Transcript WP4 - Tomsett

Molecular Analysis of Flavour
biosynthesis in garlic
Angela Tregova and Jill Hughes
Hamish Collin, Rick Cosstick, Meriel Jones, Brian Tomsett
Acknowledgement: Mark Wilkinson, protein purification facilities
Biosynthetic Pathway
SO42serine
Allyl group
serine
valine &
methacrylate
(source ?)
S-(2-carboxypropyl)-glutathione
S-allyl-γ-glu-cys
glu
S-allylcysteine
transpeptidase
oxidase
S-allyl-cysteine sulphoxide
(alliin)
S2-
cysteine
glutathione
S-allylglutathione
gly
SO32-
S-methylglutathione
gly
S-2-CP-γ-glu-cys
gly
S-methyl-γ-glu-cys
HCOOH
S-trans-1-propenyl-γ-glu-cys
glu
transpeptidase
S-trans-1-propenylcysteine
oxidase
S-trans-1-propenylcysteine sulphoxide
(isoalliin)
glu
transpeptidase
S-methylcysteine
oxidase
methiin
What we have done……
• Investigation of intermediates in the pathway
• Identification of key compounds
• Purification of a key enzyme
• Allylcysteine synthase
• The search for genes involved in flavour biosynthesis:
2 chloroplastic cysteine synthases
1 cytosolic cysteine synthase
1 S-allyl cysteine synthase
+
1 cytosolic serine acetyl transferase
Key observation
Callus converts
allyl thiol to allyl cysteine & alliin
CH2CHCH2-SH
(+ O-acetyl-serine ?)
CH2CHCH2-S-CH2CHNH2COOH
=
CH2CHCH2-S-CH2CHNH2COOH
O
But not allyl alcohol
CH2CHCH2-OH
X
But this is not
species-specific
Allyl Cysteine Synthase?
Cysteine synthase
Sulphide + O-Acetyl Serine
Cysteine
Allyl Cysteine synthase
Allyl thiol + O-Acetyl Serine
Allyl Cysteine
Is there a specific cysteine synthase homologue?
Cysteine synthases do a range of reactions
in other organisms
Protein purification: Ion Exchange chromatography
Garlic leaves were fractionated with ammonium sulphate then
separated by ion-exchange chromatography.
Cysteine synthase activity.
Q-Sepharose. 7.11.01
0.3
0.25
0.2
0.15
0.1
0.05
0
Fr
1
Fr
5
Fr
9
Fr
13
Fr
17
Fr
21
Fr
25
Fr
29
Fr
33
Only a few fractions show
allyl cysteine synthase
activity
OD
Many fractions show
cysteine synthase
activity
cysteine synthase
activity
Fraction
Protein purification: Hydrophobic Interaction Chromatography
Allyl cysteine
synthase and
cysteine synthase
activity co-elute
Phenyl sepharose fractionation
0.7
0.6
OD550
0.5
0.3
0.2
cysteine 0.1
syntase activity
Fraction
39
37
35
33
31
29
27
25
23
21
19
17
15
13
11
9
7
5
allyl cysteine
synthase
activity
3
0
1
Cysteine production was
assayed colorimetrically
and allyl cysteine by
HPLC
0.4
Protein purification
SDS-PAGE shows a distinct
band in the allyl cysteine
synthase active fractions at
approx. 34 kd
34000
Molecular weight consistent
with plant cysteine synthase
monomers found previously
Fractions
26 27 28 29 30
What is the Enzyme?
Extract 34000 band and digest with trypsin
- the resultant peptides separated by preparative HPLC
Three selected peptides were sequenced:…….FLGVMPSHYSIE………. YLGADLALTDTN…………
……………………SANPGAHYATTGP………….
A simple BLAST search of these peptides in the protein database
shows most similarity to a cysteine synthase from Oryza sativa
(Rice)
Probe for S-allyl-CSase
Peptide
1
2
3
A B C D E F G H I
cDNA fragments PCR amplified
with degenerate primer A – I from
the cDNA library
Peptide sequences:
1. FLGVMPSHYSI
2. YLGADLALTDT
3. ANPGAHYA
…. to find the gene
and related genes
AllylCSase aligns with rice sequences
Partial protein sequences relative to Arabidopsis (C) sequence
RCS2
RCS4
GCS4
IGLVLVAVQ-KGYRFIAVMPAKYSLDKQMLLRFLGAELILTDPA-IGFNG—MMDKVEEL
IGVAYNALL-KGYRFVAVMPAEYSLDKQMLLTYLGAEVILTDPT-LGFQGQ-LDKVEQI
IALAYI-GLKKGYKFLGVMPSHYSIERRMLLKYLGADLALTD-TNLGFKG-VLDKVAEL
I
KGY F VMP YS
MLL LGA
LTD
GF G
DKV
Proposed Serine Pathway
Cysteine
Acetyl CoA
2
L-Serine
Sulfide
4
OAS
1
Allyl-source
3
S-allyl-L-Cysteine
4
Alliin
Important enzymes:
1
SAT/CS complex
2
Free CSase
3
S-allyl-CSase + ?
Oxidase
cDNA library screening

