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Structure and function of endosomes in plant cells

Anthony L. Contento and Diane C. Bassham*Department of Genetics, Development and Cell Biology, Iowa State University, Ames, IA 50011, USA

*Author for correspondence (bassham@iastate.edu)

Journal of Cell Science 125, 3511–3518� 2012. Published by The Company of Biologists Ltddoi: 10.1242/jcs.093559

SummaryEndosomes are a heterogeneous collection of organelles that function in the sorting and delivery of internalized material from the cellsurface and the transport of materials from the Golgi to the lysosome or vacuole. Plant endosomes have some unique features, with anorganization distinct from that of yeast or animal cells. Two clearly defined endosomal compartments have been studied in plant cells,

the trans-Golgi network (equivalent to the early endosome) and the multivesicular body (equivalent to the late endosome), withadditional endosome types (recycling endosome, late prevacuolar compartment) also a possibility. A model has been proposed in whichthe trans-Golgi network matures into a multivesicular body, which then fuses with the vacuole to release its cargo. In addition to basictrafficking functions, endosomes in plant cells are known to function in maintenance of cell polarity by polar localization of hormone

transporters and in signaling pathways after internalization of ligand-bound receptors. These signaling functions are exemplified by theBRI1 brassinosteroid hormone receptor and by receptors for pathogen elicitors that activate defense responses. After endocytosis ofthese receptors from the plasma membrane, endosomes act as a signaling platform, thus playing an essential role in plant growth,

development and defense responses. Here we describe the key features of plant endosomes and their differences from those of otherorganisms and discuss the role of these organelles in cell polarity and signaling pathways.

Key words: Endosome, Arabidopsis, Endocytosis

IntroductionThe eukaryotic endomembrane system functions in the synthesis,

sorting, delivery and degradation of macromolecules within the

cell. The system is composed of a variety of membrane-bound

organelles that are connected either directly or through a series of

transport vesicles. The main organelles of the endomembrane

system are the endoplasmic reticulum, the Golgi complex and

trans-Golgi network (TGN), endosomes, and lysosomes or

vacuoles. Endosomes have a core sorting function within the

endomembrane system. These organelles are the first point of

fusion for endocytic vesicles, which are used to internalize

extracellular materials. They are also involved in the transport of

materials either from the Golgi to the lysosome or vacuole, or

their return from lysosomes to the Golgi. Endosomes provide

intermediate compartments where materials can be stored, sorted

and then sent to a designated target organelle within the cell

(Anitei et al., 2010; Huotari and Helenius, 2011; Otegui and

Spitzer, 2008).

In plants, lysosomes are generally absent in favor of a large

central vacuole, although there is evidence that lysosome-like

organelles can co-exist with the vacuole in some cells (Takatsuka

et al., 2011). The vacuole is responsible for degradative

functions, in common with lysosomes, and also acts as a large

storage compartment for proteins, water, ions and some defense

compounds (Marty, 1999). Appropriate sorting of proteins at the

TGN is essential for proper maintenance of the central vacuole

and cell wall (Saint-Jore-Dupas et al., 2004). In plants, as in other

organisms, endosomes serve as an entry point for the endocytosis

of external materials and an intermediate compartment in the

transport of macromolecules to the vacuole (Otegui and Spitzer,

2008). However, the organization of the endosomal system in

plants has some unique features when compared with that in

animal cells.

In animal and yeast cells, an array of different types of

endosomes have been identified that can be classified according

to their function during endocytosis. Early endosomes (EEs) are the

first site of deposition of internalized components upon

endocytosis. The internalized components might then be

transported to a recycling endosome for subsequent recycling to

the cell surface, or might enter a multivesicular body (MVB),

designated as an intermediate or late endosome (LE), for transport

to a lysosome or vacuole, often for degradation (Spang, 2009). The

MVB is generally considered to be the point at which endocytic and

biosynthetic vacuolar trafficking meet, and is therefore often also

termed the prevacuolar compartment (PVC) in both yeast and

plants (Gerrard et al., 2000; Tse et al., 2004). A third major

endosome type, the recycling endosome (RE), receives internalized

material from the EE and directs it back to the cell surface or by a

retrograde pathway to the TGN (Hsu and Prekeris, 2010). Although

endosomes perform similar functions of cargo sorting and transport

in plants, they cannot be classified using the same criteria.

