1. 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

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

3. 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

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. 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)

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
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---
RCS IGLVLVA--VQ-KGYRFIAVMPAKYSLDKQMLLRFLGAELILTDPA IG-FNG—-MMDKVEEL---
RCS IGVAYNA--LL-KGYRFVAVMPAEYSLDKQMLLTYLGAEVILTDPT LG-FQGQ--LDKVEQIKND
GCS 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