Download Plasma membrane repair in plants

TRPLSC-731; No of Pages 8
Review
Plasma membrane repair in plants
Arnaldo L. Schapire, Victoriano Valpuesta and Miguel A. Botella
Laboratorio de Bioquı́mica y Biotecnologı́a Vegetal, Departamento de Biologı́a Molecular y Bioquı́mica, Facultad de Ciencias,
Universidad de Málaga, Campus Teatinos s/n, Spain
Resealing is the membrane-repair process that enables
cells to survive disruption, preventing the loss of irreplaceable cell types and eliminating the cost of replacing
injured cells. Given that failure in the resealing process in
animal cells causes diverse types of muscular dystrophy,
plasma membrane repair has been extensively studied in
these systems. Animal proteins with Ca2+-binding
domains such as synaptotagmins and dysferlin mediate
Ca2+-dependent exocytosis to repair plasma membranes
after mechanical damage. Until recently, no components
or proof for membrane repair mechanisms have been
discovered in plants. However, Arabidopsis SYT1 is now
the first plant synaptotagmin demonstrated to participate in Ca2+-dependent repair of membranes. This
suggests a conservation of membrane repair mechanisms between animal and plant cells.
Plasma membrane resealing – a process essential for
cell survival
Cell survival depends on the maintenance of plasma membrane integrity. If an open disruption in the plasma membrane is not resealed, potential toxins that will impair
normal cell function, such as high Ca2+, flood into the
cytosol of the wounded cell and vital cytoplasmic constituents such as ions and proteins can escape [1]. The basic
underlying assumption was that the cell membrane would
spontaneously reseal if broken because lipid bilayers naturally seek the lowest energy configuration in which the
hydrophobic domains of the bilayer face each other. However, in nucleated animal cells, this view is no longer
justifiable. It is now thought that resealing of the plasma
membrane is the outcome of a dynamic and complex mechanism that requires the participation of extracellular Ca2+
and numerous cytoplasmic constituents [2].
In most animal cell types, surviving a disruption
requires the mounting of a rapid (within seconds) resealing
response [2,3]. Reflecting its biological importance, the
literature provides numerous examples of plasma membrane resealing. Free-living amoebae, such as Physarum
and Dictyostelium, can be cut in half and then observed
within seconds to resume normal behaviour [4]. Within five
seconds after ripping away >1000 mm2 of plasma membrane from a sea urchin egg, resealing to the point of
preventing further Ca2+ entry is complete, and the
wounded egg can be fertilised and is viable [5]. Regeneration of skeletal muscle cells is observed after transection
injury, and fibroblasts can, if wounded in culture (e.g. by a
needle scratch), recover and divide [6].
Corresponding author: Botella, M.A. ([email protected]).
Given such animal examples, one might wonder
whether plant cells have a similar resealing mechanism.
A negative answer generally arises from the idea that,
having a protective cell wall, plant cells would not require
such a mechanism. However, plants are sessile organisms
that are continuously subjected to stressful biotic and
abiotic conditions, such as pathogen and predator attack,
high winds, drought, soil salinity, or freezing that endanger plasma membrane integrity. Hence they need a means
of repairing damage caused by such stresses. Here, we
review molecular and biochemical data supporting a
plasma membrane resealing process in plants and we
propose other possible elements involved in repair, based
on their homology to known animal components.
Evidence for plasma membrane resealing in plant cells
The ability of a plant cell to recover after a plasma membrane injury is a well-known phenomenon that has been
demonstrated by decades of successful micromanipulation
experiments [7]. This property enabled plant biologists to
develop microinjection techniques in order to deliver
exogenous DNA [8], proteins [9] and fluorescent dyes
[10] directly into plant cells, and to take microelectrode
measurements [11] that have contributed significantly to
botanical research. Although of little scientific interest for
electrophysiologists, it is likely that the disruption of the
plasma membrane requires a resealing process that
enables the punctured cells to survive. In nature, the
repair process is also evident during aphid feeding. Most
cells along the stylet pathway are briefly (typically for
5–10 s) punctured intracellularly, but after the stylets
are withdrawn, most of the punctured cells survive with
little or no adverse effects [12].
