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Membrane Microdomains,
Rafts, and Detergent-Resistant
Membranes in Plants
and Fungi
Jan Malinsky,1,, Miroslava Opekarova,2,
Guido Grossmann,3, and Widmar Tanner4,
1

Institute of Experimental Medicine and 2 Institute of Microbiology, Academy of Sciences of


the Czech Republic, 142 20 Prague, Czech Republic; email: malinsky@biomed.cas.cz,
opekaro@biomed.cas.cz

Department for Plant Biology, Carnegie Institution for Science, Stanford,


California 94305; email: guido.grossmann@cos.uni-heidelberg.de

Institute of Cell Biology and Plant Physiology, University of Regensburg, 93053


Regensburg, Germany; email: sekretariat.tanner@biologie.uni-regensburg.de

Annu. Rev. Plant Biol. 2013. 64:50129


The Annual Review of Plant Biology is online at
plant.annualreviews.org
This articles doi:
10.1146/annurev-arplant-050312-120103
c 2013 by Annual Reviews.
Copyright 
All rights reserved

Equally contributing authors.

Corresponding authors.

Present address: Centre for Organismal


Studies/CellNetworks, Universitat Heidelberg,
69120 Heidelberg, Germany

Keywords
lateral plasma membrane compartments, pathogen response,
detergent resistance, sterol-rich domains

Abstract
The existence of specialized microdomains in plasma membranes, postulated for almost 25 years, has been popularized by the concept of
lipid or membrane rafts. The idea that detergent-resistant membranes
are equivalent to lipid rafts, which was generally abandoned after a
decade of vigorous data accumulation, contributed to intense discussions about the validity of the raft concept. The existence of membrane
microdomains, meanwhile, has been veried by unequivocal independent evidence. This review summarizes the current state of research
in plants and fungi with respect to common aspects of both kingdoms.
In these organisms, principally immobile microdomains large enough
for microscopic detection have been visualized. These microdomains
are found in the context of cell-cell interactions (plant symbionts and
pathogens), membrane transport, stress, and polarized growth, and the
data corroborate at least three mechanisms of formation. As documented in this review, modern methods of visualization of lateral membrane compartments are also able to uncover the functional relevance
of membrane microdomains.

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Contents

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INTRODUCTION . . . . . . . . . . . . . . . . . .
MEMBRANE MICRODOMAINS
AND LIPID RAFTS: A
HISTORICAL ACCOUNT . . . . . . .
MEMBRANE MICRODOMAINS IN
PLANT AND FUNGAL CELLS:
PHENOMENA AND
POSTULATED FUNCTIONAL
INVOLVEMENT . . . . . . . . . . . . . . . .
Stable Lateral Segregation of
Membrane Transporters . . . . . . . .
Steady-State Microdomains Induced
by Cell Polarization . . . . . . . . . . . . .
Microdomains Induced in Response
to Interaction with
Microorganisms . . . . . . . . . . . . . . . .
FORMATION AND FUNCTIONAL
RELEVANCE OF PLASMA
MEMBRANE MICRODOMAINS
IN PLANTS AND FUNGI . . . . . . . .
Spontaneous Lipid Demixing . . . . . . .
Energy-Dependent Directed
Membrane Flow . . . . . . . . . . . . . . . .
Protein-Mediated Restrictions of
Lateral Diffusion . . . . . . . . . . . . . . . .
Microdomain Organization Needs
Energy: Plasma Membrane of
De-energized Cells . . . . . . . . . . . . .

502

502

505
505
511

512

514
516
517
518

520

INTRODUCTION
The emergence of the plasma membrane
(PM) has been one of the most crucial steps in
evolution. This special organelle surrounding
each cell serves as an active interface between
the cell and its environment. It mediates import
and export of a multitude of molecules with
high selectivity and embeds dozens of sensors
responsible for environmental signal transfer to
the cell interior. In this way, each cell communicates with the environment, and eventually with
neighboring cells and the whole organism. For
a long time, the membrane was imagined as a
uniform envelope made up of lipids and housing
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Malinsky et al.

the proteins responsible for the processes mentioned above. Recent data indicate that the PM
is a highly heterogeneous organelle subdivided
into areas of distinct composition, structure,
and function. These areas were named lipid
or membrane rafts, detergent-resistant membranes (DRMs), micro- or nanodomains, etc.
As discussed below, some of these designations
were unfortunate (see sidebar Essential Terms).
This review summarizes current information about compartments in PMs of plants and
fungi with respect to their size, composition,
dynamics, mechanisms of formation, and functional relevance. Considering the functional
importance of this compartmentation, we focus
on the question of whether it really makes a difference to a cell if a transport or sensor protein
is homogeneously distributed in the membrane
or concentrated at a special location, and what
happens when it is translocated to a different
place within the membrane.

MEMBRANE MICRODOMAINS
AND LIPID RAFTS:
A HISTORICAL ACCOUNT
One important contribution of botany to general biology has been the discovery of the PM
and the phenomenon of osmosis. In plant cells,
which are surrounded by cell walls, plasmolysis could be observed and interpreted in terms
of the existence of a semipermeable membrane
releasing water (but not colored substances like
anthocyanins) from the cell protoplasm (28,
102, 109; reviewed in 129). It was a long time
before this postulated membrane was visualized
by high-resolution electron microscopy (114).
Although modeling of the membrane began
shortly after Gorter & Grendel (38) presented
evidence for the lipid bilayer, the rst generally accepted model (which is still largely accepted today) was Singer & Nicolsons (127)
uid mosaic. The authors modeled cellular membranes as homogeneous lipid bilayers that membrane proteins are either embedded in or attached to, resulting in a mosaic of
protein-containing and bare lipid areas. Postulation of the membrane uidity was based on

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Frye & Edidins (33) cell fusion experiment,


which demonstrated that within 40 min after the fusion of two mammalian cells, specic
membrane proteins of both cells are uniformly
distributed.
Over the years, several changes and amendments to the Singer-Nicolson model have been
broadly accepted. These include (a) the asymmetry of the lipid bilayer (16); (b) the restriction
of the free mobility of membrane proteins by
their interactions with intracellular and/or extracellular components, such as the cytoskeleton and extracellular matrix/cell wall (29, 132;
for plant cells, see 6, 91); and (c) much higher
protein density at the membrane surface than
was considered in the classical Singer-Nicolson
model. As estimated for human erythrocytes
(27) and for synaptic vesicles (138), practically
the entire membrane may actually be covered
by proteins on both sides. The transmembrane
helices of integral membrane proteins alone
occupy approximately 23% of the actual volume of the lipid core layer of the erythrocyte
PM (27). Quantitative data for plant PMs are
not available so far, but a comparable protein
density can be assumed.
The biomembrane structural model was further revised by the concept of lateral segregation of proteins and lipids into membrane
microdomains. Although these microdomains
were intensely discussed for approximately two
decades, there is no general agreement on their
nature. Nevertheless, numerous publications
support the idea that molecules in the PM are
not equally distributed, and that lateral subcompartments exist that divide the PM into
distinct microdomains (53, 87, 97, 124, 159).
These observations almost led to a paradigm
shift, and certainly did lead to signicantly increased interest in membrane and especially
in lipid research. Although Singer & Nicolson
(127) assumed a homogeneous lipid bilayer and
a random distribution of integral membrane
proteins, they did not exclude clustering, noting that wherever nonrandom distributions
are found, mechanisms must exist which are responsible for them (p. 724). To nd and elucidate these mechanisms as well as the functional

ESSENTIAL TERMS
Membrane microdomain: In the frame of this review, the
terms membrane microdomain and plasma membrane (PM) microdomain denote a lateral compartment within the plane of the
PM that exhibits a composition, structure, and biological function
distinct from the surrounding membrane, regardless of its size,
stability, or mechanism of formation. It is noteworthy that individual membrane microdomains, resulting from spontaneous
and/or energy-dependent lateral segregation of a plentitude of
PM constituents (lipids and proteins), may differ substantially
from one another in all the above-mentioned aspects.
Lipid rafts: Historically, lipid rafts were postulated as Golgi
apparatus or endoplasmic reticulum membranederived sorting platforms forming liquid-ordered microdomains in the PM
that are enriched in sphingolipids and sterols. They are now considered nanoassemblies consisting of specic proteins and lipids.
Membrane microdomains based on lipid rafts are supposed to
be highly dynamic. Their actual size, composition, and function
strongly depend on external conditionstemperature, signaling
events, etc. It is questionable, therefore, whether intact raft-based
microdomains can be detected in isolated membranes.
Detergent-resistant membranes (DRMs): DRMs, also called
detergent-insoluble membranes (DIMs), is a term used for the
nonsolubilized fraction of membranes extracted by mild detergent under dened conditions. Membrane proteins identied as
parts of DRMs are frequently denoted in the literature as raft
proteins, although no relation between the hundreds of proteins
identied in DRMs and their localization in specic membrane
microdomains has been found to date.

