RESUMEN
Members of the peptide transporter/nitrate transporter 1 (PTR/NRT1) family in plants transport a variety of substrates like nitrate, di- and tripepetides, auxin and carboxylates. We isolated two members of this family from Arabidopsis, AtPTR4 and AtPTR6, which are highly homologous to the characterized di- and tripeptide transporters AtPTR1, AtPTR2 and AtPTR5. All known substrates of members of the PTR/NRT1 family were tested using heterologous expression in Saccharomyces cerevisiae mutants and oocytes of Xenopus laevis, but none could be identified as substrate of AtPTR4 or AtPTR6. AtPTR4 and AtPTR6 show distinct expression patterns, while AtPTR4 is expressed in the vasculature of the plants, AtPTR6 is highly expressed in pollen and during senescence. Phylogenetic analyses revealed that AtPTR2, 4 and 6 belong to one clade of subgoup II, whereas AtPTR1 and 5 are found in a second clade. Like AtPTR2, AtPTR4-GFP and AtPTR6-GFP fusion proteins are localized at the tonoplast. Vacuolar localization was corroborated by co-localization of AtPTR2-YFP with the tonoplast marker protein GFP-AtTIP2;1 and AtTIP1;1-GFP. This indicates that the two clades reflect different intracellular localization at the tonoplast (AtPTR2, 4, 6) and plasma membrane (AtPTR1, 5), respectively.
Asunto(s)
Proteínas de Transporte de Anión/metabolismo , Arabidopsis/metabolismo , Regulación de la Expresión Génica de las Plantas , Membranas Intracelulares/metabolismo , Proteínas de Plantas/metabolismo , Animales , Proteínas de Transporte de Anión/clasificación , Proteínas de Transporte de Anión/genética , Arabidopsis/clasificación , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cultivo Axénico , Membrana Celular/genética , Membrana Celular/metabolismo , Clonación Molecular , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Microscopía Confocal , Oocitos/citología , Oocitos/metabolismo , Sistemas de Lectura Abierta , Filogenia , Proteínas de Plantas/clasificación , Proteínas de Plantas/genética , Polen/genética , Polen/metabolismo , Protoplastos/citología , Protoplastos/metabolismo , ARN de Planta/genética , ARN de Planta/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Vacuolas/metabolismo , Xenopus laevis/genética , Xenopus laevis/metabolismoRESUMEN
Transporters for di- and tripeptides belong to the large and poorly characterized PTR/NRT1 (peptide transporter/nitrate transporter 1) family. A new member of this gene family, AtPTR5, was isolated from Arabidopsis (Arabidopsis thaliana). Expression of AtPTR5 was analyzed and compared with tissue specificity of the closely related AtPTR1 to discern their roles in planta. Both transporters facilitate transport of dipeptides with high affinity and are localized at the plasma membrane. Mutants, double mutants, and overexpressing lines were exposed to several dipeptides, including toxic peptides, to analyze how the modified transporter expression affects pollen germination, growth of pollen tubes, root, and shoot. Analysis of atptr5 mutants and AtPTR5-overexpressing lines showed that AtPTR5 facilitates peptide transport into germinating pollen and possibly into maturating pollen, ovules, and seeds. In contrast, AtPTR1 plays a role in uptake of peptides by roots indicated by reduced nitrogen (N) levels and reduced growth of atptr1 mutants on medium with dipeptides as the sole N source. Furthermore, overexpression of AtPTR5 resulted in enhanced shoot growth and increased N content. The function in peptide uptake was further confirmed with toxic peptides, which inhibited growth. The results show that closely related members of the PTR/NRT1 family have different functions in planta. This study also provides evidence that the use of organic N is not restricted to amino acids, but that dipeptides should be considered as a N source and transport form in plants.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Dipéptidos/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Animales , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Transporte Biológico/genética , ADN Bacteriano/genética , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Prueba de Complementación Genética , Germinación , Proteínas de Transporte de Membrana/genética , Mutagénesis Insercional , Nitrógeno/metabolismo , Oocitos/metabolismo , Raíces de Plantas/genética , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/metabolismo , Polen/genética , Polen/crecimiento & desarrollo , Polen/metabolismo , ARN de Planta/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Semillas/genética , Semillas/crecimiento & desarrollo , Semillas/metabolismo , Xenopus/genética , Xenopus/metabolismoRESUMEN