The first reports describing plasma membrane repair
involved in vitro manipulations of sea urchin eggs. These
studies demonstrated that extracellular Ca2+ is essential
for plasma membrane resealing after injury [13,14]. It was
then found that the same damage induced a rapid Ca2+dependent exocytosis of intracellular vesicles at the sites of
the injured plasma membrane, providing a mechanistic
framework for the role of Ca2+ in plasma membrane resealing [15]. In plants, the requirement for Ca2+ has been
recognised in experimental techniques that depend on
plasma membrane resealing. Thus, microelectrodes can
be successfully inserted into membranes after creating
an electrical impulse through the microelectrode. In this
methodology, Ca2+ ions play a crucial role in the resealing
process of the protoplast membrane [16]. The same Ca2+dependent recovery was observed during electrofusion, a
related process consisting of the fusion of protoplasts by
application of an electrical current [17]. It is also known
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that plasma membrane damage in animal cells triggers
the repair of the cortical cytoskeleton [18]. Similarly,
cytoplasmic aggregation and actin reorganisation occur
in the plant cell below hyphae or beneath appressoria, the
tips of infectious hyphae, when a plasma membrane is
disrupted after fungal penetration [19]. Also, mechanical
stimulation applied by gently touching the surface of a
plant cell with a microneedle induces subcellular reorganisations such as actin microfilaments and peroxisomes,
which resemble those that occur during the resealing
process [20]. Finally, the most compelling evidence for
plasma membrane resealing in plants is provided by
recent molecular and biochemical data from Arabidopsis,
indicating that Synaptotagmin 1 (SYT1) has an essential
role in plasma membrane repair [21,22], as we discuss
below.
Synaptotagmin 1: the first component identified in
plasma membrane repair in plants
Synaptotagmins are membrane-trafficking proteins first
identified in synaptic vesicles [23] and defined by a specific
domain structure (Box 1). Extensive studies have been
carried out on animal synaptotagmins, such as synaptotagmin I (Syt I) and synaptotagmin VII (Syt VII), which
have been identified as the Ca2+ sensors for fast and
synchronous neurotransmitter release at synapses
[24,25] and plasma membrane repair by the exocytosis
of lysosomes [26], respectively. Synaptotagmins constitute
a family of 16 members in the mammalian genome, some of
which have been shown to function as Ca2+ sensors in
diverse membrane-vesicle fusion processes [23]. Consistent with the important in vivo roles of Syt I and Syt VII,
they are the only mammalian synaptotagmins (together
with Syt IV) that are evolutionarily conserved in Caenorhabditis elegans and Drosophila [27].
Box 1. Synaptotagmins and dysferlin proteins
Synaptotagmin 1, the prototypical mammalian synaptotagmin was
identified in a proteomic screen for synaptic proteins and is required
for neurotransmitter release [86]. After the characterisation of this
protein, synaptotagmins have been classified as proteins with
similar domain architecture (i.e. having a short sequence preceding
an N-terminal transmembrane region, a central linker and two Cterminal C2 domains) [23]. By analogy to the role of synaptotagmin
1 in neurotransmission, the other synaptotagmins might act as Ca2+
transducers, regulating other Ca2+-dependent membrane processes,
such as plasma membrane repair by Syt VII [26] and insulin
secretion in pancreatic b-cells by Syt IX [87]. Genes with a similar
domain architecture as well as sequence similarity to synaptotagmin C2 domains have also been found in plant genomes [88].
Analysis of these gene families revealed abundant and complex
variation in synaptotagmin gene expression and indicate the
presence of synaptotagmin genes in all animal and land plants
analysed [89].
