Dong 2010

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C H A P T E R N I N E

StomatalPatterningandDevelopment

JuanDongandDominiqueC.Bergmann

Contents

1. Introduction 2682. TheSignalingCascade:FromExtracellularSignalstoNuclearFactors 270

2.1. Receivingandsendingsignals 2702.2. Receptorsatthecellsurface 271

2.3.

Extracellular

ligands

2732.4. Extracellularligandsnegativeregulators 273

2.5. Extracellularligandspositiveregulators 2752.6. Modelsforstomatalpeptideligandsandmembranereceptors

andfuturequestions 2763. SignalingthroughaMAPKinaseModule 278

3.1. The YDAMAPKsignalingmoduleinstomataldevelopment 2793.2. Dissectingoutthe YDAmodule:negativeorpositive? 280

4. TranscriptionFactors 2824.1. Genesthatbreaksilence 283

4.2.

Phosphorylation

links

the

MAPK

module

to

the

transcriptional

network 2864.3. Howdoesphosphorylationaffecttranscriptionfactorfunction? 287

5. RegulationofStomatalAsymmetricCellDivision 2885.1. Modelsofasymmetriccelldivision 2885.2. Stomatalasymmetriccelldivision 2895.3. Polarizedreceptorprotein:PAN1frommaize 2895.4. Polarizedandsegregatednovelprotein:BASLinArabidopsis 290

6. ConcludingRemarks 2937. KeyConclusions 293

References

293

Abstract

Stomataareepidermalporesusedforwaterandgasexchangebetweenaplant

andtheatmosphere.Boththeentryofcarbondioxideforphotosynthesisand

theevaporationofwater thatdrives transpirationand temperatureregulation

are

modulated

by

the

activities

of

stomata.

Each

stomatal

pore

is

surrounded

by

DepartmentofBiology,StanfordUniversity,Stanford,California,USA

Current

Topics

in

Developmental

Biology,

Volume

91

2010

Elsevier

Inc.

ISSN0070-2153,DOI10.1016/S0070-2153(10)91009-0 Allrightsreserved.

267

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268 JuanDongandDominiqueC.Bergmann

two highly specialized cells calledguard cells (GCs), andmay alsobe asso-

ciatedwithneighboringsubsidiary cells; thisentireunit is referred toas the

stomatal complex. Generation of GCs requires stereotyped asymmetric and

symmetriccelldivisions,andthepatternofstomatalcomplexes intheepider-

mis

follows

a

onecellspacing

rule

(one

complex

almost

never

touches

anotherone).Bothstomatalformationandpatterningarehighlyregulatedby

anumberofgeneticcomponentsidentifiedinthelastdecade,including,butnot

limitedto,secretedpeptideligands,plasmamembranereceptorsandreceptor

likekinases,aMAPkinasemodule,andaseriesoftranscriptionfactors.

Thisreviewwillelaborateonthecurrentstateofknowledgeaboutcompo-

nentsinsignalingpathwaysrequiredforcellfateandpattern,withemphasison

(1)a familyofextracellularpeptide ligandsand their relationship to theTOO

MANYMOUTHSreceptorlikeproteinand/ormembersoftheERECTAreceptor

like

kinase

family,

(2)

three

tiers

of

a

MAP

kinase

module

and

the

kinases

that

confernovelregulatoryeffectsinspecificstomatalcelltypes,and(3)transcrip-

tion factors that generate specific stomatal cell types and the regulatory

mechanisms for modulating their activities. We will then consider two new

proteins (BASL and PAN1, from Arabidopsis and maize, respectively) thatregulatestomatalasymmetricdivisionsbyestablishingcellpolarity.

1. Introduction

Stomata

(mouths in Greek) are pores used for water and gasexchange between a plant and the atmosphere. Each stomatal pore issurrounded by two highly specialized cells called guard cells (GCs), andmay also be associated with neighboring subsidiary cells (SCs); this entireunit isreferredtoasthestomatalcomplex.Stomataarefound indifferentaerial

organs

and

in

species-specific

patterns;

nonetheless,

in

nearly

all

plants

studied,

the

generation

of

stomata

requires

asymmetric

and

symmetric

cell

divisions,

and

the

pattern

of

stomatal

complexes

in

the

epidermis

follows

a

spacing

rule

whereby

two

stomatal

complexes

are

always

separated

by

at

least

one

intervening

epidermal

cell.

Stomatal

development

inArabidopsis thalianarequiresadedicatedseries

of

asymmetric

cell

divisions

(ACDs)

followed

by

a

single

symmetric

cell

divisiontoformapairofmatureGCs(Fig.9.1;Geisleretal.,2003;NadeauandSack,2003).Inthedevelopingepidermisofyoungleaves,thestomatallineageisinitiatedfromasubsetofcommittedprotodermal(Pr)cellscalledmeristemoid mother cells (MMCs). ACD of an MMC generates two

daughter

cells:

a

small,

triangular

cell

called

a

meristemoid

and

a

larger

cell

called

a

stomatal

lineage

ground

cell

(SLGC).

This

type

of

division

is

also

referred to as an entry division, because it creates stomatal lineage cells.Meristemoidsmayfolloweitheroftwopaths(Fig.9.1);theymaytransitioninto

a

round-shaped

guard

mother

cell

(GMC)

and

divide

once

to

make

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269StomatalPatterningandDevelopment

MMC

M

SLGC

GMC

M

GMC

M

GC

Pr

PC

Entry

Amplifying

Spacing

Transition Differentiation

TMM

TMM

TMMERERL1ERL2

ERERL1

ERL2 ER

TMM

ERL1,ERL2

ERL2

Figure9.1 SchematicforstomataldevelopmentandregulationbyTMMandmembers

of

the

ERECTA

family

(ERf)

receptors.

Stomatal

development

initiates

frommeristemoid mother cells (MMCs), aset of committedprotodermal(Pr) cells. In the

stomatalentrydivision,asymmetriccelldivisionofanMMCgeneratesasmalldaughtercell,themeristemoid(M),andalargerdaughtercell,thestomatallineagegroundcell(SLGC).Ameristemoidmayimmediatelytransitionintoaguardmothercell(GMC)andfurther divide and differentiate into guard cells (GC), or it may divide againasymmetrically, up to three times, amplifying the number of cells in the stomatallineage, before transitioning into a GMC. SLGCs may divide asymmetrically in aspacing division to generate secondary meristemoids, which are always spaced awayfromtheexistingstomatalprecursorcell.RegulationofdevelopmentalprogressionbyTMMER,ERL1,andERL2ismarkedateachstepwheretheyfunction,withpositive

effects

labeled

with

black

arrows

and

negative

effects

labeled

with

gray

T-bars.

Font

sizeusedforTMMandERf indicatestherelativestrengthofeachgeneseffect,withlarger

fonts

signifying

stronger

effects.

twoGCs,ortheymaydivideagainasymmetrically.Continuedasymmetricdivisions of meristemoids (up to 3 times for each) are termed amplifyingdivisions.ThissecondpathservestoamplifythenumberofSLGCs,whichare major contributors to the population of both stomatal and pavement

cells

in

the

leaf.

SLGCs

can

directly

expand

and

differentiate

into

pavement

cells,

or

they

may

also

undergo

an

ACD

to

produce

a

secondary

meristemoid.WhenSLGCsproduce secondarymeristemoids, theyalwaysdividein

such

a

way

that

this

new

cell

is

not

next

to

existing

meristemoids

or

stomata.

This

additional

level

of

regulation

using

orientation

of

ACDs

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enforces

the

so-calledone-cell-spacingrulethattypifiesstomatalpattern

inArabidopsisandnearlyallotherplants.

Inmonocotplants,suchasmaizeandrice,stomataarealsoseparatedby

at

least

one

intervening

nonstomatal

cell,

but

the

developmental

events

that

create

and

pattern

stomata

are

quite

different

(Hernandez

et

al.,

1999).

For

example,maizestomataarelinearlyarrangedinspecificcellfilesintheleafepidermis.AnindividualmaizestomatalcomplexiscomposedofapairofGCs flanked by a pair of SCs. GC development in maize starts from anasymmetricdivisionthatgeneratesasmallerdaughtercellastheGMC.TheGMC then signals to two lateral cells from the neighboring cell files torecruit

them

to

become

subsidiary

mother

cells

(SMCs).

SMC

nuclei

become polarized by migrating toward the GMC; subsequently, the

SMCs

divide

asymmetrically

to

produce

small

SCs

that

flank

the

GMC,

and

will

become

accessory

cells

to

help

the

stomatal

pore

open

and

close.

The

pore

is

formed

in

the

next

step

when

the

GMC

divides

symmetrically

and

the

daughters

differentiate

into

a

pair

of

dumbbell-shaped

GCs.

The developmental processes and patterns that make up the stomatalsystemsinbothArabidopsisandmaizearerelativelysimple,easilyscored,andaccessibleforgeneticandexperimentalmanipulation.Also,stomataldevelopment

heavily

relies

on

ACD,

which

is

one

of

the

most

commonly

used

mechanisms for generating cellular diversity and overall morphology in

multicellular

organisms.