importance of lateral membrane compartmentation seems precisely the task of the day.
Differences between particular PM areas
have been repeatedly reported. The polar
accumulation of bacterial chemotaxis receptors is one well-documented example (84).
Another is the acid-banding phenomenon
of internodal cells in Characeae. Zones of
excess proton export alternate with zones of
OH surplus along the long axis of the cell,
which reects different activities of proton
pumps and ion transporters at special PM sites
(82). Differences in membrane composition
are known to exist in polarized cellsfor

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example, at rapidly growing cell poles (5, 17,


47). Indeed, the challenging question of how
polarized cells, such as the epithelial cells of the
small intestine, obtain their specic apical and
basolateral protein and lipid outt has started a
completely new research area. In 1988, Simons
& van Meer (126) postulated that sorting and
targeting of proteins and lipids may be directly
interlinked and may arise from the formation
of microdomains mimicking the properties of
the membranes of their destination (p. 6200).
To biochemically demonstrate an association between the locally concentrated membrane proteins and lipids, Brown & Rose (18)
described a simple detergent extraction method
for these special cells to separate the apical membrane from the basolateral one. They
found that, similar to the apical membranes,
the DRMs exhibit a lipid composition enriched
in cholesterol and glycosphingolipids. These
ndings, unfortunately generalized to any cell
type, led to the postulation that enrichment in
these lipids may result in a lateral segregation
of membrane domains of higher and lower local densities in vivo. The term lipid rafts was
coined to describe the more compact membrane entities (125). The hypothesis that, in
addition to acting as vehicles for sorting and
targeting, these rafts potentially represent signaling platforms by clustering receptors and
signal transducing proteins attracted hundreds
of scientists in a short time. Lateral immiscibility was observed in liposomes containing
cholesterol, where lipids can undergo spontaneous phase separation (9) (see also Formation
and Functional Relevance of Plasma Membrane
Microdomains in Plants and Fungi, below).
This was also recently observed in protein-rich
biomembranes, but only under special conditions, for example, when PMs were disconnected from the cytoskeleton (80).
Solubilization in 1% Triton X-100 at 4 C
became the generic assay that was widely applied to analyze the membrane fractions of any
species, tissue, or cell type. Membranes that
did not dissolve under these conditions were
called lipid rafts, membrane rafts, DRMs, or
detergent-insoluble membranes (DIMs). Be-

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Malinsky et al.

cause 10 years of research did not produce any


independent evidence that the DRM fraction
contains any intact membrane substructures,
this view could not be sustained, and in 2003 it
was dismissed by the leading groups in the eld
at a raft meeting in Tomar, Portugal. The
meeting report (161) stated, A general consensus that emerged at this meeting about the
nature of a raft in a cell membrane is summarized as follows: Considering the complexity of
the system and the poorly understood nature
of DRM formation, it is unlikely that DRMs
that are derived from cells reect some preexisting 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 DRMs
do not reect membrane substructures in
living cells, detergent extraction is still widely
used to study membrane domains in plants (see
Table 1). As in earlier studies in the animal
eld, the rst plant studies showed that a large
number of membrane proteins could not be
extracted with 1% Triton X-100 at 4 C (15, 96,
108). However, as mentioned above, the results
obtained by this procedure reect mainly
that different membrane proteinsregardless
of where they are locatedrequire different
amounts of detergent to become soluble and
cannot be considered proof of any denite
membrane structure or arrangement. Nevertheless, some of the DRM proteins were
shown to form clusters in the PM and may
therefore constitute potential membrane platforms. The PM-attached protein remorin can
be considered a prominent example. As documented by immunogold detection, remorin is
organized at the PM in patches approximately
70 nm in diameter and becomes homogeneously distributed when the sterol content is
reduced by methyl--cyclodextrin (111).
It has been argued that detergent extraction,
as a rst approach, may after all be a valuable
and helpful tool to enrich proteins of potential
interest for further membrane microdomain
oriented research. Once a protein has been established as a DRM component, Simon-Plas
et al. (123) suggested using additional methods

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for a subsequent rigorous proof of its associations with a specic membrane structure. This
approach, however, does not address two major concerns. First, proteome analysis of DRM
fractions detects hundreds of proteins (see
Table 1), of which generally very few, if any,
seem to be of special interest for the question
investigated. When employed for protein purication, this approach is therefore rather inefcient compared with other procedures available (afnity chromatography, coimmunoprecipitation, etc.). Second, and an even more serious concern, is that specic associations can
be disrupted by detergents, and protein components that are potentially much more important
in a given study might be discarded solely because they stayed in the detergent-soluble part.
In Saccharomyces cerevisiae, it has been shown
that even cytosolic proteins bind to special
membrane compartments, and these proteins
are, in part, indispensable for the establishment
of these microdomainsfor example, various
eisosomal proteins required for MCC (membrane compartment of Can1) formation (see below as well as References 11, 40, 98, and 151).
The current state of the art in the animal
eldthe guiding example for a long time
does not clarify matters; in fact, in November 2011 Science included the question do lipid
rafts exist? as one of ve cells lingering mysteries that need clarication (75). As pointed
out by Jacobson and coworkers (53), as the accuracy of the experimental approaches was continuously increasing, the sizes of rafts in mammalian cells became smaller and smaller over
time, and are now on the order of <20 nm (30).
This is the magnitude of ordinary protein complexes plus dozens of lipid molecules, in the case
of membrane protein complexes. However, the
raft attributes would then be reduced to functionally relevant interactions of specic proteins with certain lipids. In fact, this has been articulated in a way by Anderson & Jacobson (3) in
their lipid shell hypothesis. In another alternative, Kusumi et al. (68) have suggested a lateral
compartmentation of PMs based on the diffusion of certain proteins in limited membrane
areas; the meshwork of cytoskeletal elements

underlying the PM and associated proteins are


supposed to generate the limitations.
The purpose of this review is not to answer the question posed by Science (which related to animal cells anyway). Rather, we accent that the plant and fungal research communities are in the fortunate position that a
fair number of papers have been published that
document the existence of special lateral membrane compartments through various microscopic methods (see Table 2 and the sidebars Distribution, Mobility, and Interactions
and Superresolution Fluorescence Microscopy
Techniques). The direct visualization of membrane microdomains in living cells will be the
approach of choice to provide convincing evidence for their existence and biological role.
The thriving eld of superresolution techniques will further promote the discovery and
analysis of subdiffraction-size inhomogeneities
in biological membranes (42). Examples from
studies of plant and fungal cells presented in this
review document that the PM microdomains of
cell wallpossessing cells exhibit common features and differ from what the mammalian research community calls rafts.

MCC (membrane
compartment of
Can1): membrane
microdomain in yeast
PMs equivalent to the
furrow-like
invagination supported
by the cytoplasmic
protein complex
(eisosome)

MEMBRANE MICRODOMAINS IN
PLANT AND FUNGAL CELLS:
PHENOMENA AND
POSTULATED FUNCTIONAL
INVOLVEMENT
Stable Lateral Segregation of
Membrane Transporters
Among eukaryotic cells possessing a cell wall,
the rst evidence for clustering of PM constituents or membrane microdomain formation
was obtained in S. cerevisiae. Sur7a membrane protein of unknown function (155)and
the arginine/H+ cotransporter Can1 were observed to localize to discrete spotty zones enriched in ergosterol (40). These zones were
termed the MCC (85). A dozen proteins integral to the PMcomprising several specic
transporters as well as proteins of unknown
functionhave been localized to the MCC

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Malinsky et al.

ATPase PMA2, aquaporin PIP1,


remorin (24-fold enrichment)

Gel electrophoresis,
immunoanalysis, MS

Arabidopsis thaliana
Puried PM from seedlings and from
cell culture

Gel electrophoresis, MS, 14 N/15 N


metabolic labeling, quantitative
proteomics

Nicotiana tabacum
Puried PM from BY-2 cells

4 reduced, 1 enriched

37 core proteins

14 N/15 N

Arabidopsis thaliana
Puried PM from cell suspension

metabolic labeling,
methyl--cyclodextrin treatment,
quantitative proteomics

270 identied, PMAses 7-fold


enrichment

Gel electrophoresis, MS

Medicago truncatula
Puried PM from roots

ATPase PMA2, aquaporin PIP1


(24-fold enrichment)

145 enriched

Gel electrophoresis, MS

Nicotiana tabacum
Puried PM from BY-2 cells

Allium porrum
Puried PM from seedlings and from
cell culture

99

15 enriched, 15 depleted

Gel electrophoresis,
immunoanalysis, MS

Arabidopsis thaliana
Total membranes from callus

60

131

(Continued )

74

Focused on lipid analysis

70

96

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Focused on lipid analysis

15

Focused on lipid analysis

108

7 enriched in leaves, 7 enriched in


BY-2 cells

Reference

Gel electrophoresis,
immunoanalysis-specic antibody,
MS

Lipid analysis

Nicotiana tabacum
Puried PM from leaves and from
BY-2 cells

Proteins associated with DRM


6 GPIs, heterotrimeric G (often
in aggregates)

Protein-detection methods
Gel electrophoresis,
immunoanalysis, freeze-fracture
and immuno-EM

Nicotiana tabacum
Microsomal fraction from leaves

Material

Proteomic analyses of detergent-resistant membrane (DRM) fractions from plants

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Table 1

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Cold-responsive DRM proteins (6


increased and 10 decreased in
DRM)

Gel electrophoresis,
immunoanalysis, MS

Arabidopsis thaliana
Puried PM from seedlings

Abbreviations: EM, electron microscopy; MS, mass spectroscopy; PM, plasma membrane.
a
DRM observed already at early developmental stage.

