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

branching

type of branching

F-type branching,

left

F-type branching,

right

Y-type of branching

Growth!

no

yes

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

AGO

RISC

CAPAAAAA

Dicer

CAPAAAAA

CAPAAAAA

CAPAAAAA

CAPAAAAA

Nucleus

Cytosol

HASTY

HEN

HYL

AGO

RISC

pri-miRNA

pre-miRNA

Post-transcriptional regulation of gene expression

Animals and Plants: Different Sets of DICER

PLA

NTS

AN

IMA

LS

Targeted knockout of DCL1a: Drastic Effects

0.5 cm 0.5 cm 0.5 cm

WTWT

RT-PCR analysis of miRNAsRT-PCR analysis of miRNAs

DCL1aDCL1aKO1KO1

0.5 cm 0.5 cm 0.5 cm

DCL1aDCL1aKO2KO2

0.5 cm 0.5 cm 0.5 cm

WTWT KO1KO1 KO2KO2

miRNA156miRNA156

miRNA160miRNA160

miRNA166miRNA166

miRNA390miRNA390

RT-PCR analysis of target transcriptsRT-PCR analysis of target transcripts

0

0,001

0,002

0,003

0,004

0,005

0,006

0,007

SBP3 ARF1 PpC3HDZIP1 PpHB10 TAS1PpSBP3PpSBP3 PpARFPpARF PpHDZIPPpHDZIPPpHB10PpHB10 PpTAS1PpTAS1

77

66

55

44

33

22

11

00Rel.

Tra

nscri

pt

Level

Rel.

Tra

nscri

pt

Level

WTWT

KO1KO1

KO2KO2

No DCL1a: No miRNAs, enhanced TF levels,

serverely affected growth

Targeted knockout of DCL1b: Less Drastic Effects

DCL1bDCL1b

200 µm

100 µm

Wild TypeWild Type

WTWT 11 22 33 44PpDCL1b KOPpDCL1b KO

miRNA156miRNA156

miRNA160miRNA160

miRNA166miRNA166

miRNA390miRNA390

miRNA535miRNA535

miRNA538miRNA538

U6 snRNAU6 snRNA

miRNA ExpressionmiRNA Expression

miRNAs still present BUT developmental arrest

DCL1b: miRNAs Cannot Cleave Target mRNAs

Control PpGNT1Control PpGNT1(no miRNA target)(no miRNA target)

WTWTKOKO11

KOKO22

KOKO33

KOKO44

PpSBP3 (Squamosa Promoter Binding Protein)PpSBP3 (Squamosa Promoter Binding Protein)PpSBP3 5’ U 3’ GUGCUC CUCUCUUCUGUCA CACGAG GAGAGAAGACAGUmiR156 3’ U 5’

PpC3HDZIP (HD leucine zipper)PpC3HDZIP (HD leucine zipper)PpC3HDZIP1 5’CU U 3’ GG AUGAAGCCUGGUCCGG CC UACUUCGGACCAGGCUmiR165 3’CC C 5’

PpHB10 (PpHB10 (HD leucine zipperHD leucine zipper))PpHB10 5’CU U 3’ GG AUGAAGCCUGGUCCGG CC UACUUCGGACCAGGCUmiR165 3’CC C 5’

miRNA Target Genes Silenced in DCL1b Mutant

miRNAs cannot cleave target mRNAs in the mutant,

nevertheless transcript levels of target genes reduced.

Novel feed-back loop: Transcriptional control by miRNAs

miRNA target transcriptsmiRNA target transcripts

Controls (no miRNA targets)Controls (no miRNA targets)EF1EF1

PpHDZIP1PpHDZIP1

PpHB10PpHB10

PpARFPpARF

PpGNT1PpGNT1

WTWT 11 22 33 44

PpDCL1b-MutantsPpDCL1b-Mutants

PpSBP3PpSBP3

• Arabidopsis: 21 ARF-Gene

• Reis: 24 ARF-Gene

• Physcomitrella: 15 ARF-Gen

Gruppe 2

Gruppe 1

Gruppe 3

PpARF3-1

PpARF3-2 (Tandemduplikation)

