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Antioxidant Enzyme Activities During Tomato Fruit Ripening and in Response to Phosphorous Nutrients. T. Ahn, A. Schofield, and G. Paliyath Department of Food Science University of Guelph. Outline. Background Development Experiment Methods Results and Discussion Phosphorous Experiment
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Antioxidant Enzyme Activities During Tomato Fruit Ripening and in Response to Phosphorous Nutrients. T. Ahn, A. Schofield, and G. Paliyath Department of Food Science University of Guelph
Outline • Background • Development Experiment • Methods • Results and Discussion • Phosphorous Experiment • Methods • Results and Discussion • Conclusions
e– (Monovalent Reduction) ·O-O: ·O-O· (Dismutation Reaction) Fe2+ :OH– + Fe3+ (Fenton Reaction) Reactive oxygen species ·O-O· Triplet Oxygen (ground state) ·O-O: Superoxide (·O2–) H:O-O:H Hydrogen Peroxide (H2O2) H:O· Hydroxyl Radical (HO·)
Antioxidant pathway • Reactive Oxygen Species (ROS) damage: lipids, proteins, and DNA • ROS formed: • chloroplast • mitochondria • endoplasmic reticulum • microbodies • plasma membranes • cell walls
Defence Mechanisms • Chemicals • ascorbic acid, glutathione, tocopherol, carotenoids, phenolics • enzymes ·O2– H2O2 H2O + O2 superoxide dismutase catalase guaiacol peroxidase ascorbate peroxidase
Experiment 1: Changes in antioxidant enzyme activities during the development cherry tomato fruit. • fresh market, cherry tomato (Lycopersicon esculentum Mill. cv. Favorita) • hydroponically • developmental stages: young, intermediate, mature green, orange and red
Expt 1: fruit development Spectrophotometeric assays: • Superoxide dismutase (SOD) • photoreduction of nitroblue tetrazolium • 1 unit - 50% inhibition • Peroxidase (POX) • oxidation of guaiacol in presence of H2O2 • Ascorbate Peroxidase (APX) • monitor H2O2 decomposition • react residual H2O2 with Titanium chloride
Expt 1: fruit development Activity staining of native PAGE gels • Superoxide dismutase (SOD) • photoreduction of nitroblue tetrazolium • Peroxidase (POX) • oxidation of benzidine dihydrochloride in presence of H2O2 • Ascorbate Peroxidase (APX) • reduction of nitroblue tetrazolium by H2O2
Expt 1: fruit development SOD Y I MG O R Developmental stage
Expt 1: fruit development POX Y I MG O R Developmental stage
Expt 1: fruit development APX Y I MG O R Developmental stage
Expt 1: fruit development • Spec. assays and isozyme profiles provide different perspectives • SOD and POX activities: • generally decrease with maturation • increase at orange stage • fast-migrating SOD isozyme decreases during ripening • major APX isozyme shows increase during ripening
Experiment 2: Changes in antioxidant enzyme activities in response to phosphorous fertilization. • processing tomato, Heinz 9478 (L. esculentum Mill.) • developmental stages: mature green, orange and red • field grown under recommended levels of nitrogen and potassium
Expt 2: phosphorus fertilizationfive phosphorous treatments:
Expt 2: phosphorus fertilization • seni increased SOD activity • all P treatment increased activity of APX and POX • POX differences at MG stage only
Summary • activities and isozyme profiles of some antioxidant enzymes are developmentally regulated--and in particular up-regulated during fruit ripening. • Phosphorous supplementation increases the activity of several antioxidant enzymes • This may counter the deleterious effects of activated oxygen species and delay deterioration of fruit quality.
We gratefully acknowledge the financial support of: • Potash and Phosphate Institute • Phosyn PLC, UK. • Ontario Graduate Scholarship Program