316 unique

Not identied

MS, 14 N/15 N metabolic labeling,


quantitative proteomics

Gel electrophoresis, immunoanalysis


(anti-PM ATPase)

Arabidopsis thaliana
Puried PM from suspension cells

Zea mays
Puried PM from embryosa

Phaseolus vulgaris and Nicotiana


tabacum
Puried PM from leaves

213 identied

219 identied

Gel electrophoresis,
immunoanalysis, MS

Avena sativa
Puried PM from leaves

Focused on lipid analysis

95

59

19

137

Focused on sterol analysis

13

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Secale cereale
Puried PM from leaves

GPI-anchored proteins, glucan


synthase enrichment

Gel electrophoresis, immunoanalysis

Populus tremula Populus tremuloides


Enriched PM from hybrid cell
suspension

51 of 194 associated with innate


immunity

Gel electrophoresis,
immunoanalysis, MS

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Oryza sativa
Puried PM from cell suspension

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507

508

Specimen

Probe

Malinsky et al.

Rat OLN-93 oligodendroglial cells


Mouse embryonic broblast

Saccharomyces cerevisiae
Arabidopsis thaliana seedling
Arabidopsis thaliana seedling

RICS

ccRICS

TIRFM

VAEM

VA-TIRFM

Arabidopsis thaliana differentiating


metaxylem cells
Arabidopsis thaliana root

Living cells in seedlings

Nicotiana tabacum leaf epidermis

BiFC

SPT, FCS

Living cells in tissue


sections

Vigna unguiculata L. leaf

FRAP, FRET,
FLIM

Living cells in seedlings

Living cells in seedlings

Living cells

Living cells in culture

Living cells in culture

Living cells in seedlings

Protoplasts

Living cells in seedlings

Arabidopsis thaliana root

FRAP

Living cells in tissue


sections

Nicotiana tabacum leaf epidermis

Genetically encoded
uorescent proteins

Genetically encoded
uorescent proteins

Genetically encoded
uorescent proteins

Genetically encoded
uorescent proteins

Genetically encoded
uorescent proteins

Genetically encoded
uorescent proteins

Genetically encoded split


uorescent proteins

Genetically encoded
uorescent proteins

Genetically encoded
uorescent proteins

Genetically encoded
uorescent proteins

Observation

Uneven protein
distribution

Uneven protein
distribution

Uneven protein
distribution

Composition and
mobility of protein
complexes

Map of protein
mobility

Mobility of proteins

Uneven protein
distribution

Distribution of protein
complexes

Stable higher-order
proteolipid complexes

Mobility of proteins

Mobility of protein
clusters

Uneven protein
distribution

Uneven protein
distribution, mobility
of protein clusters

Uneven protein
distribution

(Continued )

152

64

130

24

36

79

103

65

149

83

136

112

43, 44

66

Reference(s)

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Photoactivation

Native uorescent
proteins (chlorophyll,
phycocyanin,
phycoerythrin)

Living cells

Gloeobacter violaceus

Peptide-specic antibody,
genetically encoded
uorescent proteins
Genetically encoded
uorescent proteins

Living cells in tissue


sections
Living cells in tissue
sections

CLSM

Erratum

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Medicago truncatula root

Cells
Solanum tuberosum stem, Solanum
lycopersicum pollen tube

Method

Table 2 Methods used for direct detection of membrane microdomains

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Fixed tissue section

Pig enterocytes
Living cells

Fixed cells

Nicotiana tabacum BY-2 cells

Saccharomyces cerevisiae

High-pressure-frozen,
freeze-substituted tissue
sections

Nicotiana tabacum var. Xanthi root


apex

Adapted PM protein

None

Contrasted product of
enzymatic activity

Peptide-specic antibody

Distribution of protein
clusters

Domains of increased
membrane thickness

Uneven protein
distribution

Uneven protein
distribution

Uneven protein
distribution

Distribution of lipidand protein-specic


membrane
microdomains

Distribution of
protein-specic
membrane
microdomains

Distribution of specic
membrane
microdomains

Distribution of
lipid-specic
microdomains

Distribution of protein
clusters

Distribution of protein
clusters

45

67

77

111

111

71

31

31

30

122, 147

105

Plant references are listed wherever available. Abbreviations: 3D-SIM, three-dimensional structured illumination microscopy; AFM, atomic force microscopy; BiFC, bimolecular uorescence
complementation; ccRICS, cross-correlation raster image correlation spectroscopy; CLSM, confocal laser scanning microscopy; dSTORM, direct stochastic optical reconstruction microscopy;
FCS, uorescence correlation spectroscopy; FLIM, uorescence lifetime imaging; FRAP, uorescence recovery after photobleaching; FRET, Forster
resonance energy transfer; PALM,

photoactivated localization microscopy; RICS, raster image correlation spectroscopy; SPT, single-particle tracking; STED, stimulated emission depletion; TEM, transmission electron
microscopy; TIRFM, total internal reection uorescence microscopy; VAEM, variable-angle epiuorescence microscopy; VA-TIRFM, variable-angle total internal reection uorescence
microscopy.

AFM

Puried PMs

Genetically encoded
uorescent proteins

Living cells in tissue


sections

Glycolipid- and
peptide-specic
antibodies

Callose-specic antibody

Fluorescent lipid analogs

Genetically encoded
uorescent proteins

Fixed tissue sections

Nicotiana tabacum var. Xanthi leaf

Nicotiana tabacum var. Xanthi


petiole

3D-SIM

Living cells in culture

Fixed cells

Kangaroo rat PtK2 kidney


epithelial cells

STED/FCS

Living cells in culture

Genetically encoded
uorescent proteins

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Borrelia burgdorferi

Rat PC12 neuroendocrine cells

STED

Fixed cells in culture

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TEM

Human HeLa cervical cancer cells

PALM, dSTORM

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DISTRIBUTION, MOBILITY, AND


INTERACTIONS
Versatile uorescence microscopy approaches can detect plasma
membrane microdomains; direct visualization of the uneven distribution of membrane constituents is only the most straightforward. The presence of microdomains also affects the mobility
of membrane-associated molecules. This can be quantied by
single-particle tracking (SPT); uorescence recovery after photobleaching (FRAP) and its modications, in particular through
use of photoactivatable uorescent proteins; or uorescence correlation spectroscopy (FCS). FRAP-like approaches reveal mobility and the immobile fraction of uorophores by marking their
localized subset. FCS monitors only the mobile fraction of uorophores, analyzing point uctuations in uorescence intensity.
Lateral membrane diffusion is slow enough to be monitored by
a currently described FCS modication, raster image correlation
spectroscopy (RICS), which incorporates the time delay between
parts of the scanned microscopic image into the analysis. The
presence of microdomains is also indicated by intermolecular interactions, detected as cross-correlated uctuations of two uorescent markers [a technique known as cross-correlation RICS
(ccRICS)]; Forster
resonance energy transfer (FRET), which oc
curs at the nanometer scale; and bimolecular uorescence complementation (BiFC). FRET is manifested as loss of donor uorescence intensity, gain of acceptor intensity, or shortening of
donor uorescence lifetime monitored by uorescence lifetime
imaging (FLIM). A complete uorescent protein formed from
two nonuorescent tags reports localization of stable complexes
in BiFC experiments.

