Post on 15-Jul-2016
ORIGINAL ARTICLE
Localization of arabinogalactan-proteins in different stagesof embryos and their role in cotyledon formationof Nicotiana tabacum L.
Yuan Qin Æ Jie Zhao
Received: 22 July 2007 / Revised: 9 September 2007 / Accepted: 25 September 2007 / Published online: 31 October 2007
� Springer-Verlag 2007
Abstract Arabinogalactan proteins (AGPs) have been
implicated in plant development including sexual plant
reproduction. In this paper, the expression of AGPs and the
effects of b-glucosyl Yariv reagent (bGlcY, which binds
arabinogalactan proteins) in embryo development and
cotyledon formation were investigated. Immunofluores-
cence assay displayed that the expression of AGPs labeled
with antibody JIM13 was developmentally regulated. In
early stages, AGPs were evenly distributed in the whole
embryo, except for a short polar expression in the basal
suspensor cell. In the globular stage of embryo, AGPs were
condensed in the embryo proper (EP), apex of the EP, and
at the juncture of the EP and suspensor. In heart-shaped
embryo, APGs were only present at the juncture of the EP
and suspensor. Immunogold labeling assay showed that the
strong expression of AGPs at the juncture of the EP and
suspensor was localized in the cell wall. Provision of
bGlcY to the in vitro ovule culture medium caused delayed
growth of embryos, cotyledon defect and abnormal vena-
tion pattern. Consequently, bGlcY induced the death of
defective seedlings with the characteristics of deformed or
irregular single cotyledon. Our results suggested that AGPs
play functional roles in embryo development, cotyledon
formation and seedling morphology establishment in
Nicotiana tabacum L.
Keywords Nicotiana tabacum L. �Arabinogalactan proteins � Embryo � Cotyledon
Abbreviations
AGPs Arabinogalactan proteins
DAP Days after pollination
EP Embryo proper
Mb Monoclonal antibodies.
Introduction
Angiosperm embryogenesis initiates from a single cell
zygote, which subsequently divides into two asymmetrical
cells with different developmental fates. The two-celled
embryo in dicots develops through a series of stages that
have been defined morphologically as preglobular, globu-
lar, heart, torpedo and mature stages (Vroemen et al. 1999).
During embryogenesis, the bilateral symmetry structure of
embryo becomes arranged along apical–basal axis. The
attainment of bilateral symmetry from axial symmetry in
embryogenesis occurs at the time of the transition from the
globular to the heart-shaped stage (Tykarska 1979). How-
ever, the mechanism underlying this change is far from
clear, although there has been progress from the traditional
experimental embryology to the genetic dissection of
embryo development by the isolation and characterization
of mutants.
It is generally accepted that plant embryogenesis is an
extremely complex process dependent on the coordination
of numerous specific genetic programs as well as com-
munication between cells. Many genes play critical roles in
the developmental events involved in pattern formation and
Communicated by Scott Russell.
Y. Qin � J. Zhao (&)
Key Laboratory of the Ministry of Education for Plant
Developmental Biology, College of Life Sciences,
Wuhan University, Wuhan 430072, China
e-mail: jzhao@whu.edu.cn
123
Sex Plant Reprod (2007) 20:213–224
DOI 10.1007/s00497-007-0058-4
cell differentiation. TWN1 influences apical pattern and
morphology in the embryo proper (EP). The twn1 mutant
disrupts cotyledon number, arrangement and morphology
(Vernon et al. 2001). The RSH gene is essential for normal
embryo development, especially for correct positioning of
the cell plate during cytokinesis in cells of the developing
embryo. The embryos of rsh mutant are defective mor-
phologically and have irregular cell shapes and sizes, and
cannot establish the body plan of bilateral symmetry (Hall
and Cannon 2002). In raspberry mutant, globular-stage
embryo could not transit into heart-stage embryo (Yadegari
et al. 1994). The following genes MP (Berlth and Jurgens
1993), KN (Lukowitz et al. 1996), and FASS (Torres-Ruiz
and Jurgens 1994) are indicated to be associated with the
establishment of the basal embryo pole. And the CUP-
SHAPED COTYLEDON genes (CUC1–3), which encode
transcription factors of the NAC family, are required for
cotyledon separation and SAM formation (Aida et al. 1997;
Takada et al. 2001; Vroemen et al. 2003).
Arabinogalactan-proteins (AGPs) are a class of plant
extracellular-matrix proteins, which are believed to par-
ticipate in a variety of plant development processes
including sexual plant reproduction (Cheung et al. 1993;
Cheung and Wu 1999; Majewska-Sawka and Nothnagel
2000; Qin and Zhao 2004). AGPs have also been impli-
cated in somatic embryogenesis. The correlation of the
presence of JIM4 epitopes with certain stages of somatic
embryogenesis supports the role of AGPs in embryo
growth and differentiation (Stacey et al. 1990). Moreover,
AGPs secreted from the culture cells or extracted from the
seeds affect the induction and development of somatic
embryos (Kreuger and van Holst 1993; Egertsdotter and
von Arnold 1995). Since bGlcY can specifically bind AGPs
and perturb their biological activity, it has also been used to
explore the role of AGPs in embryogenesis. Embryogenic
carrot cell suspension grown in the presence of bGlcY
could not produce normal embryos (Thompson and Knox
1998). The effect of bGlcY on inhibition of Cichorium
somatic embryogenesis was concentration-dependent and
reversible (Chapman et al. 2000).
Because plant embryos reside deeply inside various
sporophytic tissues of ovules and ovaries, it has been
technically difficult to explore the functions of AGPs in
embryo development. There were only a few reports that
show temporal and spatial expression of AGPs in devel-
oping embryos. One example is that, during oilseed rape
embryo development, the expression of the JIM8 epitope
occurred in the two-celled stage (Pennell et al. 1991). A
second example is that, in developing Arabidopsis thaliana
embryos, JIM13 epitope was present in the embryo proper,
shoot apex meristem and basal part of suspensor (Hu et al.