gsat1 - cytosolic SATase

gcs1 - putative plastidic CSase
(pseudogene)

gcs2 - putative plastidic CSase

gcs3 - cytosolic CSase

gcs4 - putative S-allyl-CSase
What next ?
•
Where are the genes expressed in garlic?
• Northerns
•
Does the gene encode allylcysteine synthase?
• How do we prove it?
•
What does it do in planta?
•
Transformation
Northern blot analysis
1
2
3 4
5
gcs4
 S-allyl CSase and
the SATase
gcs3
gcs2
gsat1
18s
1.
2.
3.
4.
5.
7 degree C stored clove
RT stored clove
Sprouting clove
Leaf
Root
are expressed in most tissues
examined.
 The cytosolic CSase is root
specific.
 Expression for the putative
plastidic CSase is uniformly low.
Is this allylcysteine synthase?
Proof requires expression of the gene and phenotypic testing
•
Garlic?
Ideal Choice ?
This would be best but…..time ?
A quick assessment could mean that we can plan the alternative
• If
E.wecoli?
Does it function
alone?
vitro
use ethanol-regulated
expression,
thenInwe
can testing
test the only?
effect
on the cellular phenotype of the expression of the
allylcysteine synthase vs. its absence !
• Heterologous plant system?
Time ? Arabidopsis ?
•
Plant tissue culture?
Quick and could form complexes
allowing tests in planta
Why ethanol-regulated expression?
pCAMV35S
cDNA
alcR
AlcR
AlcR E
palcA
t
+ Ethanol
E
alc is a simple two component system
transgene
t
Does it work?
Real time Luciferase Imaging
in Arabidopsis
LUC 1-12
wt
AGS
LUC 1-12
wt
AGS
1 hour before induction
LUC 1-12
wt
AGS
Time of induction
LUC 1-12
wt
AGS
30 minutes after induction
LUC 1-12
wt
AGS
1 hour after induction
LUC 1-12
wt
AGS
1.5 hour after induction
LUC 1-12
wt
AGS
2 hours after induction
LUC 1-12
wt
AGS
2.5 hours after induction
LUC 1-12
wt
AGS
3 hours after induction
LUC 1-12
wt
AGS
3.5 hours after induction
LUC 1-12
wt
AGS
4 hours after induction
LUC 1-12
wt
AGS
4.5 hours after induction
LUC 1-12
wt
AGS
5 hours after induction
LUC 1-12
wt
AGS
6 hours after induction
LUC 1-12
wt
AGS
7 hours after induction
LUC 1-12
wt
AGS
7.5 hours after induction
LUC 1-12
wt
AGS
8 hours after induction
LUC 1-12
wt
AGS
11 hours after induction
LUC 1-12
wt
AGS
13 hours after induction
Real time Arabidopsis
Luciferase Imaging
LUC 1-12
wt
AGS
Time of induction
LUC 1-12
wt
AGS
8 hours after induction
Functional analysis of
plant cell cycle genes
At progeny of AmcycA20 x alcRalcAGUS
AmcycA20 PCR
1
2
A
3
B C
4 5
D
6
E
7
F G H I
8 9 10 11
J K L
12 13 14 15 16
1.6 kb
1 kb
A
G
B
H
C
I
D
J
E
K
F
L
Induced
GUS PCR
1
A
B
C
D
E
F
G
H
I
J
K
L
2
A
3
B C
4 5
D
6
E F G H I
7 8 9 10 11
J K L
12 13 14 15 16
1 kb
500 bp
Non-induced
alcR PCR
1
2
A
3
B C D
4 5 6
E
7
F G H I J
K L
8 9 10 11 12 13 14 15 16
1 kb
500 bp
RT-PCR of AmcycA20 & controls
plus RT
1
A
2
B
3
C
4
Induced plants
D
5
E
6 7
A
8
B C
9 10
D E
11 12 13 14
1 kb
Total RNA extracted
from plants of
A = cyclin A20
B = HA-tagged cyclin A20
C = sibling
D = wild type
E = AGS-1-3
Induced RNA minus RT
1
A
B
C
D
E
2
3
4
5
6
7
8
Uninduced plants
1
A
2
B
3
C
4
D
5
E A
B
6 7
8
C D E
9 10 11 12 13 14
1 kb
1 kb
cycA20 message is specific to
induced plants containing both