Endosomes serve functions beyond the sorting of cargo from

endocytosis and biosynthesis. Plant endosomes are important in

the maintenance of the vacuole and for cell growth, including the

growth of specialized cells such as pollen tubes (Samaj et al.,

2006; Wang et al., 2010; Richter et al., 2012). The composition

and structure of the plasma membrane and cell wall require

proper endosomal sorting function (Bonifacino and Jackson,

2003; Chow et al., 2008; Toyooka et al., 2009). Endosomes have

also been linked to activation of several signaling pathways (Bar

and Avni, 2009; Geldner et al., 2007; Kang et al., 2010; Robatzek

et al., 2006). Here, we describe the classification and structure of

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endosomes in plant cells, and discuss how they differ from those

in animal cells. The functions of endosomes in plant cells are

then discussed, with examples of the growing number of

pathways in which they have been found to participate,

including roles in generating cell polarity and in hormonal and

defense signaling.

Endosomal compartmentsIn animal cells, endosomes have been classified as early

endosomes, which are composed of tubules that are dynamic in

nature; late endosomes (also categorized as multivesicular bodies),

which are typically spherical and contain internal vesicles; and

recycling endosomes, another tubular compartment located close

to the microtubule-organizing center (Huotari and Helenius, 2011).

These compartments can be distinguished by the presence of

specific marker proteins, in particular Rab GTPases (Huotari and

Helenius, 2011). For example, early endosomes might be

detectable by the presence of RAB4 and RAB5, late endosomes

can contain RAB7 or mannose 6-phosphate receptors, and

recycling endosomes contain RAB11. Both early and late

endosomes can recycle their contents back to the plasma

membrane, potentially by a recycling endosome, whereas late

endosomes can either recycle components to the TGN or deliver

them to the lysosome for degradation (Russell et al., 2006).

Transport through the endosomal system in general occurs through

a maturation of endosomes, for example, maturation of an EE into

a LE by removal of EE-specific components, which is

accompanied by morphological changes in the structure of the

endosome (Huotari and Helenius, 2011).

Although the general functions of endosomes in plant cells are

similar to those in animal cells, the organization of the endosomal

trafficking pathways has some distinct features (see Fig. 1).

Endocytosis has commonly been traced in plant cells using the

fluorescent styryl dye FM4-64 (and related compounds); it labels

the plasma membrane upon addition to intact cells, is taken up by

endocytosis, transported through endosomal compartments and

eventually reaches the vacuolar membrane (Bolte et al., 2004).

Organelles on the endocytic route are therefore sequentially

labeled with fluorescent dye during a time course of uptake.

Various inhibitors that block distinct steps in the endocytic and

endosomal trafficking pathways have allowed the dissection of

trafficking routes and the analysis of proteins potentially

involved in these pathways (see Table 1). To date, there appear

to be two clearly defined and studied endosomal compartments in

plant cells, the TGN or EE and MVB or LE (Fig. 2). Additional

types of endosomes might be present, but their identity awaits

further experimental confirmation.

