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Calcium (Ca(2+)) plays essential roles in generative reproduction of angiosperms, but the sites and mechanisms of Ca(2+) storage and mobilization during pollen-pistil interactions have not been fully defined. Both external and internal Ca(2+) stores are likely important during male gametophyte communication with the sporophytic and gametophytic cells within the pistil. Given that calreticulin (CRT), a Ca(2+)-buffering protein, is able to bind Ca(2+) reversibly, it can serve as a mobile store of easily releasable Ca(2+) (so called an exchangeable Ca(2+)) in eukaryotic cells. CRT has typical endoplasmic reticulum (ER) targeting and retention signals and resides primarily in the ER. However, localization of this protein outside the ER has also been revealed in both animal and plant cells, including Golgi/dictyosomes, nucleus, plasma membrane/cell surface, plasmodesmata, and even extracellular matrix. These findings indicate that CRT may function in a variety of different cell compartments and specialized structures. We have recently shown that CRT is highly expressed and accumulated in the ER of plant cells involved in pollen-pistil interactions in Petunia, and we proposed an essential role for CRT in intracellular Ca(2+) storage and mobilization during the key reproductive events. Here, we demonstrate that both CRT and exchangeable Ca(2+) are localized in the intra/extracellular peripheries of highly specialized plant cells, such as the pistil transmitting tract cells, pollen tubes, nucellus cells surrounding the embryo sac, and synergids. Based on our present results, we propose that extracellularly located CRT is also involved in Ca(2+) storage and mobilization during sexual reproduction of angiosperms.
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ORIGINAL ARTICLE
Calreticulin localizes to plant intra/extracellular peripheries
of highly specialized cells involved in pollen-pistil interactions
Piotr Wasąg
1
&Anna Suwińska
2
&Przemysław Zakrzewski
2
&Jakub Walczewski
3
&
Robert Lenartowski
1
&Marta Lenartowska
2
Received: 21 March 2017 /Accepted: 5 June 2017 / Published online: 15 June 2017
#The Author(s) 2017. This article is an open access publication
Abstract Calcium (Ca
2+
) plays essential roles in generative
reproduction of angiosperms, but the sites and mechanisms of
Ca
2+
storage and mobilization during pollen-pistil interactions
have not been fully defined. Both external and internal Ca
2+
stores are likely important during male gametophyte commu-
nication with the sporophytic and gametophytic cells within
the pistil. Given that calreticulin (CRT), a Ca
2+
-buffering pro-
tein, is able to bind Ca
2+
reversibly, it can serve as a mobile
store of easily releasable Ca
2+
(so called an exchangeable
Ca
2+
) in eukaryotic cells. CRT has typical endoplasmic retic-
ulum (ER) targeting and retention signals and resides primar-
ily in the ER. However, localization of this protein outside the
ER has also been revealed in both animal and plant cells,
including Golgi/dictyosomes, nucleus, plasma membrane/
cell surface, plasmodesmata, and even extracellular matrix.
These findings indicate that CRT may function in a variety
of different cell compartments and specialized structures. We
have recently shown that CRT is highly expressed and accu-
mulated in the ER of plant cells involved in pollen-pistil
interactions in Petunia, and we proposed an essential role
for CRT in intracellular Ca
2+
storage and mobilization during
the key reproductive events. Here, we demonstrate that both
CRT and exchangeable Ca
2+
are localized in the intra/
extracellular peripheries of highly specialized plant cells, such
as the pistil transmitting tract cells, pollen tubes, nucellus cells
surrounding the embryo sac, and synergids. Based on our
present results, we propose that extracellularly located CRT
is also involved in Ca
2+
storage and mobilization during sex-
ual reproduction of angiosperms.
Keywords Cell wall .Exchangeable Ca
2+
.Filiform
apparatus .Plasmodesmata .Pollen tube .Style transmitting
tissue
Abbreviations
Ca
2+
Calcium/calcium ions
Ca
2+
ppts Ca
2+
-antimonate precipitates
Cal MAb Monoclonal antibody against callose
CRT Calreticulin
CRT PAb Polyclonal antibody against plant CRT
ER Endoplasmic reticulum
Introduction
CRT is a Ca
2+
-binding/buffering protein implicated in many
cellular functions, including lectin-like chaperoning, Ca
2+
storage and signaling, regulation of gene expression, cell ad-
hesion, regeneration, immunity, and apoptosis (see reviews by
Michalak et al. 2009; Jia et al. 2009;Thelinetal.2011). This
multifunctional protein promotes folding and quality control
of newly synthesized glycoproteins in the ER via the CRT/
calnexin cycle. CRT is also involved in ER Ca
2+
capacity and
P. Wa s ąg and A. Suwińska contributed equally to this work
Handling Editor: Liwen Jiang
*Marta Lenartowska
mlenart@umk.pl
1
Laboratory of Isotope and Instrumental Analysis, Faculty of Biology
and Environmental Protection, Nicolaus Copernicus University in
Tor uń, Toruń,Poland
2
Laboratory of Developmental Biology, Faculty of Biology and
Environmental Protection, Nicolaus Copernicus University in Toruń,
Tor uń, Poland
3
Department of Plant Pathology, Plant Breeding and Acclimatization
Institute, National Research Institute, Radzików, Poland
Protoplasma (2018) 255:5767
DOI 10.1007/s00709-017-1134-8
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
thus stabilization of Ca
2+
homoeostasis in the cell cytoplasm.
Since CRT has typical ER targeting and retention signals, it
resides primarily in the ER lumen. However, localization of
this protein outside the ER has also been observed in eukary-
otic cells. For example in animals, CRT was detected in the
nucleus, nuclear matrix, on the surface of mitotic chromo-
somes, and in the extracellular matrix (see review by
Michalak et al. 2009). Plant CRT was found in many different
compartments and structures, including dictyosomes/vesicles
(Borisjuk et al. 1998; Navazio et al. 2002; Lenartowska et al.
