RESUMO
The promoters of the Arabidopsis (Arabidopsis thaliana) cytochrome c genes, Cytc-1 and Cytc-2, were analyzed using plants transformed with fusions to the beta-glucuronidase coding sequence. Histochemical staining of plants indicated that the Cytc-1 promoter directs preferential expression in root and shoot meristems and in anthers. In turn, plants transformed with the Cytc-2 promoter fusions showed preferential expression in vascular tissues of cotyledons, leaves, roots, and hypocotyls, and also in anthers. Quantitative measurements in extracts prepared from different organs suggested that expression of Cytc-1 is higher in flowers, while that of Cytc-2 is higher in leaves. The analysis of a set of deletions and site-directed mutants of the Cytc-1 promoter indicated that a segment located between -147 and -156 from the translation start site is required for expression and that site II elements (TGGGCC/T) located in this region, coupled with a downstream internal telomeric repeat (AAACCCTAA), are responsible for the expression pattern of this gene. Proteins present in cauliflower nuclear extracts, as well as a recombinant protein from the TCP-domain family, were able to specifically bind to the region required for expression. We propose that expression of the Cytc-1 gene is linked to cell proliferation through the elements described above. The fact that closely located site II motifs are present in similar locations in several genes encoding proteins involved in cytochrome c-dependent respiration suggests that these elements may be the target of factors that coordinate the expression of nuclear genes encoding components of this part of the mitochondrial respiratory chain.
Assuntos
Arabidopsis/enzimologia , Arabidopsis/genética , Citocromos c/genética , Flores/genética , Regulação da Expressão Gênica de Plantas , Genes de Plantas/genética , Meristema/genética , Sequência de Bases , Compostos de Benzil , Brassica/química , Brassica/citologia , Citocromos c/metabolismo , Flores/anatomia & histologia , Flores/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Cinetina/farmacologia , Meristema/metabolismo , Dados de Sequência Molecular , Especificidade de Órgãos , Extratos Vegetais/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica , Estrutura Terciária de Proteína , Purinas , Deleção de Sequência/genética , Sacarose/farmacologiaRESUMO
Cell suspension cultures of Brassica napus were obtained under different hormonal conditions, using 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin as growth regulators. They were analyzed as a culture system for peroxidase production in vitro to avoid many of the problems that affect the production from field-grown roots. Total peroxidase specific activities reached a maximum at the end of exponential growth phase of the cultures. Cultures obtained with 4 mg/l of 2,4-D an without kinetin or with 1 mg/l of 2,4-D and the same amount of kinetin produced twice the total activity of root extracts and, in addition, they released peroxidases to the culture medium, which would be advantageous for the commercial production of the enzyme. Peroxidase patterns, obtained by isoelectric focusing of cell extracts and of culture medium of cell suspension cultures, differed from those of root crude extracts from field-grown plants with additional bands of higher isoelectric points. These cultures showed interesting properties and could be considered an alternative source of peroxidases for commercial production and/or to be applied as a model for physiological research.
Assuntos
Brassica/enzimologia , Peroxidases/metabolismo , Brassica/citologia , Brassica/efeitos dos fármacos , Células Cultivadas/citologia , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/enzimologia , Ácidos Indolacéticos/farmacologiaRESUMO
Cell suspension cultures of Brassica napus were obtained under different hormonal conditions, using 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin as growth regulators. They were analyzed as a culture system for peroxidase production in vitro to avoid many of the problems that affect the production from field-grown roots. Total peroxidase specific activities reached a maximum at the end of exponential growth phase of the cultures. Cultures obtained with 4 mg/l of 2,4-D an without kinetin or with 1 mg/l of 2,4-D and the same amount of kinetin produced twice the total activity of root extracts and, in addition, they released peroxidases to the culture medium, which would be advantageous for the commercial production of the enzyme. Peroxidase patterns, obtained by isoelectric focusing of cell extracts and of culture medium of cell suspension cultures, differed from those of root crude extracts from field-grown plants with additional bands of higher isoelectric points. These cultures showed interesting properties and could be considered an alternative source of peroxidases for commercial production and/or to be applied as a model for physiological research.(AU)