RESUMEN
We describe the isolation of two Catharanthus roseus cDNAs encoding proteins putatively involved in the final steps of a 'histidine-to-aspartate' phosphorelay in cytokinin (CK) signaling. The expression of one of these genes, CrRR1, was specifically up-regulated by CKs in C. roseus cell suspensions. We used this system as a biological model to test the activity of bacterial histidine kinase inhibitors. Our data demonstrate that these inhibitors are active on the CK transduction pathway and represent powerful chemical tools to study hormone signal transduction in plants. Moreover, these data suggest a strong conservation of functional features between prokaryotic and plant signaling pathways utilizing histidine kinases.
Asunto(s)
Bacterias/enzimología , Catharanthus/fisiología , Citocininas/genética , Inhibidores Enzimáticos/farmacología , Inhibidores de Proteínas Quinasas , Proteínas Quinasas , Transducción de Señal/fisiología , Secuencia de Aminoácidos , Arabidopsis/genética , Catharanthus/genética , Clonación Molecular , Citocininas/antagonistas & inhibidores , Citocininas/química , ADN Complementario , Histidina Quinasa , Cinética , Modelos Biológicos , Datos de Secuencia Molecular , Proteínas de Plantas/antagonistas & inhibidores , Proteínas de Plantas/química , Proteínas de Plantas/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transducción de Señal/efectos de los fármacosRESUMEN
We previously reported that cytokinins (CK) induce the fast and specific transcription of CrRR1, a gene encoding a type A response regulator in Catharanthus roseus cell cultures. Here, we characterized the CrHPt1 gene that encodes a histidine-containing phosphotransfer domain. CrHPt1 was silenced through RNA interference (RNAi) to test its possible implication in the CK signalling pathway. In transgenic lines stably transformed with an intron-spliced construct, the degradation of CrHPt1 transcripts abolishes the CK inductive effect on CrRR1 transcription. These result give a new in vivo functional argument for the crucial role of HPt proteins in the CK signalling pathway leading to the expression of the genes encoding type A response regulators. They also show that RNAi is a powerful strategy to turn off the CK signalling circuitry.
Asunto(s)
Catharanthus/metabolismo , Citocininas/metabolismo , Histidina/metabolismo , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Catharanthus/citología , Catharanthus/genética , Células Cultivadas , Citocininas/genética , Flores/citología , Silenciador del Gen , Genes de Plantas , Hojas de la Planta/citología , Tallos de la Planta/citología , Plantas Modificadas Genéticamente , Estructura Terciaria de Proteína , ARN Interferente Pequeño/químicaRESUMEN
Two full-length cDNAs named CrRR2 and CrRR3 have been isolated by PCR from a C. roseus cDNA library. The first one encodes a 154 amino acid putative protein with a high percentage of identity with the Arabidopsis thaliana response regulators ARR16 and ARR17, and is not expressed in C. roseus organs and cell cultures. The second one encodes a 188 amino acid ORF sharing the highest homologies with the A. thaliana ARR8 and ARR9 response regulators. Its expression is root-specific and the transcripts are transiently up-regulated after trans-zeatin treatment in C. roseus suspension cells. CrRR3 protein might be involved in the cytokinin-enhanced alkaloid production in C. roseus cell cultures.