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