Uniparental activity of ribosomal RNA genes (rDNA) in interspecific hybrids is known as nucleolar dominance (ND). To see if difference in rDNA intergenic spacers (IGS) might be correlated with ND, we have used artificial Solanum allopolyploids and back-crossed lines. Combining fluorescence in situ hybridization and quantification of the level of the rRNA precursor by real-time PCR, we demonstrated that an expression hierarchy exists: In leaves, roots, and petals of the respective allopolyploids, rDNA of S lycopersicum (tomato) dominates over rDNA of S. tuberosum (potato), whereas rDNA of S. tuberosum dominates over that of the wild species S. bulbocastanum . Also in a monosomic addition line carrying only one NOR-bearing chromosome of tomato in a potato background the dominance effect was maintained. These results demonstrate that there is possible correlation between transcriptional dominance and number of conservative elements downstream of the transcription start in the Solanum rDNA. In anthers and callus tissues under-dominant rDNA was slightly (S. lycopersicum/S. tuberosum) or strongly (S. tuberosum/S. bulbocastanum) expressed indicating developmental modulation of ND. In leaves and petals, repression of the respective parental rDNA correlated with cytosine methylation at certain sites conserved in the IGS, whereas activation of under-dominant rDNA in anthers and callus tissues was not accompanied by considerable changes of the methylation pattern.
Asunto(s)
Metilación de ADN , ADN Ribosómico/genética , Perfilación de la Expresión Génica , Poliploidía , Solanum/genética , Secuencia de Bases , Cruzamientos Genéticos , ADN de Plantas/química , ADN de Plantas/genética , ADN de Plantas/aislamiento & purificación , ADN Ribosómico/metabolismo , ADN Espaciador Ribosómico/genética , Regulación de la Expresión Génica de las Plantas , Hibridación Fluorescente in Situ , Datos de Secuencia Molecular , ARN Ribosómico/genética , ARN Ribosómico/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , Homología de Secuencia de Ácido NucleicoRESUMEN
The 5(') external transcribed spacer (ETS) region of ribosomal DNA of 30 species of Solanum sect. Petota and the European Solanum dulcamara were compared. Two structural elements can be distinguished in the ETS: (i). a variable region (VR), demonstrating significant structural rearrangements and (ii). a conservative region (CR), evolving mainly by base substitutions. In VR, a conservative element (CE) with similarity to the ETS of distantly related Nicotiana is present. The ancestral organization of ETS (variant A) was found for non-tuber-bearing species of ser. Etuberosa, tuber-bearing wild potatoes of Central American ser. Bulbocastana, Pinnatisecta, and Polyadenia and S. dulcamara. Duplication of CE took place in the ETS of species from ser. Commersoniana and Circaeifolia (variant B). South American diploids and Mexican polyploids from superser. Rotata also possess two CE, and additionally two duplications around CE1 are present in VR (variant C). Three major lineages could be distinguished: non-tuber-bearing species of ser. Etuberosa, tuber-bearing Central American diploids and all South American species radiated from a common ancestor at early stages of evolution, indicating a South American origin of the tuber-bearing species. Later, Central and South American diploids evolved further as independent lineages. South American species form a monophyletic group composed of series with both stellata and rotata flower morphology. Solanum commersonii represents a sister taxon for all rotata species, whereas ser. Circaeifolia diverged earlier. Two main groups, C1 and C2, may be distinguished for species possessing ETS variant C. C1 contains ser. Megistacroloba, Conicibaccata, Maglia, and Acaulia, whereas all diploids of ser. Tuberosa are combined into C2. A closer relationship of Solanum chacoense (ser. Yungasensa) to the C2 group was found. The origin of polyploid species Solanum maglia, Solanum acaule, Solanum tuberosum, Solanum iopetalum, and Solanum demissum is discussed.