membrane microdomains, rafts, and detergent-resistant ......lipid or membrane rafts. the idea that...
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Membrane Microdomains,Rafts, and Detergent-ResistantMembranes in Plantsand FungiJan Malinsky,1,!,!! Miroslava Opekarova,2,!
Guido Grossmann,3,!!! and Widmar Tanner4,!!
1Institute of Experimental Medicine and 2Institute of Microbiology, Academy of Sciences ofthe Czech Republic, 142 20 Prague, Czech Republic; email: [email protected],[email protected] for Plant Biology, Carnegie Institution for Science, Stanford,California 94305; email: [email protected] of Cell Biology and Plant Physiology, University of Regensburg, 93053Regensburg, Germany; email: [email protected]
Annu. Rev. Plant Biol. 2013. 64:501–29
The Annual Review of Plant Biology is online atplant.annualreviews.org
This article’s doi:10.1146/annurev-arplant-050312-120103
Copyright c! 2013 by Annual Reviews.All rights reserved
!Equally contributing authors.
!!Corresponding authors.
!!!Present address: Centre for OrganismalStudies/CellNetworks, Universitat Heidelberg,69120 Heidelberg, Germany
Keywordslateral plasma membrane compartments, pathogen response,detergent resistance, sterol-rich domains
AbstractThe existence of specialized microdomains in plasma membranes, pos-tulated for almost 25 years, has been popularized by the concept oflipid or membrane rafts. The idea that detergent-resistant membranesare equivalent to lipid rafts, which was generally abandoned after adecade of vigorous data accumulation, contributed to intense discus-sions about the validity of the raft concept. The existence of membranemicrodomains, meanwhile, has been verified by unequivocal indepen-dent evidence. This review summarizes the current state of researchin plants and fungi with respect to common aspects of both kingdoms.In these organisms, principally immobile microdomains large enoughfor microscopic detection have been visualized. These microdomainsare found in the context of cell-cell interactions (plant symbionts andpathogens), membrane transport, stress, and polarized growth, and thedata corroborate at least three mechanisms of formation. As docu-mented in this review, modern methods of visualization of lateral mem-brane compartments are also able to uncover the functional relevanceof membrane microdomains.
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ContentsINTRODUCTION . . . . . . . . . . . . . . . . . . 502MEMBRANE MICRODOMAINS
AND LIPID RAFTS: AHISTORICAL ACCOUNT . . . . . . . 502
MEMBRANE MICRODOMAINS INPLANT AND FUNGAL CELLS:PHENOMENA ANDPOSTULATED FUNCTIONALINVOLVEMENT . . . . . . . . . . . . . . . . 505Stable Lateral Segregation of
Membrane Transporters . . . . . . . . 505Steady-State Microdomains Induced
by Cell Polarization . . . . . . . . . . . . . 511Microdomains Induced in Response
to Interaction withMicroorganisms . . . . . . . . . . . . . . . . 512
FORMATION AND FUNCTIONALRELEVANCE OF PLASMAMEMBRANE MICRODOMAINSIN PLANTS AND FUNGI. . . . . . . . 514Spontaneous Lipid Demixing . . . . . . . 516Energy-Dependent Directed
Membrane Flow . . . . . . . . . . . . . . . . 517Protein-Mediated Restrictions of
Lateral Diffusion. . . . . . . . . . . . . . . . 518Microdomain Organization Needs
Energy: Plasma Membrane ofDe-energized Cells . . . . . . . . . . . . . 520
INTRODUCTIONThe emergence of the plasma membrane(PM) has been one of the most crucial steps inevolution. This special organelle surroundingeach cell serves as an active interface betweenthe cell and its environment. It mediates importand export of a multitude of molecules withhigh selectivity and embeds dozens of sensorsresponsible for environmental signal transfer tothe cell interior. In this way, each cell communi-cates with the environment, and eventually withneighboring cells and the whole organism. Fora long time, the membrane was imagined as auniform envelope made up of lipids and housing
the proteins responsible for the processes men-tioned above. Recent data indicate that the PMis a highly heterogeneous organelle subdividedinto areas of distinct composition, structure,and function. These areas were named lipidor membrane rafts, detergent-resistant mem-branes (DRMs), micro- or nanodomains, etc.As discussed below, some of these designationswere unfortunate (see sidebar Essential Terms).
This review summarizes current informa-tion about compartments in PMs of plants andfungi with respect to their size, composition,dynamics, mechanisms of formation, and func-tional relevance. Considering the functionalimportance of this compartmentation, we focuson the question of whether it really makes a dif-ference to a cell if a transport or sensor proteinis homogeneously distributed in the membraneor concentrated at a special location, and whathappens when it is translocated to a differentplace within the membrane.
MEMBRANE MICRODOMAINSAND LIPID RAFTS:A HISTORICAL ACCOUNTOne important contribution of botany to gen-eral biology has been the discovery of the PMand the phenomenon of osmosis. In plant cells,which are surrounded by cell walls, plasmoly-sis could be observed and interpreted in termsof the existence of a semipermeable membranereleasing water (but not colored substances likeanthocyanins) from the cell protoplasm (28,102, 109; reviewed in 129). It was a long timebefore this postulated membrane was visualizedby high-resolution electron microscopy (114).Although modeling of the membrane beganshortly after Gorter & Grendel (38) presentedevidence for the lipid bilayer, the first gener-ally accepted model (which is still largely ac-cepted today) was Singer & Nicolson’s (127)“fluid mosaic.” The authors modeled cellu-lar membranes as homogeneous lipid bilay-ers that membrane proteins are either embed-ded in or attached to, resulting in a mosaic ofprotein-containing and bare lipid areas. Postu-lation of the membrane fluidity was based on
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Frye & Edidin’s (33) cell fusion experiment,which demonstrated that within 40 min af-ter the fusion of two mammalian cells, specificmembrane proteins of both cells are uniformlydistributed.
Over the years, several changes and amend-ments to the Singer-Nicolson model have beenbroadly accepted. These include (a) the asym-metry of the lipid bilayer (16); (b) the restrictionof the free mobility of membrane proteins bytheir interactions with intracellular and/or ex-tracellular components, such as the cytoskele-ton and extracellular matrix/cell wall (29, 132;for plant cells, see 6, 91); and (c) much higherprotein density at the membrane surface thanwas considered in the classical Singer-Nicolsonmodel. As estimated for human erythrocytes(27) and for synaptic vesicles (138), practicallythe entire membrane may actually be coveredby proteins on both sides. The transmembranehelices of integral membrane proteins aloneoccupy approximately 23% of the actual vol-ume of the lipid core layer of the erythrocytePM (27). Quantitative data for plant PMs arenot available so far, but a comparable proteindensity can be assumed.
The biomembrane structural model was fur-ther revised by the concept of lateral segre-gation of proteins and lipids into membranemicrodomains. Although these microdomainswere intensely discussed for approximately twodecades, there is no general agreement on theirnature. Nevertheless, numerous publicationssupport the idea that molecules in the PM arenot equally distributed, and that lateral sub-compartments exist that divide the PM intodistinct microdomains (53, 87, 97, 124, 159).These observations almost led to a paradigmshift, and certainly did lead to significantly in-creased interest in membrane and especiallyin lipid research. Although Singer & Nicolson(127) assumed a homogeneous lipid bilayer anda random distribution of integral membraneproteins, they did not exclude clustering, not-ing that “wherever nonrandom distributionsare found, mechanisms must exist which are re-sponsible for them” (p. 724). To find and eluci-date these mechanisms as well as the functional
ESSENTIAL TERMS
Membrane microdomain: In the frame of this review, theterms membrane microdomain and plasma membrane (PM) mi-crodomain denote a lateral compartment within the plane of thePM that exhibits a composition, structure, and biological functiondistinct from the surrounding membrane, regardless of its size,stability, or mechanism of formation. It is noteworthy that in-dividual membrane microdomains, resulting from spontaneousand/or energy-dependent lateral segregation of a plentitude ofPM constituents (lipids and proteins), may differ substantiallyfrom one another in all the above-mentioned aspects.
Lipid rafts: Historically, lipid rafts were postulated as Golgiapparatus– or endoplasmic reticulum membrane–derived sort-ing platforms forming liquid-ordered microdomains in the PMthat are enriched in sphingolipids and sterols. They are now con-sidered nanoassemblies consisting of specific proteins and lipids.Membrane microdomains based on lipid rafts are supposed tobe highly dynamic. Their actual size, composition, and functionstrongly depend on external conditions—temperature, signalingevents, etc. It is questionable, therefore, whether intact raft-basedmicrodomains can be detected in isolated membranes.
Detergent-resistant membranes (DRMs): DRMs, also calleddetergent-insoluble membranes (DIMs), is a term used for thenonsolubilized fraction of membranes extracted by mild deter-gent under defined conditions. Membrane proteins identified asparts of DRMs are frequently denoted in the literature as raftproteins, although no relation between the hundreds of proteinsidentified in DRMs and their localization in specific membranemicrodomains has been found to date.
importance of lateral membrane compartmen-tation seems precisely the task of the day.
