Intercellular trafficking via plasmodesmata: molecular layers of complexity

Abstract

Plasmodesmata are intercellular pores connecting together most plant cells. These structures consist of a central constricted form of the endoplasmic reticulum, encircled by some cytoplasmic space, in turn delimited by the plasma membrane, itself ultimately surrounded by the cell wall. The presence and structure of plasmodesmata create multiple routes for intercellular trafficking of a large spectrum of molecules (encompassing RNAs, proteins, hormones and metabolites) and also enable local signalling events. Movement across plasmodesmata is finely controlled in order to balance processes requiring communication with those necessitating symplastic isolation. Here, we describe the identities and roles of the molecular components (specific sets of lipids, proteins and wall polysaccharides) that shape and define plasmodesmata structural and functional domains. We highlight the extensive and dynamic interactions that exist between the plasma/endoplasmic reticulum membranes, cytoplasm and cell wall domains, binding them together to effectively define plasmodesmata shapes and purposes.

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Cellular and Molecular Life Sciences (2021) 78:799–816
https://doi.org/10.1007/s00018-020-03622-8
REVIEW
Intercellular trafficking viaplasmodesmata: molecular layers
ofcomplexity
ZiqiangPatrickLi1 · AndreaPaterlini2 · MarieGlavier1 · EmmanuelleM.Bayer1
Received: 15 April 2020 / Revised: 28 July 2020 / Accepted: 13 August 2020 / Published online: 12 September 2020
© The Author(s) 2020
Abstract
Plasmodesmata are intercellular pores connecting together most plant cells. These structures consist of a central constricted
form of the endoplasmic reticulum, encircled by some cytoplasmic space, in turn delimited by the plasma membrane, itself
ultimately surrounded by the cell wall. The presence and structure of plasmodesmata create multiple routes for intercellular
trafficking of a large spectrum of molecules (encompassing RNAs, proteins, hormones and metabolites) and also enable local
signalling events. Movement across plasmodesmata is finely controlled inorder to balance processes requiring communica-
tion with those necessitating symplastic isolation. Here, we describe the identities and roles of the molecular components
(specific sets of lipids, proteins and wall polysaccharides) that shape and define plasmodesmata structural and functional
domains. We highlight the extensive and dynamic interactions that exist between the plasma/endoplasmic reticulum mem-
branes, cytoplasm and cell wall domains, binding them together to effectively define plasmodesmata shapes and purposes.
Keywords Plants· Cell–cell communication· Plasmodesmata· ER–PM contacts· Nanodomains· Cell wall
Introduction
“I tried to explain as much as I could—Poppet says.
I think I made an analogy about cake Well that must
have worked—Widget says. Who doesn’t like a good
cake analogy?”
E. Morgenstern—The Night Circus (2011).
Unicellular and multicellular organisms share—among
other traits—the fundamental need for communication. This
is not to be intended in its verbal connotation but rather as
the diverse array of molecular mechanisms used to coordi-
nate biological processes within and between organisms. A
high order classification divides signalling into intracrine
(happening within a cell), autocrine (secretion of molecules
that act on the secreting cell itself), juxtacrine (between
physically touching cells), paracrine (aimed at cells in the
vicinity of the signalling source) and endocrine (the signal
produced can travel to distant cells) (reviewed in [1]). This
classification is more widely employed in animal research
but we feel it similarly carries value for research in other
organisms, albeit with conceptual adjustments for their spe-
cific biology.
Our focus is more closely aligned with a type of jux-
tacrine (and also possibly aspects of intracrine, paracrine
and endocrine as explained at various stages in this review)
signalling as we study plasmodesmata (PD) pores that put
in direct contact the cytoplasm of two neighbouring cells
(Fig.1). These should not be mistaken for passive chan-
nels as continuous and extensive regulation is operated
upon them (reviewed in [2] in the context of horticultural
applications). Direct cytosolic cell–cell signalling strate-
gies are observed throughout the kingdom of life albeit with
significant differences in their molecular composition and
their mode of action. Septal junctions connecting filament-
forming cyanobacteria were recently structurally resolved
as a multimeric protein complex [3]. Gap junctions between
animal cells are also known to be proteinaceous in nature
(reviewed in [4]). Tunneling nanotubes that bridge neuronal
Cellular andMolecular Life Sciences
Ziqiang Patrick Li and Andrea Paterlini contributed equally to this
work.
* Emmanuelle M. Bayer
emmanuelle.bayer@u-bordeaux.fr
1 Univ. Bordeaux, CNRS, Laboratoire de Biogenèse
Membranaire, UMR 5200, F-33140Villenaved’Ornon,
France
2 Sainsbury Laboratory, University ofCambridge, Cambridge,
UK
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800 Z.P.Li et al.
1 3
cells are actin enriched membranous protrusions with open
ends [5]. Conversely, in plants, PD include a continuous
plasma membrane (PM) traversing the cell wall between
neighbouring cells and a constricted form of the endoplas-
mic reticulum (ER), the desmotubule, spanning the pore in
its center (reviewed in [6]) (Fig.1c). At PD, the ER and PM
are tethered together by protein elements, leaving a space,
termed the cytoplasmic sleeve between the two. Unique
to PD is, therefore, the duplex endomembrane continuity
between cells, in addition to the cytoplasm one.
Together with other cell–cell communication mecha-
nisms, PD play a central role in plant development and
physiology. They enable metabolite fluxes between cells
([7] as an example in the context of plants with different
photosynthetic strategies), they contribute to the distribu-
tion of key plant hormones involved in development ([8]
as an example for auxin) and they control the movement of
RNA/proteins acting as developmental regulators ([9] as an
example in the context of plant stem cell maintenance). PD
are also fundamental for long distance transport of resources
and signals to distant organs, a function that might be remi-
niscent of endocrine signalling. PD influence the loading
[10], translocation [11] and ultimate release of substances
[12] from the phloem, the specialised conduit connecting
distal organs within the plant.
PD provide four potential routes for intercellular traffick-
ing: a main symplastic one across the cytoplasmic sleeve,
two membrane ones along either the PM or the ER and a
luminal one within the desmotubule. The cytoplasmic sleeve
one has long been and still is regarded as the main route of
transport for hydrophilic, soluble mobile factors (reviewed
in [13, 14]). Molecules with strong hydrophobic proper-
ties (or with domains displaying such properties) can con-
versely in theory take advantage of the ER/PM surface route.
These factors would be anchored in the membranes [15, 16].
