Molecular Analysis of Flavour
biosynthesis in garlic
Jill Hughes and Angela Tregova
Hamish Collin, Rick Cosstick, Meriel Jones, Brian Tomsett
Acknowledgement: Mark Wilkinson, protein purification facilities
To refresh your memories …
The search for genes involved in flavour biosynthesis:
Partial sequences obtained for potential:
2 chloroplastic cysteine synthases
1 cytosolic cysteine synthase
1 S-allyl cysteine synthase
1 cytosolic serine acetyl transferase
Investigation of intermediates in the biosynthetic
pathway:
Gradient hplc method brought into use
Improved resolution
Separation of gamma-glutamyl compounds possible
Since Spring 2002 …
1. Expression of garlic sequences for cysteine
synthase and serine acetyl transferase to obtain
protein – to test activity
2. Analysis of expression of CSases and SATase in
garlic tissues
3. Inter-clove variation in flavour precursors
4. Preliminary analysis for glutathione-S-transferases
– another route for flavour biosynthesis?
1. Expression of CSase and
SATase
Tobacco cell suspension culture
• Plant cell system
• Regulated, high level of expression using the alc system
P
alcR
T
palcA Garlic gene
Inducer
Express
EtOH
ALCR
Transcription
Factor
T
ALCR
Garlic protein
Transformation of tobacco
cells for protein expression
RB
t35S
Garlic gene
Transformed
sub-cultured
palcA
Untransformed
pnos
nptII
pAg7
Transformed
LB
Transformation of tobacco
cells for protein expression
• The tobacco cells will be multiplied in liquid culture
• Induce protein expression
• Assay for CSase and SATase activity
• Determine kinetic parameters – Km, Vmax
2. Expression of GCS4 in garlic
1
2
3
4
1.
7 degree C stored clove
2.
RT stored clove
3.
Sprouting clove
4.
Leaf
5.
Root
5
The potential S-allylcysteine synthase is
expressed in most tissues
examined.
Quantitation of this result,
and Northern analysis of the
three further cysteine
synthase and one serineacetyl transferase will be
carried out.
3. Inter-clove variation in
flavour compounds
Further investigation of the biosynthetic pathway
•
Inter-clove variation
•
Effect of low temperature storage
•
Statistical analysis of data
Result: Hplc profiles differed significantly
• clove location
• bulb storage conditions
Inter-clove variation
Representative HPLC traces of Outer Cloves
100
Bulbs stored at
room
temperature
80
GLUAC
Alliin
isoGLUAC
60
40
Isoalliin
20
0
0.00
10.00
20.00
30.00
40.00
100
Bulbs stored at
40C for 6 weeks
Alliin
GLUAC
80
60
Isoalliin
isoGLUAC
40
20
0
0.00
10.00
20.00
30.00
40.00
Inter-clove variation
In summary: which changes are significant?
P values location
storage
within bulb temperature
peak1
peak2
alliin
isoalliin
peak5
peak6
peak7
GLUAC
isoGLUAC
peak10
peak11
Alliin level is influenced by
location.
Isoalliin, GLUAC and
isoGLUAC are influenced
by both location and
storage temperature
Location within bulb
Cold storage
Location and storage
4. Glutathione-S-transferases
SO42serine
SO32-
Allyl group
(unknown sources)
S-allylglutathione
SO22valine &
methacrylate
cysteine
glutathione
(γ-glu-cys-gly)
S-(2-carboxypropyl)-glutathione
gly
S-allyl-γ-glu-cys
S-methylglutathione
gly
S-2-CP-γ-glu-cys
gly
S-methyl-γ-glu-cys
HCOOH
glu
transpeptidase
S-allylcysteine
S-allylcysteine
oxidase
oxidase
S-allyl-cysteine sulphoxide
(alliin)
S-trans-1-propenyl-γ-glu-cys
transpeptidase
glu
S-trans-1-propenylcysteine
oxidase
S-trans-1-propenylcysteine sulphoxide
(isoalliin)
glu
transpeptidase
S-methylcysteine
oxidase
methiin
Glutathione-S-transferases
•This family of enzymes is widespread in plants
•The Arabidopsis genome has approx. 50 GSTs
•Extensive literature on GST biochemistry and
analysis
•GSTs conjugate glutathione to many compounds
Glutathione-S-transferase
•Garlic leaf proteins - glutathione affinity matrix
•Single step gives substantial purification
Fractions on SDS gel
Fractions on 2D gel
25 kDa
5.81 5.96
pI
Mol wt
Glutathione-S-transferase
• 1-chloro-2,4-dinitrobenzene - GST activity present
• Potential substrates and products were incubated
with total protein or the GST fraction
• Products were analysed by hplc
substrate
glutathione
propyl alcohol
+
allyl alcohol
+
methacrylic acid
+
allyl thiol
+
metabolite soup
+
allyl glutathione *
carboxypropyl glutathione *
propyl glutathione
No clear potential
GST substrate
* synthesised in Liverpool
Deliverables

Genes for CSO synthesis enzymes (36m)

Publication on regulation of S biochemistry in
garlic (36m)

Paper on characterising enzymes in alliin
biosynthesis, and alliinase expression, and
regulation of sulphur biochemistry in garlic (48m)

Paper on S pathway genes on production of
flavour precursors in garlic (48m)