Early endosomes

EEs are defined as the first compartments that receive endocytic

cargo after internalization from the cell surface, and are labeled

very rapidly by FM4-64 (Bolte et al., 2004). There is now strong

TGNor EE

Nucleus

GC

ER

1

1

2

4

3

Vacuole

LE or MVBor PVC

RE

LPVC

RabA4bRabA5dRabA1eRabA1g

SYP41SYP42Ara-4Pra3Rab-A2Rab-A3SCAMP1

SYP21BP80RabF2a (Rha1)RabF2b (Ara7)ESCRTCHMP1AtVSRs

Fig. 1. The endocytic pathway in plants. In plants, components of the endocytic pathway are involved in biosynthetic, degradative and recycling transport. The

trans-Golgi network and early endosomes act as the point of organization for these three pathways. This diagram displays the organelles involved in endomembrane

trafficking: nucleus, endoplasmic reticulum (ER), Golgi complex (GC), trans-Golgi network (TGN) or early endosome (EE), late endosome (LE) or multivesicular

body (MVB) or prevacuolar compartment (PVC), late prevacuolar compartment (LPVC), vacuole surrounded by the tonoplast, recycling endosome (RE). The colored

arrows designate potential traffic between organelles. The green arrows indicate pathway 1, the biosynthetic transport route to the plasma membrane that

passes through the TGN and sometimes the MVB. The blue arrow shows a pathway (2) that is taken by plasma membrane components (red ovals) as they are

internalized into endocytic vesicles and move through the TGN. Proteins associated with the TGN are listed (Bassham et al., 2000; Chow et al., 2008; Inaba et al.,

2002; Lam et al., 2007; Sanderfoot et al., 2001; Ueda et al., 1996). The thick, red arrows represent a recycling pathway (3), by which plasma membrane components

might be returned to the plasma membrane through a specialized RE. Proteins associated with the RE are shown (Geldner et al., 2009; Preuss et al., 2006; Rutherford

and Moore, 2002). The orange arrows designate the transport pathway (4) for newly synthesized vacuolar components, as well as cellular materials destined for

degradation in the vacuole. Proteins associated with MVBs are shown (Bottanelli et al., 2012; da Silva Conceicao et al., 1997; Jiang and Rogers, 1998; Lee et al.,

2004; Li et al., 2002; Paris et al., 1997; Rutherford and Moore, 2002; Sanderfoot et al., 1998; Sanderfoot et al., 1999; Sohn et al., 2003; Spitzer et al., 2009). Transport

from the TGN to MVBs is designated by a black arrow. Retrograde transport from the VAC to the MVB is represented by curved black arrows.

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evidence that, rather than existing as a separate organelle as in

animal cells, in plants, the TGN takes on the function of an early

endosome and is the first site for delivery of endocytosed

material. This was initially proposed by Dettmer and colleagues,

who, using immunogold electron microscopy and fluorescent

labeling, showed that the vacuolar proton–ATPase subunit VHA-

a1 localizes to the TGN (Dettmer et al., 2006). Surprisingly, the

same marker was also shown to colocalize with FM4-64 very

rapidly after addition of the dye, a hallmark of an EE. Inhibition

of the vacuolar ATPase with concanamycin A (Table 1), which

leads to a block in vesicle trafficking, causes the accumulation of

both secretory and endocytic cargo at the TGN (Dettmer et al.,

2006). Endocytosed plasma membrane proteins, secretory

proteins and polysaccharides can all be found in the same TGN

(Viotti et al., 2010), suggesting that these two pathways merged

at this site, now often termed the TGN/EE. Similar results were

obtained using the secretory carrier membrane protein 1

(SCAMP1) protein from rice, which also localized to the TGN

Table 1. Examples of inhibitors used to study endosomal trafficking in plants

Inhibitor Effect on plant cells References

Brefeldin A Blocks trafficking from endosomes to plasma membraneCauses formation of endosomal aggregates (BFA compartment)Causes redistribution of Golgi proteins to ER

(Geldner et al., 2003; Nebenfuhr et al., 2002; Richter et al., 2007;Robinson et al., 2008b; Teh and Moore, 2007; Tse et al., 2006)

Wortmannin Inhibits PI3KCauses enlarged PVC/MVB

(Jaillais et al., 2006; Kasai et al., 2011; Matsuoka et al., 1995; Miaoet al., 2006; Tse et al., 2004; Vermeer et al., 2006)