Therefore,

specific

studies

of

the

molecular

mechanisms

underlying

stomatal

development

can

facilitate

our

general

understanding of many fundamental biological issues such as intracellularand

intercellular

communication,

signal

transduction

cascades,

cell

fate

determination,

establishment

of

cell

polarity,

and

the

information

for,

and

mechanics

of,

ACD.

2. TheSignalingCascade:FromExtracellular

Signals

to

Nuclear

Factors

2.1.

Receiving

and

sending

signals

Plant

growth

and

development

requires

a

tremendous

amount

of

cell-to-cell

signaling

and

communication.

Crosstalk

mediated

by

extracellular

peptide

ligands

and

cell

surface

receptors

is

a

key

component,

especially

in

maintainingthebalancebetweenstemcellpopulationsanddifferentiatedcellsintheshoot

and

root

apical

meristems

and

vascular

cambium

(Byrne etal., 2003;

Gray

et

al.,

2008;

Stahl

and

Simon,

2009).

In

plants,

the

term

stem

cells

is

typically

used

to

refer

to

a

small

population

of

undifferentiated

cells

at

the

root

and/orshootapicalmeristem.Bydividingasymmetrically,stemcellsgenerateonedaughterthatwillbecomeoneofthedifferentiatedcellsdrivingorganformation, but the other daughter will retain stem cell properties, thus

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maintaining

a

constant

size

to

the

stem

cell

pool.

Peptide

ligands,

such

as

the

ones

containing

the

conserved

CLAVATA/ESR

(CLE)

motif,

are

secreted

from the meristematic cells and perceived by a set of receptors in the

neighboring

cells

(Fiers

et

al., 2007).

The

receptors

are

typically

Leucine

rich

repeat

(LRR)

receptor-like

proteins

and/or

kinases

(LRR-RLPs

and/

orLRR-RLKs).Ligandbindingisthoughttoactivatethereceptorsandtheirdownstreamsignalingcascadetopromotedifferentiationandtherebypreventthe neighbor cells from adopting stem cell fate and expanding the pool(chapters3and4;Fletcheretal.,1999;Itoetal., 2006).

Two types of cells in the stomatal lineage population, MMCs andmeristemoids,

behave

in

a

stem

cell-like

manner,

such

that

these

cells

producestwodaughtercells,oneofwhichcanmaintainitsMMCproperty

of

further

division

potential

and

the

other

which

will

generally

differentiate

into

a

pavement

cell.

In

contrast

to

the

stem

cells

in

the

apical

meristems,

however,

stomatal

MMCs

and

meristemoids

are

not

in

niches.

Instead,

they

are

dispersed

on

the

leaf

epidermis

and

their

stem

cell

activity

is

transient.

Therefore,itisnotsurprisingthatsignalingcomponents,includingligandsand receptors, modulate stomatal development and patterning, but theexact ligandsandreceptorsaredistinct from thosepreviouslydescribed inapical

meristems.

Newly

discovered

intercellular

signals

include

the

small

secreted proteins EPIDERMAL PATTERNING FACTOR 1 (EPF1)

(Hara

et

al.,

2007),

EPF2

(Hara

et

al.,

2009;

Hunt

and

Gray,

2009),

CHALLAH

(CHAL)

(Abrash

and

Bergmann,

2010),

and

STOMAGEN

(Kondo et al., 2009; Sugano et al., 2009), all of which belong to a smallsubfamily

only

distantly

related

to

the

CLE

family.

2.2.

Receptors

at

the

cell

surface

While

the

ligands

are

quite

recent

discoveries,

the

presumptive

receptors

wereamongthefirstcomponentsshowntoregulatestomataldevelopment

and

patterning

(Yang

and

Sack,

1995).

They

include

a

receptor-like

protein

TOO

MANY

MOUTHS

(TMM)

(Nadeau

and

Sack,

2002)

and

three

receptor-like kinases, ERECTA (ER), ERECTA-LIKE 1 (ERL1), andERECTA-LIKE 2 (ERL2) (Shpak et al., 2005), all containing an extracellularLRRdomain.ThethreekinasesarealsocollectivelyreferredtoastheERECTA family (ERf).These fourputative receptorsarecommonlydescribed as negative regulators of stomatal development, mainly becauseloss

of

function

of TMM and ERf genes results in overproliferation of

stomataintheleafepidermis.

The

source

of

the

excessive

number

of

stomata

in

the

tmm

mutant

was

carefully

analyzed

and

found

to

be

a

combination

of

three

general

defects

including

(1)

more

cells

became

MMCs

and

underwent

entry

divisions;

(2)

each

meristemoid

underwent

fewer

amplifying

divisions,

thereby

becoming

a GC more rapidly; and (3) divisions that create secondary meristemoids

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were

misoriented,

often

placing

a

new

meristemoid

right

next

to

a

GC

pair

or

a

precursor

(Nadeau

and

Sack,

2002).

TMM

expression

is

strong

in

the

early stages of the stomatal lineage, with the highest level in theyoung

MMCs

and

meristemoids

and

a

lower,

but

detectable

level

in

SLGCs.

TMM

is

absent

from

mature

GCs.

These

mutant

phenotypes

and

expression

patternsleadtothehypothesisthatTMMintheMMCsandmeristemoidsfunctions in an autocrine loop to inhibit divisions in these cells, whereasTMMintheSLGCsperceivespositionalcuesonthecellsurfacetocontrolandorientthesecondarymeristemoidformingdivisions(NadeauandSack,2002).

The

TMM

protein

contains

extracellular

and

transmembrane

domains,

butlacksanintracellularkinasedomain,suggestingthatTMMmightparti

cipate

in

signal

transduction

through

interaction

with

other

partners

at

the

plasma

membrane.

The

TMM-interacting

kinase

partners

are

likely

to

be

the

ERf

LRR-RLKs,

which

were

also

characterized

as

a

set

of

key

regulators

in

stomatal

production

and

patterning

(Shpaketal.,2005). Incontrastto tmm

whoseapparentmutantphenotypesarerestricted tostomataldevelopment,theerfmutantshavepleiotropicphenotypes,implicatingtheminthecontrolofcellproliferationforpatterningandorgandevelopment,inplantresponsesto

environmental

stresses,

and

even

in

phytohormone

perception

(reviewed

in van Zanten et al., 2009). In terms of stomatal development, the triple

mutant

er;erl1;erl2

exhibits

severe

stomatal

overproliferation

and

spacing

defects

(Shpak

et

al., 2005).

The

three

ERf

genes

largely

function

redundantly

and synergistically in regulation of stomatal divisions, since these dramaticphenotypes

are

only

revealed

in

the

triple

mutant.

However,

examination

of

the

phenotypes

of

single

and

combinations

of

double

mutants

ofer,erl1, and

erl2 in the leaf epidermis shows each family member plays subtly differentroles(Fig.9.1),forexample,ERispredominantlyinvolvedininhibitionofMMC

divisions,

but

may

also

promote

meristemoid

differentiation

into

GMCs

(and

eventually

GCs);ERL1generallyinhibitsmeristemoiddifferen

tiation

and

ERL2

appears

to

promote

continued

asymmetric

division

of

the

SLGCs

(Shpak

et

al., 2005).

ThefunctionsofTMMandERf,and,especially,theirgenetic interactions, are fairly complicated. One difficulty is that different genes playdifferent rolesdependingon specificorgans examined (NadeauandSack,2002; Shpak et al., 2005). For example, the tmm mutant has an elevatednumber of stomata on leaves (consistent with a negative role in stomataldevelopment),yettmmisdevoidofstomataonstems(indicatingapositiverole,

Yang

and

Sack,

1995).

These

roles

are

not

completely

opposite;

in

stems,

the

absence

of

stomata

is

due

to

transdifferentiation

of

meristemoids

into

pavement

cells,

not,

as

might

be

expected

from

the

phenotype

in

leaves,becauseMMCdivisionsareabsent (Bhaveetal.,2009).CombinationsofthetmmmutationwithmutationsintheERfmembersalsoresultinunpredictable

organ-specific

phenotypes.

For

example,

elimination

of

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ERL1inatmmmutantbackgroundrestoresstomatatothetmmstems,butelimination

ofER in tmmsiliquesnearlyeliminates stomatalproduction,

althougheach singlemutant tends to increase thenumberof stomata in

this

organ

(Shpak

et

al., 2005).

A

model

derived

from

phenotypic

analysis

of

various

TMM

and

ERf

mutant

combinations

in

various

organs

was published by Shpak et al. (2005); we will revisit this model, incorporating new information from studies of ligands, in a later section ofthis review.

2.3.

