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Exploration of novel phytochemicals
using mammalian hepatoma cells
Regulation of glucosinolate biosynthesis and beyond
R2R3 MYBs regulate the biosynthesis of
aliphatic and indolic glucosinolates
Incoming signal: touch, wounding,
selective activation
by MeJa, SA, ABA,
glucose….
Activation of MYB target genes
Activation of
glucosinolate
biosynthesis
genes by MYBs
DHS1
ASA1
TSB1
CYP79B2/
CYP79B3
CYP83B1
UGT74B1
C-S lyase
AtST5a
Induction of TFs: MYB51,
MYB122,
MYB34
MYB28…
35S:MYB28 I3M
4M
OI3
M
8M
SO
O
5M
SO
P
4MSOB
3M
SO
P
4MTB
4-
Me
thylp
en
thyl G
LS
5-
Me
thylh
exyl G
LS
Met-GS Trp- GS Leu-GS
MYB51
MYB122
MYB34
MYB29
MYB76
MYB28
Protein-protein interaction in the regulation of GS
Yeast two hybrid assay
At4g19700
bHLH-HFs
At3g49570
At3g45900
At4g26930
At1g79280
MYBs:bHLH-HFs
bHLH-HF1
bHLH-HF2
bHLH-HF3
bHLH-HF4 Transient expression in N. bent.
MYB:SPYNE; bHLH-HF:SPYCE
mustard
oil
Plastidic transporters in GS biosynthesis
Intercompartmental metabolite signalling
Increased resistance
toward plant enemies
mustard
oil
Increased production of gluocosinolates
Pull-down experiment demonstrating an
interaction of bHLH-HFs with R2R3 MYBs
Glucosinolate levels are strongly diminished in
the double and triple bhlh-hf1/hf2/hf3 mutants
R2R3 MYBs
bHLH-HFs
Are MYBs regulated posttranslationally?
Are they phosphorylated or ubiquitinated ?
Do biotic or other stimuli affect this process?
How phosphorylation of MYBs affect:
- DNA binding activity?
- trans-activation potential?
- sub-cellular localisation?
MYB51-GFP
active
MYB51-GFP
inactive
Post-translational regulation of R2R3 MYBs
Induction of EpRE:TK:GFP
In human hepatoma cells
Induction of EpRE:GST:LUX
in murine hepatome cells
Induction of Phase II Detox.
Enzyme activity: QR
Screening of extracts of
5000 activation tagged lines for
chemoprotective activities
SO42-
Aldoxime
S-Alkyl-thiohydroximate
Aci-Nitro-compound
Desulfo-glucosinolate
Glucosinolate
CYP79F1/F2
CYP83A1
C-S lyase
AtST5b/c
UGT74C1
Methionine
a-Keto acid
MAM
BCAT4
Chain-elongated Met
2-Malat derivative
3-Malat derivative
a-Keto acid BCAT3
IMDH
IPMI
GSH-conjugate GST
thiohydroximate
GPP
+GSH
Cy
top
las
m
Vacuole
PAPS
APS
sulfate
APK
ATPS
2
CYTOPLASMA
CHLOROPLAST
SO42
- SO4
2- APS SO32- S2- Cystein
Methionine
PAPS APK1
APK2
Sulfation
PAPS
+
AtSt5 a,b, c
glucosinolates desulfoglucosinolates
OH
PAP
PAP +
A model for the PAPS/PAP antiport in A.thaliana
A.The GFP-fusion protein of PAPS/PAP antiporter
is localised in envelops; B. The double homozygous
A.thaliana knock-out mutant is lethal
B A
Plastid
Mitochond. PAP
Nucleus
PAP
XRN2/3
De-repression of
nuclear-encoded
stress-responsive
genes
A model for the intercompartmental
PAP signalling in A.thaliana
SAL1
FRY1
ALX8
AMP + Pi
PAP
Gene silencing
PAPS APS
PAP
sulfate APK1
APK2
ATPS3 ATPS1
PAPS
PAP
AMP + Pi
Cytosol
Plastid
SO3 2-
S 2-
Cystein
Mitochondrium
ATP, ADP
PAPT1
PAPT2
XRN2/3
Nucleus
SAL1
FRY1
ALX8
A prediction for the
subcellular
localisation of FRY1 A PAP level is increased in
fry1 knock-out mutant
fry1 Col-0
Appearance of fry1 knock-out mutant
In comparison to wild-type, fry1 is
retarded in growth, reveals delayed
bolting time, but also increased resistance
to high light and drought stresses
fry1 Col-0
G
l u
c
o
s i n
o
l a
t
e
s
pyk10-1D
metabolic profiling
Generation and analysis
of recapitulation lines
1MOI3M
Isolation and analysis of
homozygous knock-out line
substrate?
glycosilated
compounds
Metabolite profiling of
pyk10-1D (51.14) mutant
Using UPLC-ESI-QTOF
PYK10
700 x
T-DNA insertion in 51.14 caused
an activation of a PYK10 gene
resulting in pym10-1D EpRE EpRE TK/GST LUX/GFP
NOVEL
PHYTOCHEMICALS
Nrf2
Nucleus
3MSOP
4MSOB
5MSOP
8MSOO
I3M
4MOI3
M
1MOI3
M
µm
ol/g T
G
0,0
0,5
1,0
1,5
5,07,5
10,0
Col-0
bhlh-hf1/2
bhlh-hf1/2/3
nd*
nd nd nd nd nd
*
*
* ** *
* nd
BHLH-HF1
BHLH-HF2
BHLH-HF3
BHLH-HF4
Re
lative
lu
cife
rase
activity (
%)
0
5
10
15
20
25
RLuc-MYB51+ProtA-BHLH(x)
RLuc-leer+ProtA-BHLH(x)
RLuc-MYB51+ProtA-leer