Concanamycin A Inhibits vacuolar ATPaseBlocks transport out of TGN/EEPrevents vacuolar degradation

(Dettmer et al., 2006; Matsuoka et al., 1997)

Filipin Binds plasma membrane sterolsInhibits endocytosis

(Grebe et al., 2003; Kleine-Vehn et al., 2006)

Tyrphostin A23 Inhibits clathrin-mediated endocytosis (Fujimoto et al., 2010; Ortiz-Zapater et al., 2006)Endosidin1 Causes formation of endosomal aggregates

Blocks endocytic trafficking(Robert et al., 2008)

Fig. 2. Use of three-dimensional electron

tomography to distinguish different

regions of the endomembrane system.

(A) Transmission electron microscopy

(TEM) image of a prepared tomographic slice

(4.3 mm thick) through a region of the Golgi

complex and surrounding cytoplasm. Take

note of the division of the Golgi stack from

cis-Golgi (cis) to medial-Golgi (med) to

trans-Golgi (trans) and the trans-Golgi

network (TGN). Specialized labeling

techniques can be used to help distinguish

different cisternae. A non-coated vesicle

(NCV) and a multivesicular body (MVB) are

also labeled. (B) Here, cis-, medial- and

trans-Golgi cisternae seen in A are visualized

as a three-dimensional image, displaying the

divisions of the Golgi stack, as well as a

distinct TGN with associated non-coated and

clathrin-coated vesicles (CCVs). MVBs are

also clearly visible as white bodies attached

to the TGN. (C) Top-down or face-on views

of the Golgi stack. 3D electron tomography

allows for the virtual dissection of the Golgi

stack, from top to bottom (from top, cisternae

are indicated: cis1, cis 2, medial 1, medial 2,

medial 3, trans 2, TGN). The colors for each

cisterna are as in B. This top-down view also

allows visualization of the formation of

protein aggregates (arrows) and CCVs (CC).

Scale bars: 100 nm. Figure reprinted from

Otegui et al. (Otegui et al., 2006)

with permission.

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and colocalized with FM4-64 at early time points (Lam et al.,

2007), confirming that the previous findings are not dependent onthe TGN marker used.

Evidence exists that the TGN in plants might containspecialized subdomains with distinct functions. Using

immunogold electron microscopy, TGN marker proteins havebeen shown to localize to separate domains of the same TGN(Bassham et al., 2000). Rab-A2 and Rab-A3 GTPases partially

co-localize with a TGN marker at an organelle that also acts as anEE (Chow et al., 2008). However, unlike the TGN marker VHA-a1, these GTPases are found at the cell plate during cytokinesis,

suggesting that they label an organelle that contributes tosecretory processes (Chow et al., 2008), possibly suggestingfunctional differentiation of the TGN or EE during cell division.Recently, an Arabidopsis mutant lacking a TGN-localized

component was described that is defective in secretory but notendocytic trafficking, indicating a separation of these twofunctions of the TGN (Gendre et al., 2011). Electron

tomography indicates that the TGN is differentiated into earlyand late compartments (Kang et al., 2011; Staehelin and Kang,2008); how these relate to functionally specialized regions is not

yet entirely clear. This differentiation was further analyzed bylive-cell imaging using the secreted protein SCAMP2 as amarker. SCAMP2 could be seen at the TGN, which was

associated with the Golgi and also could be labeled with FM4-64, indicating that it was on the endocytic route (Toyooka et al.,2009). It was subsequently found in a cluster of vesicles derivedfrom the TGN that contain secretory cargo, including protein and

polysaccharides. This cluster separates from the Golgi, moves tothe plasma membrane, where it fuses and releases its contentsinto the cell wall (Toyooka et al., 2009), in a process that possibly

also involves the Rab GTPase ARA6 (Ebine et al., 2011).Tagging of Golgi and TGN with fluorescent markers followed bylive-cell imaging has demonstrated that the TGN is not

permanently associated with a Golgi stack, but that rather,individual TGNs move rapidly within the cell and can dissociatefrom their associated Golgi and even reassociate with a different

Golgi stack (Viotti et al., 2010). Whether some of these mobileTGN structures correspond to the secretory clusters described byToyooka and co-workers (Toyooka et al., 2009) is unknown.

