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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 SO42serine Allyl group serine valine & methacrylate (source ?) S-(2-carboxypropyl)-glutathione S-allyl-γ-glu-cys glu S-allylcysteine transpeptidase oxidase S-allyl-cysteine sulphoxide (alliin) S2- cysteine glutathione S-allylglutathione gly SO32- S-methylglutathione gly S-2-CP-γ-glu-cys gly S-methyl-γ-glu-cys HCOOH S-trans-1-propenyl-γ-glu-cys glu transpeptidase S-trans-1-propenylcysteine oxidase S-trans-1-propenylcysteine sulphoxide (isoalliin) glu transpeptidase S-methylcysteine oxidase methiin 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 Key observation Callus converts allyl thiol to allyl cysteine & alliin CH2CHCH2-SH (+ O-acetyl-serine ?) CH2CHCH2-S-CH2CHNH2COOH = CH2CHCH2-S-CH2CHNH2COOH O But not allyl alcohol CH2CHCH2-OH X But this is not species-specific Allyl Cysteine Synthase? Cysteine synthase Sulphide + O-Acetyl Serine Cysteine Allyl Cysteine synthase Allyl thiol + O-Acetyl Serine Allyl Cysteine Is there a specific cysteine synthase homologue? Cysteine synthases do a range of reactions in other organisms Protein purification: Ion Exchange chromatography Garlic leaves were fractionated with ammonium sulphate then separated by ion-exchange chromatography. Cysteine synthase activity. Q-Sepharose. 7.11.01 0.3 0.25 0.2 0.15 0.1 0.05 0 Fr 1 Fr 5 Fr 9 Fr 13 Fr 17 Fr 21 Fr 25 Fr 29 Fr 33 Only a few fractions show allyl cysteine synthase activity OD Many fractions show cysteine synthase activity cysteine synthase activity Fraction Protein purification: Hydrophobic Interaction Chromatography Allyl cysteine synthase and cysteine synthase activity co-elute Phenyl sepharose fractionation 0.7 0.6 OD550 0.5 0.3 0.2 cysteine 0.1 syntase activity Fraction 39 37 35 33 31 29 27 25 23 21 19 17 15 13 11 9 7 5 allyl cysteine synthase activity 3 0 1 Cysteine production was assayed colorimetrically and allyl cysteine by HPLC 0.4 Protein purification SDS-PAGE shows a distinct band in the allyl cysteine synthase active fractions at approx. 34 kd 34000 Molecular weight consistent with plant cysteine synthase monomers found previously Fractions 26 27 28 29 30 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) Probe for S-allyl-CSase Peptide 1 2 3 A B C D E F G H I cDNA fragments PCR amplified with degenerate primer A – I from the cDNA library Peptide sequences: 1. FLGVMPSHYSI 2. YLGADLALTDT 3. ANPGAHYA …. to find the gene and related genes AllylCSase aligns with rice sequences Partial protein sequences relative to Arabidopsis (C) sequence RCS2 RCS4 GCS4 IGLVLVAVQ-KGYRFIAVMPAKYSLDKQMLLRFLGAELILTDPA-IGFNG—MMDKVEEL IGVAYNALL-KGYRFVAVMPAEYSLDKQMLLTYLGAEVILTDPT-LGFQGQ-LDKVEQI IALAYI-GLKKGYKFLGVMPSHYSIERRMLLKYLGADLALTD-TNLGFKG-VLDKVAEL I KGY F VMP YS MLL LGA LTD GF G DKV Proposed Serine Pathway Cysteine Acetyl CoA 2 L-Serine Sulfide 4 OAS 1 Allyl-source 3 S-allyl-L-Cysteine 4 Alliin Important enzymes: 1 SAT/CS complex 2 Free CSase 3 S-allyl-CSase + ? Oxidase cDNA library screening gsat1 - cytosolic SATase gcs1 - putative plastidic CSase (pseudogene) gcs2 - putative plastidic CSase gcs3 - cytosolic CSase gcs4 - putative S-allyl-CSase 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 Northern blot analysis 1 2 3 4 5 gcs4 S-allyl CSase and the SATase gcs3 gcs2 gsat1 18s 1. 2. 3. 4. 5. 