant photomorphogenesis in darkness. However, COP1 plays distinct roles in the photoreceptor UVR8 pathway in Arabidopsis thaliana. COP1 interacts with ultraviolet B (UV-B)-activated UVR8 monomers and promotes their retention and accumulation in the nucleus. Moreover, COP1 has a function in UV-B signaling, which involves the binding of its WD40 domain to UVR8 and HY5 via conserved Val-Pro (VP) motifs of these proteins. UV-B-activated UVR8 interacts with COP1 via both the core domain and the VP motif, leading to the displacement of HY5 from COP1 and HY5 stabilization. However, it remains unclear whether the function of COP1 in UV-B signaling is solely dependent on its VP motif binding capacity and whether UV-B regulates the subcellular localization of COP1. Based on published structures of the COP1 WD40 domain, we generated a COP1 variant with a single amino acid substitution, COP1C509S , which cannot bind to VP motifs but retains the ability to interact with the UVR8 core domain. UV-B only marginally increased nuclear YFP-COP1 levels and significantly promoted YFP-COP1 accumulation in the cytosol, but did not exert the same effects on YFP-COP1C509S . Thus, the full UVR8-COP1 interaction is important for COP1 accumulation in the cytosol. Notably, UV-B signaling including activation of HY5 transcription was obviously inhibited in the Arabidopsis lines expressing YFP-COP1C509S , which cannot bind VP motifs. We conclude that the full binding of UVR8 to COP1 leads to the predominant accumulation of COP1 in the cytosol and that COP1 has an additional function in UV-B signaling besides VP binding-mediated protein destabilization.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Transducción de Señal , Ubiquitina-Proteína Ligasas , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Núcleo Celular/metabolismo , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Regulación de la Expresión Génica de las Plantas , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Rayos UltravioletaRESUMEN
Light regulates the subcellular localization of plant photoreceptors, a key step in light signaling. Ultraviolet-B radiation (UV-B) induces the plant photoreceptor UV RESISTANCE LOCUS 8 (UVR8) nuclear accumulation, where it regulates photomorphogenesis. However, the molecular mechanism for the UV-B-regulated UVR8 nuclear localization dynamics is unknown. With fluorescence recovery after photobleaching (FRAP), cell fractionation followed by immunoblotting and co-immunoprecipitation (Co-IP) assays we tested the function of UVR8-interacting proteins including CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1), REPRESSOR OF UV-B PHOTOMORPHOGENESIS 1 (RUP1) and RUP2 in the regulation of UVR8 nuclear dynamics in Arabidopsis thaliana. We showed that UV-B-induced rapid UVR8 nuclear translocation is independent of COP1, which previously was shown to be required for UV-B-induced UVR8 nuclear accumulation. Instead, we provide evidence that the UV-B-induced UVR8 homodimer-to-monomer photo-switch and the concurrent size reduction of UVR8 enables its monomer nuclear translocation, most likely via free diffusion. Nuclear COP1 interacts with UV-B-activated UVR8 monomer, thereby promoting UVR8 nuclear retention. Conversely, RUP1and RUP2, whose expressions are induced by UV-B, inhibit UVR8 nuclear retention via attenuating the UVR8-COP1 interaction, allowing UVR8 to exit the nucleus. Collectively, our data suggest that UV-B-induced monomerization of UVR8 promotes its nuclear translocation via free diffusion. In the nucleus, COP1 binding promotes UVR8 monomer nuclear retention, which is counterbalanced by the major negative regulators RUP1 and RUP2.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Proteínas de Arabidopsis/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Transducción de Señal , Arabidopsis/metabolismo , Fotorreceptores de Plantas/metabolismo , Rayos Ultravioleta , Ubiquitina-Proteína Ligasas/metabolismo , Regulación de la Expresión Génica de las PlantasRESUMEN
