RESUMEN
The molecular mechanism underlying the regulation of the expression of Apxl, the gene encoding cytosolic ascorbate peroxidase, as well as other antioxidant enzymes, was studied during the progression of drought stress and following recovery from drought. Increase in steady-state transcript levels of the cytosolic isozymes of ascorbate peroxidase and Cu/Zn-superoxide dismutase paralleled the increase in stomatal resistance during drought stress, but was even more dramatically enhanced following recovery from drought. Cytosolic Cu/Zn-superoxide dismutase and ascorbate peroxidase and chloroplastic Cu/Zn-superoxide dismutase protein and activity increased during drought stress and following recovery. In contrast, catalase activity increased during drought stress but returned to normal levels following recovery. During recovery from drought stress, cytosolic ascorbate peroxidase expression was regulated post-transcriptionally at the level of protein synthesis. The transcription rate of the Apxl gene, as determined by nuclear run-on assay, increased during drought stress and at 10 h following rewatering.
Asunto(s)
Citosol/enzimología , Fabaceae/enzimología , Regulación Enzimológica de la Expresión Génica , Peroxidasas/genética , Plantas Medicinales , Ascorbato Peroxidasas , Secuencia de Bases , Catalasa/metabolismo , Desastres , Datos de Secuencia Molecular , Peroxidasas/metabolismo , Polirribosomas , Biosíntesis de Proteínas , ARN Mensajero/análisis , Ribulosa-Bifosfato Carboxilasa/genética , Ribulosa-Bifosfato Carboxilasa/metabolismo , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Transcripción Genética , Agua/fisiologíaRESUMEN
A gene encoding cytosolic ascorbate peroxidase (ApxI) from pea (Pisum sativum L.) was isolated and its nucleotide sequence determined. By homologous alignment between the ApxI cDNA (Mittler, R., and Zilinskas, B. (1991) FEBS Lett. 289, 257-259) and the genomic clone, positions of introns and exons were determined. The isolated ApxI gene was found to contain 9 introns, the first of which was located within the 5'-untranslated region of the mRNA. Southern blot analysis of pea genomic DNA suggests that in pea cytosolic ascorbate peroxidase is encoded by a single copy gene. Steady state ApxI transcript levels were found to increase in response to several stresses imposed by drought, heat, and application of ethephon, abscisic acid, and the superoxide-generating agent paraquat. Increases in ascorbate peroxidase activity in response to stresses were less marked than changes observed in transcript levels; cytosolic ascorbate peroxidase protein levels measured by immunoblot analysis remained unchanged.
Asunto(s)
Citosol/enzimología , Fabaceae/enzimología , Genes de Plantas , Peroxidasas/genética , Plantas Medicinales , Secuencia de Aminoácidos , Ascorbato Peroxidasas , Secuencia de Bases , Southern Blotting , Clonación Molecular , ADN , Datos de Secuencia Molecular , Peroxidasas/metabolismo , Empalme del ARN , Mapeo Restrictivo , Homología de Secuencia de Aminoácido , Transcripción GenéticaRESUMEN
A cDNA clone encoding the cytosolic ascorbate peroxidase of pea (Pisum sativum L.) was isolated and its nucleotide sequence determined. While ascorbate peroxidase shares limited overall homology with other peroxidases, significant homology with all known peroxidases was found in the vicinity of the putative active site.
Asunto(s)
Fabaceae/genética , Peroxidasas/genética , Plantas Medicinales , Secuencia de Aminoácidos , Ascorbato Peroxidasas , Secuencia de Bases , Clonación Molecular/métodos , Citosol/enzimología , ADN/genética , Fabaceae/enzimología , Biblioteca de Genes , Datos de Secuencia Molecular , ARN Mensajero/genética , Homología de Secuencia de Ácido NucleicoRESUMEN
A cDNA encoding the chloroplastic copper/zinc-superoxide dismutase of pea (Pisum sativum L.) was isolated from a cDNA library constructed in lambda gt11 from leaf mRNA. Nucleotide sequence analysis of the 875-base-pair clone revealed that it contained the complete coding sequence of the mature superoxide dismutase isozyme subunit, along with sequence information for a 48-amino acid N-terminal transit peptide. The deduced amino acid sequence of the mature subunit proved to be 64-87% homologous with amino acid sequences of copper/zinc-superoxide dismutases from other plant species. In vitro transcription, followed by cell-free translation, of the cDNA resulted in the formation of a 23.5-kDa precursor polypeptide, which, upon incubation with isolated pea chloroplasts, was imported and processed to its mature subunit molecular mass of 17.4 kDa.