Differences between particular PM areashave been repeatedly reported. The polaraccumulation of bacterial chemotaxis recep-tors is one well-documented example (84).Another is the acid-banding phenomenonof internodal cells in Characeae. Zones ofexcess proton export alternate with zones ofOH" surplus along the long axis of the cell,which reflects different activities of protonpumps and ion transporters at special PM sites(82). Differences in membrane compositionare known to exist in polarized cells—for
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example, at rapidly growing cell poles (5, 17,47). Indeed, the challenging question of howpolarized cells, such as the epithelial cells of thesmall intestine, obtain their specific apical andbasolateral protein and lipid outfit has started acompletely new research area. In 1988, Simons& van Meer (126) postulated that sorting andtargeting of proteins and lipids may be directlyinterlinked and may arise from the “formationof microdomains mimicking the properties ofthe membranes of their destination” (p. 6200).
To biochemically demonstrate an associa-tion between the locally concentrated mem-brane proteins and lipids, Brown & Rose (18)described a simple detergent extraction methodfor these special cells to separate the api-cal membrane from the basolateral one. Theyfound that, similar to the apical membranes,the DRMs exhibit a lipid composition enrichedin cholesterol and glycosphingolipids. Thesefindings, unfortunately generalized to any celltype, led to the postulation that enrichment inthese lipids may result in a lateral segregationof membrane domains of higher and lower lo-cal densities in vivo. The term lipid rafts wascoined to describe the more compact mem-brane entities (125). The hypothesis that, inaddition to acting as vehicles for sorting andtargeting, these rafts potentially represent sig-naling platforms by clustering receptors andsignal transducing proteins attracted hundredsof scientists in a short time. Lateral immis-cibility was observed in liposomes containingcholesterol, where lipids can undergo sponta-neous phase separation (9) (see also Formationand Functional Relevance of Plasma MembraneMicrodomains in Plants and Fungi, below).This was also recently observed in protein-richbiomembranes, but only under special condi-tions, for example, when PMs were discon-nected from the cytoskeleton (80).
Solubilization in 1% Triton X-100 at 4#Cbecame the generic assay that was widely ap-plied to analyze the membrane fractions of anyspecies, tissue, or cell type. Membranes thatdid not dissolve under these conditions werecalled lipid rafts, membrane rafts, DRMs, ordetergent-insoluble membranes (DIMs). Be-
cause 10 years of research did not produce anyindependent evidence that the DRM fractioncontains any intact membrane substructures,this view could not be sustained, and in 2003 itwas dismissed by the leading groups in the fieldat a “raft meeting” in Tomar, Portugal. Themeeting report (161) stated, “A general con-sensus that emerged at this meeting about thenature of a raft in a cell membrane is summa-rized as follows: Considering the complexity ofthe system and the poorly understood natureof DRM formation, it is unlikely that DRMsthat are derived from cells reflect some preexist-ing structure or organization of the membrane”(p. 1119). Some of the main critical arguments(45, 100) have recently been summarized (140).
Despite the above agreement that DRMsdo not reflect membrane substructures inliving cells, detergent extraction is still widelyused to study membrane domains in plants (seeTable 1). As in earlier studies in the animalfield, the first plant studies showed that a largenumber of membrane proteins could not beextracted with 1% Triton X-100 at 4#C (15, 96,108). However, as mentioned above, the resultsobtained by this procedure reflect mainlythat different membrane proteins—regardlessof where they are located—require differentamounts of detergent to become soluble andcannot be considered proof of any definitemembrane structure or arrangement. Nev-ertheless, some of the “DRM proteins” wereshown to form clusters in the PM and maytherefore constitute potential membrane “plat-forms.” The PM-attached protein remorin canbe considered a prominent example. As docu-mented by immunogold detection, remorin isorganized at the PM in patches approximately70 nm in diameter and becomes homoge-neously distributed when the sterol content isreduced by methyl-!-cyclodextrin (111).
It has been argued that detergent extraction,as a first approach, may after all be a valuableand helpful tool to enrich proteins of potentialinterest for further membrane microdomain–oriented research. Once a protein has been es-tablished as a DRM component, Simon-Plaset al. (123) suggested using additional methods
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MCC (membranecompartment ofCan1): membranemicrodomain in yeastPMs equivalent to thefurrow-likeinvagination supportedby the cytoplasmicprotein complex(eisosome)
for a subsequent rigorous proof of its associa-tions with a specific membrane structure. Thisapproach, however, does not address two ma-jor concerns. First, proteome analysis of DRMfractions detects hundreds of proteins (seeTable 1), of which generally very few, if any,seem to be of special interest for the questioninvestigated. When employed for protein pu-rification, this approach is therefore rather in-efficient compared with other procedures avail-able (affinity chromatography, coimmunopre-cipitation, etc.). Second, and an even more se-rious concern, is that specific associations canbe disrupted by detergents, and protein compo-nents that are potentially much more importantin a given study might be discarded solely be-cause they stayed in the detergent-soluble part.In Saccharomyces cerevisiae, it has been shownthat even cytosolic proteins bind to specialmembrane compartments, and these proteinsare, in part, indispensable for the establishmentof these microdomains—for example, variouseisosomal proteins required for MCC (mem-brane compartment of Can1) formation (see be-low as well as References 11, 40, 98, and 151).
The current state of the art in the animalfield—the guiding example for a long time—does not clarify matters; in fact, in Novem-ber 2011 Science included the question “do lipidrafts exist?” as one of five “cell’s lingering mys-teries” that need clarification (75). As pointedout by Jacobson and coworkers (53), as the ac-curacy of the experimental approaches was con-tinuously increasing, the sizes of rafts in mam-malian cells became smaller and smaller overtime, and are now on the order of <20 nm (30).This is the magnitude of ordinary protein com-plexes plus dozens of lipid molecules, in the caseof membrane protein complexes. However, theraft attributes would then be reduced to func-tionally relevant interactions of specific pro-teins with certain lipids. In fact, this has been ar-ticulated in a way by Anderson & Jacobson (3) intheir lipid shell hypothesis. In another alterna-tive, Kusumi et al. (68) have suggested a lateralcompartmentation of PMs based on the diffu-sion of certain proteins in limited membraneareas; the meshwork of cytoskeletal elements
underlying the PM and associated proteins aresupposed to generate the limitations.
The purpose of this review is not to an-swer the question posed by Science (which re-lated to animal cells anyway). Rather, we ac-cent that the plant and fungal research com-munities are in the fortunate position that afair number of papers have been published thatdocument the existence of special lateral mem-brane compartments through various micro-scopic methods (see Table 2 and the side-bars Distribution, Mobility, and Interactionsand Superresolution Fluorescence MicroscopyTechniques). The direct visualization of mem-brane microdomains in living cells will be theapproach of choice to provide convincing ev-idence for their existence and biological role.The thriving field of superresolution tech-niques will further promote the discovery andanalysis of subdiffraction-size inhomogeneitiesin biological membranes (42). Examples fromstudies of plant and fungal cells presented in thisreview document that the PM microdomains ofcell wall–possessing cells exhibit common fea-tures and differ from what the mammalian re-search community calls rafts.
MEMBRANE MICRODOMAINS INPLANT AND FUNGAL CELLS:PHENOMENA ANDPOSTULATED FUNCTIONALINVOLVEMENT
Stable Lateral Segregation ofMembrane Transporters
Among eukaryotic cells possessing a cell wall,the first evidence for clustering of PM con-stituents or membrane microdomain formationwas obtained in S. cerevisiae. Sur7—a mem-brane protein of unknown function (155)—andthe arginine/H+ cotransporter Can1 were ob-served to localize to discrete spotty zones en-riched in ergosterol (40). These zones weretermed the MCC (85). A dozen proteins in-tegral to the PM—comprising several specifictransporters as well as proteins of unknownfunction—have been localized to the MCC
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Tab
le1
Pro
teom
ican
alys
esof
dete
rgen
t-re
sist
antm
embr
ane
(DR
M)f
ract
ions
from
plan
ts
Mat
eria
lP
rote
in-d
etec
tion
met
hods
Pro
tein
sas
soci
ated
wit
hD
RM
Lip
idan
alys
isR
efer
ence
Nico
tiana
taba
cum
Mic
roso
mal
frac
tion
from
leav
esG
elel
ectr
opho
resis
,im
mun
oana
lysis
,fre
eze-
frac
ture
and
imm
uno-
EM
6G
PIs,
hete
rotr
imer
icG
!(o
ften
inag
greg
ates
)+
108
Nico
tiana
taba
cum
Puri
fied
PMfr
omle
aves
and
from
BY-2
cells
Gel
elec
trop
hore
sis,
imm
unoa
naly
sis-s
peci
fican
tibod
y,M
S
7en
rich
edin
leav
es,7
enri
ched
inBY
-2ce
llsFo
cuse
don
lipid
anal
ysis
96
Ara
bido
psis
thal
iana
Tot
alm
embr
anes
from
callu
sG
elel
ectr
opho
resis
,im
mun
oana
lysis
,MS
$15
enri
ched
,$15
depl
eted
+15
Nico
tiana
taba
cum
Puri
fied
PMfr
omBY
-2ce
llsG
elel
ectr
opho
resis
,MS
145
enri
ched
"99
Ara
bido
psis
thal
iana
Puri
fied
PMfr
omse
edlin
gsan
dfr
omce
llcu
lture
Gel
elec
trop
hore
sis,
imm
unoa
naly
sis,M
SA
TPa
sePM
A2,
aqua
pori
nPI
P1,
rem
orin
(2–4
-fol
den
rich
men
t)Fo
cuse
don
lipid
anal
ysis
70
Alli
umpo
rrum
Puri
fied
PMfr
omse
edlin
gsan
dfr
omce
llcu
lture
AT
Pase
PMA
2,aq
uapo
rin
PIP1
(2–4
-fol
den
rich
men
t)
Med
icago
trun
catu
laPu
rifie
dPM
from
root
sG
elel
ectr
opho
resis
,MS
270
iden
tified
,PM
Ase
s$7-
fold
enri
chm
ent
Focu
sed
onlip
idan
alys
is74
Ara
bido
psis
thal
iana
Puri
fied
PMfr
omce
llsu
spen
sion
14N
/15N
met
abol
icla
belin
g,m
ethy
l-!