The transcription factor ELONGATED HYPOCOTYL5 (HY5) plays critical roles in plant photomorphogenesis. Previous studies on HY5 have mainly focused on the seedling stage in Arabidopsis (Arabidopsis thaliana), and its functions in other plant species have not been well characterized, particularly at adult stages of development. In this report, we investigated the functions of tomato (Solanum lycopersicum) HY5 (SlHY5) from seedlings to adult plants with a focus on fruits. Genome-edited slhy5 mutants exhibited typical compromised photomorphogenesis in response to various light conditions. The slhy5 mutants showed reduced primary root length and secondary root number, which is associated with altered auxin signaling. SlHY5 promoted chlorophyll biosynthesis from seedling to adult stages. Notably, the promotive role of SlHY5 on chlorophyll accumulation was more pronounced on the illuminated side of green fruits than on their shaded side. Consistent with this light-dependent effect, we determined that SlHY5 protein is stabilized by light. Transcriptome and metabolome analyses in fruits revealed that SlHY5 has major functions in the regulation of metabolism, including the biosynthesis of phenylpropanoids and steroidal glycoalkaloids. These data demonstrate that SlHY5 performs both shared and distinct functions in relation to its Arabidopsis counterpart. The manipulation of SlHY5 represents a powerful tool to influence the two vital agricultural traits of seedling fitness and fruit quality in tomato.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Solanum lycopersicum , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Clorofila/metabolismo , Frutas/genética , Frutas/metabolismo , Regulación de la Expresión Génica de las Plantas , Luz , Solanum lycopersicum/metabolismo , Desarrollo de la Planta , Plantones/metabolismoRESUMEN
In tomato (Solanum lycopersicum) and other plants, the photoreceptor UV-RESISTANCE LOCUS 8 regulates plant UV-B photomorphogenesis by modulating the transcription of many genes, the majority of which depends on the transcription factor ELONGATED HYPOCOTYL 5 (HY5). HY5 transcription is induced and then rapidly attenuated by UV-B. However, neither the transcription factors that activate HY5 transcription nor the mechanism for its attenuation during UV-B signaling is known. Here, we report that the tomato B-BOX (BBX) transcription factors SlBBX20 and SlBBX21 interact with SlHY5 and bind to the SlHY5 promoter to activate its transcription. UV-B-induced SlHY5 expression and SlHY5-controlled UV-B responses are normal in slbbx20 and slbbx21 single mutants, but strongly compromised in the slbbx20 slbbx21 double mutant. Surprisingly, UV-B responses are also compromised in lines overexpressing SlBBX20 or SlBBX21. Both SlHY5 and SlBBX20 bind to G-box1 in the SlHY5 promoter. SlHY5 outcompetes SlBBX20 for binding to the SlHY5 promoter in vitro, and inhibits the association of SlBBX20 with the SlHY5 promoter in vivo. Overexpressing 35S:SlHY5-FLAG in the WT background inhibits UV-B-induced endogenous SlHY5 expression. Together, our results reveal the critical role of the SlBBX20/21-SlHY5 module in activating the expression of SlHY5, the gene product of which inhibits its own gene transcription under UV-B, forming an autoregulatory negative feedback loop that balances SlHY5 transcription in plants.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Solanum lycopersicum , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Retroalimentación , Regulación de la Expresión Génica de las Plantas/genética , Solanum lycopersicum/genética , Solanum lycopersicum/metabolismo , Factores de Transcripción/metabolismo , Rayos UltravioletaRESUMEN
UV RESISTANCE LOCUS 8 (UVR8) is a photoreceptor that regulates UV-B photomorphogenesis in plants. UV-B photon perception promotes UVR8 homodimer dissociation into monomer, which is reverted to homodimer post UV-B, forming a complete photocycle. UVR8 monomer interacts with CONSTITUTIVELY PHOTOMORPHOGENEIC 1 (COP1) to initiate UV-B signaling. The function and mechanism of Arabidopsis UVR8 (AtUVR8) are extensively investigated, however, little is known about UVR8 and its signaling mechanisms in other plant species. Tomato is a widely used model plant for horticulture research. In this report we tested whether an ortholog of AtUVR8 in Tomato (SIUVR8) can complement Arabidopsis uvr8 mutant and whether the above-mentioned key signaling mechanisms of UVR8 are conserved. Heterologous expressed SIUVR8 in an Arabidopsis uvr8 null mutant rescued the uvr8 mutant in the tested UV-B responses including hypocotyl elongation, UV-B target gene expression and anthocyanin accumulation, demonstrating that the SIUVR8 is a putative UV-B photoreceptor. Moreover, in response to UV-B, SIUVR8 forms a protein complex with Arabidopsis COP1 in plants, suggesting conserved signaling mechanism. SIUVR8 exhibits similar photocycle as AtUVR8 in plants, which highlights conserved photoreceptor activation and inactivation mechanisms.