Movement within the desmotubule lumen has been more
controversial, being largely ruled out in face of the extreme
constriction of the ER membranes at PD and evidence sup-
porting a lack of movement of luminal marker [17–19]
Fig. 1 Whole organism to single-cell multiscale view, emphasising
cell–cell connectivity via plasmodesmata (PD). a Schematic rep-
resentation of Arabidopsis thaliana at flowering stage. b Two plant
cells showing their cytoplasmic contents (nucleus in yellow, mito-
chondria in red, vacuole in grey, cell wall in coral, endoplasmic
reticulum(ER) in blue, peroxisome in violet and ribosomes in purple,
cytoplasm in light yellow, chloroplast in green) and displaying PD
at their cell–cell interfaces. c PD are plasma membrane (PM;pink)
lined, cell wall (coral) spanning pores that enable transport of mol-
ecules (red and light blue circles), mostly across the cytoplasmic
sleeve (light yellow space). PD neck constriction via deposition of
the wall polysaccharide callose (red) can reduce trafficking across
the pores. The proposed model is through reduced ER–PM spacing
(left side of panel c). Physical continuity of the endomembrane sys-
tem (ER and PM) is also observable in panels b and c with theER
becoming highly constricted within PD and largely preventing lume-
nal transport of macromolecules. Examples of potential directional
transports are shown by the coloured arrows. Differences in PD den-
sities between cellular interfaces (basal vs lateral sides in panel b) are
also represented. Scale bars: 50μm in (b) and 50nm in (c). Abbrevia-
tions:ER, endoplasmic reticulum. PD, plasmodesmata. PM, plasma
membrane.
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801Intercellular trafficking viaplasmodesmata: molecular layers ofcomplexity
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Overall, in this review, we employ the analogy of a multi-
layered cake to deliver a series of key messages regarding
PD. In the same way as a cake is a mixture of different ingre-
dients, PD are careful assemblages of selected molecular
components. Such assemblage is not coincidental, but rather
the result of evolutional pressures and selection [20]. We
also point out that how the different components are mixed
together and relate to each other is essential, bakery being
considered a science of precision. Lastly, as elaborate cakes
present multiple different tiers, PD also encompass multiple
structural layers (ER, cytoplasmic sleeve, PM and cell wall)
that are physically and functionally interconnected. How-
ever, the imagery of a baked cake should not provide a false
static image of PD as these structures undergo extensive and
dynamic remodelling. Overall, as all analogies, ours also
carries points of strength and weakness, but it is primarily
meant to convey some important concepts in an engaging
manner.
Warming theoven withsome key concepts:
control ofPD symplastic conductivity
A lingering narrative in PD research seems to postulate an
“open” resting status of PD. This status is extrapolated as
the natural opposite of the observed cases when PD were
actively “closed” in response to external clues. The cyto-
solic cell–cell continuity enabled by PD and the presence
of a continuous symplastic space is for instance a double-
edged sword when exploited by invading pathogenic organ-
isms (reviewed in [21]). One of the physiological responses
of the cells to such challenges (and similarly upon abiotic
stresses) tends to be the closure of PD via over-accumula-
tion of callose, a polysaccharide lining the cell walls of PD
([22, 23]as examples) (Fig.1c). Callose can be detected
via specific antibodies or stains ([24] as an example of both
approaches). PD accumulating callose have been viewed as
classical “closed” situations.
Boxing the conductivity of PD into resting (open) vs
stressed/attacked (closed) statuses is, however, too simplis-
tic and rather a more complex and nuanced picture exists.
For instance, in an environmental context, conductivity was
recently shown to vary during the day, being more prominent
in presence of light and being conversely gated by circadian
clock mechanisms at night [25]. Similarly, photoperiodic
control of bud growth has also been related to PD closure,
isolating the structure from growth signals specifically dur-
ing winter [26]. In a developmental context, cotton fibres
require a transient and reversible closure of PD and a switch
to apoplastic loading specifically during their elongation
phase, boosting osmotic and turgor pressure in the cell
[27]. Similarly, during stages close to the final lateral root
emergence, a transient isolation domain is established in the
primordium [28]. Whether a PD is open or closed may there-
fore very much depend on when we ask this question. In
addition to the timing, the specific location also seems to be
a central aspect. For instance, lateral root primordia progres-
sion is accompanied by a temporally regulated PD closure
in the specific tissues overlying the primordia [29]. It is still
unclear why this induction occurs as it negatively correlates
with lateral root emergence. However, if the degree of PD
closure is quantitative rather than absolute, the mechanism
described in [29] could be viewed as a point of regulation
for the extent of root branching.
We should keep in mind that indeed cell–cell connectivity
at a given interface does not depend on a single PD but rather
on a population of them, adding further quantitative aspects
([7] as an example for metabolic fluxes in leaves). Intercel-
lular transport will indeed depend on the overall status of a
PD population, while individual PD may display different
transport capacities. For instance, in addition to callose, the
permeability of a PD also depends on its structure [30, 31]
and perhaps on other yet unidentified factors. So far, the
tools used in the field to assess symplastic transport include
small injected/applied fluorescent dyes [32, 33], proteins
expressed from endogenous tissues [34, 35] or bombarded
on the same [36]. In most cases we only study an overall
visible effect on the movement of molecules (very often non-
native substances) across entire interfaces. Having access to
the transport status of individual PD will be informative and
will help, for instance, to understand whether fast coordina-
tion of responses occurs between PD. It is however experi-
mentally challenging to address this particular point due to
the nanoscopic size of individual PD, way beyondthe light
diffraction limit. In this regard, modelling approaches may
provide an alternative way to appreciate whether or not the
timing and speed of PD state changes is relevant for overall
cell–cell interface connectivity.
In addition, the frequency and distribution of PD across
tissues, even within different sides of the same cell, can be
widely different ([37] as an example), adding extra levels of
complexity to the system. This asymmetrical arrangement
together with differing transport capacities of individual PD
can result in directional transport across several cell layers
by creating a channelling effect. Permeability differences
have for instance been observed between lateral versus
apico-basal interfaces in roots [35]. Functional impacts of
such asymmetric flow were recently reported in [8, 38]. In an
ideal situation, all of these parameters (distribution, density,
structure, transport status) should be taken into account to
accurately and comprehensively map the symplastic intercel-
lular network. Combining experimental data and modelling
approaches can, in principle, achieve this.
We already mentioned approaches to study interface
permeabilities, to instead focus on the other parameters,
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802 Z.P.Li et al.
1 3
different microscopy techniques can be informative. Confo-
cal microscopy, for instance, addressed the occurrence of
different types of PD at cellular interfaces of the leaf epider-
mis [39]; immunolocalization and scanning electron micros-
copy (EM) focused on PD densities and their surface occu-
pancy at various leaf internal interfaces [7]; transmission
electron microscopy on sections provided comprehensive
PD maps for the root [40]; serial block EM was informa-
tive for PD densities at root vascular interfaces [11, 37] and
electron tomography resolved the fine structures of PD in
root cap and vascular cells [37, 41]. The spatial distribu-
tion of PD can be extracted from these datasets [42, 43] as
it was shown to have again impacts on flow between cells,
according to computational models (see in [44]). Temporal
changes in PD frequencies and arrangements have also been
uncovered with such techniques ([11, 40, 43] as examples).
The concept of PD “openness”/”closure” is also itself
relative as it varies according to the substance being dis-
cussed. Different levels of permeability across PD depend
on the shape, size and possibly electrostatic charge of the
molecules attempting to cross. This combination of factors is
routinely defined as the size exclusion limit of PD (SEL) [45,
46]. This differential permeability is not surprising. Indeed
having a tight control on the movement of proteins that carry
developmental programmes (via PD and other intracellular
mechanisms) might be essential to maintain cell identity
despite abundant connections ([47] using the SHORT ROOT
(SHR) transcription factor as anexample).