PpARF1-1

PpARF1-2

PpARF1-3

PpARF1-4

PpARF1-5

PpARF1-6

PpARF1-7

PpARF1-8

PpARF1-9

PpARF1-10

PpARF1-11

PpARF1-12

Phypa_61245

Group 3

PpARF3-1PpARF3-2Tandem duplication

• Arabidopsis: 21 ARF Genes

• Rice: 24 ARF-Genes

• Physcomitrella: 15 ARF Genes

PpARF3-1

PpARF1-1

PpARF1-2

PpARF1-3

PpARF1-4

PpARF1-5

PpARF1-6

PpARF1-7

PpARF1-8PpARF1-

9

PpARF1-10

PpARF1-11

PpARF1-12

Phypa_61245

Gro

up

1G

rou

p 3

Gro

up

2

Tandem duplication

PpARF3-2

miRNA160 regulates ARF Transcription Factors

miRNA160: Part of Multilevel Regulatory Network

Wild type ARFm Mutant

ARFARFmiRNAmiRNA160160

AuxinAuxin

GH3-2GH3-2

ARFARFmiRNAmiRNA160160

AuxinAuxin

GH3-2GH3-2

Precise Manipulation of miRNA160 Network

Targeted base-specific mutation of Targeted base-specific mutation of endogenous gene!endogenous gene!

t ggc atg cag ggg gcc agg ca G M Q G A R

t ggc atg caa ggc gcg cga ca

Pau I

G M Q G A R

Abolish miRNA160 binding site Abolish miRNA160 binding site without changing amino acids in the transcription factorwithout changing amino acids in the transcription factor

Dynamic Modelling of 4-Level Regulatory Network

EndogenousEndogenousauxin synthesisauxin synthesis

Impact of GH3-2Impact of GH3-2on [Auxin]on [Auxin]AuxinAuxin

applicationapplication

Auxin:Auxin:

miR160:miR160:

Activation of miR160Activation of miR160by auxinby auxin

Activation of miR160Activation of miR160by ARFby ARF

GH3-2:GH3-2:

Activation of GH3-2Activation of GH3-2by ARFby ARF

ARF:ARF:

Activation of ARFActivation of ARFby auxinby auxin Inhibition of ARFInhibition of ARF

by miR160by miR160

33=0=0in thein the

ARFARFm mutantm mutant

Expectations from Dynamic Modelling

ARFMutant

2.02.0

1.51.5

1.01.0

0.50.5

001010 2020 3030 4040 5050

[Exp

ressio

n]

[Exp

ressio

n]

[Time][Time]

GH3-2GH3-2

GH3-2GH3-2miRNAmiRNA

miRNAmiRNA

ARFARF

ARFARF

Steady-Steady-StateState

AuxinAuxinapplicationapplication

Wildtype

Mutant: Elevated ARF mRNA levels

ARF transcript levels respond to auxinARF transcript levels respond to auxin

WT ARFm Mutant

0

0,2

0,4

0,6

0,8

1

1,2

1,4

1,6

Control 1h 4h 8h 12h 24h

Auxin treatment

Rela

tive E

xp

ressio

n

Elevated Transcript Levels of Downstream Gene

GH3 conjugates (= inactivates) auxinGH3 conjugates (= inactivates) auxin

GH3-2GH3-2 mRNA levels enhanced by auxin mRNA levels enhanced by auxinR

ela

tive E

xp

ressio

n

0

0,5

1

1,5

2

2,5

control 1h 4h 8h 12h 24h

Auxin treatment

WT ARFm Mutant

Elevated Levels of Conjugated (= Inactive) Auxin

AuxinAuxin

0

0,5

1

1,5

2

2,5

control 1h 4h 8h 12h 24h

NAA treatments

rela

tive

exp

ress

ion

WT Mutant

GH3-2:Auxin conjugating enzyme

Free & conjugated Auxin

Free

Conjugated

WT ARFMutant

NA

A +

IA

A [

ng

/g F

resh

Weig

ht]

Mutant is Hyposensitive to Auxin

WT

ARFmMutant

[Auxin][Auxin]00 0.1 µM0.1 µM 0.5 µM0.5 µM 1 µM1 µM

ARFARFm mutant confirms m mutant confirms prediction.prediction.

AuxinAuxin

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