SignalingNetworkinPlantCells

PlantSignalTransductionPathways
Signal

Receptor

?
?

Responses

Transcription

PlantSignalTransduction
AnIntegratedApproach
Genes
Proteins

Cells

Transgenics

Mutants

ArabidopsisMesophyllProtoplastsAreViableandResponsive

HighTransfectionEfficiencyofMesophyllProtoplasts

Maize
Arabidopsis

Maize

PEG-Calcium

Electroporation

FunctionalGenomicAnalyses
of
PlantSignalTransduction
in
Arabidopsis

Light&GlucoseSignalinginMaizeMesophyllProtopalsts

Dark

Light

Light & Glucose

ABASignalinginMaizeMesophyllProtoplasts

Control

ABA

CDPK

AuxinSignalinginMaizeMesophyllProtoplasts

CytokininSignalinginArabidopsisMesophyllProtoplasts

H2O2Signaling

inArabidopsis
MesophyllProtoplasts

Flg22SignalinginArabidopsisMesophyllProtoplasts

Functional Analysis of Plant MAPK Cascades

in Stress and Hormonal Signaling

Jen Sheen, MGH

Frederick M. Ausubel, MGH
Kan Wang, ISU

Plant Systems
Arabidopsis, maize, soybean

Sponsor
NSF Plant Genome Research Project

Why MAPK cascades?

MAPK cascades are central signaling components
that connect diverse receptors/sensors
to cellular and nuclear activities in plants

Stress
wounding, H 2O2, O3, cold, heat, salinity, drought/osmotic

Hormones

ethylene, ABA, auxin

Pathogens

virus, bacterial and fungal elicitors

Objectives
Developprotoplastassays
CloneallArabidopsisMAPKcascadegenes
Functionalanalysisinprotoplasts
FunctionalanalysisintransgenicArabidopsis
Functionalanalysisintransgenicmaize&soybean
Webaccessibledatabase

People&Assignments
JenSheenBinaryconstructs,TransgenicArabidopsis&Affychips
WanLingChiuMAKPs&PTPs
GuillaumeTenaMAPKKKs,MAPKKs&MAPKs
TsuneakiAsaiEarlyresponsegenes&Transcriptionfactors
JouliaPlotnikovaFungalinfection&TransgenicArabidopsis
SylviaShouTransgenicmaize
KanWangTransgenicsoybean
SenthilRamuGTPase&Receptors
CheriChenWebsite&Database
ShuichiYanagisawaTranscriptionfactors
RahulPatharkarMAPKsubstrates,Scaffoldproteins&Microarrays

Stimuli
Sensors/Receptors
ArabidopsisGenes

Mitogen
A ctivated
P rotein
K inase
C ascades

/Groups

MAPKKKs

>25

MAPKKs

MAPKs

23

TranscriptionFactors
PrimaryResponseGenes
SecondaryResponseGenes

FunctionalGenomicApproach

NPK1(MAPKKK)SuppressesAuxinSignaling

ProteinkinaseActivityAssay

Protein
Expression

Kinase
Activity

MKPBlocksNPK1RepressionofAuxinSignaling

ANP1(MAPKKK)SuppressesAuxinSignaling

EmbryogenesisDefectinNPK1TransgenicPlants

H2O2ActivatesTranscription&MAPKs

RedundantANPs

Mediate
H2O2Signaling
&MAPKActivation

ANP1ActivatesH2O2ResponsivePromoters

Cross Protection Phenomena in Plants

Cold
Heat
Drought
Wounding
Ozone
Pathogens
Common Messengers
H2O2
Calcium

DroughtResistance

TransgenicNPK1
PlantsDisplay
Salt,Freezing&Heat
Tolerance

Crosstalk between Stress & Auxin Signaling

AUXIN

H2O2, Heat

MAPKKK (ANP1-3, NPK1)

COP9
Signalosome
MAPKK ?
SCF (TIR1)
MAPK (AtMPK3 & 6)
PKs
IAA/AUX
ARFs

auxin genes

stress genes

(GH3, IAAs)

(GST6, HSP18.2)

