Garlic Sulphur Biochemistry

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

2. Objective: 2 To identify developmental control points for CSO synthesis • First year harvests and analysis completed. • Second year experimental material planted Milestones Feb. 2001 - 2002

3. Second Year Growth Study • Two varieties grown: • Printanor • Messidrome • Two growth media • Hydroponic • Pot-grown in a soil based compost

4. Hydroponic verses Pot-grown Is there a difference?

6. Which is most like field-grown? • Can compare mean maximum number of leaves per Printanor plant • Hydroponic-grown 19 • Pot-grown 10 • Field-grown (Remi) 13 • Messidrome similar

9. Mean Bulb weights • Pot-grown Printanor 38g • Pot-grown Messidrome 32g • Hydroponic-grown Printanor 101g • Hydroponic-grown Messidrome 53g • These differences are significant

10. Analysis completed • Determined Fresh and Dry weights of Root (R), Leaf (L), and Bulb (B) • Determined Protein content (R, L, B) • Determined Sulphur content (R, L, B) • Determined Nitrogen content (R, L, B) • Determined Carbon content (R, L, B) • Determination of CSO content (R, L, B) • Have now got complete data set

11. Hydroponic Printanor - Sulphur

12. Hydroponic Printanor - Nitrogen

13. Hydroponic Printanor - Protein

14. Hydroponic Printanor - CSOs

15. Hydroponic Messidrome - CSOs

16. Hydroponic Printanor - Leaf CSO composition

17. Hydroponic Messidrome - Leaf CSO composition

18. Hydroponic Printanor - Clove CSO composition

19. Hydroponic Printanor - Root CSO composition

20. Questions on CSO synthesis • Is the main Pre-CSO sulphur stored in the root? • Do the roots synthesis (the) CSO’s • What form is the sulphur in prior to CSO synthesis? • Is Isoalliin an intermediate or product? • Is there a Propiin to Alliin and Methiin conversion take place?

21. Hydroponic Printanor - Carbon

22. Can define 4 stages of growth • Stage 1. Early growth phase • Stage 2. Late growth phase • Stage 3. Bulb initiation • Stage 4. Bulb maturity

23. Stage One • Day: 0-40 hydroponic, 0-70 pot-grown • Plants rely on stored nutrients • Difference between systems, due to higher hydroponic temperature? • Does differences in temperature explain variation between hydroponic, pot and field-grown plants?

24. Stage two • Day: 40-150 hydroponic, 70-150 pot • Root and leaf tissue increase rapidly • leaf tissue is twice fresh weight of root • Root and leaf S, N, C, protein and CSO’s maximal • Carbon accumulates in leaf and is not lost until leaf senescence. Probably structural • Protein synthesis mainly in leaves supported by fact that leaves have 40% more N than root • S content is same in root and leaf • Since root has a third dry matter of the leaf suggesting this is the major S store

25. Stage three • Day: 150 - 200 hydroponic & pot-grown • Initiation of bulbing. Independent of plant size • temperature and day-length response • garlic plants clove 140-160 days after planting. • S, N, C, protein and CSO's decline in leaf and root • S, N, C, protein and CSO's rapidly accumulate in bulb • S & N totals do not change implying no root uptake • Root death at end of period • Dramatic rise in CSO synthesis

26. Stage four • Day: 200-bulb maturity hydroponic & pot-grown • Leaves are senescing and S, N, C and protein levels fall • Equivalent rise of S, N, C in bulb • Leaf turgor lost due to root death • The aerial parts of the plant senesce • Neck closure • Bulb matures

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

28. A B C D Hydroponic Printanor • A=50% total sulphur (d. 102, end March) • B=Start bulbing (d. 130, 20 Apr) • C=Start of senescence of root & leaf (d. 170, end May) • D=Harvest (d. 230, end Jul)

29. 34S Hydroponic Printanor Distribution and remobalisation of sulphur taken up early * * * * * * * * * * * every 14d Distribution and remobalisation of sulphur taken up late * * * * * * * * * * * every 14d A B C D 32S

30. What we want to know • When does nutrient uptake from the medium stop • When does bulb filling start • What proportion of S (and N) comes from the roots, leaves and hydroponic medium • The form of the S in the root and leaf

31. Objective: 3b To isolate and characterise alliinase cDNA clones • cDNA clones encoding alliinase isolated and sequenced Milestones Feb. 2001 - 2002

32. Clustering relationship of Messidrome alliinase PCR fragment sequences l=Leaf Clone b=Bulb Clone

33. Alliinase cDNA clone isolation • Have used PCR fragments to identify alliinase clones in the library • Have isolated 56 alliinase containing plaques and re-amplified • Have developed a method of identifying near full-length clones • Are screening for full-length clone • Expect to be sequencing soon

34. Objective: 3b To isolate and characterise alliinase cDNA clones • Analysis of second-year field experiment completed • Whole plant labelling studies completed Milestones Feb. 2002 - 2003

35. Expression analysis • Will use Single Nucleotide Primer Externsion • Based on detecting single nucleotide differences between alliinase clones • Bulb Alliinase B6 • Bulb Alliinase B5 • Bulb Alliinase B4 • Bulb Alliinase B3 • Have successfully used this approach on onion

36. SNuPE analysis of Sweet Onion RT-PCR E L E L E L IV III I II Bulb Leaf Root E = Early L = Late