Sex Plant Reprod (2004) 16:227–233
DOI 10.1007/s00497-003-0198-0
ORIGINAL ARTICLE
Jie Zhao · Jean-Claude Mollet · Elizabeth M. Lord
Lily (Lilium longiflorum L.) pollen protoplast adhesion is increased
in the presence of the peptide SCA
Received: 11 August 2003 / Accepted: 10 November 2003 / Published online: 6 December 2003
Springer-Verlag 2003
Abstract The style of lily produces a specialized extracellular matrix (ECM) in the transmitting tract epidermis
that functions to guide pollen tubes to the ovary. This
adhesive ECM contains low esterified pectins and a
peptide, SCA (stigma/stylar cysteine-rich adhesin). Together they form a matrix to which pollen tubes adhere as
they grow through the style. Pollen tubes also adhere to
each other but only when grown in vivo, not in vitro.
Pollen does not produce detectable SCA, but when SCA
is added to an in vitro growth medium, it binds to pollen
tubes that have esterified and low-esterified pectins in
their walls. Since adhesion of the pollen tube to the stylar
matrix requires tip growth, we hypothesized that the
pectin wall at the pollen tube tip interacted with the SCA
protein to initiate adhesion with the stylar pectin [Lord
(2000) Trends Plant Sci 5:368–373]. Here, we use a
pollen protoplast system to examine the effect of SCA on
protoplast adhesion when it is added to the growth
medium in the absence of the stylar pectin. We found that
SCA induces a 2-fold increase in protoplast adhesion
when it is added at the start of protoplast culture. This
effect is less when SCA is added to the medium after the
cell wall on the protoplast has begun to regenerate. We
show that among the first components deposited in the
new wall are arabinogalactan proteins (AGPs) and highly
esterified pectins. We see no labeling for low esterified
pectins even after 3 days of culture. In the pollen
J. Zhao
Key Laboratory of MOE for Plant Developmental Biology,
College of Life Science, Wuhan University,
430072 Wuhan, China
J.-C. Mollet · E. M. Lord ())
Center for Plant Cell Biology,
Department of Botany and Plant Sciences,
University of California, Riverside, CA 92521, USA
e-mail: [email protected]
Fax: +1-909-7874437
Present address:
J.-C. Mollet, Laboratoire de Glycobiologie et Physiologie Vgtale,
Facultie Jean Perrin, Universit d’Artois,
62307 Lens Cedex, France
protoplast culture, adhesion occurs in the absence of the
low esterified pectin. The newly formed wall on the
protoplast mirrors that of the pollen tube tip in lily, which
is rich in AGPs and highly esterified pectins. Thus, the
protoplast system may be useful for isolating the pollen
partner for SCA in this adhesion event.
Keywords Adhesion · LTP · Pectins · Pollen protoplasts ·
SCA
Introduction
Morphogenesis requires adhesion between somatic cells
and, in reproduction, cell-cell adhesion initiates signaling
events in pollination and fertilization (Lord and Russell
2002). The loss of adhesion that occurs during ripening,
abscission, and dehiscence of plant organs is an active
process and the molecules involved have informed us
about the identity of the adhesion molecules themselves
(Roberts et al. 2002). Pectinases and cellulases are
routinely used to separate cells of an organ and produce
protoplasts that, when cultured, promptly produce a new
wall or extracellular matrix (ECM) composed of glucans,
pectins and other wall molecules. Early work with
transgenic tobacco plants, where a tomato polygalacturonase (PG) gene was over-expressed, showed no effect on
cell separation in the leaf (Osteryoung et al. 1990), but
recent studies using transgenic apple and Arabidopsis
show that the PG from apple, when over-expressed, is
capable of causing a phenotype where cell separation is
evident (Atkinson et al. 2002). Many previous studies
including the use of biological assays had implicated
pectins in cell-cell adhesion but it was not until the recent
discovery of a pectin glucuronyltransferase gene that it
could be said with certainty that pectins were essential for
somatic cell adhesion (Iwai et al 2002; Lord and Mollet
2002). An in vitro biological assay had previously
implicated pectins in adhesion during pollination (Mollet
et al. 2000) but here a second molecule was involved as
well, a peptide called SCA (stigma/stylar cysteine-rich
228
adhesin) (Park et al. 2000). In combination, these two
molecules from the style of lily form an ECM that induces
pollen tube adhesion. The SCA and pectin reside on the
surface of the transmitting tract epidermis (TTE) of the
stylar canal and act to track the pollen tubes to the ovary
(Lord 2000). Pollen tubes grown in a liquid medium do
not adhere to one another but in vivo they adhere to the
TTE and to each other during their journey to the ovary.
