Development of the Endosperm of Myrsine laetevirens (Myrsinaceae). I.

Cellularization and
Deposition of Cell-Wall Storage Carbohydrates
Author(s): Marisa Otegui, Carlos Lima, Sara Maldonado, Rosa M. de Lederkremer
Source: International Journal of Plant Sciences, Vol. 160, No. 3, (May, 1999), pp. 491-500
Published by: The University of Chicago Press
Stable URL: http://www.jstor.org/stable/2995776
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Int. J. Plant Sci. 160(3):491-500. 1999.
C) 1999 by The University of Chicago. All rights reserved.
1058-5893/99/6003-0005$03.00

DEVELOPMENT
OF THE ENDOSPERMOF MYRSINELAETEVIRENS
(MYRSINACEAE).
1.
AND DEPOSITIONOF CELL-WALL
CELLULARIZATION STORAGECARBOHYDRATES
Marisa Otegui,* Carlos Lima,t Sara Maldonado,t and Rosa M. de Lederkremer't

*Laboratorio
de MorfologfaVegetal,Facultadde CienciasNaturalesy Museo, UniversidadNacionalde La Plata,Paseo del Bosques/n,
1900 La Plata, Argentina; +CIHIDECAR(CONICET),Departamento de Qufmica Organica, Facultad de Ciencias Exactas y Naturales,
Universidad de Buenos Aires, Pabell6n 2, Ciudad Universitaria, 1428 Buenos Aires, Argentina;
and tinstituto de Recursos Biol6gicos, INTA, 1712 Castelar, Argentina

Myrsine laetevirens has a nuclear type of endosperm development. Vacuole formation in peripheral coe-
nocytic cytoplasm precedes cytokinesis and compartmentalizes the cytoplasm. Cellularization starts as a series
of anticlinal walls projecting centripetally. Periclinal cell walls are formed from expanding cell plates following
nuclear divisions. The developing walls are flanked by endoplasmic reticulum cisternae with dilated and short
profiles and few ribosomes. Subsequent anticlinal wall growth and periclinal divisions elongate the endosperm
into the central vacuole, resulting, finally, in the convergence of the advancing tissue. After that, the endosperm
increases in size by anticlinal, periclinal, and oblique divisions in all cells of the endosperm and involving
phragmoplast and cell plate. Carbohydrate deposition in the cell walls begins 40 d after pollination (DAP)
with deposition of pectins in the cell layers next to the embryo. At ca. 50 DAP, the cell walls of the bulk
endosperm begin to thicken, showing the presence of xyloglucans. The deposition of new cell-wall material
is achieved by the activity of the Golgi stacks located near the cell walls. Neutral sugars were determined after
two conditions of acid hydrolysis. Monosaccharides were identified by high-performance anion exchange
chromatography (Dionex, Sunnyvale, Calif.). Arabinose, xylose, glucose, and galactose were the main mono-
saccharides, but their ratio changes during development. Methylation analysis of cell-wall polysaccharides
accords with the occurrence of significant amounts of xyloglucans. The presence of pectic polysaccharides can
be inferred from the amounts of uronic acids, galactose, arabinose, and rhamnose.

Keywords: carbohydrates, endosperm cellularization, endosperm development, Myrsine laetevirens,

xyloglucans.

Introduction seeds have been chemicallyand histologicallycharacterized.