Extracellular

ligands

TheArabidopsis genome contains more than 200 LRR - RLKs which

mediate

signaling

through

association

of

their

extracellular

LRR

domainswithachemicallyheterogeneouscollectionof ligandmoleculesincluding steroid hormones, peptides, and small proteins (Torii, 2004).TMMand/ortheERfare likelyengagedbyfour ligandsfromthesameprotein subfamilyEPF1, EPF2, CHAL, and STOMAGEN (Abrashand Bergmann, 2010; Hara et al., 2007, 2009; Hunt and Gray, 2009;Kondo et al., 2009; Sugano et al., 2009). As we detail below, thesepeptide ligands enrich our understanding of the potential regulatoryspectrum;

they

may

signal

from

within

the

stomatal

lineage

or

from

underlying

tissues,

they

may

function

as

positive

or

negative

regulatorsof

stomata,

and

they

display

different

requirements

for

the

TMM

and

ERf receptors.

2.4.

Extracellular

ligandsnegative

regulators

Both

EPF1

and

EPF2

were

discovered

by

their

effects

on

stomatal

development and patterning in a genome-wide overexpression screen of pre

dicted

small

secreted

proteins

(Hara

et

al.,

2007,

2009)

or

by

co-expression

with

known

stomatal

regulators

(Hunt

and

Gray,

2009).

Cauliflower

mosaic

virus35S(35S)promoter-drivenoverexpression(OX)ofEPF1(EPF1-OX)or EPF2 (EPF2-OX) reduces stomata density in the leaf epidermis, butEPF2-OXinhibitstheearliestasymmetricdivisions,resultinginanepidermiscomposedentirelyofpavementcells(Haraetal.,2009;HuntandGray,2009).EPF1-OXinhibitsstomataformationatalaterstagethanEPF2-OX;although

EPF1-OX

leaves

also

lack

mature

stomata,

asymmetric

divisions

canbeobserved(Haraetal.,2007).

Consistent

with

their

negative

roles

in

the

control

of

stomatal

production,

the

loss-of-function

mutants

epf1

and

epf2

displayed

elevated

numbers

of

stomata.

Careful

examination

of

the

mutant

phenotypes

showed

that EPF1 acts mainly on orientation of the spacing

division, so that without EPF1, the stomatal one-cell-spacing rule is

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disrupted

and

a

slightly

higher

number

of

GC

clusters

are

generated

(Hara et al., 2007).EPF2 acts earlier to restrict Pr cells from acquiring

stomatal lineagecell fates; epf2mutantsdisplaymoreentrydivisionsand

mutant

leaves

feature

clusters

of

small

stomatal

lineage

cells

that

do

not

always

become

stomata

(Hara

et

al.,

2009;

Hunt

and

Gray,

2009).

The

cell-type-specificexpressionpatternsofthese ligandscontributestotheirspecific cellular functions. EPF2 is expressed at earlier stages (MMCsand meristemoids) than EPF1 (meristemoids, GMCs, Fig. 9.2A) (Haraet al., 2007, 2009; Hunt and Gray, 2009). BothTMM andERf mutations are fully or partially epistatic to epf1 and epf2 and can block theoverexpression

effects

of

EPF1

and

EPF2,

supporting

the

idea

that

EPF1 and EPF2 function through these receptors.

EPF1

and

EPF2

can

explain

how

to

regulate

two

lineage

decisions

in

many

organs.

However,

they

do

not

solve

the

mystery

of

tmms opposite

phenotypes

in

leaves

versus

stems.

Identification

and

characterization

of

their

paralog

CHAL/EPFL6,

however,

may

provide

a

reasonable

explanationforthesetmmmutantphenotypes.Thechalmutantwasidentifiedasatmm suppressor that restored stomata to tmm hypocotyls and stems. Thephenotypesofchalare genotype specific, in that they are only revealed in a tmmbackground,andarelargelyorganspecific,inthatthephenotypesareobservedinhypocotylsandstems,butnotinleaves.LikeEPF1andEPF2,

35S-driven

CHAL-OX

reduces

stomatal

density

in

all

organs.

Also

in

parallel to EPF1 and

EPF2, mutations

in

ERf

are

genetically

epistatic

to

CHAL.Overexpressionexperiments inERffamilydoublemutants suggest

thatER,ERL1, and ERL2areallcapableoftransducingtheCHAL

signal

(Abrash

and

Bergmann,

2010).

Where

CHAL

differs

from

EPF1

and

EPF2

is

in

its

expression

pattern and relationship with TMM. Together these two features helpexplain

why

no

stomata

are

produced

in tmm stems. CHAL is not

expressed

in

stomatal

lineage

cells

but

is

produced

in

internal

tissues,

especially

in

cells

surrounding

the

vasculature

(Abrash

and

Bergmann,

2010).

This

internal

expression

reaches

a

maximum

in

stems

and

in

embryonic hypocotyls. CHAL does not appear to need TMM for itsfunction; in fact TMM appears to inhibit CHAL signaling because theability of CHAL-OX to repress stomatal formation is dramaticallyenhanced in a tmm background.

The current model is that CHAL is normally a ligand for the ERfs;in

stems,

the

high

level

of

CHAL

would

engage

these

receptors

to

inhibit

stomatal

production;

however,

TMM

normally

acts

toprotect

the

ERfs

from

CHAL,

allowing

stomata

to

be

produced

(Fig.

9.2A and

B). When TMM

is

absent, CHAL overactivates

its ERf

receptors and

stomatal production in stems is eliminated (Abrash and Bergmann,2010).

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(A)

M

SLGC

Leaf

EPF1

Orien-tateddivision

Internalcell

Epidermal cells

Stem

CHAL

STOMAGEN

EPF1 EPF2

SLGC

MMC/MMMC

Pr

EPF2

Stomatal lineageproduction

STOMAGEN

(Mesophyll)

STOMAGEN

(Mesophyll)

Leaf

Stomatal lineageproduction

Stomatallineageproduction

EPF1/2 STOMAGENCHAL

Stomataproduction

(B)ERfTMM

Signal Signal

No signal No signal

Signal Signal


Figure9.2 Speculativemodelsforinteractionsamongstomatalligandsandreceptors.(A)Inleavesandstems,ligandsdifferentiallyregulatestomatalcellfates.Protodermal(Pr)

cells

or

the

stomatal

lineage

ground

cell

(SLGCs)

in

leaves

receive

EPF1

and

EPF2

signals from the neighboring MMC or meristemoid, inhibiting the acquisition ofstomatal lineage fate. These cells also receive the STOMAGEN signal from theunderlying mesophyll thatpromotes stomatal lineage cell fate. In stems, SLGCs areadditionally subject to inhibitory CHAL signals coming from internal tissues. (B)Possible

regulatory

interactions

among

ligands

and

receptors.

If

the

receptors

all

act

as

negative regulators of stomatal development, then EPF1 and EPF2 could activate

homo- or

heterodimers

of

the

ERf

and

TMM.

TMM

could

dampen

the

CHALERf

inhibition of stomatal production activity in stems by sequestering CHAL in anonfunctional complex, or by the CHALTMM complex antagonizing activeCHALERfs. STOMAGEN, as a positive regulator, might compete with negativeregulators, EPF1 and EPF2, to bind receptor complexes. However, if TMMERfreceptors also act aspositive regulators, then STOMAGEN could stimulate stomatal

productionbyassociatingdirectlywiththeTMMERfcomplexes.

2.5.

Extracellular

ligandspositive

regulators

The

negative

regulators

EPF1,

EPF2,

and

CHAL

can

explain

much

about

stomatal development and pattern. But are all the intercellular signalsguiding this developmental pathway negative? The answer appears to be

7/25/2019 Dong 2010

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communication

among

these

players

orchestrated

to

determine

whether

a

given

cell

will

ultimately

become

a

stomatal

GC?

Thereare similaritiesand importantdifferences in the structure,beha

vior,

and

expression

patterns

of

the

ligands.

Although

clearly

part

of

the

same

protein

family,

there

are

sequences

unique

to

each

of

EPF1,

EPf2,

CHAL,andSTOMAGEN,mostnotablyintheregionoftheproteinthatformsasurface-exposedloopinSTOMAGEN(Kondoetal.,2009).Thesedifferences could lead to differential binding of receptors (CHAL vs. theTMMligands)ordifferentconsequencesofbinding(STOMAGENvs.thenegative regulators). The ligands also show unique expression patterns:EPF1

and

EPF2

are

expressed

in

different

stomatal

lineage

cell

types

but

STOMAGENandCHALarenotexpressedinthestomatallineage;instead,

they

are

secreted

from

mesophyll

cells

of

aerial

organs

and

the

internal

tissue

of

stems,

respectively,

to

regulate

epidermal

cell

fate

(Fig.

9.2A)

(Kondo

etal., 2009;Sugano etal.,2009;AbrashandBergmann,2010).Promoterswaps

between

EPF1

and

EPF2

suggest

that

some,

but

not

all

of

the

differencesintheirfunctionareduetoexpressionpattern(Haraetal.,2009).Before any ligands were identified, the models for TMM and ERf

functions were that ligand-bound ERf actively transduced a negativeregulatory

signal

and

that

TMM

could

modulate

ERf

functions

by

either dimerizing with ERfs to form a malfunctional receptor complex

or

by

binding

to

and

titrating

away

a

limiting

pool

of

ERf

ligands

(Shpak

et

al., 2005).

