1. WP4: Garlic sulphur biochemistry (P2,P3) zP2: HRI Wellesbourne yBrian Thomas, Lol Trueman, Linda Brown, Brian Smith, Gareth Griffiths zP3: The University of Liverpool, UK yHamish Collin, Rick Cosstick, Brian Tomsett, Meriel Jones yAngela Tregova, Jill Hughes, Jon Milne yMark Wilkinson, Gloria van der Werff

2. WP4: Objectives z1. Identify intermediates in alliin biosynthetic pathway (P3) z2. Identify developmental control points on CSO synthesis and translocation (P2) z3. Identify genes with altered expression and/or involved in alliicin synthesis (P2,P3)

3. 1. Identify intermediates in alliin biosynthetic pathway zReview knowledge of alliin biosynthesis zBring improved HPLC methodology into use in our laboratory yStandards – purchase, synthesis, gifts, mass spectrometry yGradient elution zDevelop experimental protocols yTissue culture yGarlic cloves

4. Biosynthetic pathway for garlic flavour precursors SO 4 2- SO 3 2- S 2- cysteine glutathione (γ-glu-cys-gly) S-methyl-γ-glu-cys gly S-methylcysteine methiin glu trans- peptidase oxidase S-2-CP-γ-glu-cys gly S-trans-1-propenyl-γ-glu-cys S-trans-1-propenylcysteine oxidase trans- peptidase glu HCOOH S-trans-1-propenylcysteine sulphoxide (isoalliin) S-methylglutathione S-(2-carboxypropyl)-glutathione S-allylglutathione S-allyl-γ-glu-cys gly S-allylcysteine glu trans- peptidase oxidase S-allyl group (unknown sources) valine & methacrylate serine oxidase S-allylcysteine S-allyl-cysteine sulphoxide (alliin)

5. Biosynthetic capacity of garlic callus Conclusion: These experiments suggest that in vivo the general reaction shown may occur:- Alk(en)yl thiol Alk(en)yl cysteine Alk(en)yl CSO

6. Glutathione-S-transferases Garlic leaf proteins - glutathione affinity matrix Single step gives substantial purification Fractions on SDS gel 25 kDa No clear potential GST substrate

7. 2. Identify developmental control points on CSO synthesis and translocation zBaseline data on garlic development zResource allocation during development zDeveloped and tested theories: yWhether roots are an important source of S for developing bulbs yWhether CSOs are synthesised in leaves and transported to bulbs

8. Identify developmental control points on CSO synthesis and translocation zGrowth studies of garlic (Messidrome, Printanor) yhydroponic versus pots ySO 4 2- uptake using isotope labelling yeffects of root and leaf removal

9. For controlled growth, greenhouse (and UK climate) Measurements during growth Leaf number, bulb weight N, S, C, protein, CSO

10. Garlic growth and S partition 1234 1234

11. Four stages in bulb development zEarly growth phase: Day 0 – 40/70 yUses stored nutrients zLate growth phase: Day 40/70 - 150 yroots, leaves grow rapidly yC, protein accumulate in leaves yS stored in roots

12. Four stages in bulb development zBulb initiation: Day 150 – 200 ytemperature and day-length dependent yS, N, C, protein and CSOs decline in roots and leaves but accumulate in bulbs yrise in CSO synthesis yroots die

13. Four stages in bulb development zBulb maturity: Day 200 yTurgor loss as leaves and roots senesce yS, N, C, protein fall in leaves, roots, and rise in bulbs yNeck closure and bulb matures.

14. Sulfur uptake and distribution in more detail zgrow hydroponically zuse isotope labelled sulfur ystable heavy isotope sulfur-34 zMeasure total S, 34/32 S ratio (delta value)

15. Distribution and remobilization of sulphur taken up early Distribution and remobilization of sulphur taken up late * * * * * * * * * * * 34 S 32 S A B Growth pattern in Year 2 experiment Sulfur labelling design

16. 34 S 32 S Year 3 hydroponic garlic

17. A: 34 S then 32 SB: 32 S then 34 S S pools in root, leaf, bulb increase while root takes up S After S uptake by roots cease, it is exported to bulb Roots therefore appear an important S source for the bulb 32 34 32

18. Effects of root and leaf removal on bulbing zTo test: yAre roots an important source of S for bulbs? yAre all CSOs synthesised in leaves and transported to bulbs? zplants grown hydroponically zat start of bulbing, remove most of either roots or leaves zcompare data from this and end-point

19. Normal development: bulb: x10 fold mass increase leaf: x 2.5 fold mass increase root: unchanged Leaves removed: bulb: 0.5 mass leaf: mass almost fully recovers roots: 0.5 mass Roots removed: bulb: mass almost unaffected leaf: x 3.5 fold mass increase roots: no recovery Severe virus infection during growth Measurements on S being done

20. 3. Identify genes with altered expression and/or involved in alliicin synthesis zAlliinase zOther genes from earlier part of biosynthetic pathway ycysteine synthase yserine acetyl transferase

