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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 biosynthesisBring 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
SO42- SO3
2- S2- cysteine
glutathione(γ-glu-cys-gly)
S-methyl-γ-glu-cys
gly
S-methylcysteine
methiin
glu
trans-peptidase
oxidase
S-2-CP-γ-glu-cys
gly
S-trans-1-propenyl-γ-glu-cys
S-trans-1-propenylcysteineoxidase
trans-peptidaseglu
HCOOH
S-trans-1-propenylcysteine sulphoxide(isoalliin)
S-methylglutathioneS-(2-carboxypropyl)-glutathioneS-allylglutathione
S-allyl-γ-glu-cys
gly
S-allylcysteine
glu trans-peptidase
oxidase
S-allyl group(unknown sources)
valine & methacrylateserine
oxidase
S-allylcysteine
S-allyl-cysteine sulphoxide(alliin)
Biosynthetic capacity of garlic callus
alliin allyl cysteine isoalliin propiin propyl cysteineallyl thiol 10,1;10,1 10; 10,1propyl thiol 10; 1;10allyl cysteine 10;10,1propenyl cysteine 1;10,1propyl cysteine 10,1;10,1
Incubation for 5 days with 10mM or 1mM substrateIncubation for 12/15 days with 10mM or 1mM substrate
Conclusion:
These experiments suggest that in vivo the general reaction shown may occur:-
Alk(en)yl thiol 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 glutathionepropyl alcohol +allyl alcohol +methacrylic acid +allyl thiol +metabolite soup +allyl glutathionecarboxypropyl glutathionepropyl glutathione
No clear potential GST substrate
2. Identify developmental control points on CSO synthesis and translocation
Baseline data on garlic developmentResource allocation during
developmentDeveloped 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
Hydroponic vpot-grown Printanor - Leaf weight
0
5
10
15
20
25
0 50 100 150 200 250
Days after planting
Mea
n m
ass
of le
af (g
, n=3
)
Hydoponic-grown Printanor
Pot-grown Printanor
Hydroponic-grown garlic - comparison of bulb formation
0
20
40
60
80
100
120
140
160
0 50 100 150 200 250
Days after planting
Fres
h w
eigh
t of c
love
Printanor clove
Messidrome Clove
Garlic growth and S partition
0.0
0.1
0.2
0.3
29 56 77 109 141 169 203Days after planting
Tota
l Su
lph
ur
Co
nte
nt
(g) Root
Leaf
Clove
0
500000
1000000
1500000
2000000
56 109 141 169 203Days after planting
CS
O c
on
ten
t Root
Leaf
Clove
1 2 3 4 1 2 3 4
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)
0
50
100
150
200
0 25 50 75 100 125 150 175 200 225
Days after planting
Fre
sh w
eig
ht
(g) Clove
Leaf
Root
Distribution and remobilizationof sulphur taken up early
Distribution and remobilizationof sulphur taken up late
* * * * * * * * * * *
* * * * * * * * * * *
34S32S
A
B
Growth pattern in Year 2 experiment
Sulfur labelling design
Sulpur accumulation in system A plants
0
50
100
150
200
250
05/0
4/02
12/0
4/02
19/0
4/02
26/0
4/02
03/0
5/02
10/0
5/02
17/0
5/02
24/0
5/02
31/0
5/02
07/0
6/02
14/0
6/02
21/0
6/02
28/0
6/02
05/0
7/02
12/0
7/02
Date
Tota
l m
ass
in m
g
Clove
Leaf
Root
Total
34S 32S
Year 3 hydroponic garlic
0
50
100
150
200
25005
/04/
02
19/0
4/02
03/0
5/02
17/0
5/02
31/0
5/02
14/0
6/02
28/0
6/02
12/0
7/02
26/0
7/02
d v
alu
e
Bulb
Leaf
Root
0
50
100
150
200
05/0
4/02
19/0
4/02
03/0
5/02
17/0
5/02
31/0
5/02
14/0
6/02
28/0
6/02
12/0
7/02
26/0
7/02
d v
alu
e
Bulb
Leaf
Root
