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Angela Tregova and Jill Hughes

Molecular Analysis of Flavour biosynthesis in garlic. Angela Tregova and Jill Hughes. Hamish Collin, Rick Cosstick, Meriel Jones, Brian Tomsett. Acknowledgement: Mark Wilkinson, protein purification facilities. Biosynthetic Pathway. SO 4 2-. serine. SO 3 2-. S 2-. cysteine.

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Angela Tregova and Jill Hughes

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  1. Molecular Analysis of Flavour biosynthesis in garlic Angela Tregova and Jill Hughes Hamish Collin, Rick Cosstick, Meriel Jones, Brian Tomsett Acknowledgement:Mark Wilkinson, protein purification facilities

  2. Biosynthetic Pathway SO42- serine SO32- S2- cysteine Allyl group (source ?) serine valine & methacrylate glutathione S-allylglutathione gly S-(2-carboxypropyl)-glutathione S-methylglutathione S-allyl-γ-glu-cys gly gly S-2-CP-γ-glu-cys S-methyl-γ-glu-cys glu HCOOH trans- peptidase glu trans- peptidase S-trans-1-propenyl-γ-glu-cys S-allylcysteine trans- peptidase glu oxidase S-methylcysteine S-trans-1-propenylcysteine oxidase S-allyl-cysteine sulphoxide (alliin) oxidase S-trans-1-propenylcysteine sulphoxide (isoalliin) methiin

  3. What we have done…… • Investigation of intermediates in the pathway • Identification of key compounds • Purification of a key enzyme • Allylcysteine synthase • The search for genes involved in flavour biosynthesis: • 2 chloroplastic cysteine synthases • 1 cytosolic cysteine synthase • 1 S-allyl cysteine synthase • + 1 cytosolic serine acetyl transferase

  4. = O X Key observation Callus converts allyl thiol to allyl cysteine & alliin CH2CHCH2-SH (+ O-acetyl-serine ?) CH2CHCH2-S-CH2CHNH2COOH CH2CHCH2-S-CH2CHNH2COOH But not allyl alcohol CH2CHCH2-OH But this is not species-specific

  5. Allyl Cysteine Synthase? Cysteine synthase Sulphide + O-Acetyl Serine Cysteine Allyl thiol + O-Acetyl Serine Allyl Cysteine Allyl Cysteine synthase Is there a specific cysteine synthase homologue? Cysteine synthases do a range of reactions in other organisms

  6. Protein purification: Ion Exchange chromatography Garlic leaves were fractionated with ammonium sulphate then separated by ion-exchange chromatography. Many fractions show cysteine synthase activity Only a few fractions show allyl cysteine synthase activity

  7. Protein purification:Hydrophobic Interaction Chromatography Allyl cysteine synthase and cysteine synthase activity co-elute Cysteine production was assayed colorimetrically and allyl cysteine by HPLC

  8. 34000 Fractions 26 27 28 29 30 Protein purification SDS-PAGE shows a distinct band in the allyl cysteine synthase active fractions at approx. 34 kd Molecular weight consistent with plant cysteine synthase monomers found previously

  9. What is the Enzyme? Extract 34000 band and digest with trypsin - the resultant peptides separated by preparative HPLC Three selected peptides were sequenced:- …….FLGVMPSHYSIE………. YLGADLALTDTN………… ……………………SANPGAHYATTGP…………. A simple BLAST search of these peptides in the protein database shows most similarity to a cysteine synthase from Oryza sativa (Rice)

  10. Probe for S-allyl-CSase • cDNA fragments PCR amplified with degenerate primer A – I from the cDNA library • Peptide sequences: • FLGVMPSHYSI • YLGADLALTDT • ANPGAHYA Peptide 1 2 3 A B C D E F G H I …. to find the gene and related genes

  11. AllylCSase aligns with rice sequences Partial protein sequences relative to Arabidopsis (C) sequence RCS2 IGLVLVAVQ-KGYRFIAVMPAKYSLDKQMLLRFLGAELILTDPA-IGFNG—MMDKVEEL RCS4 IGVAYNALL-KGYRFVAVMPAEYSLDKQMLLTYLGAEVILTDPT-LGFQGQ-LDKVEQI GCS4 IALAYI-GLKKGYKFLGVMPSHYSIERRMLLKYLGADLALTD-TNLGFKG-VLDKVAEL I KGY F VMP YS MLL LGA LTD GF G DKV

  12. Important enzymes: 1 SAT/CS complex 2 Free CSase 3S-allyl-CSase + ? 4Oxidase Proposed Serine Pathway Cysteine Sulfide Acetyl CoA 2 L-Serine OAS 1 Allyl-source 3 S-allyl-L-Cysteine 4 Alliin

  13. cDNA library screening • gsat1 - cytosolic SATase • gcs1 - putative plastidic CSase (pseudogene) • gcs2 - putative plastidic CSase • gcs3 - cytosolic CSase • gcs4 - putative S-allyl-CSase

