RESUMO
In plants, the ERF/EREBP family of transcriptional regulators plays a key role in adaptation to various biotic and abiotic stresses. These proteins contain a conserved AP2 DNA-binding domain and several uncharacterized motifs. Here, we describe a short motif, termed 'EDLL', that is present in AtERF98/TDR1 and other clade members from the same AP2 sub-family. We show that the EDLL motif, which has a unique arrangement of acidic amino acids and hydrophobic leucines, functions as a strong activation domain. The motif is transferable to other proteins, and is active at both proximal and distal positions of target promoters. As such, the EDLL motif is able to partly overcome the repression conferred by the AtHB2 transcription factor, which contains an ERF-associated amphiphilic repression (EAR) motif. We further examined the activation potential of EDLL by analysis of the regulation of flowering time by NF-Y (nuclear factor Y) proteins. Genetic evidence indicates that NF-Y protein complexes potentiate the action of CONSTANS in regulation of flowering in Arabidopsis; we show that the transcriptional activation function of CONSTANS can be substituted by direct fusion of the EDLL activation motif to NF-YB subunits. The EDLL motif represents a potent plant activation domain that can be used as a tool to confer transcriptional activation potential to heterologous DNA-binding proteins.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Ativação Transcricional , Motivos de Aminoácidos , Sequência de Aminoácidos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Clonagem Molecular , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Flores/metabolismo , Flores/fisiologia , Genes de Plantas , Genes Reporter , Dados de Sequência Molecular , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Plantas Geneticamente Modificadas/fisiologia , Regiões Promotoras Genéticas , Estrutura Terciária de Proteína , Protoplastos/citologia , Protoplastos/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Tempo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , TransfecçãoRESUMO
Transcription factors are proposed as suitable targets for the control of traits such as yield or food quality in plants. This study reports the results of a functional genomics research effort that identified ATHB17, a transcription factor from the homeodomain-leucine zipper class II family, as a novel target for the enhancement of photosynthetic capacity. It was shown that ATHB17 is expressed natively in the root quiescent centre (QC) from Arabidopsis embryos and seedlings. Analysis of the functional composition of genes differentially expressed in the QC from a knockout mutant (athb17-1) compared with its wild-type sibling revealed the over-representation of genes involved in auxin stimulus, embryo development, axis polarity specification, and plastid-related processes. While no other phenotypes were observed in athb17-1 plants, overexpression of ATHB17 produced a number of phenotypes in Arabidopsis including enhanced chlorophyll content. Image analysis of isolated mesophyll cells of 35S::ATHB17 lines revealed an increase in the number of chloroplasts per unit cell size, which is probably due to an increase in the number of proplastids per meristematic cell. Leaf physiological measurements provided evidence of improved photosynthetic capacity in 35S::ATHB17 lines on a per unit leaf area basis. Estimates of the capacity for ribulose-1,5-bisphosphate-saturated and -limited photosynthesis were significantly higher in 35S::ATHB17 lines.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Cloroplastos/metabolismo , Proteínas de Homeodomínio/metabolismo , Zíper de Leucina , Fotossíntese , Fatores de Transcrição/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/efeitos da radiação , Proteínas de Arabidopsis/genética , Cloroplastos/efeitos da radiação , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Genes de Plantas/genética , Proteínas de Homeodomínio/genética , Hibridização In Situ , Luz , Células do Mesofilo/citologia , Células do Mesofilo/metabolismo , Células do Mesofilo/efeitos da radiação , Mutação/genética , Fenótipo , Fotoperíodo , Fotossíntese/efeitos da radiação , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Transcrição/genéticaRESUMO
A B-box zinc finger protein, B-BOX32 (BBX32), was identified as playing a role in determining hypocotyl length during a large-scale functional genomics study in Arabidopsis (Arabidopsis thaliana). Further analysis revealed that seedlings overexpressing BBX32 display elongated hypocotyls in red, far-red, and blue light, along with reduced cotyledon expansion in red light. Through comparative analysis of mutant and overexpression line phenotypes, including global expression profiling and growth curve studies, we demonstrate that BBX32 acts antagonistically to ELONGATED HYPOCOTYL5 (HY5). We further show that BBX32 interacts with SALT TOLERANCE HOMOLOG2/BBX21, another B-box protein previously shown to interact with HY5. Based on these data, we propose that BBX32 functions downstream of multiple photoreceptors as a modulator of light responses. As such, BBX32 potentially has a native role in mediating gene repression to maintain dark adaptation.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Proteínas de Transporte/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Transdução de Sinal Luminoso/efeitos da radiação , Luz , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Arabidopsis/efeitos da radiação , Proteínas de Arabidopsis/genética , Proteínas de Transporte/genética , Escuridão , Perfilação da Expressão Gênica , Genes de Plantas/genética , Hipocótilo/crescimento & desenvolvimento , Hipocótilo/efeitos da radiação , Transdução de Sinal Luminoso/genética , Modelos Biológicos , Ligação Proteica/efeitos da radiaçãoRESUMO
