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WP4: Garlic sulphur biochemistry (P2,P3)

WP4: Garlic sulphur biochemistry (P2,P3). P2: HRI Wellesbourne Brian Thomas, Lol Trueman, Linda Brown, Brian Smith, Gareth Griffiths P3: The University of Liverpool, UK Hamish Collin, Rick Cosstick, Brian Tomsett, Meriel Jones Angela Tregova, Jill Hughes, Jon Milne

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WP4: Garlic sulphur biochemistry (P2,P3)

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

  2. WP4: Objectives • 1. Identify intermediates in alliin biosynthetic pathway (P3) • 2. Identify developmental control points on CSO synthesis and translocation (P2) • 3. Identify genes with altered expression and/or involved in alliicin synthesis (P2,P3)

  3. 1. Identify intermediates in alliin biosynthetic pathway • Review knowledge of alliin biosynthesis • Bring improved HPLC methodology into use in our laboratory • Standards – purchase, synthesis, gifts, mass spectrometry • Gradient elution • Develop experimental protocols • Tissue culture • Garlic cloves

  4. SO42- SO32- S2- cysteine Biosynthetic pathway for garlic flavour precursors valine & methacrylate serine S-allyl group (unknown sources) glutathione (γ-glu-cys-gly) S-(2-carboxypropyl)-glutathione S-methylglutathione S-allylglutathione gly gly gly S-2-CP-γ-glu-cys S-methyl-γ-glu-cys S-allyl-γ-glu-cys HCOOH trans- peptidase glu glu S-trans-1-propenyl-γ-glu-cys trans- peptidase trans- peptidase glu S-allylcysteine S-allylcysteine S-methylcysteine S-trans-1-propenylcysteine oxidase oxidase oxidase oxidase S-trans-1-propenylcysteine sulphoxide (isoalliin) methiin 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. Fractions on SDS gel 25 kDa Glutathione-S-transferases • Garlic leaf proteins - glutathione affinity matrix • Single step gives substantial purification No clear potential GST substrate

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

  8. Identify developmental control points on CSO synthesis and translocation • Growth studies of garlic (Messidrome, Printanor) • hydroponic versus pots • SO42-uptake using isotope labelling • effects 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 1 2 3 4 1 2 3 4

  11. Four stages in bulb development • Early growth phase: Day 0 – 40/70 • Uses stored nutrients • Late growth phase: Day 40/70 - 150 • roots, leaves grow rapidly • C, protein accumulate in leaves • S stored in roots

  12. Four stages in bulb development • Bulb initiation: Day 150 – 200 • temperature and day-length dependent • S, N, C, protein and CSOs decline in roots and leaves but accumulate in bulbs • rise in CSO synthesis • roots die

  13. Four stages in bulb development • Bulb maturity: Day 200 • Turgor loss as leaves and roots senesce • S, N, C, protein fall in leaves, roots, and rise in bulbs • Neck closure and bulb matures.

  14. Sulfur uptake and distribution in more detail • grow hydroponically • use isotope labelled sulfur • stable heavy isotope sulfur-34 • Measure total S, 34/32S ratio (delta value)

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

  16. 34S 32S Year 3 hydroponic garlic

  17. A: 34S then 32S B: 32S then 34S 34 34 32 32 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

  18. Effects of root and leaf removal on bulbing • To test: • Are roots an important source of S for bulbs? • Are all CSOs synthesised in leaves and transported to bulbs? • plants grown hydroponically • at start of bulbing, remove most of either roots or leaves • compare 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 • Alliinase • Other genes from earlier part of biosynthetic pathway • cysteine synthase • serine 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 • Identify genes coding for enzymes involved in alliin biosynthesis - Novel enzymes - Known enzymes with novel functions • Evidence from literature and tissue culture experiments for synthesis of cysteine derivatives by cysteine synthase • several CSase genes in all plants • including S-allyl cysteine

  24. Isolation of cysteine synthases from garlic • Two strategies: • Screening a garlic cDNA library for sequences with homology to known CSase • Identify a protein with S-allyl CSase activity and screen garlic cDNA library for it • Confirm function of CSase genes through expression of the protein

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

  26. Obtained CSase and SATase from garlic • Five full-length cDNAs isolated and sequenced: • GSAT1 – cytosolic SATase • GCS1 – potential plastidic CSase (contains frameshift - pseudogene ?) • GCS2 – chloroplastic CSase • GCS3 – cytosolic CSase • GCS4 – S-allyl-CSase (based on protein isolated)

  27. Northern blot analysis 1 2 3 4 5 • 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. gcs4 gcs3 gcs2 gsat1 18s • 7 degree C stored clove • RT stored clove • Sprouting clove • Leaf • Root

  28. Substrate: Na2S GCS2 GCS3 GCS4 Substrate: allyl mercaptan 0 10 0 10 0 10 min GCS2 GCS3 GCS4 Expression of gcs2 gcs3gcs4 in vitro • Results • Background activity from E. coli proteins subtracted • All three genes gcs2 gcs3gcs4 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

  29. Transformation of Arabidopsis with garlic genes • Transformed with gcs3, gcs4, gsat1 • Plants also carry GUS reporter gene • Expression should not be constitutive • Expression of both garlic and GUS genes are induced by ethanol • Seed produced from plants carrying each transgene has been analysed (ie T1 plants) • PCR to detect transgenes in genome • RT-PCR and staining (for GUS) to detect expression of transgenes • Spectrophotometric and hplc analysis for cysteine and allyl cysteine

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

  31. 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 A. thaliana containing gcs3 or gcs4 RT-PCR for gcs4 transgene expression 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 uninduced induced Some plants show expression of the inducible gcs4 transgene

  32. Arabidopsis with garlic genes • A. thaliana containing gcs3 or gcs4 • Plants did not show a phenotype none silenced express

  33. TIP and Annual reports • TIP • Completed by P2 and P3 • Fourth Annual report • Completed by P2 and P3 • Final report • Being written by P2 • Completed by P3

  34. Deliverables • DP. 8: Analytical methods for labeling and analysis (P2, P3) • DP. 9: A cDNA library from garlic (P2) • DP. 16: Pathway intermediates identified (P3) • DP. 17: First sulphur budget for garlic (P2) • DP. 18: Clones for alliinase (P2) DP. 23: Publication on alliin biosynthesis and sulphur partitioning (P2, P3) • Synthesis of alliin in garlic and onion tissue cultures – submitted to Phytochemistry • DP. 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) • Functional 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 • 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 • Effect of storage on the flavour precursors in garlic – being written

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