RESUMO
UV-B light initiates photomorphogenic responses in plants. Arabidopsis UV RESISTANCE LOCUS8 (UVR8) specifically mediates these responses by functioning as a UV-B photoreceptor. UV-B exposure converts UVR8 from a dimer to a monomer, stimulates the rapid accumulation of UVR8 in the nucleus, where it binds to chromatin, and induces interaction of UVR8 with CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1), which functions with UVR8 to control photomorphogenic UV-B responses. Although the crystal structure of UVR8 reveals the basis of photoreception, it does not show how UVR8 initiates signaling through interaction with COP1. Here we report that a region of 27 amino acids from the C terminus of UVR8 (C27) mediates the interaction with COP1. The C27 region is necessary for UVR8 function in the regulation of gene expression and hypocotyl growth suppression in Arabidopsis. However, UVR8 lacking C27 still undergoes UV-B-induced monomerization in both yeast and plant protein extracts, accumulates in the nucleus in response to UV-B, and interacts with chromatin at the UVR8-regulated ELONGATED HYPOCOTYL5 (HY5) gene. The UV-B-dependent interaction of UVR8 and COP1 is reproduced in yeast cells and we show that C27 is both necessary and sufficient for the interaction of UVR8 with the WD40 domain of COP1. Furthermore, we show that C27 interacts in yeast with the REPRESSOR OF UV-B PHOTOMORPHOGENESIS proteins, RUP1 and RUP2, which are negative regulators of UVR8 function. Hence the C27 region has a key role in UVR8 function.
Assuntos
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Regulação da Expressão Gênica de Plantas/fisiologia , Transdução de Sinais/fisiologia , Sequência de Aminoácidos , Imunoprecipitação da Cromatina , Proteínas de Fluorescência Verde , Imunoprecipitação , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Técnicas do Sistema de Duplo-Híbrido , Ubiquitina-Proteína Ligases , Raios Ultravioleta , LevedurasRESUMO
Arabidopsis thaliana UV RESISTANCE LOCUS8 (UVR8) is a UV-B-specific signaling component that mediates low fluence, photomorphogenic responses to UV-B. It is required for UV-B-induced expression of the gene encoding the ELONGATED HYPOCOTYL5 (HY5) transcription factor. HY5 is a key effector of responses mediated by UVR8. In mature leaf tissue, HY5 transcript accumulation occurred rapidly in response to a brief UV-B treatment and no induction was observed in a uvr8 mutant over a broad range of UV wavelengths. In response to monochromatic light, maximal transcript accumulation occurred in wild-type plants at wavelengths 280-300 nm. HY5 transcript accumulation showed reciprocity between the fluence rate and duration of UV-B exposure, and on this basis conditions were chosen to generate an action spectrum for the UVR8 signaling pathway. Dose-response curves were produced for a range of UV wavelengths using 20 min exposure to UV and harvesting tissue 2 h after the start of illumination. Experiments using mutants defective in sinapate ester and flavonoid biosynthesis indicated that the presence of UV-absorbing compounds did not affect the construction of an action spectrum under the conditions employed. The action spectrum for the induction of HY5 by the UVR8 pathway showed a main peak at 280 nm with a smaller peak at 300 nm. The data are discussed in relation to the proposed mechanisms of UV-B photoreception.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/efeitos da radiação , Fatores de Transcrição de Zíper de Leucina Básica/genética , Proteínas Nucleares/genética , RNA Mensageiro/metabolismo , Raios Ultravioleta , Arabidopsis/genética , Arabidopsis/metabolismo , Sequência de Bases , Primers do DNA , Folhas de Planta/metabolismo , Folhas de Planta/efeitos da radiação , Reação em Cadeia da Polimerase , RNA Mensageiro/genéticaRESUMO
UV-B signaling is an important but poorly understood aspect of light responsiveness in plants. Arabidopsis (Arabidopsis thaliana) UV RESISTANCE LOCUS8 (UVR8) is a recently identified UV-B-specific signaling component that regulates UV-protective responses. Using the uvr8 mutant, we defined genetically distinct UVR8-dependent and UVR8-independent pathways that stimulate different sets of genes in mature Arabidopsis leaf tissue. Both pathways operate at 1 micromol m(-2) s(-1) UV-B and above, but the UVR8-dependent pathway is able to stimulate UV-protective genes even in response to 0.1 micromol m(-2) s(-1) UV-B. Both pathways function in mutants lacking phytochromes, cryptochromes, or phototropins. Genes encoding the ELONGATED HYPOCOTYL5 (HY5) and HY5 HOMOLOG (HYH) transcription factors are induced at low UV-B fluence rates (0.1 micromol m(-2) s(-1)). Experiments with hy5 and hyh mutants reveal that both these factors mediate responses of the UVR8-dependent pathway, acting with partial or complete redundancy to stimulate expression of particular genes. Furthermore, evidence is presented that all UVR8 pathway genes are likely to be regulated by HY5/HYH and that these transcription factors do not mediate UV-B responses independent of UVR8. Finally, we highlight the functions of HY5 and HYH in UV protection and show that HY5 plays the more critical role. This research provides evidence that, in UV-B signaling, UVR8, HY5, and HYH act together in a photoregulatory pathway and demonstrates a new role for HYH in UV-B responses.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Proteínas de Transporte/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Nucleares/metabolismo , Transdução de Sinais/fisiologia , Raios Ultravioleta , Arabidopsis/efeitos da radiação , Proteínas de Arabidopsis/genética , Fatores de Transcrição de Zíper de Leucina Básica/genética , Proteínas de Transporte/genética , Proteínas Cromossômicas não Histona/genética , Criptocromos , Proteínas de Ligação a DNA , Flavoproteínas/genética , Flavoproteínas/metabolismo , Regulação da Expressão Gênica de Plantas/fisiologia , Dados de Sequência Molecular , Mutação , Proteínas Nucleares/genética , Fitocromo/genética , Fitocromo/metabolismo , Folhas de Planta/metabolismo , Folhas de Planta/efeitos da radiação , Transdução de Sinais/efeitos da radiaçãoRESUMO
UV-B radiation in sunlight has diverse effects on humans, animals, plants, and microorganisms. UV-B can cause damage to molecules and cells, and consequently organisms need to protect against and repair UV damage to survive in sunlight. In plants, low nondamaging levels of UV-B stimulate transcription of genes involved in UV-protective responses. However, remarkably little is known about the underlying mechanisms of UV-B perception and signal transduction. Here we report that Arabidopsis UV RESISTANCE LOCUS 8 (UVR8) is a UV-B-specific signaling component that orchestrates expression of a range of genes with vital UV-protective functions. Moreover, we show that UVR8 regulates expression of the transcription factor HY5 specifically when the plant is exposed to UV-B. We demonstrate that HY5 is a key effector of the UVR8 pathway, and that it is required for survival under UV-B radiation. UVR8 has sequence similarity to the eukaryotic guanine nucleotide exchange factor RCC1, but we found that it has little exchange activity. However, UVR8, like RCC1, is located principally in the nucleus and associates with chromatin via histones. Chromatin immunoprecipitation showed that UVR8 associates with chromatin in the HY5 promoter region, providing a mechanistic basis for its involvement in regulating transcription. We conclude that UVR8 defines a UV-B-specific signaling pathway in plants that orchestrates the protective gene expression responses to UV-B required for plant survival in sunlight.
