RESUMO
Plants adapt to abiotic stresses by complex mechanisms involving various stress-responsive genes. Here, we identified a DEAD-box RNA helicase (RH) gene, AtRH17, in Arabidopsis, involved in salt-stress responses using activation tagging, a useful technique for isolating novel stress-responsive genes. AT895, an activation tagging line, was more tolerant than wild type (WT) under NaCl treatment during germination and seedling development, and AtRH17 was activated in AT895. AtRH17 possesses nine well-conserved motifs of DEAD-box RHs, consisting of motifs Q, I, Ia, Ib, and II-VI. Although at least 12 orthologs of AtRH17 have been found in various plant species, no paralog occurs in Arabidopsis. AtRH17 protein is subcellularily localized in the nucleus. AtRH17-overexpressing transgenic plants (OXs) were more tolerant to high concentrations of NaCl and LiCl compared with WT, but no differences from WT were detected among seedlings exposed to mannitol and freezing treatments. Moreover, in the mature plant stage, AtRH17 OXs were also more tolerant to NaCl than WT, but not to drought, suggesting that AtRH17 is involved specifically in the salt-stress response. Notably, transcriptions of well-known abscisic acid (ABA)-dependent and ABA-independent stress-response genes were similar or lower in AtRH17 OXs than WT under salt-stress treatments. Taken together, our findings suggest that AtRH17, a nuclear DEAD-box RH protein, is involved in salt-stress tolerance, and that its overexpression confers salt-stress tolerance via a pathway other than the well-known ABA-dependent and ABA-independent pathways.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/efeitos dos fármacos , Arabidopsis/enzimologia , RNA Helicases DEAD-box/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , RNA Helicases DEAD-box/genética , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/genética , Tolerância ao Sal , Cloreto de Sódio/farmacologiaRESUMO
MAIN CONCLUSION: AtNAP , an Arabidopsis NAC transcription factor family gene, functions as a negative regulator via transcriptional repression of AREB1 in salt stress response. AtNAP is an NAC family transcription factor in Arabidopsis and is known to be a positive regulator of senescence. However, its exact function and underlying molecular mechanism in stress responses are not well known. Here, we investigated functional roles of AtNAP in salt stress response. AtNAP expression significantly increased at the seedling stage, with higher expression in both shoots and roots under NaCl, mannitol, and ABA treatments. T-DNA insertional loss-of-function mutants of AtNAP were more tolerant to salt stress than wild type (WT), whereas AtNAP-overexpressing transgenic plants (OXs) were more sensitive to salt stress than WT during germination, seedling development, and mature plant stage. Transcript levels of stress-responsive genes in the ABA-dependent pathway, such as AREB1, RD20, and RD29B, were significantly higher and lower in atnap mutants and AtNAP OXs, respectively, than in WT under salt stress conditions, suggesting that AtNAP might negatively regulate the expression of those genes under salt stress conditions. Indeed, AtNAP repressed the promoter activity of AREB1 under normal and salt stress conditions. These results indicate that AtNAP functions as a negative regulator in the salt stress response. Our results, together with previous studies, suggest that AtNAP functions as a negative regulator in osmotic stress responses, whereas it functions as a positive regulator in senescence.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Estresse Fisiológico/genética , Arabidopsis/efeitos dos fármacos , Proteínas de Arabidopsis/genética , Fatores de Transcrição de Zíper de Leucina Básica/genética , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas e Peptídeos de Choque Frio/genética , Proteínas e Peptídeos de Choque Frio/metabolismo , Regulação da Expressão Gênica de Plantas , Pressão Osmótica , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas , Plântula/genética , Plântula/crescimento & desenvolvimento , Cloreto de Sódio/farmacologiaRESUMO