-cyc
lode
xtri
ntr
eatm
ent,
quan
titat
ive
prot
eom
ics
37co
repr
otei
ns+
60
Nico
tiana
taba
cum
Puri
fied
PMfr
omBY
-2ce
llsG
elel
ectr
opho
resis
,MS,
14N
/15N
met
abol
icla
belin
g,qu
antit
ativ
epr
oteo
mic
s
4re
duce
d,1
enri
ched
+13
1
(Con
tinue
d)
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Ory
zasa
tiva
Puri
fied
PMfr
omce
llsu
spen
sion
Gel
elec
trop
hore
sis,
imm
unoa
naly
sis,M
S51
of19
4as
soci
ated
with
inna
teim
mun
ity"
34
Popu
lust
rem
ula
%Po
pulu
stre
mul
oide
sE
nric
hed
PMfr
omhy
brid
cell
susp
ensio
n
Gel
elec
trop
hore
sis,i
mm
unoa
naly
sisG
PI-a
ncho
red
prot
eins
,glu
can
synt
hase
enri
chm
ent
+13
Ave
nasa
tiva
Puri
fied
PMfr
omle
aves
Gel
elec
trop
hore
sis,
imm
unoa
naly
sis,M
S21
9id
entifi
ed"
137
Seca
lece
real
ePu
rifie
dPM
from
leav
es21
3id
entifi
ed
Phas
eolu
svul
gari
sand
Nico
tiana
taba
cum
Puri
fied
PMfr
omle
aves
Gel
elec
trop
hore
sis,i
mm
unoa
naly
sis(a
nti-
PMA
TPa
se)
Not
iden
tified
Focu
sed
onst
erol
anal
ysis
19
Zea
may
sPu
rifie
dPM
from
embr
yosa
Ara
bido
psis
thal
iana
Puri
fied
PMfr
omsu
spen
sion
cells
MS,
14N
/15N
met
abol
icla
belin
g,qu
antit
ativ
epr
oteo
mic
s31
6un
ique
"59
Ara
bido
psis
thal
iana
Puri
fied
PMfr
omse
edlin
gsG
elel
ectr
opho
resis
,im
mun
oana
lysis
,MS
Col
d-re
spon
sive
DR
Mpr
otei
ns(6
incr
ease
dan
d10
decr
ease
din
DR
M)
Focu
sed
onlip
idan
alys
is95
Abb
revi
atio
ns:E
M,e
lect
ron
mic
rosc
opy;
MS,
mas
sspe
ctro
scop
y;PM
,pla
sma
mem
bran
e.a D
RM
obse
rved
alre
ady
atea
rly
deve
lopm
enta
lsta
ge.
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Tab
le2
Met
hods
used
for
dire
ctde
tect
ion
ofm
embr
ane
mic
rodo
mai
ns
Met
hod
Cel
lsSp
ecim
enP
robe
Obs
erva
tion
Ref
eren
ce(s
)C
LSM
Sola
num
tube
rosu
mst
em,S
olanu
mly
cope
rsicu
mpo
llen
tube
Liv
ing
cells
intis
sue
sect
ions
Pept
ide-
spec
ific
antib
ody,
gene
tical
lyen
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508 Malinsky et al.
Erratum
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of F
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PP64CH21-Tanner ARI 24 March 2013 13:8
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DISTRIBUTION, MOBILITY, ANDINTERACTIONS
Versatile fluorescence microscopy approaches can detect plasmamembrane microdomains; direct visualization of the uneven dis-tribution of membrane constituents is only the most straightfor-ward. The presence of microdomains also affects the mobilityof membrane-associated molecules. This can be quantified bysingle-particle tracking (SPT); fluorescence recovery after pho-tobleaching (FRAP) and its modifications, in particular throughuse of photoactivatable fluorescent proteins; or fluorescence cor-relation spectroscopy (FCS). FRAP-like approaches reveal mo-bility and the immobile fraction of fluorophores by marking theirlocalized subset. FCS monitors only the mobile fraction of fluo-rophores, analyzing point fluctuations in fluorescence intensity.Lateral membrane diffusion is slow enough to be monitored bya currently described FCS modification, raster image correlationspectroscopy (RICS), which incorporates the time delay betweenparts of the scanned microscopic image into the analysis. Thepresence of microdomains is also indicated by intermolecular in-teractions, detected as cross-correlated fluctuations of two fluo-rescent markers [a technique known as cross-correlation RICS(ccRICS)]; Forster resonance energy transfer (FRET), which oc-curs at the nanometer scale; and bimolecular fluorescence com-plementation (BiFC). FRET is manifested as loss of donor fluo-rescence intensity, gain of acceptor intensity, or shortening ofdonor fluorescence lifetime monitored by fluorescence lifetimeimaging (FLIM). A complete fluorescent protein formed fromtwo nonfluorescent tags reports localization of stable complexesin BiFC experiments.
Image restoration:mathematical methodof increasing imageresolution byincluding informationabout the imagingproperties of themicroscope; also calledimage deconvolution
(39), which adopts a characteristic structure offurrow-like invaginations (134). Other proteinsassociate with it from the cytosolic side in alarge, supposedly hemitubular heterocomplex(58, 160) called the eisosome (39, 151).
The most abundant PM protein, Pma1, isexcluded from the MCC and forms its ownmesh-like subcompartment, the MCP (mem-brane compartment of Pma1). The separationof the two compartments is remarkably sta-ble, and the compartment domains are largelyimmobile (40, 86). Patterns of lateral segrega-tion of some other transporters, like the generalamino acid permease Gap1 and the hexose per-mease Hxt1, are not resolvable by diffraction-
limited fluorescence microscopy (72, 85). How-ever, total internal reflection fluorescence mi-croscopy combined with either image restora-tion or structured illumination microscopy(see sidebar Superresolution Fluorescence Mi-croscopy Techniques) has revealed uneven dis-tributions of all PM proteins, including Gap1and Hxt1, reflecting individual and (to variousextents) overlapping microdomains in the PM.Higher-than-random overlap of four membersof the hexose transporter family possessinghighly similar transmembrane domains—Hxt1,Hxt2, Hxt3, and Hxt6—suggests that these mi-crodomains could execute specific functions inthe yeast PM (130).
Immunostaining has revealed accumulationof the hexose/H+ symporter HUP1 in a punc-tate pattern in the native PM of Chlorella kessleri.Interestingly, HUP1 accumulates in the MCCwhen functionally expressed in yeast (41). Thisobservation suggests that lateral microdomainsin yeast PMs could reflect an evolutionarilyconserved principle of PM organization. In-deed, specific structures resembling the char-acteristic furrows of the MCC have been re-ported in many organisms, including bacteria,fungi, and plants (134 and references therein).
Krugel et al. (66) showed that in plant sieve-element cells, the native sucrose transporterSUT1 also localizes to the PM in a punctate pat-tern. They suggested that the redox-dependentdimerization and microdomain association ofSUT1 regulate sugar transport. The potassiumchannel KAT1 forms distinct clusters that arerandomly distributed in the PMs of leaf epi-dermis cells, whereas in guard cells, this chan-nel is ordered in radially oriented linear do-mains. KAT1 clusters are probably linked toother cellular structures, as they show a highdegree of positional stability (136). The lineardomains in guard cells depend on turgor pres-sure, as they disappear under hypertonic con-ditions. The orientation of KAT1 domains co-incides with the orientations of cellulose fibrilsand the cortical array of microtubules, point-ing to an interaction between KAT1 and ei-ther the cell wall or the cortical microtubules(50).
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Flotillins/reggies:widely expressed andevolutionarilyconserved proteinsinvolved in membraneshaping, pathogenesis,and symbiotic events
Sterol-rich domains(SRDs): large,steady-state PMmicrodomainsenriched in sterols,reflecting the dynamicbalance betweendirected exocytosis andlateral diffusion
Aquaporin PIP2;1 is another plant proteinthat has been shown to form clusters in thePM (79). Each cluster comprises up to fourmolecules, which is consistent with a crystallog-raphy study predicting that aquaporins residein the PM as tetramers (144). Under normalconditions, PIP2;1 is removed from the PM viaclathrin-mediated endocytosis, whereas underhigh-salt conditions, the protein follows an ad-ditional internalization route (79) that coincideswith its increased association with flotillins suchas Flot1—a class of proteins that has beenpreviously reported to play roles in clathrin-independent endocytosis (37).
