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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

Production and purification of the recombinant B6T173 (ZmPrx35) protein in E. coli (using the vector pET19b). (A) Sequential staining (guaiacol- H2O2 + Coomassie R-250) of the POD fractions obtained by IMAC affinity purification. (B) Coomassie R-250 staining of the POD active fractions. (C) Guaiacol-H2O2 staining of the same POD fractions. In all cases, 20 μL of protein solution were loaded. Arrows indicate bands with POD activity.
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Figure 4: Production and purification of the recombinant B6T173 (ZmPrx35) protein in E. coli (using the vector pET19b). (A) Sequential staining (guaiacol- H2O2 + Coomassie R-250) of the POD fractions obtained by IMAC affinity purification. (B) Coomassie R-250 staining of the POD active fractions. (C) Guaiacol-H2O2 staining of the same POD fractions. In all cases, 20 μL of protein solution were loaded. Arrows indicate bands with POD activity.

Mentions: The recombinant active protein obtained from the pET19b construct expression has an approximate MW of 34 kDa, which is congruent with the B6T173 amino acid sequence (Figure 4). NanoLC-MS/MS based identification verified the presence of B6T173_MAIZE (ZmPrx35) in the band with POD activity from the 1D gel with semi-purified fractions of recombinant E. coli production (see Table 4). Since no POD activity is detected in non-induced cultures (Supplementary Figure S4A), the results provide strong evidence for the heterologous production of active ZmPrx35 POD. The presence of activity after production in a bacterial host suggests that B6T173_MAIZE activity is independent from glycosylation. Similar findings were reported for the soybean cytosolic ascorbate POD (Dalton et al., 1996) and catalase (Ray et al., 2012).


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)

Production and purification of the recombinant B6T173 (ZmPrx35) protein in E. coli (using the vector pET19b). (A) Sequential staining (guaiacol- H2O2 + Coomassie R-250) of the POD fractions obtained by IMAC affinity purification. (B) Coomassie R-250 staining of the POD active fractions. (C) Guaiacol-H2O2 staining of the same POD fractions. In all cases, 20 μL of protein solution were loaded. Arrows indicate bands with POD activity.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: Production and purification of the recombinant B6T173 (ZmPrx35) protein in E. coli (using the vector pET19b). (A) Sequential staining (guaiacol- H2O2 + Coomassie R-250) of the POD fractions obtained by IMAC affinity purification. (B) Coomassie R-250 staining of the POD active fractions. (C) Guaiacol-H2O2 staining of the same POD fractions. In all cases, 20 μL of protein solution were loaded. Arrows indicate bands with POD activity.
Mentions: The recombinant active protein obtained from the pET19b construct expression has an approximate MW of 34 kDa, which is congruent with the B6T173 amino acid sequence (Figure 4). NanoLC-MS/MS based identification verified the presence of B6T173_MAIZE (ZmPrx35) in the band with POD activity from the 1D gel with semi-purified fractions of recombinant E. coli production (see Table 4). Since no POD activity is detected in non-induced cultures (Supplementary Figure S4A), the results provide strong evidence for the heterologous production of active ZmPrx35 POD. The presence of activity after production in a bacterial host suggests that B6T173_MAIZE activity is independent from glycosylation. Similar findings were reported for the soybean cytosolic ascorbate POD (Dalton et al., 1996) and catalase (Ray et al., 2012).

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