RESUMO
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
The last step in the synthesis of lignin and suberin has been proposed to be catalyzed by peroxidases, although other proteins may also be involved. To determine which peroxidases are involved in the synthesis of lignin and suberin, five peroxidases from tomato (Lycopersicon esculentum) roots, representing the majority of the peroxidase activity in this organ, have been partially purified and characterized kinetically. The purified peroxidases with isoelectric point (pI) values of 3.6 and 9.6 showed the highest catalytic efficiency when the substrate used was syringaldazine, an analog of lignin monomer. Using a combination of transgenic expression and antibody recognition, we now show that the peroxidase pI 9.6 is probably encoded by TPX1, a tomato peroxidase gene we have previously isolated. In situ RNA hybridization revealed that TPX1 expression is restricted to cells undergoing synthesis of lignin and suberin. Salt stress has been reported to induce the synthesis of lignin and/or suberin. This stress applied to tomato caused changes in the expression pattern of TPX1 and induced the TPX1 protein. We propose that the TPX1 product is involved in the synthesis of lignin and suberin.
Assuntos
Lignina/biossíntese , Lipídeos de Membrana/biossíntese , Peroxidases/metabolismo , Solanum lycopersicum/enzimologia , Cinética , Lipídeos , Peroxidases/genéticaRESUMO
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.
RESUMO
Peroxidases with syringaldazine oxidase activity have been claimed to be specifically involved in lignification. The use of syringaldazine as a substrate presents problems of color diffusion and fading. We report a method for timing color development of syringaldazine peroxidase reactions in tissue prints, and conditions under which diffusion is minimized. The best time interval in which the tissue print and background gave significant contrast was determined using ELISA plates in which impressions of the tissue could be made, mimicking reactions on a membrane. This technique permitted simultaneous reading of a large number of samples and blanks, rendering enough data points for statistical analysis. Optimum development time for syringaldazine peroxidase reactions was between 10 and 20 min. Color diffusion and smearing of syringaldazine peroxidase reactions in tissue prints were minimized when prints were covered with a strip of membrane before adding the substrate mixture.