Steady-State Microdomains Inducedby Cell PolarizationUneven distribution of PM components duringcell polarization is well documented in manymodel organisms. As cell polarization–inducedPM domains are usually of micrometer scale,they can be well resolved by wide-field fluo-rescence microscopy. Their dynamic charac-ter, however, prevents their detection in puri-fied membranes. It should be emphasized that,in addition to the above-mentioned argumentsagainst DRM as a tool for detecting PM mi-crodomains, DRM analysis fails to detect theseparticular domains for this fundamental reason.
In fungal PMs, sterol-rich domains (SRDs)form during polarized growth at the elongat-ing cell tips and locations of septum forma-tion (Schizosaccharomyces pombe) (150), at the hy-phal tips of pathogenic yeast (Candida albicans)(89) and filamentous fungi (Aspergillus nidulans)(139), and at the mating projections (shmoos) ofpheromone-stimulated cells (S. cerevisiae) (5).SRDs show strong filipin staining, reflectinga high concentration of sterols, and accumu-late specific proteins (78, 88, 110, and manyother studies). Analysis of Laurdan fluorescenceshowed that, compared with the overall PM, thelipid bilayer at the shmoo tip is more condensedand ordered (110).
In plants, Liu et al. (81) visualized SRDformation by using filipin fluorescence and aphase-sensitive dye, di-4-ANEPPDHQ, at the
SUPERRESOLUTION FLUORESCENCEMICROSCOPY TECHNIQUES
The finite resolution of fluorescence microscopy resulting fromthe wave nature of light remains a limiting factor in optical de-tection of membrane microdomains. Therefore, methods break-ing the diffraction barrier have become popular in membranebiology. Membrane microdomains have been successfully re-solved by structured illumination microscopy (SIM), stimulatedemission depletion (STED), direct stochastic optical reconstruc-tion microscopy (dSTORM), and photoactivated localization mi-croscopy (PALM). SIM uses excitation by a spatially modulatedbeam, which enables reconstruction of high-resolution fluores-cence signals. In STED, only fluorophores in part of the excitedfocal volume are allowed to fluoresce; the rest are forced to relaxvia spectrally distinct stimulated emission. dSTORM and PALMare stochastic approaches that use iterative light-induced acti-vation of sparse subsets of fluorophores, allowing their accuratelocalization. This is a time-consuming process, however, whichseriously limits the use of stochastic approaches in vivo.
Superresolution techniques are frequently combined withtotal internal reflection fluorescence microscopy (TIRFM).Evanescent wave-exciting fluorescence in TIRFM penetrates nofurther than $200–300 nm into the sample, making the use ofTIRFM on membranes covered by a cell wall difficult but notimpossible. A compromise between wide-field fluorescence mi-croscopy and TIRFM in this respect represents variable-angleepifluorescence microscopy (VAEM) or variable-angle TIRFM(VA-TIRFM) with adjustable penetration depth.
tube apex of a rapidly growing pollen tube ofPicea meyeri. NADPH oxidase clustered to thisSRD, which restricted reactive oxygen speciesproduction to the pollen growth point. Thisstudy also showed that sterol-based associationis required for both NADPH oxidase clusteringand activity.
PIN-FORMED (PIN) proteins are effluxcarriers that play an important role in cell po-larity by controlling the directionality of auxinflow toward the place of growth. PIN2 local-izes to distinct clusters in the PM in a sterol-dependent manner. Kleine-Vehn et al. (61)showed that cluster association restricts PIN2mobility, which contributes (along with polar
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Reactive oxygenspecies: modulatorsof cell wall elasticityand pathogen defensethat respond to bioticand abioticenvironmental stimuli
Rac/Rop GTPases:regulators in plantsignal transductionthat act as molecularswitches, regulating avariety of processessuch as polarized cellgrowth
deposition and specific lateral internalization)to its almost exclusive accumulation at the apicalmembrane of cortical and epidermal cells of theroot elongation zone. In roots, PIN1 localizesmostly to the basal membrane of the stele, per-icycle, and endodermis (117). Although not yetresolved by fluorescence microscopy, PIN1’sorganization in microdomains has also beensuggested (143).
Specific interactions between PINs andlipids take part in tropic responses to polar hor-mone transport. Loss of SMT1 (sterol methyl-transferase 1) activity altered PIN1 and PIN3localization, which resulted in severe develop-mental defects (153). Patchy organization ofPIN2 was affected in a null mutant of CPI1 (cy-clopropylsterol isomerase 1), catalyzing a latestep of sterol biosynthesis. In this mutant, en-docytosis of the membrane dye FM4-64 and ofPIN2 was influenced, causing PIN2 mislocal-ization and affecting gravitropism (94). In con-trast to PIN2 and PIN3, PIN1 by itself doesnot exhibit an intrinsic affinity to sterols. To as-sociate with PM microdomains, it requires aninteraction partner, the auxin ABC transporterABCB19, as suggested by the higher suscepti-bility of PIN1 to detergents when ABCB19 ismissing (143).
Microdomains Induced in Response toInteraction with MicroorganismsInitial indications of microbe interaction–induced rearrangement of PM constituentswere obtained from studies of changes in thedetergent solubility of membrane proteins. Al-though detergent extraction as a method fordescribing the real situation in living cells wasdismissed, this approach is still useful as away to indicate that the protein distributionwithin the membrane plane is not constant.The techniques of 14N/15N metabolic label-ing and global quantitative proteomics haverevealed differences in the amounts of sev-eral proteins in the detergent-resistant frac-tion obtained from BY-2 (Nicotiana tabacumcv. Bright Yellow 2) cells treated with crypto-gein, an elicitor of hypersensitive defense re-
sponse, as compared with control cells (131).An immediate change in PM protein solu-bility in response to the bacterial pathogen-associated molecular pattern was documentedusing the same technique. In reaction to flag-ellin treatment of Arabidopsis thaliana cell sus-pension, the DRM protein composition pro-foundly changed. The amounts of 10 receptor-like kinases (e.g., FLS2 and FER), 4 PM H+-ATPases, and 2 Ca2+-ATPases were signifi-cantly augmented in DRMs. Surprisingly, 10 of14 identified subunits of two V-ATPases werealso enriched in DRMs upon flg22 treatment(59). Exposition of rice suspension-culture cellsto a chitin elicitor evoked a shift in the dis-tribution of two proteins from the soluble tothe detergent-resistant fraction; these were Os-Rac1 (a member of the Rac/Rop GTPase fam-ily) and its effector RACK1A (34). Further-more, cold acclimation elicited marked changesin DRM composition from the PMs of Ara-bidopsis seedlings: P-type H+-ATPases, aqua-porins, and endocytosis-related proteins wereaugmented in the DRM fraction, whereas tubu-lins, actins, and V-type H+-ATPase subunitswere reduced (95).
The above data, obtained by quantitativeanalyses before and after elicitation, reflectchanges in the lipid environment of the mon-itored proteins. However, these observationscan be interpreted in several ways. Besides themost commonly accepted explanation that theprotein changes its lipid environment withinthe PM, a protein can also acquire detergent re-sistance following its stimuli-controlled releasefrom cortically located endogenous membranesto the sterol-rich PM. In addition, a proteincan acquire detergent resistance by a physicalassociation with another protein stably incor-porated in the PM, and a protein modificationmay change its affinity for certain lipids. And,of course, all of the above mechanisms may actin concert.
In response to external stimuli, specific pro-teins of a host cell are typically delivered to aplace of physical contact with the elicitor. Con-sequently, the specific proteins accumulate andputatively mutually interact, which results in
512 Malinsky et al.
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the formation of defined compartments withinthe plane of the PM and/or in the cell cor-tex. These events can be directly observed us-ing fluorescence labeling technology and high-resolution live cell imaging. Below we presentthe most recent studies that include microscopydocumentations of plant PM compartmenta-tion in response to interaction with pathogenicor symbiotic microorganisms.
In 2005, Bhat et al. (14) published thefirst study directly observing pathogen-initiatedprotein rearrangement in living cells. Usingfluorescence microscopy, they showed that en-try of the pathogen Blumeria graminis f. sp.hordei into host (Hordeum vulgare) epidermalcells coincided with accumulation of the in-tegral PM proteins MLO, ROR2, PEN1, cy-tochrome b561, and SYP132-related syntaxinbeneath fungal appressoria. Filipin staining ofsterols documented the sterol rearrangement inthe invading fungus. In the fungus’s nonger-minating conidiospores, sterols accumulated atopposite poles of the spores; after germination,the sterol focal spot diffused and polarized tothe tips of appressorial germ tubes.