Asunto(s)
Fotorreceptores de Plantas/genética , Proteínas de Plantas/genética , Solanum lycopersicum/genética , Antocianinas/metabolismo , Arabidopsis , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/fisiología , Proteínas Cromosómicas no Histona/metabolismo , Proteínas Cromosómicas no Histona/fisiología , Secuencia Conservada/genética , Luz , Solanum lycopersicum/metabolismo , Fotorreceptores de Plantas/metabolismo , Fotorreceptores de Plantas/fisiología , Proteínas de Plantas/metabolismo , Proteínas de Plantas/fisiología , Plantas Modificadas Genéticamente , Reacción en Cadena en Tiempo Real de la Polimerasa , Técnicas del Sistema de Dos Híbridos , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/fisiologíaRESUMEN
Plant UV-B responses are mediated by the photoreceptor UV RESISTANCE LOCUS 8 (UVR8). In response to UV-B irradiation, UVR8 homodimers dissociate into monomers that bind to the E3 ubiquitin ligase CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1). The interaction of the C27 domain in the C-terminal tail of UVR8 with the WD40 domain of COP1 is critical for UV-B signaling. However, the function of the last 17 amino acids (C17) of the C-terminus of UVR8, which are adjacent to C27, is unknown, although they are largely conserved in land plants. In this study, we established that Arabidopsis thaliana UVR8 C17 binds to full-length UVR8, but not to COP1, and reduces COP1 binding to the remaining portion of UVR8, including C27. We hypothesized that overexpression of C17 in a wild-type background would have a dominant negative effect on UVR8 activity; however, C17 overexpression caused strong silencing of endogenous UVR8, precluding a detailed analysis. We therefore generated YFP-UVR8N423 transgenic lines, in which C17 was deleted, to examine C17 function indirectly. YFP-UVR8N423 was more active than YFP-UVR8, suggesting that C17 inhibits UV-B signaling by attenuating binding between C27 and COP1. Our study reveals an inhibitory role for UVR8 C17 in fine-tuning UVR8-COP1 interactions during UV-B signaling.
Asunto(s)
Aminoácidos/química , Arabidopsis/efectos de la radiación , Rayos Ultravioleta , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas Cromosómicas no Histona/química , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Regulación de la Expresión Génica de las Plantas/efectos de la radiación , Transducción de Señal/fisiología , Transducción de Señal/efectos de la radiación , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
UV RESISTANCE LOCUS 8 (UVR8) is a UV-B photoreceptor that regulates various aspects of plant photomorphogenesis. Physiological functions of UVR8 have been extensively investigated in Arabidopsis. However, functions of Tomato UVR8 (SlUVR8) are largely unknown. To analyze physiological functions of SlUVR8, we generated sluvr8 knock-out mutant lines with CRISPR-CAS9 gene editing approach. At seedling stage, SlUVR8 regulates hypocotyl elongation and anthocyanin accumulation under UV-B. Moreover, SlUVR8 regulates acclimation to low dose UV-B and promotes tolerance to elevated UV-B stress. These results revealed pivotal roles of SlUVR8 in the regulation of Tomato seedling development and UV-B stress tolerance. The manipulation of photoreceptor SlUVR8 may represent a powerful tool to improve Tomato plant performance in nature where high dose UV-B is present.