  14. What next ? • Where are the genes expressed in garlic? • Northerns • Does the gene encode allylcysteine synthase? • How do we prove it? • What does it do in planta? • Transformation

  15. 1 2 3 4 5 gcs4 gcs3 gcs2 gsat1 18s Northern blot analysis • S-allyl CSase and the SATase are expressed in most tissues examined. • The cytosolic CSase is root specific. • Expression for the putative plastidic CSase is uniformly low. • 7 degree C stored clove • RT stored clove • Sprouting clove • Leaf • Root

  16. Is this allylcysteine synthase? Ideal Choice ? A quick assessment could mean that we can plan the alternative If we use ethanol-regulated expression, then we can test the effect on the cellular phenotype of the expression of the allylcysteine synthase vs. its absence ! Proof requires expression of the gene and phenotypic testing • Garlic? This would be best but…..time ? • E. coli? Does it function alone? In vitro testing only? • Heterologous plant system? Time ? Arabidopsis ? • Plant tissue culture? Quick and could form complexes allowing tests in planta

  17. AlcR E + Ethanol AlcR E Why ethanol-regulated expression? cDNA pCAMV35S alcR t palcA transgene t alc is a simple two component system

  18. Does it work? Real time Luciferase Imagingin Arabidopsis

  19. LUC 1-12 wt AGS

  20. LUC 1-12 wt AGS 1 hour before induction

  21. LUC 1-12 wt AGS Time of induction

  22. LUC 1-12 wt AGS 30 minutes after induction

  23. LUC 1-12 wt AGS 1 hour after induction

  24. LUC 1-12 wt AGS 1.5 hour after induction

  25. LUC 1-12 wt AGS 2 hours after induction

  26. LUC 1-12 wt AGS 2.5 hours after induction

  27. LUC 1-12 wt AGS 3 hours after induction

  28. LUC 1-12 wt AGS 3.5 hours after induction

  29. LUC 1-12 wt AGS 4 hours after induction

  30. LUC 1-12 wt AGS 4.5 hours after induction

  31. LUC 1-12 wt AGS 5 hours after induction

  32. LUC 1-12 wt AGS 6 hours after induction

  33. LUC 1-12 wt AGS 7 hours after induction

  34. LUC 1-12 wt AGS 7.5 hours after induction

  35. LUC 1-12 wt AGS 8 hours after induction

  36. LUC 1-12 wt AGS 11 hours after induction

  37. LUC 1-12 wt AGS 13 hours after induction

  38. Real time ArabidopsisLuciferase Imaging

  39. LUC 1-12 wt AGS Time of induction

  40. LUC 1-12 wt AGS 8 hours after induction

  41. Functional analysis of plant cell cycle genes

  42. A B C D E F G H I J K L 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 A B C D E F G H I J K L 1.6 kb 1 kb Induced A B C D E F G H I J K L Non-induced A B C D E F G H I J K L 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 1 kb 500 bp A B C D E F G H I J K L 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 1 kb 500 bp At progeny of AmcycA20 x alcRalcAGUS AmcycA20 PCR GUS PCR alcR PCR

  43. A B C D E A B C D E 1 2 3 4 5 6 7 8 9 10 11 12 13 14 A B C D E A B C D E 1 2 3 4 5 6 7 8 9 10 11 12 13 14 1 kb 1 kb RT-PCR of AmcycA20 & controls plus RT Induced plants Total RNA extracted from plants of A = cyclin A20 B = HA-tagged cyclin A20 C = sibling D = wild type E = AGS-1-3 Induced RNA minus RT A B C D E 1 2 3 4 5 6 7 8 Uninduced plants 1 kb Total DNA extracted from Induced plants of A = cyclin A20 B = HA-tagged cyclin A20 C = sibling (cyc+;GUS-) D = wild type E = AGS-1-3 13 = A.majus genomic DNA There is no DNA contamination cycA20 message is specific to induced plants containing both T-DNAs

  44. Western Blots of HA tagged cycA20 WT I N WT I N 48 kDa HA-CycA20 Probe = antibody to HA tag

  45. Rosette leaves 1 2 Phenotypic analysis Leaf cell density, primary leaf area, rosette leaf number, trichomes and flowering time. Plants were grown for six weeks. Vertically grown A.thaliana plants, growing in a tissue culture square plate. Root growth experiments (after 15 days) and fresh weight measurements (after four weeks).

  46. Fresh Weight

  47. Root Length – AmcycA20 expression WT 25 26 2728 29 3031 32 AGS G A A

  48. Root length

  49. Cyclin A20 sibling Leaf number and area Leaf number remains constant after AMcycA20 expression Plus ethanol Minus ethanol Leaf area is bigger after induction in AmcycA20 expressing lines Minus ethanol Plus ethanol

  50. Leaf Area

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