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).

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