2002,2009; Hsieh and Huang 2005; Nardi et al. 2006;
Lenartowski et al. 2015;Niedojadłoetal.2015), the cytosol
(Lenartowska et al. 2002; Jia et al. 2008), protein bodies
(Torres et al. 2001;Šamaj et al. 2008), nucleus (Denecke
et al. 1995;Napieretal.1995; Lenartowska et al. 2002;
Lenartowski et al. 2015), plasma membrane/cell surface
(Borisjuk et al. 1998; Lenartowska et al. 2002; Navazio
et al. 2002;Šamaj et al. 2008), plasmodesmata (Baluška
et al. 1999; Laporte et al. 2003; Bayer et al. 2004;Chen
et al. 2005; Lenartowska et al. 2009; Bilska and Sowiński
2010; Christensen et al. 2010), and the cell wall
(Lenartowska et al. 2002,2009; Lenartowski et al. 2015;
Luczak et al. 2015; Niedojadłoetal.2015). These varied
locations suggest that CRT may function in different plant cell
compartments and specialized structures, including extracel-
lular regions.
Sexual reproduction of angiosperms involves complicated
pollen-pistil interactions during directional growth of pollen
tubes through the pistil transmitting tract, pollen tube entry to
the ovule, and then the male and female gametophytes com-
munication. Ca
2+
haslongbeenrecognizedtoplayessential
signaling, physiological, and regulatory roles in these repro-
ductive events (see reviews by Ge et al. 2007; Dresselhaus and
Franklin-Tong 2013; Steinhorst and Kudla 2013). Given that
CRT is able to bind Ca
2+
reversibly (exchangeable Ca
2+
), its
Ca
2+
-buffering activity has the potential to be involved in
modulation of Ca
2+
concentrations in the ER and consequent-
ly in the cytosol. Recent work from our lab provided evidence
that CRT is highly expressed and accumulated in the ER of
Petunia cells during pistil transmitting tract maturation, the
progamic phase and early embryogenesis (Lenartowski et al.
2014,2015). Since the ER cisternae and Golgi stacks are
known to be the effective Ca
2+
stores in eukaryotic cells (see
review by Vandecaetsbeek et al. 2011), we proposed an essen-
tial role for CRT in intracellular Ca
2+
storage and mobilization
during these key reproductive events (Lenartowski et al. 2014,
2015). We have also found that CRT is located on the cell
membrane/surface and in the apoplast of highly specialized
plant cells involved in pollen-pistil interactions
(Lenartowska et al. 2002,2009; Lenartowski et al. 2015).
Localization of CRT outside the protoplast in different plant
cells has been also confirmed by other authors (Borisjuk et al.
1998; Navazio et al. 2002;Šamaj et al. 2008;Luczaketal.
2015;Niedojadłoetal.2015). Since both internal and exter-
nal Ca
2+
stores are likely important during communication
of the male gametophyte and the female sporophyte/
gametophyte cells (see reviews by Ge et al. 2007;
Dresselhaus and Franklin-Tong 2013; Steinhorst and
Kudla 2013), in this report we focus on CRT located in
intra/extracellular peripheries in the context of its probable
role/s in mobile Ca
2+
storage during pollen-pistil interac-
tions in angiosperms.
Materials and methods
Plant material
Commercial cultivars of Petunia hybrida and Haemanthus
albiflos were grown at room temperature, and whole pistils
were dissected from unpollinated and pollinated flowers.
Semithin sections of styles (Petunia)andovules(Petunia
and Haemanthus) were prepared according to standard proto-
cols, stained with 0.1% methylene blue and observed by light
microscopy. Then, selected tissue samples of styles and ovules
were prepared for immunocytochemical and cytochemical
(potassium antimonate precipitation) studies according to the
protocols as described below. All experiments were repeated
many times during several growing seasons with similar
results.
Immunocytochemical studies
Immunofluorescence and immunogold localizations of CRT
were performed according to protocols described previously
(Lenartowska et al. 2009; Lenartowski et al. 2015). In brief,
samples of styles and ovules were fixed with 4% (v/v)form-
aldehyde and 0.25% (v/v) glutaraldehyde in phosphate-
buffered saline (PBS, pH 7.2) for 1 h at room temperature
(slight vacuum infiltration) followed by overnight fixation at
4 °C. Fixed samples were dehydrated in graduated ethanol
concentrations, embedded in LR Gold resin (Fluka) according
to the standard protocol, and then semithin and ultrathin lon-
gitudinal or cross-sections were collected on microscope
slides covered with Biobond (BBInternational) or Formvar-
coated nickel grids. After blocking in 35% bovine serum
albumin (Sigma-Aldrich), sections were incubated with a rab-
bit polyclonal antibody against maize CRT (CRT PAb, Napier
et al. 1995) and then with goat anti-rabbit IgG Cy3
®
second-
ary antibody (Sigma-Aldrich) or with 1020 nm diameter
gold-conjugated goat anti-rabbit IgG antibody
(BBInternational). As a final step, DNA was stained with
2μg/ml 4, 6-diamidino2-phenylindole (DAPI, Fluka). In
the controls, incubations with the CRT PAb were omitted.
Images were acquired using an Olympus BX50 fluorescence
microscope, Olympus Xc50 digital color camera, and CellB
58 Lenartowska et al.
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
software (Olympus Soft Imaging Solutions gmbH); ultrathin
sections were examined by transmission electron microscopy
(Jeol EM 1010) at 80 kV.
For CRT and callose immunolocalizations, a double-
labeling technique was performed as previously described
(Lenartowska et al. 2009). After blocking with 5% BSA
(Sigma-Aldrich), ultrathin sections were treated with two
kinds of primary antibodies: CRT PAb and monoclonal
anti-(1 3)-β-glucan antibody (Cal MAb, Biosupplies).
