Effect of salinity stress on watermelon [ Citrullus lanatus (Thunb . ) Matsum & Nakai]

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

2. The aim: • effects of salt stress onsuperoxide dismutase activity in watermelonsgraftedonto 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: • fruit shape • skin colour • skin or rind smoothness • flex texture and colour • rind thickness • yield • degree of environmental effectstolerance (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. 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) 2dS m-1 4 dS m-1 6 dS m-1 • different levels of salt stress

10. Salt stress • Disrupts: • homeostasis in ion distribution • water potential • plant growth • productivity • Mechanism of salt tolerance include: • efficient uptake selectivityof 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 • 1O2singlet 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 • 2.5μl • 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 colouredformazan 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. % of inhibition = (sample A560 – control A560) / control A560 Ve= volume of enzyme at 50% of inhibition estimated graphically Calculation

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

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-1EC solution • difference in salt ion accumulation in the shoot

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

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

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. Emphasisgrafted 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: • 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: • preventstoxic effects induced by salinity • differentiatesaline ion accumulation in the scion

29. 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 studiesto select rootstock adapted for growth under saline conditions

30. Thank You! ivana.bogic@st.htnet.hr