Asunto(s)
Fotorreceptores de Plantas/metabolismo , Plantones/crecimiento & desarrollo , Solanum lycopersicum/crecimiento & desarrollo , Rayos Ultravioleta/efectos adversos , Antocianinas/metabolismo , Solanum lycopersicum/efectos de la radiación , Plantones/efectos de la radiación , Estrés Fisiológico/efectos de la radiaciónRESUMEN
PHYTOCHROME-INTERACTING FACTOR 3 (PIF3) is a basic âhelix-loop-helix transcription factor with critical roles in light signaling. Recent work identified PIF3 as a negative regulator of arabidopsis (Arabidopsis thaliana) freezing tolerance.
Asunto(s)
Proteínas de Arabidopsis/genética , Fitocromo/genética , Arabidopsis , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Regulación de la Expresión Génica de las Plantas , Luz , TemperaturaRESUMEN
PHYTOCHROME-INTERACTING FACTOR 4 (PIF4) is a key transcriptional regulator promoting plant responses to elevated ambient temperatures. A recent study reported that the ultraviolet B radiation (UV-B) photoreceptor UVR8 signaling pathway inhibits PIF4 via multiple mechanisms to repress plant responses to high ambient temperatures.
Asunto(s)
Arabidopsis/metabolismo , Arabidopsis/efectos de la radiación , Rayos Ultravioleta , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Regulación de la Expresión Génica de las Plantas/genética , Regulación de la Expresión Génica de las Plantas/fisiología , Transducción de Señal/genética , Transducción de Señal/efectos de la radiaciónRESUMEN
Ultraviolet-B radiation (UV-B) is an intrinsic part of the solar radiation that reaches the Earth's surface and affects the biosphere. Plants have evolved a specific UV-B signaling pathway mediated by the UVR8 photoreceptor that regulates growth, development, and acclimation. Major recent advances have contributed to our understanding of the UVR8 photocycle, UV-B-responsive protein-protein interactions, regulation of UVR8 subcellular localization, and UVR8-regulated physiological responses. Here, we review the latest progress in our understanding of UVR8 signaling and UV-B responses, which includes studies in the unicellular alga Chlamydomonas reinhardtii and the flowering plant Arabidopsis.
Asunto(s)
Rayos Ultravioleta , Arabidopsis/metabolismo , Arabidopsis/efectos de la radiación , Chlamydomonas/metabolismo , Chlamydomonas/efectos de la radiación , Regulación de la Expresión Génica de las Plantas/efectos de la radiación , Fotorreceptores de Plantas/metabolismo , Transducción de Señal/efectos de la radiaciónRESUMEN
The UV-B photoreceptor UV RESISTANCE LOCUS 8 (UVR8) promotes UV-B acclimation and tolerance in Arabidopsis thaliana UVR8 localizes to both cytosol and nucleus, but its main activity is assumed to be nuclear. UV-B photoreception stimulates nuclear accumulation of UVR8 in a presently unknown manner. Here, we show that CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1) is required for UV-B-induced nuclear accumulation of UVR8, but bypassing the COP1 requirement for UVR8 nuclear accumulation did not rescue the cop1 mutant UV-B phenotype. Using a glucocorticoid receptor (GR)-based fusion protein system to conditionally localize GR-UVR8 to the nucleus, we have demonstrated that both photoactivation and nuclear localization of UVR8 are required for UV-B-induced photomorphogenic responses. In contrast, there was no UV-B response when UV-B-activated UVR8 was artificially retained in the cytosol. In agreement with a predominantly nuclear activity, constitutively active UVR8(W285A) accumulated in the nucleus also in the absence of UV-B. Furthermore, GR-COP1 expression lines suggested that UV-B-activated UVR8 can be coimported into the nucleus by COP1. Our data strongly support localization of UVR8 signaling in the nucleus and a dual role for COP1 in the regulation of UV-B-induced UVR8 nuclear accumulation and in UVR8-mediated UV-B signaling.