Signals were detected with the following secondary anti-
bodies: 20 nm diameter gold-conjugated goat anti-rabbit
IgG (for CRT) and 10 nm diameter gold-conjugated goat
anti-mouse IgG (for callose), both from BBInternational.
To quantitate the amount of CRT localized intra/
extracellularly in different Petunia cells involved in
pollen-pistil interactions, the average number of gold
traces was determined in different cell sections of
compartments/structures (min. 20) labeled with CRT PAb
conjugated with immunogold secondary antibody. In the
negative control, incubation with the primary antibodies
was omitted. To verify if the CRT PAb specifically bound
to protein epitopes, the immunolocalizaton was performed
on ultrathin sections pretreated by incubation with a pro-
teinase K solution (Lenartowska et al. 2002). Finally, the
sections were stained with 2.5% (w/v) uranyl acetate and
0.4% (w/v) lead citrate solutions and examined by trans-
mission electron microscopy as above. The specificity of
maize CRT PAb in Petunia and Haemanthus was previous-
ly verified by immunoblotting (Lenartowska et al. 2009;
Lenartowskietal.2015).
Visualization of loosely bound Ca
2+
by potassium
antimonate precipitation
Localization of exchangeable Ca
2+
was performed according
to the protocol described previously (Lenartowska et al. 2009;
Lenartowski et al. 2015). In brief, samples of styles and ovules
dissected from unpollinated/pollinated pistils were fixed with
freshly prepared 2% (w/v) potassium antimonate, 2% (v/v)
glutaraldehyde, and 2% (v/v) formaldehyde in 0.1 M phos-
phate buffer (KH
2
PO
4
, pH 7.8) for 4 h at room temperature,
and then subsequently postfixed with 1% (v/v) osmium tetrox-
ide (OsO
4
) in the same buffer-antimonate solution for 12 h at
4 °C. Next, samples were dehydrated in graduated ethanol
concentrations and embedded in Poly/Bed 812 resin
(Polysciences) according to the standard protocol. Ultrathin
longitudinal or cross-sections were collected on copper grids,
stained with 2.5% (w/v)uranylacetateand0.4%(w/v)lead
citrate solutions, and examined by transmission electron mi-
croscopy as above. The presence of Ca
2+
in the precipitates
was confirmed previously using energy-dispersive X-ray mi-
croanalysis (Bednarska et al. 2005).
Results
CRT is present in intra/extracellular peripheries
of the stylar transmitting tissue and pollen tubes
We first wished to determine if CRT is extracellularly local-
ized in the stylar transmitting tract linking the stigma with the
ovary. To investigate this, samples of unpollinated and polli-
nated Petunia styles were processed for immunogold labeling
and visualized by electron microscopy. As shown in semithin
sections stained with methylene blue, Petunia has a solid style
with highly specialized transmitting tissue composed of secre-
tory cells (Fig. 1a, b). The extracellular matrix of this tissue is
enriched with exudates and formsthe appropriate physical and
nutritional medium for pollen tube growth in vivo (Fig. 1c).
Within the cytoplasm of transmitting cells, CRT was typi-
cally localized in the ER, both in unpollinated and pollinated
pistils (Fig. 1d, e, respectively). However, before pollination,
numerous gold traces were also detected along the edge of
these cells, on the border between the protoplast and the cell
wall (Fig. 1d, arrows). After pollination, CRT was frequently
observed at the cellular peripheries (Fig. 1f, arrows) and ac-
cumulated in the plasma membrane-attached patches (Fig. 1f).
Consistent with CRT being a Ca
2+
-binding/buffering protein,
Ca
2+
-antimonate precipitates (Ca
2+
ppts corresponding to ex-
changeable Ca
2+
) were observed in the same localizations
where CRT was found; there were the ER (Fig. 1g) and several
patches adjacent to the cell wall of the transmitting cells
(Fig. 1h). It should be noted that Ca
2+
ppts were predominant-
ly observed in the ER enriched peripheral cytoplasm (Fig. 1g,
arrows). Epitopes binding CRT PAb were also found at plas-
modesmata connecting transmitting cells. The specific linear
pattern of the labeling is likely to correspond to ER in the
cytoplasmic sleeve - an essential component of these narrow
channels (Fig. 1i, arrow). Moreover, double-labeling experi-
ments using both CRT PAb and Cal MAb clearly showed that
CRTco-localized tightly with callose at the neck region of the
plasmodesmata (Fig. 1i, k). Numerous Ca
2+
ppts were found
in the cortical ER attached to plasmodesmata (Fig. 1j, l) as
well as with their central cavity and neck regions (Fig. 1j, l,
arrows).
After pollination, CRT labeling was confirmed in pollen
tubes growing between the transmitting cells. Intracellularly,
the protein was localized to the most prominent organelles in
the pollen tube subapical zone, such as dictyosomes (Fig. 2a)
and ER (Fig. 2b). Some gold traces were also found in the
peripheral cytoplasm of the tube adjacent to the cell wall
(Fig. 2a). Such CRT labeling often corresponded with the
position of the ER within the cytoplasm (Fig. 2b). However,
epitopes binding CRT PAb were also identified in the inner
cell wall of the pollen tube (Fig. 2b). Double staining demon-
strated that, similar to plasmodesmata, peripheral and extra-
cellular CRT localizations in elongated pollen tubes were
Calreticulin localizes to plant intra/extracellular peripheries 59
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tightly correlated with callose deposition (Fig. 2c) and plug
formation in the shank of highly elongated tube (Fig. 2d). It
should be noted that CRT labeling in the inner callose wall
was predominantly associated with several vesicles containing
electron-dense cores; they were present in both the tube cyto-
plasm and the callose depositions (Fig. 2c, d, arrows). In con-
trast, the fibrillar outer cell wall of the pollen tube was devoid
of CRT PAb and Cal MAb labeling (Fig. 2ad). As we ex-
pected, numerous Ca
2+
ppts were found in cellular peripheries
and the extracellular space of elongated pollen tubes, includ-
ing vesicles undergoing exocytosis (Fig. 2e, arrows) and the
callosic cell wall of the tube (Fig. 2e, f).