The dysferlin gene encodes a 230-kDa protein containing seven C2
domains. Mutation in this gene causes a hereditary disease
consisting of two important genetic disorders, Miyoshi myopathy
[90] and limb-girdle muscular dystrophy type 2B [91]. Immunohistochemical studies showed that, in skeletal muscle, dysferlin is
mainly located at the plasma membrane and accumulates at the site
of damage [39]. Dysferlin is a type II transmembrane protein with a
membrane topology suggesting that it anchors to the plasma
membrane by its C-terminal transmembrane domain, whereas the
N-terminal part of the protein resides in the cytoplasm.
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SYT1, the first plant synaptotagmin characterised to
date, belongs to a five-member family in Arabidopsis. The
first evidence of its role in abiotic stress responses came
after its identification as a cold-responsive plasma membrane protein [28]. It is known that freezing causes irreversible damage in the plasma membrane [29] and that
cold acclimation enhances the freezing tolerance of the
plant, minimising the occurrence of freeze-induced plasma
membrane lesions [30]. These results led to further investigations demonstrating that freezing tolerance involved a
Ca2+-dependent membrane resealing process that depends
on SYT1 [22]. An independent study identified SYT1 from
a genetic screen for Arabidopsis mutants showing hypersensitivity to salt stress [21]. The authors of these studies
[21,22] concluded that SYT1 is essential for plasma membrane integrity under abiotic stresses such as salt, osmotic
stress and freezing. All of these reports highlight SYT1 as a
protein involved in a mechanism that protects plasma
membrane integrity, particularly under stressful conditions. Moreover, the fact that SYT1 has functional phospholipid and Ca2+-binding domains known as C2 domains
(Box 2) together with its preferential plasma membrane
localisation, suggest that the repair mechanism mediated
by SYT1 involves a Ca2+-mediated fusion of vesicles to the
plasma membrane [21]. The ubiquitous expression of
SYT1, together with the reduced plasma membrane integrity observed in different tissues of the syt1 mutant, indicated that SYT-mediated plasma membrane resealing
must be maintained in most cells [21].
Other components that might be involved in plasma
membrane repair in plants
The endomembrane system and many regulatory and
structural proteins involved in membrane trafc are well
conserved between plants and animals [31–34]. One
example is the identification of SYT1 as a constituent of
the resealing machinery. This functional conservation is
expected to extend to other components of the plasma
membrane repair machinery identified in animals that
contain plant homologues. These proteins include soluble
N-ethymaleimide-sensitive factor attachment protein
receptors (SNAREs) and annexins, whereas other components involved in animal plasma membrane repair, such
caveolin 3 and calpain 3, do not have clear homologues in
plants [3,18].
Box 2. C2 domains
The C2 domain is a Ca2+-binding motif of 130 residues in length,
originally identified in the Ca2+-dependent isoforms of protein
kinase C [92]. Single and multiple copies of C2 domains have been
described in an increasing number of eukaryotic signalling proteins
that interact with cellular membranes and mediate a broad array of
crucial intracellular processes, such as insulin secretion and
neurotransmitter release [93–95]. As a group, C2 domains display
the remarkable property of binding a variety of different ligands and
substrates, including Ca2+, phospholipids, inositol polyphosphates
and intracellular proteins. Effective activation of the C2 domain by
intracellular Ca2+ signals requires high Ca2+ selectivity to exclude the
prevalent physiological metal ions K+, Na+ and Mg2+. Expanding this
functional diversity is the finding that, as is the case for the SYT1C2B domain, not all C2 domains are regulated by Ca2+ [96].
Therefore, some C2 domains might have a purely structural role.
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SNAREs
The involvement of SNAREs, a family of transmembrane
proteins essential in intracellular membrane fusion
might be anticipated, not only because these proteins
constitute the core exocytosis machinery [35,36], but also
because Syt VII [37] and otoferlin, a close dysferlin
homologue [38] interact functionally with SNARE
proteins in animal cells. Dysferlin is a C2-containing
protein that has been shown to be required for plasma
membrane repair (see Box 1) [39,40]. The essential role of
SNAREs in membrane fusion has attracted much attention and many reviews on these proteins in plants are
available [33,36,41,42].
