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WP4: Garlic sulphur
biochemistry (P2,P3)
P2: HRI Wellesbourne
Brian Thomas, Lol Trueman, Linda Brown, Brian Smith,
Gareth Griffiths
P3: The University of Liverpool, UK
Hamish Collin, Rick Cosstick, Brian Tomsett, Meriel Jones
Angela Tregova, Jill Hughes, Jon Milne
Mark Wilkinson, Gloria van der Werff
WP4: Objectives
1. Identify intermediates in alliin
biosynthetic pathway (P3)
2. Identify developmental control points
on CSO synthesis and translocation (P2)
3. Identify genes with altered expression
and/or involved in alliicin synthesis
(P2,P3)
1. Identify intermediates in
alliin biosynthetic pathway
Review knowledge of alliin biosynthesis
Bring improved HPLC methodology into use in
our laboratory
Standards – purchase, synthesis, gifts, mass
spectrometry
Gradient elution
Develop experimental protocols
Tissue culture
Garlic cloves
Biosynthetic pathway for garlic
flavour precursors
SO42serine
SO32-
S2-
S-allyl group
(unknown sources)
S-allylglutathione
valine &
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
Biosynthetic capacity of
garlic callus
alliin
allyl cysteine isoalliin
10,1;10,1 10; 10,1
allyl thiol
propyl thiol
allyl cysteine
10;10,1
propenyl cysteine
propyl cysteine
Conclusion:
propyl cysteine
10;
1;10
1;10,1
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 reaction shown may
occur:Alk(en)yl thiol
propiin
Alk(en)yl cysteine
Alk(en)yl CSO
Glutathione-S-transferases
•Garlic leaf proteins - glutathione affinity matrix
•Single step gives substantial purification
Fractions on SDS gel
25 kDa
substrate
propyl alcohol
allyl alcohol
methacrylic acid
allyl thiol
metabolite soup
allyl glutathione
carboxypropyl glutathione
propyl glutathione
No clear potential GST substrate
glutathione
+
+
+
+
+
2. Identify developmental control
points on CSO synthesis and
translocation
Baseline data on garlic development
Resource allocation during development
Developed and tested theories:
Whether roots are an important source of S
for developing bulbs
Whether CSOs are synthesised in leaves and
transported to bulbs
Identify developmental control points
on CSO synthesis and translocation
Growth studies of garlic (Messidrome,
Printanor)
hydroponic versus pots
SO42-uptake using isotope labelling
effects of root and leaf removal
For controlled growth,
greenhouse (and UK climate)
Measurements during
growth
•Leaf number, bulb weight
•N, S, C, protein, CSO
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
Four stages in bulb development
Bulb initiation: Day 150 – 200
temperature and day-length dependent
S, N, C, protein and CSOs decline in
roots and leaves but accumulate in bulbs
rise in CSO synthesis
roots die
Four stages in bulb
development
Bulb maturity: Day 200
Turgor loss as leaves and roots senesce
S, N, C, protein fall in leaves, roots, and rise
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
Distribution and remobilization
of sulphur taken up early
A
*
*
*
*
*
*
*
*
*
*
*
Distribution and remobilization
of sulphur taken up late
B
*
*
*
*
*
*
*
*
*
*
*
34S
32S
Growth pattern in Year 2 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
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
150
03/05/02
200
26/04/02
19/04/02
12/04/02
05/04/02
Total mass in mg
34S
32S
Year 3 hydroponic garlic
Sulpur accumulation in system A plants
250
Clove
Leaf
Root
Total
100
50
0
A: 34S then 32S
B: 32S then 34S
200
250
Bulb
200
150
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
d value
d value
Leaf
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
Effects of root and leaf
removal on bulbing
To test:
Are roots an important source of S for bulbs?
Are all CSOs synthesised in leaves and
transported to bulbs?
plants grown hydroponically
at start of bulbing, remove most of either
roots or leaves
compare data from this and end-point
Normal development:
Fresh weight
Mass in g
400
Early Control
Leafless
Rootless
300
Late Control
200
bulb: x10 fold mass increase
leaf: x 2.5 fold mass increase
root: unchanged
100
Leaves removed:
0
Clove
Leaf
Root
bulb: 0.5 mass
leaf: mass almost fully recovers
Dry Weight
Early Control
Leafless
120
100
80
60
40
20
0
Mass in g
Rootless
Late Control
roots: 0.5 mass
Roots removed:
bulb: mass almost unaffected
leaf: x 3.5 fold mass increase
Clove
Leaf
Root
Severe virus infection during
growth
roots: no recovery
Measurements on S being
done
3. Identify genes with altered
expression and/or involved in alliicin
synthesis
Alliinase
Other genes from earlier part of
biosynthetic pathway
cysteine synthase
serine acetyl transferase
Alliinase – sequence obtained
Clustering of alliinase fragments
from leaf (l) and bulb(b)
97% identity among all clones
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
Other genes in
biosynthetic pathway
Identify genes coding for enzymes
involved in alliin biosynthesis
-
Novel enzymes
-
Known enzymes with novel functions
Evidence from literature and tissue culture
experiments for synthesis of cysteine derivatives
by cysteine synthase
 several CSase genes in all plants
 including S-allyl cysteine
Isolation of cysteine
synthases from garlic
Two strategies:
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
Purification of an allyl cysteine
synthase from garlic leaves
Phenyl sepharose fractionation
0.7
0.6
OD550
0.5
0.4
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 and SATase
from garlic
Five full-length cDNAs isolated and
sequenced:
GSAT1 – cytosolic SATase
GCS1 – potential plastidic CSase
(contains frameshift - pseudogene ?)