Image restoration:
mathematical method
of increasing image
resolution by
including information
about the imaging
properties of the
microscope; also called
image deconvolution

510

(39), which adopts a characteristic structure of


furrow-like invaginations (134). Other proteins
associate with it from the cytosolic side in a
large, supposedly hemitubular heterocomplex
(58, 160) called the eisosome (39, 151).
The most abundant PM protein, Pma1, is
excluded from the MCC and forms its own
mesh-like subcompartment, the MCP (membrane compartment of Pma1). The separation
of the two compartments is remarkably stable, and the compartment domains are largely
immobile (40, 86). Patterns of lateral segregation of some other transporters, like the general
amino acid permease Gap1 and the hexose permease Hxt1, are not resolvable by diffractionMalinsky et al.

limited uorescence microscopy (72, 85). However, total internal reection uorescence microscopy combined with either image restoration or structured illumination microscopy
(see sidebar Superresolution Fluorescence Microscopy Techniques) has revealed uneven distributions of all PM proteins, including Gap1
and Hxt1, reecting individual and (to various
extents) overlapping microdomains in the PM.
Higher-than-random overlap of four members
of the hexose transporter family possessing
highly similar transmembrane domainsHxt1,
Hxt2, Hxt3, and Hxt6suggests that these microdomains could execute specic functions in
the yeast PM (130).
Immunostaining has revealed accumulation
of the hexose/H+ symporter HUP1 in a punctate pattern in the native PM of Chlorella kessleri.
Interestingly, HUP1 accumulates in the MCC
when functionally expressed in yeast (41). This
observation suggests that lateral microdomains
in yeast PMs could reect an evolutionarily
conserved principle of PM organization. Indeed, specic structures resembling the characteristic furrows of the MCC have been reported 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 transporter
SUT1 also localizes to the PM in a punctate pattern. They suggested that the redox-dependent
dimerization and microdomain association of
SUT1 regulate sugar transport. The potassium
channel KAT1 forms distinct clusters that are
randomly distributed in the PMs of leaf epidermis cells, whereas in guard cells, this channel is ordered in radially oriented linear domains. KAT1 clusters are probably linked to
other cellular structures, as they show a high
degree of positional stability (136). The linear
domains in guard cells depend on turgor pressure, as they disappear under hypertonic conditions. The orientation of KAT1 domains coincides with the orientations of cellulose brils
and the cortical array of microtubules, pointing to an interaction between KAT1 and either the cell wall or the cortical microtubules
(50).

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Aquaporin PIP2;1 is another plant protein


that has been shown to form clusters in the
PM (79). Each cluster comprises up to four
molecules, which is consistent with a crystallography study predicting that aquaporins reside
in the PM as tetramers (144). Under normal
conditions, PIP2;1 is removed from the PM via
clathrin-mediated endocytosis, whereas under
high-salt conditions, the protein follows an additional internalization route (79) that coincides
with its increased association with otillins such
as Flot1a class of proteins that has been
previously reported to play roles in clathrinindependent endocytosis (37).

Steady-State Microdomains Induced


by Cell Polarization
Uneven distribution of PM components during
cell polarization is well documented in many
model organisms. As cell polarizationinduced
PM domains are usually of micrometer scale,
they can be well resolved by wide-eld uorescence microscopy. Their dynamic character, however, prevents their detection in puried membranes. It should be emphasized that,
in addition to the above-mentioned arguments
against DRM as a tool for detecting PM microdomains, DRM analysis fails to detect these
particular domains for this fundamental reason.
In fungal PMs, sterol-rich domains (SRDs)
form during polarized growth at the elongating cell tips and locations of septum formation (Schizosaccharomyces pombe) (150), at the hyphal tips of pathogenic yeast (Candida albicans)
(89) and lamentous fungi (Aspergillus nidulans)
(139), and at the mating projections (shmoos) of
pheromone-stimulated cells (S. cerevisiae) (5).
SRDs show strong lipin staining, reecting
a high concentration of sterols, and accumulate specic proteins (78, 88, 110, and many
other studies). Analysis of Laurdan uorescence
showed that, compared with the overall PM, the
lipid bilayer at the shmoo tip is more condensed
and ordered (110).
In plants, Liu et al. (81) visualized SRD
formation by using lipin uorescence and a
phase-sensitive dye, di-4-ANEPPDHQ, at the

SUPERRESOLUTION FLUORESCENCE
MICROSCOPY TECHNIQUES
The nite resolution of uorescence microscopy resulting from
the wave nature of light remains a limiting factor in optical detection of membrane microdomains. Therefore, methods breaking the diffraction barrier have become popular in membrane
biology. Membrane microdomains have been successfully resolved by structured illumination microscopy (SIM), stimulated
emission depletion (STED), direct stochastic optical reconstruction microscopy (dSTORM), and photoactivated localization microscopy (PALM). SIM uses excitation by a spatially modulated
beam, which enables reconstruction of high-resolution uorescence signals. In STED, only uorophores in part of the excited
focal volume are allowed to uoresce; the rest are forced to relax
via spectrally distinct stimulated emission. dSTORM and PALM
are stochastic approaches that use iterative light-induced activation of sparse subsets of uorophores, allowing their accurate
localization. This is a time-consuming process, however, which
seriously limits the use of stochastic approaches in vivo.
Superresolution techniques are frequently combined with
total internal reection uorescence microscopy (TIRFM).
Evanescent wave-exciting uorescence in TIRFM penetrates no
further than 200300 nm into the sample, making the use of
TIRFM on membranes covered by a cell wall difcult but not
impossible. A compromise between wide-eld uorescence microscopy and TIRFM in this respect represents variable-angle
epiuorescence microscopy (VAEM) or variable-angle TIRFM
(VA-TIRFM) with adjustable penetration depth.

tube apex of a rapidly growing pollen tube of


Picea meyeri. NADPH oxidase clustered to this
SRD, which restricted reactive oxygen species
production to the pollen growth point. This
study also showed that sterol-based association
is required for both NADPH oxidase clustering
and activity.
PIN-FORMED (PIN) proteins are efux
carriers that play an important role in cell polarity by controlling the directionality of auxin
ow toward the place of growth. PIN2 localizes to distinct clusters in the PM in a steroldependent manner. Kleine-Vehn et al. (61)
showed that cluster association restricts PIN2
mobility, which contributes (along with polar

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Flotillins/reggies:
widely expressed and
evolutionarily
conserved proteins
involved in membrane
shaping, pathogenesis,
and symbiotic events
Sterol-rich domains
(SRDs): large,
steady-state PM
microdomains
enriched in sterols,
reecting the dynamic
balance between
directed exocytosis and
lateral diffusion

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Reactive oxygen
species: modulators
of cell wall elasticity
and pathogen defense
that respond to biotic
and abiotic
environmental stimuli
Rac/Rop GTPases:
regulators in plant
signal transduction
that act as molecular
switches, regulating a
variety of processes
such as polarized cell
growth

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deposition and specic lateral internalization)


to its almost exclusive accumulation at the apical
membrane of cortical and epidermal cells of the
root elongation zone. In roots, PIN1 localizes
mostly to the basal membrane of the stele, pericycle, and endodermis (117). Although not yet
resolved by uorescence microscopy, PIN1s
organization in microdomains has also been
suggested (143).
Specic interactions between PINs and
lipids take part in tropic responses to polar hormone transport. Loss of SMT1 (sterol methyltransferase 1) activity altered PIN1 and PIN3
localization, which resulted in severe developmental defects (153). Patchy organization of
PIN2 was affected in a null mutant of CPI1 (cyclopropylsterol isomerase 1), catalyzing a late
step of sterol biosynthesis. In this mutant, endocytosis of the membrane dye FM4-64 and of
PIN2 was inuenced, causing PIN2 mislocalization and affecting gravitropism (94). In contrast to PIN2 and PIN3, PIN1 by itself does
not exhibit an intrinsic afnity to sterols. To associate with PM microdomains, it requires an
interaction partner, the auxin ABC transporter
ABCB19, as suggested by the higher susceptibility of PIN1 to detergents when ABCB19 is
missing (143).

Microdomains Induced in Response to


Interaction with Microorganisms
Initial indications of microbe interaction
induced rearrangement of PM constituents
were obtained from studies of changes in the
detergent solubility of membrane proteins. Although detergent extraction as a method for
describing the real situation in living cells was
dismissed, this approach is still useful as a
way to indicate that the protein distribution
within the membrane plane is not constant.
The techniques of 14 N/15 N metabolic labeling and global quantitative proteomics have
revealed differences in the amounts of several proteins in the detergent-resistant fraction obtained from BY-2 (Nicotiana tabacum
cv. Bright Yellow 2) cells treated with cryptogein, an elicitor of hypersensitive defense re512

Malinsky et al.

sponse, as compared with control cells (131).


