Papers by Remedios Crespillo
Interaction of carbon an nitrogen in a C4 plant (Sorghum bicolor L)
Physiologia Plantarum, Aug 1, 2004
Glutamine synthetase (GS) genes, GS1a and GS1b, in pine (Pinus sylvestris L.) are differentially ... more Glutamine synthetase (GS) genes, GS1a and GS1b, in pine (Pinus sylvestris L.) are differentially regulated in tissue specificity and during seedling development. To gain insight into the regulatory mechanisms controlling their expression, we have analysed the 5 0 -flanking sequences of the gene GS1a using a transient expression system in pine protoplasts. Structural analysis of this region revealed the presence of putative regulatory elements including two AT-rich elements and a poly CT consensus sequence. A series of 5 0 -and 3 0 -deletions of the untranslated region covering the three putative elements, À800 to À626, À626 to À427 and 1118 to 1177 were analysed to demonstrate the functional implications of these elements in gene regulation. An electrophoretic mobilityshift assay showed that nuclear proteins prepared from pine cotyledons interact with both AT-rich regions (À800 to À427). Interestingly, no protein binding was detected when the untranslated region (1118 to 1177) was included, even if deletion of that region suppressed promoter activity in the transient expression experiments conducted. However, simultaneous deletion of both types of cis elements, A/T and CT, resulted in a recovery of promoter activity of 50%. These results suggest a key regulatory role of the CT box by the interaction with A/T stretches in the distal part of the promoter and possibly with the proximal region (À427 to À1).
Regulación diferencial de la expresión génica de glutamina sintetasa de pino por Dof5
Functional expression in E coli and kinetic characterization of glutamine synthetase isoenzymes from Pinus sylvestris
Plant Biology (Rockville), Oct 12, 2001
Minireview: Molecular physiology of glutamine and glutamate biosynthesis in developing seedlings of conifers [review]
Physiologia Plantarum (Denmark), 1998
Plant Molecular Biology Reporter, Dec 1, 2001
Establishment of protoplast techniques for conifers is of crucial importance to forest biotechnol... more Establishment of protoplast techniques for conifers is of crucial importance to forest biotechnology. Protoplasts can be used to regenerate whole trees and to study transcriptional regulation in woody plants by promoter analysis. Here we describe a technique for isolating protoplasts from Pinus pinaster shoots. Protoplast viability was determined by fluorescein diacetate staining. Transient gene expression was studied after electroporation of protoplasts with plasmid DNA containing a b-glucuronidase (GUS) reporter gene. The 35S promoter and a pine (cytosolic glutamine synthetase) promoter both were able to drive GUS expression, indicating applicability of conifer protoplasts as a model to study gene promoters using transient expression. Isolation of gymnosperm shoot protoplasts Gómez-Maldonado et al.
Plant Journal, Apr 4, 2006
In this paper, we report the identification of genes from pine (PpAAT), Arabidopsis (AtAAT) and r... more In this paper, we report the identification of genes from pine (PpAAT), Arabidopsis (AtAAT) and rice (OsAAT) encoding a novel class of aspartate aminotransferase (AAT, EC 2.6.1.1) in plants. The enzyme is unrelated to other eukaryotic AATs from plants and animals but similar to bacterial enzymes. Phylogenetic analysis indicates that this prokaryotic-type AAT is closely related to cyanobacterial enzymes, suggesting it might have an endosymbiotic origin. Interestingly, most of the essential residues involved in the interaction with the substrate and the attachment of pyridoxal phosphate cofactor in the active site of the enzyme were conserved in the deduced polypeptide. The polypeptide is processed in planta to a mature subunit of 45 kDa that is immunologically distinct from the cytosolic, mitochondrial and chloroplastic isoforms of AAT previously characterized in plants. Functional expression of PpAAT sequences in Escherichia coli showed that the processed precursor is assembled into a catalytically active homodimeric holoenzyme that is strictly specific for aspartate. These atypical genes are predominantly expressed in green tissues of pine, Arabidopsis and rice, suggesting a key role of this AAT in nitrogen metabolism associated with photosynthetic activity. Moreover, immunological analyses revealed that the plant prokaryotic-type AAT is a nuclear-encoded chloroplast protein. This implies that two plastidic AAT co-exist in plants: a eukaryotic type previously characterized and the prokaryotic type described here. The respective roles of these two enzymes in plant amino acid metabolism are discussed.
