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Identification of B6T173 (ZmPrx35) as the prevailing peroxidase in highly insect-resistant maize (Zea mays, p84C3) kernels by activity-directed purification.

López-Castillo LM, López-Arciniega JA, Guerrero-Rangel A, Valdés-Rodríguez S, Brieba LG, García-Lara S, Winkler R - Front Plant Sci (2015)

Bottom Line: Contrary to their biological importance, only very few plant PODs have been proven on protein level, because their low abundance makes them difficult to detect in standard proteomics work-flows.A statistically significant positive correlation between POD activity and post-harvest insect resistance has been found for maize (Zea mays, p84C3) kernels.Our findings support the screening for insect resistant maize variants and the construction of genetically optimized maize plants.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Biochemical and Instrumental Analysis, Department of Biotechnology and Biochemistry, Cinvestav Unidad Irapuato Irapuato, Mexico ; Laboratorio Nacional de Genómica para la Biodiversidad, Unidad de Genómica Avanzada del Centro de Investigación y de Estudios Avanzados - Instituto Politécnico Nacional Irapuato, Mexico.

ABSTRACT
Plant peroxidases (PODs) are involved in diverse physiological processes, including defense against pathogens and insects. Contrary to their biological importance, only very few plant PODs have been proven on protein level, because their low abundance makes them difficult to detect in standard proteomics work-flows. A statistically significant positive correlation between POD activity and post-harvest insect resistance has been found for maize (Zea mays, p84C3) kernels. In combining activity-directed protein purification, genomic and proteomic tools we found that protein B6T173 (ZmPrx35) is responsible for the majority of the POD activity of the kernel. We successfully produced recombinant ZmPrx35 protein in Escherichia coli and demonstrate both, in vitro activity and the presence of a haem (heme) cofactor of the enzyme. Our findings support the screening for insect resistant maize variants and the construction of genetically optimized maize plants.

No MeSH data available.


Related in: MedlinePlus

Purification of protein with POD activity from p84C3 maize kernels. (A) Coomassie R-250 staining of the POD fractions of the different purification steps. (B) Sequential staining of POD fractions (guaiacol-H2O2 + Coomassie R-250). (C) Guaiacol staining of the same fractions. In all cases, 20 μL of protein of the indicated fraction was loaded on each lane. Arrows indicate bands with POD activity.
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Figure 2: Purification of protein with POD activity from p84C3 maize kernels. (A) Coomassie R-250 staining of the POD fractions of the different purification steps. (B) Sequential staining of POD fractions (guaiacol-H2O2 + Coomassie R-250). (C) Guaiacol staining of the same fractions. In all cases, 20 μL of protein of the indicated fraction was loaded on each lane. Arrows indicate bands with POD activity.

Mentions: Activity-directed partial purification of the most active POD from p84C3 seeds revealed a rarefied, yet highly active, protein (Figure 2). The apparent MW of the semi-purified enzyme is congruent with the spot location of the POD active protein in the 2D analysis.


Identification of B6T173 (ZmPrx35) as the prevailing peroxidase in highly insect-resistant maize (Zea mays, p84C3) kernels by activity-directed purification.

López-Castillo LM, López-Arciniega JA, Guerrero-Rangel A, Valdés-Rodríguez S, Brieba LG, García-Lara S, Winkler R - Front Plant Sci (2015)

Purification of protein with POD activity from p84C3 maize kernels. (A) Coomassie R-250 staining of the POD fractions of the different purification steps. (B) Sequential staining of POD fractions (guaiacol-H2O2 + Coomassie R-250). (C) Guaiacol staining of the same fractions. In all cases, 20 μL of protein of the indicated fraction was loaded on each lane. Arrows indicate bands with POD activity.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4553411&req=5

Figure 2: Purification of protein with POD activity from p84C3 maize kernels. (A) Coomassie R-250 staining of the POD fractions of the different purification steps. (B) Sequential staining of POD fractions (guaiacol-H2O2 + Coomassie R-250). (C) Guaiacol staining of the same fractions. In all cases, 20 μL of protein of the indicated fraction was loaded on each lane. Arrows indicate bands with POD activity.
Mentions: Activity-directed partial purification of the most active POD from p84C3 seeds revealed a rarefied, yet highly active, protein (Figure 2). The apparent MW of the semi-purified enzyme is congruent with the spot location of the POD active protein in the 2D analysis.

Bottom Line: Contrary to their biological importance, only very few plant PODs have been proven on protein level, because their low abundance makes them difficult to detect in standard proteomics work-flows.A statistically significant positive correlation between POD activity and post-harvest insect resistance has been found for maize (Zea mays, p84C3) kernels.Our findings support the screening for insect resistant maize variants and the construction of genetically optimized maize plants.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Biochemical and Instrumental Analysis, Department of Biotechnology and Biochemistry, Cinvestav Unidad Irapuato Irapuato, Mexico ; Laboratorio Nacional de Genómica para la Biodiversidad, Unidad de Genómica Avanzada del Centro de Investigación y de Estudios Avanzados - Instituto Politécnico Nacional Irapuato, Mexico.

ABSTRACT
Plant peroxidases (PODs) are involved in diverse physiological processes, including defense against pathogens and insects. Contrary to their biological importance, only very few plant PODs have been proven on protein level, because their low abundance makes them difficult to detect in standard proteomics work-flows. A statistically significant positive correlation between POD activity and post-harvest insect resistance has been found for maize (Zea mays, p84C3) kernels. In combining activity-directed protein purification, genomic and proteomic tools we found that protein B6T173 (ZmPrx35) is responsible for the majority of the POD activity of the kernel. We successfully produced recombinant ZmPrx35 protein in Escherichia coli and demonstrate both, in vitro activity and the presence of a haem (heme) cofactor of the enzyme. Our findings support the screening for insect resistant maize variants and the construction of genetically optimized maize plants.

No MeSH data available.


Related in: MedlinePlus