Now

that

we

know

four

ligands,

we

can

revisit

this

model, modifying the possibilities based on new data. We must nowconsider

that

receptors

mediate

signaling

that

results

in

opposite

outcomes,

as

with

EPF1,

EPF2,

and

STOMAGEN.

How

might

this

work?

If

we

assume

that

the

receptors

always

produce

a

stomatal

repressive

signal when activated, and that EPF1/2 are active ligands, then aninactive STOMAGEN could compete with them for associationwith

the

receptors,

thereby

preventing

the

receptors

from

sending

a

repressive

signal

(Fig.

9.2B).

Alternatively,

activated

receptors

might

be

able

to

produce

both

stomatal

promoting

and

stomatal

repressing

signals

depending on which receptors and which ligands are in a particularcomplex (Fig. 9.2B). The data on CHAL also make it clear that not allligands will bear the same relationships to all receptors. If CHAL is a ligand for each of the ERf members, but does not act through TMM,then two reasonable regulatory scenarios are that CHAL binds toTMM,but this formsanonfunctionalassociation that serves todampenthe

stimulatory

ligandreceptor interaction between CHAL and ERf

(Fig.

9.2B),

or

that

ERf

ERf

and

ERf

TMM

complexes

compete

and

CHAL

only

participates

in

signaling

through

ERfERf

complexes;

the

absenceofTMMwould shift thebalanceofreceptorcomplexes towardmoreCHAL-responsiveones (Fig.9.2B).Note that in thesemodelswehave

not

yet

addressed

organ-type

specificity.

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It

is

exciting

that

secreted

peptide

ligands

from

the

same

subfamily

are

stomatal

regulators

with

a

range

of

signaling

properties.

This

greatly

improves our understanding of how cellcell communication determines

cell

fate.

But

there

are

still

quite

a

few

pieces

missing

from

the

puzzle,

for

example,

the

genetic

interaction

between

STOMAGEN

and

the

ERfs

has

notyet been investigated, nor have all combinations of receptors beentestedwithEPF1,EPF2,orCHAL.Otherquestionsoffutureinterestare:

DothecharacterizedligandsindeedphysicallyinteractwithTMMand/orERf,

as

CLV3

does

with

CLV1?

How

many

(if

any)

versions

of

receptor

complexesareformedamongTMMandtheERf?Whatarethepreferredpartners?Biochemicalmethods,suchasinvitroassaysforbindingofCLV3toCLV1(Ogawaetal.,2008),orcell-basedassayssuchasFRETmaybe

needed

to

rigorously

identify

and

test

the

relevant

physical

associations.

Areallofthesepeptideligandspost-translationallyprocessedandmodified?If

so,

how?

The

functional

domains

of

some

CLE

proteins

can

be

restricted

totheshortconservedCLEmotif,itselfhydroxylatedattwoprolineresidues(Itoetal.,2006;Kondoetal.,2006).ThematureformsofCHAL,EPF1,andEPF2

have

not

been

elucidated

yet,

but

biochemical

methods

used

on

STOMAGEN

would

be

ideal

for

characterizing

these

ligands.

DootherEPFsorotherligandclassesparticipateinstomataldevelopment?SDD1,asubtilisin-likeserineprotease,hasbeenknownformanyyearsto

regulate

stomatal

development

and

pattern

(Berger

and

Altmann,

2000;VonGrolletal.,2002).Thisclassof subtilaseshasbeen shown, inother

cases,

to

process

extracellular

ligands.

However,

based

on

genetic

analysis,

EPF1, EPF2, STOMAGEN, and CHAL act independently of SDD1(Abrash and Bergmann, 2010; Hara et al., 2007, 2009; Kondo et al.,2009). It would be intriguing to uncover the substrates of SDD1, someofwhichcouldrepresentnewligandsactingthroughTMMandERf,andmay

also

provide

some

hints

about

ligand

processing.

AreTMMandEPfreceptors sufficient tomediateallstomatal signaling

events?

Based

on

studies

of

signal

transduction

in

the

shoot

apicalmeristem, another class of receptors, those possessing a kinase domain

but

no

extracellular

domains,

and

exemplified

by

CORYNE,

form

complexes

with

the

TMM-like

RLPs

(Mller et al., 2008). The

possibility exists for the CORYNE type (or other unknown receptors)toalsoparticipateinstomataldevelopmentsignals.

3. SignalingthroughaMAPKinaseModule

Regardless of the details of ligand and receptor interactions, theinformation

originating

at

the

cell

surface

must

be

relayed

to

targets

inside.

InArabidopsis, someof theclearestandmostcomplete signal transduction

information

comes

from

studies

of

plant

innate

immune

responses.

Here,

a

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mitogen-activated

protein

kinase

(MAPK)

signaling

cascade

lies

downstream

of

a

LRR-RLK,

FLS2,

whose

activity

is

triggered

by

binding

flagellin peptide. Ultimately, the responses involve altered transcription,

trafficking,

and

growth

(Gomez-Gomez

and

Boller,

2002).

Recent

research

progress

reveals

that

the

paradigm

of

MAPK

signaling

cascades

functioning

downstream of LRR receptors applies to stomatal development as well(Bergmannetal.,2004;Lampardetal.,2009;Wangetal.,2007).

3.1.

The

YDA

MAPK

signaling

module

in

stomatal

development

MAPK

cascades

are

evolutionary

conserved

across

eukaryotes

and

play

fundamental roles regulating numerous biological processes, including

growth,

development,

and

stress

responses

(reviewed

in

Colcombet

and

Hirt,

2008;

Widmann

et

al., 1999).

The

typical

three-step

phosphorylation

relayproceedsfromaMAPKkinasekinase(MAPKKK),toaMAPKkinase(MAPKK),

and

to

a

MAP

kinase

(MAPK)

to

regulate

the

activity

of

target

proteins

by

phosphorylation.

TheArabidopsisgenomeencodesalargenumber

ofcomponentsintheMAPKmodule,themajorityofwhichareassociatedwith plant responses to stresses (Colcombet and Hirt, 2008). Interestingly,MAPK

modules

consisting

of

one

MAPKKK,

YODA

(YDA),

four

MAPKKs

(MKK4/5/7/9),

and

two

MAPKs

(MPK3/6)

appear

to

regulate

stomatal

development

as

well

as

contributing

to

stress

responses

(Colcombet

and

Hirt,

2008).

The

existence

of

this

shared

module

may

reflect

one

of

the

innatesystemsthatenablearapidandflexiblemodulationofplantdevelopmentalprocessesinresponsetoexternalstresses.

Theconnectionbetween individualMAPKsignalingcomponentsandstomatal development came from several experimental sources. TheMAPKKKYODA (YDA) was identified in a screen for stomatal patterndefects (Bergmann et al., 2004). Soon after, MAPKK4/5 and MAPK3/6were

identified

by

a

group

interested

in

stress

responses

who

noticed

the

stomatal

phenotypes

in

double

mutants

(Wang

et

al.,

2007).

Two

additional

MAPKKs

(MAPKK7/9)

were

identified

later

in

a

stomatal-targeted

constitutive activity screen (Lampard et al., 2009). The phenotypes of ydamutants,

the

double

mutant

ofmpk3;mpk6, or simultaneous downregula

tion

of

MKK4

and

MKK5

by

RNA

interference

(RNAi)

are

similar

and

resemble

those

of

the er;erl1;erl2 mutant. Each results in an excessive

number of clustered stomata, indicating that MAPK signaling regulatesboth stomatal density and distribution. Consistent with this, expressingconstitutively active versions ofYDA (CA-YDA, Bergmann, 2004) or of

NtMek2

(a

MKK4/5

homolog,

Wang

et

al.,

2007)

completely

represses

stomatal

production.

Genetic

analysis

places

YDA

downstream

of

TMM,

which is based upon the fact that one copy of CA-YDA (which has nophenotypeon itsown) suppresses tmm stomataloverproliferationandpatterning defects (Bergmann, 2004). Based on similar genetic evidence,MKK4/5

and

MPK3/6

act

downstream

of YDA (Wang et al., 2007;

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Lampard etal.,2009), andall these factors functionasnegativeplayers in

stomatal

development.

It

is

interesting

to

note

that

only

YDA

has

a

noticeable effect on stomatal development as a single mutant. The redundant

activities

of

MPK3

and

MPK6

(as

MAPKs)

or

MKK4

and

MKK5

(as

MAPKKs)

were

first

documented

in

regard

to

regulation

of

plant

responses

tobioticandabioticstresses,anditappearsthatthisisalsotrueforregulationofstomataldevelopmentandpatterning.

3.2.

Dissecting

out

the

YDA

module:

negative

or

positive?

