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Molecular analysis of flavour biosynthesis in garlic Angela Tregova Jill Hughes, Jonothan Milne, Hamish Collin, Meriel Jones, Rick Cosstick, Brian Tomsett EU Framework 5 Garlic and Health Objectives • Identify genes coding for enzymes involved in alliin biosynthesis - Novel enzymes - Known enzymes with novel functions • Analysis of flavour precursors Cysteine synthase (CSase) L-Serine Acetyl Co-A SAT/CS ? OAS Free CS Sulfide Pyrazol Allyl-mercaptan Cyanide -PA S-allyl-L-Cysteine Cysteine Free CAS/CS 3-Cyano-L-Ala Clone cysteine synthase • Two strategies • Screening a garlic cDNA library for sequences with homology to known CSase • Identify a protein with S-allyl CSase activity and screen garlic cDNA library for it Results • Five full-length cDNAs isolated and sequenced: • GSAT1 – cytosolic SATase • GCS1 – potential plastidic CSase (contains frameshift - pseudogene ?) • GCS2 – chloroplastic CSase • GCS3 – cytosolic CSase • GCS4 – S-allyl-CSase Northern blot analysis 1 2 3 4 5 gcs4 gcs3 gcs2 • The potential S-allyl CSase and the SATase are expressed in most tissues examined. • The cytosolic CSase is root specific. gsat1 18s 1. 2. 3. 4. 5. 7 degree C stored clove RT stored clove Sprouting clove Leaf Root • Expression for the putative plastidic CSase is uniformly low. Phylogenetic Tree A. thaliana [8] A. thaliana [1] A. thaliana [9] A. thaliana [7] Watermelon A. thaliana [5] A. thaliana [2] Spinach A. thaliana [3, 10] GCS3 A. thaliana [6] GCS2 GCS4 RCS2 A. thaliana [4] RCS4 GCS4 related to two isoforms identified from rice that form a new CSase family. Expression of CSase and SAT Transgenic tobacco BY2 cells and A. thaliana RB P Garlic gene t35S alcR palcA pnos nptII T palcA Garlic gene Inducer Transcription Factor T Express EtOH ALCR pAg7 ALCR Garlic protein LB Transformation of tobacco cells for protein expression RB t35S Garlic gene Transformed sub-cultured palcA Untransformed pnos nptII pAg7 Transformed LB Unexpected results • SDS – PAGE • No detectable increase in protein products • Cysteine synthase assays • No detectable increase in cysteine • S-allylcysteine synthase assays (HPLC) • No detectable S-allylcysteine synthase • Northern blot analysis • No detectable transgene expression 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! In vitro protein biosynthesis • Rapid Translation System RTS 100 E. coli HY kit (Roche) • Cell-free in vitro transcription/translation protein expression system based on E. coli lysate • Suggested by Rolf at February 2003 meeting - thanks Rolf! pIVEX expression vectors Garlic genes: 5’ T7P RBS Garlic gene His-tag T7T 3’ His-tag T7T 3’ gsat1 TAA 5’ T7P RBS Garlic gene gcs2; gcs3 gcs4 • PCR cloning strategy to remove 5’ and 3’UTRs. • Hi-fidelity PCR enzyme mix introduced 1 mutation into gsat1 and 2 mutations into gcs4. • All mutations corrected. In vitro cysteine biosynthesis In vitro CSase activity Results µmol cys min-1 ml-1 35 30 • Background activity from E. coli proteins subtracted 25 20 15 • All three genes gcs2 gcs3 gcs4 are functional to transcribe and translate CSase 10 5 0 GCS2 GCS3 GCS4 Substrate: Na2S • GCS4 shows the highest activity in cysteine biosynthesis Is GSC4 an S-allyl-CS? 35000 Results 30000 Peak area 25000 • Background activity from E. coli proteins subtracted • GCS4 functions as S-allylCSase • GCS2 and GCS3 can act weakly as S-allyl-CSase 20000 15000 10000 5000 0 1GCS210 1 GCS3 10 GSC2 GCS3 Substrate: allyl mercaptan 1GCS4 10 GCS4 min While this was going on ….. • Transformation of A. thaliana as in vivo strategy to assess activity of GCS3, GSC4 and GSAT1 • Used constructs already created for transformation of tobacco BY2 cells • Used A. thaliana line containing: • AlcR transcription factor on 35S promoter • GUS reporter gene on AlcA promoter • Checks for AlcR expression • GUS and garlic transgenes only expressed in presence of inducer (ethanol) Transformation of A. thaliana • Uses Agrobacterium tumefaciens • Flower heads dipped into detergent and bacterial mixture weekly for 3 weeks • Allow seeds to set (~4 weeks) • Collect seeds • Used 432 plants per construct • Several g seeds per construct Screen seeds for transformants • Kanamycin selection on phytogel plates • ~200,000 seeds screened per construct • Seedlings that survived transferred to soil • When plants large enough, leaf DNA preps screened for garlic transgene by PCR Results • Transgenic A. thaliana 16 plants contain gcs4 7 plants contain gcs3 6 plants contain gsat1 • No obvious phenotype in non-induced plants, as expected • These transgenic plants (To) have been selffertilized to obtain seeds (T1) The final step Analyse T1 plants for: • Presence of transgenes – PCR • Expression of alcR – GUS staining • Expression of transgenes - RT-PCR • Activity of cysteine synthase - spectrophotometry • Activity of S-allyl cysteine synthase - HPLC A. thaliana HPLC profile alliin Young leaves isoalliin 100 S-allylcysteine 80 mV 60 40 20 0 0 10 20 time (min) 30 Deliverables: by December 2003 • DP. 23 Papers on alliin biosynthesis and sulphur partitioning • Synthesis of alliin in garlic and onion tissue cultures – draft on project website • DP. 29 Papers on the characterisation of key enzymes in alliin biosynthesis and alliinase expression and the regulation of sulphur biochemistry in garlic • Functional analysis of a novel garlic cysteine synthase in Arabidopsis thaliana Deliverables: by December 2003 • DP. 33 Paper on S pathway genes on the production of flavour precursors in garlic • Biosynthesis of the flavour precursors of onion and garlic, invited review for special issue on Sulphur Metabolism in Plants, Journal of Experimental Botany – in preparation • DP. 36 Paper on the regulation of sulphur biochemistry in garlic • Induction of the pattern of flavour precursors in garlic – in preparation Other publications • Poster presented at Seventh International Congress of Plant Molecular Biology, Barcelona, June 2003. ‘Molecular analysis of cysteine synthase and allylcysteine synthase from garlic, and their contributions to garlic flavour precursor biosynthesis’