An immediate change in PM protein solubility in response to the bacterial pathogenassociated molecular pattern was documented
using the same technique. In reaction to agellin treatment of Arabidopsis thaliana cell suspension, the DRM protein composition profoundly changed. The amounts of 10 receptorlike kinases (e.g., FLS2 and FER), 4 PM H+ ATPases, and 2 Ca2+ -ATPases were signicantly augmented in DRMs. Surprisingly, 10 of
14 identied subunits of two V-ATPases were
also enriched in DRMs upon g22 treatment
(59). Exposition of rice suspension-culture cells
to a chitin elicitor evoked a shift in the distribution of two proteins from the soluble to
the detergent-resistant fraction; these were OsRac1 (a member of the Rac/Rop GTPase family) and its effector RACK1A (34). Furthermore, cold acclimation elicited marked changes
in DRM composition from the PMs of Arabidopsis seedlings: P-type H+ -ATPases, aquaporins, and endocytosis-related proteins were
augmented in the DRM fraction, whereas tubulins, actins, and V-type H+ -ATPase subunits
were reduced (95).
The above data, obtained by quantitative
analyses before and after elicitation, reect
changes in the lipid environment of the monitored proteins. However, these observations
can be interpreted in several ways. Besides the
most commonly accepted explanation that the
protein changes its lipid environment within
the PM, a protein can also acquire detergent resistance following its stimuli-controlled release
from cortically located endogenous membranes
to the sterol-rich PM. In addition, a protein
can acquire detergent resistance by a physical
association with another protein stably incorporated in the PM, and a protein modication
may change its afnity for certain lipids. And,
of course, all of the above mechanisms may act
in concert.
In response to external stimuli, specic proteins of a host cell are typically delivered to a
place of physical contact with the elicitor. Consequently, the specic proteins accumulate and
putatively mutually interact, which results in

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the formation of dened compartments within


the plane of the PM and/or in the cell cortex. These events can be directly observed using uorescence labeling technology and highresolution live cell imaging. Below we present
the most recent studies that include microscopy
documentations of plant PM compartmentation in response to interaction with pathogenic
or symbiotic microorganisms.
In 2005, Bhat et al. (14) published the
rst study directly observing pathogen-initiated
protein rearrangement in living cells. Using
uorescence microscopy, they showed that entry of the pathogen Blumeria graminis f. sp.
hordei into host (Hordeum vulgare) epidermal
cells coincided with accumulation of the integral PM proteins MLO, ROR2, PEN1, cytochrome b561, and SYP132-related syntaxin
beneath fungal appressoria. Filipin staining of
sterols documented the sterol rearrangement in
the invading fungus. In the funguss nongerminating conidiospores, sterols accumulated at
opposite poles of the spores; after germination,
the sterol focal spot diffused and polarized to
the tips of appressorial germ tubes.
Using confocal laser scanning microscopy,
Koh et al. (62) visualized subcellular events
occurring on infection of Arabidopsis plants
by Erysiphe cichoracearum. In host epidermal
cells, various green uorescent protein (GFP)
tagged organelles moved toward penetration
sites and accumulated near penetration pegs.
Interestingly in terms of PM compartmenting,
some GFP-tagged PM marker proteins aggregated into rings around penetration sites. The
formation of the extrahaustorial membrane,
which separates the haustorium from the host
cytoplasm, was accompanied by the exclusion of
eight PM markers that remained in a collar-like
formation around the haustorial neck. These
observations document the formation of a complex and unique specialized membrane that is
different from the host PM.
Screening barley sequence databases for potential interacting proteins with RACB (a barley ROP protein involved in susceptibility to
the fungal pathogen B. graminis f. sp. hordei )
revealed a 171-amino-acid protein, RIC171.

Whereas RACB is a PM-associated protein (via


its prenylation), RIC171 lacks any known motifs for membrane targeting. After the pathogen
attack, the uorescence of the two proteins accumulated at the sites of infection; the activated
RACB obviously recruits RIC171 into distinct
microdomains at the cell periphery (118).
Peroxidase-dependent oxidative bursts play
an important role in Arabidopsis basal resistance. Generated hydrogen peroxide is required
for wild-type levels of pathogen-triggered
immunity-associated responses, one of which
results in the synthesis of callose and its distinct deposition at the cell cortex. Aniline blue
staining revealed a distinct callose deposition
pattern in mature Arabidopsis leaves exposed to
Flg22, Elf26, or FoCWE (agellin, elongation
factor Tu, and cell wall elicitor preparations,
respectively) (21). This pattern was lacking in
untreated cells and cells with knockdowns of
peroxidase ( prx34). In Arabidopsis, 12 genes encode putative callose synthase, all containing
multiple transmembrane domains. The visualization of their product documents the formation of pathogen-activated callose-synthase microdomains.
Ligand-induced receptor endocytosis can
serve as another example of PM compartmentation or microdomain formation. The binding
of elicitor g22 to GFP-tagged FLS2 induces
receptor internalization, which was visualized
in discrete puncta in the cell cortex (113). The
accumulation of the FLS2/g22 complex together with proteins of the endocytic machinery
obviously must occur in dened microdomains.
Similarly, transmission electron microscopy
has revealed a specic stimulation of clathrincoated pit formation a few minutes after the
addition of cryptogein to tobacco BY-2 cells.
The process is dependent on reactive oxygen
species production by the NADPH oxidase
NtrbohD (73). The localization of this protein in microdomains was elegantly demonstrated by electron microscopy using a method
based on the generation of cerium perhydroxides. The observed stained patches at the cell
cortex reected the activity of the PM-located
NtrbohD (77).

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Sinorhizobium melilotitreated root hairs

Buffer-treated root hairs

LYK3:GFP

FLOT4:mCherry

Merged

LYK3:GFP

FLOT4:mCherry

Merged

2 m

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2 m

Figure 1
Redistribution of plant membrane proteins after bacterial treatment. Symbiotic rhizobia trigger a change in the localization of the
Medicago truncatula lysine motif receptor-like kinase LYK3 and the otillin FLOT4. Both proteins accumulate at the root hair cell tips
after bacterial inoculation. In addition, colocalization of LYK3 and FLOT4 increases in inoculated root hairs. (a) Codistributions of
FLOT4:mCherry and LYK3:GFP signals in 3D space. (b) Higher magnication of the images in panel a. Adapted from Reference 44;
c 2011 by the American Society of Plant Biologists.
copyright 

In 2010, Haney & Long (43) reported a


strong upregulation of two otillin-like proteins, FLOT2 and FLOT4, during early symbiotic events in nitrogen xation and showed
that these two proteins play crucial, nonredundant roles in the process. The two proteins
distribute in distinct patches at the cortices of
noninoculated root hair cells. Upon Sinorhizobium meliloti inoculation, the FLOT4:GFP
patches (but not the FLOT2:GFP patches) polarly localize to the root hair tips. Fluorescently labeled LYK3, a lysine motif receptorlike kinase, forms dynamic patches located at
the cortices of root epithelial cells and root
hairs. On symbiotic onset, the LYK3:GFP dynamics decrease and, as shown in Figure 1,
its patches signicantly overlap with those of
FLOT4:mCherry (44). These two studies explicitly document a protein-mediated recruitment of another protein into transiently formed
microdomains. Recruitment of the symbiosisspecic protein PUB1 (a putative PUB E3
ubiquitin ligase) to the microdomains above is
mediated by a direct interaction with LYK3
(93).
514

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FORMATION AND FUNCTIONAL


RELEVANCE OF PLASMA
MEMBRANE MICRODOMAINS IN
PLANTS AND FUNGI
Published explanations of PM microdomain
formation have so far dealt with several mechanisms that could be involved in this process
(Figure 2). Notably, all of these mechanisms
are conserved throughout the phylogenic tree,
including in plants and fungi. In a living cell,
the PM should be considered a compartment in
which virtually all of the proposed mechanisms
can be effective at the same time. The temporal
and local prevalence of some of them then denes the size, stability, composition, and structure of particular membrane microdomains.
One of the main tasks of future membrane biology research is to uncover how a cell can regulate individual mechanisms of membrane domain formation in order to accomplish various
biological functions.
To study the potential functional relevance
of lateral PM compartmentation, it is necessary to consider the following: A phenotype
observed in a mutant lacking a protein that is

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Lipid demixing

Protein scaffolds

Cell wall

Plasma
membrane

Lipids
Cytoskeletal network

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Endoplasmic reticulum Hemitubular eisosome

Kinetic polarization

Protein fences and cell wall

Integral protein

Integral protein
Cytoskeletal network
Attached protein

Figure 2
Mechanisms of plasma membrane (PM) microdomain formation. A PM accompanied by a cell wall is shown at various scales. (a) In a
mixture of a large number of different lipids participating in the PM structure (colored circles in panels a and b), one or several specic
lipid species spontaneously aggregate. The resulting nanoscale microdomains exhibit various degree of order. (b) Fast directed vesicular
transport (thick arrow) contributes to microdomain PM organization by forming large, temporary microdomains continuously
dissipated by slow lateral diffusion of the delivered membrane material (thin arrows). (c) Curved membrane domains are supported by
protein scaffolds. For example, furrow-like PM invaginations of the MCC, scaffolded by a hemitubular eisosome, remain apart from
cytoskeletal networks and interfere with the lateral motion of the cortical endoplasmic reticulum. (d ) The PM surface is divided into
small areas by cytoskeletal networks attached to the PM. Proteins attached or integral to the PM, as well as membrane lipids (not
shown), therefore exhibit caged or hop lateral diffusion. Some of the proteins are additionally connected to the cell wall.

normally located in a PM microdomain conveys information about the function of this protein but not about the potential importance of
its localization in a special membrane environment. Just a few casesall in fungal cellsare
currently known where a protein localized in a
specic PM microdomain becomes dislocalized

or homogeneously distributed owing to specic treatments or mutations. Only when the


amount of the protein in question per cell stays
the same under these two conditions can any
phenotype observed be attributed to the localization of this protein. For such new regulatory mechanisms, we propose the designation

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Control by change in
location (CCL):
a proposed regulatory
mechanism related to
the compartmentation
of membrane
components and their
induced lateral change
in position

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control by change in location (CCL). Below,


we discuss a few examples of the corresponding
phenomena documented so far.