Components of the MAPK cascade are generally expressed at low levelsthroughouttheplant.Somesubstantiallyimpactplantgrowthanddevelop

ment

at

early

stages;

for

example,

yda

mutants

form

malformed

embryos

and

the

plants

are

severely

dwarfed

and

sterile

(chapter

1;

Lukowitz

et

al., 2004) and

thedoublemutantsofmpk3;mpk6areembryolethal(Wangetal., 2007).ThepleiotropicphenotypesgeneratedbysystemicmanipulationofMAPKsignaling

(for

example,

via

loss-of-function

mutants,

gene

overexpression,

or

expression

of

constitutive-active

versions)

may

hinder

researchers

from

dissecting

the

roles

of

individual

MAPK

components

in

specific

plant

developmental

contexts.Recently, Lampard et al. (2009) assayed the impact of the MAPK

cascade

members

on

specific

stomatal

cell

types

by

utilizing

a

set

of

stomatal

lineage

promoters.

These

promoters

were

based

on

a

set

of

transcription

factors(SPEECHLESS(SPCH),MUTE,andFAMA)whosespecificfunctionswillbediscussedindetailbelowinsection4.1.Briefly,thesepromoters drive expression in (1) cells that undergo entry divisions (SPCHpro),(2)cellstransitioningfrommeristemoidtoGMC(MUTEpro),and(3)cellstransitioning

from

GMC

to

GCs

(FAMApro).

By

this

strategy,

the

altered

activity

of

MAP

kinase

components

is

limited

to

particular

subset

of

stomatal cells, therefore substantially reducing phenotypes that are linked to

other

developmental

events.

Lampard

et

al.

first

assayed

two

variants

of

YDA,

including

a

dominant

negative

form

and

a

constitutively

active

form,

and

constitutively

active

versionsof six selected MAPKKs drivenbycell-type specificpromoters toexamine their function in stomatal development at each aforementionedstage.Asexpected, when expressed at theearliest stage (SPCHpro),YDA,MKK4,andMKK5behavedasnegativeregulators in the stomatal lineage,specifically

inhibiting

stomatal

formation

without

introducing

developmental

defects outside of the epidermis (Lampard et al., 2009). This study then

expanded

our

understanding

of

MAPK

cascade

control

of

stomatal

development

by

demonstrating

that

YDA

negatively

regulates

stomatal

formation

at

both

entry

and

amplification

stages,

but

then

functions

as

a

positive

regulator

at

the

final

stage

of

development

where

the

GMC

differentiates

into

paired

GCs.

Activated

MKK4

and

MKK5

do

not

have

any

effects

on

stomatal

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development

at

this

final

stage,

but

the

study

revealed

that

MKK7

and

MKK9

do

(Lampardetal.,2009).MKK7/9signalingmirrorsYDAatthreediscrete

steps,negativeforthefirsttwotransitionsandpositiveatthelaststage(Fig.9.3

and

Lampard

et

al.,

2009).

This

positive

function

of

YDA

and

MKK7/9

at

the

final

stage

of

stomatal formation was unexpected however; perhaps it is not entirelysurprising.ItmayreflectthatastomatallineagecellpreferstoformamatureGC once it has progressed to the GMC stage, rather than being arrestedmidway through thisprocess.Fromobservationofmutants,meristemoidsand GMCs appear to have different commitments to becoming stomata.

TMM, ERf

MMCM

SLGC

GMCGC

MUTE FAMASPCH

FLP/MYB

SCRM/SCRM2

MAPKKK

MAPKK

MAPK

SPCH

P

? ?

YDA

MKK4/5 MKK4/5MKK7/9 MKK7/9 MKK7/9

MPK3/6 MPK3/6 MPK3/6 MPK ?

?

Figure 9.3 Components of the YDA MAPK cascade regulating of stomataldevelopment (modified from Lampard et al., 2009). At the base of the figure, bHLHtranscriptionfactorspromotethreesequentialstepsofstomatalcellfatetransition.SPCHisneededfortheMMCtoMtransition,MUTEfortheMtoGMCtransition,andFAMAto transit from GMC to GC. FLP/MYB88 acts contemporaneous with FAMA toterminate

divisions

of

the

GMC

to

form

a

single

pair

of

GCs.

SCRM/SCRM2

are

expressed throughout the stomatal lineage and could form heterodimers with SPCH,MUTEandFAMA,tofacilitateprogressionthrougheachstep.Abovethetranscriptionfactors,threetiersoftheYDAMAPKcascademodulatethesamethreestomatalcellfatetransitions.

SPCH

has

been

experimentally

shown

to

be

phosphorylated

by

MPK3/6.

YDA

and

MKK4/5/7/9

negatively

regulate

the

first

and

second

fate

transition.

The

third

transition,however,ispositivelyregulatedbyYDAandMKK7/9.MPKsareinvolvedateach transition, as MPK3/6 were demonstrated for the first, with additional, currentlyunidentified,MPKsrequiredespeciallyatthethirdstep.

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Arrested

GMCs

are

rarely

seen,

but

meristemoids

do

not

always

complete

the

pathway;

meristemoids

can

arrest,

as

seen

in

the

leaves

of

theermutant

(Shpaketal.,2005),ordedifferentiateintoaSLGC,asobservedonstemsof

the

tmm

mutant

(Bhave

et

al.,

2009).

Multiple

control

points

during

stomatal

development

might

provide

a

way

to

naturally

expand

the

flexibility

of

a

plantcellconfrontingenvironmentalchallenges.Manyenvironmentalstresses induce an elevated number of stomata (Baena-Gonzalez et al., 2007;Baena-Gonzalez and Sheen, 2008), and ithas also been known for sometimethatmanyplantspeciesrespondtohigherlevelsofCO2byproducingalterednumbersofstomata,suggestingbothpositiveandnegativeregulatorsinterpret theCO2

effect for stomataldevelopment (Woodward,1987). Itwould

be

intriguing

to

know

whether

YDA

and

MKK4/5/7/9

are

reg

ulators

that

integrate

CO2

signals

with

developmental

control.

Like

with

ligandreceptor

interactions,

new

information

raises

new

questionsandboth logicalandtechnicalchallenges.Inthenextfewyears,we

imagine

several

new

directions

for

MAPK

signaling.

These

include

elucidating

complete

MAPK

cascades

in

relevant

cell

types

and

monitoring

their

activities

in

real

time.

Some

questions

of

particular

interest

are

as

follows:

What is the connection between the highest level kinase (MAPKKKYDA)

and

the

receptor-like

kinases

at

the

membrane?

MAPKKKs

are

often

activated

by

phosphorylation

and

require

scaffold

proteins

for

specificity. In the embryo,a cytoplasmic receptor-like kinase, SHORTSUSPSENSOR

(SSP),

acts

upstream

of

YDA,

but

does

not

require

its

kinase

activity

to

function

(Bayeretal.,2009).CouldSSPoritshomologs

be

a

bridge

between

YDA

and

the

ER

family

kinases?

Whichofthe20MAPKsarerequireddownstreamofidentifiedMKKsatthe different stages of stomatal development? Are some requiredredundantly? In addition, MAPKs are also regulated by phosphatases,andagain,genesencodingtheseproteinsarenumerous inplants.Using

the

information

from

many

large-scale

screens

such

as

protein

arrays

andexpressionprofilesmayhelpreducethecandidatestoatestablenumber.

4. TranscriptionFactors

HowcouldthelinearMAPkinasecascade,simplybyphosphorylationrelays,

oppositely

promote

or

repress

stomatal

cell

fate

at

different

transitions

in

the

stomatal

lineage?

We

have

seen

how

different

ligands

can

trigger

different

responses

in

different

tissues.

It

is

equally

plausible

that

the

YDA

MAPK module phosphorylates distinct transcription factors, which aredifferentially expressed at specific cell types or stages, thereby switchingonoroffkeydevelopmentalprocesses.

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How

might

these

transcriptional

targets

of

MAPKs

be

identified?

Recently,

Popescuetal. (2009)utilizedprotein-microarray technology to

revealthepotentialsubstratesofanumberofMAPKs,includingMPK3and

MPK6

(activated

by

each

of

MKK4/5/7/9).

The

array

is

an

incomplete

proteome,

but

among

570

identified

targets

of

MAPK

phosphorylation,

manytranscriptionfactorswereidentified(Popescuetal.,2009).Thearrayisbydesignaninvitroapproachleavingopenthequestionofhowmanyofthesetargetswouldberelevantforstomataldevelopment.However,comparingthetranscriptionfactorsknowntoregulatestomataldevelopmenttothe array data may be a very fruitful enterprise. Here we describe severalrelated

master regulator transcription factors in stomatal development;onehasalreadybeenexperimentallydemonstratedtobephosphorylatedby

the

downstream

kinases

in

the

YDA

MAPK

signaling

pathway

(Lampard

et

al.,

2008),

and

others

may

well

incorporate

phosphorylation

as

part

of

their

regulatory

strategies.

4.1.

Genes

that

break

silence

SPCH,

MUTE,

and

FAMA

are

three

key

basic

helixloophelix (bHLH)factorscontrollingthreediscreteandsequentialcellfatetransitionsinstomataldevelopment;eachisabsolutelyessentialfortheplanttomakestomata(Fig.9.4;reviewed

in

Barton, 2007; Pillitteri and

Torii,

2007).