Development of the endosperm has been studied in many
families of angiosperms. In Myrsinaceae, the endosperm de-
velopment conforms to the nuclear type (Johri et al. 1992),
i.e., the endosperm is initially coenocytic, and cellularization
occurs later to form a solid tissue (Vijayaraghavan and Prab-
hakar 1984; Werker 1997). This information comes mainly
from three studies: one on Aegiceras majus, a mangrove with
exalbuminous seeds (Carey and Fraser 1932), and the other
two on Ardisia crispa (De Jongh 1938; Miller 1990). On the
other hand, all investigated species of the Primulales, the order
to which Myrsinaceae belongs, are characterized by the pres-
ence of xyloglucans in the mature endosperm cell walls (Kooi-
man 1960; Hegnauer 1969, 1973; Reid 1985). In Myrsinaceae,
on the basis of the reaction with iodine-potassium iodide, xy-
loglucans have been detected in the endosperm cell walls of
species of Ardisia, Maesa, and the Myrsine-Rapanea complex
(Kooiman 1960; Hegnauer 1969, 1973). Up to now, only in
Myrsine laetevirens, cell walls of the endosperm of mature
1 Author for correspondence and reprints; e-mail lederk@
qo.fcen.uba.ar;fax 5411576-3346.
ManuscriptreceivedSeptember1998; revisedmanuscriptreceivedDecember
1998.
(Otegui et al. 1998). However,xyloglucandepositionin the
endospermcell walls has not been studiedin speciesof Myr-
sinaceaeor in speciesbelongingto otherfamiliesof Primulales.
The aim of the presentwork was to investigatethe endo-
spermdevelopmentin M. laetevirensto establishwhethercell-
wall formationandsubsequentdepositionof xyloglucansagree
with previousfindingsin other speciesof angiosperms.Struc-
turalinvestigationusinghistochemicalmethodsandTEMwas
complementedwith chemical analyses. This investigationis
part of a monographictreatmentof the genus Myrsine.
Material and Methods
Plant Material
Developingand mature seeds were harvestedduringFeb-
ruarythroughApril,in 1996 and 1997, froma wild population
of Myrsine laetevirens (=Rapanea laetevirens). The vouchers
(Otegui 53, 55, 56) are deposited in LP: Argentina,Prov.
BuenosAires,PuntaLara.
Seedswere chemicallyanalyzedat 10, 20, 30, 40, 50, 60,
70, 80, and 90 d after pollination(DAP).