In brief, complementary subsets of SNAREs are found
at both vesicle (vSNARE) and target membranes
(tSNARE) that pair to form a tetrameric bundle of coiled
helices that draws the membrane surfaces together for
docking and fusion. To confirm the role of SNARE proteins
in vesicle fusion, two different pharmacokinetic
approaches have been used in animals and have recently
been applied to plants [43,44]. The first uses Clostridium
botulinum neurotoxins, compounds that act as endopeptidases to cleave selectively SNARE proteins, causing the
blocking of vesicle fusion [45–48]. The second strategy uses
dominant negative inhibitors complementary to a selected
SNARE [43,49]. Using this approach, an ectopic expression
of a SNARE fragment produces the binding to the protein
partners, thereby preventing SNARE interactions and
vesicle fusion [42]. However, this approach can result in
non-specific binding to many SNARE complexes, making
difficult the identification of the specific SNAREs involved
in plasma membrane repair [50,51].
There are several approaches that can be used to
identify those SNAREs involved in plasma membrane
repair in plants. Syt VII interacts in vitro with the
Ca2+-triggered SNARE complex formed by VAMP7,
SNAP-23 and syntaxin 4 [37]. Therefore, obvious SNARE
candidates in plants involved in plasma membrane
repair could be their closest plant homologues. However,
this is doubtful because of both the functional diversification between the SNAREs of plants and animals
and the different membrane localisation of SYT1 and
Syt VII.
Alternative strategies would be to perform systematic
analysis of SNARE mutants and identify which one of
these show defective plasma membrane resealing.
However, this could be hampered if SNARE proteins
operate redundantly, in particular in secretory pathways, as has been shown for VAMP721 and VAMP722
[52]. SNAREs involved in plasma membrane repair are
expected to interact with SYT1 and therefore they could
be identified using the various protein–protein interaction techniques already available, such as yeast twohybrid, fluorescence resonance energy transfer (FRET)
or immunoprecipitation [53]. An important advantage
that will enable identification of the vSNAREs involved
in plasma membrane repair is that, once their membrane localisation is established, it would provide valuable information about the vesicle pool responsible for
resealing in plants, an aspect that still is controversial in
animals [18,54,55].
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Annexins
Other proteins that are essential in plasma membrane
resealing in animal cells are the annexins [56,57]. These
are widely expressed Ca2+- and phospholipid-binding
proteins that are implicated in membrane trafficking,
transmembrane channel activity, inhibition of phospholipase A2 and cell–matrix interactions [58,59]. The annexin
domain responsible for Ca2+ and phospholipid binding is
not homologous to C2 domains and binding occurs via
formation of a ternary complex between annexin, Ca2+
and the membranes [60]. Although many of their functions
are still unknown, annexins A1 and A2 have been shown to
aggregate intracellular vesicles and lipid rafts in a Ca2+dependent manner at the cytosolic surface of the plasma
membrane [61,62]. The first insight pointing to a role in
plasma membrane repair for animal annexins A1 and A2
came with the observation that their expression was coregulated in mice affected with limb-girdle muscular dystrophy caused by a mutation in the dysferlin gene [57].
Further experiments confirmed that dysferlin is enriched
in membrane patches near the disruption site and associates with both annexins A1 and A2 in a Ca2+- dependent
and membrane injury-dependent manner [57]. Functional
demonstration of a role for annexin A1 in plasma membrane repair came with the resealing inability of cell
cultures with the use of annexin A1 antibodies, a peptide
competitor, and a dominant-negative annexin A1 mutant
protein incapable of Ca2+ binding [56].