Loss-of-function

of

any

onegivesrisetoaplantdevoidofmaturestomata,thoughtheydifferatwhichstagesthestomatallineageisblocked.WithoutSPCH,theepidermisconsistsonly of pavement cells (MacAlister etal., 2007), indicating that the primaryfunction of SPCH is to initiate the stomatal lineage by promoting ACDs.The mute mutant, in contrast to spch, undergoes entry divisions to formmeristemoids;

however,

the

meristemoids

are

not

able

to

transition

from

meristemoid

to

GMC

and

after

excessive

rounds

of

amplifying

divisions,

the

cellsarrest(PillitteriandTorii,2007).ThekeyfunctionofMUTE,therefore,isto

terminate

asymmetric

divisions

and

allow

the

second

phase

transition

in

the

stomatal

lineage.

Thefamamutantproducescaterpillar-liketumorscomposed

of

cells

with

GMC

identity

instead

of

forming

recognizable

stomata,

implicating

FAMA in the termination of the GMC divisions and in finalizing thedifferentiationoftheGCs(Ohashi-Itoetal., 2006).Combinedwithexpressionanalysisandgain-of-functionphenotypes,thepicturethatemergesisthateachof these three bHLHs sequentially turn on and regulate a single differentdevelopmental

transition

in

the

stomatal

lineage

(Fig.

9.3).

MYBtranscriptionfactorsoftenformcomplexeswithbHLHproteinsinplants

(Payne

et

al.,

2000).

Mutations

in

the

MYB

gene

FOUR

LIPS

(FLP)

or double mutants of FLP and its close paralogMYB88 generate plantswhose

epidermis

contains

clusters

of

GCs

and

GMCs,

suggesting

that

they

redundantly

function

to

terminate

symmetric

GMC

divisions,

thereby

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GMC

ACD ACD

PAN1

SMC

SMC

SM

PAN1 in

maizeGMC

SMC

SMC

EntryAmplifying

ACDACD

BASL BASL

BASL in

Arabidopsis

Cell types that are involved in stomatal ACD

Meristemoid mother cell (MMC)

Meristemoid (M)

Meristemoid + stomatal lineage ground cell

(M + SLGC)

BASLACD

Spacing

Figure9.4 (Continued)

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allowing

cells

to

complete

the

final

step

in

GC

differentiation

(Lai et al.,

2005).

These

stomatal

arrest

phenotypes

are

similar,

but

weaker

thanfamas,

and along withexpression data indicating high expression in GMCs, it is

tempting

to

consider

FLP/MYB88

as

partners

for

FAMA.

However,

genetic

interaction

results

do

not

support

a

model

of

FLP/MYB88

working

togetherwithFAMA,nordotheyphysicallyinteractwithFAMA insplitGFPassays(Ohashi-Itoetal.,2006).

If not complexed with MYB or other proteins, bHLH transcriptionfactorscanalsoformhomo- orheterodimerswithbHLHproteins.SeveralinteractionpartnersofSPCH,MUTE,and/orFAMAhavebeenreported(Ohashi-Ito

et al., 2006; Kanaoka et al., 2008); the most likely to bebiologically relevantare the ICE/SCREAM (SCRM) proteins (Fig.9.3,

Kanaoka

et

al.,

2008).

Originally

characterized

as

a

bHLH

involved

in

response

to

cold

stress,

ICE1

(Chinnusamy

et

al.,

2003)

was

also

recognized

by

its

effects

on

stomatal

development

(Kanaokaetal.,2008).Adominant

mutation

inICE1(scrmD)thatreplacesarginine236withhistidineleads

to an epidermis composed nearly completely of GCs. ICE1/SCRMencodes a bHLH-leucine zipper protein expressed during several stagesof stomatal development. Its paralog SCRM2 is also expressed in thestomatal

lineage,

and

together

they

appear

to

regulate

all

three

key

transitionstepsinstomataldevelopment(Kanaokaetal.,2008).Thishypothesis

is

supported

genetically

by

the

phenotypes

of

scrm,

scrm;scrm2/

, and

scrm;

scrm2,

which

display

defects

comparable

to

those

in

fama,

mute, and

spch

mutants,respectively.Yeast-two-hybridandsplit-GFPassaysindicatethatwhile

SPCH,

MUTE,

and

FAMA

do

not

strongly

homodimerize,

each

physically

interacts

with

SCRM-D

and

(to

a

lesser

extent)

with

SCRM

(Kanaokaetal.,2008).ThesedataleadtothemodelthatSCRM/SCRM2

proteins act in a dosage-dependent manner as partners with SPCH,MUTE,

and

FAMA

(Kanaokaetal.,2008andFig.9.3).

Figure9.4 PolarizedproteinsPAN1andBASLinstomataldevelopment.Asymmetriccell divisions (ACD) are labeled and localization of PAN1 and BASLproteins areindicated

by

gray

shading.

(Top)

In

maize,

the

guard

mother

cell

(GMC)

is

generated

fromanACDinaspecificlineage(firstpanel,dashedline).GMCsprovideapositionalcue interpreted by the neighboring subsidiary mother cells (SMCs), inducing theirnuclei to migrate toward the GMC inpreparation for a highly asymmetric division.ThesmallerdaughtersoftheSMCdivisionbecomesubsidiarycells(SCs)that,togetherwith

the

GCs,

comprise

the

stomatal

complex.

In

SMCs,

PAN1

appears

in

patches

at

the

GMCcontactsites.(Bottom)ArabidopsisBASLfirstappearsinthenucleusoftheMMC,and then as a crescent in the cellperiphery. After the MMC divides, only the larger

daughter

cell

inherits

the

peripheral

BASL.

The

nuclear

BASL

initially

present

in

both

daughtercellsmaydisappearcausingthecelltoexitanasymmetricdivisionphase;lossofnuclearBASLfromSLGCsisassociatedwithdifferentiationintopavementcells.Inspacing

divisions,

the

BASL

peripheral

crescent

relocates

to

maintain

its

position

distal

tothenewlyformedmeristemoid.

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4.2.

Phosphorylation

links

the

MAPK

module

to

the

transcriptional

network

While

most

of

the

protein

domains

of

SPCH,

MUTE,

and

FAMA

are

highly

conserved,

SPCH

contains

a

unique

region

that

neither

MUTE,

nor

FAMA, nor any other bHLH possesses. This domain, named the MAPKtargetdomain(MPKTD),isenrichedwithpredictedMAPKphosphorylation target sequences. An in vitro kinase assay confirmed that the SPCHMPKTD was phosphorylated by MPK3 and MPK6. Deletion of thisdomain

removed

all

detectable

phosphorylation in vitro and in vivo and

resulted in the accumulation of SPCH protein, excess stomatal lineagedivisions, and overproduction of stomatal lineage cells (Lampard et al.,2008).

It

was

hypothesized

that

in

the

plant

epidermis,

phosphorylation

of

SPCH

by

MPK3/6

would

reduce

the

level

of

active

SPCH

(Fig.

9.3).

To

test

this,

SPCH

variants

bearing

mutations

in

some

of

the

predicted

phosphorylationsites(weaklyoveractive)weremonitoredinthebackgroundof differentMAPKand receptormutations; in thesebackgrounds,SPCHoveractivitywasenhanced,indicatingthatthepreviouslyidentifiedstomatalsignaling

components

could

work

upstream

of

SPCH

and

modulate

its

activity.Disruptionofonespecificphosphorylationsiteresulted inaformofSPCHthatwasincapableofproducingstomata,whilenotaffectingtheproteinsabilitytopromoteasymmetricdivision.Itappears,therefore,that

MAPK-mediated

phosphorylation

not

only

generally

downregulates

SPCH,

but

it

may

also

be

required

to

activate

SPCHscellfatepromotingactivity.

How

this

complex

regulation

is

achieved

is

not

yet

known

(Lampard et al., 2008), but it is interesting to consider in light of theopposite effects MAPK signaling can have on the different stomataltransitions

described

in

the

previous

section.

Could

the

YDA

MAPK

module

regulate

stomatal

transcription

factors

otherthanSPCH?Basedoninvitrophosphorylationdata,theconclusionisprobablyyes(Fig.9.3).ICE1/SCRM,FLP,andMYB88containconsensus

MAPK

phosphorylation

sites

(Blom

et

al., 1999).

On

protein

array-based

kinase assays, MUTE and MYB88 are in vitro targets of MAPKs, thoughinterestingly,

MUTE

is

a

target

of

MAPKs

other

than

MPK3/6

(Feilneretal.,

2005;Popescuetal.,2009).Itshouldbenotedthattheseproteinarraysdonotincludeallthetranscriptionfactorsknowntobeinvolvedinstomataldevelopment (forexample,SPCH isnotpresent).Althoughpromising,thusfar,the

biological

relevance

of

phosphorylation

has

not

been

demonstrated

yet.

Confirmation

of

phosphorylation

sites

by

mass

spectrometry

andinvivoassays

on

variants

of

SCRM/SCRM2,

MUTE,

and

MYB88

containing

point

mutations

that

disrupt

the

putative

phosphorylation

sites

will

be

necessary

todeterminewhetherphosphorylationaffectstheirfunctionandwhethertheeffects

seen

by

altering

MAPK

signaling

in

different

stomatal

cell

types

can

be

explained

by

phosphoregulation

of

these

known

transcription

factors.

