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

2. To refresh your memories … The search for genes involved in flavour biosynthesis: Partial sequences obtained for potential: 2 chloroplastic cysteine synthases 1 cytosolic cysteine synthase 1 S-allyl cysteine synthase 1 cytosolic serine acetyl transferase Investigation of intermediates in the biosynthetic pathway: Gradient hplc method brought into use Improved resolution Separation of gamma-glutamyl compounds possible

3. Since Spring 2002 … 1.Expression of garlic sequences for cysteine synthase and serine acetyl transferase to obtain protein – to test activity 2.Analysis of expression of CSases and SATase in garlic tissues 3. Inter-clove variation in flavour precursors 4. Preliminary analysis for glutathione-S-transferases – another route for flavour biosynthesis?

4. 1. Expression of CSase and SATase Tobacco cell suspension culture Plant cell system Regulated, high level of expression using the alc system alcR P T ALCR Transcription Factor EtOH Inducer ALCR Garlic gene T Express Garlic protein palcA

5. Transformation of tobacco cells for protein expression RBLB t35SpalcA pAg7nptIIpnosGarlic gene Transformed Untransformed Transformed sub-cultured

6. Transformation of tobacco cells for protein expression The tobacco cells will be multiplied in liquid culture Induce protein expression Assay for CSase and SATase activity Determine kinetic parameters – K m, V max

7. 2. Expression of GCS4 in garlic 1.7 degree C stored clove 2.RT stored clove 3.Sprouting clove 4.Leaf 5.Root 1 2 3 4 5 The potential S-allyl- cysteine synthase is expressed in most tissues examined. Quantitation of this result, and Northern analysis of the three further cysteine synthase and one serine- acetyl transferase will be carried out.

8. 3. Inter-clove variation in flavour compounds Further investigation of the biosynthetic pathway Inter-clove variation Effect of low temperature storage Statistical analysis of data Result: Hplc profiles differed significantly clove location bulb storage conditions

9. Inter-clove variation Representative HPLC traces of Outer Cloves 0.00 10.00 20.00 30.00 40.00 0 20 40 60 80 100 0.00 10.00 20.00 30.00 40.00 0 20 40 60 80 100 Bulbs stored at room temperature Bulbs stored at 4 0 C for 6 weeks Alliin Isoalliin GLUAC isoGLUAC Isoalliin Alliin isoGLUAC GLUAC

10. Inter-clove variation In summary: which changes are significant? Alliin level is influenced by location. Isoalliin, GLUAC and isoGLUAC are influenced by both location and storage temperature Location within bulb Cold storage Location and storage

11. 4. Glutathione-S-transferases SO 4 2- SO 3 2- SO 2 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 Allyl group (unknown sources) valine & methacrylate serine oxidase S-allylcysteine S-allyl-cysteine sulphoxide (alliin)

12. Glutathione-S-transferases This family of enzymes is widespread in plants The Arabidopsis genome has approx. 50 GSTs Extensive literature on GST biochemistry and analysis GSTs conjugate glutathione to many compounds

13. Glutathione-S-transferase Garlic leaf proteins - glutathione affinity matrix Single step gives substantial purification 5.815.96 Fractions on SDS gel 25 kDa Fractions on 2D gel Mol wt pI

14. Glutathione-S-transferase 1-chloro-2,4-dinitrobenzene - GST activity present Potential substrates and products were incubated with total protein or the GST fraction Products were analysed by hplc No clear potential GST substrate * synthesised in Liverpool * *

15. Deliverables Genes for CSO synthesis enzymes (36m) Genes for CSO synthesis enzymes (36m) Publication on regulation of S biochemistry in garlic (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 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) Paper on S pathway genes on production of flavour precursors in garlic (48m)