Spontaneous Lipid Demixing


Depending foremost on temperature, lipids in
a bilayer can adopt either solid-phase (gel; So )
or liquid-phase behavior. In mixed bilayers containing cholesterol or its analogs, liquid ordered
(Lo ) and liquid disordered (Ld ) phases are further distinguished. In the So phase, lipids are
tightly packed and do not move toward each
other. In liquid phases, lipid molecules can laterally diffuse through the bilayer, with the acyl
chains either extended (in the Lo phase) or
showing rotational conformation variations (in
the Ld phase) (52).
The theory of lipid rafts has been based
on the assumption that biological membranes
represent a mixture of Lo areas (rafts) enriched
in sterols and sphingolipids (SLs) and Ld areas
enriched in phosphoglycerols, both containing
specic sets of embedded and associated proteins. In such a mixture, areas of the same phase
are able to coalesce or disaggregate rapidly depending on the actual conditions. This feature
facilitates exible local adaptation of raftcontaining membranes; for example, bipartite
division of the PM into rafts and nonraft areas
is able to partition the surfaces of epithelial
cells into apical and basolateral regions facing
two different environments. However, taking
into account the great variety of biological
functions that membrane microdomains are
supposed to administer, such as trafcking and
numerous types of signaling, a much broader
diversity of microdomains with respect to lipid
and protein composition has to be expected.
Therefore, a much more complex mechanism
of membrane raft formation, based mainly
on specic lipid-protein and protein-protein
interactions, has been recently suggested (124).
Sterols and SLs may indeed interact in forming microdomains in the PM, as is often suggested in the context of lipid rafts, but their relative abundance and interaction with other lipids
may diverge, resulting largely in the formation
of a variety of microdomain types with distinct
Malinsky et al.

lipid compositions (Figure 2a) and thus providing individual microenvironments for membrane proteins. For example, in S. cerevisiae,
Pma1 has been shown to prefer an SL-rich
microenvironment (145) but also to avoid the
sterol-rich MCC (85). In addition, independent indications of SL-rich microdomains in
yeast PMs have recently been published (4). Although the distribution of PM SLs has not yet
been shown by direct visualization, these observations indicate at least a partial spatial separation of sterols and SLs in yeast PMs.
It seems evident that the transmembrane domains of integral membrane proteins associate
with specic lipids and that this lipid-protein
interaction determines the protein localization.
Point mutation analysis has shown that a single
transmembrane domain of the plasmodesmatalocated protein PDLP1a determines the
targeting of this membrane protein to plasmodesmata in A. thaliana, and that it can also target
heterologous proteins to this location (141).
Membrane distribution of the yeast ferro-O2 oxidoreductase Fet3, required for iron uptake,
was changed by replacing its transmembrane
domain with the only transmembrane domain
of the small regulatory subunit of the PM
H+ -ATPase Pmp1. The chimera not only
nonrandomly overlapped with Pmp1 but also
lost the Fet3 function. It is unclear whether the
functional defect was caused by protein delocalization from the specic lipid milieu or whether
the transmembrane domain exchange itself
affected the enzymatic activity of the protein.
The MCC-specic arginine permease Can1
has been dislocated to the PM microdomain occupied by the H+ -ATPase Pma1 by direct binding of Can1-GFP to Pma1 tagged with GFP
binder (GB), a monomeric, high-afnity antiGFP antibody. Similarly to Fet3, Can1-GFP
loses its function upon binding to Pma1-GB.
Importantly, however, its transport activity is
preserved when bound to the MCC resident
Sur7-GB, indicating that protein localization
and not the GB binding itself might determine
the effect (130). In wild-type cells, Can1 consistently localizes in the specic ergosterol-rich
lipid milieu of the MCC (40). Furthermore,

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MCC accumulation and the transport activity


of the hexose transporter HUP1 were reduced
in mutants defective in ergosterol biosynthesis (41), pointing to the participation of specic
lipids in this proteins localization and function.
It is worth mentioning that microdomains
of specic lipid composition are formed even
in PMs lacking sterols. Spiral-shaped microdomains enriched in negatively charged
cardiolipin and phosphatidylglycerol were detected by 10-N-nonyl-acridine orange and Fei
Mao (FM) dyes in Bacillus subtilis PMs (7). Even
though FM dyes alter the PM structure (54),
the lipid spirals seem to be stain independent:
The FM staining did not affect the distribution
of MinD, an ATPase involved in septation,
which to a high extent colocalized with these
microdomains in the bacterial membrane (7).
Spiral-shaped microdomains were also reported in Escherichia coli PMs (121).
The necessity of lipid phase separation
for spontaneous demixing of membrane components remains to be veried. For example, in PMs of the cyanobacterium Gloeobacter violaceus, quantitative differences in the
lipid composition of two functionally distinct,
protein-specic microdomains differing in relative amounts of mono- and digalactosyldiacylglycerol were not considered sufcient to induce phase separation (112).
The existence of So -phase domains, which
are not addressed by the lipid raft hypothesis, in otherwise liquid biological membranes
has been reported by time-resolved uorescence spectroscopy studies since the 1980s
(57). Highly ordered, ergosterol-free, and SLenriched gel domains were recently found in
yeast PMs (4). These domains clearly differ
from lipid rafts in both composition and degree of order. The functional relevance and
protein settlement of highly packed, rigid gelphase (So -phase) membrane areas are still unclear. They may play a role in yeast adaptation
to oxidative stress (107).
In the mammalian eld, the most prominent concept concerning the role of lipid rafts
has been their potential involvement as signaling platforms (132). A signicant improvement

in signal output, especially at low signal intensities, is due to the cooperative association
of interdependent signaling components; this
has been shown, for example, in RAS signaling (142). T cell signaling, however, requires
not raft-based mechanisms but rather direct
protein-protein interactions, which of course
are always a prerequisite for signaling processes
(25). Thus, the specic role of lipid rafts in signaling remains a matter of dispute. In the plant
eld, the importance of rafts in signaling has
been repeatedly stated (102); however, we are
not aware of any convincing evidence for this.

Energy-Dependent Directed
Membrane Flow
For many purposes, including cell growth,
reproduction, sensing, and adaptation to a
wide range of environmental stimuli, the PM is
continuously rearranged. To a large extent, this
membrane ow is accomplished through vesicular transport. The endocytosed membrane can
be directionally recycled to specic sites of the
cell surface (1). This spatial separation of endoand exocytosis, in combination with limited
lateral diffusion of the reinserted membrane
components, is able to maintain steady-state
gradients of PM constituents (Figure 2b).
This was rst demonstrated on peripheral
accumulations of transferrin receptors in the
PMs of human-derived broblasts (17). The
premise of slow lateral diffusion is especially
valid in the PMs of plants and fungi (146).
Therefore, kinetic polarization achieved by
vesicle recycling seems to be a dominant
mechanism of PM domain formation in these
organisms (reviewed in 156).
For example, the formation and maintenance of SRDs in plant and fungal PMs require
a functional secretory pathway. Sterol-rich
membranes of secretory vesicles, routed by
cytoskeletal structures, fuse with the PM at
sites of intensive assembly of the new cell
surface. The spreading of sterol molecules
in the PM is too slow to reach an even
sterol distribution over the cell surface, and a
steady-state sterol gradient is established by

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coupling this polarized sterol delivery with


ongoing nonpolarized endocytosis (146).
Alvarez et al. (2) have reviewed the potential biological roles of fungal SRDs. In the
pathogenic yeast C. albicans, for example, many
glycophosphatidylinositol-anchored virulence
factors have been found to accumulate at SRDs.
In addition, ergosterol promotes the accumulation of the signaling lipid phosphatidylinositol
4,5-bisphosphate [PI(4,5)P2 ] at mating projections of S. cerevisiae. The SL-free (and therefore
lipid raftindependent) membrane pool of ergosterol in SRDs was also shown to promote
membrane fusion during mating (55).
Endocytic membrane recycling also plays a
role in keeping the apical PM domain enriched
in PI(4,5)P2 and diacylglycerol (DAG) during
pollen tip growth in N. tabacum. PI(4,5)P2 is
synthesized at the growing tip in a Rac/Roptype Rho-family small-GTPase-dependent
manner. The spreading of PI(4,5)P2 from the
tip is prevented by laterally targeted phospholipase C, which hydrolyzes PI(4,5)P2 to
DAG and inositol 1,4,5-trisphosphate. DAG
is consequently endocytosed and delivered
back to the apex, retrograde to the ow of
PI(4,5)P2 spreading out from the apical area.
This PI(4,5)P2 /DAG accumulation at the tip
is essential for pollen tip growth (47). The
presumable dependence of PI(4,5)P2 /DAG
microdomains on membrane sterols has not
been tested.
In contrast, however, membrane sterols are
essential for polar localization of PIN2 in Arabidopsis (94). Newly synthesized PINs are delivered to the entire PM but then rapidly cycle between the PM and endosomes (22) (Figure 3).
Kleine-Vehn et al. (61) found that endocytosed
PIN1 and PIN2 are targeted to the center of
the polar PM domain. Computer modeling of
polar domain dynamics revealed that polar deposition of recycled PIN proteins was not sufcient to maintain the observed PIN polarity,
and suggested that a contribution of spatially
conned endocytosis could be required in this
case as well. The enrichment of the crucial endocytic factor clathrin at lateral cell sides has
been accordingly documented (61).