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4.3.

How

does

phosphorylation

affect

transcription

factor

function?

In

general,

phosphorylation

has

the

potential

to

change

many

properties

of

proteins

including

their

stability,

subcellular

localization,

activity,

and

proteinprotein or proteinDNA interactions. One interesting model toconsider for stomatal development is how phosphorylation could modulate the relationship between SPCH and SCRM/SCRM2. The phenotype of scrmD homozygous mutant plantsoverproduction of stomataand

stomatal

lineage

cellsmimicsthatgeneratedbyexpressingMPKTD-deleted SPCH in plants. Consistent with this, the double mutant scrm;scrm2exhibitsnostomatalproductionintheepidermis,resemblingthespchmutant

(Kanaoka et al., 2008). SCRM and SCRM2 appear to weakly

bind

to

SPCH,

but

the

SCRM-D

mutant

protein

shows

a

strikingly

increased

affinity

for

SPCH

(Kanaoka et al., 2008). If a partnership

between SCRM/SCRM2 and SPCH promotes initiation of stomataldevelopmentandSPCHisnormallydownregulatedbyMAPKphosphor

ylation, then the stimulatory effect of SCRM/SCRM2 on SPCH couldbe

achieved

by

these

proteins

also

serving

as

MAPK

substrates.

SCRM/

SCRM2 could (1) serve as competitive substrates (shielding SPCH fromphosphorylation)and (2)change theiractivityuponphosphorylation suchthat they bind SPCH and stabilize it, or interfere with the MPK3/6

directed

SPCH

phosphorylation

and

degradation.

Of

course,

these

models

require

more

rigorous

tests

of

whether

SCRM/SCRM2

phosphorylation

takes

place invivo,andbetterassaystodeterminetheeffectsofphosphor

ylationon stomatal lineage transcription factors.Although many transcription factors have now been identified, the

targets

of

these

transcription

factors

are

not

yet

known.

Based

on

their

ability

toreprogramcellfateandfunctionalconservationbetweenArabi-

dopsisandriceproteins (Ohashi-Itoetal.,2006;PillitteriandTorii,2007;Kanaokaetal.,2008;Liuetal.,2009), it is likely that thebHLHswillbe

high-level

controllers,

regulating

many

other

transcriptional

regulators

as

wellascellcycleanddifferentiationgenes.Severaloutstandingquestionsareas

follows:

Whatarethetargetsofeachtranscriptionfactorateachstomataltransitionphase? Do putative partners (e.g., SPCH and SCRM/SCRM2) haveoverlapping regulatory profiles? Information from large-scaletranscriptomics,

proteomics,

and

phosphoproteomics

may

be

especially

useful,incombinationwithforwardgeneticsandcellularorbiochemical

analysis,

to

establish

biologically

relevant

targets.What other transcription factors classes participate in stomatal cell fate,

and

how

are

they

regulated?

For

example,

the

MADS-box

protein

AGL16

appears

to

affect

stomatal

development

and

it

is

targeted

by

a

microRNA

(Kutteretal.,2007).Conventionalforwardgeneticswillstill

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be

valuable

for

isolating

new

mutants,

and

screening

for

enhancers

or

suppressors

of

the

existing

stomatal

mutants

may

identify

regulators

with

moresubtleeffects.

5. RegulationofStomatalAsymmetric

CellDivision

We

have

described

the

individual

stages

in

stomatal

development

and

someofthesignalsandintrinsicfactorsthatregulatethem.Wenowturntothe

mechanism

of

ACD

that

establishes

different

identities

and

division

behaviors

in

these

different

cell

types.

ACD

refers

to

divisions

that

generate

two

daughter

cells

with

distinctive

cell

fates;

in

the

case

of

meristematic

or

stemcells,oneofthedaughtercellsadoptsthefateofitsmother,whiletheothertakesonadivergentfate.ACDisaprimarymechanismthatleadstocell-type diversity and is fundamental for growth and development ofmulticellular organisms including animals, fungi, and plants (Horvitz andHerskowitz,

1992;

Caussinus

and

Hirth,

2007;

Gonczy,

2008).

5.1.

Models

of

asymmetric

cell

division

In

general,

ACD

requires

the

generation

of

cellular

asymmetry

in

(or

polarization

of)

the

progenitor

cell;

this

includes

physical

changes

in

cell

shape

and

asymmetricdistributionofcellularcomponentssuchasorganelles,proteins,andRNAs.Followingcellularpolarization,apreciselyorienteddivisionplanewillbeestablishedanddaughtercellfatesspecified. In animals, two basic mechanisms are thought to confer distinguishable daughter cell fates. Differentialdistribution of protein complexes, most notably the conserved PARaPKCmembrane

kinase

complex,

can

unequally

segregate

internal

fate

determinants

(Goldstein

and

Macara,

2007).

Alternatively,

extrinsic

mechanisms

may

be

employed

in

which

initially

equivalent

daughters

obtain

unequal

cell

fates

depending

on

their

physical

location

post

division.

In

many

cases,

a

combination

of

both

intrinsic

and

extrinsic

mechanisms

may

be

used

(Munro,

2006;

Knoblich,

2008).

Extensive effort has been applied to study the molecular mechanismsunderlying ACD inArabidopsis, with emphasis on early embryogenesis,stomatalproduction, root morphogenesis,andmalegametophytegeneration

(reviewed

in

Heidstra,

2007).

Both

internal

factors

and

external

cues,

especially

for

stomatal

development,

are

thought

to

play

crucial

roles.

For

example,

in

early

leaf

development,

MMCs

divide

asymmetrically

and

generate two physically and functionally distinct cells, but their divisionplane is not obviously oriented by any external information (Serna et al.,2002).

However,

later,

when

the

secondary

meristemoids

are

generated

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(Fig.

9.1),

external

signals

from

GCs,

GMCs,

or

meristemoids

orient

the

direction

of

the

new

ACDs,

so

that

the

newly

formed

meristemoid

is

placed

distalfromthem(NadeauandSack,2002).Inthisreview,wewillpointout

where

signals

and

transcription

factors

regulate

stomatal

lineage

ACDs,

but

will

consider

in

more

depth

two

newly

identified

regulators

that

appear

to

becriticalinternalpolarityfactors.

5.2.

Stomatal

asymmetric

cell

division

ManygenesaffectthenumberandpositionofstomatallineageACDs,suchas the ligands (EPF1,EPF2,CHAL,STOMAGEN),membrane receptors(TMMandERf),MAPKmodule,andbHLHtranscriptionfactors(SPCH,

MUTE,

and

SCRM1/2)

previously

described.

This

reflects

an

integration

of

external

signaling

and

internal

control

in

stomatal

development

(Petricka

etal.,2009).While it is indeed true that all thesegenes regulate stomatalACDs,mostdosobyspecifyingcellfatesinwhichasymmetricdivisionsarepartoftheir identities.Noneofthesegenes,fromacellularperspective,islikely

to

be

part

of

the

machinery

that

creates

a

physically

and

functionally

asymmetric

division.

Based

on

studies

in

plant

roots

and

embryos,

it

appeared that plants relied primarily on extrinsic information to generateACDs

(Song et al., 2006; Cui et al., 2007; Willemsen et al., 2008; Bayer

et

al.,

2009).

Recently,

however,

identification

of

two

polarly

localized

proteins,

PANGLOSS1

(PAN1)

and

BREAKING

OF

ASYMMETRY

INTHESTOMATALLINEAGE(BASL),suggeststhatplantsmightalsoutilize polarized protein localization as a mechanism to generate ACDs(Cartwrightetal.,2009;Dongetal.,2009).

5.3.

Polarized

receptor

protein:

PAN1from

maize

In grasses, both GC and SC formation require asymmetric divisions

(Fig.

9.4;

Gallagher

and

Smith,

2000).

The

maize

genes

controlling

the

first

asymmetric

division

that

creates

GMCs

are

still

unknown,

but

several

mutations that affect SMC divisions have been identified (Gallagher andSmith, 2000; Cartwright et al., 2009; Wright et al., 2009). Two of thesegenes,PAN1andPAN2,areinvolvedinSMCACDandSCfatespecification(GallagherandSmith,2000;Cartwrightetal.,2009).PAN1encodesaLRR-RLK

and

is

expressed

in

SMCs.

Immunolocalization

of

PAN1

in

maize

leaves

indicated

that

this

protein

forms

strikingly

polarizedpatches

atsiteswheretheSMCscontactGMCs(Fig.9.4).Inacorrectlypatterned

ACD,

actin

patches

form

and

SMC

nuclei

migrate

to

these

same

contact

sites.

Absence

of

PAN1

patches

in

the

pan1

mutant

leads

to

defects

in

both

actin

and

nuclear

recruitment

to

contact

site,

eventually

leading

to

incorrectly

placed

division

planes

and

incorrectly

specified

SCs

(Cartwrightetal.,

2009).PAN1,asareceptororaco-receptorintheSMCs,mayrespondto

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the

GMC-derived

signals

that

promote

SMC

polarization

in

preparation

for

asymmetric

divisions.