2006). By provision of bGlcY, a synthetic reagent that
specifically binds to AGPs, into the A. thaliana ovule in
vitro culture medium, we showed that AGPs were involved
in A. thaliana embryo differentiation and shoot meristem
formation (Hu et al. 2006). It is interesting that in another
species, Streptocarpus prolixus, addition of bGlcY induced
variation of cotyledon during the process of seed germi-
nation (Rauh and Basile 2003). Therefore, we raised the
question of whether the function of AGPs in embryo dif-
ferentiation and cotyledon formation are non species-
specific or not. Since our previous studies showed that the
AGPs played roles in tobacco fertilization and zygote
division (Qin and Zhao 2006), a reasonable next step is to
now better define the functions of AGPs in tobacco embryo
development and cotyledon formation. In the present
paper, we further investigated the expression of AGPs in
different developmental stages of tobacco embryo and used
an in vitro ovule culture system supplemented with bGlcY
to disturb the function of AGPs. The results showed that
bGlcY influenced tobacco embryo development and coty-
ledon formation, which led to embryo morphologic defects.
Thus we deduced that some of AGPs’ functions are to
establish tobacco embryo bilateral symmetry and form
morphologically normal seedlings. Taken together with the
previous studies, which revealed AGPs function in aiding
embryo differentiation and cotyledom formation of A.tha-
liana and Streptocarpus prolixus, we hypothesize that
AGPs functions in these events are universal.
Materials and methods
Plant materials
Nicotiana tabacum L. (cv. Xanthi and SR1) plants were
grown under standard greenhouse conditions for 16 h in
daylight and 8 h in dark at 25�C. Flowers were artificially
pollinated during anthesis.
Isolation of embryos
Isolation of embryos was performed according to the
methods reported previously (Qin and Zhao 2006).
Tobacco ovules were dissected from ovaries and placed
into enzyme solution containing 6–13% mannitol, 1%
cellulose R-10 and 0.8% macerozyme R-10 (YAKULT
HONSHA CO., Japan), pH 5.7, then vibrationally incu-
bated for 30–60 min at 30�C on an oscillator (ZW-A,
FU-HUA). After washing three times with the same con-
centration of mannitol solution without the enzymes, the
macerated ovules were gently ground with a small glass
pestle. Embryos were released from ovules and collected
with a micropipette under an inverted microscope (Olym-
pus CK40, Japan).
214 Sex Plant Reprod (2007) 20:213–224
123
Localization of AGPs by immunofluorescence labeling
Isolated embryos were fixed in 50 mM PIPES buffer
pH 6.7 including 1.5% paraformaldehyde, 2 mM MgSO4�7H2O, 2 mM EGTA and 6–13% mannitol for 1 h at room
temperature. After rinsing three times with 50 mM PIPES
buffer (pH 6.7) containing 2 mM MgSO4�7H2O and 2 mM
EGTA and one time with 100 mM PBS pH 7.4, the fixed
samples were incubated in the primary Mb JIM13 diluted
at 1/10 in 100 mM PBS (pH 7.4) for 2 h at room temper-
ature. After rinsing three times with 100 mM PBS
(pH 7.4), the samples were then incubated for 1 h in
the dark with the secondary antibody, anti-rat-IgG-FITC
conjugate (Sigma) diluted at 1/100 with 100 mM PBS
(pH 7.4). The samples were rinsed three times with
100 mM PBS (pH 7.4) before microscopic examination
(Zhao et al. 2004). In the control tests, the samples were
incubated in 100 mM PBS (pH 7.4) instead of the primary
antibody Mb JIM13, and without the second antibody anti-
rat-IgG-FITC conjugate. Observation and photography
were made under a microscope (LEICA, DM IRB,
Germany) equipped with a charge coupled device (CCD,
OPTRONICS, USA).
Labeling of AGPs by bGlcY
Isolated embryos were fixed for 1 h at room temperature in
1.5% paraformaldehyde and 6–13% mannitol. After rinsing
three times with distilled water, the fixed samples were
incubated in 50 lM�bGlcY (Biosupplies Pty, Australia) for
7 h at room temperature. After rinsing three times with
distilled water, the samples were observed and photo-
graphed under a microscope (LEICA, DM IRB, Germany)
equipped with a charge coupled device (CCD, OPTRON-
ICS, USA).
Immunolocalization of AGPs by transmission electron
microscopy
The isolated embryos were fixed in a mixture of 3%
paraformaldehyde and 1% glutaraldehyde in 10 mM PBS,
pH 7.2 under vacuum for 2 h at room temperature and then
kept in fresh fixatives at 4�C overnight. After fixation, the
samples were rinsed three times with 10 mM PBS
(pH 7.2), and then dehydrated gradually in a series of
increasing concentration of ethanol at 10 (at 4�C), 30 (at
4�C and the following steps after 30% ethanol were con-
ducted at @20�C), 50, 70, 90 and 95% for 30 min at each
step, and 100% for 60 min for the last two steps of dehy-
dration. The samples were infiltrated with increasing ratio
of ethanol: Lowicryl K4M at 2:1, 1:1, and 1:2 for 12 h at
each step, followed by Lowicryl K4M without ethanol for
12 h at @20�C. The infiltration was finished with fresh
Lowicryl K4M for 1 day at @20�C. The samples were
transferred into capsules containing fresh Lowicryl K4M,
and cured under two 15-watt ultraviolet lamps (360 nm)
for at least 24 h at @20�C, and then curing continued under
UV light for 2 days at room temperature.
Ultrathin sections (60 nm) were prepared using a
Sorvall MT-6000 ultramicrotome and collected onto
Formvar-coated nickel grids. The sections were incubated
in the PBST buffer (60 mM PBS, 0.1% Tween-20, 0.02%
NaN3, pH 7.2) containing 0.2 M glycin and 1% BSA for
20 min to block non-specific binding and then incubated
with AGPs antibody JIM13 at 37�C for 3 h. The sections
were rinsed three times with PBST, and then incubated
with goat anti-rat IgG conjugated to 15-nm gold particles
(Sigma) at 1:100 dilutions for 1 h at 37�C. After washing
three times with PBST and three times with ddH2O, the
sections on grids were air-dried and post-stained with sat-
urated uranyl acetate for 15 min. Control sections were
treated similarly except that primary antibody was omitted.
