RESUMO
Phosphoinositides (PIs) are essential metabolites which are generated by various lipid kinases and rapidly respond to a variety of environmental stimuli in eukaryotes. One of the precursors of important regulatory PIs, phosphatidylinositol (PtdIn) 4-phosphate, is synthesized by PtdIns 4-kinases (PI4K). Despite its wide distribution in eukaryotes, its role in plants remains largely unknown. Here, we show that the activity of AtPI4Kγ3 gene, an Arabidopsis (Arabidopsis thaliana) type II PtdIn 4-kinase, is regulated by DNA demethylation and some abiotic stresses. AtPI4Kγ3 is targeted to the nucleus and selectively bounds to a few PtdIns. It possessed autophosphorylation activity but unexpectedly had no detectable lipid kinase activity. Overexpression of AtPI4Kγ3 revealed enhanced tolerance to high salinity or ABA along with inducible expression of a host of stress-responsive genes and an optimal accumulation of reactive oxygen species. Furthermore, overexpressed AtPI4Kγ3 augmented the salt tolerance of bzip60 mutants. The ubiquitin-like domain of AtPI4Kγ3 is demonstrated to be essential for salt stress tolerance. Besides, AtPI4Kγ3-overexpressed plants showed a late-flowering phenotype, which was caused by the regulation of some flowering pathway integrators. In all, our results indicate that AtPI4Kγ3 is necessary for reinforcement of plant response to abiotic stresses and delay of the floral transition.
Assuntos
1-Fosfatidilinositol 4-Quinase/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Arabidopsis/fisiologia , Flores/fisiologia , Estresse Fisiológico , 1-Fosfatidilinositol 4-Quinase/química , 1-Fosfatidilinositol 4-Quinase/genética , Ácido Abscísico/farmacologia , Adaptação Fisiológica/efeitos dos fármacos , Adaptação Fisiológica/genética , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Metilação de DNA/efeitos dos fármacos , Metilação de DNA/genética , Flores/efeitos dos fármacos , Flores/genética , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genes de Plantas , Peróxido de Hidrogênio/farmacologia , Modelos Biológicos , Mutação/genética , Fotoperíodo , Domínios Proteicos , Transporte Proteico/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Salinidade , Transdução de Sinais/efeitos dos fármacos , Cloreto de Sódio/farmacologia , Estresse Fisiológico/efeitos dos fármacos , Estresse Fisiológico/genética , Relação Estrutura-Atividade , Frações Subcelulares/metabolismo , Especificidade por Substrato/efeitos dos fármacosRESUMO
RNA silencing is an evolutionarily conserved mechanism triggered by double-stranded RNA that is processed into 21- to 24-nt small interfering (si)RNA or micro (mi)RNA by RNaseIII-like enzymes called Dicers. Gene regulations by RNA silencing have fundamental implications in a large number of biological processes that include antiviral defense, maintenance of genome integrity and the orchestration of cell fates. Although most generic or core components of the various plant small RNA pathways have been likely identified over the past 15 years, factors involved in RNAi regulation through post-translational modifications are just starting to emerge, mostly through forward genetic studies. A genetic screen designed to identify factors required for RNAi in Arabidopsis identified the serine/threonine protein kinase, TOUSLED (TSL). Mutations in TSL affect exogenous and virus-derived siRNA activity in a manner dependent upon its kinase activity. By contrast, despite their pleiotropic developmental phenotype, tsl mutants show no defect in biogenesis or activity of miRNA or endogenous trans-acting siRNA. These data suggest a possible role for TSL phosphorylation in the specific regulation of exogenous and antiviral RNA silencing in Arabidopsis and identify TSL as an intrinsic regulator of RNA interference.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Proteínas Serina-Treonina Quinases/metabolismo , Interferência de RNA , Arabidopsis/enzimologia , Arabidopsis/metabolismo , Plantas Geneticamente Modificadas/genética , Pequeno RNA não Traduzido/metabolismo , Complexo de Inativação Induzido por RNA/metabolismoRESUMO
Although heat-shock transcription factors are well characterized in the heat stress-related pathway, they are poorly understood in other stress responses. Here, we functionally characterized AtHsfA6a in the presence of exogenous abscisic acid (ABA) and under high salinity and dehydration conditions. AtHsfA6a expression under normal conditions is very low, but was highly induced by exogenous ABA, NaCl and drought. Unexpectedly, the levels of AtHsfA6a transcript were not significantly altered under heat and cold stresses. Electrophoretic mobility shift assays and transient transactivation assays indicated that AtHsfA6a is transcriptionally regulated by ABA-responsive element binding factor/ABA-responsive element binding protein, which are key regulators of the ABA signalling pathway. Additionally, fractionation and protoplast transient assays showed that AtHsfA6a was in cytoplasm and nucleus simultaneously; however, under conditions of high salinity the majority of AtHsfA6A was in the nucleus. Furthermore, at both seed germination and seedlings stage, plants overexpressing AtHsfA6a were hypersensitive to ABA and exhibited enhanced tolerance against salt and drought stresses. Finally, the microarray and qRT-PCR analyses revealed that many stress-responsive genes were up-regulated in the plants overexpressing AtHsfA6a. Taken together, the data strongly suggest that AtHsfA6a acts as a transcriptional activator of stress-responsive genes via the ABA-dependent signalling pathway.