Antioxidant Enzyme Activities During Tomato Fruit Ripening

and in Response to Phosphorous Nutrients.

T. Ahn, A. Schofield, and G. PaliyathDepartment of Food Science

University of Guelph

OutlineBackgroundDevelopment Experiment

Methods Results and Discussion

Phosphorous Experiment Methods Results and Discussion

Conclusions

Reactive oxygen species

(Fenton Reaction)Fe2+

:OH– + Fe3+

·O-O· Triplet Oxygen (ground state)

·O-O: Superoxide (·O2–)

H:O· Hydroxyl Radical (HO·)

H:O-O:H Hydrogen Peroxide (H2O2)

·O-O:

·O-O·(Dismutation Reaction)

e–(Monovalent Reduction)

Antioxidant pathwayReactive Oxygen Species (ROS)

damage: lipids, proteins, and DNAROS formed:

chloroplast mitochondria endoplasmic reticulum microbodies plasma membranes cell walls

Defence MechanismsChemicals

ascorbic acid, glutathione, tocopherol, carotenoids, phenolics

enzymes

·O2–

superoxidedismutase

H2O2

catalaseguaiacol peroxidase

ascorbate peroxidase

H2O + O2

Experiment 1:Changes in antioxidant enzyme activities during the development cherry tomato

fruit.fresh market, cherry tomato (Lycopersicon esculentum Mill. cv. Favorita)

hydroponicallydevelopmental 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 gelsSuperoxide dismutase (SOD)

photoreduction of nitroblue tetrazoliumPeroxidase (POX)

oxidation of benzidine dihydrochloride in presence of H2O2

Ascorbate Peroxidase (APX) reduction of nitroblue tetrazolium by H2O2

Expt 1: fruit development

Y I MG O R

SOD

Developmental stage

Expt 1: fruit development

d

a

ccdb

0102030405060

Y I MG O RDevelopmental stage

Tot

al a

ctiv

ity S

OD

(uni

ts /

gfw

)SOD

Expt 1: fruit development

Y I MG O R

POX

Developmental stage

Expt 1: fruit development

a

decb

500

550

600

650

700

Y I MG O RDevelopmental stage

Tota

l act

ivity

PO

X(n

mol

Gua

iaco

l /m

in /

gfw

)POX

Expt 1: fruit development

Y I MG O R

APX

Developmental stage

Expt 1: fruit development

05

10152025

Y I MG O RDevelopmental stage

Tota

l act

ivity

APX

(um

ol H

2O2 /

10 m

in /

gfw

)APX

Expt 1: fruit developmentSpec. assays and isozyme profiles

provide different perspectivesSOD 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 fertilization five phosphorous treatments:

none No

soil regular50 kg / hectare before Reg

soil low170 kg / hectare x-plant Low

foliar spray hydrophos

8g P 1.2g Mg in 4L 4x at Hydro

foliar spray seniphos

11.6g P1.5g Ca in 4L

2-week intervals Seni

Expt 2: phosphorus fertilization

bb b ab a

01020304050

No Reg Low Hydro Seni

Phosphorous treatment

Tota

l act

ivity

SO

D(u

nits

/ gf

w)

Expt 2: phosphorus fertilization

cbc b

a a

0

100

200

300

400

No Reg Low Hydro Seni

Phosphorous treatment

Tota

l act

ivity

PO

X(n

mol

Gua

iaco

l /m

in /

gfw

)

Expt 2: phosphorus fertilization

0100200300400500600700

No Reg Low Hydro Seni

Phosphorus treatment

Tota

l act

ivity

PO

X (n

mol

Gua

iaco

l /m

in /

gfw

)MG O R

Expt 2: phosphorus fertilization

abbb

c

05

1015202530

No Reg Low Hydro Seni

Phosphorous treatment

Tota

l act

ivity

APX

(um

ol H

2O2 /

10 m

in /

gfw

)

Expt 2: phosphorus fertilizationseni increased SOD activity

all P treatment increased activity of APX and POX POX differences at MG stage only

Summaryactivities 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 InstitutePhosyn PLC, UK. Ontario Graduate Scholarship Program