Late endosomes

Endocytosed material has several possible destinations afterarriving at an EE. It might be recycled back to the plasmamembrane, maintained in the EE as it matures into a LE, or

possibly transported to earlier compartments of the secretorypathway. Unlike EEs, LEs have a multivesicular structure, inwhich the external limiting membrane of the endosome buds

inward to produce internal vesicles. Functionally, this structureallows delivery of membrane to both the lysosomal or vacuolarmembrane by fusion of the external endosomal membrane with

the lysosomal or vacuolar membrane and to the lumen of thelysosome or vacuole for degradation in the internal vesicles. Ithas therefore been predicted that membrane proteins that aredestined for degradation would be concentrated into the internal

vesicles of LEs, whereas those destined for recycling wouldremain on the limiting outer membrane (Babst, 2011).

Evidence that MVBs, which were identified morphologically

by electron microscopy, act as LEs in plants was provided by thedemonstration in tobacco cell culture, by FM4-64 labeling, thatthese organelles are on the endocytic pathway (Tse et al., 2004).

MVBs also contain vacuolar sorting receptors and vacuolarproteins that are en route to the vacuole, suggesting that they are

also intermediates in biosynthetic trafficking to the vacuole(Bottanelli et al., 2011; Jaillais et al., 2008; Miao et al., 2008; Moet al., 2006; Tse et al., 2004). Several Rab GTPases (Ebine et al.,2011; Ueda et al., 2004; Ueda et al., 2001) were shown to localize

to MVBs and to also colocalize with a PVC marker, but not anEE or TGN marker, confirming that MVBs observed by electronmicroscopy correspond functionally to both a LE and a PVC

(Haas et al., 2007; Miao et al., 2008). Plasma membrane proteinsare found in internal vesicles of MVBs and are absent from thelimiting membrane, indicating that they are en route to the

vacuole for degradation and that recycling to the plasmamembrane must occur from an earlier compartment, either theTGN or a separate RE (Viotti et al., 2010).

The lumenal vesicles of MVBs are generated through the

endosomal sorting complex required for transport (ESCRT)machinery. This machinery consists of several protein complexes(ESCRT-0, -I, -II, -III) that function in the invagination of the

limiting endosomal membrane and release of the vesicles formed,and in targeting of vacuole-destined plasma membrane proteinsinto these vesicles (Henne et al., 2011). Although most of the

ESCRT proteins identified in yeast and animals are conserved inplants, the ESCRT-0 cargo-sorting complex appears to bemissing in the majority of eukaryotic lineages, including plants

(Leung et al., 2008), and other components might therefore act incargo recognition in these organisms, with the Tom1 proteinsbeing likely candidates based on their interaction with ESCRTsubunits (Richardson et al., 2011). On the basis of protein

localization and the phenotypes of mutants in a few of the plantESCRT proteins, in plants, the ESCRT proteins probablyfunction in LE maturation and sorting of cargo proteins for

degradation (see Fig. 3) (Haas et al., 2007; Ibl et al., 2012;Katsiarimpa et al., 2011; Scheuring et al., 2011; Shahriari et al.,2011; Spitzer et al., 2009). An example of regulated transport in

which a normally plasma-membrane-localized protein, the BOR1boron transporter, is internalized and sorted by the ESCRTcomplex in response to environmental conditions has recentlybeen described (Takano et al., 2005) (see Box 1 for more details),

and additional examples probably exist.