The

molecular

identity

or

nature

of

the

GMC-derived

cuethatpositionsPAN1proteininSMCsisstillunknown,buttheabilityto

detect

PAN1

will

provide

an

excellent

tool

to

identify

genes

or

processes

required

for

its

polarization

in

the

future.

Once

PAN1

is

polarized,

how

it

transmitsthisinformationtodownstreamprocesseslikeactinpatchformationalsoneedsfurtherinvestigation.Althoughannotatedasareceptor-likekinase,thePAN1sequencedoesnotcontainthekeyresidueforkinaseactivityandPAN1 failed to phosphorylate a generic substrate in in vitro kinase assays(Cartwrightetal., 2009).

Although

they

all

encode

LRR-containing

transmembrane

proteins,

the

asymmetric localizationofPAN1distinguishes thisprotein fromArabidopsis

TMM

and

the

ERf

LRR-RLKs.

A

TMM

GFP

fusion

expressed

under

the

TMM

promoter

was

generally

plasma

membrane

localized

in

stomatal

lineage

cells

(Nadeau

and

Sack,

2002),

and

the

subcellular

localizations

of

the

Erf

members

have

not

yet

been

described.

Considering

the

large

collection

of

LRR-RLKs (~220) in theArabidopsis genome (Torii, 2004), it is plausiblethatotherLRR-RLKsfunctionlikePAN1byaccumulatingasymmetricallyinresponsetoexternalcuesandbypolarizingcellsinadvanceofasymmetricdivision.

It

is

also

possible

that

since

the

specific

developmental

event,

the

recruitmentofneighboringcells to formSCsof the stomatalcomplexes, is

not

found

in

Arabidopsis,

there

will

not

be

LRR-RLKs

with

comparable

polarized

expression

or

functions.

5.4.

Polarized

and

segregated

novel

protein:

BASLinArabidopsis

PAN1

is

clearly

a

polarized

protein

in

plant

cells;

however,

there

is

no

evidence that it is segregated during an ACD toyield daughter cells of

different

fates.

A

novel

protein

that

does

appear

to

be

segregated

is

Arabidopsis

BASL.

Like

PAN1,

BASL

regulates

asymmetric

divisions

in

stomatagenesisandexhibitsa strikinglypolarized subcellulardistribution(Dong et al., 2009). The basl mutant loses asymmetries associated withstomatal divisions; marker expression is inappropriately segregated, andthedaughtercellsizesandidentitiesaresimilartoeachother(Dongetal.,2009).BASLproteinhasnopredictedfunctionaldomainsandisencodedonly

in

the

genomes

of

dicot

plants.

GFPBASL exhibits a dynamicsubcellular localization pattern and is found in a crescent at the cell

periphery,

a

localization

reminiscent

of

the

PAR/aPKC

complex

in

animals

(Fig.

9.4).

In

asymmetrically

dividing

stomatal

lineage

cells,

BASL

first

appears

in

the

interphase

nucleus

and

begins

to

accumulate

in

a

crescent

at

the

cell

periphery

prior

to

the

cell

exhibiting

any

morphologicalasymmetry.Afteratypicalasymmetricdivision,theBASLperipheral

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crescent

is

always

inherited

by

only

the

larger

daughter

cell

(the

SLGC,

Fig.

9.4).

Immediately

after

division,

both

daughter

cells

have

BASLGFP in their nuclei, but nuclear BASL does not always persist.

Disappearance

of

BASL

from

the

nucleus

correlates

with

cell

differentiation;

if

the

cell

did

not

inherit

peripheral

BASL,

it

will

become

a

GMC

andeventuallyGCs,butifthecelldoespossessBASLatthecellperiphery,loss of nuclear BASL correlates with adoption of pavement cell fate(Fig. 9.4). Interestingly, peripheral BASL is more than a convenientasymmetrically segregatedmarker.Thisregion is the siteofBASLfunctionas demonstrated by the expression of an N-terminal-deleted variant ofBASL

(BASL-IC)

that

is

localized

to

a

crescent

in

the

cell

periphery

(but

not the nucleus),yet it fully rescues the basl mutant phenotypes (Dong

et

al., 2009).

In

contrast

to

PAN1,

BASL

seems

to

be

an

internal

regulator

of

stomatal

ACDs.

The

orientation

of

the

BASL

crescent

does

not

appear

to

respond

to

external

cues

such

as

EPF1

coming

from

GCs,

nor

does

BASL

pattern

changeinatmmmutant.ItwashypothesizedthatBASLmaytrigger localcellexpansiontoinducelocalphysicalasymmetries.Indeed,whenectopically expressed in nonstomatal lineage cells of the hypocotyl, the BASLperipheral

crescent

overlays

areas

of

abnormal

cell

outgrowth

(Dongetal.,

2009). The BASL crescent, to some extent, also coordinates cell fate

determination;

it

is

invariably

in

the

larger

daughter

cell

resulting

from

an

asymmetric

division

and

the

division

and

fate

of

each

sister

can

be

predicted

by monitoring BASL in the nucleus and at the periphery (Dong et al.,2009).

Together

with

evidence

that

BASL

is

polarized

before

any

outward

physical

asymmetries

can

be

detected

in

stomatal

lineage

cells,

it

appears

that

BASL

plays

a

role

very

similar

to

that

of

the

PARaPKC complexes inanimalcells.

Discovery

of

PAN1

and

BASL,

polarized

proteins

that

regulate

asymmetric

division,

suggested

that

the

establishment

of

cell

polarity

by

accumulation

of

proteins

in

distinct

subcellular

regions

is

a

mechanism

used

not

only

by

animals,

but

by

plants,

for

asymmetric

division

control.

Still, there are differences between plants and animals; the molecularidentities of BASL and PAN1 as well as the lack of genes encodinghomologs of animal polarity proteins indicate that each group recruitedadifferentsetofproteins.

It seems likely thatthewaythatBASL(andpossiblyPAN1)promotesasymmetricdivisionmustalsobedifferentfromwhatisknownfromanimalsystems.

Classical

morphology

and

cell

biology

already

showed

that

there

are

different

mechanisms

for

division

plane

specification

in

animals

and

plants.

In

animal

cells,

the

position

of

the

centrosomes

and

the

mitotic

spindle determine the orientation of the division plane (Cowan andHyman, 2004). In an animal asymmetric division, polarity proteins (suchas

the

PAR/aPKC

complex),

coordinating

with

microtubules,

adjust

the

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spindle

position

and

the

division

plane

is

set

when

the

membrane

pinches

down

from

the

periphery

to

the

midzone

of

the

spindle

in

apurse-string

closure cytokinesis. By contrast, the division plane of a plant cell is

predicted

by

a

specialized

ring

of

cortical

cytoskeleton

elements

(microtubules

and

F-actin)

named

the

preprophase

band

(PPB).

The

PPB

appears

duringinterphaseandistransient,beinglargelydisassembledbeforemitosis;however,areminderofitspositionismaintainedandthephragmoplastandnewcellwalldividingthedaughtercellswillformatthissite.Whatistheconnection between PAN1 and BASL and PPB orientation? Does eitherprotein orient PPB placement directly, and if so, how? PAN1 appears toestablish

cellular

asymmetry

by

recruiting

actin,

so

it

is

possible

that

it

creates

aplatformfororientationofthePPBthroughthispolymer.However,the

placement

of

the

actin

patch

in

SMCs

is

not

where

the

PPB

would

assemble,

so

it

is

unlikely

that

PAN1

is

a

direct

scaffold.

Interphase

nuclear

position

also

influences

PPB

placement

(Murata

and

Wada,

1991),

though

the

precise

mechanism

is

still

unknown.

Since

BASL

is

expressed

in

the

nucleusbeforePPBformation,mightnuclearBASLdirecttheplacementofthe PPB? Alternatively, nuclear BASL might act in coordination withpolarizedperipheralBASLtofacilitatePPBformationataspecificsite.

Polarity

generation,

though

known

from

decades

of

morphological

studies to be a critical part of stomatal development and pattern, isjust

now

beginning

to

be

studied

at

a

molecular

level.

We

anticipate

many

new

discoveries

in

the

coming

decade

and

that

many

of

these

will

translate

to

polarity issues outside of the stomatal lineage. Some of the outstandingquestions

are

as

follows:

How are the PAN1 and BASL proteins brought to and maintained attheirpolarizedperipherallocations?MuchworkonplantcellpolarityhasfollowedthelocalizationofthetransmembraneauxintransportersofthePIN

family.

Will

the

trafficking

elements

required

for

PIN

localization

also

be

used

for

PAN1

and

BASL,

or

are

the

mechanisms

different?

Experiments

comparing

PIN

and

BASL

localization

in

roots

suggestthat theyaredifferent(Dongetal.,2009), leavingopenthequestionof

what

is

required

for

the

dynamic

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Dong 2010