1. Sulfur biochemistry of garlic: the biosynthesis of flavour precursors Hamish A Collin, Jill M Hughes, Angela Tregova, Jonathan GC Milne, Gloria van der Werff, Mark Wilkinson, Rick Cosstick, Meriel G Jones and A Brian Tomsett The School of Biological Sciences, The University of Liverpool Laurence Trueman, Tim Crowther, Linda Brown and Brian Thomas Warwick HRI, The University of Warwick, Wellesbourne, UK

2. Project objectives: Garlic flavour zImproved understanding of S allocation and translocation during garlic development zIdentify genes and intermediates involved in alliicin synthesis

3. For controlled growth in the UK climate - hydroponic and pot culture in a glasshouse Measurements during growth Leaf number, bulb weight N, S, C, protein, CSO SO 4 2- uptake using stable isotope labelling Improved understanding of S allocation and translocation during garlic development

4. Garlic growth and S partition 1234 1234

5. Four stages in bulb development zEarly growth phase: Day 0 – 40/70 yuses stored nutrients zLate growth phase: Day 40/70 - 150 yroots, leaves grow rapidly yC, protein accumulate in leaves; S stored in roots zBulb initiation: Day 150 – 200 yS, N, C, protein and CSOs decline in roots and leaves but accumulate in bulbs yrise in CSO synthesis zBulb maturity: Day 200 yturgor loss as leaves and roots senesce yS, N, C, protein falls in leaves, roots, and rises in bulbs yneck closure and bulb matures

6. Sulfur uptake and distribution in more detail zgrow hydroponically zuse isotope labelled sulfur ystable heavy isotope sulfur-34 zmeasure total S, 34/32 S ratio (delta value)

7. Distribution and remobilization of sulphur taken up early Distribution and remobilization of sulphur taken up late * * * * * * * * * * * 34 S 32 S A B Growth pattern in earlier experiment Sulfur labelling design

8. 34 S 32 S Hydroponic garlic in isotopically labelled sulfur Sulphur accumulation in system A plants ( 34 S then 32 S)

9. A: 34 S then 32 SB: 32 S then 34 S S pools in root, leaf, bulb increase while root takes up S After S uptake by roots cease, it is exported to bulb Roots therefore appear an important S source for the bulb 3234 32

10. To identify genes and intermediates in flavour precursor biosynthesis zAlliinase zOther genes from earlier part of biosynthetic pathway

11. Sequence obtained Relative alliinase expression during development Alliinase

12. Biosynthetic pathway for garlic flavour precursors SO 4 2- SO 3 2- S 2- cysteine glutathione (γ-glu-cys-gly) S-methyl-γ-glu-cys gly S-methylcysteine S-methylcysteine sulphoxide (methiin) glu trans- peptidase oxidase S-2-CP-γ-glu-cys gly S-trans-1-propenyl-γ-glu-cys S-trans-1-propenylcysteine oxidase trans- peptidase glu HCOOH S-trans-1-propenylcysteine sulphoxide (isoalliin) S-methylglutathione S-(2-carboxypropyl)-glutathione S-allylglutathione S-allyl-γ-glu-cys gly S-allylcysteine glu trans- peptidase oxidase S-allyl group (unknown source) valine & methacrylate serine oxidase S-allylcysteine S-allyl-cysteine sulphoxide (alliin) Lancaster and Shaw 1989; Granroth 1970

13. Is cysteine synthase involved in garlic flavour precursor biosynthesis? zO-acetyl serine + sulphide cysteine zcytoplasmic, plastid and mitochondrial forms znon-protein amino acids synthesised

14. Non-protein aminoacid synthesis by CSases z serine z z SAT/CSase z Complex z z O-acetyl serine z H 2 S CH 2 =CH-CH 2 -SH methyl-SH 3,4-dihydroxy- pyrazole z Free CSase pyridine z z L-cysteine S-allyl-L-cysteine S-methyl- mimosine  -pyrazol-1-yl alanine z L-cysteine z z Free CAS HCN z 3-cyano-L-ala watermelonMimosa pudica CSase cysteine synthase; CAS  -cyanoalanine synthase Pea (Pisum sativum) Leucaena leucocephala watermelon Leucaena leucocephala Lathyrus latifolius Ikegami and Murakoshi 1994; Warrilow and Hawkesford 2002

15. Biosynthetic capacity of garlic callus Conclusion: These experiments suggest that in vivo the general reactions shown may occur:- alk(en)yl thiol alk(en)yl cysteine alk(en)yl CSO

16. Isolation of cysteine synthases from garlic zStrategy: yScreening a garlic cDNA library for sequences with homology to known CSase yIdentify a protein with S-allyl CSase activity and screen garlic cDNA library for it yConfirm function of CSase genes through expression of the protein

17. Screening using homology to known CSases zThree full-length sequences from garlic cDNA library yGCS1, GCS2 xGCS1 – frameshift; truncated 202 aa, 22 kDa xGCS2 – 332 aa, 35 kDa x51 aa predicted transit peptide - plastid yGCS3 x323 aa, 34 kDa xNo transit peptide - cytosol

18. Purification of an allyl cysteine synthase from garlic leaves …….FLGVMPSHYSIE………. YLGADLALTDTN………… SANPGAHYATTGP…………. Sequence of peptides from this protein 34 kDa

19. Obtained CSase from garlic zFour full-length cDNAs isolated and sequenced: zGCS1 – potential plastidic CSase (frameshift) zGCS2 – potential plastidic CSase zGCS3 – potential cytosolic CSase zGCS4 – potential S-allyl-CSase (based on protein data)

20. Phylogenetic tree of garlic cysteine synthases 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] 50 changes PAUP version 4.0b 10 100 78 97 100 28 72 46 45 99 100

21. 1 2 3 4 5 gcs4 gcs3 gcs2 18s 1.7 o stored clove 2.20 o stored clove 3.Sprouting clove 4.Leaf 5.Root Low expression of putative plastidic CSase gcs2 Root expression of cytosolic CSase gcs3 Most tissues expressed potential S-allyl CSase gcs4 Northern blot analysis

22. 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 GCS4 functions as S-allyl- CSase Substrate: Na 2 S GCS2 GCS3 GCS4 0 10 0 10 0 10 min Substrate: allyl mercaptan GCS2 GCS3 GCS4 Expression of gcs2 gcs3 gcs4 in vitro Peak area

23. Summary zS allocation and re-mobilisation during garlic development zAlliinase ySequence obtained yExpression during development zCould a cysteine synthase be involved in flavour precursor biosynthesis in garlic? zSequences of three cysteine synthases obtained, all expressed in garlic yFunctional in vitro xcysteine synthesis – GCS2, GCS3, GCS4 xS-allyl cysteine synthesis – GCS4 yRole in planta?

24. Acknowledgements The Garlic and Health project partners EU FP5 Quality of Life program: Garlic and Health project QLK1-CT-1999-00498