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Jill Hughes and Angela Tregova

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

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Jill Hughes and Angela Tregova

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

  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 … • 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 T P T alcR palcA Garlic gene Inducer EtOH Express ALCR ALCR Transcription Factor Garlic protein

  5. Transformation of tobacco cells for protein expression t35S palcA RB Garlic gene pnos nptII pAg7 LB TransformedUntransformed 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 – Km, Vmax

  7. 1 2 3 4 5 2. Expression of GCS4 in garlic 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. • 7 degree C stored clove • RT stored clove • Sprouting clove • Leaf • Root

  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 storageconditions

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

  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. SO42- SO32- SO22- cysteine 4. Glutathione-S-transferases valine & methacrylate serine 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-methylcysteine S-allylcysteine S-trans-1-propenylcysteine oxidase oxidase oxidase oxidase S-trans-1-propenylcysteine sulphoxide (isoalliin) methiin 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. 25 kDa 5.81 5.96 Glutathione-S-transferase • Garlic leaf proteins - glutathione affinity matrix • Single step gives substantial purification Fractions on SDS gel 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) • 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)

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