1. Garlic Sulphur Biochemistry Partner 2: Horticulture Research International Laurence Trueman, Brian Thomas, Linda Brown & Brian Smith Workpackage Four

2. To identify developmental control points for CSO synthesis Objective: 2 Analysis of second-year field experiment completed Whole plant labelling studies completed Milestones Feb. 2002 - 2003

3. Distribution and remobilization of sulphur taken up early Distribution and remobilization of sulphur taken up late * * * * * * * * * * * every 14d Hydroponic Printanor 34 S 32 S * * * * * * * * * * * every 14d

4. Analysis of second year field trial Fresh weight - completed Dry weight - completed Bulbing index - completed Sulphur content - completed Nitrogen content - completed Carbon content - completed CSO analysis - in progress

5. Comparison with last year This years plants were larger, bulbed later and produced smaller bulbs This may be explained by the fact that S, N and C accumulation was initiated 2-3 weeks later, but peaked at the same time as last years plants The general pattern of growth however was similar and the four stages of growth proposed last year is still valid

6. 34 S labelling experiment System A labelled in the first half of the growth period System B labelled late in second half of the growth period

7. Pattern of sulphur accumulation

8. What are delta (d) values The results are expressed in delta values The ratio of 34 S/ 32 S in the plant tissue divided by the ratio of 34 S/ 32 S in a universal standard minus 1 and expressed as ‰ Basically –The greater a value is above 0 the more 34 S it contains

9. System A delta values 34 S 32 S

10. System B delta values 34 S 32 S

11. Changes in delta values Changing delta values can indicate –Import of S compounds with a different d value –Export of S compounds with a d value different to the mean of that tissue Unchanging delta values can indicate –No import/Export of S compounds –Import/Export of S compounds with a d value equal to the mean of that tissue

12. Absolute Amounts To understand what is happening we have to look at actual amounts of sulphur imported/exported from a tissue over time Delta values and total sulphur contents allow us to calculate actual amounts of 34 S and 32 S This allows us to compare rates of change of the 34 S and 32 S pools

13. Rates of change of actual amounts - system B

14. Comparison of 34 S content

15. It is easier to plot the ratio δ 34 S divided by δ 32 S A result greater than 1 indicates a greater change in the 34 S pool A result less than than 1 indicates a greater change in the 32 S pool

16. System A 34 S 32 S

17. System B 34 S 32 S

18. Conclusions The Sulphur pools in root, leaf and bulb tissue increase whilst the root are actively taking up sulphur These sulphur pools appear to be fluid between root and leaf The Sulphur may actually cycle between the leaf and root pools Bulb delta values are always intermediate to root and leaf values suggesting that the bulb sulphur does not all pass through the leaf

19. Conclusions After uptake of sulphur by the root ceases a phase of sulphur export to the bulb commences This appears to involve the release of sulphur stored early in the growing period in to the pool of cycling sulphur This may be programmed or simply due to root death

20. To isolate and characterise alliinase cDNA clones Objective: 3b Start Expression Analysis Milestones Feb. 2002 - 2003

21. Garlic Alliinase Expression relative to total rRNA content

23. 2002 - Hydroponic data Bulb Index Fresh weight Dry Weight

24. System A Nitrogen Sulphur Carbon

25. System B Nitrogen Sulphur Carbon

26. mRNA Expression - Method