RESUMEN
Plant plasma membrane H+-ATPases (PM H+-ATPase) are essential for establishing a proton electrochemical gradient across the cell plasma membrane. Their regulation is poorly understood, except for the role of 14-3-3 proteins, which relieve autoinhibition from the C-terminal domain. A novel protein interacting with this domain was recently identified in Arabidopsis and named PPI1 (Proton Pump Interactor 1). PPI1 stimulates PM H+-ATPase activity in vitro. Here, we analyse the expression pattern of Ppi1 using beta-glucuronidase as a reporter. Expression is strong in root and shoot vascular systems, particularly in meristematic and sink tissues, as well as in pollen, stigmas and siliques, but not in developing embryos. Removal of the first intron decreased GUS expression 45-fold. We also analysed the transcription of Ppi2, another gene in the family, and demonstrated that Ppi2 is expressed in seedlings, cultured cells and flowers. We reassessed Ppi2 gene structure based on RT-PCR amplifications, cDNA data and similarity to other Ppi genes. Insertional mutants for both Ppi1 and Ppi2 were isolated. Two different mutants of Ppi1 showed aberrant mRNAs and lacked any detectable protein and are therefore true knockouts. Interestingly, one mutation inhibited the splicing of one intron at a considerable distance (>700 bp) from the T-DNA insertion site, but not the splicing of a proximal intron (29 bp) or of any other intron. At the plant level, neither of the single mutants nor the double ppi1ppi2 mutant showed an altered phenotype in standard growth conditions under acid load or salt stress.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Proteínas Portadoras/genética , Mutación , Arabidopsis/citología , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Secuencia de Bases , Northern Blotting , Western Blotting , Proteínas Portadoras/metabolismo , Células Cultivadas , Flores/genética , Flores/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Intrones/genética , Modelos Genéticos , Datos de Secuencia Molecular , Mutagénesis Insercional , Plantas Modificadas Genéticamente , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Plantones/genética , Plantones/metabolismoRESUMEN
A mutagenised population of seeds of Arabidopsis thaliana was allowed to germinate in the presence of the positively charged aminoglycoside hygromycin (4 µg/ml) and the fungal toxin fusicoccin (5×10-6 M). This hygromycin concentration, which is non-toxic by itself, becomes toxic when used together with fusicoccin, which stimulates cation uptake. Seeds that had germinated after 3-5 days and produced seedlings with green cotyledons were potentially resistant to fusicoccin and were therefore transferred into sterile Magenta vessels containing 1/2-strength Murashige and Skoog medium. This selection procedure is non-destructive, i.e. it allows the recovery of viable seedlings and their growth into adult plants thus permitting direct physiological characterisation.
RESUMEN
An actin-related protein (ACLA) has been identified in the cellular slime mould Dictyostelium discoideum. The complete cDNA sequence indicates that within the actin superfamily it belongs to the ARP3 family of actin-related proteins together with Arp66B from Drosophila melanogaster, Actin2 from Bos taurus, act2 from Schizosaccharomyces pombe and possibly act2 from Caenorhabditis elegans. The ACLA mRNA is regulated during development, showing a maximum between 2 and 4 h after starvation. The protein has been expressed in E. coli and antibodies raised against it. Immunofluorescence microscopy shows that ACLA protein co-localises with mitochondria; the protein copurifies with Dictyostelium mitochondria.