T-DNAs
Total DNA extracted from
Induced plants of
A = cyclin A20
B = HA-tagged cyclin A20
C = sibling (cyc+;GUS-)
D = wild type
E = AGS-1-3
13 = A.majus genomic DNA
There is no DNA
contamination
Western Blots of HA tagged cycA20
WT
I
N
WT
I
N
48 kDa
HA-CycA20
Probe = antibody to HA tag
Phenotypic analysis
Rosette leaves
1 2
Leaf cell density, primary leaf area,
rosette leaf number, trichomes and
flowering time.
Plants were grown for six weeks.
Vertically grown A.thaliana plants,
growing in a tissue culture square
plate.
Root growth experiments (after 15
days) and
fresh weight measurements (after
four weeks).
Fresh Weight
100
80
60
40
20
0
WT
AGS
SIBLING
NON-INDUCED
CYCA20-HA
INDUCED
CYCA20
Root Length – AmcycA20 expression
WT 25 26 27 28 29 30 31 32 AGS
G
A
A
Root length
9
WT
AGS
SIBLING
CYCA20-HA
CYCA20
WT
AGS
SIBLING
CYCA20-HA
CYCA20
Root length (cm)
8
7
6
5
4
3
2
1
0
0
5
10
Days growth
15
Leaf number and area
Leaf number remains constant
after AMcycA20 expression
Plus ethanol
Minus ethanol
sibling
Leaf area is bigger
after induction in
AmcycA20 expressing lines
Minus ethanol
Plus ethanol
Cyclin A20
Leaf Area
2.50
2.00
1.50
1.00
0.50
0.00
WT
AGS
Sibling
Non-induced
CycA20-HA
Induced
CycA20
Cell Size and density
Uninduced
Induced
HA-tagged
cyclin A20
There appear to be less cells per unit area - cells are larger
Trichomes on rosette leaves
Days
Flowering time of cycA20 and controls
25
20
15
10
5
0
WT
AGS
Sibling
Non-induced
CycA20 CycA20
Induced
Mean flowering time (days) of twenty seedlings of each of HA-tagged cyclin A20, cyclin A20,
wild type (Columbia), AGS-1-3 and sibling plants in comparison between non-induced and
induced conditions.
The plants were induced after 5 days of germination, when the plants reached the 2 leafstage, and were checked regularly until the appearance of the first visible flower bud.
Tobacco transformation for protein expression
kanR
RB
t35S
Transformed
Garlic gene
palcA
Untransformed
pnos
nptII
pAg7
Transformed
LB
Tobacco transformation for protein expression
• The tobacco cells can be multiplied in liquid culture
• Induce protein expression
• Determine whether –SH content of cells has increased
• Assay for allylCSase activity
• Use HPLC to look for allylcysteine and ….?
Does tobacco possess an oxidase to make alliin?
Diagnostic PCRs for transgenic BY2 lines
1 2 3 4 5 6 7
Lane 1 = untransformed BY2
Lane 2 = gcs3 plasmid control
Lane 3 = gcs3 transformant
Lane 4 = gcs4 plasmid control
Lane 5 = gcs4 transformant
Lane 6 = gsat1 plasmid control
Lane 7 = gsat1 transformant
PCR primers:
1.palcA forward
2. t35S reverse
However…….
 Cysteine
synthase assays
No detectable increase in cysteine. Time course assays
and assay optimisations failed.
 S-allyl-cysteine
synthase assay (HPLC)
No detectable levels of S-allyl-cysteine.
Northern blot analysis
gcs3
 RNA extracted from transgenic
tobacco cells after 1, 3 and 6
days induction.
gcs4
 Northern blots show no
transgene expression, except
gcs3 that was detected after
several days induction.
gsat1
Garlic RNA
Tobacco
RNA
Why are the transgenes not expressed?