Multicellular organs are composed of distinct cell types with unique assemblages of translated mRNAs. Here, ribosome-associated mRNAs were immunopurified from specific cell populations of intact seedlings using Arabidopsis thaliana lines expressing a FLAG-epitope tagged ribosomal protein L18 (FLAG-RPL18) via developmentally regulated promoters. The profiling of mRNAs in ribosome complexes, referred to as the translatome, identified differentially expressed mRNAs in 21 cell populations defined by cell-specific expression of FLAG-RPL18. Phloem companion cells of the root and shoot had the most distinctive translatomes. When seedlings were exposed to a brief period of hypoxia, a pronounced reprioritization of mRNA enrichment in the cell-specific translatomes occurred, including a ubiquitous rise in 49 mRNAs encoding transcription factors, signaling proteins, anaerobic metabolism enzymes, and uncharacterized proteins. Translatome profiling also exposed an intricate molecular signature of transcription factor (TF) family member mRNAs that was markedly reconfigured by hypoxia at global and cell-specific levels. In addition to the demonstration of the complexity and plasticity of cell-specific populations of ribosome-associated mRNAs, this study provides an in silico dataset for recognition of differentially expressed genes at the cell-, region-, and organ-specific levels.
Assuntos
Arabidopsis/citologia , Arabidopsis/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Biossíntese de Proteínas , Hipóxia Celular , Especificidade de Órgãos , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , RNA Mensageiro/isolamento & purificação , RNA Mensageiro/metabolismo , Ribossomos/metabolismo , Estresse Fisiológico , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
CONSTANS is an evolutionarily-conserved central component of the genetic pathway that controls the onset of flowering in response to daylength. However, the specific biochemical mechanism by which the CONSTANS protein regulates the expression of its target genes remains largely unknown. *By using a combination of cell-based expression analysis and in vitro DNA binding studies, we have demonstrated that CONSTANS possesses transcriptional activation potential and is capable of directly binding to DNA. *CONSTANS was found to bind DNA via a unique sequence element containing a consensus TGTG(N2-3)ATG motif. This element is present in tandem within the FLOWERING LOCUS T promoter and is sufficient for CO binding and activity. The conserved CCT (CONSTANS, CONSTANS-like and TOC1) domain of CONSTANS was shown to be required for its recruitment to the DNA motif and other CCT-containing proteins were also found to have the ability to regulate gene expression via this element. *The CCAAT box, which has been previously hypothesized as a recruitment site for complexes containing the CONSTANS protein, potentiated CONSTANS-mediated activation but was not essential for CONSTANS recruitment to a target promoter or for its activity as a transcriptional factor.
Assuntos
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas de Ligação a DNA/metabolismo , Flores/genética , Regiões Promotoras Genéticas/genética , Fatores de Transcrição/metabolismo , Motivos de Aminoácidos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Sequência de Bases , Proteínas de Ligação a DNA/química , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Genes Reporter , Glutamina/metabolismo , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Elementos de Resposta/genética , Fatores de Tempo , Fatores de Transcrição/química , Ativação Transcricional/genéticaRESUMO
ATHB17 (AT2G01430) is an Arabidopsis gene encoding a member of the α-subclass of the homeodomain leucine zipper class II (HD-Zip II) family of transcription factors. The ATHB17 monomer contains four domains common to all class II HD-Zip proteins: a putative repression domain adjacent to a homeodomain, leucine zipper, and carboxy terminal domain. However, it also possesses a unique N-terminus not present in other members of the family. In this study we demonstrate that the unique 73 amino acid N-terminus is involved in regulation of cellular localization of ATHB17. The ATHB17 protein is shown to function as a transcriptional repressor and an EAR-like motif is identified within the putative repression domain of ATHB17. Transformation of maize with an ATHB17 expression construct leads to the expression of ATHB17Δ113, a truncated protein lacking the first 113 amino acids which encodes a significant portion of the repression domain. Because ATHB17Δ113 lacks the repression domain, the protein cannot directly affect the transcription of its target genes. ATHB17Δ113 can homodimerize, form heterodimers with maize endogenous HD-Zip II proteins, and bind to target DNA sequences; thus, ATHB17Δ113 may interfere with HD-Zip II mediated transcriptional activity via a dominant negative mechanism. We provide evidence that maize HD-Zip II proteins function as transcriptional repressors and that ATHB17Δ113 relieves this HD-Zip II mediated transcriptional repression activity. Expression of ATHB17Δ113 in maize leads to increased ear size at silking and, therefore, may enhance sink potential. We hypothesize that this phenotype could be a result of modulation of endogenous HD-Zip II pathways in maize.