Both nucellus cell peripheries and filiform apparatus
of the synergid accumulate CRT
Based on the results obtained for Petunia stylar transmitting
tract, we hypothesized that CRTwould be also a component of
the extracellular space in the ovule. To test this idea, longitu-
dinal sections through the ovules dissected from unpollinated/
pollinated Petunia and Haemanthus pistils were prepared and
processed for immunogold or immunofluorescence labeling.
We prepared ultrathin or semithin sections because of different
size of the ovulessmall in Petunia andmuchbiggerin
Haemanthus. Dissected ovules were also fixed for visualiza-
tion of exchangeable Ca
2+
. As shown in semithin sections
stained with methylene blue, the synergid cell wall forms a
highly thickened structure called the filiform apparatus at the
micropylar end, consisting of numerous finger-like projec-
tions into the cytoplasm between sister synergids (Figs. 3a
and 4a, d). Thishighly specialized structure was clearly visible
before pollination and well-preserved during the progamic
phase (Fig. 4a, d, respectively).
In Petunia ovules, the presence of CRT in intra/
extracellular peripheries has been confirmed for the nucellus
surrounding the embryo sac (Fig. 3b, d) and for the synergid
cell (3f, h). Although it was difficult to discern whether CRT
labeling in nucellus cells is limited to the protoplast/cell mem-
brane adjacent to the cell wall or occurs in the apoplast, the
localization pattern of CRT was comparable before and after
pollination (Fig. 3b, d, respectively). An extremely high level
of CRT was found in the synergid filiform apparatus where
gold traces were uniformly distributed along the electron-
dense fibrils (Fig. 3f, h). Intracellularly, CRT was found in
the ER-rich cytoplasm of this cell (Fig. 3f, i). Such intense
Fig. 1 Immunogold localization
of CRT (df,i,k) and distribution
of exchangeable Ca
2+
(g,h,j,l)in
Petunia transmitting cells. ac
Methylene blue stained cross-
sections of the pistil style showing
transmitting tissue before (a,b)
and after pollination (c). d,g,i,j
Distributions of CRT and loosely
bound Ca
2+
in transmitting cells
before pollination. e,f,h,k,l
Distributions of CRT and loosely-
bound Ca
2+
in transmitting cells
after pollination. cx cortex, d
dictyosome, ecm extracellular
matrix, er endoplasmic reticulum,
mmitochondria, pl
plasmodesmata, tt transmitting
tissue, ttc transmitting tissue cells,
pt pollen tube, vb vascular bundle.
Bars 50 μm(ac), 500 nm (d,e,g,
j,l), 200 nm (f,h,i,k)
60 Lenartowska et al.
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CRT PAb labeling of the synergid was observed both before
and after pollination. In contrast, we found clear differences in
the level, size and localization of Ca
2+
ppts in both the nucel-
lus and the synergid cells between the unpollinated and polli-
nated pistils. Before pollination, numerous but minor Ca
2+
ppts were detected in the nucellus. They were localized mainly
in the cortical ER (Fig. 3c, arrows) and dictyosomes (Fig. 3c),
while the apoplast was devoid of ppts. After pollination, Ca
2+
ppts strongly labeled the cell walls and several cell membrane-
associated patches (Fig. 3e). The labeling was also found in-
tracellularly, in dictyosomes and nuclei with associated ER
(Fig. 3e). Similar to the nucellus, we did not find accumulation
of Ca
2+
ppts within the synergid before pollination (Fig. 3g).
However, a drastic increase of exchangeable Ca
2+
was observed
in the synergid during the progamic phase (Fig. 3j, l, m).
Extracellularly, Ca
2+
ppts were evident in the filiform apparatus
Fig. 2 Immunogold localization
of CRT (ad) and visualization of
exchangeable Ca
2+
(e,f)in
Petunia pollen tubes growing in
situ. ccytoplasm, ccw callosic cell
wall, ddictyosome, e
endoplasmic reticulum, fcw
fibrillar cell wal, m
mitochondria, plug callose
plug, va vacuole. Bars 1μm(e,f),
500 nm (a,b), 200 nm (c,d)
Calreticulin localizes to plant intra/extracellular peripheries 61
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
(Fig. 3jm) along which the pollen tube elongated (Fig. 3m),
and in the cell wall separating the embryo sac from the nucellus
(Fig. 3k, arrows). Ca
2+
labeling was also prominent in the syn-
ergid cell cytoplasm, including the ER (Fig. 3jl), dictyosomes
(Fig. 3l), and the nucleus (Fig. 3k). Since the presence of CRT
in the extracellular space is somewhat controversial, the speci-
ficity of immunogold reaction was verified in two ways. First,
no background was observed when control sections were incu-
bated with only secondary gold-labeled antibodies; only single
gold traces were found in the negative control (Fig. 3n, arrow).