491
492 INTERNATIONAL JOURNAL OF PLANT SCIENCES
Light and Electron Microscopy
Small blocks of seed tissues were fixed for 3 h in 2.5%
glutaraldehyde in 0.1 M phosphate buffer pH 7.5 at 4?C. Fresh
tissues and those that had been embedded for TEM were also
used for light microscopy. The staining procedures used on
fresh and fixed tissues included toluidine blue 0 (Feder and
O'Brien 1968), periodic acid-Schiff reagent (PAS) for poly-
saccharides (O'Brien and McCully 1981), ruthenium red for
pectic substances (O'Brien and McCully 1981), and iodine-
potassium iodide for xyloglucans (Kooiman 1960). For TEM,
tissues were postfixed in 1% OS04 for 4 h, dehydrated in a
graded ethanol-acetone series, and embedded in Spurr's resin.
Sections were mounted on grids, stained with uranyl acetate
followed by lead citrate, and examined in a Turbo Zeiss EM
109.
Chemical Analysis
Endosperm was separated from 70 to 75 seeds of each stage.
The tissues were dried in a vacuum desiccator over NaOH
pellets for 7 d, until they reached a constant weight, and were
ground to a fine powder in a mortar. Lipids were extracted as
described in a preceding article (Otegui et al. 1998), and pro-
teins were extracted following the method of Gifford et al.
(1982).
Neutral sugar and uronic acid analyses. The delipidated
and deproteinated pellets, composed mainly of cell-wall car-
bohydrates, were washed with water and dried. Neutral sugars
were determined by the phenol-sulfuric acid method, with glu-
cose as a standard (Dubois et al. 1956). Samples of endosperm
(2 mg) from each stage were treated with 2 mL of 99% tri-
fluoroacetic acid at 37?C for 18 h with stirring. An aliquot
(0.5 mL) was transferred to a glass vial and diluted with water
(1 mL); inositol (2 mg/mL, 0.5 mL) was added. This sample,
for hemicellulose hydrolysis, was heated at 100?C for 30 min
and then evaporated to dryness (condition a). A second aliquot
(0.5 mL) for cellulose hydrolysis was diluted with water (0.1
mL), inositol was added as above, and heated for 30 min. The
addition of water (0.1 mL) was repeated at 30-min intervals
until a total volume of 0.5 mL was added, and heating con-
tinued for a further 2 h (condition b) (Morrison 1988). Samples
were evaporated under reduced pressure with repeated addi-
tions of water and suspended in 1 mL of water. After centrif-
ugation, 10 ,uL of the supernatants were used for analysis by
high-performance anion exchange chromatography (HPAEC)
(Dionex, Sunnyvale, Calif.) equipped with pulsed ampero-
metric detection. For the separation of monosaccharides, a
Carbopac PA-1 column with a guard column was used. Elution
was performed isocratically with a 20 mM solution of NaOH.
Carbohydrate peaks were identified by comparison with re-
tention times of standards and by cochromatography. The data
were analyzed using A-I-450 chromatography software
(Dionex).
Uronic acids were determined by the sulfamate/m-hydroxy-
diphenyl method (Filisetti-Cozzi and Carpita 1991) with gal-
acturonic acid as a standard.
Methylation analysis. Samples were methylated by the
method of Ciucanu and Kerek (1984), with modification de-
scribed by Ferguson (1993). Hydrolysis was performed with
2N trifluoroacetic acid at 110?C for 3 h. Partially methylated
sugars were reduced with NaBH4, acetylated with acetic an-
hydride/pyridine, and separated by gas-liquid chromatography
on a capillary column SP2330 (0.25 mm x 30 m; Supelco,
Bellefonte, Pa.) with a temperature program of 1600-2100C at
2?C/min, then from 2100 to 2400C at 5?C/min (Shea and Car-
pita 1988). The injector temperature was 2250C, and the de-
tector temperature was 2500C. Gas-liquid chromatogra-
phy-mass spectrometry was performed using a TRIO-2VG
Masslab at 70 eV, with a SP2330 column similar to that de-
scribed above. The temperature program was from 1400 (2
min) to 2350C at 8?C/min;the injector temperature was 2400C,
and the detector temperature, 2500C.
Results
General Changes during Development
Three major phases could be recognized in the endosperm
development of Myrsine laetevirens: (1) free nuclear division,
(2) cellularization, and (3) reserve deposition.
Following fertilization, the primary endosperm nucleus and
its derivatives underwent nuclear divisions. After several nu-
clear divisions, the coenocytic endosperm became arranged in
a peripheral cytoplasm surrounding the large central vacuole
(figs. 1.1, 1.2). Nuclear divisions were followed by vacuolation
of the cytoplasm that became organized as thin strands (fig.
1.3). At 6 DAP, the peripheral layer contained ca. 400 nuclei.
At this time, two events occurred almost simultaneously: (1)
cellularization started as a series of free-growing anticlinal
walls projecting centripetally, and (2) the first periclinal nuclear
divisions were initiated (fig. 1.4). Unlike the anticlinal cell
walls, most observed periclinal cell walls were formed from
expanding cell plates following nuclear divisions. Subsequent
periclinal divisions elongated the endosperm into the central
vacuole, resulting finally in the convergence of the advancing
tissue at 20 DAP (fig. 1.5).
Electron microscopy of the anticlinal cell walls revealed very
thin and crooked cell walls extending along the cytoplasmic
strands. The developing walls were flanked by endoplasmic
reticulum cisternae that showed dilated and short profiles with
few ribosomes. Developing walls were also associated with the
presence of Golgi bodies with five to seven cisternae and nu-
merous electron-lucent Golgi-derived vesicles (figs. 2.6, 2.7).
The cytoplasm strands also contained numerous vesicles, vac-
uoles, mitochondria, free ribosomes, and smooth endoplasmic
reticulum (fig. 2.8). The walls had a fibrillar middle lamella
(figs. 2.6, 2.7, 2.9) and contained many membrane-bound ves-
icles (figs. 2.9, 2.10) that seemed to incorporate their contents
into the developing wall (fig. 2.9). Then the endosperm in-
creased in size by anticlinal, periclinal, and oblique divisions
involving phragmoplast and cell plates occurring in all cells of
the endosperm (fig. 3.12). Cell walls were PAS negative at this
stage. At 30 DAP, cell division was confined to the outer
regions, adjacent to the integument. These outer endosperm
cells were smaller than the inner ones (fig. 3.13). At this stage,
endosperm cell walls showed positive PAS reaction, and the
inner layers of the integument were digested (fig. 3.13).
Following completion of division (40 DAP), the endosperm
consisted of highly vacuolated cells with very thin primary
walls (fig. 3.14), and deposition of cell-wall storage carbo-
 OTEGUI ET AL.-DEVELOPMENT OF ENDOSPERM CELL WALLS 493