A model for endosome formation and maturation has beenproposed, in which, instead of vesicle transport moving cargo

between endosomes, the TGN matures into a MVB, which thenfuses with the vacuole to release its cargo (Scheuring et al.,2011). The authors of this study were able to capture MVBs

fusing with the vacuole, suggesting that this transport step doesnot involve vesicle trafficking. They also imaged MVBs that areconnected to tubules, which they hypothesized to correspond to

immature MVBs derived from the TGN. In support of theirmodel, ESCRT components are found distributed on the TGN aswell as on MVBs, and disruption of endosome maturation leadsto increased colocalization of TGN and MVB markers (Scheuring

et al., 2011).

An extension of this model is implicated in work by Forestiand colleagues, in which a mutant vacuolar-sorting receptor is

shown to accumulate in a compartment that is distinct from theGolgi, TGN and MVB (Foresti et al., 2010). A soluble vacuolarmarker also accumulates in these structures, which contains

members of the Rab5 family of small GTPases (Bottanelli et al.,2012; Foresti et al., 2010). These structures are thereforesuggested to be an intermediate compartment, termed the late

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PVC (LPVC), between the MVB and vacuole. It is proposed that

the MVB matures into the LPVC by recycling of vacuolar sorting

receptors, and presumably other components of the trafficking

machinery, back to earlier compartments before fusion of the

LPVC with the vacuole. The retromer complex is likely to be a

key component in this recycling process, consisting in yeast

of two subcomplexes that function in cargo recognition

and membrane association and deformation, respectively

(Schellmann and Pimpl, 2009). These subcomplexes are

conserved in plants (Oliviusson et al., 2006; Pourcher et al.,

2010) and might function in recycling of vacuolar-sorting

receptors during endosome maturation (Shimada et al., 2006;

Yamazaki et al., 2008; Jaillais et al., 2007). Although the exact

site of recycling of vacuolar-sorting receptors is controversial,

with different localization of the retromer complex reported by

different laboratories (Jaillais et al., 2006; Jaillais et al., 2007;

Kleine-Vehn et al., 2008; Niemes et al., 2010a; Niemes et al.,

2010b; Oliviusson et al., 2006; Phan et al., 2008; Pourcher et al.,

2010; Yamazaki et al., 2008), the general principles of the

endosome maturation model are likely to stand.

Role of endosomes in plantsThe primary role of the endosome in all eukaryotes is in

endocytosis. Endocytosis has been studied most extensively in

yeast and mammalian systems, partly because of its association

with a number of diseases (Durieux et al., 2010; Li and Difiglia,

2011; Zhang et al., 2009). In plants, endosomes serve these same

basic functions of uptake of extracellular materials to the interior

of the cell (Meckel et al., 2004), and recycling and maintenance

of plasma membrane receptors (Altenbach and Robatzek, 2007).

In addition, other possible roles for endosomes in plant systems

have also been identified in cell polarity and signaling pathways

(Geldner, 2004; Geldner and Robatzek, 2008).

Plasma membrane maintenance and cell polarity are both

linked to the EE or TGN in plants. Plant cell polarity is important

in maintaining overall plant structure, organogenesis and

tropisms. One family of plant proteins with a role in cell

polarity are PIN proteins, plasma-membrane-localized auxin

efflux carriers that are involved in the transport of the plant

hormone auxin. They are important for determining the direction

and extent of growth of individual cells and therefore for the

maintenance of plant cell polarity (Grunewald and Friml, 2010).

