RESUMEN
The nucleotide sequence of a 20.5-kb genomic region harboring nif genes was determined and analyzed. The fragment was obtained from Frankia sp. EuIK1 strain, an indigenous symbiont of Elaeagnus umbellata. A total of 20 ORFs including 12 nif genes were identified and subjected to comparative analysis with the genome sequences of 3 Frankia strains representing diverse host plant specificities. The nucleotide and deduced amino acid sequences showed highest levels of identity with orthologous genes from an Elaeagnus-infecting strain. The gene organization patterns around the nif gene clusters were well conserved among all 4 Frankia strains. However, characteristic features appeared in the location of the nifV gene for each Frankia strain, depending on the type of host plant. Sequence analysis was performed to determine the transcription units and suggested that there could be an independent operon starting from the nifW gene in the EuIK strain. Considering the organization patterns and their total extensions on the genome, we propose that the nif gene clusters remained stable despite genetic variations occurring in the Frankia genomes.
Asunto(s)
Elaeagnaceae/microbiología , Frankia/genética , Familia de Multigenes , Nitrogenasa/genética , Hibridación Genómica Comparativa , ADN Bacteriano/genética , Frankia/enzimología , Genes Bacterianos , Anotación de Secuencia Molecular , Fijación del Nitrógeno/genética , Sistemas de Lectura Abierta , Operón , Análisis de Secuencia de ADN , SimbiosisRESUMEN
Seedling-lethal phenotypes of Arabidopsis (Arabidopsis thaliana) mutants that are defective in early steps in the sterol biosynthetic pathway are not rescued by the exogenous application of brassinosteroids. The detailed molecular and physiological mechanisms of seedling lethality have yet to be understood. Thus, to elucidate the underlying mechanism of lethality, we analyzed transcriptome and proteome profiles of the cyp51A2 mutant that is defective in sterol 14alpha-demethylation. Results revealed that the expression levels of genes involved in ethylene biosynthesis/signaling and detoxification of reactive oxygen species (ROS) increased in the mutant compared with the wild type and, thereby, that the endogenous ethylene level also increased in the mutant. Consistently, the seedling-lethal phenotype of the cyp51A2 mutant was partly attenuated by the inhibition of ethylene biosynthesis or signaling. However, photosynthesis-related genes including Rubisco large subunit, chlorophyll a/b-binding protein, and components of photosystems were transcriptionally and/or translationally down-regulated in the mutant, accompanied by the transformation of chloroplasts into gerontoplasts and a reduction in both chlorophyll contents and photosynthetic activity. These characteristics observed in the cyp51A2 mutant resemble those of leaf senescence. Nitroblue tetrazolium staining data revealed that the mutant was under oxidative stress due to the accumulation of ROS, a key factor controlling both programmed cell death and ethylene production. Our results suggest that changes in membrane sterol contents and composition in the cyp51A2 mutant trigger the generation of ROS and ethylene and eventually induce premature seedling senescence.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Etilenos/biosíntesis , Especies Reactivas de Oxígeno/metabolismo , Plantones/fisiología , Esteroles/metabolismo , Arabidopsis/fisiología , Proteínas de Arabidopsis/genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas/fisiología , Mutación , Transducción de SeñalRESUMEN
Citrus sunki (Jinkyool) is a medicinal landrace citrus belonging to the Rutaceae family. We determined the complete chloroplast genome (160,699 bp) of C. sunki CRS0085 in Jeju Island, Korea. The genome is composed of four distinct parts; a large single copy of 87,918 bp, a small single copy of 21,355 bp, and a pair of inverted repeat regions of 25,713 bp. A total of 134 genes including 89 protein-coding genes, 37 tRNA genes, and eight rRNA genes were identified. The phylogenetic tree showed that C. sunki CRS0085 has the closest relationship with C. reticulata within genus Citrus.
RESUMEN
Root nodule formation is controlled by plant hormones such as auxin. Auxin-repressed protein (ARP) genes have been identified in various plant species but their functions are not clear. We have isolated a full-length cDNA clone (EuNOD-ARP1) showing high sequence homology to previously identified ARP genes from root nodules of Elaeagnus umbellata. Genomic Southern hybridization showed that there are at least four ARP-related genes in the genome of E. umbellata. The cDNA clone encodes a polypeptide of 120 amino acid residues with no signal peptide or organelle-targeting signals, indicating that it is a cytosolic protein. Its cytosolic location was confirmed using Arabidopsis protoplasts expressing a EuNOD-ARP1:smGFP fusion protein. Northern hybridization showed that EuNOD-ARP1 expression was higher in root nodules than in leaves or uninoculated roots. Unlike the ARP genes of strawberry and black locust, which are negatively regulated by exogenous auxin, EuNOD-ARP1 expression is induced by auxin in leaf tissue of E. umbellata. In situ hybridization revealed that EuNOD-ARP1 is mainly expressed in the fixation zone of root nodules.
