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Sulfur biochemistry of garlic: the
biosynthesis of flavour precursors
Hamish A Collin, Jill M Hughes, Angela Tregova,
Jonathan GC Milne, Gloria van der Werff, Mark Wilkinson, Rick
Cosstick, Meriel G Jones and A Brian Tomsett
The School of Biological Sciences, The University of
Liverpool
Laurence Trueman, Tim Crowther, Linda Brown and Brian
Thomas
Warwick HRI, The University of Warwick, Wellesbourne, UK
Project objectives: Garlic flavour
Improved understanding of S allocation
and translocation during garlic
development
Identify genes and intermediates involved
in alliicin synthesis
Improved understanding of S
allocation and translocation during
garlic development
Measurements during
growth
•Leaf number, bulb weight
•N, S, C, protein, CSO
•SO42-uptake using stable
isotope labelling
For controlled growth in the UK climate hydroponic and pot culture in a glasshouse
Garlic growth and S partition
Hydroponic-grown garlic - comparison of bulb formation
Hydroponic v
pot-grown Printanor - Leaf weight
160
Printanor clove
Messidrome Clove
140
Hydoponic-grown Printanor
Pot-grown Printanor
20
1
15
10
2
3
Fresh weight of clove
Mean mass of leaf (g, n=3)
25
4
120
100
1
80
60
2
3
4
40
5
20
0
0
0
50
100
150
200
250
0
50
100
Days after planting
0.3
200
250
2000000
Root
CSO content
Total Sulphur
Content (g)
150
Days after planting
Leaf
0.2
Clove
0.1
0.0
1500000
1000000
Root
Leaf
Clove
500000
0
29
56
77 109 141 169
Days after planting
203
56
109
141
169
Days after planting
203
Four stages in bulb development
 Early growth phase: Day 0 – 40/70
uses stored nutrients
 Late growth phase: Day 40/70 - 150
roots, leaves grow rapidly
C, protein accumulate in leaves; S stored in roots
 Bulb initiation: Day 150 – 200
S, N, C, protein and CSOs decline in roots and leaves
but accumulate in bulbs
rise in CSO synthesis
 Bulb maturity: Day 200
turgor loss as leaves and roots senesce
S, N, C, protein falls in leaves, roots, and rises in bulbs
neck closure and bulb matures
Sulfur uptake and
distribution in more detail
grow hydroponically
use isotope labelled sulfur
stable heavy isotope sulfur-34
measure total S,
34/32S
ratio (delta value)
Sulfur labelling design
A
Distribution and remobilization
of sulphur taken up early
*
*
*
*
*
*
*
*
*
*
*
Distribution and remobilization
of sulphur taken up late
B
*
*
*
*
*
*
*
*
*
*
*
34S
32S
Growth pattern in earlier experiment
Fresh weight (g)
200
Clove
150
Leaf
Root
100
50
0
0
25
50
75
100
125
150
Days after planting
175
200
225
32
34S
S
Hydroponic garlic in isotopically labelled sulfur
34S then
SulphurSulpur
accumulation
in system
A plantsA(plants
accumulation
in system
32S)
250
200
Leaf
Root
150
Total
100
50
Date
12/07/02
05/07/02
28/06/02
21/06/02
14/06/02
07/06/02
31/05/02
24/05/02
17/05/02
10/05/02
03/05/02
26/04/02
19/04/02
12/04/02
0
05/04/02
Total mass in mg
Clove
A: 34S then 32S
B: 32S then 34S
200
250
Bulb
Leaf
150
Root
100
50
100
Bulb
Leaf
50
Root
32
32
26/07/02
12/07/02
28/06/02
14/06/02
31/05/02
17/05/02
03/05/02
19/04/02
0
05/04/02
26/07/02
12/07/02
28/06/02
14/06/02
31/05/02
17/05/02
03/05/02
19/04/02
05/04/02
0
34
150
d value
d value
200
34
S pools in root, leaf, bulb increase while root takes up S
After S uptake by roots cease, it is exported to bulb
Roots therefore appear an important S source for the bulb
To identify genes and
intermediates in flavour precursor
biosynthesis
Alliinase
Other genes from earlier part of
biosynthetic pathway
Alliinase
Sequence obtained
Relative alliinase expression
Relative alliinase expression during development
1
0.8
0.6
0.4
Bulb
0.2
0
08/02/01
Leaf
10/03/01
09/04/01
09/05/01
08/06/01
Biosynthetic pathway for garlic
flavour precursors
SO42serine
SO32-
S2-
S-allyl group
(unknown source)
S-allylglutathione
cysteine
valine &
methacrylate
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
glu
transpeptidase
S-methylcysteine
oxidase
S-trans-1-propenylcysteine sulphoxide S-methylcysteine sulphoxide
(isoalliin)
(methiin)
Lancaster and Shaw 1989; Granroth 1970
Is cysteine synthase involved in
garlic flavour precursor
biosynthesis?