Despite increasing reports that CCCH zinc finger proteins function in plant development and stress responses, the functions and molecular aspects of many CCCH zinc finger proteins remain uncharacterized. Here, we characterized the biological and molecular functions of AtC3H17, a unique Arabidopsis gene encoding a non-tandem CCCH zinc finger protein. AtC3H17 was ubiquitously expressed throughout the life cycle of Arabidopsis plants and their organs. The rate and ratio of seed germination of atc3h17 mutants were slightly slower and lower, respectively, than those of the wild type (WT), whereas AtC3H17-overexpressing transgenic plants (OXs) showed an enhanced germination rate. atc3h17 mutant seedlings were smaller and lighter than WT seedlings while AtC3H17 OX seedlings were larger and heavier. In regulation of flowering time, atc3h17 mutants showed delayed flowering, whereas AtC3H17 OXs showed early flowering compared with the WT. In addition, overexpression of AtC3H17 affected seed development, displaying abnormalities compared with the WT. AtC3H17 protein was localized to the nucleus and showed transcriptional activation activity in yeast and Arabidopsis protoplasts. The N-terminal region of AtC3H17, containing a conserved EELR-like motif, was necessary for transcriptional activation activity, and the two conserved glutamate residues in the EELR-like motif played an important role in transcriptional activation activity. Real-time PCR and transactivation analyses showed that AtC3H17 might be involved in seed development via transcriptional activation of OLEO1, OLEO2 and CRU3. Our results suggest that AtC3H17 has pleiotropic effects on vegetative development such as seed germination and seedling growth, flowering and seed development, and functions as a nuclear transcriptional activator in Arabidopsis.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Núcleo Celular/metabolismo , Flores/crescimento & desenvolvimento , Pleiotropia Genética , Sementes/crescimento & desenvolvimento , Transativadores/metabolismo , Dedos de Zinco , Motivos de Aminoácidos , Sequência de Aminoácidos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Sequência Conservada , Flores/genética , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Germinação/genética , Ácido Glutâmico/metabolismo , Mutação/genética , Especificidade de Órgãos/genética , Fenótipo , Regiões Promotoras Genéticas/genética , Domínios Proteicos , Sementes/genética , Transativadores/química , Transativadores/genética , Ativação Transcricional/genéticaRESUMO
KEY MESSAGE: AtSFT12, an Arabidopsis Qc-SNARE protein, is localized to Golgi organelles and is involved in salt and osmotic stress responses via accumulation of Na (+) in vacuoles. To reduce the detrimental effects of environmental stresses, plants have evolved many defense mechanisms. Here, we identified an Arabidopsis Qc-SNARE gene, AtSFT12, involved in salt and osmotic stress responses using an activation-tagging method. Both activation-tagged plants and overexpressing transgenic plants (OXs) of the AtSFT12 gene were tolerant to high concentrations of NaCl, LiCl, and mannitol, whereas loss-of-function mutants were sensitive to NaCl, LiCl, and mannitol. AtSFT12 transcription increased under NaCl, ABA, cold, and mannitol stresses but not MV treatment. GFP-fusion AtSFT12 protein was juxtaposed with Golgi marker, implying that its function is associated with Golgi-mediated transport. Quantitative measurement of Na(+) using induced coupled plasma atomic emission spectroscopy revealed that AtSFT12 OXs accumulated significantly more Na(+) than WT plants. In addition, Na(+)-dependent fluorescence analysis of Sodium Green showed comparatively higher Na(+) accumulation in vacuoles of AtSFT12 OX cells than in those of WT plant cells after salt treatments. Taken together, our findings suggest that AtSTF12, a Golgi Qc-SNARE protein, plays an important role in salt and osmotic stress responses and functions in the salt stress response via sequestration of Na(+) in vacuoles.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/fisiologia , Osmose/efeitos dos fármacos , Proteínas Qc-SNARE/genética , Cloreto de Sódio/farmacologia , Sódio/metabolismo , Estresse Fisiológico/genética , Vacúolos/metabolismo , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genes de Plantas , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/metabolismo , Mutação/genética , Especificidade de Órgãos/efeitos dos fármacos , Especificidade de Órgãos/genética , Plantas Geneticamente Modificadas , Transporte Proteico/efeitos dos fármacos , Proteínas Qc-SNARE/metabolismo , Estresse Fisiológico/efeitos dos fármacos , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/metabolismo , Fatores de Tempo , Transcrição Gênica/efeitos dos fármacos , Vacúolos/efeitos dos fármacosRESUMO