21. Alliinase – sequence obtained Clustering of alliinase fragments from leaf (l) and bulb(b) 97% identity among all clones

22. Relative alliinase expression during development

23. Other genes in biosynthetic pathway zIdentify genes coding for enzymes involved in alliin biosynthesis -Novel enzymes -Known enzymes with novel functions zEvidence from literature and tissue culture experiments for synthesis of cysteine derivatives by cysteine synthase y several CSase genes in all plants y including S-allyl cysteine

24. Isolation of cysteine synthases from garlic zTwo strategies: yScreening a garlic cDNA library for sequences with homology to known CSase yIdentify a protein with S-allyl CSase activity and screen garlic cDNA library for it yConfirm function of CSase genes through expression of the protein

25. Purification of an allyl cysteine synthase from garlic leaves …….FLGVMPSHYSIE………. YLGADLALTDTN………… SANPGAHYATTGP…………. Sequence of peptides from this protein

26. Obtained CSase and SATase from garlic zFive full-length cDNAs isolated and sequenced: yGSAT1 – cytosolic SATase yGCS1 – potential plastidic CSase (contains frameshift - pseudogene ?) yGCS2 – chloroplastic CSase yGCS3 – cytosolic CSase yGCS4 – S-allyl-CSase (based on protein isolated)

27. 1 2 3 4 5 gcs4 gcs3 gcs2 gsat1 18s 1.7 degree C stored clove 2.RT stored clove 3.Sprouting clove 4.Leaf 5.Root The potential S-allyl CSase gcs4 and the SATase gsat1 are expressed in most tissues examined. The cytosolic CSase gcs3 is root specific. Expression for the putative plastidic CSase gcs2 is uniformly low. Northern blot analysis

28. Results Background activity from E. coli proteins subtracted All three genes gcs2 gcs3 gcs4 are functional to transcribe and translate CSase GCS4 shows the highest activity in cysteine biosynthesis GCS4 functions as S-allyl- CSase GCS2 and GCS3 can act weakly as S-allyl-CSase Peak area Substrate: Na 2 S GCS2 GCS3 GCS4 0 10 0 10 0 10 min Substrate: allyl mercaptan GCS2 GCS3 GCS4 Expression of gcs2 gcs3 gcs4 in vitro

29. Transformation of Arabidopsis with garlic genes zTransformed with gcs3, gcs4, gsat1 zPlants also carry GUS reporter gene zExpression should not be constitutive zExpression of both garlic and GUS genes are induced by ethanol zSeed produced from plants carrying each transgene has been analysed (ie T 1 plants) yPCR to detect transgenes in genome yRT-PCR and staining (for GUS) to detect expression of transgenes ySpectrophotometric and hplc analysis for cysteine and allyl cysteine

30. A. thaliana containing gcs3 or gcs4 Histochemical staining for GUS Uninduced After induction with ethanol Background line Some plants show activity of the inducible GUS transgene

31. A. thaliana containing gcs3 or gcs4 GCS4-2-M GCS4-2-J GCS4-2-I GCS4-2-G GCS4 – F GCS4-2-E GCS4-2-D GCS4-2-C GCS4-2-B GCS4-2-A AGS1-3 Control 1.6 kbp 1.0 kbp GCS4-2-B GCS4-2-A AGS1-3 GCS4-2-I GCS4-2-G GCS4-2-F GCS4-2-E GCS4-2-D GCS4-2-B GCS4-2-A AGS1-3 Control RT-PCR for gcs4 transgene expression uninduced induced Some plants show expression of the inducible gcs4 transgene

32. Arabidopsis with garlic genes zA. thaliana containing gcs3 or gcs4 zPlants did not show a phenotype none silenced express

33. TIP and Annual reports zTIP yCompleted by P2 and P3 zFourth Annual report yCompleted by P2 and P3 zFinal report yBeing written by P2 yCompleted by P3

34. Deliverables 4DP. 8: Analytical methods for labeling and analysis (P2, P3) 4DP. 9: A cDNA library from garlic (P2) 4DP. 16: Pathway intermediates identified (P3) 4DP. 17: First sulphur budget for garlic (P2) 4DP. 18: Clones for alliinase (P2) DP. 23: Publication on alliin biosynthesis and sulphur partitioning (P2, P3) ySynthesis of alliin in garlic and onion tissue cultures – submitted to Phytochemistry 4DP. 24: Genes for key CSO synthesis enzymes (P2,P3) DP. 29 Papers on the characterisation of key enzymes in alliin biosynthesis and alliinase expression and the regulation of sulphur biochemistry in garlic (P2, P3, P5) yFunctional analysis of a novel garlic cysteine synthase in Arabidopsis thaliana – being written

35. Deliverables: DP. 33 Paper on S pathway genes on the production of flavour precursors in garlic y Biosynthesis of the flavour precursors of onion and garlic – submitted to Journal of Experimental Botany DP. 35 Publication on the regulation of alliinase expression (P2) DP. 36 Paper on the regulation of sulphur biochemistry in garlic yEffect of storage on the flavour precursors in garlic – being written