The sections were examined and photographed under a
JEM 100/II transmission electron microscope.
Ovule culture
Ovaries with ovules at early globular embryo stage
(5 DAP) were sterilized with 70% ethanol for 0.5–1 min
and then with 2% NaOCl for 4 min. After rinsing 3–4 times
with sterile distilled water, the ovaries were cultured in the
MS medium supplemented with 6% sucrose and 2.5 g/l
phytagel, pH 5.8 at 25�C in the dark. The test samples were
cultured in medium with 10 or 100 lM bGlcY and the
controls were cultured in medium with or without 100 lM
bManY (a Yariv reagent incapable of binding AGPs). After
10 days of culture, ovules were moved to fresh MS med-
ium supplemented with 2% sucrose and 2.5 g/l phytagel,
pH 5.8 and then continued to culture till maturity at 25�C
in dark. To evaluate the embryos inside, some ovules were
placed into enzyme solution for isolating embryos. The
number of the different staged embryos was separately
counted and their percentages were calculated. The ovules
were germinated and the seedlings formed in medium and
then transplanted to soil in pots for tracking their continued
growth. Each experiment was repeated at least three times,
and the standard errors were calculated.
Transparentizing of cotyledons
To further investigate cotyledon venation patterns, we
transparentized cotyledon referring to the methods
Sex Plant Reprod (2007) 20:213–224 215
123
mentioned by Bougourd et al. (2000). The cotyledons were
dehydrated through an ethanol series (15, 50, 70 and 96%,
and two changes of 100% ethanol), for 15 min at each
concentration. After the samples were left in fresh 100%
ethanol at 4�C overnight, they were rehydrated in a similar
way with 96, 70, 50 and 15% ethanol for 15 min each time,
and finally with two changes of distilled water for 15 min
each time. The samples were stained in 0.5% aniline blue
solution for 30 min and then soaked in two changes of
water for 15 min each time. The cotyledons were soaked in
Hover’s solution (30 g arabic gum, 200 g chloral hydrate,
20 g glycerol, 50 ml water) till taking off the color of
aniline blue, and transferred onto clean slides. Then a cover
slip was gently lowered on to the mountant, taking care to
minimize the creation of air bubbles around the samples.
Observation and photography were performed under a
microscope (Olympus SZX13) equipped with a charge
coupled device (CCD, OPTRONICS, USA).
Results
Immunolocalization of AGPs in developing embryos
In our previous paper, we used a western blotting assay
with different monoclonal antibodies (Mb) JIM4, JIM13
and LM2, which showed that JIM13 reacted best with total
protein extracts from 5–8 DAP tobacco ovules (Qin and
Zhao 2006). Mb JIM13 was therefore used as a probe to
examine the distribution of AGP epitope in different
developmental stages of tobacco embryos. The results
revealed that the expression of AGPs changed along with
the development of embryos (Fig. 1). The two-celled
embryo, resulting from asymmetric zygote division was
evenly labeled by AGPs monoclonal antibody JIM13
(Fig. 1A). Then, a polar distribution pattern of AGPs was
detected in three-celled embryo. The fluorescence in the
basal cell of suspensor was stronger than that in the apical
cell of suspensor (Fig. 1B). In the 6-celled embryo, the
labeling fluorescence was again present in an even distri-
bution pattern (Fig. 1C). Through several cell divisions, the
several-celled embryo developed into an early stage of
globular embryo, which appeared in a polar fluorescent
distribution once more. Intense fluorescence of AGPs was
localized in the embryo proper (EP) (Fig. 1D). At the late
globular stage of embryo, the fluorescent labeling was even
more concentrated in the apex of the EP and at the juncture
of the EP and suspensor (Fig. 1E). When the embryo
developed into the heart-shaped embryo, the cotyledon
primordia formed. The fluorescence disappeared in the
apex of the EP, and was present only at the juncture of
the EP and suspensor (Fig. 1F). In torpedo-shaped embryo,
the fluorescence was mainly observed in the shortened
suspensor (Fig. 1G). Two types of control embryos un-
labeled with primary Mb JIM13 or secondary antibody
showed no green fluorescence (data not shown). In addi-
tion, bGlcY, which can react with AGPs and form red
deposit, was used as another tool for the localization of
AGPs in embryos. We found that the bGlcY staining
of developing embryos was consistent with the JIM13
immunofluorescent labeling. The late globular embryo
presented a red deposit in the apex of the EP and at the
juncture of the EP and suspensor (Fig. 1H, arrows). The
shortened suspensor of torpedo-shaped embryo also pre-
sented red labeling (Fig. 1I, arrow).
To detect the precise subcellular localization of AGPs
within cells, we applied immunogold labeling and TEM
techniques. Using Mb JIM13, in the late stage of globular
embryo, numerous gold particles were observed in the cell
wall of the EP adjacent to the suspensor and the incrassated
cell wall at the juncture of the EP and suspensor (Fig. 2A–
D). The abundant amount of gold particles was also
detected in the cell wall of the juncture of the EP and
suspensor of heart-shaped embryo (Fig. 2E–G). The con-
trol sections without incubation with Mb JIM13 showed no
gold particles (data not shown). Since the abundance of
gold particles was detected only in the cell wall of the
juncture of the EP and suspensor, it is confirmed that the
immunogold labeling result is consistent with the immu-
nofluorescent observation.
Effects of bGlcY on embryo development
and cotyledon formation in ovule culture
To examine the effects of AGPs on embryo development,
5 DAP undifferentiated ovules at early globular embryo
were cultured in medium supplemented with 10 and
100 lM bGlcY, and 100lM bManY (an isomer of
bGlcY, the isomerization of the hydroxyl group at carbon
atom 2 of the sugar, is non reactive with AGPs),
respectively. After 10 days of culture, the developmental
situation of embryos in ovules was divided into three
types: globular embryo, heart-shaped embryo and tor-
pedo-shaped embryo. The embryos in control without
bGlcY treatment, mostly developed into heart-shaped
embryos (Fig. 3a) and torpedo-shaped embryos (Fig. 3b)
with the frequencies 52 and 36%, respectively (Table 1).