GCS2 – chloroplastic CSase
GCS3 – cytosolic CSase
GCS4 – S-allyl-CSase (based on protein isolated)
Northern blot analysis
1
2
3 4
5
gcs4
gcs3
gcs2
• The potential S-allyl CSase
gcs4 and the SATase gsat1
are expressed in most
tissues examined.
• The cytosolic CSase gcs3 is
root specific.
gsat1
18s
1.
2.
3.
4.
5.
7 degree C stored clove
RT stored clove
Sprouting clove
Leaf
Root
• Expression for the putative
plastidic CSase gcs2 is
uniformly low.
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
• GCS2 and GCS3 can act
weakly as S-allyl-CSase
5000
0
0GCS2
10
GCS2
0 GCS3
10
GCS3
0GCS4
10
GCS4
min
Transformation of Arabidopsis
with garlic genes
 Transformed with gcs3, gcs4, gsat1
 Plants also carry GUS reporter gene
 Expression should not be constitutive
 Expression of both garlic and GUS genes are induced by
ethanol
 Seed produced from plants carrying each transgene has
been analysed (ie T1 plants)
PCR to detect transgenes in genome
RT-PCR and staining (for GUS) to detect expression of transgenes
Spectrophotometric and hplc analysis for cysteine and allyl cysteine
A. thaliana containing gcs3 or gcs4
Histochemical staining for GUS
Uninduced
Background line
After induction with ethanol
Some plants show activity of the
inducible GUS transgene
A. thaliana containing gcs3 or gcs4
RT-PCR for gcs4 transgene expression
induced
uninduced
GCS4-2-M
GCS4-2-J
GCS4-2-I
GCS4-2-G
GCS4 – F
GCS4-2-E
GCS4-2-D
GCS4-2-C
GCS4-2-B
GCS4-2-A
AGS1-3
Control
GCS4-2-B
GCS4-2-A
AGS1-3
GCS4-2-I
GCS4-2-G
GCS4-2-F
GCS4-2-E
GCS4-2-D
GCS4-2-B
GCS4-2-A
AGS1-3
Control
1.6 kbp
1.0 kbp
Some plants show expression of the inducible
gcs4 transgene
Arabidopsis with garlic genes
A. thaliana containing gcs3 or gcs4
Plants did not show a phenotype
GCS4 line 2
µm ol/cysteine/m in/mg total
protein
0.60
AGS1-3 (un)
0.50
AGS1-3 (in)
0.40
GCS4-2-D (un)
GCS4-2-D (in)
0.30
GCS4-2 silenced (un)
0.20
GCS4-2 silenced (in)
0.10
GCS2-2 expressed (un)
0.00
GCS4-2 expressed (in)
none
silenced
express
TIP and Annual reports
TIP
Completed by P2 and P3
Fourth Annual report
Completed by P2 and P3
Final report
Being written by P2
Completed by P3
Deliverables
DP. 8: Analytical methods for labeling and analysis (P2, P3)
DP. 9: A cDNA library from garlic (P2)
DP. 16: Pathway intermediates identified (P3)
DP. 17: First sulphur budget for garlic (P2)
DP. 18: Clones for alliinase (P2)
DP. 23: Publication on alliin biosynthesis and sulphur partitioning
(P2, P3)
Synthesis of alliin in garlic and onion tissue cultures – submitted
to Phytochemistry
DP. 24: Genes for key CSO synthesis enzymes (P2,P3)
DP. 29 Papers on the characterisation of key enzymes in alliin
biosynthesis and alliinase expression and the regulation of sulphur
biochemistry in garlic (P2, P3, P5)
Functional analysis of a novel garlic cysteine synthase in
Arabidopsis thaliana – being written
Deliverables:
DP. 33 Paper on S pathway genes on the production of flavour precursors
in garlic
Biosynthesis of the flavour precursors of onion and garlic –
submitted to Journal of Experimental Botany
DP. 35 Publication on the regulation of alliinase expression (P2)
DP. 36 Paper on the regulation of sulphur biochemistry in garlic
Effect of storage on the flavour precursors in garlic – being written