Download Auxin

Moss Systems Biology for
Translational Research
Ralf Reski
Moss Physcomitrella patens: A New Model Plant
 Small
 Few cell types
 Cell lineage
 Tightly controlled growth conditions
 Predominant generation haploid
 Fully sequenced genome
 Efficient homologous recombination
 targeted gene knockout / replacement
unique to higher eukaryotes
Modelling Plant Development 1: Light Microscope
Modelling Plant Development 2: Pattern Recognition
Modelling Plant Development 3: Simulation
no
branching
yes
type of
branching
F-type
branching,
left
F-type
branching,
right
Growth!
Y-type of
branching
miRNAs Regulate Gene Expression Qickly
micro RNAs:

not translated

more efficient than synthesis of
regulatory proteins (e. g. repressors)

faster regulatory circuits
Biogenesis and Function of micro RNAs
Dicer
RISC
pri-miRNA
AGO
HASTY
HYL
pre-miRNA
RISC
HEN
CAP
Nucleus
AGO
AAAAA
Cytosol
CAP
AAAAA
CAP
AAAAA
Post-transcriptional regulation of gene expression
PLANTS
ANIMALS
Animals and Plants: Different Sets of DICER
Targeted knockout of DCL1a: Drastic Effects
RT-PCR analysis of miRNAs
WT
KO1
WT
0.5 cm
KO2
miRNA156
miRNA160
0.5 cm
miRNA166
0.5 cm
miRNA390
No DCL1a: No miRNAs, enhanced TF levels,
DCL1a
KO1
serverely affected
growth
7
0.5 cm
0,007
Rel. Transcript Level
0.5 cm
RT-PCR analysis of target transcripts
WT
0,0066
0,0055
KO1
0,0044
DCL1a
KO2
0.5 cm
KO2
0,0033
0,0022
0,0011
00
SBP3
PpSBP3
ARF1
PpARF
PpC3HDZIP1
PpHDZIP
PpHB10
TAS1
PpHB10
PpTAS1
Targeted knockout of DCL1b: Less Drastic Effects
Wild Type
miRNA Expression
PpDCL1b KO
WT 1
2 3 4
200 µm
miRNA156
miRNA160
DCL1b
miRNA166
100 µm
miRNA390
miRNA535
miRNA538
U6 snRNA
miRNAs still present BUT developmental arrest
DCL1b: miRNAs Cannot Cleave Target mRNAs
KO KO KO KO
WT 1 2 3 4
PpSBP3 (Squamosa Promoter Binding Protein)
PpSBP3
miR156
5’
U
3’
GUGCUC CUCUCUUCUGUCA
CACGAG GAGAGAAGACAGU
3’
U
5’
PpC3HDZIP (HD leucine zipper)
PpC3HDZIP1 5’CU
miR165
U
3’
GG AUGAAGCCUGGUCCGG
CC UACUUCGGACCAGGCU
3’CC
C
5’
PpHB10 (HD leucine zipper)
PpHB10
miR165
5’CU U
3’
GG AUGAAGCCUGGUCCGG
CC UACUUCGGACCAGGCU
3’CC C
5’
Control PpGNT1
(no miRNA target)
miRNA Target Genes Silenced in DCL1b Mutant
PpDCL1b-Mutants
WT
1
2
3
4
PpARF
PpHDZIP1
miRNA target transcripts
PpHB10
PpSBP3
PpGNT1
Controls (no miRNA targets)
EF1
miRNAs cannot cleave target mRNAs in the mutant,
nevertheless transcript levels of target genes reduced.
Novel feed-back loop: Transcriptional control by miRNAs
Gruppe 1
miRNA160 regulates ARF Transcription Factors
PpARF1-1
PpARF1-10
PpARF1-2
PpARF1-11
PpARF1-3
PpARF1-4
PpARF1-12
PpARF1-5
Arabidopsis: 21 ARF Genes
Rice: 24 ARF-Genes
Physcomitrella: 15 ARF Genes
Gruppe 2
PpARF1-6
PpARF1-7
PpARF1-8
Group 1
•
•
•
PpARF1-9
PpARF1-9
PpARF1-10
PpARF1-11
PpARF1-12
Group
Gruppe
33
Group 2
PpARF3-1
PpARF3-1
Phypa_61245
PpARF3-2
PpARF3-2
(Tandemduplikation)
Tandem duplication
PpARF3-2
Phypa_61245
Tandem duplication
Group 3
PpARF3-1
miRNA160: Part of Multilevel Regulatory Network
Wild type
ARFm Mutant
Auxin
miRNA
160
Auxin
ARF
GH3-2
miRNA
160
ARF
GH3-2
Precise Manipulation of miRNA160 Network
Abolish miRNA160 binding site
without changing amino acids in the transcription factor
t ggc atg cag ggg gcc agg ca
G
M
Q
G
A
R
Pau I
t ggc atg caa ggc gcg cga ca
G
M
Q
G
A
R
Targeted base-specific mutation of endogenous gene!
Dynamic Modelling of 4-Level Regulatory Network
Endogenous
auxin synthesis
Auxin
application
Impact of GH3-2
on [Auxin]
Auxin:
Activation of miR160
by auxin
Activation of miR160
by ARF
miR160:
Activation of ARF
by auxin
Inhibition of ARF
by miR160
ARF:
Activation of GH3-2
by ARF
GH3-2:
3=0
in the
ARFm mutant
Expectations from Dynamic Modelling
[Expression]
2.0
Auxin
application
SteadyState
GH3-2
ARF
1.5
miRNA Mutant
ARF
1.0
GH3-2
miRNA
Wild
type
0.5
ARF
0
10
20
30
[Time]
40
50
Mutant: Elevated ARF mRNA levels
Relative Expression
ARF transcript levels respond to auxin
1,6
1,4
1,2
1
0,8
0,6
0,4
0,2
0
Control
1h
4h
8h
12h
Auxin treatment
WT
ARFm Mutant
24h
Elevated Transcript Levels of Downstream Gene
GH3 conjugates (= inactivates) auxin
Relative Expression
GH3-2 mRNA levels enhanced by auxin
2,5
2
1,5
1
0,5
0
control
1h
4h
8h
12h
Auxin treatment
WT
ARFm Mutant
24h
Auxin
GH3-2:
Auxin conjugating enzyme
relative expression
2,5
2
1,5
1
0,5
0
control
1h
4h
8h
NAA treatments
WT
Mutant
12h
24h
Weight]
Fresh
IAA [ng/g(ng/g
NAA
Menge
an +
IAA+NAA
Frischgewicht)
Elevated Levels of Conjugated (= Inactive) Auxin
freie und konjugierte Auxine
Free & conjugated
(IAA+NAA)Auxin
500
450
400
350
300
250
200
150
100
50
0
Free
Conjugated
freie
konjugierte
KOH NAA KOH NAA
WT
ARF
WT
A-B44
Mutant
Mutant is Hyposensitive to Auxin
Auxin
ARFm mutant confirms prediction.
WT
ARFm
Mutant
0
0.1 µM
0.5 µM
1 µM [Auxin]
Extended Model to Include Diurnal Changes
Current Modelling Approaches
The Players
Gotelinde Seumel
Basel Kraiwesh
Asif Arif
Erika Lang
Philipp Rödel
Marta Tomek
Jutta Ludwig-Müller
Olaf Rönneberger
Stefan Jansen
Andreas Schlosser
Daniel Lang
Marco Vervliet-Scheebaum
Wolfgang Frank
Jens Timmer