Molecular analysis of flavour biosynthesis in garlic

1. 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

2. Objectives • Identify genes coding for enzymes involved in alliin biosynthesis - Novel enzymes - Known enzymes with novel functions • Analysis of flavour precursors

3. Cysteine synthase (CSase) L-Serine Acetyl Co-A SAT/CS ? OAS Sulfide Pyrazol Allyl-mercaptan Free CS Cysteine Cyanide -PA S-allyl-L-Cysteine Free CAS/CS 3-Cyano-L-Ala

4. 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

5. 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

6. Northern blot analysis 1 2 3 4 5 • The potential 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. gcs4 gcs3 gcs2 gsat1 18s • 7 degree C stored clove • RT stored clove • Sprouting clove • Leaf • Root

7. A. thaliana [8] A. thaliana [9] A. thaliana[1] A. thaliana [7] Watermelon A. thaliana [5] Spinach A. thaliana [2] A. thaliana [3, 10] GCS3 A. thaliana [6] GCS2 GCS4 A. thaliana [4] RCS2 RCS4 Phylogenetic Tree GCS4related to two isoforms identified from rice that form a new CSase family.

8. LB RB t35S Garlic gene palcA pnos nptII pAg7 Expression of CSase and SAT • Transgenic tobacco BY2 cells and A. thaliana T P T alcR palcA Garlic gene Inducer EtOH Express ALCR ALCR Transcription Factor Garlic protein

9. LB RB t35S Garlic gene palcA pnos nptII pAg7 Transformation of tobacco cells for protein expression TransformedUntransformed Transformed sub-cultured

10. 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

11. 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!

12. 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!

13. Garlic genes: gsat1 gcs2; gcs3 gcs4 RBS Garlic gene 3’ T7P T7T 5’ pIVEX expression vectors 5’ RBS Garlic gene His-tag 3’ T7P T7T TAA His-tag • 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.

14. In vitro cysteine biosynthesis • Results • Background activity from E. coli proteins subtracted • All three genes gcs2 gcs3gcs4 are functional to transcribe and translate CSase • GCS4 shows the highest activity in cysteine biosynthesis GCS2 GCS3 GCS4 Substrate: Na2S

15. Is GSC4 an S-allyl-CS? • Results • Background activity from E. coli proteins subtracted • GCS4 functions as S-allyl-CSase • GCS2 and GCS3 can act weakly as S-allyl-CSase Peak area 1 10 1 10 1 10 min GSC2 GCS3 GCS4 Substrate: allyl mercaptan

16. 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)

17. 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

18. 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

19. 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 self-fertilized to obtain seeds (T1)

20. 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

21. A. thaliana HPLC profile alliin Young leaves isoalliin 100 S-allylcysteine 80 60 mV 40 20 0 20 30 10 0 time (min)

22. 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

23. 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

24. 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’