Despite some structural differences, proteins with
similar characteristics to animal annexins have been
identified in plants [63,64]. The annexin protein family
in Arabidopsis consists of seven or eight members [63] and,
similar to animal annexins, their primary physiological
roles are still poorly understood. Based on their biochemical properties and what is known in animals, they
have been implicated in plants in processes related to Ca2+regulated membrane dynamics, such as membrane organisation and trafficking, interactions with the cytoskeleton,
and secretion [63,64]. Despite their involvement in regulating Ca2+ signalling, such as the activation of Ca2+permeable non-selective cation channels [65], the broadest
consensus about the physiological role of plant annexins is
their implication in stress tolerance. Not only is there
stress-regulated expression of annexin genes [64,66], but
an increasing number of functional studies have been
performed demonstrating their role on stress tolerance
[67–69]. Interestingly, among the possible functions
assigned to plant annexins, a role in plasma membrane
resealing has not been previously proposed, despite the
functional demonstration of a role in plasma membrane
repair in animal cells. One possible complication determining whether a particular annexin is involved in resealing
could be a functional redundancy owing to the high degree
of homology among these proteins [63,64]. However, it is
conceivable that, as occurs in the case of SYT1, this function could be primarily performed by a particular isoform, a
possibility that requires further research.
Caveolin 3 and calpain 3
Caveolin 3 and calpain 3 are muscle-specific proteins
required for plasma membrane repair whose mutations
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cause two different forms of muscular dystrophy (LGMD1C
and LGMD2A, respectively) [70]. Caveolin 3 does not have
a homologue in plants and its function in plasma membrane repair in animals is not clear. However, one recent
study indicates that caveolin 3 is important for retaining
dysferlin at the plasma membrane, and is therefore likely
to have an indirect role in plasma membrane repair [71].
During the transport of vesicles for fusion to the disruption
site, local dissolution of the filamentous actin is mediated
by the Ca2+-activated calpain 3, a cysteine protease. This is
necessary for the removal of the physical barrier resulting
from the presence of actin filaments in the pathway of
fusion events [18]. The importance of the cytoskeleton has
been previously highlighted in this article and is expected
to have an important role in plasma repair events also in
plant. A search for Arabidopsis proteins similar to calpain
3 only identified one sequence that shares significant
homology. The gene identified (At1g55350) encodes the
unique plant-specific calpain-like protein DEK1 (defective
kernel 1) that is essential for the correct development of the
embryo [72]. Recently, it was shown that, although fulllength DEK1 protein localises to membranes, it undergoes
intramolecular autolytic cleavage that releases the calpain
domain into the cytoplasm [73]. What is intriguing about
this result is the association of the cleaved DEK-calpain
with membranes, suggesting a phospholipid-binding
activity for this protein. Therefore, it would be interesting
to investigate whether DEK1, in addition to embryo development, functions in plasma membrane resealing
through modification of the actin cytoskeleton.
A model for plasma membrane repair in plants
To explain the role of Ca2+-triggered exocytosis in membrane resealing, two mechanistic models have been proposed [2]. The first of these proposes that Ca2+ influx
through wounds in the plasma membrane triggers homotypic fusion of intracellular vesicles, leading to a reparative
‘patch’ that then fuses with the plasma membrane surrounding the injured site. This model, known as the ‘patch
hypothesis’, is suggested to be mainly responsible for the
repair of large lesions. The second model suggests that the
primary role of exocytosis, instead of forming a patch over
the lesion, is to reduce plasma membrane tension. This
process could promote membrane resealing by facilitating
spontaneous bilayer resealing and so would preferentially
repair small wounds.
In both cases, Syt VII has been proposed to be the sensor
for Ca2+-dependent vesicle exocytosis. In contrast to the
vesicle (lysosomal) localisation of Syt VII [26], SYT1 is
mainly localised at the plasma membrane, which indicates
mechanistic differences in plasma membrane resealing
between animals and plants. However, Arabidopsis
SYT1 is not the only plasma membrane-localised synaptotagmin involved in membrane repair, as the yeast Tcb3,
which also shows plasma membrane localisation, has a role
in membrane resealing during the mating process [74].