Second, binding of the CRT PAb to the cell wall was inhibited
when the sections were pretreated with proteinase K to digest
some protein epitopes (Fig. 3o). In this case, only Cal MAb
labeling was preserved in the inner cell wall of the pollen tube
Fig. 3 Immunogold localization of CRT (b,d,f,h,i,m) and distribution
of exchangeable Ca
2+
(c,e,g,j-m)inPetunia micropylar pole of the
ovule. aMethylene blue stained longitudinal section of the embryo sac
micropylar pole before pollination. b,c,f,g,j,kDistributions of CRT and
loosely-bound Ca
2+
in the micropylar pole of the ovule form unpollinated
pistil. d,e,h,i,l,mDistributions of CRT and looselybound Ca
2+
in the
micropylar pole of the ovule form pollinated pistil. nNegative
immunocytochemical control. oProteinase K control. pPotassium
antimonate precipitation control. ccytoplasm, ccw callosic cell wall, cc
central cell, cw cell wall, ddictyosome, ec egg cell, er endoplasmic
reticulum, fa filiform apparatus, fcw fibrillar cell wal, mmitochondria, n
nucleus, nc nucellus, pt pollen tube, sy synergid, va vacuole. Bars 25 μm
(a), 1 μm(j,k,m,p), 500 nm (c,e,fi,l), 250 nm (b,d,n,o)
62 Lenartowska et al.
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(Fig. 3o, arrowheads). Thus, presence of CRT PAb within the
pollen tube callose wall appears to be specific. The same control
results were observed in the filiform apparatus (data not
showed). Furthermore, a negative control for potassium
antimonate precipitation showed Ca
2+
ppts were absent in ana-
lyzed cells (Fig. 3p).
Finally, we investigated Haemanthus ovules processed for
immunofluorescence labeling. We confirmed presence of
CRTin the cytoplasm of nucellus cells and between these cells
both before and after pollination (Fig. 4b, e, respectively). In
the synergid, the strongest signals detected were associated
with the filiform apparatus (Fig. 4c, f), the cell wall bordering
tween synergids (Fig. 4f, arrow and double arrow), and the
cytoplasm (Fig. 4c, arrow). During the late progamic phase,
when pollen tubes penetrated the micropylar pole of the em-
bryo sac, enrichment of CRT was still observed in the filiform
apparatus (Fig. 4g) and in the cortical cytoplasm of the syner-
gid (Fig. 4g, double arrow). This cytoplasmic region was ex-
tremely rich in ER strongly labeled by the CRT PAb (Fig. 4h,
arrows). To obtain higher-resolution visualization of Ca
2+
ppts
in the synergid cell, we performed electron microscopy of
ultrathin sections prepared for potassium antimonate precipi-
tation. As we expected, a large mass of Ca
2+
ppts was ob-
served occupying the filiform apparatus (Fig. 4i) and the syn-
ergid cytoplasm (Fig. 4i, double arrow). Thus, we conclude
that similar patterns of CRT and exchangeable Ca
2+
localiza-
tions are present in the micropylar pole of the ovules dissected
from Petunia and Haemanthus ovaries.
Fig. 4 Immunofluorescence
localization of CRT (b,c,eh)in
Haemanthus micropylar pole of
the ovule and visualization of
exchangeable Ca
2+
(i)in
Haemanthus filiform apparatus of
the synergid. a,dMethylene blue
stained longitudinal section of the
embryo sac micropylar pole
before (a) and after (d)
pollination. ccytoplasm, ec egg
cell, er endoplasmic reticulum, fa
filiform apparatus, nnucleus, nc
nucellus, pt pollen tube, sy
synergid. Bars 20 μm(b,c,e,f),
10 μm(a,d,gi)
Calreticulin localizes to plant intra/extracellular peripheries 63
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Discussion
Here, we have demonstrated that both CRT and exchangeable
Ca
2+
are localized to the intra/extracellular peripheries of
highly specialized plant cells, such as transmitting cells, pollen
tubes, nucellus cells surrounding the embryo sac, and syner-
gids. Although, some previous work from our lab and others
indicated location of CRT in several cell wall patches, at the
cell surface and in the cell wall of various cell types, these
findings were somewhat controversial (Borisjuk et al. 1998;
Lenartowska et al. 2002,2009;Šamaj et al. 2008;
Lenartowski et al. 2015;Niedojadłoetal.2015). However,
recent elegant work of Luczak et al. (2015) provided clear
evidence that CRT, similar to several other proteins, is always
present in the cell walls of few plant species including maize,
Lupinus and Arabidopsis. These results prompted us to per-
form a detailed analysis of CRT and exchangeable Ca
2+
dis-
tributions in cellular peripheries and in the apoplast during
pollen-pistil interactions. Our observations were supplement-
ed by simple quantitative analysis of immunogold particles
(summarized in Table 1) on the light of exchangeable Ca
2+
distribution in investigated cells. To the best of our knowl-
edge, this is the first study focused on extracellularly located
CRTincontextitsprobableroleinmobileCa
2+
storage during
sexual reproduction in angiosperms.
CRT and exchangeable Ca
2+
are localized
to intra/extracellular peripheries in the pistil transmitting
tissue before and after pollination
We have demonstrated that CRT and exchangeable Ca
2+
are
typically located in the ER of transmitting cells, both in
unpollinated and pollinated Petunia pistils. However, numer-
ous gold traces (corresponding to CRT), as well as Ca
2+
ppts
(corresponding to exchangeable Ca
2+
potentially bound by
CRT), were also found in the cortical cytoplasm, the plasma
membrane/wall attached patches and plasmodesmata. It
should be noted that long ER cisternae commonly occurred
in the peripheral cytoplasm of transmitting cells and were
associated with intercellular connections. It has been also long
recognized that plasmodesmata of higher plants contain a cen-
tral strand of tightly compressed ER, called the rod or
desmotubule, that creates the cytosolic sleeve providing con-
tinuity of cytoplasm between adjacent cells (see review by
Kitagawa and Jackson 2017). Thus, localization of CRT in
the cortical cytoplasm and plasmodesmata most likely corre-
sponds with the ER. However, our double labeling experi-
ments clearly showed that CRT co-localized also with callose
at the neck region of plasmodesmata in Petunia transmitting
cells, as we previously revealed for Haemanthus transmitting
tract epidermis cells (Lenartowska et al. 2009). CRT was fre-
quently shown to localize to plasmodesmta, though not all
them accumulate this protein (Baluška et al. 2003;Laporte
et al. 2003; Bayer et al. 2004;Chenetal.2005;
Lenartowska et al. 2009;BilskaandSowiński 2010;
Christensen et al. 2010; Demchenko et al. 2014).