Using confocal laser scanning microscopy,Koh et al. (62) visualized subcellular eventsoccurring on infection of Arabidopsis plantsby Erysiphe cichoracearum. In host epidermalcells, various green fluorescent protein (GFP)–tagged organelles moved toward penetrationsites and accumulated near penetration pegs.Interestingly in terms of PM compartmenting,some GFP-tagged PM marker proteins aggre-gated into rings around penetration sites. Theformation of the extrahaustorial membrane,which separates the haustorium from the hostcytoplasm, was accompanied by the exclusion ofeight PM markers that remained in a collar-likeformation around the haustorial neck. Theseobservations document the formation of a com-plex and unique specialized membrane that isdifferent from the host PM.
Screening barley sequence databases for po-tential interacting proteins with RACB (a bar-ley ROP protein involved in susceptibility tothe fungal pathogen B. graminis f. sp. hordei )revealed a 171-amino-acid protein, RIC171.
Whereas RACB is a PM-associated protein (viaits prenylation), RIC171 lacks any known mo-tifs for membrane targeting. After the pathogenattack, the fluorescence of the two proteins ac-cumulated at the sites of infection; the activatedRACB obviously recruits RIC171 into distinctmicrodomains at the cell periphery (118).
Peroxidase-dependent oxidative bursts playan important role in Arabidopsis basal resis-tance. Generated hydrogen peroxide is requiredfor wild-type levels of pathogen-triggeredimmunity-associated responses, one of whichresults in the synthesis of callose and its dis-tinct deposition at the cell cortex. Aniline bluestaining revealed a distinct callose depositionpattern in mature Arabidopsis leaves exposed toFlg22, Elf26, or FoCWE (flagellin, elongationfactor Tu, and cell wall elicitor preparations,respectively) (21). This pattern was lacking inuntreated cells and cells with knockdowns ofperoxidase ( prx34). In Arabidopsis, 12 genes en-code putative callose synthase, all containingmultiple transmembrane domains. The visual-ization of their product documents the forma-tion of pathogen-activated callose-synthase mi-crodomains.
Ligand-induced receptor endocytosis canserve as another example of PM compartmen-tation or microdomain formation. The bindingof elicitor flg22 to GFP-tagged FLS2 inducesreceptor internalization, which was visualizedin discrete puncta in the cell cortex (113). Theaccumulation of the FLS2/flg22 complex to-gether with proteins of the endocytic machineryobviously must occur in defined microdomains.
Similarly, transmission electron microscopyhas revealed a specific stimulation of clathrin-coated pit formation a few minutes after theaddition of cryptogein to tobacco BY-2 cells.The process is dependent on reactive oxygenspecies production by the NADPH oxidaseNtrbohD (73). The localization of this pro-tein in microdomains was elegantly demon-strated by electron microscopy using a methodbased on the generation of cerium perhydrox-ides. The observed stained patches at the cellcortex reflected the activity of the PM-locatedNtrbohD (77).
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Bu!er-treated root hairs
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LYK3:GFP FLOT4:mCherry Merged LYK3:GFP FLOT4:mCherry MergedSinorhizobium meliloti–treated root hairs
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Figure 1Redistribution of plant membrane proteins after bacterial treatment. Symbiotic rhizobia trigger a change in the localization of theMedicago truncatula lysine motif receptor-like kinase LYK3 and the flotillin FLOT4. Both proteins accumulate at the root hair cell tipsafter bacterial inoculation. In addition, colocalization of LYK3 and FLOT4 increases in inoculated root hairs. (a) Codistributions ofFLOT4:mCherry and LYK3:GFP signals in 3D space. (b) Higher magnification of the images in panel a. Adapted from Reference 44;copyright c! 2011 by the American Society of Plant Biologists.
In 2010, Haney & Long (43) reported astrong upregulation of two flotillin-like pro-teins, FLOT2 and FLOT4, during early sym-biotic events in nitrogen fixation and showedthat these two proteins play crucial, nonredun-dant roles in the process. The two proteinsdistribute in distinct patches at the cortices ofnoninoculated root hair cells. Upon Sinorhi-zobium meliloti inoculation, the FLOT4:GFPpatches (but not the FLOT2:GFP patches) po-larly localize to the root hair tips. Fluores-cently labeled LYK3, a lysine motif receptor-like kinase, forms dynamic patches located atthe cortices of root epithelial cells and roothairs. On symbiotic onset, the LYK3:GFP dy-namics decrease and, as shown in Figure 1,its patches significantly overlap with those ofFLOT4:mCherry (44). These two studies ex-plicitly document a protein-mediated recruit-ment of another protein into transiently formedmicrodomains. Recruitment of the symbiosis-specific protein PUB1 (a putative PUB E3ubiquitin ligase) to the microdomains above ismediated by a direct interaction with LYK3(93).
FORMATION AND FUNCTIONALRELEVANCE OF PLASMAMEMBRANE MICRODOMAINS INPLANTS AND FUNGIPublished explanations of PM microdomainformation have so far dealt with several mech-anisms that could be involved in this process(Figure 2). Notably, all of these mechanismsare conserved throughout the phylogenic tree,including in plants and fungi. In a living cell,the PM should be considered a compartment inwhich virtually all of the proposed mechanismscan be effective at the same time. The temporaland local prevalence of some of them then de-fines the size, stability, composition, and struc-ture of particular membrane microdomains.One of the main tasks of future membrane bi-ology research is to uncover how a cell can reg-ulate individual mechanisms of membrane do-main formation in order to accomplish variousbiological functions.
To study the potential functional relevanceof lateral PM compartmentation, it is neces-sary to consider the following: A phenotypeobserved in a mutant lacking a protein that is
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Lipid demixing
Kinetic polarization Protein fences and cell wall
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Figure 2Mechanisms of plasma membrane (PM) microdomain formation. A PM accompanied by a cell wall is shown at various scales. (a) In amixture of a large number of different lipids participating in the PM structure (colored circles in panels a and b), one or several specificlipid species spontaneously aggregate. The resulting nanoscale microdomains exhibit various degree of order. (b) Fast directed vesiculartransport (thick arrow) contributes to microdomain PM organization by forming large, temporary microdomains continuouslydissipated by slow lateral diffusion of the delivered membrane material (thin arrows). (c) Curved membrane domains are supported byprotein scaffolds. For example, furrow-like PM invaginations of the MCC, scaffolded by a hemitubular eisosome, remain apart fromcytoskeletal networks and interfere with the lateral motion of the cortical endoplasmic reticulum. (d ) The PM surface is divided intosmall areas by cytoskeletal networks attached to the PM. Proteins attached or integral to the PM, as well as membrane lipids (notshown), therefore exhibit caged or hop lateral diffusion. Some of the proteins are additionally connected to the cell wall.
normally located in a PM microdomain con-veys information about the function of this pro-tein but not about the potential importance ofits localization in a special membrane environ-ment. Just a few cases—all in fungal cells—arecurrently known where a protein localized in aspecific PM microdomain becomes dislocalized
or homogeneously distributed owing to spe-cific treatments or mutations. Only when theamount of the protein in question per cell staysthe same under these two conditions can anyphenotype observed be attributed to the local-ization of this protein. For such new regula-tory mechanisms, we propose the designation
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Control by change inlocation (CCL):a proposed regulatorymechanism related tothe compartmentationof membranecomponents and theirinduced lateral changein position
“control by change in location” (CCL). Below,we discuss a few examples of the correspondingphenomena documented so far.
Spontaneous Lipid DemixingDepending foremost on temperature, lipids ina bilayer can adopt either solid-phase (gel; So)or liquid-phase behavior. In mixed bilayers con-taining cholesterol or its analogs, liquid ordered(Lo) and liquid disordered (Ld) phases are fur-ther distinguished. In the So phase, lipids aretightly packed and do not move toward eachother. In liquid phases, lipid molecules can lat-erally diffuse through the bilayer, with the acylchains either extended (in the Lo phase) orshowing rotational conformation variations (inthe Ld phase) (52).
The theory of lipid rafts has been basedon the assumption that biological membranesrepresent a mixture of Lo areas (rafts) enrichedin sterols and sphingolipids (SLs) and Ld areasenriched in phosphoglycerols, both containingspecific sets of embedded and associated pro-teins. In such a mixture, areas of the same phaseare able to coalesce or disaggregate rapidly de-pending on the actual conditions. This featurefacilitates flexible local adaptation of raft-containing membranes; for example, bipartitedivision of the PM into rafts and nonraft areasis able to partition the surfaces of epithelialcells into apical and basolateral regions facingtwo different environments. However, takinginto account the great variety of biologicalfunctions that membrane microdomains aresupposed to administer, such as trafficking andnumerous types of signaling, a much broaderdiversity of microdomains with respect to lipidand protein composition has to be expected.Therefore, a much more complex mechanismof membrane raft formation, based mainlyon specific lipid-protein and protein-proteininteractions, has been recently suggested (124).
Sterols and SLs may indeed interact in form-ing microdomains in the PM, as is often sug-gested in the context of lipid rafts, but their rela-tive abundance and interaction with other lipidsmay diverge, resulting largely in the formationof a variety of microdomain types with distinct
lipid compositions (Figure 2a) and thus pro-viding individual microenvironments for mem-brane proteins. For example, in S. cerevisiae,Pma1 has been shown to prefer an SL-richmicroenvironment (145) but also to avoid thesterol-rich MCC (85). In addition, indepen-dent indications of SL-rich microdomains inyeast PMs have recently been published (4). Al-though the distribution of PM SLs has not yetbeen shown by direct visualization, these obser-vations indicate at least a partial spatial separa-tion of sterols and SLs in yeast PMs.