Based on traffic models, we can also categorize molecules
into two types, those whose intercellular movement is non-
targeted and those for which it is. The non-targeted mol-
ecules follow SEL requirements and are assumed to pass
through PD cytoplasmic sleeve (reviewed in [48]). Both
diffusion and advection might drive non-targeted molecular
flow across PD [49]. The former is caused by concentration
differences between cells for a given solute. This type of
transport is directional: from region of high concentration
to region of low concentration. However, this only applies
to the specific solute displaying the concentration difference
([49]). Advective movement instead refers to mechanical
transport through bulk motion (reviewed in [50]). Examples
of this are pressure driven bulk flow in the phloem (reviewed
in [50]) or cytoplasmic streaming (reviewed in [51]). In that
case, all substances would be dragged along with the water
flux, which sets the direction of transport. Evidence of trans-
port unidirectionality indeed exists from trichome studies
[52] and batch unloading in the root [12]. Additional, yet
unexplored biophysical processes may also influence inter-
cellular movement. For instance, surface fluctuation can
modify the transport capacity of nanochannels by modify-
ing diffusion and advection locally [53]. In all scenarios, PD
geometry (such as pressure of a desmotubule, central cavity,
constricted neck) but also internal membrane electrostatics
are expected to be determinant. The relative contritubion of
these different transport processes is then likely to depend
on the cellular and environmental context.
In contrast to non-targeted transport, a number of plant
native mobile factors have been shown to gate PD and
modify SEL to facilitate their own transport across the cell
border (reviewed in [13]). Non-native proteins produced by
invading plant viruses (reviewed in [54], fungi [55] and bac-
teria [56] similarly exploit these gating strategies to enable
spread of the pathogens, which would be normally impaired
at PD resting state ([57] as an example of compromised
movement of a virus lacking a functioning movement pro-
tein). The actual mechanisms of targeted movement, which
can occur with or without basal SEL modification, most
likely relates to interactions with local PD factors ([58] as
an example). Additional or alternative modification of the
mobile protein/mRNA might be necessary [59, 60].
However, it is also important to point out that PD are
not isolated cellular structures but rather part of the broader
organellar environment of the cell. Mechanisms affecting
intracellular sorting of molecules would therefore feed
into the subsequent intercellular strategies. Mobile factors
indeed need to reach (and subsequently move away from)
PD ([61, 62] as examples). In addition, PD permeability can
also be influenced by other organelles (such as chloroplasts
and mitochondria) for instance by affecting reactive oxy-
gen species levels in the cell ([63, 64] as examples). The
ultimate effect of this is likely largely callose dependent. A
more recent example showed that a mitochondrial protein,
in this case related to TARGET OF RAPAMYCIN (TOR)
metabolic signalling, can also influence permeability [65].
Overall, for cells, flow or “leakage” avoidance may be
equally important aspects of permeability control and the
relative balance between the two likely depends on the sub-
stances moving across, the specific tissues being crossed,
the overall developmental status, the environmental con-
text (encompassing both biotic and abiotic factors) and the
intracellular partitioning mechanisms. The balance of these
mechanisms might altogether enable retention of cell iden-
tity while also enabling extensive communication with sur-
rounding cells.
Molecular ingredients ofthePD cake
The nature of PD as membrane-lined pores spanning a wall
naturally introduces three molecular structural components
of relevance: lipids, proteins (embedded/anchored to a
membrane or in the polysaccharide matrix) and polysaccha-
rides. We do not include in this framework proteins that are
transiting across PD as part of their cell signalling function
but are not resident at PD. We highlight that in our vision
these three components are all relevant for PD function as
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803Intercellular trafficking viaplasmodesmata: molecular layers ofcomplexity
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experimental perturbation of any of the threeclasses can
lead to phenotypes.
Lipids
Lipids are critical components of cellular membranes. They
display a diversity of structures and physical properties that
have direct consequences on membrane organisation and
function, including at PD ([37, 66, 67] for PD and reviewed
for general membranes in [68]). Based on their chemical
structures, membrane lipids are classified into three main
classes: sterols, glycerophospholipids (GPLs) and sphin-
golipids. Each group is further subdivided into subspecies
that present variation in the nature of their polar heads, fatty
acid tails (length/saturation) and steryl moieties, creating
a vast collection of lipids with distinct physicochemical
properties.
Despite cell–cell continuity of the PM across PD,
some structural lipids segregate from the bulk PM and are
enriched at PD, creating a membrane microdomain with a
unique lipid environment. In the first study of the PD mem-
branes [66], the main lipid subspecies for each of the three
major lipid classes were conserved between PD and the
bulk PM (for instance phosphatidylcholine and phosphati-
dylethanolamine for the GPLs, glucosylceramide (Glucer)
and glycosyl inositol phospho ceramides (GIPCs) for sphin-
golipids and sitosterol for sterols). However PD-associated
GPLs presented a higher saturation level in their fatty acyl
chains [66]. The relative proportion between the three lipid
classes was also different, with sterols and GIPCs being sig-
nificantly enriched at PD. In agreement with the lipidome
results from Grison, Brocard etal. 2015[66], another study
further characterized the PD sphingolipid backbones and
found they were enriched with phytoshinganine (t18:0) long
chain bases (LCB) [67]. In the lipid analyses of both stud-
ies the ER and PM membranes could not be separated so
the PD lipid signatures cannot be unequivocally assigned to
either compartment. However, in face of the likely quantita-
tively larger contribution of the PM to the lipid pool (more
extensive surface volume at PD) [66], the low abundance of
sterols in the ER (reviewed in [69]) and the modification of
sphingolipids in the Golgi apparatus (reviewed in [70]), the
lipidomic results might indeed better reflect PM composi-
tion. In plants, sphingolipids and sterols are considered as a
functional pair, and their interaction has been documented at
both chemical and genetic levels ([71] and reviewed in [72]).
Sterols present a strong affinity to sphingolipids (and to a
lesser extent GPLs) driving, in model membranes and pos-
sibly biological membranes, lateral segregation through lipid
clustering and leading to the formation of ordered domains
(reviewed in [68]) (Fig.2). A similar process has been sug-
gested to occur at PD [66, 73].
Even within a single membrane compartment, heteroge-
neity inside the PD pores is likely to exist. For instance,
the PD-PM (PM domain lining the PD pores)often adopts
positive curvature at the neck region and negative curvature
in the central cavity [41], which could drive lateral segrega-
tion of lipids with different structural properties [74]. The
PM bilayer is also strongly asymmetrical, with GIPC sphin-
golipids being preferentially located in the outer leaflet while
phosphatidylserine and phosphoinositides are inserted in
the inner leaflet (reviewed in [68]). This asymmetrical lipid
distribution confers contrasting biochemical properties to
the inner and outer leaflets, leading to different functional
specificities on cytosolic and extracellular sides [75]. In the
context of PD, sterols and certain species of sphingolipids
arefunctionally linked to callose deposition in the outer leaf-
let [67, 76, 77], while phosphoinositides may help recruit
elements for PM-ER tethering at the inner leaflet [78]. The
multiple combinations of lipid identities (hence physico-
chemical properties) and distributions (along the PD-PM,
inner/outer leaflet) collectively create the unique membrane
properties of the PD pores.
