RESUMO
Plants germinating under subterranean darkness assume skotomorphogenesis, a developmental program strengthened by ethylene in response to mechanical pressure of soil. Upon reaching the surface, light triggers a dramatic developmental transition termed de-etiolation that requires immediate termination of ethylene responses. Here, we report that light activation of photoreceptor phyB results in rapid degradation of EIN3, the master transcription factor in the ethylene signaling pathway. As a result, light rapidly and efficiently represses ethylene actions. Specifically, phyB directly interacts with EIN3 in a light-dependent manner and also physically associates with F box protein EBFs. The light-activated association of phyB, EIN3, and EBF1/EBF2 proteins stimulates robust EIN3 degradation by SCFEBF1/EBF2 E3 ligases. We reveal that phyB manipulates substrate-E3 ligase interactions in a light-dependent manner, thus directly controlling the stability of EIN3. Our findings illustrate a mechanistic model of how plants transduce light information to immediately turn off ethylene signaling for de-etiolation initiation.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Etilenos/metabolismo , Proteínas F-Box/metabolismo , Proteínas Nucleares/metabolismo , Fitocromo B/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/efeitos da radiação , Proteínas de Ligação a DNA , Proteínas F-Box/genética , Luz , Modelos Biológicos , Mutação , Proteínas Nucleares/genética , Processos Fototróficos , Fitocromo B/efeitos da radiação , Plantas Geneticamente Modificadas , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica/efeitos da radiação , Proteólise/efeitos da radiação , Transdução de Sinais , Fatores de Transcrição/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
Alpha subunits of heterotrimeric G proteins (Gα) are involved in a variety of cellular functions. Here we report an optogenetic strategy to spatially and temporally manipulate Gα in living cells. More specifically, we applied the blue light-induced dimerization system, known as the Magnet system, and an alternative red light-induced dimerization system consisting of Arabidopsis thaliana phytochrome B (PhyB) and phytochrome-interacting factor 6 (PIF6) to optically control the activation of two different classes of Gα (Gαq and Gαs). By utilizing this strategy, we demonstrate successful regulation of Ca2+ and cAMP using light in mammalian cells. The present strategy is generally applicable to different kinds of Gα and could contribute to expanding possibilities of spatiotemporal regulation of Gα in mammalian cells.
Assuntos
Subunidades alfa de Proteínas de Ligação ao GTP/efeitos da radiação , Animais , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/efeitos da radiação , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/efeitos da radiação , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/efeitos da radiação , Células COS , Sinalização do Cálcio/efeitos da radiação , Chlorocebus aethiops , AMP Cíclico/metabolismo , Dimerização , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Células HeLa , Humanos , Luz , Optogenética , Fitocromo B/genética , Fitocromo B/metabolismo , Fitocromo B/efeitos da radiação , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/efeitos da radiaçãoRESUMO
It has been reported that Arabidopsis phytochrome (phy) A and phyB are crucial photoreceptors that display synergistic and antagonistic action during seedling de-etiolation in multiple light signaling pathways. However, the functional relationship between phyA and phyB is not fully understood under different kinds of light and in response to different intensities of such light. In this work, we compared hypocotyl elongation of the phyA-211 phyB-9 double mutant with the wild type, the phyA-211 and phyB-9 single mutants under different intensities of far-red (FR), red (R), blue (B) and white (W) light. We confirmed that phyA and phyB synergistically promote seedling de-etiolation in B-, B plus R-, W- and high R-light conditions. The correlation of endogenous ELONGATED HYPOCOTYL 5 (HY5) protein levels with the trend of hypocotyl elongation of all lines indicate that both phyA and phyB promote seedling photomorphogenesis in a synergistic manner in high-irradiance white light. Gene expression analyses of RBCS members and HY5 suggest that phyB and phyA act antagonistically on seedling development under FR light.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/efeitos da radiação , Hipocótilo/efeitos da radiação , Fitocromo A/metabolismo , Fitocromo B/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/efeitos da radiação , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Escuridão , Relação Dose-Resposta à Radiação , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Hipocótilo/genética , Hipocótilo/crescimento & desenvolvimento , Hipocótilo/metabolismo , Luz , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fitocromo A/genética , Fitocromo A/efeitos da radiação , Fitocromo B/genética , Fitocromo B/efeitos da radiaçãoRESUMO
Phytochromes are red/far-red photoreceptors encoded by a small gene family in higher plants. Differences in phenotype among mutants suggest distinct functions among phytochrome subfamilies. We attempted to find distinct functions among phytochromes by oligo-microarray analysis of single, double, and triple mutants in rice. In most cases, gene expression was redundantly regulated by phytochromes A and B after irradiation by a red light pulse in etiolated rice shoots. However, we found that several genes were specifically regulated by phytochromes A and C. Most of them were expressed immediately after the red light pulse in a transient manner. They are stress-related genes that may be involved in resistance to light stress when etiolated seedlings are exposed to light. These genes were not expressed in green leaves after the red light pulse, suggesting that they have a function specific to etiolated seedlings.
Assuntos
Regulação da Expressão Gênica de Plantas , Luz , Oryza/efeitos da radiação , Fitocromo A/metabolismo , Plântula/efeitos da radiação , Perfilação da Expressão Gênica , Genes de Plantas , Análise de Sequência com Séries de Oligonucleotídeos , Oryza/genética , Oryza/metabolismo , Fenótipo , Fitocromo A/genética , Fitocromo A/efeitos da radiação , Fitocromo B/genética , Fitocromo B/metabolismo , Fitocromo B/efeitos da radiação , Folhas de Planta/genética , Folhas de Planta/metabolismo , Folhas de Planta/efeitos da radiação , Brotos de Planta/genética , Brotos de Planta/metabolismo , Brotos de Planta/efeitos da radiação , Plântula/genética , Plântula/metabolismo , Estresse Fisiológico , Fatores de TempoRESUMO
The phytochrome (phy)A and phyB photoreceptors mediate three photobiological response modes in plants; whereas phyA can mediate the very-low-fluence response (VLFR), the high-irradiance response (HIR) and, to some extent, the low fluence response (LFR), phyB and other type II phytochromes only mediate the LFR. To investigate to what level a rice phyA can complement for Arabidopsis phyA or phyB function and to evaluate the role of the serine residues in the first 20 amino acids of the N-terminus of phyA, we examined VLFR, LFR, and HIR responses in phyB and phyAphyB mutant plants transformed with rice PHYA cDNA or a mutant rice PHYA cDNA in which the first 10 serine residues were mutated to alanines (phyA SA). Utilizing mutants without endogenous phyB allowed the evaluation of red-light-derived responses sensed by the rice phyA. In summary, the WT rice phyA could complement VLFR and LFR responses such as inhibition of hypocotyl elongation under pulses of FR or continuous R light, induction of flowering and leaf expansion, whereas the phyA SA was more specific for HIR responses (e.g. inhibition of hypocotyl elongation and anthocyanin accumulation under continuous far-red light). As the N-terminal serines can no longer be phosphorylated in the phyA SA mutant, this suggests a role for phosphorylation discriminating between the different phyA-dependent responses. The efficacy of the rice phyA expressed in Arabidopsis was dependent upon the developmental age of the plants analyzed and on the physiological response, suggesting a stage-dependent downstream modulation of phytochrome signaling.