It seems evident that the transmembrane do-mains of integral membrane proteins associatewith specific lipids and that this lipid-proteininteraction determines the protein localization.Point mutation analysis has shown that a singletransmembrane domain of the plasmodesmata-located protein PDLP1a determines thetargeting of this membrane protein to plasmo-desmata in A. thaliana, and that it can also targetheterologous proteins to this location (141).Membrane distribution of the yeast ferro-O2-oxidoreductase Fet3, required for iron uptake,was changed by replacing its transmembranedomain with the only transmembrane domainof the small regulatory subunit of the PMH+-ATPase Pmp1. The chimera not onlynonrandomly overlapped with Pmp1 but alsolost the Fet3 function. It is unclear whether thefunctional defect was caused by protein delocal-ization from the specific lipid milieu or whetherthe transmembrane domain exchange itselfaffected the enzymatic activity of the protein.
The MCC-specific arginine permease Can1has been dislocated to the PM microdomain oc-cupied by the H+-ATPase Pma1 by direct bind-ing of Can1-GFP to Pma1 tagged with GFPbinder (GB), a monomeric, high-affinity anti-GFP antibody. Similarly to Fet3, Can1-GFPloses its function upon binding to Pma1-GB.Importantly, however, its transport activity ispreserved when bound to the MCC residentSur7-GB, indicating that protein localizationand not the GB binding itself might determinethe effect (130). In wild-type cells, Can1 con-sistently localizes in the specific ergosterol-richlipid milieu of the MCC (40). Furthermore,
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MCC accumulation and the transport activityof the hexose transporter HUP1 were reducedin mutants defective in ergosterol biosynthe-sis (41), pointing to the participation of specificlipids in this protein’s localization and function.
It is worth mentioning that microdomainsof specific lipid composition are formed evenin PMs lacking sterols. Spiral-shaped mi-crodomains enriched in negatively chargedcardiolipin and phosphatidylglycerol were de-tected by 10-N-nonyl-acridine orange and FeiMao (FM) dyes in Bacillus subtilis PMs (7). Eventhough FM dyes alter the PM structure (54),the lipid spirals seem to be stain independent:The FM staining did not affect the distributionof MinD, an ATPase involved in septation,which to a high extent colocalized with thesemicrodomains in the bacterial membrane (7).Spiral-shaped microdomains were also re-ported in Escherichia coli PMs (121).
The necessity of lipid phase separationfor spontaneous demixing of membrane com-ponents remains to be verified. For exam-ple, in PMs of the cyanobacterium Gloeobac-ter violaceus, quantitative differences in thelipid composition of two functionally distinct,protein-specific microdomains differing in rel-ative amounts of mono- and digalactosyldiacyl-glycerol were not considered sufficient to in-duce phase separation (112).
The existence of So-phase domains, whichare not addressed by the lipid raft hypothe-sis, in otherwise liquid biological membraneshas been reported by time-resolved fluores-cence spectroscopy studies since the 1980s(57). Highly ordered, ergosterol-free, and SL-enriched gel domains were recently found inyeast PMs (4). These domains clearly differfrom lipid rafts in both composition and de-gree of order. The functional relevance andprotein settlement of highly packed, rigid gel-phase (So-phase) membrane areas are still un-clear. They may play a role in yeast adaptationto oxidative stress (107).
In the mammalian field, the most promi-nent concept concerning the role of lipid raftshas been their potential involvement as signal-ing platforms (132). A significant improvement
in signal output, especially at low signal in-tensities, is due to the cooperative associationof interdependent signaling components; thishas been shown, for example, in RAS signal-ing (142). T cell signaling, however, requiresnot raft-based mechanisms but rather directprotein-protein interactions, which of courseare always a prerequisite for signaling processes(25). Thus, the specific role of lipid rafts in sig-naling remains a matter of dispute. In the plantfield, the importance of rafts in signaling hasbeen repeatedly stated (102); however, we arenot aware of any convincing evidence for this.
Energy-Dependent DirectedMembrane FlowFor many purposes, including cell growth,reproduction, sensing, and adaptation to awide range of environmental stimuli, the PM iscontinuously rearranged. To a large extent, thismembrane flow is accomplished through vesic-ular transport. The endocytosed membrane canbe directionally recycled to specific sites of thecell surface (1). This spatial separation of endo-and exocytosis, in combination with limitedlateral diffusion of the reinserted membranecomponents, is able to maintain steady-stategradients of PM constituents (Figure 2b).This was first demonstrated on peripheralaccumulations of transferrin receptors in thePMs of human-derived fibroblasts (17). Thepremise of slow lateral diffusion is especiallyvalid in the PMs of plants and fungi (146).Therefore, kinetic polarization achieved byvesicle recycling seems to be a dominantmechanism of PM domain formation in theseorganisms (reviewed in 156).
For example, the formation and mainte-nance of SRDs in plant and fungal PMs requirea functional secretory pathway. Sterol-richmembranes of secretory vesicles, routed bycytoskeletal structures, fuse with the PM atsites of intensive assembly of the new cellsurface. The spreading of sterol moleculesin the PM is too slow to reach an evensterol distribution over the cell surface, and asteady-state sterol gradient is established by
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coupling this polarized sterol delivery withongoing nonpolarized endocytosis (146).
Alvarez et al. (2) have reviewed the poten-tial biological roles of fungal SRDs. In thepathogenic yeast C. albicans, for example, manyglycophosphatidylinositol-anchored virulencefactors have been found to accumulate at SRDs.In addition, ergosterol promotes the accumula-tion of the signaling lipid phosphatidylinositol4,5-bisphosphate [PI(4,5)P2] at mating projec-tions of S. cerevisiae. The SL-free (and thereforelipid raft–independent) membrane pool of er-gosterol in SRDs was also shown to promotemembrane fusion during mating (55).
Endocytic membrane recycling also plays arole in keeping the apical PM domain enrichedin PI(4,5)P2 and diacylglycerol (DAG) duringpollen tip growth in N. tabacum. PI(4,5)P2 issynthesized at the growing tip in a Rac/Rop-type Rho-family small-GTPase-dependentmanner. The spreading of PI(4,5)P2 from thetip is prevented by laterally targeted phos-pholipase C, which hydrolyzes PI(4,5)P2 toDAG and inositol 1,4,5-trisphosphate. DAGis consequently endocytosed and deliveredback to the apex, retrograde to the flow ofPI(4,5)P2 spreading out from the apical area.This PI(4,5)P2/DAG accumulation at the tipis essential for pollen tip growth (47). Thepresumable dependence of PI(4,5)P2/DAGmicrodomains on membrane sterols has notbeen tested.
In contrast, however, membrane sterols areessential for polar localization of PIN2 in Ara-bidopsis (94). Newly synthesized PINs are deliv-ered to the entire PM but then rapidly cycle be-tween the PM and endosomes (22) (Figure 3).Kleine-Vehn et al. (61) found that endocytosedPIN1 and PIN2 are targeted to the center ofthe polar PM domain. Computer modeling ofpolar domain dynamics revealed that polar de-position of recycled PIN proteins was not suf-ficient to maintain the observed PIN polarity,and suggested that a contribution of spatiallyconfined endocytosis could be required in thiscase as well. The enrichment of the crucial en-docytic factor clathrin at lateral cell sides hasbeen accordingly documented (61).
Spatially confined endocytosis has onlyrecently been described in yeast as well. Local-ization of MCC domains (39) and the actual dis-tribution of the cortical endoplasmic reticulum(133) were found to determine the positioningof endocytic events in the S. cerevisiae PM.Restriction of an endocytic site initiation, andpossibly of other interactions between solublecytoplasmic factors and the PM, to membraneareas not associated with the endoplasmicreticulum could generate kinetic polarizationof the PM similar to that described in the ex-amples above and define additional functionalmicrodomains at the cell surface. This wasobserved in S. pombe cells, in which the corticalendoplasmic reticulum delimits an area in thePM where the actomyosin-ring-organizingprotein Mid1 can bind and where the plane ofcytokinesis is consequently established (157).
Protein-Mediated Restrictionsof Lateral DiffusionThe free lateral diffusion of PM constituentsis slowed and obstructed by membrane-integrated or membrane-associated protein as-semblies. Based on the mechanisms of theirfunctions, these assemblies can be catego-rized as membrane-shaping scaffolds (coats)(Figures 2c and 4), which generate curva-tures in the membrane that represent uniqueenvironments preferred by specific lipid andprotein species, and membrane-cytoskeletonfences (corrals) (Figure 2d ), which partitionthe entire PM into small regions with crossableborders.