The low esterified, anionic pectin and the basic peptide
SCA bind each other in a pH-dependent fashion (Mollet et
al. 2000) and, in vitro, can induce pollen tube adhesion to
a glass slide coated with the SCA/pectin matrix. The
adhesion activity in vitro is comprised of pollen tubes
induced to adhere to each other as well as to the SCA/
pectin matrix. The pollen partner in this adhesion event is
unknown. The pollen tube cell does not produce detectable SCA protein or mRNA but can bind the SCA
peptide in culture (Park et al. 2000). The lily pollen tube
has an outer pectin wall composed primarily of low
esterified pectins similar to those in the TTE that bind
SCA (Jauh and Lord 1996). Pollen tubes grown in vivo
adhere to one another but do not show this adhesion in
vitro. Presumably, the TTE-secreted low esterified pectin
and SCA are necessary to induce pollen tube adhesion to
the TTE and to each other. To test this hypothesis we used
the lily pollen protoplast system with SCA added to the
medium. We find that SCA is capable of inducing pollen
protoplast adhesion in the absence of stylar pectin and
that increased adhesion occurs when SCA is added to the
culture at an early stage in protoplast wall formation.
Materials and methods
Plant material
Plants of lily (Lilium longiflorum Thunb. cv Nellie White) were
grown in the greenhouse at the University of California, Riverside.
Lily anthers were collected 1–2 days after anthesis.
Isolation, purification and incubation of pollen protoplasts
Pollen protoplasts were isolated from six anthers by immersion in
2 ml mixed enzyme solution that contained 0.8% cellulase (ICN),
0.8% pectinase (Sigma, St. Louis, Mo.), 0.5% hemicellulase
(Sigma), 12–13% mannitol and Murashige-Skoog (MS) basic salt
medium. Pollen was incubated at room temperature in darkness for
2–3 h with shaking at a speed of 50–60 rpm. After isolation, the
protoplasts were rinsed and centrifuged (500 rpm, 3 min) three
times with enzyme-free MS medium containing 16% sucrose. The
pollen protoplast suspension was placed on a discontinuous sucrose
gradient and centrifuged 8–10 min at a speed of 500 rpm (Tanaka et
al. 1987). Purified protoplasts were collected from the interface of
the two layers with a pipette and incubated in MS medium
supplemented with 16% sucrose, 0.2 mll 2,4-dichlorophenoxyacetic acid (2,4-D), Km8p (Sigma) mixed organic acids and Km8p
(Sigma) mixed sugars. The isolated protoplasts were stained with
50 mg ml1 fluorescein diacetate (FDA) (Aldrich, Milwaukee, Wis.)
for 5 min to determine their viability.
Pollen protoplast adhesion assay
At the beginning of culture (0 h) and after 5 or 15 h of culture, a
native preparation of SCA proteins (Park and Lord 2003) (0.05–
0.40 mg ml1) was added to 100 ml medium containing the isolated
protoplasts. Controls were grown in medium without SCA. The
culture density was 1.5–3.0104 protoplasts ml1, optimized to
avoid agglutination of protoplasts but to allow for protoplast
interaction. Protoplasts were incubated for 1–96 h at room
temperature in darkness. A 10 ml drop of protoplast suspension
was taken after 0, 24 and 48 h incubation and the percent adhesion
calculated by counting the number of adhered protoplasts in 10 ml.
The length of the adhesion zone was measured on images of
protoplasts from 24, 48 and 96 h of culture. These measurements
were taken on samples treated with SCA at the initiation of culture
as well as on controls. From three to five 10 ml samples were taken
from each experiment and the experiments were repeated 2–3
times.
Fluorescence assay of cell wall production
with Calcofluor White-M2R
Samples of pollen protoplasts incubated with or without SCA for 1–
24 h were treated with Calcofluor White (CW)-M2R (1 mg/ml)
(Polysciences, Warrington, Pa.). Protoplasts were then incubated in
a moist, dark, box for 20–30 min at room temperature before
observation.