PlantDefenseMechanisms
RecognizeEnemies
GeneforGeneInteractions

HostReceptor:Rgenes(Pto,N,Rps2,Cf9,Xa21)
PathogenElicitor:Avrgenes(avrPto,Avr9)

GeneralInteractions

HostReceptor:FLS2
PathogenElicitor:Flagellin(Flg22),OS,LP

PlantDefenseMechanisms
SignalTransduction
GeneralElicitors

SpecificElicitors
LRR R

R LRR

LRR

ConvergentSignaling

Ca++,PKsH2O2,ionflux

EarlyGenes:
TFs,SA,ET,JA

LateGenes:
PRs,PL

Flg22SignalingCascadein
PlantPathogenDefense

Convergent
Signaling ?
Cascade
?

Defense

Transcription

ArabidopsisLeafProtoplastSystem

Flg22SignalinginArabidopsisMesophyllProtoplasts

Flg22SignalingIsMediatedby
LRRReceptorKinaseFLS2

Flg22ActivationofMAPKsIsFLS2Dependent

Flg22 MAPK Signaling Cascade in

Plant Pathogen Defense
Flg22 peptide

FLS2 (LRR-RLK)

MAPKKK
MAPKK
MAPK

Defense

MAPK
WRKY
Transcription

Arabidopsis

Mammals
LRR Receptor

LRR Receptor

Elicitors

Drosophila
LRR Receptor

TLR2, 4, 5, 9

FLS2

Toll

Other LRR
Receptors
?

IRAK Kinase

FLS2 Kinase
?

Pelle Kinase

MEKK

TAK

AtMKK4/AtMKK5

MEK

NIK

AtMPK3/AtMPK6

JNK
ERK,p38

IKK

JNK
p38

AtMEKK1

Jun

WRKY
22/29

W W W

IkB

R?
Fos

NF-kB

Cactus
Dif/Rel

Immune Response Gene Transcription

Positive Feedback Control by WRKY 22/29

Disease Resistance

PlantMAPKSignalingCascades2001
Arabidopsis

Plant
Signal

Bacterial
Elicitor

Sensor

FLS2?

MAPKKK

AtMEKK1

MAPKK

MAPK

Oxidative
Stress

Alfalfa

Arabidopsis

Tobacco

Fungal
Elicitor

Salt
Stress

Abiotic
Sresses

Mitosis
Cytokinesis

AtHK1?

AtANP1
(NtNPK1)

AtMKK4/5
(NtMEK2)
(MsSIMKK)

AtMPK3/6

Tobacco

AtMPK3/6

NtNPK1

AtMKK1/2

NtMEK1**

(AtANP1)

NtMEK2

MsSIMKK

NtSIPK/WIPK

MsSIMK
(AtMPK6)
(NtSIPK)

(MsMMK2)

AtMPK4

NtNtf6**

(AtMKK4/5)
(MsSIMKK)

(NtWIPK/SIPK)
(MsSAMK/SIMK*)

AtMEKK1

(AtMPK3/6)
(MsSAMK/SIMK*)

(AtMKK4/5)
(NtMEK2)

(NtSIPKK)

(MsMMK3)

Target
Genes

PAL(+)

GST6,HSP18.2(+)
GH3,ER7()

HMGR(+)
PAL(+)

Negative
Regulator

AtMKP1?
AtPTP1?

MsMP2C?

AtMKP1?
AtPTP1?

AntagonisticInteractionsbetweenMAPKCascades
H2O2
Stress

Auxin

Unknown
Signal

Ethylene

AtANP1
MAPK
cascade

Unknown
MAPK
cascade

Signalintegration

etr1
mutant

OUTPUT

mpk4
mutant

NtSIPK
AtMPK6
MAPK
cascade

AtCTR1
MAPK
cascade

Signalintegration

AtMPK4
MAPK
cascade

Signalintegration

OUTPUT

JA

ctr1
mutant

PD98059
U0126

Unknown
MAPK
cascade

SA

OUTPUT

Summary
MAPK cascades play a central
role in intracellular signaling
MAPK cascades are involved in
stress and hormonal signaling
MAPK cascades mediate both
positive and negative controls
Manipulation of MAPK cascades
confers multiple stress protection

MAPKSignalingGroupNSF/DIP