Sulfur biochemistry of garlic: the biosynthesis of flavour precursors Hamish A Collin, Jill M...
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Transcript of Sulfur biochemistry of garlic: the biosynthesis of flavour precursors Hamish A Collin, Jill M...
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 TomsettThe School of Biological Sciences, The University of Liverpool
Laurence Trueman, Tim Crowther, Linda Brown and Brian ThomasWarwick 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
For controlled growth in the UK climate - hydroponic and pot culture in a glasshouse
Measurements during growth
•Leaf number, bulb weight
•N, S, C, protein, CSO
•SO42-uptake using
stable isotope labelling
Improved understanding of S allocation and translocation during garlic development
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
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)
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 earlier 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
Hydroponic garlic in isotopically labelled sulfur
Sulphur accumulation in system A plants (34S then 32S)
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
To identify genes and intermediates in flavour precursor biosynthesis
Alliinase
Other genes from earlier part of biosynthetic pathway
Sequence obtained
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
Alliinase
Biosynthetic pathway for garlic flavour precursors
SO42- SO3
2- S2- cysteine
glutathione(γ-glu-cys-gly)
S-methyl-γ-glu-cys
gly
S-methylcysteine
S-methylcysteine sulphoxide(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 source)
valine & methacrylateserine
oxidase
S-allylcysteine
S-allyl-cysteine sulphoxide(alliin) 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 O-acetyl serine H2S CH2=CH-CH2-SH methyl-SH 3,4-dihydroxy-
pyrazole Free CSase pyridine L-cysteine S-allyl-L-cysteine S-methyl- mimosine -pyrazol-
1-yl alanine L-cysteine Free CAS HCN
3-cyano-L-ala
watermelonMimosa pudica
CSase cysteine synthase; CAS -cyanoalanine
synthase
Pea (Pisum sativum)
Leucaena leucocephala
watermelon
Leucaena leucocephala
Lathyrus latifolius
Ikegami and Murakoshi 1994; Warrilow and Hawkesford 2002
Biosynthetic capacity of garlic callus
allyl cysteine alliin isoalliin propyl cysteine propiinallyl thiol 10; 10,1 10,1;10,1 allyl cysteine 10;10,1propenyl cysteine 1;10,1propyl thiol 1;10 10;propyl 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 reactions shown may occur:-
alk(en)yl thiol alk(en)yl cysteine alk(en)yl CSO
Isolation of cysteine synthases from garlicStrategy:
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 kDa51 aa predicted transit peptide - plastid
GCS3323 aa, 34 kDaNo transit peptide - cytosol
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
34 kDa
Obtained CSase from garlic
Four full-length cDNAs isolated and sequenced:
GCS1 – potential plastidic CSase (frameshift)
GCS2 – potential plastidic CSase GCS3 – potential cytosolic CSaseGCS4 – potential S-allyl-CSase (based on
protein data)
Phylogenetic tree of garlic cysteine synthases
Spinach
A. thaliana [3, 10]
A. thaliana [6]
GCS2
A. thaliana [4]
RCS4RCS2
GCS4
GCS3
A. thaliana [2]
A. thaliana [5]
Watermelon
A. thaliana [1]
A. thaliana [8]A. thaliana [9]
A. thaliana [7]
50 changes
PAUP version 4.0b 10
100
78
97
100 100
100
100
100 28
72
7246
45
99
100
1 2 3 4 5
gcs4
gcs3
gcs2
18s
1. 7o stored clove
2. 20o stored clove3. Sprouting clove4. Leaf5. Root
• Low expression of putative plastidic CSase gcs2
• Root expression of cytosolic CSase gcs3
• Most tissues expressed potential S-allyl CSase gcs4
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
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
Pea
k ar
ea
SummaryS 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, GCS4S-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