Fig. 1 Lightmicrographsof the developingendospermof Myrsine laetevirens (stainedwith toluidineblue 0). CS = cytoplasmicstrands;
I = integument;P = placentae;V = vacuole.Fig. 1.1, Longitudinalsectionof an ovule afterfertilization(4 DAP).The coenocyticendospermis
arrangedin a peripheralcytoplasm(arrow)surroundingthe centralvacuole.Bar= 100 rim.Fig. 1.2, Two telophasesduringfree-nucleardivision
stage (4 DAP). Mitotic spindles(arrows)can be easily seen. Bar= 10 tim. Fig. 1.3, Coenocyticendospermis vacuolatedand surroundsthe
centralvacuole;peripheralcytoplasmis organizedin thin strands(6 DAP).Bar= 10 zm. Fig. 1.4, Endospermafterthe firstpericlinaldivision.
Anticlinalwalls are freelyformingalong the cytoplasmicstrands(10 DAP).Bar= 10 ,um.Fig. 1.5, Endospermafterseveralpericlinaldivisions
(20 DAP). Bar= 10,usm.

hydratewas initiatedin a thin zone immediatelyaroundthe
embryo (figs. 2.11, 3.15). In this area, cell walls showed a
strongpositivereactionwith rutheniumred,denotingthe pres-
enceof pectins.At 50 DAP,the cell wallsof the bulkendosperm
began to thickenand showed a positivereactionwith iodine-
potassiumiodide, indicatingxyloglucans.The wall thickening
started at the corner of the cells (fig. 3.16) and continued
throughoutthe entirecell walls, except for the regionsof plas-
modesmata(fig.3.17). The depositionof cell-wallmaterialwas
associatedwith activityof the Golgi stacks (figs. 4.18, 4.19)
located near the cell walls (fig. 4.19) that producedvesicles
with electron-densefibrillarcontents (figs. 4.18-4.20). These
contentswere incorporatedinto the thickeningwall (fig.4.21),
in which plasmodesmatacould be easily distinguished(fig.
 A ER Qv"' ~~~A4

v~

6 ~~~~~~~~~~~~~~~~~~~~~~~~,.~~~~~~~~~~~~~Q

Fig. 2 Transmission electron micrographs of developing endosperm. Fig. 2.6, Developing cell wall with a fibrillar middle lamella (arrows).
Cell walls are flanked by endoplasmic reticulum cisternae (ER) and associated with Golgi stacks (G) and numerous electron-lucent Golgi-derived
vesicles (20 DAP); cytoplasm is rich in free ribosomes. Bar = 0.2 ym. Fig. 2.7, Detail of figure 2.6. Bar = 0.2 ,um. Fig 2.8, Cytoplasm showing
numerous vesicles and some vacuoles (V); smooth endoplasmic reticulum cisternae (ER) is in a concentric arrangement (20 DAP). Bar = 0.1
ym. Fig. 2.9, Membrane-bound vesicle (arrow) incorporating its content into the developing wall (20 DAP). Bar = 0.2 ,sm. Fig. 2.10, Several
vesicles are included in a developing cell wall (20 DAP). Bar = 0.2 ytm. Fig. 2.11, Detail of an endosperm cell facing the embryo; very thick cell
walls (CW) and lipid bodies (L) can be observed (40 DAP). Bar = 1 ym.
 -~~~~ 5, jrw

Fig. 3 Light micrographs of sections of the developing endosperm. Sections were stained with toluidine blue 0. Fig. 3.12, Detail of a division
in a central endosperm cell. At this time (25 DAP), endosperm is completely cellularized, and further cell divisions involve phragmoplast and
cell plate (arrow). Bar = 10 jim. Fig. 3.13, Detail of the integument and the endosperm (30 DAP). Cell division is occurring mainly in the outer
cells (arrows). Integument is partially digested. E, endosperm; I, integument. Bar = 100 jim. Fig. 3.14, Detail of a cell with a central nucleous,
several vacuoles, and thin cell walls (40 DAP); cells have reached the definitive size. Bar = 10 ,um. Fig. 3.15, Detail of the cell layers next to the
embryo; thick cell walls rich in pectins can be observed in the inner endosperm cells (IEC) (40 DAP). Bar = 10 jim. Fig. 3.16, Detail of the
central cells of the endosperm. Deposition of storage cell-wall carbohydrates has been initiated. The wall thickening started at the corner of the
cells (arrows) (50 DAP). Bar = 10 jim. Fig. 3.17, Detail of central cells of the endosperm showing very thick cell walls (CW).Plasmodesmata
are indicated by arrows. Bar = 10 jim.
 ... Im~~~~~~~~~~~~~~~~

e ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~
4~~~~~~~~~~~~~~~~~4~~~4

Fig.4 Transmissionelectronmicrographsof cell-wallcarbohydratedeposition.Fig.4.18, Golgistackandvesicleswith electron-dense fibrillar

contentscan be seen. Bar= 0.1 ,um.Fig. 4.19, Golgi stack (G) locatednearthe cell walls (CW).Bar= 0.1 p4m.Fig. 4.20, Golgi-derivedvesicles
with electron-densefibrillarcontents. Bar= 0.1 Z.m.Fig. 4.21, Golgi-derivedvesicles incorporatingtheir contents into the cell wall (CW).
Bar = 0.1 psm.Fig. 4.22, Primarypit field with threeplasmodesmata(arrows)is shown in a thick endospermcell wall (CW).Bar= 0.5 ,um.
 OTEGUI ET AL.-DEVELOPMENT OF ENDOSPERM CELL WALLS 497

4.22). The carbohydrate depositions were complete at 60-70

16 *.*endosperm weight(mg) 800
DAP, as walls reach final thickness. The integument was di-
gested and compacted in very thin layers by the time the en- 14 -U*-mg neutralsugars/ seed
-1-- mg neutralsugars/ g 600
dosperm reached the final volume (70-80 DAP). The endo-
sperm-weight variation during endosperm development is D 10 t 600
shown in figure 5a. 8 -400 .

Neutral Sugarsand Uronic Acids

4 200
Total neutral sugars were determined in the delipidated and
deproteinated pellets during endosperm development (fig. Sa). 2
Both, on a milligram-per-gram dry weight or milligram-per-
a0 0
50 ; 500
seed basis, neutral sugars increased until 75-80 DAP, after
which the level was maintained to maturity (90 DAP). The 40 ..400
highest quantities of neutral sugars per seed were reached at
80-90 DAP, when the carbohydrate fraction amounted to L" 30--- 300
64.7% of the endosperm weight.
HPAEC analysis showed that glucose, xylose, galactose, and 2 20 > 200
arabinose are the main monosaccharides in the endosperm cell /:C~~~~~-C- pg uronic acids/ g
walls during seed development under both conditions of acid 10 _ dryweight - 100
hydrolysis (table 1). Under condition a (hemicellulose hydro-
b__ _- -g uronicacids/ seed