Lastly, the lipid analyses performed so far at PD (and
this might also apply to protein and cell wall studies) prob-
ably only captured part of the existing diversity. It is fair to
speculate that the range of PD structures associated with
different tissues, different developmental stages and even
different connectivity statuses may very well require lipid
changes in their membranes. Likewise, we currently have lit-
tle understanding as to how specific lipids are clustered and
adjusted at PD. Most likely this would operate through local
enzymatic activity and targeted vesicular and non-vesicular
transport.
Proteins
Presence of proteins at PD is an implicit corollary to the
membranous nature of these structures, as a complete exclu-
sion of transmembrane or lipid-associated proteins would
be highly unlikely. However, presence of resident proteins,
exclusively localising or displaying increased abundance at
PD, is a different expectation that more closely matches the
specialised functions of PD.
Proteomic approaches are those that more significantly
contributed to capturing the diversity of proteins at PD
in a number of species [78–82]. The overlap between the
available proteomes and confirmed localisation of some
of the detected proteins well substantiate an actual local
enrichment of specific sets of proteins at PD. In Arabidop-
sis, the most recent and curated protein list was provided
by Brault, Petit etal., 2019 [78]. The list of 115 proteins
well reflects the structural and functional diversity of
PD. Cell wall functions are well represented with around
twenty members: from enzymes involved in direct callose
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804 Z.P.Li et al.
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Fig. 2 Model of cell wall, lipid and protein distributions and inter-
actions within PD. The upper opening area of a PD is shown in the
figure. PD are built up through intimate connections and interactions
between the cell wall, the PM and the ER. All these compartments
exhibit unique molecular signatures (in terms of lipids, proteins and
polysaccharides) at PD. Scale bar: 5nm. Abbreviations: CalSs, cal-
lose synthases. GIPC, glycosyl inositol phospho ceramides. GluCer,
glucosylceramide. LYK4, LysM-containing receptor-like kinase 4.
LYM2, LysM domain-containing glycosylphosphatidylinositol-
anchored protein 2. MCTPs, Arabidopsis multiple C2 domain and
transmembrane region proteins. PdBGs, plasmodesmal-localized
β-1,3-glucanases. PDCBs, plasmodesmata callose-binding proteins.
PDLPs, plasmodesmata-located proteins. PI4P, phosphatidylinositol
4-phosphate. REM, remorin. RTNLB, reticulon-like protein B. SYT,
synaptotagmin.
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805Intercellular trafficking viaplasmodesmata: molecular layers ofcomplexity
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turnover (such as CALLOSE SYNTHASES, CalSs, and
degrading enzymes, PLASMODESMATAL-LOCALIZED
β-1,3 GLUCANASES, PdBGs, [83, 84] for experimental
validation) (Fig.2), to those binding to wall components
or those modifying other polysaccharides (cellulose, pec-
tin and xyloglucans). We know significantly less about
the latter class compared to the former. Proteins with the
potential to affect lipids also seem to be present, such as
ceramidases, phospholipases, lipases, acyl-esterases, phos-
phodiesterase, phosphatases. However, confirmed localisa-
tions and functional evidence for these at PD is altogether
lacking. Proteins that might contribute to the structure and
function of PD are also present with for instance several
members of the MULTIPLE C2 DOMAIN AND TRANS-
MEMBRANE REGION PROTEINS (MCTPs) [58, 78,
85], a family sitting at the boundary between the desmo-
tubule and the PM (Fig.2), which will be discussed in
more detail in later sections.
In our description so far, we have narrowly ascribed the
function of PD to the direct translocation of molecules. This
is, however, incorrect as these structures also operate them-
selves as signalling hubs. This is the intracrine-like aspect of
PD we had hinted at in the introduction. Presence of resident
receptor-like proteins at PD, presumably at the PM, is well
supported by proteomic studies [78–81]. Functional studies
validated the presence of STRUBBELIG/SCRAMBLED
(SUB/SCM), a receptor-like pseudo-kinase involved in tis-
sue patterning and morphogenesis [85, 86]; LYSIN MOTIF
DOMAIN CONTAINING GLYCOSYLPHOSPHATI-
DYLINOSITOL ANCHORED PROTEIN 2 (LYM2) recep-
tor-like protein, playing a role in fungal pathogen perception
[87]; in association with the other RLKs shown in [88]), and
the cysteine-rich receptor-like protein PD-LOCATED PRO-
TEIN5 (PDLP5), important for innate immune responses
[89] (Fig.2). More recent studies even provided examples
of kinases dynamically re-localising to PD in response to
biotic [88] or abiotic [76, 90] stimuli. CLAVATA1 (CLV1)
and ARABIDOPSIS CRINKLY4 (ACR4), form a receptor
kinase complex, which localizes at PD, and is important for
root meristem maintenance and stem cell signalling [91].
The specific reason for the enrichment at PD of stem cell
signalling receptors remains partially unclear. A number of
biological processes in meristems do indeed rely on non-
cell autonomous signalling ([9] as an example). Presence
of these receptors could naturally relate to the positioning
of PD at the junction between cells (and indeed some of the
downstream signalling effects likely operate to limit PD con-
ductivity, see [87] as an example) but also it might be due
to the close contact of various cellular compartments and
components (ER-PM-wall) at PD, facilitating coordinated
responses. While the ability to perceive external stimuli
would be advantageously spread across the entire mem-
brane of a plant cell (detection across a larger surface area),
differential and targeted responses across the cell might be
subsequently required. This has for instance been shown for
the fungal elicitor chitin [88].
So far, most of the listed proteins at PD reside in the PM
(as examples [84, 89, 91–94]). Only a handful are embed-
ded in the ER: Calnexins [19], the reticulon-like family
[95] and the multiple C2 and transmembrane region protein
family [58, 78, 85] (Fig.2). The latter family actually ends
up spanning the cytoplasmic sleeve [78]. Members of the
calreticulin family have also been reported as resident in
the ER lumen [96, 97]. Overall, we know very little about
the function of desmotubule and associated proteins in the
context of PD functionality.
Lastly, cytoskeleton related proteins (actin, myosin, tro-
pomyosin, formins, Arabidopsis networked superfamily
of actin binding proteins and other actin related proteins)
have been localised to PD by immunological and func-
tional studies (reviewed in [98] with more recent additions
in [99, 100]). From this list, actin clearly seems to play a
role at—and most likely within—PD. Conversely, this
does not seem to be the case for microtubules (reviewed in
[98]). Actin microfilament fibers might not fit within PD
in their traditional conformation (discussed in [101], argu-
ing against models like the one in [102]) so the specific
roles of the cytoskeleton and its organisation at PD are, to
this day, a debated topic. Formins are suggested to anchor
actin filaments to PD, PM and possibly cell wall [100, 103].
Cytoskeletal inhibitor treatments reduced, increased or left
transport unaltered across PD depending on species and tis-
sues being studied (reviewed in [98]). The ultrastructure of
PD in columella and suspension cells was recently shown to
be unaffected by actin inhibitory drugs [41]. This was rel-
evant as the spokes between PM-ER (first reported in [104])
were at some stages suggested to be cytoskeletal in nature (
[102] as an example). This idea has been superseded by the
concept of PD as membrane contact sites (reviewed in [48]).
