sulfur biochemistry of garlic: the biosynthesis of flavour precursors hamish a collin, jill m...
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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 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
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Project objectives: Garlic flavour
Improved understanding of S allocation and translocation during garlic development
Identify genes and intermediates involved in alliicin synthesis
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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
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Hydroponic vpot-grown Printanor - Leaf weight
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Days after planting
Mea
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Hydoponic-grown Printanor
Pot-grown Printanor
Hydroponic-grown garlic - comparison of bulb formation
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Days after planting
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t of c
love
Printanor clove
Messidrome Clove
Garlic growth and S partition
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29 56 77 109 141 169 203Days after planting
Tota
l Su
lph
ur
Co
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(g) Root
Leaf
Clove
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500000
1000000
1500000
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56 109 141 169 203Days after planting
CS
O c
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t Root
Leaf
Clove
1 2 3 4 1 2 3 4
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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
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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)
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Days after planting
Fre
sh w
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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
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Sulpur accumulation in system A plants
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l m
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Clove
Leaf
Root
Total
34S 32S
Hydroponic garlic in isotopically labelled sulfur
Sulphur accumulation in system A plants (34S then 32S)
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/04/
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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
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To identify genes and intermediates in flavour precursor biosynthesis
Alliinase
Other genes from earlier part of biosynthetic pathway
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Sequence obtained
Relative alliinase expression during development
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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
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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
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Is cysteine synthase involved in garlic flavour precursor biosynthesis?
O-acetyl serine + sulphide cysteine
cytoplasmic, plastid and mitochondrial forms
non-protein amino acids synthesised
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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
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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
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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
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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
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Purification of an allyl cysteine synthase from garlic leaves
Phenyl sepharose fractionation
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Fraction
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cysteinesyntase activity
allyl cysteinesynthaseactivity
…….FLGVMPSHYSIE………. YLGADLALTDTN………… SANPGAHYATTGP………….
Sequence of peptides from this protein
34 kDa
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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)
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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
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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
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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
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Substrate: allyl mercaptan
GCS2 GCS3 GCS4
Expression of gcs2 gcs3 gcs4 in vitro
Pea
k ar
ea
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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?
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Acknowledgements
The Garlic and Health project partners
EU FP5 Quality of Life program: Garlic and Health project QLK1-CT-1999-00498