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