T. Ahn, A. Schofield, and G. Paliyath Department of Food Science University of Guelph

1. 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

2. Outline • Background • Development Experiment • Methods • Results and Discussion • Phosphorous Experiment • Methods • Results and Discussion • Conclusions

3. 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·)

4. Antioxidant pathway • Reactive Oxygen Species (ROS) damage: lipids, proteins, and DNA • ROS formed: • chloroplast • mitochondria • endoplasmic reticulum • microbodies • plasma membranes • cell walls

5. Defence Mechanisms • Chemicals • ascorbic acid, glutathione, tocopherol, carotenoids, phenolics • enzymes ·O2– H2O2 H2O + O2 superoxide dismutase catalase guaiacol peroxidase ascorbate peroxidase

6. 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

7. 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

8. 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

9. Expt 1: fruit development SOD Y I MG O R Developmental stage

10. Expt 1: fruit development SOD

11. Expt 1: fruit development POX Y I MG O R Developmental stage

12. Expt 1: fruit development POX

13. Expt 1: fruit development APX Y I MG O R Developmental stage

14. Expt 1: fruit development APX

15. 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

16. 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

17. Expt 2: phosphorus fertilizationfive phosphorous treatments:

18. Expt 2: phosphorus fertilization

19. Expt 2: phosphorus fertilization

20. Expt 2: phosphorus fertilization

21. Expt 2: phosphorus fertilization

22. Expt 2: phosphorus fertilization • seni increased SOD activity • all P treatment increased activity of APX and POX • POX differences at MG stage only

23. 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.

24. We gratefully acknowledge the financial support of: • Potash and Phosphate Institute • Phosyn PLC, UK. • Ontario Graduate Scholarship Program