Molecular genetic analysis of alliin biosynthesis in garlic, Allium sativum L.

1. Molecular genetic analysis of alliin biosynthesis in garlic, Allium sativum L. WP 4: Angela Tregova Hamish Collin, Meriel Jones, Brian Tomsett, Jill Hughes EU Garlic and Health QLK1-CT-1999-00498

2. Objectives • Identify genes coding for enzymes involved in alliin biosynthesis • Novel enzymes • Known enzymes with novel functions

3. cysteine SO4 SO3 SO2 serine allyl source (unknown) glutathione glutathione conjugate S-allyl-cysteine -glutamyl conjugates alliin alliin Alliin biosynthesis ? Proposed Pathways for Alliin Biosynthesis • Proposed pathways are highly hypothetical • No molecular evidence

4. Cysteine synthase (CS) 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

5. Which enzyme(s) ? • Multiple CSases in plants: • Compartment-specific (cytosol, chloroplasts, mitochondria) • Tissue-specific (e.g. root hairs) • Developmentally regulated (e.g. early seed development) • In A. thaliana 10 genes identified that code for potential CSases

6. Two strategies • Isolate multiple CSase isoforms from a cDNA library and test for S-allyl-CSase activity in vivo • Identify S-allyl-CSase protein (HPLC analysis, PAGE, partial protein sequencing, screen cDNA library, confirm activity in vivo)

7. CS1 CS2 CS5 R R L L C C R R L L C C R R L L C C C/M CH/M C Probes for different CSases • CSase cDNA fragments amplified by RT-PCR with degenerate primer pair CS1, CS2, and CS5 : • from root (R), • leaf (L), • and callus (C) tissues • Cytosolic / Mitochondrial (C/M) • Chloroplastic / Mitochondrial(CH/M) • Cytosolic (C)

8. A probe for SATase SAT 1 Cytosolic SATase cDNA fragments amplified by RT-PCR with degenerate primer SAT 1 from bulb (B), leaf (L) and root (R) tissues. B L R

9. A probe for S-allyl-CSase • cDNA fragments PCR amplified with degenerate primer A – I from the cDNA library • Peptide sequences: • FLGVMPSHYSI • YLGADLALTDT • ANPGAHYA Peptide 1 2 3 A B C D E F G H I

10. cDNA Library Screening • Library construction from garlic root, leaf and clove tissues • Multiple library enrichments: • 4 different CSase isoforms • 1 SATase • Library screening

11. Results • Five full-length cDNAs isolated and sequenced: • GSAT1 – cytosolic SATase • GCS1 – potential chloroplastic CSase (pseudogene ?) • GCS2 – potential chloroplastic CSase • GCS3 – cytosolic CSase • GCS4 – S-allyl-CSase

12. CSase protein alignments Partial protein sequences relative to Arabidopsis (C) sequence GCS1 (B) IALAFVAAS---KGYK-----------------------LILTMPASMSMERRVLFKAFGAELVLTDAAKG--- GCS2 (B) IALAFVAAS---KGYK-----------------------LILTMPASMSMERRVLFKAFGAELVLTDAAKG--- GCS3 (A) IGLAFIAAA---KGYK-----------------------LIITMPASFSLERRIIIKAFGGQLVLTDPLLG--- Arab. (C) IGLAFIAASRGY--------------------------RLILTMPASMSMERRVLLKAFGAELVLTDPAKG--- Spinach (B) IGLAFIAAARGYKIT------------------------L—-TMPASMSMERRVILKAFGAELVLTDPAKG—-- Watermelon.(A) IGLAFIAAA---KGYR-----------------------LIICMPASMSLERRTILRAFGAELVLTDPARG--- RCS2 IGLVLVA--VQ-KGYRFIAVMPAKYSLDKQMLLRFLGAELILTDPA---------IG-FNG—-MMDKVEEL--- RCS4 IGVAYNA--LL-KGYRFVAVMPAEYSLDKQMLLTYLGAEVILTDPT---------LG-FQGQ--LDKVEQIKND GCS4 IALAYI---GLKKGYKFLGVMPSHYSIERRMLLKYLGADLALTD-TN--------LG-FKG--VLDKVAEL---

13. Future work • Express all identified cDNAs in a suitable expression system (E. coli, yeast or plant cell-line) to confirm S-allyl-CSase activity in vivo • Northern Blot analysis