lysis) and condition b (cellulose hydrolysis), the molar pro-

portions of glucose and xylose increased with the endosperm
development, while those of fucose, arabinose, and rhamnose
decreased. Only a small fraction of glucose was released under
condition a. Under condition b, galactose decreased from 33 %
at 10 DAP to 15%-18% in the next stages. However, under
condition a, galactose increased from 12%-17% in the first
stages of development to almost 45% at 90 DAP.
Uronic acids (fig. Sb) increased on a per-gram dry weight
basis during the first stages (10-30 DAP) and then decreased
until ca. 70 DAP, after which minor change occurred. On a
per-seed basis, this fraction increased following a sigmoidal
pattern until ca. 80 DAP; this level was maintained to maturity
(90 DAP).
MethylationAnalysis
Complete methylation of the cell-wall preparations was in-
dicated by the absence of undermethylated monosaccharides
in the hydrolysates. The type of linkages, inferred by gas-liquid
chromatography-mass spectrometry analysis of the partially
methylated alditols, was similar in all developmental stages of
the endosperm (table 2). The bulk of the glucosyl residues were
4-0-linked in the first stages (10-30 DAP) with significant
amounts of 4,6-di-O-linked and terminal residues. In the fol-
lowing stages (40-90 DAP), the most abundant glucosyl res-
idues were 4,6-di-O-linked. Xylosyl residues were mainly ter-
minal and 2-0-linked. The content of terminal xylosyl residues
was highest at 40 DAP (37%), after which the relative pro-
portion decreased until 12%-25%. Galactosyl residues were
mainly terminal, and 2-0-linked units were detected as traces.
Arabinofuranosyl residues were terminal and 5-0-linked.
Discussion
As in Aegiceras (Carey and Fraser 1932) and Ardisia (de
Jongh 1938; Miller 1990), the endosperm development in Myr-
sine laetevirens corresponds to the nuclear type.
The organelles found in the endosperm of M. laetevirens
during the first stages of development, i.e., abundant endo-
10 20 30 40 50 60 70 80 90
Days after pollination
Fig.5 Quantitativechangesin endospermweightandneutralsug-
ars (a) and uronic acids (b) duringthe developmentof Myrsine lae-
tevirens endosperm. Each plotted value is the mean of three
determinations? SD.
plasmic reticulum, membrane-bound vesicles, high quantities
of ribosomes, very active Golgi stacks, and mitochondria, are
essentially the same as those reported in developing endosperm
of Stellaria media (Newcomb and Fowke 1973), Gossypium
hirsutum (Jensen et al. 1977), Ipomoea purpurea, Cytinus by-
pocistis (Ponzi and Pizzolongo 1984), and Euphorbia dulcis
(Gori 1987). In fact, they are typical of a rapidly growing
system. We have found in the developing endosperm of M.
laetevirens endoplasmic reticulum cisternae in close proximity
to the developing cell walls; Gori (1987) reported the same
observation in the endosperm of Euphorbia dulcis and related
it to the trapping of Golgi-derived vesicles. Dute and Peterson
(1992) suggest that endoplasmic reticulum cisternae in Glycine
max are associated with the formation of plasmodesmata. The
origin and development of walls of the endosperm were studied
by various authors (Mares et al. 1975, 1977; Morrison and
O'Brien 1976; Jensen et al. 1977; Morrison et al. 1978; Olson
1981; Fineran et al. 1982; Vijayaraghavan and Prabhakar
1984; Gori 1987; Dute and Peterson 1992; Olsen et al. 1992;
Brown et al. 1996). The first-forming walls that compartmen-
talize the endosperm are often described as "free growing" to
indicate that they do not form from a cell plate (Brown et al.
1996). Despite numerous studies, the process itself remains
largely unexplained. The periclinal cell-wall formation is also
a topic of discussion. Some investigators (Morrison and
O'Brien 1976; Newcomb 1978; Chamberlin and Horner 1990)
suggested that periclinal walls may originate from branching
and fusing of free-growing anticlinal walls. Brown et al.
(1996), on the basis of studies in cereals and dicots, have re-
ported that there is a general and unique pattern of develop-
498 INTERNATIONAL JOURNAL OF PLANT SCIENCES

Table 1

Sugars from Endosperm Cell Walls at Different Stages of Development

Sugars and hydroly- DAP

sis conditions 10 20 30 40 50 60 70 80 90
Fucose:
Condition a ...... 2.48 4.02 2.73 7.28 1.79 2.44 1.75 2.31 0.98
Condition b ...... 1.27 1.38 1.02 1.09 0.53 1.14 0.91 0.62 0.42
Rhamnose:
Condition a ...... 1.02 0.91 1.14 1.55 0.50 0.48 tr tr tr
Condition b ...... 1.59 1.27 0.83 0.84 0.32 0.32 tr tr tr
Arabinose:
Condition a ...... 58.53 54.04 35.87 36.74 14.79 18.35 11.29 11.75 7.88
Condition b ...... 12.04 8.82 8.10 7.11 2.42 1.89 2.51 0.70 0.92
Xylose:
Condition a ...... 17.22 23.10 29.91 28.14 40.26 38.11 40.72 39.75 37.28
Condition b ...... 9.49 11.48 20.90 19.49 22.10 21.27 25.14 20.41 24.27
Galactose:
Condition a ...... 17.37 12.89 27.49 25.22 41.87 38.55 44.43 44.83 44.93
Condition b ...... 33.08 22.92 17.11 14.81 15.73 16.29 18.24 17.20 17.73
Glucose:
Condition a ...... 3.37 5.05 2.87 1.06 0.81 2.08 1.81 1.37 8.92
Condition b ...... 42.53 54.12 52.04 56.66 58.90 59.11 53.19 61.07 56.65
Note. DAP = days after pollination. tr = traces. Abundance of sugars is given in molar proportion (mol %). Conditions a and b are defined
in "Material and Methods."