Sobreproducción de la glutamina sintetasa de pino: Análisis funcional de las enzimas salaje y mutante C249A
Identificación de una nueva forma de aspartato aminotransferasa en plantas
Plant and Cell Physiology, Jul 15, 2002
Two glutamine synthetase isogenes, GS1a and GS1b, isolated from pine have been functionally expre... more Two glutamine synthetase isogenes, GS1a and GS1b, isolated from pine have been functionally expressed in E. coli and the characteristics of individual gene products compared. When bacteria were grown at 37°°°°C most pine GS1 protein was found in the insoluble fraction but lowering of the expression temperature increased yield of both GS1 polypeptide and activity in the soluble fraction. High levels of functionally active GS1a (309± ± ± ±35 nkat mg -1 ) and GS1b (1,166± ± ± ±65 nkat mg -1 ) enzymes were obtained by decreasing the expression temperature to 10°°°°C. Purification and characterization of recombinant products showed that pine GS1 polypeptides are assembled in octameric GS holoenzymes showing structural and kinetic differences. The results are discussed with regard to the specific localization of GS1a and GS1b in different cell types of pine seedlings. The isoform GS1a may control the assimilation of the high levels of ammonium released in photosynthetic tissues, whereas GS1b enzyme could mitigate oscillations in glutamate availability providing a constant flux of glutamine for nitrogen transport in vascular cells.
Abstract. Establishment of protoplast techniques for conifers is of crucial importance to forest ... more Abstract. Establishment of protoplast techniques for conifers is of crucial importance to forest biotechnology. Protoplasts can be used to regenerate whole trees and to study transcriptional regulation in woody plants by promoter analysis. Here we describe a tech-nique for isolating protoplasts from Pinus pinaster shoots. Protoplast viability was deter-mined by fluorescein diacetate staining. Transient gene expression was studied after electroporation of protoplasts with plasmid DNA containing a b-glucuronidase (GUS) re-porter gene. The 35S promoter and a pine (cytosolic glutamine synthetase) promoter both were able to drive GUS expression, indicating applicability of conifer protoplasts as a model to study gene promoters using transient expression. Isolation of gymnosperm shoot protoplasts Gómez-Maldonado et al.
Regulación diferencial de la expresión génica de glutamina sintetasa de pino por Dof5
Interaction of carbon an nitrogen in a C4 plant (Sorghum bicolor L)
Functional expression in E coli and kinetic characterization of glutamine synthetase isoenzymes from Pinus sylvestris
Identificación de una nueva forma de aspartato aminotransferasa en plantas
La asimilación de amonio en plántulas de pino utiliza dos glutamina sintetasas citosólicas con distribución espacial y temporal diferentes
Análisis molecular de la región promotora del gen GS1b de "P. sylvestris
Minireview: Molecular physiology of glutamine and glutamate biosynthesis in developing seedlings of conifers [review]
La asimilación de amonio en plántulas de pino utiliza dos glutamina sintetasas citosólicas con distribución espacial y temporal diferentes
Avances En El Metabolismo Del Nitrogeno Bioquimica Fisiologia Y Biologia Molecular 2000 Isbn 84 7496 842 9 Pags 225 232, 2000
Sobreproducción de la glutamina sintetasa de pino: Análisis funcional de las enzimas salaje y mutante C249A
Avances En El Metabolismo Del Nitrogeno De La Fisiologia a La Biologia Molecular Iv Reunion Nacional Marbella 1997 1998 Isbn 84 472 0402 2 Pags 535 542, 1998
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Papers by Remedios Crespillo