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

Biosynthetic Pathway SO 4 2- SO 3 2- S 2- cysteine glutathione 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-methylglutathioneS-(2-carboxypropyl)-glutathione valine & methacrylate S-allylglutathione S-allyl-γ-glu-cys gly glu trans- peptidase Allyl group (source ?) serine oxidase S-allylcysteine S-allyl-cysteine sulphoxide (alliin) serine

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 CH 2 CHCH 2 -SH (+ O-acetyl-serine ?) CH 2 CHCH 2 -S-CH 2 CHNH 2 COOH But not allyl alcohol CH 2 CHCH 2 -OH = O X But this is not species-specific

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

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

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

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 Fractions

What is the Enzyme? Extract 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 A B C D E F G H I Peptide 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 RCS2IGLVLVAVQ-KGYRFIAVMPAKYSLDKQMLLRFLGAELILTDPA-IGFNG—MMDKVEEL RCS4IGVAYNALL-KGYRFVAVMPAEYSLDKQMLLTYLGAEVILTDPT-LGFQGQ-LDKVEQI GCS4IALAYI-GLKKGYKFLGVMPSHYSIERRMLLKYLGADLALTD-TNLGFKG-VLDKVAEL I KGY F VMP YS MLL LGA LTD GF G DKV

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

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 gcs4 gcs3 gcs2 gsat1 18s 1.7 degree C stored clove 2.RT stored clove 3.Sprouting clove 4.Leaf 5.Root  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.

Is this allylcysteine synthase? 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 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 !

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

Does it work? Real time Luciferase Imaging in Arabidopsis

wt AGS LUC 1-12

1 hour before induction wt AGS LUC 1-12

Time of induction wt AGS LUC 1-12

30 minutes after induction wt AGS LUC 1-12

1 hour after induction wt AGS LUC 1-12

1.5 hour after induction wt AGS LUC 1-12

2 hours after induction wt AGS LUC 1-12

2.5 hours after induction wt AGS LUC 1-12

3 hours after induction wt AGS LUC 1-12

3.5 hours after induction wt AGS LUC 1-12

4 hours after induction wt AGS LUC 1-12

4.5 hours after induction wt AGS LUC 1-12

5 hours after induction wt AGS LUC 1-12

6 hours after induction wt AGS LUC 1-12

7 hours after induction wt AGS LUC 1-12

7.5 hours after induction wt AGS LUC 1-12

8 hours after induction wt AGS LUC 1-12

11 hours after induction wt AGS LUC 1-12

13 hours after induction wt AGS LUC 1-12

Real time Arabidopsis Luciferase Imaging

Time of induction wt AGS LUC 1-12

8 hours after induction wt AGS LUC 1-12

Functional analysis of plant cell cycle genes

At progeny of AmcycA20 x alcRalcAGUS A B C D E F G H I J K L A B C D E F G H I J K L Induced Non-induced A B C D E F G H I J K L 1.6 kb 1 kb A B C D E F G H I J K L 1 kb 500 bp A B C D E F G H I J K L kb 500 bp AmcycA20 PCR GUS PCR alcR PCR

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

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

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

Root Length – AmcycA20 expression WT AGS G A A

Root length

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

Leaf Area

Cell Size and density HA-tagged cyclin A20 Uninduced Induced There appear to be less cells per unit area - cells are larger

Trichomes on rosette leaves

Flowering time of cycA20 and controls Days 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 leaf- stage, and were checked regularly until the appearance of the first visible flower bud.

Tobacco transformation for protein expression RB t35SpalcA Garlic geneLB pAg7nptIIpnos Transformed Untransformed Transformed kan R

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 PCR primers: 1.palcA forward 2. t35S reverse 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

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 gcs4 gsat1 Garlic RNATobacco RNA  RNA extracted from transgenic tobacco cells after 1, 3 and 6 days induction.  Northern blots show no transgene expression, except gcs3 that was detected after several days induction.

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? Lane 1 = alcR control (genomic DNA) Lane 2 = gcs3 BY-2 transformant Lane 3 = gcs4 BY-2 transformant Lane 4 = gsat1 BY-2 transformant RT-PCR results:  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 Positive RT-PCR controls using degenerate primers that anneal to SAT RT-PCR results: Lane 1 = No RT control Lane 2 = alcR control (genomic DNA) Lane 3 = alcR expression in the new cells  Again, no detectable alcR expression in the new cell line!

RT-PCRs using a highly sensitive detection 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 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 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.SO 2 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