O-acetyl serine + sulphide
cysteine
cytoplasmic, plastid and mitochondrial forms
non-protein amino acids synthesised
Non-protein aminoacid
synthesis by CSases
serine
















SAT/CSase
Complex
Pea (Pisum sativum)
Leucaena leucocephala Leucaena leucocephala
watermelon
Lathyrus latifolius
Mimosa pudica watermelon
O-acetyl serine
Free CSase
H2 S
L-cysteine
Free CAS
CH2=CH-CH2-SH
S-allyl-L-cysteine
methyl-SH
S-methylL-cysteine
3,4-dihydroxypyridine
pyrazole
mimosine -pyrazol-1-yl alanine
HCN
3-cyano-L-ala
Ikegami and Murakoshi 1994; Warrilow and Hawkesford 2002
CSase cysteine synthase; CAS -cyanoalanine synthase
Biosynthetic capacity of
garlic callus
allyl cysteine alliin
isoalliin propyl cysteine
allyl thiol
10; 10,1
10,1;10,1
allyl cysteine
10;10,1
propenyl cysteine
1;10,1
propyl thiol
1;10
propyl cysteine
Conclusion:
propiin
10;
10,1;10,1
Incubation for 5 days with 10mM or 1mM substrate
Incubation for 12/15 days with 10mM or 1mM substrate
These experiments suggest that in vivo
the general reactions shown may occur:alk(en)yl thiol
alk(en)yl cysteine
alk(en)yl CSO
Isolation of cysteine
synthases from garlic
Strategy:
Screening a garlic cDNA library for sequences
with homology to known CSase
Identify a protein with S-allyl CSase activity
and screen garlic cDNA library for it
Confirm function of CSase genes through
expression of the protein
Screening using homology
to known CSases
Three full-length sequences from garlic
cDNA library
GCS1, GCS2
GCS1 – frameshift; truncated 202 aa, 22 kDa
GCS2 – 332 aa, 35 kDa
51 aa predicted transit peptide - plastid
GCS3
323 aa, 34 kDa
No transit peptide - cytosol
Purification of an allyl cysteine
synthase from garlic leaves
Phenyl sepharose fractionation
0.7
0.6
OD550
0.5
0.4
34 kDa
0.3
0.2
cysteine 0.1
syntase activity
39
37
35
33
31
29
27
25
23
21
19
17
15
13
11
9
7
5
3
1
0
allyl cysteine
synthase
activity
Fraction
Sequence of peptides from this protein
…….FLGVMPSHYSIE………. YLGADLALTDTN…………
SANPGAHYATTGP………….
Obtained CSase from garlic
Four full-length cDNAs isolated and
sequenced:
GCS1
GCS2
GCS3
GCS4
data)
–
–
–
–
potential
potential
potential
potential
plastidic CSase (frameshift)
plastidic CSase
cytosolic CSase
S-allyl-CSase (based on protein
Phylogenetic tree of garlic
cysteine synthases
A. thaliana [8]
99
A. thaliana [1]
A. thaliana [9]
100
A. thaliana [7]
100
100
Watermelon
A. thaliana [5] 72
A. thaliana [2] 100 28
Spinach
100
100
45
46
GCS3
97
A. thaliana [3, 10]
72
A. thaliana [6]
GCS2
100
GCS4
A. thaliana [4]
78
PAUP version 4.0b 10
RCS2
50 changes
RCS4
Northern blot analysis
1
2
3 4
gcs2
5
• Low expression of putative
plastidic CSase gcs2
• Root expression of
cytosolic CSase gcs3
gcs3
gcs4
• Most tissues expressed
potential S-allyl CSase gcs4
18s
1.
2.
3.
4.
5.
7o stored clove
20o stored clove
Sprouting clove
Leaf
Root
Expression of gcs2 gcs3 gcs4 in vitro
In vitro CSase activity
Results
µmol cys min-1 ml-1
35
30
25
Substrate: Na2S
• Background activity from E.
coli proteins subtracted
20
15
10
• All three genes gcs2 gcs3 gcs4
are functional to transcribe
and translate CSase
5
0
GCS2 GCS3 GCS4
35000
• GCS4 shows the highest
activity in cysteine biosynthesis
30000
Substrate: allyl mercaptan
Peak area
25000
• GCS4 functions as S-allylCSase
20000
15000
10000
5000
0
0GCS2
10
GCS2
0 GCS3
10
GCS3
0GCS4
10
GCS4
min
Summary
S allocation and re-mobilisation during garlic
development
Alliinase
Sequence obtained
Expression during development
Could a cysteine synthase be involved in flavour
precursor biosynthesis in garlic?
Sequences of three cysteine synthases obtained, all
expressed in garlic
Functional in vitro
cysteine synthesis – GCS2, GCS3, GCS4
S-allyl cysteine synthesis – GCS4
Role in planta?
Acknowledgements
The Garlic and Health project partners
EU FP5 Quality of Life program: Garlic and Health
project QLK1-CT-1999-00498