RESUMEN
Recently we described the novel nodulin gene VfENOD18, whose corresponding transcripts were restricted to the nitrogen-fixing zone III of broad bean root nodules. To characterize VfENOD18 on the protein level, polyclonal antibodies were generated using the purified recombinant VfENOD18 protein produced in Escherichia coli by employing the pMAL-c expression system. These antibodies recognized immunoreactive proteins isolated from indeterminate nodules of different leguminous plants, but also from non-symbiotic tissues of Glycine max and from tissues of Arabidopsis thaliana and Zea mays. Using immunogold labelling the nodulin VfENOD18 was localized to the cytoplasm of infected cells in the nitrogen-fixing zone of broad bean nodules. Due to the homology of the VfENOD18 sequence to that of the ATP-binding protein MJ0577 from the hyperthermophile Methanococcus jannaschii the recombinant VfENOD18 protein was tested for ATP-binding. Using the biotin photoaffinity ATP analogue 8N3ATP[gamma]biotin it could be demonstrated that VfENOD18 is an ATP-binding protein. PCR experiments revealed that the amino acid sequences of the putative C-terminal ATP-binding sites of the VfENOD 18 homologues from Lens culinaris, Vicia hirsuta, Vicia sativa and Vicia villosa were conserved. We propose that VfENOD18 is a member of a novel family of ATP-binding proteins in plants.
Asunto(s)
Adenosina Trifosfato/metabolismo , Fabaceae/genética , Proteínas de la Membrana , Proteínas de Plantas/genética , Raíces de Plantas/genética , Secuencia de Aminoácidos , Anticuerpos/inmunología , Arabidopsis/inmunología , Arabidopsis/metabolismo , Western Blotting , Fabaceae/citología , Fabaceae/microbiología , Methanococcus/genética , Microscopía Inmunoelectrónica , Datos de Secuencia Molecular , Proteínas de Plantas/inmunología , Proteínas de Plantas/metabolismo , Raíces de Plantas/microbiología , Raíces de Plantas/ultraestructura , Unión Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Rhizobium leguminosarum/crecimiento & desarrollo , Homología de Secuencia de Aminoácido , Glycine max/inmunología , Glycine max/metabolismo , Simbiosis , Zea mays/inmunología , Zea mays/metabolismoRESUMEN
Full-length transcript sequences were isolated from broad bean root nodules, which encode a novel nodulin designated VfENOD18. The corresponding transcripts were detected in early and in late stages of nodule development and were localized exclusively in the nitrogen-fixing zone III. The VfENOD18 sequence is not only homologous to a number of ESTs from various mono- and dicotyledonous plants, but also to the ATP-binding protein MJ0577 from Methanococcus jannaschii and to a range of bacterial proteins that belong to the MJ0577 superfamily. Hence, VfENOD18 is a member of a ubiquitous family of plant proteins that might function as ATP-binding proteins or ATPases. On the genomic level, VfENOD18 genes can be divided into two groups on the basis of differences in their 5' UTRs. One group lacks the 5' UTR region including the ATG initiation codon, whereas the second group contained the complete 5' UTR region. Further upstream of this VfENOD18 gene, a retrotransposon sequence was identified. The -14/-964 VfENOD18 promoter fragment was devoid of complete organ-specific elements known from other nodulin gene promoters. Nevertheless, this region was able to mediate full promoter activity in the central region of transgenic Vicia hirsuta root nodules.
Asunto(s)
Genes de Plantas , Proteínas de la Membrana , Familia de Multigenes , Proteínas de Plantas/genética , Raíces de Plantas/genética , Regiones no Traducidas 5' , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Secuencia de Bases , Northern Blotting , Codón Iniciador , ADN Complementario/genética , ADN Complementario/metabolismo , Etiquetas de Secuencia Expresada , Fabaceae/genética , Biblioteca de Genes , Methanococcus/genética , Modelos Genéticos , Datos de Secuencia Molecular , Fijación del Nitrógeno , Hibridación de Ácido Nucleico , Plantas Modificadas Genéticamente/genética , Plantas Medicinales , Regiones Promotoras Genéticas , ARN Mensajero/genética , Proteínas Recombinantes/genética , Retroelementos , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Factores de Tiempo , Distribución TisularRESUMEN
Nodulin gene transcripts isolated from a broad bean (Vicia faba L.) root nodule cDNA library and designated VfNOD32 are detectable in the nitrogen-fixing zone III of nodules and in much smaller amounts in flowers. In nodules, these transcripts are detectable for the first time 7 d after inoculation, at least 1 d before leghemoglobin gene transcription starts. Two putative full-length cDNAs representing different transcript sequences of 92.5% identity were sequenced. The corresponding broad bean genes were termed VfNOD32-A1 and VfNOD32-A2, and the encoded proteins were termed Nvf32-A1 and Nvf32-A2. The derived amino acid sequences of the Nvf32 proteins are highly homologous to the Vicia narbonensis (alpha/beta)8-barrel seed protein narbonin. Considering this homology, Nvf32 is assumed to have a similar structure consisting of beta-sheets forming a central barrel surrounded by alpha-helices. The two Nvf32 sequences also contain two conserved amino acid motifs that are characteristic of class-III chitinases. Several amino acids demonstrated to be essential for chitinase activity are conserved in both regions, whereas one essential glutamic acid was changed to glycine in the Nvf32-A1 isoform but not in the Nvf32-A2 isoform.