ment of periclinalcell walls that, like meristematiccells, in-
volves the presenceof an expandingphragmoplastfollowing
mitosis in the alveoli. Our observationsindicate that in M.
laetevirensendosperm,most periclinalcell walls developfrom
a plate cell. However,the formationof cell walls and nuclear
divisionsdoes not seem to follow a strictlysynchronouspat-
tern, as in cereals(Olsenet al. 1992, 1995; Brownet al. 1996;
Olsen 1998)
One of the majorfunctionsof the Golgi apparatusin plants
is the biosynthesisof polysaccharidesfor the cell walls, the
two majorclassesbeingxyloglucansand the acidicpecticpoly-
saccharides.Accordingto Staehelinand Moore (1995), the
biosynthesisof polysaccharidesis confinedexclusivelyto the
Golgi stack-trans-Golginetwork units. All polysaccharides
follow the default pathway to the cell surface, where they
become integratedinto the extracellularmatrix.Accordingly,
we have observedthat during the initial phases of cell-wall
formation,when deposition of storage cell-wall polysaccha-
rides takes place, the Golgi stacks are numerous and show
intensive activity. However, the Golgi-derived vesicles showed
contents with different aspects: during the phase of cell-wall
formation, vesicle contents were electron lucent, but during
deposition of storage polysaccharides, they were fibrillar and
electron dense.
The presence of xyloglucans in the cell walls could be in-
ferred from the identification of 4-0-linked and 4,6-di-0-
linked glucosyl residues, terminal and 2-0-linked xylosyl res-
idues, and terminal galactosyl residues that are all character-
istic glycosidic linkages of xyloglucans (Reid 1985; Redgewell
and Selvendran 1986; York et al. 1990; Cutillas-Iturralde et
al. 1998). In the first stages of development (10-20 DAP), most
of the glucosyl residues are 4-0-linked, showing that they de-

Table 2
Methylation Analysis of the Cell Wall from Different Stages of Endosperm Development
DAP
0-methyl sugars Linkage types 10 20 30 40 50 60 70 80 90
2,3,5-arabinose ....... Terminal 5.08 7.45 3.45 4.94 1.16 2.67 1.93 1.75 2.21
2,3-arabinose .......... 5-0-linked 0.71 2.42 3.70 1.58 0.72 0.78 0.46 1.46 1.67
2,3,4-xylose ........... Terminal 11.18 11.42 11.07 37.39 12.48 25.72 18.24 20.96 17.48
3,4-xylose ............. 2-0-linked 15.93 12.38 9.14 13.05 9.11 11.40 13.61 12.40 14.16
2,3,4,6-galactose ...... Terminal 12.20 7.69 8.03 19.05 13.11 18.92 13.36 13.28 13.80
3,4,6-galactose ........ 2-0-linked tr tr tr tr tr tr tr tr tr
2,3,4,6-glucose ........ Terminal 7.12 5.41 11.28 3.53 1.62 3.40 2.31 2.97 3.13
2,3-glucose ............ 4,6-di-O-linked 5.08 8.05 19.81 11.29 35.95 19.41 34.68 27.78 27.78
2,3,6-glucose .......... 4-0-linked 42.70 45.19 33.52 9.17 25.84 17.71 15.41 20.61 19.87
Note. DAP = days after pollination; tr = traces. Abundance of partially methylated sugars is given in molar proportion (mol %).
 OTEGUI ET AL.-DEVELOPMENT
rive from celluloseor from a xyloglucanwith low branching
by xylosyl units. The increaseof 4,6-di-O-linkedglucosylres-
idues along the endospermdevelopmentwould indicate the
progressivebranchingwith xylosyl residuesand furtherdep-
osition of xyloglucan.
Accordingto Waldronand Selvendran(1990), the presence
of pectic polysaccharidescan be inferredfrom the amountof
uronicacids, galactose,arabinose,and rhamnose,with rham-
nose diagnosticfor pectins.We found rhamnose,althougha
very minor component, in the monosaccharidecomposition
analysisand differencesin the amountsof galactoseand arab-
inose betweenmonosaccharidecompositionand methylation
analyses.Both facts could be explainedconsideringthat the
methylationof pectic polysaccharidesis often difficultdue to
degradationon prolongedexposureto the strongalkalinecon-
ditions, and some of the degradedpectic fragmentsmay be
lost duringthe process.This resultsin underestimatingsome
oligosaccharidesidechainscontaininggalactoseandarabinose
linkedto the galacturonanbackbone(Waldronand Selvendran
1990).
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