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Núcleo Celular/metabolismo , Fotorreceptores de Plantas/metabolismo , Transporte Activo de Núcleo Celular/efectos de la radiación , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/efectos de la radiación , Proteínas de Arabidopsis/genética , Núcleo Celular/efectos de la radiación , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Regulación de la Expresión Génica de las Plantas , Immunoblotting , Mutación , Fotorreceptores de Plantas/genética , Plantas Modificadas Genéticamente , Transducción de Señal/genética , Ubiquitina-Proteína Ligasas , Rayos UltravioletaRESUMEN
Plants perceive UV-B, an intrinsic component of sunlight, via a signaling pathway that is mediated by the photoreceptor UV RESISTANCE LOCUS8 (UVR8) and induces UV-B acclimation. To test whether similar UV-B perception mechanisms exist in the evolutionarily distant green alga Chlamydomonas reinhardtii, we identified Chlamydomonas orthologs of UVR8 and the key signaling factor CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1). Cr-UVR8 shares sequence and structural similarity to Arabidopsis thaliana UVR8, has conserved tryptophan residues for UV-B photoreception, monomerizes upon UV-B exposure, and interacts with Cr-COP1 in a UV-B-dependent manner. Moreover, Cr-UVR8 can interact with At-COP1 and complement the Arabidopsis uvr8 mutant, demonstrating that it is a functional UV-B photoreceptor. Chlamydomonas shows apparent UV-B acclimation in colony survival and photosynthetic efficiency assays. UV-B exposure, at low levels that induce acclimation, led to broad changes in the Chlamydomonas transcriptome, including in genes related to photosynthesis. Impaired UV-B-induced activation in the Cr-COP1 mutant hit1 indicates that UVR8-COP1 signaling induces transcriptome changes in response to UV-B. Also, hit1 mutants are impaired in UV-B acclimation. Chlamydomonas UV-B acclimation preserved the photosystem II core proteins D1 and D2 under UV-B stress, which mitigated UV-B-induced photoinhibition. These findings highlight the early evolution of UVR8 photoreceptor signaling in the green lineage to induce UV-B acclimation and protection.
Asunto(s)
Chlamydomonas reinhardtii/metabolismo , Chlamydomonas reinhardtii/efectos de la radiación , Rayos Ultravioleta , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Chlamydomonas reinhardtii/genética , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Regulación de la Expresión Génica de las Plantas/genética , Regulación de la Expresión Génica de las Plantas/efectos de la radiación , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Transducción de Señal/efectos de la radiación , Ubiquitina-Proteína LigasasRESUMEN
BACKGROUND: Plants perceive UV-B through the UV RESISTANCE LOCUS 8 (UVR8) photoreceptor and UVR8 activation leads to changes in gene expression such as those associated with UV-B acclimation and stress tolerance. Albeit functionally unrelated, UVR8 shows some homology with RCC1 (Regulator of Chromatin Condensation 1) proteins from non-plant organisms at the sequence level. These proteins act as guanine nucleotide exchange factors for Ran GTPases and bind chromatin via histones. Subsequent to the revelation of this sequence homology, evidence was presented showing that UVR8 activity involves interaction with chromatin at the loci of some target genes through histone binding. This suggested a UVR8 mode-of-action intimately and directly linked with gene transcription. However, several aspects of UVR8 chromatin association remained undefined, namely the impact of UV-B on the process and how UVR8 chromatin association related to the transcription factor ELONGATED HYPOCOTYL 5 (HY5), which is important for UV-B signalling and has overlapping chromatin targets. Therefore, we have investigated UVR8 chromatin association in further detail. RESULTS: Unlike the claims of previous studies, our chromatin immunoprecipitation (ChIP) experiments do not confirm UVR8 chromatin association. In contrast to human RCC1, recombinant UVR8 also does not bind nucleosomes in vitro. Moreover, fusion of a VP16 activation domain to UVR8 did not alter expression of proposed UVR8 target genes in transient gene expression assays. Finally, comparison of the Drosophila DmRCC1 and the Arabidopsis UVR8 crystal structures revealed that critical histone- and DNA-interaction residues apparent in DmRCC1 are not conserved in UVR8. CONCLUSION: This has led us to conclude that the cellular activity of UVR8 likely does not involve its specific binding to chromatin at target genes.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Fotorreceptores de Plantas/metabolismo , Genes de Plantas , Regiones Promotoras Genéticas , Unión ProteicaRESUMEN
UV-B photon reception by the Arabidopsis thaliana homodimeric UV RESISTANCE LOCUS8 (UVR8) photoreceptor leads to its monomerization and a crucial interaction with CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1). Relay of the subsequent signal regulates UV-B-induced photomorphogenesis and stress acclimation. Here, we report that two separate domains of UVR8 interact with COP1: the ß-propeller domain of UVR8 mediates UV-B-dependent interaction with the WD40 repeats-based predicted ß-propeller domain of COP1, whereas COP1 activity is regulated by interaction through the UVR8 C-terminal C27 domain. We show not only that the C27 domain is required for UVR8 activity but also that chemically induced expression of the C27 domain is sufficient to mimic UV-B signaling. We further show, in contrast with COP1, that the WD40 repeat proteins REPRESSOR OF UV-B PHOTOMORPHOGENESIS1 (RUP1) and RUP2 interact only with the UVR8 C27 domain. This coincides with their facilitation of UVR8 reversion to the ground state by redimerization and their potential to interact with UVR8 in a UV-B-independent manner. Collectively, our results provide insight into a key mechanism of photoreceptor-mediated signaling and its negative feedback regulation.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Fotorreceptores de Plantas/metabolismo , Rayos Ultravioleta , Regulación de la Expresión Génica de las Plantas , Unión Proteica , Transducción de Señal/efectos de la radiación , Ubiquitina-Proteína LigasasRESUMEN
Polar auxin transport (PAT) plays key roles in the regulation of plant growth and development. Flavonoids have been implicated in the inhibition of PAT. However, the active flavonoid derivative(s) involved in this process in vivo has not yet been identified. Here, we provide evidence that a specific flavonol bis-glycoside is correlated with shorter plant stature and reduced PAT. Specific flavonoid-biosynthetic or flavonoid-glycosylating steps were genetically blocked in Arabidopsis thaliana. The differential flavonol patterns established were analyzed by high-performance liquid chromatography (HPLC) and related to altered plant stature. PAT was monitored in stem segments using a radioactive [(3)H]-indole-3-acetic acid tracer. The flavonoid 3-O-glucosyltransferase mutant ugt78d2 exhibited a dwarf stature in addition to its altered flavonol glycoside pattern. This was accompanied by reduced PAT in ugt78d2 shoots. The ugt78d2-dependent growth defects were flavonoid dependent, as they were rescued by genetic blocking of flavonoid biosynthesis. Phenotypic and metabolic analyses of a series of mutants defective at various steps of flavonoid formation narrowed down the potentially active moiety to kaempferol 3-O-rhamnoside-7-O-rhamnoside. Moreover, the level of this compound was negatively correlated with basipetal auxin transport. These results indicate that kaempferol 3-O-rhamnoside-7-O-rhamnoside acts as an endogenous PAT inhibitor in Arabidopsis shoots.