Asunto(s)
Elaeagnaceae/metabolismo , Ácidos Indolacéticos/farmacología , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Nódulos de las Raíces de las Plantas/efectos de los fármacos , Homología de Secuencia de Aminoácido , Regulación hacia Arriba/efectos de los fármacos , Secuencia de Aminoácidos , Southern Blotting , Elaeagnaceae/efectos de los fármacos , Elaeagnaceae/genética , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Genes de Plantas , Genoma de Planta/efectos de los fármacos , Datos de Secuencia Molecular , Familia de Multigenes , Filogenia , Proteínas de Plantas/química , Transporte de Proteínas/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Nódulos de las Raíces de las Plantas/citologíaRESUMEN
Ionotropic glutamate receptors (iGluRs) are ligand-gated nonselective cation channels that mediate fast excitatory neurotransmission. Although homologues of the iGluRs have been identified in higher plants, their roles are largely unknown. In this work we isolated a full-length cDNA clone (RsGluR) encoding a putative glutamate receptor from small radish. An RsGluR: mGFP fusion protein was localized to the plasma membrane. In Arabidopsis thaliana overexpressing the full-length cDNA, glutamate treatment triggered greater Ca2+ influx in the root cells of transgenic seedlings than in those of the wild type. Transgenic plants exhibited multiple morphological changes such as necrosis at their tips and the margins of developing leaves, dwarf stature with multiple secondary inflorescences, and retarded growth, as previously observed in transgenic Arabidopsis overexpressing AtGluR3.2 [Kim et al. (2001)]. Microarray analysis showed that jasmonic acid (JA)-responsive genes including defensins and JA-biosynthetic genes were up-regulated. RsGluR overexpression also inhibited growth of a necrotic fungal pathogen Botrytis cinerea possibly due to up-regulation of the defensins. Based on these results, we suggest that RsGluR is a glutamate-gated Ca2+ channel located in the plasma membrane of higher plants and plays a direct or indirect role in defense against pathogen infection by triggering JA biosynthesis.
Asunto(s)
Arabidopsis/genética , Calcio/metabolismo , Membrana Celular/metabolismo , Ácido Glutámico/metabolismo , Raphanus/metabolismo , Raphanus/microbiología , Receptores de Glutamato/metabolismo , Secuencia de Aminoácidos , Arabidopsis/metabolismo , Arabidopsis/microbiología , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Clonación Molecular , Ciclopentanos/metabolismo , Regulación de la Expresión Génica de las Plantas , Análisis por Micromatrices , Datos de Secuencia Molecular , Oxilipinas , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/microbiología , Plantas Modificadas Genéticamente , Raphanus/genética , Receptores de Glutamato/genética , Fracciones SubcelularesRESUMEN
The nifV gene from the Frankia EuIK1 strain, a symbiont of Elaeagnus umbellata, was cloned and a complementation test using the Klebsiella pneumoniae nifV mutant was performed to verify its function. The nifV ORF consists of 1245 bp, which encodes 414 amino acids. However, the putative promoter and Shine-Dalgarno sequences were not found in the 5' region of the ORF. The Frankia EuIK1 nifV ORF showed about a 70% nucleotide identity and 80% amino acid similarity with that of Frankia sp. FaC1. In the upstream region of the nifV, a putative ORF that showed a 51% nucleotide identity with the afcD gene from Burkholderia cepacia BC11 was found. The other partial ORF that showed a 59% identity with the pkaD gene from Streptomyces coelicolor A(3) was found in the downstream region. In this respect, Frankia EuIK1 nifV has an unusual location on the genome, considering the nif gene organization. A phylogenetic analysis revealed that the NifV from Frankia EuIK1 was close to those from two Alnus-infective Frankia species, and they were grouped with those of the alpha-class proteobacteria, supporting the vertical descent of nifV. The transcription and function of Frankia EuIK1 nifV were verified by a RT-PCR analysis and complementation test with the K. pneumoniae mutant, respectively. These results suggested that Frankia EuIK1 nifV is a functional gene.
Asunto(s)
Proteínas Bacterianas/genética , Frankia/genética , Fijación del Nitrógeno/genética , Oxo-Ácido-Liasas/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/fisiología , Composición de Base , Clonación Molecular , Codón/genética , Genes Bacterianos , Prueba de Complementación Genética , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/metabolismo , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Oxo-Ácido-Liasas/fisiología , Filogenia , Proteínas Recombinantes de Fusión/fisiología , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Transcripción GenéticaRESUMEN
For high throughput screening of root nodule-enhanced genes, cDNA libraries specific for three different developmental stages of soybean root nodules were constructed after inoculation with Bradyrhizobium japonicum USDA110. 5,469 cDNA clones were sequenced and grouped into 2,511 non-redundant (nr) ESTs consisting of 769 contigs and 1,742 singletons. Using similarity searches against several public databases we constructed a functional classification of the ESTs into root nodule-specific nodulin genes, stress-responsive genes and genes related to carbon and nitrogen metabolism. We also constructed a cDNA microarray with 382 selected clones that appeared to be up-regulated in the root nodule. Using the microarray we compared the transcript levels of uninfected roots and root nodules from four developmental stages. We identified 81 genes that were differentially expressed, and grouped them into seven clusters according to the similarity of their expression profiles, using a hierarchical clustering algorithm. Clusters 1, 2, 3, and 6, comprised of 58 genes, showed root nodule-enhanced expression. The information from this study will be used to analyze the roles of root nodule-specific genes and signaling pathways during root nodule development.