Are there mistakes in the binary constructs?
Re-sequencing verified correct assemblies.

Is the alcR cDNA present in the tobacco cell-line?
alcR confirmed by PCR.

Is alcR expressed?
Is alcR expressed?
RT-PCR results:
1 2 3 4
Lane 1 = alcR control (genomic DNA)
Lane 2 = gcs3 BY-2 transformant
Lane 3 = gcs4 BY-2 transformant
Lane 4 = gsat1 BY-2 transformant
 No alcR expression
detected in any of the
transformed cell lines!
Repeat BY-2 transformation

New BY-2 cell-lines from the John Innes Centre

Transformations have been repeated and we are
currently waiting for new transformants to grow

But is alcR expressed in the new cell-line?
alcR expression in the new cell-line
1 2 3
RT-PCR results:
Lane 1 = No RT control
Lane 2 = alcR control (genomic DNA)
Lane 3 = alcR expression in the new cells
Positive RT-PCR
controls using
degenerate primers that
anneal to SAT.
 Again, no detectable alcR
expression in the new
cell line!
RT-PCRs using a highly sensitive detection
1 2 3 4 5 6 7 8 9
RT-PCR results:
Lane 1 = untransformed BY-2
Lane 2 = gcs3 BY-2 transformant
Lane 3 = gcs4 BY-2 transformant
Lane 4 = gsat1 BY-2 transformant
Lane 5-8 = No RT controls
Lane 9 = alcR control (genomic DNA)
RT-PCRs using a highly sensitive detection
1 2 3 4 5 6 7 8 9
RT-PCR results:
Lane 1 = untransformed BY-2
Lane 2 = gcs3 BY-2 transformant
Lane 3 = gcs4 BY-2 transformant
Lane 4 = gsat1 BY-2 transformant
Lane 5-8 = No RT controls
Lane 9 = alcR control (genomic DNA)
RT-PCRs using a highly sensitive detection
1 2 3 4 5 6 7 8 9
RT-PCR results:
Lane 1 = untransformed BY-2
Lane 2 = gcs3 BY-2 transformant
Lane 3 = gcs4 BY-2 transformant
Lane 4 = gsat1 BY-2 transformant
Lane 5-8 = No RT controls
Lane 9 = alcR control (genomic DNA)
Future ?
The longer route looks more attractive !
• E. coli –
his-tagged protein
purification
assay in vitro
•
Arabidopsis - test for expression
assay in vivo
phenotype
Expression of a wheat CSase in tobacco
A. Transgenic tobacco
shows 2-fold higher Cys
content.
B. SO2 fumigation increased
thiol levels.
Deliverables
•
Genes for CSO synthesis enzymes (36m)
•
Publication on regulation of S biochemistry in garlic
(36m)
•
Paper on characterising enzymes in alliin biosynthesis,
and alliinase expression, and regulation of sulphur
biochemistry in garlic (48m)
•
Paper on S pathway genes on production of flavour
precursors in garlic (48m)
Thanks to ……..
Liverpool
Angela Tregova
Jill Hughes
Piyarat Parinyapong
Hairul Roslan
Chris Wood
Mike White
Mark Caddick
Brian Tomsett
Jealott’s Hill
Jackie Paine
Mary Knight
Susan Wright
Justin Sweetman
Alberto Martinez
Wolfgang Schuch
Andy Greenland
Ian Jepson
ICI Agrochemicals
ICI Seeds
Zeneca Seeds
Zeneca Agrochemicals
Syngenta
JIC
John Doonan
and his lab
Funding
BBSRC
EU FP5 Garlic &
Health