RESUMEN
Developing new strategies for crop plants to respond to drought is crucial for their innovative breeding. The down-regulation of nuclear cap-binding proteins in Arabidopsis renders plants drought tolerant. The CBP80 gene in the potato cultivar Desiree was silenced using artificial microRNAs. Transgenic plants displayed a higher tolerance to drought, ABA-hypersensitive stomatal closing, an increase in leaf stomata and trichome density, and compact cuticle structures with a lower number of microchannels. These findings were correlated with a higher tolerance to water stress. The level of miR159 was decreased, and the levels of its target mRNAs MYB33 and MYB101 increased in the transgenic plants subjected to drought. Similar trends were observed in an Arabidopsis cbp80 mutant. The evolutionary conservation of CBP80, a gene that plays a role in the response to drought, suggests that it is a candidate for genetic manipulations that aim to obtain improved water-deficit tolerance of crop plants.
Asunto(s)
Sequías , Proteínas de Plantas/metabolismo , Solanum tuberosum/metabolismo , Regulación de la Expresión Génica de las Plantas/genética , Regulación de la Expresión Génica de las Plantas/fisiología , Proteínas de Plantas/genética , Solanum tuberosum/genéticaRESUMEN
Virus-coded VPg protein of Potato virus Y (PVY) does not have homologs apart from other VPgs. Since VPg is indispensable for the potyvirus life cycle, it appeared a good candidate for eliciting pathogen-derived resistance to PVY. Following agroinfection used to obtain PVY VPg-transgenic Arabidopsis thaliana plants, only few transgenic seeds were recovered giving rise to six transgenic plants that contained the VPg gene with the correct sequence. They generated VPg mRNA, but VPg protein was not detected. Some plants were immune to PVY infection suggesting post-transcriptional gene silencing. However, the likely PVY VPg toxicity exerted at an early stage of transformed seeds development precludes its use for engineering pathogen-derived resistance.
Asunto(s)
Arabidopsis/genética , Ribonucleoproteínas/genética , Proteínas no Estructurales Virales/genética , Arabidopsis/crecimiento & desarrollo , Arabidopsis/inmunología , Arabidopsis/virología , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/virología , Plantas Modificadas Genéticamente/genética , Potyvirus/genética , Potyvirus/patogenicidad , ARN Mensajero/análisis , Ribonucleoproteínas/metabolismo , Proteínas no Estructurales Virales/metabolismoRESUMEN
The cap-binding protein complex (CBC) binds to the caps of all RNA polymerase II transcripts, and plays an important role in RNA metabolism. We characterized interactions, localization and nuclear-cytoplasmic transport of two subunits of the Arabidopsis thaliana cap-binding protein complex (AtCBC): AtCBP20 and AtCBP80. Using CFP/YFP-tagged proteins, we show that transport of AtCBC from the cytoplasm to the nucleus in the plant cell is different from that described in other eukaryotic cells. We show that the smaller subunit of the complex, AtCBP20, plays a crucial role in the nuclear import of AtCBC. The C-terminal part of AtCBP20 contains two functionally independent nuclear localization signals (NLSs). At least one of these two NLSs is required for the import of CBC into the plant nucleus. The interaction between the A. thaliana CBP20 and CBP80 was also analyzed in detail, using the yeast two-hybrid system and fluorescence resonance energy transfer (FRET) assays. The N-terminal part of AtCBP20 is essential for interaction with AtCBP80. Furthermore, AtCBP80 is important for the protein stability of the smaller subunit of CBC. Based on these data, we propose a model for the nuclear-cytoplasmic trafficking of the CBC complex in plants.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas de Unión a Caperuzas de ARN/metabolismo , Proteínas de Unión al ARN/metabolismo , Transporte Activo de Núcleo Celular , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Núcleo Celular/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Señales de Localización Nuclear/genética , Señales de Localización Nuclear/metabolismo , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Transporte de Proteínas , Proteínas de Unión a Caperuzas de ARN/genética , ARN de Planta/metabolismo , Proteínas de Unión al ARN/genética , Técnicas del Sistema de Dos HíbridosRESUMEN
In this study we investigated whether in plants, like in mammals, components of the nuclear cap-binding protein complex (CBC) are involved in nonsense-mediated mRNA decay (NMD). We selected several genes producing at least two alternatively spliced mRNA variants: one with a premature termination codon (PTC+) and another without it (PTC-). For each gene the PTC+/PTC- ratio was calculated using RT-PCR and direct sequencing in four Arabidopsis thaliana lines: wild type, the NMD mutant atupf3-1 and two CBC mutants: cbp20 and abh1. Whereas in the NMD mutant the ratios of PTC+/PTC- splice variants were higher than in wild-type plants, the two CBC mutants investigated showed no change in the PTC+/PTC- ratios. Our results suggest that neither CBP20 nor CBP80 is involved in NMD in A. thaliana.