A: 34S then 32S B: 32S then 34S
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
3234 3432
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 hydroponicallyat start of bulbing, remove most of
either roots or leavescompare data from this and end-point
Dry Weight
020406080
100120
Clove Leaf Root
Ma
ss
in g
Early Control
Leafless
Rootless
Late Control
Fresh weight
0
100
200
300
400
Clove Leaf Root
Ma
ss
in g
Early Control
Leafless
Rootless
Late Control
Normal development:
bulb: x10 fold mass increase
leaf: x 2.5 fold mass increase
root: unchanged
Leaves removed:
bulb: 0.5 mass
leaf: mass almost fully recovers
roots: 0.5 mass
Roots removed:
bulb: mass almost unaffected
leaf: x 3.5 fold mass increase
roots: no recoverySevere virus infection during growth
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 during development
0
0.2
0.4
0.6
0.8
1
08/02/01 10/03/01 09/04/01 09/05/01 08/06/01Rel
ativ
e al
liin
ase
exp
ress
ion
Bulb
Leaf
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 garlicTwo 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
0.1
0.2
0.3
0.4
0.5
0.6
0.7
1 3 5 7 9
11
13
15
17
19
21
23
25
27
29
31
33
35
37
39
Fraction
OD
55
0
cysteinesyntase activity
allyl cysteinesynthaseactivity
…….FLGVMPSHYSIE………. YLGADLALTDTN………… SANPGAHYATTGP………….
Sequence of peptides from this protein
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)
1 2 3 4 5
gcs4
gcs3
gcs2
gsat1
18s
1. 7 degree C stored clove
2. RT stored clove3. Sprouting clove4. Leaf5. Root
• The potential S-allyl CSase gcs4 and the SATase gsat1 are expressed in most tissues examined.
• The cytosolic CSase gcs3 is root specific.
• Expression for the putative plastidic CSase gcs2 is uniformly low.
Northern blot analysis
Results
• Background activity from E. coli proteins subtracted
• All three genes gcs2 gcs3 gcs4 are functional to transcribe and translate CSase
• GCS4 shows the highest activity in cysteine biosynthesis
• GCS4 functions as S-allyl-CSase
• GCS2 and GCS3 can act weakly as S-allyl-CSase
Pea
k ar
ea
In vitro CSase activity
0
5
10
15
20
25
30
35
µm
ol c
ys
min
-1 m
l-1
Substrate: Na2S
GCS2 GCS3 GCS4
0
5000
10000
15000
20000
25000
30000
35000
GCS2 GCS3 GCS4 0 10 0 10 0 10 min
Substrate: allyl mercaptan
GCS2 GCS3 GCS4
Expression of gcs2 gcs3 gcs4 in vitro
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 After induction with ethanol
Background line Some plants show activity of the inducible GUS transgene
A. thaliana containing gcs3 or gcs4
GCS4-2-MGCS4-2-JGCS4-2-IGCS4-2-GGCS4 – FGCS4-2-EGCS4-2-DGCS4-2-CGCS4-2-BGCS4-2-AAGS1-3Control
1.6 kbp1.0 kbp
GCS4-2-BGCS4-2-AAGS1-3GCS4-2-IGCS4-2-GGCS4-2-FGCS4-2-EGCS4-2-DGCS4-2-BGCS4-2-AAGS1-3Control
RT-PCR for gcs4 transgene expression
uninduced induced
Some plants show expression of the inducible gcs4 transgene
Arabidopsis with garlic genes
A. thaliana containing gcs3 or gcs4Plants did not show a phenotype
GCS4 line 2
0.00
0.10
0.20
0.30
0.40
0.50
0.60
µmo
l/cys
tein
e/m
in/m
g t
ota
l p
rote
in
AGS1-3 (un)
AGS1-3 (in)
GCS4-2-D (un)
GCS4-2-D (in)
GCS4-2 silenced (un)
GCS4-2 silenced (in)
GCS2-2 expressed (un)
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
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