Highly curved areas in biological mem-branes are usually enriched in specific lipids.Lipids alone, however, are not likely to gener-ate and maintain this curvature (56). It is, rather,generated either by membrane-integrated butnot membrane-spanning coat proteins (forexample, caveolins and flotillin/reggie pro-teins; reviewed in Reference 8) or by pro-teins attached to the cytoplasmic membranesurface either directly [for example, proteinscontaining the BAR (Bin/Amphiphysin/Rvs-homology) domain; reviewed in Reference 92]
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a 0 min 2 min
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Figure 3Mechanism of PIN polarity generation in Arabidopsis roots. (a) The two steps that restore polar plasmamembrane (PM) localization of PIN1-YFP ( first subpanel ): After complete photobleaching of PIN1-YFPfluorescence (second subpanel ), the newly synthesized protein is first delivered to the PM in a nonpolarmanner (third subpanel ). Later, polarity is established by spatially confined endocytic recycling ( fourthsubpanel ). Arrowheads point to nonpolar or basal (rootward) PIN1-YFP localization. (b) Ratio of polar tolateral PIN1-YFP intensity prior to the bleaching (left bar) and at the indicated time points of the recovery offluorescent protein distribution (middle and right bars). Adapted from Reference 23; copyright c! 2008 by theNature Publishing Group.
or via adaptor proteins (clathrin; see Reference106).
Besides proteins involved in clathrin-mediated endocytosis from yeast to plants andmammals (amphiphysins and epsins; see Refer-ence 49), novel BAR domain–containing pro-teins were recently identified in the yeast S.cerevisiae. Homodimers of Pil1 and Lsp1 adopta serpent or banana shape typical for the BARdomain protein superfamily (104), bind to thePM, and form eisosomes, which underlie thefurrow-like invaginations of the yeast PM, i.e.,the MCC (98, 120, 134) (Figure 4). The bio-logical role of the MCC is not fully understood.Among other functions suggested, several ob-servations indicate its involvement in the re-sponse to various stress stimuli in fungi (11, 26,32, 51, 107, 154).
A recent study of Berchtold et al. (11)suggests a feedback loop in which membranestress—caused by a drop in SL levels, hypo-osmotic shock, or direct mechanical stretch-ing of the PM—induces the release of Slm1/2proteins from the MCC-associated eisosometo activate TORC2 (target of rapamycin ki-nase complex 2) localized in another PM do-
main, the MCT (membrane compartment ofTORC2). The activated TORC2 then reg-ulates SL metabolism via the Ypk1 pathwayby phosphorylating evolutionarily conservedOrm proteins (135) to mediate compensatorychanges. In this respect, invaginated membraneareas of the MCC resemble mammalian caveo-lae, which disappear in conditions of acute me-chanical stress (128) and release caveolin-1 to
Pil1/Lsp1
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Figure 4Model for the assembly of yeast eisosomal proteins at the plasma membrane(PM) in the form of a presumably hemitubular scaffold shaping the furrow-likemembrane invagination of the MCC. Adapted from Reference 58.
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activate the Ypk1 homolog protein kinase B(Akt) (119). The flattening of MCC furrowsin response to membrane stress has not beenreported; however, cells lacking the proposedSL sensor, MCC-accumulated Nce102 (32), doexhibit a flat MCC (134). Both of these ob-servations represent excellent examples of theCCL principle—after the stimulus, the effector(Slm1, caveolin-1) must be laterally relocalizedin the PM to execute the function.
Other Ypk-activating kinases—namely, thePDK1 (3-phosphoinositide-dependent proteinkinase 1) homologs Pkh1 and -2, which are keyplayers in SL-mediated signaling (158)—alsoaccumulate in eisosomes, making the MCC atarget of protein kinase inhibitor–based anti-fungal drugs (10). Eisosomal Pil1 and Lsp1,which are phosphorylated by Pkh1 and -2, werefound to downregulate resistance to heat stressand, with respect to their wide evolutionaryconservation, suggested to function as nega-tive regulators of PDK-like protein kinases andtheir downstream cellular pathways that controlcell growth and survival (158). It should be men-tioned here that PM invaginations that are mor-phologically highly similar to the MCC werealso reported in freeze-tolerant (but not freeze-sensitive) Chlamydomonas and Chloromonas uni-cellular algae (20). In the above context, thisobservation accents the involvement of thesestructures in an evolutionarily conserved mech-anism of adaptation to dehydration stress.
Proteins and lipids in a PM with cytoskele-ton fences undergo diffusion confined to indi-vidual fence-defined membrane microdomainscombined with hop diffusion across their bor-ders (69). In mammals, the size of fence-definedmembrane microdomains was estimated to varybetween 30 and 230 nm depending on thecell line (101). In plants, much larger fence-bordered microdomains were suggested onlyrecently. Oda & Fukuda (103) showed that intobacco leaf epidermis, a scattered network ofcortical microtubules restricts the localizationof the PM-anchored MIDD1-ROP11 complexinto micrometer-size PM areas. To analyzemembrane protein diffusion in detail, Gross-mann et al. (G. Grossmann, J.J. Lindeboom,
W.B. Frommer & D.W. Ehrhardt, unpub-lished results) performed fluorescence recov-ery after photobleaching (FRAP)/fluorescenceloss induced by photobleaching (FLIP) mea-surements and particle tracking of individualprotein clusters in Arabidopsis tissue and wall-less protoplasts and found that cortical micro-tubules have a general impact on the dynam-ics at the PM by defining diffusion barriers andthereby subdividing the PM into large-scale dif-fusional domains.
Membrane-associated septins, which areGTP-binding proteins assembled into rods andfilaments in eukaryotic cells from protists tomammals (but not, however, in higher plants),partition the PM during cytokinesis (116). InS. cerevisiae, the septin ring consists ofmembrane-anchored circumferential pairs oftightly associated septin filaments intercon-nected by simple axial filaments intersecting thecircumferential filaments with 30-nm spacing(12). During budding, this hourglass-shapedseptin network effectively prevents the mixingof mother-cell and bud membranes.
In cells possessing cell walls, the mobility ofPM constituents can also be constrained fromthe outer PM surface. For example, AtFH1(A. thaliana actin nucleation protein formin 1)has been shown to form a bridge across thePM, connecting the actin cytoskeleton with thecell wall (90). But even in the absence of di-rect interactions between PM proteins and cellwall components, the presence of a cell wallconstrains the lateral diffusion of PM proteins(91, 103).
Microdomain Organization NeedsEnergy: Plasma Membrane ofDe-energized CellsThe MCC patterning of H+ symporters andergosterol in the yeast PM was lost within sec-onds when the PM potential (!") was re-duced by uncoupling agents. Even partial PMdepolarization due to HUP1-mediated sym-port of the nonmetabolized glucose analog 6-deoxyglucose plus protons into S. cerevisiae cellswas enough to release HUP1-GFP from the
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MCC patches (40). As an analogy, Can1 leavesthe MCC with an excess of arginine (39). It hasbeen suggested that accumulation of H+ sym-porters in an endocytotically silent area of theMCC prevents the untimely internalization ofthese proteins. According to the proposed CCLprinciple, the symporters, after their function isno longer required, are released from the pro-tected area of the MCC to the surrounding PMin order to enable their rapid internalizationand degradation (40).
The above observations led to the postula-tion of a crucial role of !" in the molecularorder of the membrane. Interestingly, the PMsof energized and “de-energized” cells differin their detergent susceptibility. Uncoupler-treated cells are much more resistant to deter-gents (40). A similar observation had also previ-ously been reported for algae and bacteria (63).
De-energized cells are also much less sus-ceptible to polyenes, like nystatin—indicatingreduced sterol accessibility—and to oligopep-tide antibiotics, like histatin 5 (63, 115). A40-fold higher concentration of the polyeneamphotericin, for example, had to be used forstarved or 2,4-dinitrophenol (DNP)–treatedC. albicans cells to obtain the same toxic effectas for a growing culture (35). In addition, anincreased resistance of nongrowing, starvedbacteria to several classes of antibiotics (76) maybe related to the same phenomenon. A change
in !" has also been shown to affect membraneorder in liposomes of synthetic lipids (48). Theexact mechanism of these changes in detergentsusceptibility, sterol accessibility, and generalpermeability are not understood. It seems thatthe PM is somehow more tightly organized andless accessible to detergents and polyenes oncethe membrane potential is reduced. Indeed,a considerably lower amount of H3 TritonX-100 binds to such membranes (63). As analternative interpretation, it has been suggestedthat the membrane order might be increasedby cytoskeletal rearrangements following ATPdepletion (148 and references therein).
Whatever the case may be, it is evident thatdifferent energy-dependent states of membraneorder can be distinguished in fungi, algae, andbacteria (higher plants have not been investi-gated). One state may correspond to a more ac-tive, optimally organized, and compartmentedstate of the membrane, allowing a certain pas-sive exchange of small molecules when cells areenergized and growing well; another may cor-respond to a tighter, less detergent-accessiblestate adapted to cells at rest. To investigatethis most likely general phenomenon in higherplants and elucidate its exact mechanism in anyorganism should be an interesting problemto be solved in the future. Until the answeris known, we do not fully understand thePM.
SUMMARY POINTS
1. A large body of evidence supports the existence of membrane microdomains in bacteria,fungi, and plants. They are mostly stable entities and differ from the highly dynamic lipidor membrane rafts postulated to exist in mammalian cells.