Cell wall polysaccharides
Plant cells are encased by rigid cell walls performing both
structural and developmental functions (reviewed in an
evolutionary context in [105]). PD span the wall separating
two neighboring cells and, similar to the desmotubule and
PD-PM, the wall around PD seem to also possess unique
molecular signatures, in particular in terms of wall polysac-
charides. We know much less about proteins that might also
localise in the wall matrix (some examples are reviewed in
[106]). In this section we highlight in particular the bio-
physical properties that such composition might confer.
A more marked presence of β-(1 → 3)-glucan, com-
monly known as callose, at the PD wall is well established
([107, 108] as examples). The levels of this polysaccharide
inversely correlate with PD permeability, as hinted in the
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806 Z.P.Li et al.
1 3
introduction (Fig.1c). However, in addition to a direct role in
modulating PD aperture, callose presence has been reported
to reduce stiffness and increase the elastic properties of cel-
lulose hydrogels [109]. It remains to be determined if these
physiochemical properties also apply to the in-planta wall.
A developmental correlation between callose deposition
and cessation of cell wall thickening has also been reported
in modified forms of PD important for long distance trans-
port [110, 111]. Cell wall thickness would determine the
length of the path molecules need to cross before enter-
ing the neighbouring cell. A reduction in cellulose content
seems indeed characteristic of areas rich in PD—so called
pitfields [43]. This might underpin the thinner cell walls
observed at PD [42] for a new quantitative visualisation of
this aspect). Cell wall thickness is also now starting to be
integrated in complex cell–cell permeability models [44].
In addition to callose, differential abundances of pectin
polysaccharides have been noted using specific antibodies.
Homogalacturonans are abundant at PD clusters, although
also present more broadly [112–114]. (1 → 5)-α-arabinan
containing pectins are enriched in the cell walls surround-
ing PD clusters [43, 112] while (1 → 4)-β-galactan contain-
ing ones are excluded from those areas [112, 113]. Overall,
pectins form an interlinked gel-like matrix that—suppos-
edly—might be more amenable to dynamic modifications
of PD aperture, in comparison to stiffer cellulose microfi-
brils, although models for cell wall structure are an evolving
topic (discussed for instance in [115]). The specific types
of pectins detected at PD might then impart additional
mechanical properties: arabinans containing pectins have
for instance been associated with cell wall flexibility [116]
while galactan containing ones to stiffness [117]. However,
PD specific research in this regard remains to be seen.
The specificity of the cell wall environment at PD (or
modulation of the same) might be co-opted by invading
viruses to target (and favour) their own spread. Interactions
with pectin-modifying factors were for example shown to
be important for viral movement protein trafficking [118].
Overall, polysaccharide signatures might also relate to
PD formation and elaboration. Indeed, while primary PD are
generated by the encapsulation of ER strains in the cell wall
septum formed during cell division, secondary de-novo PD
formation in elongating cells likely requires some form of
cell wall modification. Cell wall modifications are also likely
to occur when the morphology of existing PD increases in
complexity (reviewed in [119]). However, whether a specific
cell wall composition and associated biophysical properties
are a prerequisite for these processes or an effect of the same
is unclear. In most cases we cannot forecast the positioning
of PD to assay the cell wall in advance, despite some spatial
rules having been suggested [43, 120]. In principle, mecha-
nisms to generate cell wall micro-domains in plants do exist.
These tend to operate at the level of the cytoskeleton, via
membrane-anchored proteins that locally promote assembly
or dis-assembly of microtubules in particular [120, 121].
Cellulose synthase enzymes travel along the microtubules
and determine the positioning of the fibrils on the outside
of the cell [122]. Ultimately, these molecular mechanisms
were shown to regulate the positioning and size of cell wall
pits (areas devoid of secondary cell wall deposition) in
xylem cells (reviewed in [123]). Whether similar mecha-
nisms might be at play at PD is unknown, yet a fascinating
prospect.
This last example is useful to highlight the actual inter-
connection between lipids, proteins and polysaccharides. We
will explore this in the context of PD in the next section.
Mixing recipes andmolecular interactions
We have so far presented for simplicity the molecular com-
ponents of PD in isolation and highlighted the potential
importance of each of them in some contexts. We now want
to stress how these three components are, however, inter-
linked and together determine PD function.
The first hint of this comes from the proteomic approaches
to study PD. Although the goal of these approaches is to
enrich for a PD fraction while avoiding cell wall and orga-
nelle contamination (besides the desmotubular ER and prox-
imal cell wall themselves) (reviewed in [124]), this is never
fully achieved as PD can never be separated from the cell
wall fraction itself. They have been “baked” together in an
undissolvable bond. The relative enrichment for PD proteins
versus general cell wall protein relies on a careful level of
enzymatic cell wall digestion on the extracted fraction [125].
In some studies what is termed a PD fraction is actually the
full cell wall fraction [126]. The recalcitrance of the mem-
branous parts of the PD to separate from the cell wall is
likely to reflect direct binding between the two components.
The sphingolipid GIPCs, which sit in the outer leaflet of the
PM were for instance found to be directly boron bridged
to pectins in Rosa cultured cells [127] (Fig.2). Whether
this applies to PD, remains to be established. Changes in
sphingolipid levels in biosynthetic mutants seemed to cor-
relate with thicker cell walls at PD [42]. Some PD mem-
brane proteins (besides those directly involved in cell wall
biosynthesis or modification) also have polysaccharide
binding properties. PLASMODESMATA CALLOSE-
BINDING PROTEINS (PDCBs), which are inserted in the
outer leaflet of the PM and thereby face the extracellular
space [92] (Fig.2), possess an X8 domain known to bind
carbohydrates and more specifically callose [128]. PDLPs’
extracellular domains have structural homology to fungal
lectins, also capable of binding carbohydrates [129] (Fig.2).
Interestingly, changes in cell wall have been shown to affect
the mobility and nanocluster size of proteins in the PM
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807Intercellular trafficking viaplasmodesmata: molecular layers ofcomplexity
1 3
membrane (albeit not at PD) ([130] via protoplasting; [131]
via treatment with cellulose or pectin inhibitors). A similar
scenario might be at play at PD.
PD, when they undergo harsh mechanical shredding
during isolation or plasmolysis, still retain the ER strand
inside, highlighting a tight connection also between lipid
membranes [66, 132]. PD proteins indeed tightly engage
with local lipids. Glycosylphosphatidylinositol (GPI) lipid
anchors serve as minimal sorting signals for preferential
insertion in the PM outer leaflet of PD [133]. Proteins can
also be directly recruited through their lipid-binding mod-
ules. PDLP5 targeting to PD seems to require an interaction
between its transmembrane domain and phytoshinganine
(t18:0) based sphingolipids, enriched at PD [67]. Interest-
ingly, PDLP1, another PD protein from the same family did
not show direct in-vitro binding, suggesting that specific-
ity might reside in the structure and sequence of the trans-
membrane domains (TMs). In a similar manner, MCTP4
PD enrichment may partially rely on their phosphoinositide-
binding C2 domains [78] (Fig.2). Overall, different PD pro-
teins may utilize different lipid species for targeting to dif-
ferent subdomains within PD.
Aside from direct lipid binding, proteins can also recog-
nize membrane properties (such as thickness and curvature)
to facilitate their enrichment at specific areas of the cellular
membrane (reviewed in [68]) which may also apply to PD.