Furthermore, a comparison of the immunolocalization of
CRT and callose in these structures favors a location of CRT
in the ER. Baluška et al. (1999,2003) originally suggested
that CRT within plasmodesmata could gate their permeability
via modulation of local/actual Ca
2+
level as both CRT and
plant-specific myosin VIII (likely regulated by Ca
2+
)are
enriched at sink plasmodesmata. However, CRT was also
found to be strongly expressed and accumulated near the neck
region of closed channels in response to different stresses,
irrespective of callose deposition (Bilska and Sowiński
2010; Demchenko et al. 2014). It was also shown that plas-
modesmata connecting mature infected cells (in contrast to
connecting uninfected or young infected cells) did not accu-
mulate CRT or callose (Demchenko et al. 2014). These au-
thors speculated that loss of callose and presumably also
desmotubules leads to plasmodesmata becoming open chan-
nels and improves metabolite exchange between adjacent
cells. Their observations led to hypothesis that CRT represents
a universal mediator of fast plasmodesmata closure that plays
a key role in cell-to-cell transport, as it was originally
Tabl e 1 The estimated amount
of CRT localized intra/
extracellularly in different
Petunia cells and their organelles
or structures
Localization Amount high +++ Amount medium ++ Amount Low +
Transmitting cells Cell wall patches ER
Cellular periphery
Cytoplasm
(including dictyosomes)
Plasmodesmata
Pollen tubes Callosic wall/plugs ER
Cellular periphery
Cytoplasm
(including dictyosomes)
Dense-core vesicles
Nucellus cells ER
Cellular/extracellular peripheries
Cytoplasm
(including dictyosomes)
Synergids Filiform apparatus ER Cytoplasm
(including dictyosomes)
64 Lenartowska et al.
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
suggested (Baluška et al. 1999,2003). In the light of these
results, we argue that CRT localized within the plasmodesma-
ta of transmitting cells regulates the architecture of intercellu-
lar connections via its Ca
2+
-binding/buffering capacity.
Therefore, this protein seems to be involved in cell-to-cell
communications within the style transmitting tract that inten-
sify during the progamic phase.
In contrast, we did not find ER cisternae in the cell
membrane-attached patches of transmitting cells, where both
CRT and exchangeable Ca
2+
were particularly concentrated
after pollination. This observation suggests that CRTaccumu-
lation in cellular peripheries outside the ER, in several patches
adjacent to the cell wall, is induced by pollination. Our find-
ings are consistent with only one report of such CRT-rich
patches in dividing Nicotiana protoplasts which exhibit high
metabolic activity required for cell proliferation and the cell
wall biosynthesis (Borisjuk et al. 1998). These authors spec-
ulated that CRT localized to cellular peripheries may function
indirectly insignal perception (as an effective Ca
2+
store), and/
or in cell adhesion during the cell shape formation. Directional
growth of the pollen tube in vivo involves both the tube cell
adhesion to the style transmitting tract and diverse cell signal-
ing pathways that regulate complicated pollen-pistil interac-
tions (see review by Dresselhaus and Franklin-Tong 2013).
Therefore, we suggest that CRT located at the cellular periph-
eries of the style transmitting cells may function as a mobile
Ca
2+
store involved in these Ca
2+
-dependent cellular process-
es during the progamic phase.
After pollination, CRT labeling was confirmed in pollen
tubes that elongated within the extracellular matrix of
Petunia stylar transmitting tract. The protein was identified
in the peripheral ER-rich cytoplasm of the tube adjacent to
the callosic wall and in this inner cell wall. In this region,
CRT was usually associated with vesicles containing
electron-dense cores in the tube cytoplasm and the callosic
cell wall. As expected, numerous Ca
2+
ppts were found in
intra/extracellular peripheries of elongated Petunia pollen
tubes, including vesicles undergoing exocytosis and the
calosic cell wall of the tube. Similar results we found previ-
ously in Haemanthus pollen tubes (Lenartowska et al. 2009).
Thus, we argue that localization pattern of CRT in the inner
cell wall is universal in pollen tubes elongating in anatomical-
ly different pistil styles (solid in Petunia and hollow in
Haemantus). Other studies have demonstrated that a lily
pollen-specific protein LP2 and two pistil-specific proteins,
120 kDa and PELPIII glycoproteins, were also located in the
callosic cell wall of developing pollen and pollen tubes grow-
ing in situ (Lind et al. 1996;Mogamietal.2002; Graaf et al.
2003). In the light of these reports and based on the controls
presented here, CRT labeling in the callose depositions is not
an artifact. Despite these findings, it still remains unclear how
CRTcould leave the ER and move into the cell wall. The most
likely explanations include existence of splice variants that
can localize to different cell compartments, post-
trancriptional modifications such as glycosylation, enzymatic
modification of the glycan complexity, or degradation of the
ER-retention signal (see reviews by Johnson et al. 2001;
Michalak et al. 2009). Therefore, it is possible that CRT is
translocated from the ER/dictyosomes to the cell periphery
and then to the inner cell wall where it may play a role in
external Ca
2+
storage of the elongatinging pollen tube.
Although the precise mechanism is still unclear, stabilization
of a tip-focused Ca
2+
gradient is critical for pollen germination
and pollen tube growth (see reviews by Hepler et al. 2012;
Steinhorst and Kudla 2013). We previously showed that in
Petunia pollen tubes growing in vitro, CRT is translated on
ER membrane-bound ribosomes and accumulated in the ER at
the subapical zone of the tube, where plays a role in stabilizing
Ca
2+
homeostasis that is required for actomyosin-dependent
cytoplasmic streaming, organelle positioning, vesicle traffick-
ing, and cell wall biogenesis (Suwińska et al. 2015,2017).