Asunto(s)
Arabidopsis/metabolismo , Ácidos Indolacéticos/metabolismo , Quempferoles/fisiología , Arabidopsis/crecimiento & desarrollo , Transporte Biológico , Quempferoles/metabolismo , Fenotipo , Brotes de la Planta/genética , Brotes de la Planta/metabolismoRESUMEN
Arabidopsis thaliana UV RESISTANCE LOCUS 8 (UVR8) is a UV-B photoreceptor that initiates photomorphogenic responses underlying acclimation and UV-B tolerance in plants. UVR8 is a homodimer in its ground state, and UV-B exposure results in its instantaneous monomerization followed by interaction with CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1), a major factor in UV-B signaling. UV-B photoreception by UVR8 is based on intrinsic tryptophan aromatic amino acid residues, with tryptophan-285 as the main chromophore. We generated transgenic plants expressing UVR8 with a single amino acid change of tryptophan-285 to alanine. UVR8(W285A) appears monomeric and shows UV-B-independent interaction with COP1. Phenotypically, the plants expressing UVR8(W285A) exhibit constitutive photomorphogenesis associated with constitutive activation of target genes, elevated levels of anthocyanins, and enhanced, acclimation-independent UV-B tolerance. Moreover, we have identified COP1, REPRESSOR OF UV-B PHOTOMORPHOGENESIS 1 and 2 (RUP1 and RUP2), and the SUPPRESSOR OF PHYA-105 (SPA) family as proteins copurifying with UVR8(W285A). Whereas COP1, RUP1, and RUP2 are known to directly interact with UVR8, we show that SPA1 interacts with UVR8 indirectly through COP1. We conclude that UVR8(W285A) is a constitutively active UVR8 photoreceptor variant in Arabidopsis, as is consistent with the crucial importance of monomer formation and COP1 binding for UVR8 activity.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Proteínas Cromosómicas no Histona/genética , Fenotipo , Fotorreceptores de Plantas/genética , Antocianinas/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromatografía en Gel , Cromatografía Liquida , Electroforesis en Gel de Poliacrilamida , Ingeniería Genética , Inmunoprecipitación , Mutación Missense/genética , Plantas Modificadas Genéticamente , Reacción en Cadena en Tiempo Real de la Polimerasa , Espectrometría de Masas en Tándem , Técnicas del Sistema de Dos Híbridos , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Ultraviolet-B radiation (UV-B) is an intrinsic part of sunlight that is accompanied by significant biological effects. Plants are able to perceive UV-B using the UV-B photoreceptor UVR8 which is linked to a specific molecular signaling pathway and leads to UV-B acclimation. Herein we review the biological process in plants from initial UV-B perception and signal transduction through to the known UV-B responses that promote survival in sunlight. The UVR8 UV-B photoreceptor exists as a homodimer that instantly monomerises upon UV-B absorption via specific intrinsic tryptophans which act as UV-B chromophores. The UVR8 monomer interacts with COP1, an E3 ubiquitin ligase, initiating a molecular signaling pathway that leads to gene expression changes. This signaling output leads to UVR8-dependent responses including UV-B-induced photomorphogenesis and the accumulation of UV-B-absorbing flavonols. Negative feedback regulation of the pathway is provided by the WD40-repeat proteins RUP1 and RUP2, which facilitate UVR8 redimerization, disrupting the UVR8-COP1 interaction. Despite rapid advancements in the field of recent years, further components of UVR8 UV-B signaling are constantly emerging, and the precise interplay of these and the established players UVR8, COP1, RUP1, RUP2 and HY5 needs to be defined. UVR8 UV-B signaling represents our further understanding of how plants are able to sense their light environment and adjust their growth accordingly.
RESUMEN
Light-sensitive proteins are useful tools to control protein localization, activation and gene expression, but are currently limited to excitation with red or blue light. Here we report a novel optogenetic system based on the ultraviolet-B-dependent interaction of the Arabidopsis ultraviolet-B photoreceptor UVR8 with COP1 that can be performed in visible light background. We use this system to induce nuclear accumulation of cytoplasmic green fluorescent protein fused to UVR8 in cells expressing nuclear COP1, and to recruit a nucleoplasmic red fluorescent protein fused to COP1 to chromatin in cells expressing UVR8-H2B. We also show that ultraviolet-B-dependent interactions between DNA-binding and transcription activation domains result in a linear induction of gene expression. The UVR8-COP1 interactions in mammalian cells can be induced using subsecond pulses of ultraviolet-B light and last several hours. As UVR8 photoperception is based on intrinsic tryptophan residues, these interactions do not depend on the addition of an exogenous chromophore.