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Proteínas de Ligação a DNA/metabolismo , Salinidade , Fatores de Transcrição/metabolismo , Ácido Abscísico/farmacologia , Adaptação Fisiológica/efeitos dos fármacos , Adaptação Fisiológica/genética , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , DNA Bacteriano/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Desidratação , Secas , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Fatores de Transcrição de Choque Térmico , Mutagênese Insercional/genética , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Reprodutibilidade dos Testes , Saccharomyces cerevisiae/citologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genética , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/genética , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
In eukaryotes, RNA silencing, mediated by small interfering RNAs, is an evolutionarily widespread and versatile silencing mechanism that plays an important role in various biological processes. Increasing evidences suggest that various components of RNA silencing pathway are involved in plant defense machinery against microbial pathogens in Arabidopsis thaliana. Here, we show genetic and molecular evidence that Arabidopsis SDE5 is required to generate an effective resistance against the biotrophic bacteria Pseudomonas syringae pv. tomato DC3000 and for susceptibility to the necrotrophic bacteria Erwinia caratovora pv. caratovora. SDE5, encodes a putative mRNA export factor that is indispensable for transgene silencing and the production of trans-acting siRNAs. SDE5 expression is rapidly induced by exogenous application of phytohormone salicylic acid (SA), methyl jasmonate (MeJA), phytopathogenic bacteria, and flagellin. We further report that SDE5 is involved in basal plant defense and mRNA export. Our genetic data suggests that SDE5 and Nonexpressor of PR Gene1 (NPR1) may contribute to the same SA-signaling pathway. However, SDE5 over-expressing transgenic plant exhibits reduced defense responsive phenotype after flagellin treatment. Taken together, these results support the conclusion that SDE5 contributes to plant innate immunity in Arabidopsis.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/imunologia , Arabidopsis/metabolismo , Flagelina/metabolismo , Imunidade Inata , Transdução de Sinais , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Resistência à Doença/genética , Resistência à Doença/imunologia , Regulação da Expressão Gênica de Plantas , Imunidade Inata/genética , Mutação , Fenótipo , Plantas Geneticamente Modificadas , Transporte de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
The interaction of Obg (Spo0B-associated GTP-binding protein) GTPase and SpoT, which is a bifunctional ppGpp (guanosine 3',5'-bispyrophosphate) hydrolase/synthetase, is vital for the modulation of intracellular ppGpp levels during bacterial responses to environmental cues. It has been recently reported that the ppGpp level is also inducible by various stresses in the chloroplasts of plant cells. However, the function of the Obg-SpoT interaction in plants remains elusive. The results from the present and previous studies suggest that AtRSH1 is a putative bacterial SpoT homolog in Arabidopsis and that its transcription levels are responsive to wounding and salt stresses. In this study, we used a yeast two-hybrid analysis to map the regions required for the AtObgC-AtRSH1 interaction. Moreover, protein-protein docking simulations revealed reasonable geometric and electrostatic complementarity in the binding surfaces of the two proteins. The data support our experimental results, which suggest that the conserved domains in AtObgC and the N terminus of AtRSH1 containing the TGS domain contribute to their interaction. In addition, quantitative real-time PCR (qRT-PCR) analyses showed that the expression of AtObgC and AtRSH1 exhibit a similar inhibition pattern under wounding and salt-stress conditions, but the inhibition pattern was not greatly influenced by the presence or absence of light. Based on in vivo analyses, we further confirmed that the AtRSH1 and AtObgC proteins similarly localize in chloroplasts. Based on these results, we propose that the AtObgC-AtRSH1 interaction plays a vital role in ppGpp-mediated stress responses in chloroplasts.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Transdução de Sinais , Estresse Fisiológico/fisiologia , Arabidopsis/metabolismo , Simulação de Acoplamento Molecular , Ligação ProteicaRESUMO
Arabidopsis CTD-PHOSPHATASE-LIKE 1 (CPL1) is a protein phosphatase that can dephosphorylate RNA polymerase II C-terminal domain (CTD). Unlike typical CTD-phosphatases, CPL1 contains a double-stranded (ds) RNA-binding motif (dsRBM) and has been implicated for gene regulation mediated by dsRNA-dependent pathways. We investigated the role of CPL1 and its dsRBMs in various gene silencing pathways. Genetic interaction analyses revealed that cpl1 was able to partially suppress transcriptional gene silencing and DNA hypermethylation phenotype of ros1 suggesting CPL1 is involved in the RNA-directed DNA methylation pathway without reducing siRNA production. By contrast, cpl1 reduced some miRNA levels at the level of processing. Indeed, CPL1 protein interacted with proteins important for miRNA biogenesis, suggesting that CPL1 regulates miRNA processing. These results suggest that CPL1 regulates DNA methylation via a miRNA-dependent pathway.