Another C2-containing protein localised in the plasma
membrane involved in resealing is dysferlin (Box 1). This
is a large type II transmembrane protein with a predicted
intracellular portion composed of seven C2 domains and
nested repeat sequences termed DysfN and DysfC, of
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unknown function [75]. It belongs to a new family of
mammalian proteins, named ‘ferlin-1’-like proteins, predicted on the basis of structural similarity and sequence
homology [70]. Loss of dysferlin impedes Ca2+-dependent
plasma membrane repair in skeletal muscle, causing three
clinically distinct muscular dystrophies [70].
C2 domains are independent autonomously-folded
protein modules responsible for promoting Ca2+-dependent interactions with membrane phospholipids and
SNARE proteins (Box 2) [23], events that precede membrane fusion. Biochemical studies of SYT1 C2 domains
show differential phospholipid binding properties between C2A and C2B. Whereas SYT1-C2A is capable of
binding phospholipids in a Ca2+-dependent manner, the
C2B domain exhibits phospholipid binding in vitro that is
independent of Ca2+ [21]. Thus, given the biochemical
properties of the C2 domains of SYT1 and the preferential
plasma membrane localisation of this protein, it is feasible
that SYT1 is able to dock vesicles at the plasma membrane
via its C2B domain even under the low Ca2+ concentrations normally present in the cytosol. Damage to the
plasma membrane causes an influx of extracellular Ca2+,
which in turn would result in an intracellular rise of
cytosolic Ca2+ and fusion of the docked vesicles, a process
mediated by the Ca2+-dependent activity of the C2A
domain. Ca2+-activated exocytosis could function in
plasma membrane repair either by reducing the membrane tension through an increase of the membrane surface or by creating a patch that directly reseals the
membrane (Figure 1a and b). A docking mechanism has
also been proposed for the control of fast synaptic vesicle
exocytosis [76]. Interestingly, it has been recently shown
that, as in SYT1, only the C2A domain of dysferlin has
Ca2+-dependent phospholipid binding activity, whereas
the remainder of the C2 domains exhibit weaker and
Ca2+-independent binding [77]. Dysferlin homologues
have not been identified in plants. Therefore, despite
the structural differences between SYT1 and dysferlin,
both proteins could function similarly in plasma membrane repair, indicating that these models could also be
valid for plasma membrane repair in animals.
Recently, it has been proposed that endocytosis has a
role in animal plasma membrane repair, based on the
capacity of animal cells to repair rapidly lesions created
by pore-forming proteins [78]. This is supported by the
dual role of Syt I and Syt VII in simultaneously coordinating Ca2+-triggered exocytosis of intracellular vesicles
and recruiting protein complexes to promote their own
endocytosis [79]. The C2B domain of Syt I contains
binding sites for endocytic proteins, such as the clathrin
adapter protein (AP-2) [80,81] and stonin 2 [82]. Therefore, it is also possible that SYT1 has a role in endocytosis in plants, a mechanism not mutually exclusive to
that previously proposed. In this scenario, the C2B-driven constitutive lipid-binding activity of SYT1 would
cause the binding of the protein to the plasma membrane
(e.g. the cis membrane to which the protein is anchored
via its transmembrane domain). Then, the interaction of
the C2 domains of SYT1 with endocytic proteins would
promote endocytosis and plasma membrane repair
(Figure 1c).
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Figure 1. Schematic models showing SYT1-mediated plasma membrane repair. (a) Tension reduction model. (i) Intact plasma membrane showing docked vesicles
resulting from the Ca2+-independent vesicle binding of the SYT1-C2B domain. (ii) Immediately after plasma membrane damage, extracellular Ca2+ enters the cell. The local
increase in Ca2+ activates the SYT1-C2A domain, which, after binding to SNAREs, triggers vesicle–plasma-membrane fusion. (iii) This Ca2+-dependent fusion of vesicles
brings about reduction in membrane tension, thus promoting resealing. (b) The patch model. (i) Intact plasma membrane showing docked vesicles interacting with the
SYT1-C2B domain. (ii) After the damage, Ca2+ enters through the lesion. This local increase in Ca2+ triggers vesicle-vesicle homotypic fusion, forming a patch vesicle
through the activation of annexins. (iii) SYT1/SNARE-mediated fusion of the patch vesicle with the plasma membrane seals the damage. (c) Endocytic model. (i) damage to
the plasma membrane creates a local increase in intracellular Ca2+. (ii) This is rapidly followed by SYT1-mediated endocytosis by recruitment of clathrin, which internalises
the lesions. (iii) Plasma membrane repair is accomplished after the endocytosis occurs.
