Hamish Collin, Meriel Jones, Brian Tomsett, Jill Hughes 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

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

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

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

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

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)

Probes for different CSases R R L L C C R R L L C C CS1 CS2 CS5 C/M CH/M C 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)

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

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

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

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

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---

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