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 OAS Cysteine SAT/CS Free CS 3-Cyano-L-Ala Free CAS/CS Acetyl Co-A Sulfide Cyanide Allyl-mercaptanPyrazol  -PA S-allyl-L-Cysteine ?

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 gcs4 gcs3 gcs2 gsat1 18s 1.7 degree C stored clove 2.RT stored clove 3.Sprouting clove 4.Leaf 5.Root 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.

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

8. Expression of CSase and SAT alcR P T ALCR Transcription Factor EtOH Inducer ALCR Garlic gene T Express Garlic protein palcA Transgenic tobacco BY2 cells and A. thaliana LB t35SpalcA pAg7nptIIpnos RB Garlic gene

9. Transformation of tobacco cells for protein expression LB t35SpalcA pAg7nptIIpnos RB Garlic gene Transformed Untransformed 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? 1 2 3 4 RT-PCR results: 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. pIVEX expression vectors T7P 5’ RBSGarlic geneHis-tag T7T 3’ T7P RBSGarlic gene T7T 3’ 5’ Garlic genes: gsat1 gcs2; gcs3 gcs4 His-tag TAA 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 gcs3 gcs4 are functional to transcribe and translate CSase GCS4 shows the highest activity in cysteine biosynthesis Substrate: Na 2 S GCS2GCS3GCS4

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 Substrate: allyl mercaptan Peak area 1 10 1 10 1 10 min GSC2 GCS3 GCS4

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 (T o ) have been self- fertilized to obtain seeds (T 1 )

20. The final step Analyse T 1 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 0 10 20 30 0 20 40 60 80 100 mV time (min) alliin isoalliin S-allylcysteine Young leaves

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. 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 Deliverables: by December 2003

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’