7 degree C stored clove RT stored clove Sprouting clove Leaf Root are expressed in most tissues examined. The cytosolic CSase is root specific. Expression for the putative plastidic CSase is uniformly low. Is this allylcysteine synthase? Proof requires expression of the gene and phenotypic testing • Garlic? Ideal Choice ? This would be best but…..time ? A quick assessment could mean that we can plan the alternative • If E.wecoli? Does it function alone? vitro use ethanol-regulated expression, thenInwe can testing test the only? effect on the cellular phenotype of the expression of the allylcysteine synthase vs. its absence ! • Heterologous plant system? Time ? Arabidopsis ? • Plant tissue culture? Quick and could form complexes allowing tests in planta Why ethanol-regulated expression? pCAMV35S cDNA alcR AlcR AlcR E palcA t + Ethanol E alc is a simple two component system transgene t Does it work? Real time Luciferase Imaging in Arabidopsis LUC 1-12 wt AGS LUC 1-12 wt AGS 1 hour before induction LUC 1-12 wt AGS Time of induction LUC 1-12 wt AGS 30 minutes after induction LUC 1-12 wt AGS 1 hour after induction LUC 1-12 wt AGS 1.5 hour after induction LUC 1-12 wt AGS 2 hours after induction LUC 1-12 wt AGS 2.5 hours after induction LUC 1-12 wt AGS 3 hours after induction LUC 1-12 wt AGS 3.5 hours after induction LUC 1-12 wt AGS 4 hours after induction LUC 1-12 wt AGS 4.5 hours after induction LUC 1-12 wt AGS 5 hours after induction LUC 1-12 wt AGS 6 hours after induction LUC 1-12 wt AGS 7 hours after induction LUC 1-12 wt AGS 7.5 hours after induction LUC 1-12 wt AGS 8 hours after induction LUC 1-12 wt AGS 11 hours after induction LUC 1-12 wt AGS 13 hours after induction Real time Arabidopsis Luciferase Imaging LUC 1-12 wt AGS Time of induction LUC 1-12 wt AGS 8 hours after induction Functional analysis of plant cell cycle genes At progeny of AmcycA20 x alcRalcAGUS AmcycA20 PCR 1 2 A 3 B C 4 5 D 6 E 7 F G H I 8 9 10 11 J K L 12 13 14 15 16 1.6 kb 1 kb A G B H C I D J E K F L Induced GUS PCR 1 A B C D E F G H I J K L 2 A 3 B C 4 5 D 6 E F G H I 7 8 9 10 11 J K L 12 13 14 15 16 1 kb 500 bp Non-induced alcR PCR 1 2 A 3 B C D 4 5 6 E 7 F G H I J K L 8 9 10 11 12 13 14 15 16 1 kb 500 bp RT-PCR of AmcycA20 & controls plus RT 1 A 2 B 3 C 4 Induced plants D 5 E 6 7 A 8 B C 9 10 D E 11 12 13 14 1 kb 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 1 A B C D E 2 3 4 5 6 7 8 Uninduced plants 1 A 2 B 3 C 4 D 5 E A B 6 7 8 C D E 9 10 11 12 13 14 1 kb 1 kb cycA20 message is specific to induced plants containing both T-DNAs 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 Western Blots of HA tagged cycA20 WT I N WT I N 48 kDa HA-CycA20 Probe = antibody to HA tag Phenotypic analysis Rosette leaves 1 2 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). Fresh Weight 100 80 60 40 20 0 WT AGS SIBLING NON-INDUCED CYCA20-HA INDUCED CYCA20 Root Length – AmcycA20 expression WT 25 26 27 28 29 30 31 32 AGS G A A Root length 9 WT AGS SIBLING CYCA20-HA CYCA20 WT AGS SIBLING CYCA20-HA CYCA20 Root length (cm) 8 7 6 5 4 3 2 1 0 0 5 10 Days growth 15 Leaf number and area Leaf number remains constant after AMcycA20 expression Plus ethanol Minus ethanol sibling Leaf area is bigger after induction in AmcycA20 expressing lines Minus ethanol Plus ethanol Cyclin A20 Leaf Area 2.50 2.00 1.50 1.00 0.50 0.00 WT AGS Sibling Non-induced CycA20-HA Induced CycA20 Cell Size and density Uninduced Induced HA-tagged cyclin A20 There appear to be less cells per unit area - cells are larger Trichomes on rosette leaves Days Flowering time of cycA20 and controls 25 20 15 10 5 0 WT AGS Sibling Non-induced CycA20 CycA20 Induced Mean flowering time (days) of twenty seedlings of each of HA-tagged cyclin A20, cyclin A20, wild type (Columbia), AGS-1-3 and sibling plants in comparison between non-induced and induced conditions. The plants were induced after 5 days of germination, when the plants reached the 2 leafstage, and were checked regularly until the appearance of the first visible flower bud. Tobacco transformation for protein expression kanR RB t35S Transformed Garlic gene palcA Untransformed pnos nptII pAg7 Transformed LB Tobacco transformation for protein expression • The tobacco cells