S-RBP11, a chloroplast protein, which was isolated using activation tagging system, is shown to be the first Arabidopsis small RNA-binding group protein involved in oxidative and salt stress responses. Activation tagging is one of the most powerful tools in reverse genetics. In this study, we isolated S-RBP11, encoding a small RNA-binding protein in Arabidopsis, by salt-resistant activation tagging line screen and then characterized its function in the abiotic stress response. The isolated activation tagging line of S-RBP11 as well as transgenic plants overexpressing S-RBP11 showed increased tolerance to salt and MV stresses compared to WT plants, whereas s-rbp11 mutants were more sensitive to salt stresses. Transcription of S-RBP11 was elevated upon MV treatment but not NaCl or cold treatment. Interestingly, S-RBP11 protein was localized in the chloroplast and the N-terminal 34 amino acid region of S-RBP11 was necessary for its chloroplast targeting. Our results suggest that S-RBP11 is a chloroplast protein involved in the responses to salt and oxidative stresses.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/fisiologia , Proteínas de Cloroplastos/genética , Regulação da Expressão Gênica de Plantas , Proteínas de Ligação a RNA/genética , Estresse Fisiológico , Sequência de Aminoácidos , Arabidopsis/citologia , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Cloroplastos/metabolismo , Cloroplastos/metabolismo , Secas , Dados de Sequência Molecular , Mutagênese Insercional , Estresse Oxidativo , Fenótipo , Plantas Geneticamente Modificadas , Proteínas de Ligação a RNA/metabolismo , Tolerância ao Sal , Plântula/citologia , Plântula/genética , Plântula/fisiologia , Alinhamento de SequênciaRESUMO
Plants have developed various regulatory pathways to adapt to environmental stresses. In this study, we identified Arabidopsis MKKK20 as a regulator in the response to osmotic stress. mkkk20 mutants were found to be sensitive to high concentration of salt and showed higher water loss rates than wild-type (WT) plants under dehydration conditions. In addition, mkkk20 mutants showed higher accumulation of superoxide, a reactive oxygen species (ROS), compared to WT plants under high salt condition. In contrast, transgenic plants overexpressing MKKK20 displayed tolerance to salt stress. MKKK20 transcripts were increased by the treatments with NaCl, mannitol, MV, sorbitol, and cold, suggesting that MKKK20 is involved in the response to osmotic, ROS, and cold stresses. In-gel kinase assay showed that MKKK20 regulates the activity of MPK6 under NaCl, cold, and H(2)O(2) treatments. Taken together, our results suggest that MKKK20 might be involved in the response to various abiotic stresses, especially osmotic stress, through its regulation of MPK6 activity.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Temperatura Baixa , Secas , Regulação da Expressão Gênica de Plantas , Peróxido de Hidrogênio/farmacologia , Manitol/farmacologia , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/genética , Mutação , Pressão Osmótica , Fosforilação , Plantas Geneticamente Modificadas , Espécies Reativas de Oxigênio/metabolismo , Tolerância ao Sal , Transdução de Sinais , Cloreto de Sódio/farmacologia , Sorbitol/farmacologia , Estresse Fisiológico , Superóxidos/metabolismoRESUMO
Plants have developed disparate regulatory pathways to adapt to environmental stresses. In this study, we identified MKK4 as an important mediator of plant response to osmotic stress. mkk4 mutants were more sensitive to high salt concentration than WT plants, exhibiting higher water-loss rates under dehydration conditions and additionally accumulating high levels of ROS. In contrast, MKK4-overexpressing transgenic plants showed tolerance to high salt as well as lower water-loss rates under dehydration conditions. In-gel kinase assays revealed that MKK4 regulates the activity of MPK3 upon NaCl exposure. Semi-quantitative RT-PCR analysis showed that expression of NCED3 and RD29A was lower and higher in mkk4 mutants and MKK4-overexpressing transgenic plants, respectively. Taken together, our results suggest that MKK4 is involved in the osmotic-stress response via its regulation of MPK3 activity.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Secas , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Salinidade , Estresse Fisiológico , Arabidopsis/enzimologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Dioxigenases/genética , Regulação da Expressão Gênica de Plantas , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Mutação , Pressão Osmótica , Proteínas de Plantas/genética , Transcrição GênicaRESUMO