2. The detergent extraction method does not lead to the isolation of authentic PM mi-crodomains. Use of the differential detergent solubility of membrane proteins has beena standard purification method, and in this way the procedure may still be useful.
3. Various methods for the direct visualization of membrane microdomains such as super-resolution techniques of fluorescence microscopy are applicable to plants and shouldbecome the approaches of primary choice in membrane microdomain detection.
4. Several organizing principles result in membrane microdomain formation. Liquid-ordered phase separation of membrane lipids (lipid rafts) is only one of them.
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5. Membrane proteins involved in cell-cell interactions, membrane transport, stress re-sponse, and polarized growth have been shown to localize to membrane microdomains.
6. Evidence is available for the functional importance of concentrating specific membranecomponents in microdomains. Functional clustering accompanied by modulation of theactivity of PM proteins has been observed, and a new regulatory mechanism is postulated:control by change in location (CCL).
7. Membrane order, microdomain organization, and general properties like sterol accessi-bility and detergent susceptibility of the PMs in energized cells differ from those of thePMs of de-energized or starving cells.
DISCLOSURE STATEMENTThe authors are not aware of any affiliations, memberships, funding, or financial holdings thatmight be perceived as affecting the objectivity of this review.
ACKNOWLEDGMENTSThis work was supported by DFG (Priority Program 1108 and TA 36/18-1 to W.T.), Fondsder Chemischen Industrie (W.T.), grants from the Grant Agency of the Czech Republic(P302/11/0146 and P205/12/0720 to J.M.), institutional grants (AVOZ 50390703 to J.M. andRVO 61388971 to M.O.), and an EMBO long-term fellowship (to G.G.).
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Annual Review ofPlant Biology
Volume 64, 2013Contents
Benefits of an Inclusive US Education SystemElisabeth Gantt ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 1
Plants, Diet, and HealthCathie Martin, Yang Zhang, Chiara Tonelli, and Katia Petroni ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !19
A Bountiful Harvest: Genomic Insights into Crop DomesticationPhenotypesKenneth M. Olsen and Jonathan F. Wendel ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !47
Progress Toward Understanding Heterosis in Crop PlantsPatrick S. Schnable and Nathan M. Springer ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !71
Tapping the Promise of Genomics in Species with Complex,Nonmodel GenomesCandice N. Hirsch and C. Robin Buell ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !89
Understanding Reproductive Isolation Based on the Rice ModelYidan Ouyang and Qifa Zhang ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 111
Classification and Comparison of Small RNAs from PlantsMichael J. Axtell ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 137
Plant Protein InteractomesPascal Braun, Sebastien Aubourg, Jelle Van Leene, Geert De Jaeger,
and Claire Lurin ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 161
Seed-Development Programs: A Systems Biology–Based ComparisonBetween Dicots and MonocotsNese Sreenivasulu and Ulrich Wobus ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 189
Fruit Development and RipeningGraham B. Seymour, Lars Østergaard, Natalie H. Chapman, Sandra Knapp,
and Cathie Martin ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 219
Growth Mechanisms in Tip-Growing Plant CellsCaleb M. Rounds and Magdalena Bezanilla ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 243
Future Scenarios for Plant PhenotypingFabio Fiorani and Ulrich Schurr ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 267
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PP64-frontmatter ARI 25 March 2013 10:21
Microgenomics: Genome-Scale, Cell-Specific Monitoring of MultipleGene Regulation TiersJ. Bailey-Serres ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 293
Plant Genome Engineering with Sequence-Specific NucleasesDaniel F. Voytas ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 327
Smaller, Faster, Brighter: Advances in Optical Imagingof Living Plant CellsSidney L. Shaw and David W. Ehrhardt ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 351
Phytochrome Cytoplasmic SignalingJon Hughes ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 377
Photoreceptor Signaling Networks in Plant Responses to ShadeJorge J. Casal ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 403
ROS-Mediated Lipid Peroxidation and RES-Activated SignalingEdward E. Farmer and Martin J. Mueller ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 429
Potassium Transport and Signaling in Higher PlantsYi Wang and Wei-Hua Wu ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 451
Endoplasmic Reticulum Stress Responses in PlantsStephen H. Howell ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 477
Membrane Microdomains, Rafts, and Detergent-Resistant Membranesin Plants and FungiJan Malinsky, Miroslava Opekarova, Guido Grossmann, and Widmar Tanner ! ! ! ! ! ! ! 501
The EndodermisNiko Geldner ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 531
Intracellular Signaling from Plastid to NucleusWei Chi, Xuwu Sun, and Lixin Zhang ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 559
The Number, Speed, and Impact of Plastid Endosymbioses inEukaryotic EvolutionPatrick J. Keeling ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 583
Photosystem II Assembly: From Cyanobacteria to PlantsJorg Nickelsen and Birgit Rengstl ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 609
Unraveling the Heater: New Insights into the Structure of theAlternative OxidaseAnthony L. Moore, Tomoo Shiba, Luke Young, Shigeharu Harada, Kiyoshi Kita,
and Kikukatsu Ito ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 637
Network Analysis of the MVA and MEP Pathways for IsoprenoidSynthesisEva Vranova, Diana Coman, and Wilhelm Gruissem ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 665
vi Contents
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PP64-frontmatter ARI 25 March 2013 10:21
Toward Cool C4 CropsStephen P. Long and Ashley K. Spence ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 701
The Spatial Organization of Metabolism Within the Plant CellLee J. Sweetlove and Alisdair R. Fernie ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 723
Evolving Views of Pectin BiosynthesisMelani A. Atmodjo, Zhangying Hao, and Debra Mohnen ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 747
Transport and Metabolism in Legume-Rhizobia SymbiosesMichael Udvardi and Philip S. Poole ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 781
Structure and Functions of the Bacterial Microbiota of PlantsDavide Bulgarelli, Klaus Schlaeppi, Stijn Spaepen, Emiel Ver Loren van Themaat,
and Paul Schulze-Lefert ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 807
Systemic Acquired Resistance: Turning Local Infectioninto Global DefenseZheng Qing Fu and Xinnian Dong ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 839
Indexes
Cumulative Index of Contributing Authors, Volumes 55–64 ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 865
Cumulative Index of Article Titles, Volumes 55–64 ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 871
Errata
An online log of corrections to Annual Review of Plant Biology articles may be found athttp://www.annualreviews.org/errata/arplant
Contents vii
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ANNUAL REVIEWSIt’s about time. Your time. It’s time well spent.
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New From Annual Reviews:Annual Review of Statistics and Its ApplicationEditor: Stephen E. Fienberg, Carnegie Mellon UniversityAssociate Editors: Nancy Reid, University of Toronto
Stephen M. Stigler, University of ChicagoThe Annual Review of Statistics and Its Application aims to inform statisticians and quantitative methodologists, as well as all scientists and users of statistics about major methodological advances and the computational tools that
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TABLE OF CONTENTS:
What Is Statistics? Stephen E. FienbergA Systematic Statistical Approach to Evaluating Evidence from Observational Studies, David Madigan, Paul E. Stang, Jesse A. Berlin, Martijn Schuemie, J. Marc Overhage, Marc A. Suchard, Bill Dumouchel, Abraham G. Hartzema, Patrick B. RyanThe Role of Statistics in the Discovery of a Higgs Boson, David A. van DykBrain Imaging Analysis, F. DuBois BowmanStatistics and Climate, Peter GuttorpClimate Simulators and Climate Projections, Jonathan Rougier, Michael GoldsteinProbabilistic Forecasting, Tilmann Gneiting, Matthias KatzfussBayesian Computational Tools, Christian P. RobertBayesian Computation Via Markov Chain Monte Carlo,
Build, Compute, Critique, Repeat: Data Analysis with Latent Variable Models, David M. BleiStructured Regularizers for High-Dimensional Problems: Statistical and Computational Issues, Martin J. Wainwright
High-Dimensional Statistics with a View Toward Applications in Biology, Peter Bühlmann, Markus Kalisch, Lukas MeierNext-Generation Statistical Genetics: Modeling, Penalization, and Optimization in High-Dimensional Data, Kenneth Lange, Jeanette C. Papp, Janet S. Sinsheimer, Eric M. SobelBreaking Bad: Two Decades of Life-Course Data Analysis in Criminology, Developmental Psychology, and Beyond, Elena A. Erosheva, Ross L. Matsueda, Donatello TelescaEvent History Analysis, Niels Keiding
Christopher D. Steele, David J. BaldingUsing League Table Rankings in Public Policy Formation: Statistical Issues, Harvey GoldsteinStatistical Ecology, Ruth KingEstimating the Number of Species in Microbial Diversity Studies, John Bunge, Amy Willis, Fiona WalshDynamic Treatment Regimes, Bibhas Chakraborty, Susan A. MurphyStatistics and Related Topics in Single-Molecule Biophysics, Hong Qian, S.C. KouStatistics and Quantitative Risk Management for Banking and Insurance, Paul Embrechts, Marius Hofert
Access this and all other Annual Reviews journals via your institution at www.annualreviews.org.
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