The polar localization and maintenance of PIN family members

is controlled by endocytosis (Dhonukshe et al., 2007) and

transport by both ESCRT-dependent and ESCRT-independent

pathways (Spitzer et al., 2009). Several pathways have been

elucidated that control the maintenance of plasma membrane

polarity (Fig. 3), at least in part through the use of the inhibitors

Brefeldin A, which blocks the cycling of the PIN proteins

through endosomes (Kleine-Vehn et al., 2006), and wortmannin,

which inhibits transport out of endosomes (Jaillais et al., 2006)

(see Table 1). For example, localization of PIN1 involves

the endosome-localized GNOM protein, a guanine nucleotide

exchange factor for an ARF GTPase (Geldner et al., 2003;

Tanaka et al., 2009), or an alternative GNOM-like protein GNL2

in tip-growing cells that undergo rapid cell expansion (Richter

et al., 2012), whereas a separate pathway is responsible for the

internalization of PIN2, which requires GNOM-like 1 (Jaillais

et al., 2006; Pan et al., 2009; Teh and Moore, 2007). The auxin

influx carrier AUX1 is regulated by yet another distinct pathway,

but still requires endosomal trafficking (Du et al., 2011; Kleine-

Vehn et al., 2006; Robert et al., 2008). The endosome thus

regulates the location of components in different domains of the

Box 1. BOR1 borate transporter: an example ofregulated endosomal trafficking

Examples of regulated endocytic trafficking in plants have recently

come to light. One of the better studied is the BOR1 borate

exporter. Boron is an essential element for plants, required for

correct cell wall structure (O’Neill et al., 2004), but an excess of

boron is toxic. BOR1 transports borate into the xylem and

therefore controls the amount of boron in the plant (Noguchi

et al., 1997; Takano et al., 2002). Under boron-limiting conditions,

BOR1 is localized to the plasma membrane, but addition of boron

leads to uptake of BOR1 into endosomes and further transport to

the vacuole (Takano et al., 2005). Study of this protein has

provided insight into the signals required for endosomal transport.

Upon addition of boron, BOR1 is ubiquitylated on K590, and

mutation of this residue blocked MVB sorting for degradation in the

vacuole (Kasai et al., 2011). Because BOR1 is present on internal

vesicles of MVBs (Viotti et al., 2010), this raises the possibility that

ubiquitylation of BOR1 signals its transfer into internal vesicles,

presumably through the ESCRT complex.

LE or MVBor PVC

Vacuole

TGNor EE

GC

TGNor EE

RE

RE

LPVC

1

1

2

2

3

Fig. 3. The AUX and PIN auxin carrier proteins are transported by both

ESCRT-dependent and ESCRT-independent pathways. ESCRT is

essential for the degradation of auxin influx carriers (AUX, red ovals) and

pin-formed auxin efflux carriers (PIN1 or PIN2 proteins, black diamonds), but

not for their recycling back to the plasma membrane (Spitzer et al., 2009).

These auxin transporter proteins are essential for proper growth of the cell and

the determination of cellular polarity. The proper positioning of AUX and

PIN proteins at opposite poles of the cell dictates the flow of auxin through

the cell, and AUX and PIN proteins are internalized by endocytosis and

accumulate at the TGN or EE, as illustrated by the blue arrows (pathway 1).

This pathway is ESCRT dependent. The ESCRT-mediated transport pathway

directs the auxin carrier proteins to the internal vesicles of the MVB, where

they will be eventually transported into the lumen of the vacuole for

degradation. AUX and PIN proteins can also be recycled back to the plasma

membrane as illustrated by the thick red arrows (pathway 2) in an ESCRT-

independent pathway. Organelles are depicted as in Fig. 1.

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plasma membrane through endocytosis and recycling, which, in

turn, is essential for plant cell growth and polarity.