Provision of 10 lM bGlcY to the medium increased the
number of undifferentiated globular embryos and abnor-
mal differentiated embryos. Interestingly, the heart-shaped
embryos (27%) and torpedo-shaped embryo (23%) dis-
played abnormities in the number, arrangement and shape
of cotyledon (Table 1). The abnormal cotyledon primor-
dia in the embryos were described as single cotyledon
primordia (Fig. 3e, f), asymmetric cotyledon primordia
216 Sex Plant Reprod (2007) 20:213–224
123
(Fig. 3j, k) and supernumerary cotyledon primordia
(Fig. 3o, p, as shown by arrows). When 100 lM bGlcY
was added into the medium, there was an increased
frequency of undifferentiated globular embryos and
abnormal embryos, but a decreased frequency in those of
differentiated heart-shaped and torpedo-shaped embryos
(Table 1). In the control test of bManY treatment, three
types of globular, heart-shaped and torpedo-shaped
embryos displayed similar proportions to the control with-
out treatment. The results revealed that bGlcY treatment
could cause delay of embryo development and abnormity
of cotyledon formation.
In the control, the embryos germinated and formed
seedlings with two symmetric cotyledons (Fig. 3c) and two
symmetric young leaves (Fig. 3d). In the treatment of
bGlcY, the embryos could mature and germinate, but
the germinated seedlings displayed defective cotyledons.
When 10 lM bGlcY was added to medium, the frequen-
cies 2.9 and 4% of the seedlings exhibited single cotyledon
and two asymmetry cotyledons, respectively. When bGlcY
Fig. 1 Localization of AGPs at
different developmental stages
of tobacco embryos by Mb
JIM13 immunofluorescence and
bGlcY labeling. The bright-field
micrographs (a–f) and their
fluorescent images (A–F) are
shown in each picture. H and Iare the bright-field micrographs
with the red reaction of bGlcY.
A A two-celled embryo showed
uniform and strong fluorescence
in apical and basal cells. B A
three-celled embryo displayed a
polar distribution pattern.
Intense fluorescence labeling
was examined in the basal part
of the suspensor. C A six-celled
embryo showed a distribution
pattern of faint and even green
fluorescence. D An early stage
of globular embryo showed a
polar distribution pattern again.
Intense fluorescence was
labeled in the embryo proper
(EP). E In the late globular
embryo, a polar fluorescence
labeling was detected in the
apex of the EP and the juncture
between the EP and suspensor.
F In the differentiated heart-
shaped embryo, the fluorescence
in the apex of the embryo
disappeared, but the
fluorescence in the juncture of
the EP and suspensor was still
strong. G A torpedo-shaped
embryo presented strong
fluorescence in the shortened
suspensor. H A late stage
globular embryo yielded red
deposit in the apex of the EP
and the juncture of the EP and
suspensor as shown by the
arrow. I A short suspensor of
torpedo-shaped embryo
displayed a red labeling as
shown by the arrow.
Bar = 20 lm
Sex Plant Reprod (2007) 20:213–224 217
123
concentration was increased up to 100 lM, the percentage
of abnormal seedlings increased (Table 2). These defective
seedlings had single (Fig. 3g–i), asymmetric (Fig. 3l–n) or
extra cotyledons (Fig. 3q–s). When using 100 lM bManY
instead of bGlcY in medium, the percentage of germinated
seedlings was similar to that of untreated samples
(Table 2). Thus, the results showed that bGlcY affected
cotyledon formation in embryo development, and its
effects were concentration-dependent.
To further examine the development of seedlings, the
germinated seedlings were transplanted to soil. The control
seedlings (Fig. 4G-a) were bigger and taller than bGlcY-
treated seedlings (Fig. 4G-b) due to their earlier germina-
tion. In bGlcY-treated plants, some seedlings were
abnormal and could not grow up and consequently tended
to die (Fig. 4G-1–4). However, the seedlings with normal
shoot apical meristem all developed and reproduced seeds,
no matter which defect the cotyledons had (Fig. 4G-5–7).
Effects of bGlcY on the vasculature differentiation
of cotyledons
The effect of bGlcY on cotyledon formation was further
investigated by observing the cotyledon vasculature in
various types of seedlings. Cotyledon venation structure in
control seedlings was normal, which has one main vein and
several venules branching off from it (Fig. 4A), but
abnormal in bGlcY-treated seedlings which had various
accessorial venations (Fig. 4B-F). In the seedlings with
single regular (Fig. 4B) or irregular cotyledon (Fig. 4C),
some accessorial venations occurred in the cotyledon. The
larger cotyledon in two asymmetric cotyledons sometimes
displayed double main-venation patterns (Fig. 4D), and
sometimes more adjunctive main-veins (Fig. 4E), while the
smaller one contained only one central main-venation. In
the multiple cotyledon-like seedlings, vascular tissues
seemed to be more disorganized (Fig. 4F). Therefore, it
Fig. 2 TEM
immunolocalization of AGP
epitope recognized by JIM13 in
late globular embryo and heart-
shaped embryo of tobacco.
A A semithin section of late
globular embryo. B An ultrathin
section of late globular embryo
showing the region in the
juncture of the EP and
suspensor, which is a
magnification of the square in
A. C, D Magnified images of
the areas indicated by the
squares on the right (C) and left(D) side in B. Numerous gold
particles appeared in the
incrassated cell wall of the
embryo proper cell adjacent to
the suspensor. E An ultrathin
section of heart-shaped embryo
showed the juncture of the EP
and suspensor. F A magnified
image of the right square in E.
A number of gold particles were
examined in the EP cell wall
adjacent to the suspensor cell.
G A magnified image of the leftsquare in E. Abundant gold
particles were observed in the
EP outer cell wall adjacent to
the suspensor cell.
A bar = 20 lm; B–Gbar = 1 lm. A amyloplast,
C cytoplasm, CW cell wall,
EP embryo proper, EPC embryo
proper cell, S suspensor,
SC suspensor cell, SN suspensor
nucleus, V vacuole
218 Sex Plant Reprod (2007) 20:213–224
123
indicates that the disturbance of AGPs with the treatment
of bGlcY affect the organization of vascular tissues in
seedling cotyledons.