Concluding remarks
The intricate mechanisms which higher plants have
evolved to cope with adverse environmental conditions
have been widely studied and characterised [83–85]. However, the molecular mechanisms by which the plasma
membrane of a plant cell is able to recover after an injury
have not been extensively studied, perhaps because of the
assumption that the resealing is a purely physical phenomenon and not the result of a complex and highly regulated
process. Although recent studies have highlighted the
significance of plasma membrane integrity in cell viability
in plants, further characterisation of the process is needed.
By contrast, extensive work has been done in animals
largely because of the pathological implications that a
failure in this process has in mammals. This has led to
the development of many biochemical and molecular techniques with the aim of identifying new components and to
investigate the processes leading to plasma membrane
repair. These data can be used by the plant research
community to obtain valuable information about the
resealing process in plants. The recent identification of
SYT1 in Arabidopsis has opened a new avenue that is
likely to help in the identification of additional components
involved in plasma membrane resealing in plants. It is
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envisaged that some of these proteins might have a role in
other processes involving Ca2+-regulated vesicular trafficking such as pollen tube or root hair growth. The identification of plasma membrane repair in plants has
prompted several outstanding questions that require
further research (see Outstanding questions). However
new methodology, including controlled mechanical damage
and continuous monitoring of this damage, need first to be
established in plant cells. Understanding responses to
plasma membrane damage will provide new insights in
plant resistance to biotic and abiotic stresses, which in turn
might lead to an improvement in plant stress tolerance.
Outstanding questions
How important is the maintenance of SYT1-regulated plasma
membrane resealing during plant growth in the field? This would
provide information on whether plasma membrane repair in
plants is important for plant fitness in the field where they are not
growing under optimal conditions.
Is SYT1 involved in the regulation of Ca2+-dependent exocytosis,
endocytosis or both? This is important to determine the mechanism of plasma membrane repair in plants.
Does SYT1 accumulate on the membrane disruption sites similar
to other plasma membrane resealing components described in
animals? Dysferlin and other proteins having a direct role in
membrane repair accumulate at the sites of damage [39]. The
localisation of SYT1 in damage sites would provide further
evidence and provide insights on its role in plasma membrane
repair.
What other components are involved in plasma membrane
repair? Are other C2-containing proteins involved? C2-containing
proteins are highly abundant in plants. It is possible that other
proteins with C2 domains such as SYT1 homologues also have a
role in this process.
What plant proteins involved in exocytosis bind to SYT1? The
identification of these interacting proteins would likely provide
new components of the plasma membrane repair machinery in
plants.
What is the role of the plant cytoskeleton in plasma membrane
repair? In animals cells the cytoskeleton has an essential role in
membrane repair [18,97,98]. Considering the importance of the
cytoskeleton in membrane dynamics we anticipate an essential
role in plasma membrane repair in plants.
What is the nature of the cytoplasmic vesicles involved in plasma
membrane resealing in plants? The identification of the pool of
vesicles that are involved in plasma membrane repair in plants
could provide important information about this process.
Is SYT1 involved in additional Ca2+-dependent trafficking processes? Because other processes in plants require Ca2+-dependent
exocytosis it is possible that SYT1 has additional as yet unknown
roles.
Acknowledgements
Work in the authors’ laboratory was supported by grants from El
Ministerio de Ciencia e Innovación (Grant BIO2008-01709, cofinanced by
the European Regional Development Fund) and Consejerı́a de Innovación
Ciencia y Empresa - La Junta de Andalucı́a (Grant P07-CVI-03021).
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