Transcript WP4 - Jones
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 … 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 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? 1. Expression of CSase and SATase Tobacco cell suspension culture • Plant cell system • Regulated, high level of expression using the alc system P alcR T palcA Garlic gene Inducer Express EtOH ALCR Transcription Factor T ALCR Garlic protein Transformation of tobacco cells for protein expression RB t35S Garlic gene Transformed sub-cultured palcA Untransformed pnos nptII pAg7 Transformed LB 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 2. Expression of GCS4 in garlic 1 2 3 4 1. 7 degree C stored clove 2. RT stored clove 3. Sprouting clove 4. Leaf 5. Root 5 The potential S-allylcysteine synthase is expressed in most tissues examined. Quantitation of this result, and Northern analysis of the three further cysteine synthase and one serineacetyl transferase will be carried out. 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 Inter-clove variation Representative HPLC traces of Outer Cloves 100 Bulbs stored at room temperature 80 GLUAC Alliin isoGLUAC 60 40 Isoalliin 20 0 0.00 10.00 20.00 30.00 40.00 100 Bulbs stored at 40C for 6 weeks Alliin GLUAC 80 60 Isoalliin isoGLUAC 40 20 0 0.00 10.00 20.00 30.00 40.00 Inter-clove variation In summary: which changes are significant? P values location storage within bulb temperature peak1 peak2 alliin isoalliin peak5 peak6 peak7 GLUAC isoGLUAC peak10 peak11 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 4. Glutathione-S-transferases SO42serine SO32- Allyl group (unknown sources) S-allylglutathione SO22valine & methacrylate cysteine glutathione (γ-glu-cys-gly) S-(2-carboxypropyl)-glutathione gly S-allyl-γ-glu-cys S-methylglutathione gly S-2-CP-γ-glu-cys gly S-methyl-γ-glu-cys HCOOH glu transpeptidase S-allylcysteine S-allylcysteine oxidase oxidase S-allyl-cysteine sulphoxide (alliin) S-trans-1-propenyl-γ-glu-cys transpeptidase glu S-trans-1-propenylcysteine oxidase S-trans-1-propenylcysteine sulphoxide (isoalliin) glu transpeptidase S-methylcysteine oxidase methiin 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 Glutathione-S-transferase •Garlic leaf proteins - glutathione affinity matrix •Single step gives substantial purification Fractions on SDS gel Fractions on 2D gel 25 kDa 5.81 5.96 pI Mol wt 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 substrate glutathione propyl alcohol + allyl alcohol + methacrylic acid + allyl thiol + metabolite soup + allyl glutathione * carboxypropyl glutathione * propyl glutathione No clear potential GST substrate * synthesised in Liverpool 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)