Discussion
Roles of AGPs in embryo development
Several monoclonal antibodies against the carbohydrate
epitopes of AGPs have been extensively used to examine
AGPs expression and their potential functions (Knox
1997). JIM13, which was originally prepared from immu-
nizations with proteins of the embryogenic carrot
suspension cells, is a rat monoclonal antibody that recog-
nizes b-D-GlcpA-(1?3)-a-D-GalpA-(1?2)-L-Rha epitope
found on AGP core proteins (Showalter 2001). The
developmentally regulated expression pattern of AGPs in
embryos was explored in this paper using immunolocali-
zation technique with Mb JIM13 and histochemical
localization method with bGlcY. It is known that bGlcY
reacts with the majority of AGPs. Moreover, JIM13 and
bGlcY can recognize different AGPs epitopes (Roy et al.
1998). In our tests, it is interesting that the labeling of
AGPs with Mb JIM13 in developing embryos was similar
to that of bGlcY. This implies that JIM13-reactive AGPs
are the most fundamental subset of AGPs in embryos.
In the early globular embryo of tobacco, AGPs were
Fig. 3 Effects of bGlcY on
tobacco embryo development
and cotyledon formation of five
DAP ovules after 10 days of
culture and phenotypes of
germinated seedlings. a–dEmbryos cultured in the control
medium without bGlcY; e–sEmbryos cultured in the
medium with 100 lM bGlcY.
a A normal heart-shaped
embryo differentiated from
globular embryo. b A normally
formed torpedo-shaped embryo.
c A normally germinated
seedling containing two
symmetric cotyledons. d A
normal seedling containing two
symmetric leaves. e–f Abnormal
embryos possessing single
cotyledon primordia. g–iAbnormal seedlings
characterized as
monocotyledonous. j An
abnormal heart-shaped embryo
containing two asymmetric
cotyledon primordia. k An
abnormal torpedo-shaped
embryo containing two
asymmetric cotyledons. l–mAbnormal seedlings having two
asymmetric cotyledons. n An
Abnormal seedling containing
two asymmetric cotyledons and
two asymmetric leaves. o–pAbnormal embryos comprising
supernumerary cotyledon
primordia as shown by the
arrows. q–s Abnormal seedlings
containing supernumerary
cotyledons. a, b, e, f, j, k, o, pbar = 20 lm; c, d, g, h, l, n, q, sbar = 1 mm
Sex Plant Reprod (2007) 20:213–224 219
123
immunofluorescently labeled in the EP. The late globular
embryo close to differentiation presented intense distri-
bution of AGPs in the apex of the EP. The instantaneous
high level of accumulation of AGPs may result from the
recruitment of signal in the apex cells of the EP for
cotyledon initiation. During the transition of embryo dif-
ferentiation from the globular to heart-shaped stage, AGPs
may function in determining the cell fates. The provided
bGlcY disturbed the function of AGPs and caused abnor-
mal cotyledon formation. Our results indicated that AGPs
participated in the embryo developmental and differential
processes.
We also found that an abundance of AGPs was presented
at the juncture of the EP and suspensor in the globular and
heart-shaped embryos of tobacco. This polar AGPs locali-
zation reflected the importance of AGPs in the interaction
between the embryo proper and the suspensor, including
cell communication, signal transduction and material
transportation. Characterization of Arabidopsis develop-
mental mutants showed that interaction between the embryo
proper and the suspensor is of central importance for
embryo development (Vernon et al. 2001). The sus and rsp
mutants displayed aberrant embryo proper development
followed by suspensor cell proliferation and development of
inviable cell masses that resemble the mutant embryo
proper (Schwartz et al. 1994; Yadegari et al. 1994). In the
twn2 mutant, the embryo proper degenerated early in
development process, but the suspensor cells survived,
entered into embryogenic development and formed one or
more embryos (Zhang and Somerville 1997). It was clear
from the sus, rsp and twn2 phenotypes that cells of the
suspensor had embryogenic potential, and this potential was
normally suppressed by interaction with the embryo proper.
Despite the importance of the embryo proper-suspensor
interaction, little is known about the nature of cell com-
munication between these two parts of embryos in higher
plants. The ultrastructure immunocytochemistry localiza-
tion in this study showed that the numerous AGPs
accumulated in the cell wall adjacent to the suspensor in the
EP cells of late globular and heart-shaped embryos. It was
reported that the proteins involved in secretion and cell wall
synthesis were required for normal embryo development in
Arabidopsis (Scheres and Benfey 1999; Vroemem et al.
1999). AGPs, hydroxyproline-rich glycoproteins (HRGPs),
Pro-rich proteins and Gly-rich proteins are known to be the
four major classes of structural cell wall proteins (Showalter
1993; Cassab 1998). Recently, a cell wall protein, RSH,
which is one kind of HRGPs was demonstrated to be
essential for normal embryo development (Hall and Cannon
2002). The higher concentration of AGPs in the cell wall of
Table 2 Effects of bGlcY on germination of tobacco embryos at 5 DAP cultured in vitro
Treatments No. of
cultured
ovules
Percentage of germination (%)a
Total Normal cotyledon Types of abnormal cotyledons
Single cotyledon Asymmetry
cotyledon
Supernumerary
cotyledon
Control 266 80.76 : 7.60 80.46 : 7.33 0.30 : 0.52 0 0
10 lM bGlcY 171 78.79 : 2.09 71.72 : 1.82 2.93 : 0.02 4.12 : 0.27 0
100 lM bGlcY 264 76.39 : 5.83 53.01 : 13.70 6.16 : 1.99 14.66 : 6.71 3.16 : 2.18
100 lM bManY 253 76.61 : 6.41 76.61 : 6.41 0 0 0
a Experiments were repeated at least three times
Table 1 Effects of bGlcY on development of tobacco embryos at 5 DAP cultured in vitro
Treatments No. of
isolated
embryos
Percentage of
globular embryos (%)
Percentage of
heart-shaped
embryos (%)
Percentage of
torpedo-shaped
embryos (%)
Control 177 11.30 : 2.10 52.54 : 4.25 36.16 : 2.44
10 lM bGlcY 115 16.52 : 2.63 57.39 : 4.08 26.09 : 3.89a27.27 : 2.44 a23.33 : 3.57
100 lM bGlcY 102 36.27 : 5.39 39.22 : 2.56 24.51 : 5.34a32.50 : 1.69 a32.00 : 4.23
100 lM bManY 129 17.05 : 3.17 51.16 : 3.11 31.78 : 2.44
Experiments were repeated at least three timesa Presenting the abnormal percentage of the embryos
220 Sex Plant Reprod (2007) 20:213–224
123
the juncture of EP and suspensor supported a hypothesis
that AGPs functioned in the interaction of both and played a
critical role in maintaining normal embryo development.
