1. Effect of salinity stress on watermelon [Citrullus lanatus (Thunb
Effect of salinity stress on watermelon [Citrullus lanatus (Thunb.) Matsum & Nakai]
SUPERVISOR: STUDENT:
Professor Maja Pavela-Vrančič Ivana Bogić
TUTOR:
Professor Gabriella Stefania Scippa

2. The aim:
effects of salt stress on superoxide dismutase activity in watermelons grafted onto different rootstocks

3. Watermelon herbaceous plant glycophile species
tolerate small amounts of salt
low salinity
increases the yield
high salinity
damage

4. Watermelon cultivation - Croatia
River Neretva valley
watermelon - one of the most important crops
increment of soil salinity, result of :
seawater intrusion
construction of the hydroelectric dams
agricultural activities
land developmental activities
Growers demand:
higher yield
better quality

5. Grafting Improve: Rootstock selection according to: fruit shape
skin colour
skin or rind smoothness
flex texture and colour
rind thickness
yield
degree of environmental effects tolerance (salt stress...)
Rootstock selection according to:
suitability for growing season
cultivation methods
soil environment
type of crops and cultivars
salt resistance

6. Shoot:
salt sensitive
Cl- and Na+ ions damage
Root
exclusion
controled absorption
Grafted plants:
growth and reproduction altered
increase salinity resistance - salt tolerant rootstock

7. Watermelons cultivation:
Institute for Adriatic Crops and Karst Reclamation, Split
glass greenhouse = controled conditions
from May to July 2004

8. The watermelons were grafted :
4 combinations of scion and rootstock
scion: Fantasy
rootstocks: Fantasy, Strong Tosa, Emphasis and the S1

9. sea salt + standard nutrient solution
Grafted plants:
28 days irrigated with standard nutrient solution
2 weeks irrigated with saline solution:
sea salt + standard nutrient solution
3 saline solution treatments:
electrical conductivity (EC) 2 dS m-1
4 dS m-1
6 dS m-1
different levels of salt stress

10. Salt stress Disrupts: Mechanism of salt tolerance include:
homeostasis in ion distribution
water potential
plant growth
productivity
Mechanism of salt tolerance include:
efficient uptake selectivity of ions by roots and transport into the leaves
compartmentalization of ions
synthesis of compatible solutes
induction of plant hormones
induction of antioxidative enzymes
Salt ion exclusion by roots:
variable ability
influence on accumulation of salt ions in scions
Salt ion accumulation in scions:
controlled by the genotype of the rootstock
leads to salt stress  oxidative stress

11. Oxidative stress
imbalance between the oxidant and the antioxidant activity
salt stress  excess of absorbed light energy
absorbed energy + O2  reactive oxygen species
reactive oxygen species:
O2·¯ superoxide radical
H2O2 hydrogen peroxide
OH. hydroxyl radical
1O2 singlet oxygen
unspecifically react with different molecules
scavengered by antioxidant enzymes
superoxide dismutase (SOD)

12. Plant material (leaves):
sampled 2 weeks after salinisation started
stored at -80 oC
used for superoxide dismutase activity assay

13. Plant material (leaves):
homogenized in protein extraction buffer
homogenate is centrifuged
supernatant = enzyme extract
stored at -20 oC
used for:
assay of enzyme activity
protein determination
Enzyme extract:
the day after: thawed, centrifuged
diluted  enzyme solution corresponds to
2.5 μl
5.0 μl
7.5 μl
10.0 μl of the original enzyme extract

14. Superoxide dismutase activity assay
Reaction mixture:
prepared
50 mM phosphate buffer, pH 7.8
13 mM methionine
75 μM nitroblue tetrazolium (NBT)
0.1 mM EDTA
2 μM riboflavin
0.1 ml of:
enzyme solution – for reaction
protein extraction buffer – for control
shaken
placed in a light box
Reaction:
turning-on the light
10 min
light was turned-off
test tubes were covered with a black cloth

15. Control reaction – without SOD
NBT is reduced by O2·¯ into the water-insoluble blue coloured formazan that exhibits an absorbance maximum at λmax=560 nm:
O2·¯ + nitroblue tetrazolium+  O2 + nitroblue tetrazolium.
2 nitroblue tetrazolium.  nitroblue tetrazolium+ + formazan
intensity of the blue colour  absorbance of the reaction mixture at 560 nm (Lambda Bio 40 UV-Vis spectrophotometer)
Control reaction = highest blue colour intensity

16. Reaction with SOD: inhibits reduction of NBT
catalyzes dismutation of two superoxide radicals into hydrogen peroxide and molecular oxygen:
SODox + O2·¯ + H+  SODred(H+) + O2
SODred(H+) + O2·¯ + H+  SODox+ H2O2
Reaction mixture:
increasing volume of the enzyme extract  decreased blue colour intensity

17. Calculation
% of inhibition = (sample A560 – control A560) / control A560
Ve = volume of enzyme at 50% of inhibition
estimated graphically

18. Specific activity of SOD:
expressed in unit per mg protein:
SOD (U mg-1) = Ve x c
c = protein concentration
measured according to Bradford
One unit of SOD activity:
amount of enzyme required to cause 50% inhibition of the rate of nitroblue tetrazolium reduction

19. The mean value of SOD activity in watermelon grafted plants
4 grafted watermelon cultivars showed an increase in superoxide dismutase activity, after a two week exposure to salinity stress

20. SOD activity in leaves of salt-stressed watermelons grafted onto four different rootstocks

21. variation between different rootstocks in SOD activity following treatment with the control, 2 dS m-1 EC solution
difference in salt ion accumulation in the shoot

22. Fantasy non-grafted control SOD activity increased
EC value of the saline solution increased

23. S1 grafted plants SOD activity increased
EC value of the saline solution increased
6 dS m-1 EC treatment 2X control

24. S1 grafted plants significant morphological damage first died
exclusion by the root insufficient
large amounts of salt reach the leaves, accumulate and cause oxidative stress

25. Emphasis grafted plants
SOD activity increased
Emphasis grafted root - very good ion excluder

26. Strong Tosa grafted plants
SOD activity increased
last showed visible morphologycal damage
Strong Tosa root - very good ion-excluder

27. effect of salinity: salt stress: complex process dependins on:
salt concentration
plant genotype
growth stage
environmental conditions
genotype of the rootstock
salt stress:
causes oxidative stress
increases SOD activity

28. Conclusion SOD activity increases under increased salinity
Response to salt stress - depends on the rootstock genotype
Rootstock:
prevents toxic effects induced by salinity
differentiate saline ion accumulation in the scion

29. farmers - Neretva valley
Grafting:
valid strategy
amelioration of shoot growth under salinity
farmers - Neretva valley
which rootstock - better fruit yield and quality
how improve growth and development under high salt concentrations
more studies to select rootstock adapted for growth under saline conditions

30. Thank You!