Thus, we concluded that internal Ca
2+
stores involving CRT
activity are crucial for proper pollen tube elongation. Based on
our present data showing both CRTand exchangeable Ca
2+
in
the callosic cell wall of the pollen tube, we cannot exclude the
possibility that this external Ca
2+
store is equally important in
polar tip growth of the tube. It has been long suggested that
both the concentration/availability of Ca
2+
and the degree of
pectin esterification are crucial for the cell wall expansion and
the pollen tube tip growth (see review by Hepler et al. 2013).
However, the neutral polymer of 1,3-β-glucan does not bind
Ca
2+
. Thus, an attractive hypothesis suggests that excess of
Ca
2+
is translocated from the pollen tube tip cytosol not only
to the ER but also to the callosic cell wall, and bound by CRT
present there in order to maintaining the stable Ca
2+
gradient
in growing pollen tube. More precise research is required,
however, to verify this hypothesis.
Exchangeable Ca
2+
dynamics during the progamic phase
corresponds with extracellularly localized CRT
in the nucellus and the synergids
One of our most interesting observations was the continuous
location of CRT (before pollination and during the progamic
phase) between nucellus cells surrounding the embryo sac and
its preferential accumulation within the ER and the filiform
apparatus of the synergids. Similar results were observed in
Petunia and Haemantus ovules. Moreover, preferential local-
ization of CRT in the filiform apparatus during the late
progamic phase/fertilization was also confirmed in
Hyacinthus ovule (Niedojadło et al. 2015). In contrast, we
found that the level of exchangeable Ca
2+
increased signifi-
cantly during the progamic phase in Petunia;Ca
2+
ppts
strongly labeled the ER and dictyosomes as well as the intra/
extracellular peripheries of both nucellus and synergid cells.
An extremely high level of CRTwas found within the filiform
Calreticulin localizes to plant intra/extracellular peripheries 65
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
apparatus of the synergid cell. Similar results we found in
Haemanthus micropylar pole of the ovule where a large mass
of Ca
2+
ppts was observed in the synergid after pollination.
Since the probable function of CRT in the cellular peripheries
of nucellus cells is similar to the function proposed in trans-
mitting cells, the presence of CRT in the filiform apparatus
needs an additional comment.
The filiform apparatus is a thickened cell wall of heteroge-
neous structure that involves fibrillar and amorphous fractions
with numerous finger-like projections into the synergid cyto-
plasm (Płachno et al. 2014). This unique structure greatly
increases the surface area of the plasma membrane at the mi-
cropylar pole of the synergid and isalso associated with highly
expanded ER. Several functions have been proposed for the
filiform apparatus, including pollen tube guidance and recep-
tion, import of metabolites, and export of the pollen tube at-
tractants, and numerous proteins have been found to localize
in this specialized compartment (see review by Dresselhaus
and Franklin-Tong 2013). We previously revealed a
pollination-induced significant increase of CRT mRNA,
CRT, and exchangeable Ca
2+
levels in the synergid resulting
in preferential accumulation of CRT and Ca
2+
in the ER and
the filiform apparatus (Lenartowski et al. 2014,2015). This
phenomenon is consistent with reports that, of all the cells in
the embryo sac, the highest level of loosely bound Ca
2+
is in
the synergid (see review by Ge et al. 2007). It is still unknown
whether Ca
2+
is delivered from the extracellular space, the ER,
or both. We postulate that CRT accumulated in the filiform
apparatus may provide an exchangeable Ca
2+
external store in
the synergid and thus modulate the local concentration of
cytoplasmic Ca
2+
to prevent extra pollen tubes from entering
the embryo sac. Generally, only one pollen tube enters each
embryo sac in angiosperms, and precise regulation of the Ca
2+
level in the penetrated ovule determines its receptivity. Upon
pollen tube arrival at the Arabidopsis receptive synergid, Ca
2+
oscillation begins at the micropylar pole and spreads toward
the chalazal pole, and Ca
2+
reaches a maximum level at pollen
tube rupture Iwano et al. (2012). Even though recent studies
demonstrate that pollen tube entry into the Arabidopsis syner-
gid cell is observed at a site distinct from the filiform apparatus
(Lesherm et al. 2013), it remains certain that this unique struc-
ture plays an important role in male and female gametophyte
recognition. However, further studies are needed to under-
stand the functional CRTaccumulation within this highly spe-
cific structure during the progamic phase in angiosperms.
Acknowledgments The authors thank Richard Napier (University of
Warwick, Wellesbourne, UK) for providing the CRT PAb antibody and
Kathy G. Miller (Washington University in St. Louis, US) for critical
reading of the manuscript. This project was supported by statutory funds
from Ministry of Science and Higher Education (PL) for the research
programs of the Laboratory of Developmental Biology and the
Laboratory of Isotope and Instrumental Analysis (Nicolaus Copernicus
University in Toruń,PL).
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Open Access This article is distributed under the terms of the Creative
Commons Attribution 4.0 International License (http://
creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided you give appro-
priate credit to the original author(s) and the source, provide a link to the
Creative Commons license, and indicate if changes were made.
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... Previous work from our laboratory provided evidence that CRT may be involved in Ca 2+ homeostasis and molecular chaperoning during the key reproductive events in Petunia pistil, such as pistil transmitting tract maturation, pollen-pistil interactions, double fertilization, and early embryogenesis [17,18,22]. We also demonstrated that CRT has a critical role in pollen tube growth in vitro [20]. ...
... However, localization of this protein outside the ER has also been observed in eukaryotic cells. Plant CRT was detected in many different compartments and structures, including dictyosomes, vesicles, protein bodies, nucleus, plasma membrane, and the cell wall [22]. Although, some previous work from our lab and others indicated location of CRT in several extracellular regions were somewhat controversial, an elegant work of Luczak et al. [34] provided clear evidence that CRT, similar to several other proteins, is present in the cell walls of few plant species including maize, Lupinus and Arabidopsis. ...