Thus, we suggested that the supplemented bGlcY induced a
negative effect in the interaction and consequently caused
delay of embryo development.
Roles of AGPs in cotyledon formation
In this paper, defective cotyledon morphologies, including
abnormal number, asymmetry and abnormal venation
patterns, were observed in bGlcY-treated embryos and
seedlings. It was also shown that the inhibitory activity of
Fig. 4 Effects of bGlcY on the
vasculature of cotyledon and
growth of seedlings after they
were transplanted to soil. A A
normal cotyledon with a central
venation in control conditions
without the treatment of bGlcY.
B–F Phenotypes of the
vasculatures in abnormal
cotyledons induced by 100 lM
bGlcY. B A single regular
cotyledon with accessorial
main-venations (arrow). C A
single irregular cotyledon with
accessorial main-venations
(arrow). D, E In seedlings with
two asymmetric cotyledons,
the large cotyledon displayed
double venations or more
adjunctive venations (arrows).
F A multiple cotyledon seedling
showed disorganized
vasculature. G a Control
seedlings without the treatment
of bGlcY, appearing bigger and
taller than bGlcY-treated
seedlings. G b Seedlings
induced by 100 lM bGlcY.
G1–7 Magnified images of
the bGlcY-treated seedlings in
Fig. Gb. The 1–4 deformed or
irregular single cotyledon
seedlings could not grow up and
consequently died. The 5–7seedlings could revert to normal
type and survive. Bar = 1 mm.
Lcot large cotyledon, Scot small
cotyledon
Sex Plant Reprod (2007) 20:213–224 221
123
bGlcY on embryo development and organ differentiation is
concentration dependent. We presumed that AGPs act as a
guidance signal. Because we found that a suppression of
AGPs, by the addition of bGlcY, caused cotyledon defect,
which led to mislocalization of cotyledon-forming areas
and apical cells into the differentiated region. Through
manipulation of the amount or types of AGPs in the culture
medium or using the advantage of the synthetic reagent
bGlcY to perturb the function of AGPs, it was exhibited
that AGPs were involved in different embryonic stages of
various plants, such as follows, somatic embryogenesis in
carrot, cyclamen, and Norway spruce (Kreuger and van
Holst 1996). Other reports have sited AGPs participation in
zygotic embryo development in Nicotiana tabacum L.,
embryo differentiation and shoot meristem formation in
A. thaliana, and cotyledon formation in Streptocarpus
prolixus (Qin and Zhao 2006; Hu et al. 2006; Rauh and
Basile 2003). In the present paper, we demonstrated that
AGPs also function in aiding in embryo differentiation and
cotyledon formation in Nicotiana tabacum L. Taken
together, it was plausible to hypothesize that AGPs
functions in plant embryo initiation, development, differ-
entiation, and pattern formation are universal. These
characteristics of AGPs further demonstrated that these
ubiquitous complex macromolecules had a fundamental
and an important function in higher plant development.
Many Arabidopsis mutations that cause defects in
cotyledon patterns have been identified. For example,
the gurke mutant helped to identify putative ‘‘patterning
genes’’ which were essential for the proper development of
the embryo’s apical region (Torrez-Ruiz et al. 1996). The
amp1 mutation resulted in embryos with deformed or extra
cotyledons (Chaudhury et al. 1993). The pin1, pid, twn1,
xtc1 and xtc2 mutations caused variable phenotypes
including cotyledon fusion, deformities and formation of
three or more cotyledons (Okada et al. 1991; Bennett et al.
1995; Conway and Poethig 1997; Vernon et al. 2001). The
cuc mutants produced fused cotyledons or cup-shaped
cotyledon that encircles the embryonic apex (Aida et al.
1997). AGPs are present in the cotyledon tissues in Vigna
radiata, and addition of endogenous (beta)-arabinogalactan
into medium increased the frequency of shoot differentia-
tion in ‘‘Cot’’ cotyledon type of explants (Das and Pal
2004). In the process of seed germination, supplementation
of bGlcY induced variation of cotyledon in Streptocarpus
prolixus (Rauh and Basile 2003). What is the molecular
mechanism of AGP-mediate cotyledon differentiation? At
what time does AGPs begin to function in this process?
How AGP genes interact with these known genes to
manipulate cotyledon formation is still unclear.
Using the ovule culture system with the advantage to be
cultured to maturity at high efficiency combined with our
embryo dissection technique, we were able to show that the
disturbance of AGPs-induced tobacco seedling cotyledon
defects was initiated by embryo differentiation. This event
might occur at the time of the transition stage from glob-
ular embryo to heart-shaped embryo. A possible suggestion
for how AGPs can affect cotyledon formation is that AGPs
influence cell fate in the embryonic apex as a part of hor-
mone-mediating cell interaction in embryos, especially
between the EP and the suspensor and within the embryo
apex. Hormones are prime candidates for mediating cell
interactions. It was reported that auxin polar transport and
PIN genes were essential for the establishment of bilateral
symmetry and shoot apical meristem function (Liu et al.
1993; Blilou et al. 2005). However, the interactions of
AGPs and hormone molecules in embryo development are
far from understood. There are many challenging problems
to be solved. Due to lack of AGP mutants, embryo-speci-
ficity and incomplete penetrance of the mutation remain to
be tackled. Further studies are expected to elucidate the
precise molecular mechanisms underlying the functions of
AGPs in embryogenesis and cotyledon differentiation.