Integral membrane proteins are, for instance, sensitive to
local membrane thickness and lipid packing order and con-
sequently segregated into subdomains [75, 134]. PD mem-
branes, as mentioned, are enriched with sterols and very
long chain saturated fatty acid—containing lipid species,
which presumably make them thicker compared to the bulk
PM. Sorting based on transmembrane domain length might
therefore be relevant at PD. Indeed, bioinformatic analyses
of PD membrane proteins seemed to show on average larger
TMs [79]; however, direct physical measurements of mem-
brane thickness [135] are, to this day, not available for PD.
On a different note, biological membranes are by nature
heterogeneous. This heterogeneity is caused by specific
lipid-lipid but also lipid-protein interactions which alto-
gether result in the formation of microdomains differing in
composition and function ([136] as example). This has been
observed at PD as well [66, 73] (Fig.2). For example, sterol-
sphingolipid interactions may facilitate lipid nanodomain
(also called “rafts”) formation in the PM outer leaflet and
recruit GPI-anchored proteins to the PD entry region [66].
Highly ordered nanodomains mediated by sterols and PI4P
in the PD-PM inner leaflet host Remorins (REMs). These
proteins can locally affect lipid order and influence nano-
domain expansion [73, 136]. The relative size and lifetime
of PD lipid domains may further determine the repertoire
of resident proteins and how these proteins function. Pro-
tein mutants and lipid treatments can indeed phenocopy
each other (an example in [78] for MCTP mutants and PI4P
lipids). However, it would be an oversimplification to largely
categorize PD-PM into “raft” and “non-raft” domains; other
types of microdomains initiated by specific lipid-protein
pairs almost certainly exist. For instance tetraspanin proteins
have been detected at PD and other domains [80, 137] and
these are known to affect microdomains in other organisms
(reviewed in [138]).
Besides the direct physical binding mentioned before,
interactions or mutual effects between lipids and cell wall
components have also been described, mostly operating
via the specific proteins recruited to the PD microdomains.
Treatments with sterol inhibitors resulted in altered callose
levels at PDand correlated with miss-localisation of PDCB1
protein and callose degrading enzymes PdBGs [66]. In a
genetic context, a similar effect was observed in a sterol
carrier gene mutant; this time with effects on the transcrip-
tion of PdBGs and potentially PD structure [77]. Interactions
between components can also be elicited by external factors.
For instance, changes in lipids brought about by phospho-
lipase enzymes (themselves activated by osmotic-stress)
were shown to influence the relocalisation of the CYS-RICH
RECEPTOR-LIKE KINASE 2(CKR2) to PD, which in turn
interacted with a callose synthase (CalS1) and led to closure
of PD [90].
This set of examples show how lipid, protein and poly-
saccharide can all be affected by a change in one of these
elements.
Cake structural tiers generated
We conclude this review by emphasizing how these ingredi-
ent mixtures ultimately lead to clear PD domains in terms of
structure and functional properties. Indeed, as we mentioned
in the introduction, PD present elaborate architectures: they
contain a central desmotubule (ER) core, surrounded by a
cytoplasmic sleeve, in turn delimited by a PM bilayer, itself
ultimately surrounded by the cell wall. These four domains
are not freestanding: extensive interactions between them
exist and are mediated by the molecular players previously
described. Functional and dynamic interactions between
these different compartments are indeed fundamental for
PD function.
Cytoplasmic sleeve, ER‑PM contacts
andtheregulation ofsymplastic transport
The cytoplasmic sleeve is regarded as the main route of
transport within PD, and a direct correlation between the
size of the sleeve and its transport capacity is assumed in
current canonical descriptions (see for instance [139]). The
cell wall polysaccharide callose, arguably the only well
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808 Z.P.Li et al.
1 3
known regulator of PD permeability, is believed to push the
PM and ER closer together, reducing the functional sleeve
space and hence transport (Figs.1c and.2). Enlarged callose
collars at PD openings have indeed been observed in EM
images [140]. We previously mentioned several examples
of callose action and induction in biological contexts. How-
ever, mechanistically, how such an extensive change in the
cell wall is perceived and accommodated by the PD mem-
branes and transduced to the cytoplasmic sleeve remains
unresolved. However, changes in the cell wall may also trig-
ger relevant modifications in the composition or physical
properties of the PD membranes.
This direct relationship between cytoplasmic sleeve size
and transport was recently challenged by novel structural
descriptions of PD. PD populations, especially those in
cell types with specialised functions [12] are structurally
heterogeneous and diverse. Electron microscopy (for exam-
ple [30]) and some protein markers ([39] as an example)
had enabled classifications based on volume traits, such as
number of channels, shape and cavities (overall reviewed in
[141]). The use of electron tomography in recent years has
put more emphasis on the membrane contact sites nature
of PD and it introduced a type classification based on the
more subtle degree of apposition between the PM and ER
membranes. Newly formed Type I PD display a remarkably
close apposition between the two membranes leaving a tiny
gap (2–3nm) filled with electron dense material. Type II
PD, however, have the classic conformation of a clear sleeve
separating the two [41]. A developmental progression from
Type I to Type II was suggested based on the age of the cells,
pointing towards an “opening” and modification of the for-
mer into the latter [41]. This progression was also correlated
with PD protein population changes [78].
Close membranes appositions—such as those observed
at PD (in both types)—could require active tethering by
proteins (reviewed in [142]). MCTP protein family mem-
bers have been suggested to perform such a role within PD
[78] and, in addition or as a consequence of that, influence
both targeted and non-targeted transport (as examples [58,
78, 143, 144]) (Fig.2). Synaptotagmins family members
also act as ER-PM tethers and may have a more significant
contribution in the immediate proximities of PD and in par-
ticular contexts such as viral infection [145, 146] (Fig.2).
The overall linkage of the ER to PM via the tethers might
also be important for rapidly regulating PD conductivity
during osmotic shock. A pressure induced sliding of the ER
against the openings of the PD pore was recently suggested
as a mechanism for closure [147]. The model tries to resolve
decades long evidence for pressure regulation of PD (for
instance [148]).
Modification of the tether populations, in terms of pro-
tein types or conformations of the same (likely also in
combination with the underlying lipids in the membranes
and possibly in a calcium dependant manner) could regu-
late the size and conductive properties of the cytoplasmic
sleeve and possibly affect transport of substances. The mini-
mal sleevesize would be at least the physical space of the
protein tethers themselves (as exemplified in the figures of
[149]). How sleeve modifications would be allowed by the
surrounding cell wall, which would require relaxation or
some degradation to accommodate the opening sleeve, also
remains to be elucidated.
What is important in the context of transport is that type I
PD, counterintuitively, considered their reduced sleeve, were
found to be competent for transport of macromolecules such
as GFP [41] and were consistently found in tissues with high
cell–cell permeability [35, 41]. They were also ultimately
shown to be more permeable than type II PD [37]. The last
point clearly breaks the sleeve size assumption we men-
tioned earlier. How mechanistically transport might occur
within such crowded and limited space remains unclear and
resolving this will be essential.