Assuntos
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Deleção de Sequência/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Zea mays/crescimento & desenvolvimento , Zea mays/genética , Transporte Ativo do Núcleo Celular , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteínas de Arabidopsis/química , Peso Corporal/genética , Núcleo Celular/metabolismo , Sequência Consenso , Expressão Gênica , Dados de Sequência Molecular , Multimerização Proteica , Estrutura Quaternária de Proteína , Protoplastos/metabolismo , Reprodução , Fatores de Transcrição/química , Transcrição Gênica , Zea mays/citologia , Zea mays/fisiologiaRESUMO
Accumulating evidence supports a role for members of the plant Nuclear Factor Y (NF-Y) family of CCAAT-box binding transcription factors in the regulation of flowering time. In this study we have used a genetic approach to show that the homologous proteins NF-YB3 and NF-YB2 have comparable activities and play additive roles in the promotion of flowering, specifically under inductive photoperiodic conditions. We demonstrate that NF-YB2 and NF-YB3 are both essential for the normal induction of flowering by long-days and act through regulation of the expression of FLOWERING LOCUS T (FT). Using an ELISA-based in-vitro assay, we provide a novel demonstration that plant NF-YB subunits are capable of directly binding to a CCAAT-box containing region of the FLOWERING LOCUS T promoter as part of an NF-Y trimer in combination with the yeast HAP2 and HAP5 subunits. These results support an emerging model in which NF-Y complexes provide a component of the DNA target specificity for transcriptional regulators such as CONSTANS.
Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/fisiologia , Fator de Ligação a CCAAT/fisiologia , Flores/fisiologia , Fotoperíodo , Sequência de Aminoácidos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Fator de Ligação a CCAAT/genética , Fator de Ligação a CCAAT/metabolismo , DNA Bacteriano , Ensaio de Imunoadsorção Enzimática , Flores/genética , Flores/metabolismo , Regulação da Expressão Gênica de Plantas , Genótipo , Dados de Sequência Molecular , Mutagênese Insercional , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Plantas Geneticamente Modificadas/fisiologia , Ligação Proteica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/fisiologiaRESUMO
Commercially improved crop performance under drought conditions has been challenging because of the complexity of the trait and the multitude of factors that influence yield. Here we report the results of a functional genomics approach that identified a transcription factor from the nuclear factor Y (NF-Y) family, AtNF-YB1, which acts through a previously undescribed mechanism to confer improved performance in Arabidopsis under drought conditions. An orthologous maize transcription factor, ZmNF-YB2, is shown to have an equivalent activity. Under water-limited conditions, transgenic maize plants with increased ZmNF-YB2 expression show tolerance to drought based on the responses of a number of stress-related parameters, including chlorophyll content, stomatal conductance, leaf temperature, reduced wilting, and maintenance of photosynthesis. These stress adaptations contribute to a grain yield advantage to maize under water-limited environments. The application of this technology has the potential to significantly impact maize production systems that experience drought.
Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/genética , Fator de Ligação a CCAAT/fisiologia , Desastres , Proteínas de Plantas/fisiologia , Plantas Geneticamente Modificadas/genética , Fatores de Transcrição/fisiologia , Água , Zea mays/genética , Proteínas de Arabidopsis/genética , Fator de Ligação a CCAAT/genética , Genômica , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Subunidades Proteicas/genética , Subunidades Proteicas/fisiologia , Fatores de Transcrição/genética , Zea mays/crescimento & desenvolvimentoRESUMO
Previous studies have established that mutations in the NDR1 gene in Arabidopsis thaliana suppress the resistance response of three resistance proteins, RPS2, RPM1, and RPS5, to Pseudomonas syringae pv. tomato (Pst) strain DC3000 containing the cognate effector genes, avrRpt2, avrRpm1, and avrpPhB, respectively. NDR1 is a plasma membrane (PM)-localized protein, and undergoes several post-translational modifications including carboxy-terminal processing and N-linked glycosylation. Expression of NDR1 under the NDR1 native promoter complements the ndr1-1 mutation, while overexpression of NDR1 results in enhanced resistance to virulent Pst. Sequence analysis and mass spectrometry suggest that NDR1 is localized to the PM via a C-terminal glycosylphosphatidyl-inositol (GPI) anchor. GPI modification would potentially place NDR1 on the outer surface of the PM, perhaps allowing NDR1 to act as a transducer of pathogen signals and/or interact directly with the pathogen.