For further tracking the growth of the cotyledon defec-
tive seedlings induced by the treatment of bGlcY, we
transplanted them to soil. The results showed that the
severely affected irregular single cotyledon seedlings also
had a defect in their shoot apical meristem. The abortion of
this shoot apical meristem defective seedling indicates that
the shoot apical meristem is significant in plant develop-
ment. A large number of genes have been identified to be
involved in shoot meristem formation in Arabidopsis. The
SHOOT MERISTEMLESS (STM) gene is expressed in the
two cotyledon primordia and is involved in shoot meristem
organization throughout plant development (Barton and
Poethig 1993; Endrizzi et al. 1996). The CUC1, CUC2 and
NAM (NO APICAL MERISTEM) genes also affect the
initiation of shoot apical meristem (Takada et al. 2001;
Aida et al. 1997; Vroemen et al. 2003; Souer et al. 1996).
To date, a number of AGP and AGP-like genes have been
identified, but there is a lack of evidences of their functions
in embryo development, cotyledon differentiation, and
shoot meristem formation, even though it still is presum-
able that some AGP genes are involved in processes of
plant morphogenesis and interact with other genes to reg-
ulate coordinately these events.
Roles of AGPs in cotyledon venation formation
Cotyledon venation patterns in control seedlings and
bGlcY-treated seedlings were compared in this paper.
Accessorial venations were observed in the abnormal cot-
yledon of bGlcY-induced seedlings. This result suggested
that the venation pattern might assist in defining the coty-
ledon development. It was reported that xylogen is an
222 Sex Plant Reprod (2007) 20:213–224
123
AGP. It played a role in directing the cotyledon vascular
development in Arabidopsis (Motose et al. 2004). Mb
JIM13 and bGlcY both bind to xylogen. More experiments
need to be conducted to determine whether the variation of
venation patterns in tobacco cotyledon is due to the
removal of xylogen by bGlcY treatment or other AGPs. In
Arabidopsis, a complex venation pattern was also detected
in the defective cotyledon of the twn1 mutant (Vernon et al.
2001), and disorganized vascular tissue was observed in
gnom mutant (Geldner et al. 2003). Such aberrant vascular
patterns could be interpreted in two ways: they could be a
result of extensive organ fusion, or they could reflect
plasticity in vascular development. This is why abnormally
larger cotyledons developed more complex and extended
different venation patterns.
Acknowledgments The authors thank Dr J.P. Knox (Centre for
Plant Sciences, University of Leeds, UK) for the generous gifts of
the antibodies. This project was supported the Major State Basic
Research Program of China (2007CB108704) and the National
Natural Science Foundation of China (30521004, 30770132).
References
Aida M, Ishida T, Fukaki H, Fujisawa H, Tasaka M (1997) Gene
involved in organ separation in Arabidopsis: an analysis of the
cup-shaped cotyledon mutant. Plant Cell 9:841–857
Barton MK, Poethig RS (1993) Formation of the shoot apical
meristem in Arabidopsis thaliana: An analysis of development in
the wild type and in the shoot meristem mutant. Development
119:823–831
Bennett SRM, Alvarez J, Bossinger G, Smyth DR (1995) Morpho-
genesis in pinoid mutants of Arabidopsis thaliana. Plant J 8:505–
520
Berleth T, Jurgens G (1993) The role of the monopteros gene in
organising the basal body region of the Arabidopsis embryo.
Development 118:575–587
Blilou I, Xu J, Wildwater M, Willemsen V, Paponov I, Friml J,
Heldstra R, Aida M, Plame K, Scheres B (2005) The PIN auxin
efflux facilitator network controls growth and patterning in
Arabidopsis roots. Nature 433:39–44
Bougourd S, Marrison J, Haseloff J (2000) An aniline blue staining
procedure for confocal microscopy and 3D imaging of normal
and perturbed cellular phenotypes in mature Arabidopsisembryo. Plant J 24:543–550
Cassab GI (1998) Plant cell wall proteins. Annu Rev Plant Physiol
Plant Mol Biol 49:281–309
Chapman A, Blervacq AS, Vasseur J, Hilbert JL (2000) Arabinoga-
lactan-proteins in Cichorium somatic embryogenesis: effect of
b-glucosyl Yariv reagent and epitope localization during embryo
development. Planta 211:305–314
Chaudhury AM, Letham S, Craig S, Dennis ES (1993) Amp1-a
mutant with high cytokinin levels and altered embryonic pattern,
faster vegetative growth, constitutive photomorphogenesis and
precocious flowering. Plant J 4:907–916
Cheung AY, Wu HM (1999) Arabinogalactan proteins in plant sexual
reproduction. Protoplasma 208:87–98
Cheung AY, May B, Kawata EE, Gu Q, Wu HM (1993) Character-
ization of cDNAs for stylar transmitting tissue-specific proline-
rich proteins in tobacco. Plant J 3:151–160
Conway LJ, Poethig RS (1997) Mutations of Arabidopsis thalianathat transform leaves into cotyledons. Proc Natl Acad Sci USA
94:10209–10214
Das S, Pal A (2004) Differential regeneration response in two
cotyledon types of Vigna radiata: Histomorphological analysis
and effect of (beta)-arabinogalactan. J Plant Biochem Biotechnol
13(2):101–106
Egertsdotter U, Arnold SV (1995) Importance of arabinogalactan
proteins for the development of somatic embryos of Norway
spruce (Picea abies). Physiol Plant 93:334–345
Endrizzi K, Moussian B, Haecker A, Levin J, Laux T (1996) The
SHOOT MERISTEMLESS gene is required for maintenance of
undifferentiated cell in Arabidopsis shoot and floral meristems
and acts at a different regulatory level than the meristem genes