Immunolocalization of arabinogalactan proteins
with MAb JIM13 and pectins with MAb JIM5 and MAb JIM7
Pollen protoplasts incubated from 1–96 h (€SCA) were fixed for 2–
3 h with 50 mM piperazine-N, N0 -bis (2-ethanesulfonic acid)
(PIPES) buffer, pH 6.7, 2 mM MgSO4·7H2O, 2 mM EGTA, 16%
sucrose and 2.5% paraformaldehyde at room temperature. Protoplasts were rinsed three times with 50 mM PIPES buffer, pH 6.7
(containing 2 mM MgSO4·7H2O and 2 mM EGTA) and then with
phosphate-buffered saline (PBS), pH 7.4. Primary antibodies, MAb
JIM13 for labeling arabinogalactan proteins (AGPs) (Pennell et al.
1989), MAb JIM5 and MAb JIM7 for labeling low-esterified and
high-esterified pectins (Knox et al. 1990), respectively, were
diluted 1:5 with PBS (pH 7.4) and incubated for 2–3 h as previously
described (Jauh and Lord 1996). The protoplasts were rinsed three
times with PBS (pH 7.4), and then incubated overnight at 4C in
darkness with the secondary antibody, anti-rat-IgG-FITC conjugate
(Sigma) diluted at 1/100 with PBS (pH 7.4). The protoplasts were
rinsed three times with PBS (pH 7.4) before microscopic examination. In the control, protoplasts were incubated with PBS
(pH 7.4) instead of the primary antibody. Every step was carried out
in Eppendorf tubes and the waste liquid was removed by pipette
after centrifugation at 4,000–5,000 rpm for 3 min.
Protoplast observation and image capture
Samples treated with FDA were observed using a Nikon Microphot
FXA microscope equipped with a fluorescence microscopy filter
set for fluorescein isothiocyanate (FITC). Images were captured
with a SPOT Insight camera (Diagnostic Instruments, Sterling
Heights, Mich.) and Image-Pro·Plus software (Media Cybernetics,
Carlsbad, Calif.).
Protoplasts labeled with FDA, Calcofluor White and FITCconjugated secondary antibodies were observed under fluorescence
illumination and Nomarski differential interference contrast (DIC)
optics on a Nikon Microphot FXA microscope equipped with a
filter set for UV or FITC. Images were captured as before and
processed using Adobe PhotoShop software 6.0 (Adobe Systems,
Mountain View, Calif.).
229
Results
Pollen protoplast adhesion increases in the presence
of SCA in the culture medium
When SCA is added to the culture medium, the level of
protoplast adhesion increases to 30% and more (Fig. 2a–
c). When SCA is added at the beginning of protoplast
incubation, the effect is most apparent and the adhesion,
as measured by length of the adhesion zone, is tighter
(Fig. 1c, d; Table 1). In repeated trials, the adhered
protoplasts could not be separated by shaking or low
speed centrifugation (600–1,000 rpm). The concentration
of SCA in the culture medium affects the amount of
adhesion seen at 24 h, up to a threshold level (Fig. 2a).
When protoplasts are incubated for 5 or 15 h before
adding SCA and then cultured for 24 and 48 h (Fig. 2b, c),
the frequency of adhesion was increased over the control,
Fig 2a–c Effect of SCA on the adhesion of lily pollen protoplasts.
a SCA added when protoplast culture begins. b SCA added 5 h
after protoplast culture begins. c SCA added 15 h after protoplast
culture begins. Percent adhesion is measured by counting number
of adhered protoplasts in a 10 ml sample. This was repeated 3–5
times for each experiment and experiments were repeated 2–3
times
but the effect was not as strong as when SCA was added
at the beginning of culture. Some pollen protoplasts
adhere to each other in control cultures beginning after
6 h incubation, but with an extended period of culture
(24–48 h) only 10–18% of the protoplasts show adhesion
(Figs. 1a, b; 2a–c).