Asunto(s)
Regulación de la Expresión Génica de las Plantas , Glycine max/anatomía & histología , Glycine max/genética , Proteínas de Plantas/metabolismo , Raíces de Plantas/crecimiento & desarrollo , Transcripción Genética , Bases de Datos de Ácidos Nucleicos , Etiquetas de Secuencia Expresada , Perfilación de la Expresión Génica , Datos de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas de Plantas/genética , Raíces de Plantas/metabolismo , Raíces de Plantas/microbiología , Glycine max/fisiologíaRESUMEN
Remorins, a family of plant-specific proteins containing a variable N-terminal region and conserved C-terminal domain, play a role in various biotic and abiotic stresses, including host-microbe interactions. However, their functions remain to be completely elucidated, especially for the Arabidopsis thaliana remorin group 4 (AtREM4). To elucidate the role of remorins in Arabidopsis, we first showed that AtREM4s have typical molecular characteristics of the remorins, such as induction by various types of biotic and abiotic stresses, localization in plasma membrane and homo- and hetero-oligomeric interaction. Next, we showed that their loss-of-function mutants displayed reduced susceptibility to geminiviruses, Beet Curly Top Virus and Beet Severe Curly Top Virus, while overexpressors enhanced susceptibility. Moreover, we found that they interacted with SnRK1, which phosphorylated AtREM4.1, and were degraded by the 26S proteasome pathway. These results suggest that AtREM4s may be involved in the SnRK1-mediated signaling pathway and play a role as positive regulators of the cell cycle during geminivirus infection.
RESUMEN
The AT-hook motif is a small DNA-binding protein motif that has been found in the high mobility group of non-histone chromosomal proteins. The Arabidopsis genome contains 29 genes encoding the AT-hook motif DNA-binding protein (AHL). Recent studies of Arabidopsis genes (AtAHLs) have revealed that they might play diverse functional roles during plant growth and development. In this report, we mined 20 AHL genes (OsAHLs) from the rice genome database using AtAHL genes as queries and characterized their molecular features. A phylogenetic tree revealed that OsAHL proteins can be classified into 2 evolutionary clades. Tissue expression pattern analysis revealed that all of the OsAHL genes might be functionally expressed genes with 3 distinct expression patterns. Nuclear localization analysis using transgenic Arabidopsis showed that several OsAHL proteins are exclusively localized in the nucleus, indicating that they may act as architectural transcription factors to regulate expression of their target genes during plant growth and development.
Asunto(s)
Secuencias AT-Hook/genética , Genes de Plantas , Oryza/genética , Proteínas de Plantas/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Bases de Datos Genéticas , Regulación de la Expresión Génica de las Plantas , Genoma de Planta , Oryza/metabolismo , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/clasificación , Proteínas de Plantas/metabolismo , Plantas Modificadas GenéticamenteRESUMEN
Alder (Alnus glutinosa) and more than 200 angiosperms that encompass 24 genera are collectively called actinorhizal plants. These plants form a symbiotic relationship with the nitrogen-fixing actinomycete Frankia strain HFPArI3. The plants provide the bacteria with carbon sources in exchange for fixed nitrogen, but this metabolite exchange in actinorhizal nodules has not been well defined. We isolated an alder cDNA from a nodule cDNA library by differential screening with nodule versus root cDNA and found that it encoded a transporter of the PTR (peptide transporter) family, AgDCAT1. AgDCAT1 mRNA was detected only in the nodules and not in other plant organs. Immunolocalization analysis showed that AgDCAT1 protein is localized at the symbiotic interface. The AgDCAT1 substrate was determined by its heterologous expression in two systems. Xenopus laevis oocytes injected with AgDCAT1 cRNA showed an outward current when perfused with malate or succinate, and AgDCAT1 was able to complement a dicarboxylate uptake-deficient Escherichia coli mutant. Using the E. coli system, AgDCAT1 was shown to be a dicarboxylate transporter with a K(m) of 70 microm for malate. It also transported succinate, fumarate, and oxaloacetate. To our knowledge, AgDCAT1 is the first dicarboxylate transporter to be isolated from the nodules of symbiotic plants, and we suggest that it may supply the intracellular bacteria with dicarboxylates as carbon sources.