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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

2D Analysis of peroxidase (POD) activity from p84C3 maize kernels. (A) Guaiacol-H2O2 staining. (B) Sequential staining (guaiacol-H2O2 + Coomassie R-250). Arrows point to the spots with POD activity.
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Figure 1: 2D Analysis of peroxidase (POD) activity from p84C3 maize kernels. (A) Guaiacol-H2O2 staining. (B) Sequential staining (guaiacol-H2O2 + Coomassie R-250). Arrows point to the spots with POD activity.

Mentions: Separation of proteins from p84C3 maize kernel extracts with 2D electrophoresis and subsequent staining for POD activity revealed six defined active spots (Figure 1A). Densitometric analysis suggests that spot 1 accounts for about 80% of the total activity (see Table 1). This is astonishing because more than 400 PODs are suspected for Z. mays according to the UniProt8 database. Spot 1 exhibits an apparent MW of 31 kDa and a pI of 9.5. Spots 4–6 display the same apparent molecular weight on the gel and thus might be isoforms of the protein represented by spot 1. Spots 2 and 3 displayed an apparent MW of 45 kDa and pI of 9.3 and 9.5, respectively. After subsequent Coomassie staining of the gel, only one protein spot was still visible (Figure 1B). This spot corresponds to the Spot 1 of the guaiacol-H2O2 staining (Figure 1A). The protein of this spot was sliced from the gel and subjected to nanoLC-MS/MS 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)

2D Analysis of peroxidase (POD) activity from p84C3 maize kernels. (A) Guaiacol-H2O2 staining. (B) Sequential staining (guaiacol-H2O2 + Coomassie R-250). Arrows point to the spots with POD activity.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: 2D Analysis of peroxidase (POD) activity from p84C3 maize kernels. (A) Guaiacol-H2O2 staining. (B) Sequential staining (guaiacol-H2O2 + Coomassie R-250). Arrows point to the spots with POD activity.
Mentions: Separation of proteins from p84C3 maize kernel extracts with 2D electrophoresis and subsequent staining for POD activity revealed six defined active spots (Figure 1A). Densitometric analysis suggests that spot 1 accounts for about 80% of the total activity (see Table 1). This is astonishing because more than 400 PODs are suspected for Z. mays according to the UniProt8 database. Spot 1 exhibits an apparent MW of 31 kDa and a pI of 9.5. Spots 4–6 display the same apparent molecular weight on the gel and thus might be isoforms of the protein represented by spot 1. Spots 2 and 3 displayed an apparent MW of 45 kDa and pI of 9.3 and 9.5, respectively. After subsequent Coomassie staining of the gel, only one protein spot was still visible (Figure 1B). This spot corresponds to the Spot 1 of the guaiacol-H2O2 staining (Figure 1A). The protein of this spot was sliced from the gel and subjected to nanoLC-MS/MS 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