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    Overexpression of a pepper ascorbate peroxidase-like 1 gene in tobacco plants enhances tolerance to oxidative stress and pathogens

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    Abstract
    In order to determine the role of ascorbate peroxidase, an antioxidant enzyme, in the cellular responses to oxidative stress and pathogens, transgenic tobacco plants were generated, using the Capsicum annuum ascorbate peroxidase-like 1 gene (CAPOA1), under the control of the CaMV 35S promoter. High levels of CAPOA1 gene expression were observed in the transgenic plants, with a 2-fold increase in total peroxidase activity. The constitutive expression of CAPOA1 in the tobacco exhibited no morphological abnormalities, while significantly increased growth was observed in transgenic plants, as compared to control plants. The CAPOA1-overexpressed plants exhibited increased tolerance to methyl viologen-mediated oxidative stress, and also enhanced resistance to the oomycete pathogen, Phytophthora nicotianae. However, the transgenic plants were not found to be resistant to the bacterial pathogen, Pseudomonas syringae pv. tabaci, but were weakly resistant to Ralstonia solanacearum. Our results suggested that the overproduction of ascorbate peroxidase increased peroxidase activity that enhances active oxygen scavenging system, leading to oxidative stress tolerance and oomycete pathogen resistance.
    Article Outline
    1. Introduction
    2. Materials and methods
    2.1. Construction of plant expression vector and Agrobacterium transformation
    2.2. Tobacco transformation and T1 seed production
    2.3. Genomic DNA isolation and PCR screening for transgenic plants
    2.4. RNA isolation and Northern blot analysis
    2.5. Total peroxidase activity assay
    2.6. Bioassay of oxidative stress tolerance
    2.7. Pathogen inoculation procedures
    2.8. Subcellular localization of CAPOA1 in onion epidermal cells
    3. Results
    3.1. Production of transgenic plants
    3.2. Morphology and growth rates of the CAPOA1 transgenic plants
    3.3. Overexpression of CAPOA1 increases peroxidase activity
    3.4. Enhanced tolerance of transgenic plants to oxidative stress
    3.5. Enhanced resistance to pathogens
    3.6. Subcellular localization of CAPOA1 in onion epidermal cells
    4. Discussion
    Acknowledgements
    References
     

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