WUSCHEL and ZWILLE. Plant J 10:967–979
Geldner N, Richter S, Vieten1 A, Marquardt S, Torres-Ruiz RA,
Mayer U, Jurgens1 G (2003) Partial loss-of-function alleles
reveal a role for GNOM in auxin transport-related, post-
embryonic development of Arabidopsis. Development
131:389–400
Hall Q, Cannon MC (2002) The cell wall hydroxyproline-rich
glycoprotein RSH is essential for normal embryo development in
Arabidopsis. Plant Cell 14:1161–1172
Hu Y, Qin Y, Zhao J (2006) Localization of an arabinogalactan
protein epitope and the effects of Yariv phenylglycoside during
zygotic embryo development of Arabidopsis thaliana. Proto-
plasma 229:21–31
Knox JP (1997) The use of antibodies to study the architecture and
developmental regulation of plant cell walls. Int Rev Cytol
171:79–120
Kreuger M, van Holst GJ (1993) Arabinogalactan-proteins as
essential in somatic embryogenesis of Daucus carota L. Planta
189:243–248
Kreuger M, van Holst GJ (1996) Arabinogalactan proteins and plant
differentiation. Plant Mol Biol 30:1077–1086
Liu CM, Xu ZH, Chua NH (1993) Auxin polar transport is essential
for the establishment of bilateral symmetry during early plant
embryogenesis. Plant Cell 5:621–630
Lukowitz W, Mayer U, Jurgens G (1996) Cytokinesis in the
Arabidopsis embryo involves the syntaxin-related KNOLLE
gene product. Cell 84:61–71
Majewska-Sawka A, Nothnagel EA (2000) The multiple roles of
arabinogalactan proteins in plant development. Plant Physiol
122:3–9
Motose H, Sugiyama M, Fukuda H (2004) A proteoglycan mediates
inductive interaction during plant vascular development. Nature
429:873–878
Okada K, Ueda J, Komaki K, Bell CJ, Shimura Y (1991) Requirement
of the auxin polar transport system in early stages of Arabidopsisfloral bud formation. Plant Cell 3:667–684
Pennell RI, Janniche L, Kjellbom P, Scofield GN, Peart JM, Roberts
K (1991) Developmental regulation of a plasma membrane
arabinogalactan protein epitope in oilseed rape flowers. Plant
Cell 3:1317–1326
Rauh RA, Basile DV (2003) Phenovariation induced in Streptocarpusprolixus (Gesneriaceae) by b-glucosyl Yariv reagent. Can J Bot
81:338–344
Roy S, Jauh GY, Hepler PK, Lord EM (1998) Effects of Yariv
phenylglycoside on cell wall assembly in the lily pollen tube.
Planta 204:450–458
Qin Y, Zhao J (2004) The role of arabinogalactan-proteins in sexual
reproduction of angiosperms. J Plant Physiol Mol Biol 30:371–378
Qin Y, Zhao J (2006) Localization of arabinogalactan-proteins in egg
cells, zygotes and two-celled proembryos and effects of b-D-
glucosyl Yariv reagent on egg cell fertilization and zygote
division in Nicotiana tabacum L. J Exp Bot, 57:2061–2074
Sex Plant Reprod (2007) 20:213–224 223
123
Scheres B, Benfey P (1999) Asymmetric cell division in plants. Annu
Rev Plant Physiol Plant Mol Biol 50:505–537
Schwartz BW, Yeung EC, Meinke DW (1994) Disruption of
morphogenesis and transformation of the suspensor in abnormal
suspensor mutants of Arabidopsis. Development 120:3235–3245
Showalter AM (1993) Structure and function of plant cell wall
proteins. Plant Cell 5:9–23
Showalter AM (2001) Arabinogalactan-proteins: structure, expression
and function. Cell Mol Life Sci 58:1399–1417
Souer E, van Houwelingen A, Kloos D, Mol J, Koes R (1996) The NoApical Meristem gene of petunia is required for pattern
formation in embryos and flowers and is expressed at meristem
and primordia boundaries. Cell 85:159–170
Stacey NJ, Roberts K, Knox JP (1990) Patterns of expression of the
JIM4 arabinogalactan-protein epitope in cell cultures and
during somatic embryogenesis in Daucus carota L. Planta
180:285–292
Takada S, Hibara K, Ishida T, Tasaka M (2001) The CUP-SHAPEDCOTYLEDON1 gene of Arabidopsis regulates shoot apical
meristem formation. Development 128:1127–1135
Thompson HJM, Knox JP (1998) Stage-specific responses of
embryogenic carrot cell suspension cultures to arabinogalactan
protein-binding b-glucosyl Yariv reagent. Planta 205:32–38
Torres-Ruiz RA, Jurgens G (1994) Mutations in the FASS gene
uncouple pattern formation and morphogenesis in Arabidopsis
development. Development 120:2967–2978
Torres-Ruiz RA, Lohner A, Jurgens G (1996) The GURKE gene is
required for normal organization of the apical region in the
Arabidopsis embryo. Plant J 10:1005–1016
Tykarska T (1979) Rape embryogenesis. II. Development of embryo
proper. Acta Soc Bot Pol 48:391–421
Vernon DM, Hannon MJ, Le M, Forsthoefel NR (2001) An expanded
role for the TWN1 gene in embryogenesis: defects in cotyledon
pattern and morphology in the twn1 mutant of Arabidopsis(Brassicaceae). Am J Bot 88:570–582
Vroemen C, de Vries S, Quatrano R (1999) Signalling in plant
embryos during the establishment of the polar axis. Semin Cell
Dev Bio 10:157–164
Vroemen CW, Mordhorst AP, Albrecht C, Kwaaitaal MA, de Vries
SC (2003) The CUP-SHAPED COTYLEDON3 gene is required
for boundary and shoot meristem formation in Arabidopsis. Plant
Cell 15:1563–1577
Yadegari R, Depaiva GR, Laux T, Koltunow AM, Apuya N,
Zimmerman JL, Fischer RL, Harada JJ, Goldberg RB (1994)
Cell differentiation and morphogenesis are uncoupled in Ara-bidopsis raspberry embryos. Plant Cell 6:1713–1729
Zhang JZ, Somerville CR (1997) Suspensor-derived polyembryony
caused by altered expression of valyl-tRNA synthase in the twn2
mutant of Arabidopsis. Proc Nat Aca Sci USA 94:7349–7355
Zhao J, Mollet JG, Lord EM (2004) Lily (Lilium longiflorum L.)
pollen protoplast adhesion is increased in the presence of the
peptide SCA. Sex Plant Repord 16:227–233
224 Sex Plant Reprod (2007) 20:213–224
123