Endocytosis and signaling are linked through the need formaintenance and internalization of plasma membrane receptors,as well as the role of endosomes as platforms for signaling

pathways (Geldner, 2004; Geldner and Robatzek, 2008). Inaddition to more typical signaling cascades that are initiated atthe plasma membrane by membrane-localized receptors, several

examples of signaling pathways that involve endosomes are nowknown in plants (Geldner and Robatzek, 2008). Some leucine-rich repeat receptor kinases can function in signaling mechanisms

that involve the endosome. Upon extracellular ligand binding, theligand–receptor complex is internalized, which in the case ofrecognition of flagellin by the flagellin-sensitive 2 (FLS2)receptor, initiates the defense response (Gomez-Gomez and

Boller, 2000). Mutations in the gene encoding FLS2 that blockendocytosis also impair downstream signaling, indicating thatthere is a relationship between these two processes (Robatzek

et al., 2006; Salomon and Robatzek, 2006). Another example isthat of the BRI1 brassinosteroid receptor; although itsinternalization is independent of ligand binding, experimental

enhancement of its endosomal localization increasesbrassinosteroid signaling, suggesting that signaling occurspreferentially from endosomes rather than the plasma

membrane (Geldner et al., 2007).

An unusual pathway for initiation of signaling has beendescribed for the extracellular leucine-rich repeat receptor-likeprotein LeEix2 from tomato (Ron and Avni, 2004). This class of

proteins are cell-surface receptors, but lack a functionalcytoplasmic domain, which in most receptors functions insignaling to downstream components (Kruijt et al., 2005); it is

therefore unclear how the signal is transduced in the absence ofthis domain. LeEix2 induces defense responses by binding to thefungal elicitor ethylene-inducing xylanase (EIX), causing itsinternalization to endosomes (Bar and Avni, 2009; Bar et al.,

2009). Inhibitor studies demonstrated that endosomes andendocytosis are required for LeEix2-mediated initiation ofpathogen defense responses (Sharfman et al., 2011).

Surprisingly, the addition of EIX leads to changes in endosomemobility, with increased directional movement of endosomes inthe presence of the elicitor (Sharfman et al., 2011). Although the

significance of this movement is not yet understood, thefunctional implications of endosome dynamics in the defenseof plants against pathogen attack should prove a fascinating topic

of future study.

Closing remarks and future challengesIn this Commentary, we have discussed the structure of theendomembrane system in plants, with its two main classes of

endosomes, early endosomes (equivalent to the TGN) and lateendosomes (equivalent to multivesicular bodies or prevacuolarcompartments). The differences between endosome organization

in plants compared with animal cells, and in particular theadditional role of the TGN as an early endosome (Dettmer et al.,2006), highlight the need to study endosomal structure and

function in plant cells to have a complete understanding of theseimportant organelles. Recent research into plant endosomes hasfocused on the regulation of endosomal trafficking and its

relationship to polarized cell growth (Du and Chong, 2011; Ebineet al., 2011; Richter et al., 2012), the structure and maturationpathways of different types of endosomes (Kang et al., 2011;

Scheuring et al., 2011; Viotti et al., 2010), and thecharacterization of endosomal proteins such as ESCRT and

retromer components involved in transport and maturationpathways (Pourcher et al., 2010; Shahriari et al., 2011; Wanget al., 2010). Future important avenues of research will include

the clarification and further characterization of the pathways andcomponents for recycling from endosomes to the plasmamembrane (Jaillais et al., 2008; Pan et al., 2009; Richter et al.,2012) and functional analysis of proteins that are required for

endosomal trafficking and maturation (Niemes et al., 2010b;Otegui and Spitzer, 2008; Pourcher et al., 2010; Robinson et al.,2008a). The role of endosomes in signaling pathways is still

limited to a small number of examples in plants (Geldner andRobatzek, 2008; Gu and Innes, 2011; Kang et al., 2010); it islikely that this number will increase and our understanding of the

function of endosomes as signaling platforms will grow overthe next few years. As new tools are becoming available for theanalysis of endosomal structure, function and trafficking (see

Table 1 for examples), new avenues of research might beilluminated, and our understanding of these important organelleswill greatly expand.

AcknowledgementsWe thank Marisa Otegui for providing Fig. 2.

FundingThis work was supported by a grant from the National Aeronauticsand Space Administration [grant number NNX09AK78G] to D.C.B.

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