The PM influences thecytoplasmic sleeve
andthecell wall environment
The last paper we mentioned [37] highlighted another
important point, in this case related to the lipid membranes:
sphingolipid biosynthesis (in particular of those speciescon-
taining very long chain fatty acids) controlled the type tran-
sition at the phloem pole pericycle (PPP)—endodermis
(EN) interface in the root. Sphingolipids might therefore be
among the lipid classes recruiting protein factors important
for sleeve regulation at PD. Modifications of the PPP-EN
cell wall might also relate to these sphingolipids as differ-
ences were detected between mutant and wild-typeback-
grounds [42], once again further linking the ingredients of
the PD cake.
The role of lipids in shaping PD structure has also been
suggested in Huang, Sun, Ma etal., [73]. A mechanism was
proposed whereby REM proteins oligomerization, induced
by salicylic acid (SA) treatment, increases the order level of
the lipid bilayer and this, through a still unknown mecha-
nism, leads to PD closure. The mechanism was suggested to
be (at least partially) callose-independent and, therefore, act
in parallel to the known callose dependent mechanism of SA
induced PD closure [89]. REMs are known PM nanodomain
proteins [150] and, so far, group1 REMs have been corre-
lated to callose regulation at PD ([151, 152] as examples).
Huang, Sun, Ma etal., [73] found that overexpression of
REM reduced dye transport in the root but this effect could
be partially rescued with chemical treatments that depleted
membrane sterols (high order generating components) but
not by treatments impairing callose deposition. The authors
proposed that REM misexpression rigidifies the PD-PM
and reduces the size of the cytoplasmic sleeve. This is an
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809Intercellular trafficking viaplasmodesmata: molecular layers ofcomplexity
1 3
opposite correlation to the Yan, Yadav etal., [37] paper in
terms of sleeve size and transport capacities.
Differences within the same lipid class might also be
important. While in[37] the change in sphingolipids did
not affect any aspect of callose levels, another study has
shown that mutations in two other sphingolipid modifying
genes conversely lead to the direct recruitment of PDLP5,
increased callose deposition, reduced PD permeability in
leaves and enhanced resistance to pathogens [67]. A direct
comparison between the two studies is difficult but the
lipid changes in the two are, however, clearly different: the
mutation of interest in Yan, Yadav etal., [36] reduces most
sphingolipid classes while the mutations in Liu etal.,[67]
conversely increase them. Specific lipid alterations, with
likely compensatory effects from the other lipids in PD
membranes, might therefore lead to recruitment of different
PD proteins, depending on the specificity of the latter. Dif-
ferent tissues and their specificities might also contribute
to differences between the studies. It is also possible that,
since certain sphingolipid species carry signalling function
(reviewed in [153]), the change elicited in Liu etal., [67]
actually leads to a broader immune response involving cal-
lose (rather than PD structural changes). In future work, it
will be interesting to understand how callose deposition
might affect the local PD-PM organisation/properties and
impact on the ER-PM contact sites within PD.
The desmotubule andits structural andtransport
capabilities
The instinctive focus on the cytoplasmic sleeve is built on
the idea that PD mainly support cytosolic trafficking. How-
ever, another structural domain within PD is a constricted
form of the ER (in close contact with the PM). This has been
observed since the earliest identification of these channels
in plants [154].
The presence of the ER inside PD that originated from
cell division (termed primary PD) has largely been taken
as a side effect of ER strands trapping in the forming cell
plate [17]. This integration of ER tubules into the forming
cell plate [155] is well contrasted with active ER clearance
and abscission during cytokinesis in yeast and mammalian
cells (reviewed in [156]). The reason for such drastic dif-
ferences in ER network organization during cell division
remains largely elusive. In land plants, the ER seems to be
a prerequisite for building up PD pores, as fenestrae in the
cell plate only develop into PD when the ER pre-occupies
such positions [155] and no PD without a central ER have
ever been observed. The presence of the ER inside PD that
are formed independently of cytokinesis (termed secondary
PD) [157] is an additional argument against the idea that the
ER is just being passively trapped inside PD. The forma-
tion of PD (primary and secondary alike) is likely to require
tethering between the PM and the ER [41, 145, 157]. To
further support such point, genetic disruption of PD tethers
in maize, namely mutating the CARBOHYDRATE PARTI-
TIONING DEFECTIVE 33 protein (ortholog of members
of the MCTP family in Arabidopsis), results in a significant
reduction in PD at the companion cell—sieve element inter-
face in leaf veins [143].
Researchers seldomly consider the impact the desmotu-
bulehas on PD function and how the latter would be affected
in absence of the same. Some PD analogous structures in
algae lack a desmotubule (reviewed in [6]), hinting that pres-
ence of ER is not an absolute requirement for connectivity,
although, as suggested by Park etal., [147], deformation
of the desmotubule/ER might be important for regulating
trafficking through the cytoplasmic sleeve. Intriguingly, in
comparison to other membranous channels described across
the eukaryotic kingdom, only plant PD exhibit an intracel-
lular organelle as an integral part of the structure (reviewed
in [6]). This singularity of PD highlights the value of inves-
tigating the function of the ER. The desmotubule likely per-
forms some selectively advantageous purpose making such
membrane a pivotal player, rather than an entrapped victim.
At a local scale, close contact between the opposing
membranes from two organelles might facilitate non-vesic-
ular transport and intraorganellar communication (reviewed
in [158]). Within PD, it is rational to speculate that ER takes
part in establishing the unique lipid environment of PD by
recruiting proteins that modify the lipids or directly transfer
lipids between two membranes. Such actions on PM lipid
modification could have a direct impact on membrane shap-
ing, protein recrutement, organisation and even function (via
conductive and structural properties of PD).
From a geometric and fluid dynamics point of view, hav-
ing a ER tubule inside a PM channel significantly increases
transport capacity while maintaining size selectivity, as
recently suggested by mathematical modeling [44]. A PD
with a desmotubule (tubule within a tube structure), in the-
ory, provides more transport volume than a cylinder channel
without a desmotubule. This can be explained by the fact that
the cytosolic cross-sectionalarea of a PD with desmotubule
is much larger than the cross-section of a cylindrical chan-
nel with the same theoretical SEL (which corresponds to the
maximum particle radius that fits in the channel). Hence,
with a desmotubule radius of 8nm and a maximum particle
radius of 2nm, 20 cylindrical channels would be needed to
match one PD with a desmotubule. The presence of a des-
motubule also reduces the surface area in proximity to the
wall and hence steric hindrance and viscous drag. As par-
ticle centers cannot come closer to a surface that their own
radius (steric hindrance), the available area for a particle size
of ½ SEL would be 25% in a cylindrical channel and 50%
in a channel with desmotubule. Forty cylindrical channels
would then be required to match one PD with desmotubule.
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810 Z.P.Li et al.
1 3
This difference in transport capacity further increases when
considering the hindrance effects from viscous drag along
the channel surface (Eva Deinum, personal communication).
Lastly, the desmotubule could work in trans by modulat-
ing PD-PM signalling. This was recently suggested at PD
for ER protein MCTP15/QUIRKY, which promotes, through
direct physical interaction, the stabilisation and signalling of
PM localised SUB [85, 144] (Fig.2).