Detection of cell wall formation with CW
Fig. 1a–d Adhesion of lily pollen protoplasts during culture. a
Protoplasts in medium without stigma/stylar cysteine-rich adhesin
(SCA) at 24 h of incubation. b Protoplasts in medium without SCA
at 48 h of incubation. c Protoplasts in medium with 0.1 mg/ml SCA
at 48 h of incubation. d Protoplasts in medium with 0.2 mg/ml SCA
at 48 h of incubation. Bars 20 mm
Table 1 Length (mm) of adhesion sites between lily pollen
protoplasts cultured €SCA (stigma/stylar cysteine-rich adhesin)
Culture duration
(h)
SCA
24
17.16€0.49
n=12
18.76€0.55
n=16
19.32€0.52
n=11
48
96
a
+SCA (mg/ml)a
0.1
0.2
29.43€0.52
n=14
30.73€0.82
n=10
28.58€0.40
n=11
28.18€0.91
n=6
33.42€1.01
n=7
36.32€1.63
n=5
Added at beginning of protoplast culture
Protoplasts labeled with CW (glucans) 1–2 h after culture
showed a patchy signal (Fig. 3a). Labeling at 3–6 h after
culture was stronger and continuous around the whole
protoplast (Fig. 3b, c). The signal appeared to be brighter
at the site of adhesion between two protoplasts after 24 h
of culture, especially if SCA was added to the culture
(Fig. 3d–i). The presence of SCA in the culture medium
had no detectable effect on the timing of protoplast wall
regeneration.
Immunolocalization of AGPs during culture
of pollen protoplasts
Labeling of freshly isolated pollen protoplasts with MAb
JIM13 (AGPs) showed patchy fluorescence along the
protoplast membrane (Fig. 4a). At 1–3 h of incubation,
the fluorescence was stronger, but still remained in the
form of patches (Fig. 4b, c). By 6 h of incubation, the
fluorescence was continuous around the protoplasts
(Fig. 4d). At 48 h of incubation, all of the cells were
labeled with MAb JIM13 and a brighter fluorescence
appeared at the adhesive site between cells whether they
were treated with SCA or not (data not shown). Again, we
saw no effect of SCA on the localization patterns of MAb
JIM13.
230
Fig. 3a–i Cell wall formation
and adhesion during culture of
lily pollen protoplasts (€SCA
0.1 mg/ml) labeled with Calcofluor White (CW). a A patch
of signal appeared on protoplasts in medium without SCA
at 2 h of incubation. b The CW
label appeared around protoplasts in medium without SCA
at 3 h of incubation. c The
entire protoplast was labeled by
CW in medium without SCA at
6 h of incubation. d, f, h The
CW label was stronger at the
adhesive site between two protoplasts in medium with 0.1 mg/
ml SCA at 24 h of incubation. e,
g, i Differential interference
contrast (DIC) microscopy images of the samples in d, f, h,
respectively. Bars 20 mm
Immunolocalization of pectins during culture
of pollen protoplasts
Labeling with MAb JIM7 (highly esterified pectins) was
detected in pollen protoplasts incubated for 3–6 h and
appeared in the form of patches or dots (Fig. 4e, f). After
24 h of culture, labeling was around the entire protoplast
(Fig. 4g) and again was brighter at the adhesion site
between cells (Fig. 4h). Labeling of the cell wall with JIM
5 (low esterified pectins) was undetectable, even up to
68 h of culture (data not shown). No differences in pectin
localization were observed between protoplasts treated
with or without SCA.
Discussion
Lily pollen protoplasts have been shown to first adhere
and then fuse when an electrical pulse is applied (Ueda et
al. 1990) but adhesion of protoplasts in culture is typically
very low. We found that addition of SCA to the culture
medium can increase adhesion of lily pollen protoplasts 2fold and increase the length of the adhesion zone. These
effects are most evident when SCA is added to the
protoplasts at the start of culture when plasma membranes
are still exposed. Since adhesion between protoplasts
occurs to some extent in the absence of SCA, there must
be an inherent mechanism for pollen protoplast adhesion
that the addition of SCA facilitates.