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Spatially conned endocytosis has only


recently been described in yeast as well. Localization of MCC domains (39) and the actual distribution of the cortical endoplasmic reticulum
(133) were found to determine the positioning
of endocytic events in the S. cerevisiae PM.
Restriction of an endocytic site initiation, and
possibly of other interactions between soluble
cytoplasmic factors and the PM, to membrane
areas not associated with the endoplasmic
reticulum could generate kinetic polarization
of the PM similar to that described in the examples above and dene additional functional
microdomains at the cell surface. This was
observed in S. pombe cells, in which the cortical
endoplasmic reticulum delimits an area in the
PM where the actomyosin-ring-organizing
protein Mid1 can bind and where the plane of
cytokinesis is consequently established (157).

Protein-Mediated Restrictions
of Lateral Diffusion
The free lateral diffusion of PM constituents
is slowed and obstructed by membraneintegrated or membrane-associated protein assemblies. Based on the mechanisms of their
functions, these assemblies can be categorized as membrane-shaping scaffolds (coats)
(Figures 2c and 4), which generate curvatures in the membrane that represent unique
environments preferred by specic lipid and
protein species, and membrane-cytoskeleton
fences (corrals) (Figure 2d ), which partition
the entire PM into small regions with crossable
borders.
Highly curved areas in biological membranes are usually enriched in specic lipids.
Lipids alone, however, are not likely to generate and maintain this curvature (56). It is, rather,
generated either by membrane-integrated but
not membrane-spanning coat proteins (for
example, caveolins and otillin/reggie proteins; reviewed in Reference 8) or by proteins attached to the cytoplasmic membrane
surface either directly [for example, proteins
containing the BAR (Bin/Amphiphysin/Rvshomology) domain; reviewed in Reference 92]

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5
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0m
in
20

5 m

10

0m
in

0m
in

PIN1::PIN1-YFP

Polarity index

Figure 3
Mechanism of PIN polarity generation in Arabidopsis roots. (a) The two steps that restore polar plasma
membrane (PM) localization of PIN1-YFP ( rst subpanel ): After complete photobleaching of PIN1-YFP
uorescence (second subpanel ), the newly synthesized protein is rst delivered to the PM in a nonpolar
manner (third subpanel ). Later, polarity is established by spatially conned endocytic recycling ( fourth
subpanel ). Arrowheads point to nonpolar or basal (rootward) PIN1-YFP localization. (b) Ratio of polar to
lateral PIN1-YFP intensity prior to the bleaching (left bar) and at the indicated time points of the recovery of
c 2008 by the
uorescent protein distribution (middle and right bars). Adapted from Reference 23; copyright 
Nature Publishing Group.

or via adaptor proteins (clathrin; see Reference


106).
Besides proteins involved in clathrinmediated endocytosis from yeast to plants and
mammals (amphiphysins and epsins; see Reference 49), novel BAR domaincontaining proteins were recently identied in the yeast S.
cerevisiae. Homodimers of Pil1 and Lsp1 adopt
a serpent or banana shape typical for the BAR
domain protein superfamily (104), bind to the
PM, and form eisosomes, which underlie the
furrow-like invaginations of the yeast PM, i.e.,
the MCC (98, 120, 134) (Figure 4). The biological role of the MCC is not fully understood.
Among other functions suggested, several observations indicate its involvement in the response 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 membrane
stresscaused by a drop in SL levels, hypoosmotic shock, or direct mechanical stretching of the PMinduces the release of Slm1/2
proteins from the MCC-associated eisosome
to activate TORC2 (target of rapamycin kinase complex 2) localized in another PM do-

main, the MCT (membrane compartment of


TORC2). The activated TORC2 then regulates SL metabolism via the Ypk1 pathway
by phosphorylating evolutionarily conserved
Orm proteins (135) to mediate compensatory
changes. In this respect, invaginated membrane
areas of the MCC resemble mammalian caveolae, which disappear in conditions of acute mechanical stress (128) and release caveolin-1 to
PM

Pil1/Lsp1

PM

Figure 4
Model for the assembly of yeast eisosomal proteins at the plasma membrane
(PM) in the form of a presumably hemitubular scaffold shaping the furrow-like
membrane invagination of the MCC. Adapted from Reference 58.

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activate the Ypk1 homolog protein kinase B


(Akt) (119). The attening of MCC furrows
in response to membrane stress has not been
reported; however, cells lacking the proposed
SL sensor, MCC-accumulated Nce102 (32), do
exhibit a at MCC (134). Both of these observations represent excellent examples of the
CCL principleafter the stimulus, the effector
(Slm1, caveolin-1) must be laterally relocalized
in the PM to execute the function.
Other Ypk-activating kinasesnamely, the
PDK1 (3-phosphoinositide-dependent protein
kinase 1) homologs Pkh1 and -2, which are key
players in SL-mediated signaling (158)also
accumulate in eisosomes, making the MCC a
target of protein kinase inhibitorbased antifungal drugs (10). Eisosomal Pil1 and Lsp1,
which are phosphorylated by Pkh1 and -2, were
found to downregulate resistance to heat stress
and, with respect to their wide evolutionary
conservation, suggested to function as negative regulators of PDK-like protein kinases and
their downstream cellular pathways that control
cell growth and survival (158). It should be mentioned here that PM invaginations that are morphologically highly similar to the MCC were
also reported in freeze-tolerant (but not freezesensitive) Chlamydomonas and Chloromonas unicellular algae (20). In the above context, this
observation accents the involvement of these
structures in an evolutionarily conserved mechanism of adaptation to dehydration stress.
Proteins and lipids in a PM with cytoskeleton fences undergo diffusion conned to individual fence-dened membrane microdomains
combined with hop diffusion across their borders (69). In mammals, the size of fence-dened
membrane microdomains was estimated to vary
between 30 and 230 nm depending on the
cell line (101). In plants, much larger fencebordered microdomains were suggested only
recently. Oda & Fukuda (103) showed that in
tobacco leaf epidermis, a scattered network of
cortical microtubules restricts the localization
of the PM-anchored MIDD1-ROP11 complex
into micrometer-size PM areas. To analyze
membrane protein diffusion in detail, Grossmann et al. (G. Grossmann, J.J. Lindeboom,

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W.B. Frommer & D.W. Ehrhardt, unpublished results) performed uorescence recovery after photobleaching (FRAP)/uorescence
loss induced by photobleaching (FLIP) measurements and particle tracking of individual
protein clusters in Arabidopsis tissue and wallless protoplasts and found that cortical microtubules have a general impact on the dynamics at the PM by dening diffusion barriers and
thereby subdividing the PM into large-scale diffusional domains.
Membrane-associated septins, which are
GTP-binding proteins assembled into rods and
laments in eukaryotic cells from protists to
mammals (but not, however, in higher plants),
partition the PM during cytokinesis (116). In
S. cerevisiae, the septin ring consists of
membrane-anchored circumferential pairs of
tightly associated septin laments interconnected by simple axial laments intersecting the
circumferential laments with 30-nm spacing
(12). During budding, this hourglass-shaped
septin network effectively prevents the mixing
of mother-cell and bud membranes.
In cells possessing cell walls, the mobility of
PM constituents can also be constrained from
the outer PM surface. For example, AtFH1
(A. thaliana actin nucleation protein formin 1)
has been shown to form a bridge across the
PM, connecting the actin cytoskeleton with the
cell wall (90). But even in the absence of direct interactions between PM proteins and cell
wall components, the presence of a cell wall
constrains the lateral diffusion of PM proteins
(91, 103).