... Although, some previous work from our lab and others indicated location of CRT in several extracellular regions were somewhat controversial, an elegant work of Luczak et al. [34] provided clear evidence that CRT, similar to several other proteins, is present in the cell walls of few plant species including maize, Lupinus and Arabidopsis. We have previously demonstrated that both CRT and exchangeable Ca 2+ were localized to the extracellular peripheries of highly specialized plant cells, such as pollen tubes and synergids [14,16,18,22]. In pollen tubes, CRT labeling was detected in the peripheral ER-rich cytoplasm of the tube adjacent to the cell membrane and in the callosic cell wall. ...
Article
Full-text available
Background Pollen development in the anther in angiosperms depends on complicated cellular interactions associated with the expression of gametophytic and sporophytic genes which control fundamental processes during microsporo/gametogenesis, such as exo/endocytosis, intracellular transport, cell signaling, chromatin remodeling, and cell division. Most if not all of these cellular processes depend of local concentration of calcium ions (Ca ²⁺ ). Work from our laboratory and others provide evidence that calreticulin (CRT), a prominent Ca ²⁺ -binding/buffering protein in the endoplasmic reticulum (ER) of eukaryotic cells, may be involved in pollen formation and function. Here, we show for the first time the expression pattern of the PhCRT1 gene and CRT accumulation in relation to exchangeable Ca ²⁺ in Petunia hybrida developing anther, and discuss probable roles for this protein in the male gametophyte development. Results Using northern hybridization, western blot analysis, fluorescent in situ hybridization (FISH), immunocytochemistry, and potassium antimonate precipitation, we report that PhCRT1 is highly expressed in the anther and localization pattern of the CRT protein correlates with loosely bound (exchangeable) Ca ²⁺ during the successive stages of microsporo/gametogenesis. We confirmed a permanent presence of both CRT and exchangeable Ca ²⁺ in the germ line and tapetal cells, where these factors preferentially localized to the ER which is known to be the most effective intracellular Ca ²⁺ store in eukaryotic cells. In addition, our immunoblots revealed a gradual increase in CRT level from the microsporocyte stage through the meiosis and the highest CRT level at the microspore stage, when both microspores and tapetal cells show extremely high secretory activity correlated with the biogenesis of the sporoderm. Conclusion Our present data provide support for a key role of CRT in developing anther of angiosperms – regulation of Ca ²⁺ homeostasis during pollen grains formation. This Ca ²⁺ -buffering chaperone seems to be essential for pollen development and maturation since a high rate of protein synthesis and protein folding within the ER as well as intracellular Ca ²⁺ homeostasis are strictly required during the multi-step process of pollen development.
... A C-terminally truncated calreticulin was observed (CaCl2 spot 1274). Although regarded as an ER-resident protein, calreticulin was described in other subcellular compartments [43]. Animal calreticulin is linked with the recognition of tumor and apoptotic cells, and wound healing in plants it is linked to growth and stress responses [44]. ...
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... It is known that in the tip, there is a high concentration of calcium (Steinhorst and Kudla 2013); in addition, CRT has been reported in mitotic cells in the spindle apparatus and the nuclear envelope (Denecke et al. 1995). CRT could also be influencing the storage of Ca 2+ and cytosolic calcium elevations (Krause and Michalak 1997), and actively participating during the reproductive process of angiosperm plants as recently proposed by Wasąg et al. (2018). In addition, Suwińska et al. (2015) proposed that during the development of the pollen tube, the CRT keeps calcium at the tip of the PT and the suppression of this gene directly affects the cytoskeleton organization of actin (Suwińska et al. 2017). ...
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... Large numbers of CRT proteins are localized near the transmitting tract and synergid cells, cells surrounding the embryo sac, and pollen tubes. Because CRT regulates calcium storage, it may be involved in the accumulation of calcium used for pollen tube elongation (Wasag et al. 2017). CNGC18 is involved in calcium ion uptake into pollen tubes and pollen tube guidance (Gao et al. 2016). ...
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Key message In germinating pollen grains and growing pollen tubes, CRT is translated on ER membrane-bound ribosomes in the regions where its activity is required for stabilization of tip-focused Ca 2+ gradient. Abstract Pollen tube growth requires coordination of signaling, exocytosis, and actin cytoskeletal organization. Many of these processes are thought to be controlled by finely tuned regulation of cytoplasmic Ca2+ in discrete regions of the tube cytoplasm. Most notably, a mechanism must function to maintain a steep gradient of Ca2+ that exists at the tip of growing pollen tube. Several pieces of evidence point to calreticulin (CRT) as a key Ca2+-binding/-buffering protein involved in pollen germination and pollen tube growth. We previously hypothesized that in germinating pollen and growing tubes, CRT is translated on the ribosomes associated with endoplasmic reticulum (ER) in the regions where its activity might be required. In this report, we have addressed this idea by identifying the sites where CRT mRNA, CRT protein, 18S rRNA, and rough ER are localized in Petunia pollen tubes. We observed all four components in the germinal aperture of pollen grains and in subapical regions of elongating tubes. These results seem to support our idea that CRT is translated on ER membrane-bound ribosomes during pollen germination and pollen tube growth. In elongated pollen tubes, we found CRT mainly localized in the subapical zone, where ER and Golgi stacks are abundant. In eukaryotic cells, these organelles serve as mobile intracellular stores of easily releasable Ca2+, which can be buffered by proteins such as CRT. Therefore, we postulate that subapical-localized CRT is involved in pollen tube growth by maintaining the stable tip-focused Ca2+ gradient and thus modulating local Ca2+ concentration within the tube cytoplasm.
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