Wall regeneration begins immediately after formation
of the protoplast in the form of a patchy network, which is
continuous by 6 h as measured by CW staining. Several
studies of wall regeneration in pollen and somatic
protoplast culture reported that cell wall regeneration
occurred more slowly than we observed, over 1–3 days
(David et al. 1995; Majewska-Sawka et al. 2002; MikiHirosige et al. 1988; Shea et al. 1989). Typically, glucans
(either cellulose or callose) appear first on the protoplast
surface followed by esterified pectins. The de-esterification of pectins apparently does not occur until 6 days or
longer in culture (David et al. 1995; Majewska-Sawka et
al. 2002). The first detectable matrix molecules on the lily
231
Fig. 4a–h Immunolocalization
of arabinogalactan proteins
(AGPs) (MAb JIM13; a-d) and
highly esterified pectins (MAb
JIM7; e-h) during culture of
pollen protoplasts. a, b Patchy
labeling appeared on the protoplast membrane at 0 and 1 h of
incubation, respectively. c Labeling appeared continuous
around the protoplasts at 3 h. d
The protoplast was strongly labeled at 6 h. e, f Patchy labeling
of pectins appeared along protoplast membranes cultured for
3–6 h. g Labeled protoplasts at
24 h. h Labeled protoplasts at
96 h of incubation. Bars 20 mm
232
pollen protoplasts were AGPs at 3 h after culture, with
esterified pectins then evident at 6 h. We never observed
any low esterified pectin localization even after 3 days of
culture. We found no effect of SCA on the timing of wall
regeneration or on its composition as detected by
Calcofluor White or the MAbs used, but we did see an
effect of SCA on the location of wall deposition. The site
of adhesion between the protoplasts showed increased
levels of wall deposition with SCA treatment.
Pollen tubes are tip growing cells that produce new
wall material at the growing point via secretion of Golgi
vesicles that, in lily, contain AGPs and esterified pectins
(Jauh and Lord 1996; Roy et al. 1998). Lily pollen tube
walls are comprised mainly of callose and pectin with
little cellulose present. Back from the tip the pectins
become de-esterified and a secondary callosic wall forms.
So, the two major components of the newly formed lily
pollen tube wall, AGPs and esterified pectins, are among
the first to appear on the protoplast surface in vitro.
SCA is a small (9.3 kDa), basic, cysteine-rich protein
that is secreted by the transmitting tract of the stigma and
style of lily (Lord 2000). In the transmitting tract, SCA
and a low-esterified pectin from the style bind ionically to
form an adhesive matrix that functions to guide the pollen
tube to the ovary (Mollet et al. 2000). In an in vitro
adhesion assay using the SCA/pectin matrix, pollen tubes
will adhere to and grow on that matrix. Since SCA binds
low esterified pectins from the style, we originally
postulated that it also binds the low-esterified pectins of
the mature pollen tube wall and acts simply as a glue
between the two (Lord 2000). However, we found that
adhesion requires tip growth of the pollen tube (Jauh et al.
1997) so the interaction between the pollen tube and the
matrix is probably initiated where new wall formation is
occurring at the tube tip; the same site where the AGPs
and highly esterified pectins are being deposited. The fact
that SCA alone can induce pollen protoplast adhesion, in
the absence of the low esterified stylar pectin and prior to
the production of de-esterified pectin on the pollen
protoplast wall, argues for a direct interaction of SCA
with the newly forming wall or the exposed plasma
membrane. The fact that the most adhesive protoplast is
the newly formed one suggests that the interaction of SCA
is with the plasma membrane, perhaps with a receptor
there. We plan to repeat these experiments using protoplasts isolated from vegetative organs such as leaves to
determine whether the adhesion event is pollen specific.
SCA is related to a family of proteins called LTPs
(lipid transfer proteins) (Kader 1997). These small
proteins were once thought to move lipids intracellularly,
but it is now known that the proteins are secreted into the
wall and occur in nearly all plant organs. Several
functions have been proposed for these peptides, one of
which is antimicrobial since many show potent antifungal
and antibacterial activity (Garcia-Olmedo et al. 1995;
Selitrennikoff 2001). The mechanism of their action as
antimicrobial compounds is unknown but it is thought that
they bind plasma membranes (Blein et al. 2002). It is
possible that the LTPs are a multifunctional family of
signaling molecules that bind receptors in the plasma
membrane. Several such peptide signaling molecules and
their receptors have been described recently (Buhot et al.
2001; Ryan et al 2002). Our pollen protoplast adhesion
assay can now be used to explore the possibility of a
pollen tube interacting partner for SCA.
Acknowledgements This research was supported by National
Science Foundation grant IBN-0077886 to E.M. Lord and the
Major State Basic Research Program of China (2002CCA00100 to
J. Zhao. The MAbs were the generous gifts of Dr. J.P. Knox,
University of Leeds, UK and Dr. Keith Roberts, John Innes
Institute, Norwich, UK.
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