Microdomain Organization Needs


Energy: Plasma Membrane of
De-energized Cells
The MCC patterning of H+ symporters and
ergosterol in the yeast PM was lost within seconds when the PM potential () was reduced by uncoupling agents. Even partial PM
depolarization due to HUP1-mediated symport of the nonmetabolized glucose analog 6deoxyglucose plus protons into S. cerevisiae cells
was enough to release HUP1-GFP from the

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MCC patches (40). As an analogy, Can1 leaves


the MCC with an excess of arginine (39). It has
been suggested that accumulation of H+ symporters in an endocytotically silent area of the
MCC prevents the untimely internalization of
these proteins. According to the proposed CCL
principle, the symporters, after their function is
no longer required, are released from the protected area of the MCC to the surrounding PM
in order to enable their rapid internalization
and degradation (40).
The above observations led to the postulation of a crucial role of  in the molecular
order of the membrane. Interestingly, the PMs
of energized and de-energized cells differ
in their detergent susceptibility. Uncouplertreated cells are much more resistant to detergents (40). A similar observation had also previously been reported for algae and bacteria (63).
De-energized cells are also much less susceptible to polyenes, like nystatinindicating
reduced sterol accessibilityand to oligopeptide antibiotics, like histatin 5 (63, 115). A
40-fold higher concentration of the polyene
amphotericin, for example, had to be used for
starved or 2,4-dinitrophenol (DNP)treated
C. albicans cells to obtain the same toxic effect
as for a growing culture (35). In addition, an
increased resistance of nongrowing, starved
bacteria to several classes of antibiotics (76) may
be related to the same phenomenon. A change

in  has also been shown to affect membrane


order in liposomes of synthetic lipids (48). The
exact mechanism of these changes in detergent
susceptibility, sterol accessibility, and general
permeability are not understood. It seems that
the PM is somehow more tightly organized and
less accessible to detergents and polyenes once
the membrane potential is reduced. Indeed,
a considerably lower amount of H3 Triton
X-100 binds to such membranes (63). As an
alternative interpretation, it has been suggested
that the membrane order might be increased
by cytoskeletal rearrangements following ATP
depletion (148 and references therein).
Whatever the case may be, it is evident that
different energy-dependent states of membrane
order can be distinguished in fungi, algae, and
bacteria (higher plants have not been investigated). One state may correspond to a more active, optimally organized, and compartmented
state of the membrane, allowing a certain passive exchange of small molecules when cells are
energized and growing well; another may correspond to a tighter, less detergent-accessible
state adapted to cells at rest. To investigate
this most likely general phenomenon in higher
plants and elucidate its exact mechanism in any
organism should be an interesting problem
to be solved in the future. Until the answer
is known, we do not fully understand the
PM.

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 lipid
or membrane rafts postulated to exist in mammalian cells.
2. The detergent extraction method does not lead to the isolation of authentic PM microdomains. Use of the differential detergent solubility of membrane proteins has been
a standard purication method, and in this way the procedure may still be useful.
3. Various methods for the direct visualization of membrane microdomains such as superresolution techniques of uorescence microscopy are applicable to plants and should
become the approaches of primary choice in membrane microdomain detection.
4. Several organizing principles result in membrane microdomain formation. Liquidordered 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 response, and polarized growth have been shown to localize to membrane microdomains.
6. Evidence is available for the functional importance of concentrating specic membrane
components in microdomains. Functional clustering accompanied by modulation of the
activity of PM proteins has been observed, and a new regulatory mechanism is postulated:
control by change in location (CCL).

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7. Membrane order, microdomain organization, and general properties like sterol accessibility and detergent susceptibility of the PMs in energized cells differ from those of the
PMs of de-energized or starving cells.

DISCLOSURE STATEMENT
The authors are not aware of any afliations, memberships, funding, or nancial holdings that
might be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS
This work was supported by DFG (Priority Program 1108 and TA 36/18-1 to W.T.), Fonds
der 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. and
RVO 61388971 to M.O.), and an EMBO long-term fellowship (to G.G.).
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Contents

Annual Review of
Plant Biology
Volume 64, 2013

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Benets of an Inclusive US Education System


Elisabeth Gantt p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 1
Plants, Diet, and Health
Cathie Martin, Yang Zhang, Chiara Tonelli, and Katia Petroni p p p p p p p p p p p p p p p p p p p p p p p p p19
A Bountiful Harvest: Genomic Insights into Crop Domestication
Phenotypes
Kenneth M. Olsen and Jonathan F. Wendel p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p47
Progress Toward Understanding Heterosis in Crop Plants
Patrick S. Schnable and Nathan M. Springer p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p71
Tapping the Promise of Genomics in Species with Complex,
Nonmodel Genomes
Candice N. Hirsch and C. Robin Buell p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p89
Understanding Reproductive Isolation Based on the Rice Model
Yidan Ouyang and Qifa Zhang p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 111
Classication and Comparison of Small RNAs from Plants
Michael J. Axtell p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 137
Plant Protein Interactomes
Pascal Braun, Sebastien Aubourg, Jelle Van Leene, Geert De Jaeger,
and Claire Lurin p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 161
Seed-Development Programs: A Systems BiologyBased Comparison
Between Dicots and Monocots
Nese Sreenivasulu and Ulrich Wobus p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 189
Fruit Development and Ripening
Graham B. Seymour, Lars stergaard, Natalie H. Chapman, Sandra Knapp,
and Cathie Martin p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 219
Growth Mechanisms in Tip-Growing Plant Cells
Caleb M. Rounds and Magdalena Bezanilla p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 243
Future Scenarios for Plant Phenotyping
Fabio Fiorani and Ulrich Schurr p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 267
v

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Microgenomics: Genome-Scale, Cell-Specic Monitoring of Multiple


Gene Regulation Tiers
J. Bailey-Serres p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 293
Plant Genome Engineering with Sequence-Specic Nucleases
Daniel F. Voytas p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 327
Smaller, Faster, Brighter: Advances in Optical Imaging
of Living Plant Cells
Sidney L. Shaw and David W. Ehrhardt p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 351
Phytochrome Cytoplasmic Signaling
Jon Hughes p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 377
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Photoreceptor Signaling Networks in Plant Responses to Shade


Jorge J. Casal p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 403
ROS-Mediated Lipid Peroxidation and RES-Activated Signaling
Edward E. Farmer and Martin J. Mueller p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 429
Potassium Transport and Signaling in Higher Plants
Yi Wang and Wei-Hua Wu p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 451
Endoplasmic Reticulum Stress Responses in Plants
Stephen H. Howell p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 477
Membrane Microdomains, Rafts, and Detergent-Resistant Membranes
in Plants and Fungi
Jan Malinsky, Miroslava Opekarova, Guido Grossmann, and Widmar Tanner p p p p p p p 501
The Endodermis
Niko Geldner p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 531
Intracellular Signaling from Plastid to Nucleus
Wei Chi, Xuwu Sun, and Lixin Zhang p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 559
The Number, Speed, and Impact of Plastid Endosymbioses in
Eukaryotic Evolution
Patrick J. Keeling p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 583
Photosystem II Assembly: From Cyanobacteria to Plants
Jorg Nickelsen and Birgit Rengstl p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 609
Unraveling the Heater: New Insights into the Structure of the
Alternative Oxidase
Anthony L. Moore, Tomoo Shiba, Luke Young, Shigeharu Harada, Kiyoshi Kita,
and Kikukatsu Ito p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 637
Network Analysis of the MVA and MEP Pathways for Isoprenoid
Synthesis
Eva Vranova, Diana Coman, and Wilhelm Gruissem p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 665

vi

Contents

PP64-frontmatter

ARI

25 March 2013

10:21

Toward Cool C4 Crops


Stephen P. Long and Ashley K. Spence p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 701
The Spatial Organization of Metabolism Within the Plant Cell
Lee J. Sweetlove and Alisdair R. Fernie p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 723
Evolving Views of Pectin Biosynthesis
Melani A. Atmodjo, Zhangying Hao, and Debra Mohnen p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 747

Annu. Rev. Plant Biol. 2013.64:501-529. Downloaded from www.annualreviews.org


by University of Wales - Aberystwyth on 10/04/13. For personal use only.

Transport and Metabolism in Legume-Rhizobia Symbioses


Michael Udvardi and Philip S. Poole p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 781
Structure and Functions of the Bacterial Microbiota of Plants
Davide Bulgarelli, Klaus Schlaeppi, Stijn Spaepen, Emiel Ver Loren van Themaat,
and Paul Schulze-Lefert p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 807
Systemic Acquired Resistance: Turning Local Infection
into Global Defense
Zheng Qing Fu and Xinnian Dong p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 839
Indexes
Cumulative Index of Contributing Authors, Volumes 5564 p p p p p p p p p p p p p p p p p p p p p p p p p p p 865
Cumulative Index of Article Titles, Volumes 5564 p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 871
Errata
An online log of corrections to Annual Review of Plant Biology articles may be found at
http://www.annualreviews.org/errata/arplant

Contents

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