can be multiplied in liquid culture • Induce protein expression • Determine whether –SH content of cells has increased • Assay for allylCSase activity • Use HPLC to look for allylcysteine and ….? Does tobacco possess an oxidase to make alliin? Diagnostic PCRs for transgenic BY2 lines 1 2 3 4 5 6 7 Lane 1 = untransformed BY2 Lane 2 = gcs3 plasmid control Lane 3 = gcs3 transformant Lane 4 = gcs4 plasmid control Lane 5 = gcs4 transformant Lane 6 = gsat1 plasmid control Lane 7 = gsat1 transformant PCR primers: 1.palcA forward 2. t35S reverse However……. Cysteine synthase assays No detectable increase in cysteine. Time course assays and assay optimisations failed. S-allyl-cysteine synthase assay (HPLC) No detectable levels of S-allyl-cysteine. Northern blot analysis gcs3 RNA extracted from transgenic tobacco cells after 1, 3 and 6 days induction. gcs4 Northern blots show no transgene expression, except gcs3 that was detected after several days induction. gsat1 Garlic RNA Tobacco RNA Why are the transgenes not expressed? Are there mistakes in the binary constructs? Re-sequencing verified correct assemblies. Is the alcR cDNA present in the tobacco cell-line? alcR confirmed by PCR. Is alcR expressed? Is alcR expressed? RT-PCR results: 1 2 3 4 Lane 1 = alcR control (genomic DNA) Lane 2 = gcs3 BY-2 transformant Lane 3 = gcs4 BY-2 transformant Lane 4 = gsat1 BY-2 transformant No alcR expression detected in any of the transformed cell lines! Repeat BY-2 transformation New BY-2 cell-lines from the John Innes Centre Transformations have been repeated and we are currently waiting for new transformants to grow But is alcR expressed in the new cell-line? alcR expression in the new cell-line 1 2 3 RT-PCR results: Lane 1 = No RT control Lane 2 = alcR control (genomic DNA) Lane 3 = alcR expression in the new cells Positive RT-PCR controls using degenerate primers that anneal to SAT. Again, no detectable alcR expression in the new cell line! RT-PCRs using a highly sensitive detection 1 2 3 4 5 6 7 8 9 RT-PCR results: Lane 1 = untransformed BY-2 Lane 2 = gcs3 BY-2 transformant Lane 3 = gcs4 BY-2 transformant Lane 4 = gsat1 BY-2 transformant Lane 5-8 = No RT controls Lane 9 = alcR control (genomic DNA) RT-PCRs using a highly sensitive detection 1 2 3 4 5 6 7 8 9 RT-PCR results: Lane 1 = untransformed BY-2 Lane 2 = gcs3 BY-2 transformant Lane 3 = gcs4 BY-2 transformant Lane 4 = gsat1 BY-2 transformant Lane 5-8 = No RT controls Lane 9 = alcR control (genomic DNA) RT-PCRs using a highly sensitive detection 1 2 3 4 5 6 7 8 9 RT-PCR results: Lane 1 = untransformed BY-2 Lane 2 = gcs3 BY-2 transformant Lane 3 = gcs4 BY-2 transformant Lane 4 = gsat1 BY-2 transformant Lane 5-8 = No RT controls Lane 9 = alcR control (genomic DNA) Future ? The longer route looks more attractive ! • E. coli – his-tagged protein purification assay in vitro • Arabidopsis - test for expression assay in vivo phenotype Expression of a wheat CSase in tobacco A. Transgenic tobacco shows 2-fold higher Cys content. B. SO2 fumigation increased thiol levels. Deliverables • Genes for CSO synthesis enzymes (36m) • Publication on regulation of S biochemistry in garlic (36m) • Paper on characterising enzymes in alliin biosynthesis, and alliinase expression, and regulation of sulphur biochemistry in garlic (48m) • Paper on S pathway genes on production of flavour precursors in garlic (48m) Thanks to …….. Liverpool Angela Tregova Jill Hughes Piyarat Parinyapong Hairul Roslan Chris Wood Mike White Mark Caddick Brian Tomsett Jealott’s Hill Jackie Paine Mary Knight Susan Wright Justin Sweetman Alberto Martinez Wolfgang Schuch Andy Greenland Ian Jepson ICI Agrochemicals ICI Seeds Zeneca Seeds Zeneca Agrochemicals Syngenta JIC John Doonan and his lab Funding BBSRC EU FP5 Garlic & Health