Various transcription factors are involved in the response to environmental stresses in plants. In this study, we characterized AtERF71/HRE2, a member of the Arabidopsis AP2/ERF family, as an important regulator of the osmotic and hypoxic stress responses in plants. Transcript level of AtERF71/HRE2 was highly increased by anoxia, NaCl, mannitol, ABA, and MV treatments. aterf71/hre2 loss-of-function mutants displayed higher sensitivity to osmotic stress such as high salt and mannitol, accumulating higher levels of ROS under high salt treatment. In contrast, AtERF71/HRE2-overexpressing transgenic plants showed tolerance to salt and mannitol as well as flooding and MV stresses, exhibiting lower levels of ROS under high salt treatment. AtERF71/HRE2 protein was localized in the nucleus, and the C-terminal region of AtERF71/HRE2 was required for transcription activation activity. Taken together, our results suggest that AtERF71/HRE2 might function as a transcription factor involved in the response to osmotic stress as well as hypoxia.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Tolerância ao Sal/genética , Estresse Fisiológico/genética , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Anaerobiose , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Núcleo Celular/metabolismo , Manitol/farmacologia , Dados de Sequência Molecular , Osmose , Pressão Osmótica , Cloreto de Sódio/farmacologia , Fatores de Transcrição/genética , Transcrição GênicaRESUMO
Aurora A kinase has drawn considerable attention as a therapeutic target for cancer therapy. However, the underlying molecular and cellular mechanisms of the anticancer effects of Aurora A kinase inhibition are still not fully understood. Herein, we show that depletion of Aurora A kinase by RNA interference (RNAi) in hepatocellular carcinoma (HCC) cells upregulated FoxO1 in a p53-dependent manner, which induces cell cycle arrest. Introduction of an RNAi-resistant Aurora A kinase into Aurora A-knockdown cells resulted in downregulation of FoxO1 expression and rescued proliferation. In addition, silencing of FoxO1 in Aurora A-knockdown cells allowed the cells to exit cytostatic arrest, which, in turn, led to massive cell death. Our results suggest that FoxO1 is responsible for growth arrest at the G2/M phase that is induced by Aurora A kinase inhibition.
Assuntos
Fatores de Transcrição Forkhead/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Apoptose , Aurora Quinases , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Regulação para Baixo , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/antagonistas & inibidores , Fatores de Transcrição Forkhead/genética , Pontos de Checagem da Fase G2 do Ciclo Celular , Células Hep G2 , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Pontos de Checagem da Fase M do Ciclo Celular , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
The plant hormone, abscisic acid (ABA), is a main signal transducer that confers abiotic stress tolerance to plants. Although the pathway of ABA production and the genes catalyzing its biosynthesis are largely defined, the regulatory mechanism of ABA biosynthesis in response to abiotic stress remains much unknown. In this study, to identify upstream genes regulating ABA biosynthesis involved in abiotic stress signal transduction, Arabidopsis thaliana mutants with altered promoter activity of 9-cis-epoxycarotenoid dioxygenase 3 (NCED3), a key gene in ABA biosynthesis, were identified and characterized. Among selected mutants, lenc1 (for low expression of NCED3 1) after dehydration treatment had lower AtNCED3 promoter activity compared with wild type. lenc1 mutation is recessive and is located on chromosome 4. Expression analysis of AtNCED3 and quantification of ABA levels showed that both the AtNCED3 transcripts and the endogenous ABA in lenc1 were less abundant than in wild type under dehydration treatments. The lenc1 was hypersensitive to methyl viologen (MV), LiCl, NaCl and high light. The aerial part of lenc1 lost water faster than wild type possibly due to a larger stomata opening. Our results suggest LENC1 might act as a positive regulator in AtNCED3 gene expression under osmotic stress.