molecular genetic analysis of alliin biosynthesis in garlic, allium sativum l. wp 4: angela tregova...
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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 for Alliin Biosynthesis
Proposed pathways are highly hypothetical
No molecular evidenceSO4 SO3 SO2
cysteine
allyl source(unknown)
glutathioneserine
S-allyl-cysteineglutathioneconjugate
-glutamylconjugates
alliinalliin
Cysteine synthase (CS)
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
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 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
1. Cytosolic / Mitochondrial (C/M)
2. Chloroplastic / Mitochondrial (CH/M)
3. Cytosolic (C)
A probe for SATase
B L R
SAT 1
Cytosolic SATase cDNA
fragments amplified by RT-
PCR with degenerate
primer SAT 1 from bulb (B),
leaf (L) and root (R) tissues.
A probe for S-allyl-CSase
A B C D E F G H I
Peptide 1 2 3cDNA fragments PCR amplified with degenerate primer A – I from the cDNA library
Peptide sequences:
1. FLGVMPSHYSI
2. YLGADLALTDT
3. ANPGAHYA
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--LDKVEQIKNDGCS4 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