ER continuity through PD could also serve as a route for
intercellular transport, as we briefly discussed in the intro-
duction. Lipid derived signals related to plant immunity
have been suggested to propagate between cells along the
desmotubule and lipid transfer proteins located in the ER
would facilitate this [159]. Diacylglycerol was suggested
to diffuse along the ER membranes of PD when microin-
jected, while sphingolipids could not move along the PM
[15, 16]. PD membranes might, therefore, represent both
routes of exchange and isolation points. Transport of native
transmembrane proteins along PD membranes remains to
be demonstrated but targeted transport has been shown for
viral movement proteins (reviewed in [160]). Movement
within the desmotubule lumen was experimentally ruled out
for proteins the size of GFP or greater [161, 162]although
lumenal transport of 10 kDa molecules has been reported
in stem epidermal cells[163] andleaf trichome cells[164].
Preferential intercellular movement of zinc ions has also
been speculated to occur across the desmotubule in mineral
deficiency conditions [165]; evidence for this is, however,
lacking. Overall, mechanisms to regulate such flows have
not been clarified.
Similar to plants, mammalian and yeast cells share
strands of ER between cells during cell division. However,
they establish an ER diffusion barrier to prevent uncon-
trolled flow along the membrane, especially of substances
related to ER stresses and aging [166, 167]. Whether this is
also the case in plants remains largely unexplored. In addi-
tion, it is interesting to point out that, while yeasts and mam-
malian cells do not establish a barrier for transport within
the connected ER lumen, plants clearly do. The extreme
physical constriction of the desmotubule seems to largely
prevent protein transport [161, 162]. It has been shown that
both ER shaping proteins, such as reticulon-like proteins
(RTNLB) [95] and ER-PM tethers of the synaptotagmin
SYT family [145] contribute to extreme ER constriction at
PD (Fig.2). The mechanisms through which ER constriction
is specially executed within PD—and why this may be rel-
evant in the context of plant multicellularity—again remain
to be determined.
The cell wall andapoplastic /symplastic
crosstalks
Earlier in this review, we highlighted the existence of direct
receptor mediated signalling at PD and we mentioned its role
on PD permeability. Here, in the context of the structural
layers, we want to stress how this offers opportunities at
PD for signal integration between the symplast and the apo-
plast. For the role in the apoplast sensing, one could envis-
age aspects of paracrine signalling applying to PD.
Some proteins perform a direct relay of an extracellular
clue to the inside of the cell. This is for instance the case of
LYM2/RLK receptor complex, perceiving fungal chitin in
the apoplast and triggering a signalling cascade resulting in
PD closure via callose (Fig.2). LysM receptor-like kinases
4 (LYK4) is one of the kinases that conditionally associates
with LYM2 (Fig.2). The response aims to block the spread
of potential pathogen effectors and also regulate move-
ment of endogenous defence signalling molecules [87, 88,
168]. Other proteins highlight more significant integration
between the two transport pathways. ACR4 (acting in homo-
meric complexes and heteromeric ones with CLV1), for
instance, detects the secreted CLAVATA3/ESR-RELATED
(CLE) 40 peptide in the apoplast of root tips and influences
stem cell maintenance [91]. A mechanism was suggested
by which a gradient of CLE40 peptide within the root tip
would lead to a dose dependent activation of ACR4-CLV1
complexes at PD. The differential intracellular activity of
the receptors in the various cell layers would then promote
or repress the PD symplastic mobility of unknown stem cell
factors (reviewed in [169]). While experimental evidence of
this model is not yet available, the mechanism would act as
a robust positional system to balance meristem maintenance
and differentiation. A potentially similar mechanism could
involve the beta glucanase ZERZAUST (ZET). This protein
is atypical as it does not seem to actually degrade callose nor
to localise to the PM despite its GPI anchor. Conversely, it
localises to the cell wall and moves in the apoplast between
cell layers of lateral roots [170]. Mutations in this gene result
in phenotypes similar to SUB mutants [171]. SUB/SCM is a
receptor like kinase partially localising to PD and involved
in tissue morphogenesis and patterning [85, 144] likely by
affecting the movement of an unknown mobile factor. The
interaction between SUB and ZET (if any) is, however,
not direct [170]. It is possible to speculate of other cases
where signal integration might be happening. For instance,
switches between symplastic and apoplastic loading/unload-
ing of molecules, associated with developmental stages or
triggered by biotic challenges [27, 172–175], might require
some coordination. This could partially occur at PD. Sys-
temic acquired resistance (SAR), one of the long-term plant
defence responses to pathogens, might also benefit from
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811Intercellular trafficking viaplasmodesmata: molecular layers ofcomplexity
1 3
signal integration. Some of the signals required to trigger
this response indeed travel in the apoplast (salicylic acid)
while others move in the symplast (azelaic acid and glycerol-
3-phosphate). The latter two have been shown to interact
with proteins also localising to PD [159, 175] and SAR has
been shown to depend on PD function [175].
Prospects andfuture baking endeavours
Over the last decade, remarkable progress has been made in
identifying the molecular elements and building blocks of
PD pores. While this process has not exactly been a piece
of cake—but rather a complex meal to digest—the extent of
detail on protein composition of PD we have now accumu-
lated, the characterisation and emerging roles of lipids and
the continued relevance of cell wall polysaccharides have all
proved to be significant milestones. The novel functionalities
of PD as direct receptor-signalling hubs and their capac-
ity to couple symplastic and apoplastic signalling have also
added novel functional angles to these structures. Recent
work has also questioned well-accepted textbook models,
for instance, the direct relationship between ER–PM spac-
ing and the transport capacity of the cytoplasmic sleeve.
Overall, accumulating evidence clearly suggests a synergetic
action between lipids, proteins and polysaccharides. The
multiple cake layers of PD (ER–sleeve–PM–cell wall) are
also all fundamentally and functionally intertwined. Only by
embracing this close-knit molecular and architectural com-
plexity, the field will be able to further resolve the subtle and
hidden flavours of PD.
Acknowledgements We thank Yvon Jaillais,Sébastien Mongrand,
Jules D. Petit, Jessica Pérez-Sancho and Yka Helariutta for critical
review of the article prior to submission and Eva Deinum for discussion
on mathematical modelling of PD transport.
Author contributions Z.P.L and A.P. wrote the manuscript with input
from all other authors. M.G. generated the figures. E.B conceived the
manuscript.
Funding This work was supported by the National Agency for
Research (Grant ANR-18-CE13-0016 STAYING-TIGHT to E.M.B),
the European Research Council (ERC) under the European Union’s
Horizon 2020 research and innovation programme (grant agreement
No 772103-BRIDGING) to E.M.B, the EMBO Young Investigator Pro-
gram to E.M.B, and the Gatsby Foundation (GAT3395/PR3 awarded
to Yka Helariutta and supporting A.P.).
Compliance with ethical standards
Conflict of interests The authors declare no competing interests.
Open Access This article is licensed under a Creative Commons Attri-
bution 4.0 International License, which permits use, sharing, adapta-
tion, distribution and reproduction in any medium or format, as long
as you give appropriate credit to the original author(s) and the source,
provide a link to the Creative Commons licence, and indicate if changes
were made. The images or other third party material in this article are
included in the article’s Creative Commons licence, unless indicated
otherwise in a credit line to the material. If material is not included in
the article’s Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will
need to obtain permission directly from the copyright holder. To view a
copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/.
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