Download The Boron Requirement and Cell Wall Properties

Plant Physiol. (1998) 117: 1401–1410
The Boron Requirement and Cell Wall Properties of
Growing and Stationary Suspension-Cultured
Chenopodium album L. Cells1
Axel Fleischer, Christine Titel, and Rudolf Ehwald*
Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, Institut für Biologie,
Invalidenstrasse 42, 10115 Berlin, Germany
Suspension-cultured Chenopodium album L. cells are capable of
continuous, long-term growth on a boron-deficient medium. Compared with cultures grown with boron, these cultures contained
more enlarged and detached cells, had increased turbidity due to
the rupture of a small number of cells, and contained cells with an
increased cell wall pore size. These characteristics were reversed by
the addition of boric acid (>7 mM) to the boron-deficient cells. C.
album cells grown in the presence of 100 mM boric acid entered the
stationary phase when they were not subcultured, and remained
viable for at least 3 weeks. The transition from the growth phase to
the stationary phase was accompanied by a decrease in the wall
pore size. Cells grown without boric acid or with 7 mM boric acid
were not able to reduce their wall pore size at the transition to the
stationary phase. These cells could not be kept viable in the stationary phase, because they continued to expand and died as a result of
wall rupture. The addition of 100 mM boric acid prevented wall
rupture and the wall pore size was reduced to normal values. We
conclude that boron is required to maintain the normal pore structure of the wall matrix and to mechanically stabilize the wall at
growth termination.
The ultrastructure and physical properties of plant cell
walls are known to be affected by boron deficiency (Kouchi
and Kumazawa, 1976; Hirsch and Torrey, 1980; Fischer and
Hecht-Buchholz, 1985; Matoh et al., 1992; Hu and Brown,
1994; Findeklee and Goldbach, 1996). Moreover, boron is
predominantly localized in the cell wall when plants are
grown with suboptimal boron (Loomis and Durst, 1991;
Matoh et al., 1992; Hu and Brown, 1994; Hu et al., 1996). In
radish, .80% of the cell wall boron is present in the pectic
polysaccharide RG-II (Matoh et al., 1993; Kobayashi et al.,
1996), which is now known to exist as a dimer that is
cross-linked by a borate ester between two apiosyl residues
(Kobayashi et al., 1996; O’Neill et al., 1996). Dimeric RG-II
is unusually stable at low pH and is present in a large
number of plant species (Ishii and Matsunaga, 1996; Kobayashi et al., 1996, 1997; Matoh et al., 1996; O’Neill et al.,
1996; Pellerin et al., 1996; Kaneko et al., 1997). The widespread occurrence and conserved structure of RG-II (Darvill et al., 1978; O’Neill et al., 1990) have led to the sugges1
This research was supported by grant no. Eh 14471-1 from the
Deutsche Forschungsgemeinschaft, Bonn, Germany.
* Corresponding author; e-mail [email protected].
de; fax 49 –30 –20 –93– 8635.
tion that borate ester cross-linked RG-II is required for the
development of a normal cell wall (O’Neill et al., 1996;
Matoh, 1997).
One approach for determining the function of boron in
plant cell walls is to compare the responses to boron deficiency of growing plant cells that are dividing and synthesizing primary cell walls with those of growth-limited
plant cells in which the synthesis of primary cell walls is
negligible. Suspension-cultured cells are well suited for
this purpose because they may be reversibly transferred
from a growth phase to a stationary phase. Continuous cell
growth phase is maintained by frequent transfer of the cells
into new growth medium (King, 1981; Kandarakov et al.,
1994), whereas a stationary cell population is obtained by
feeding the cells with Suc and by not subculturing them.
Cells in the stationary phase are characterized by mechanically stabilized primary walls and reduced biosynthetic
activity. Here we describe the responses of suspensioncultured Chenopodium album L. cells in the growth and
stationary phases to boron deficiency. These cells have a
high specific-growth rate, no significant lag phase, and
reproducible changes in their wall pore size during the
transition from the growth phase to the stationary phase
(Titel et al., 1997).
MATERIALS AND METHODS
Cell Culture
Chenopodium album L. cells (strain C.9.1. described by
Knösche and Günther, 1988) were grown on a modified
Murashige and Skoog medium (Murashige and Skoog,
1962) containing KH2PO4 (0.4 g L21), Suc (40 g L21), myoinositol (100 mg L21), thiamine hydrochloride (0.4 mg L21),
2,4-D (0.3 mg L21), and 6-furfurylaminopurine (0.1 mg
L21). The boron concentration of the standard medium was
varied as required. Cultures (150 mL) were grown at 27°C
in 500-mL flasks under dim light on a rotary shaker at 200
rpm. Media with low boron concentrations (#7 mm) were
prepared in autoclavable polypropylene containers with
bidistilled water from concentrated stock solutions (macroelements, 20-fold; microelements, 100-fold; and Suc, 10fold) that had been assayed for the absence of boron (deAbbreviations: FAV, free apoplasmic volume; RG-II, rhamnogalacturonan II.
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Fleischer et al.
Plant Physiol. Vol. 117, 1998
tection limit, 2 mm) by inductively coupled plasma-atomic
emission spectrometry (Unicam 701, Unicam Ltd., Cambridge, UK). Suc stock solutions were passed over a column with a boron-absorbing ion-exchange resin (IRA-743,
Sigma) before autoclaving (20 volumes/bed volume). All
reagents used were of analytical grade. At subcultivations
cells grown at low boron concentration or “without boron”
remained in the same quartz vessels, and the harvested
suspension volume was replaced by fresh boron-deficient
medium. The boron content in fresh medium was determined colorimetrically using curcumin after extraction of
boric acid from the acidified medium with hexanediol/
chloroform (Mair and Day, 1972). The procedure was modified by reducing the dye concentration from 0.375% to
0.05% and by using water to extract the uncomplexed dye
from the organic phase. This method has a detection limit
of 0.03 mm boron.
Propagation Culture
Cultures that had been previously subcultivated weekly
(1:6 dilution) were subcultivated at high frequency (2:5
dilution every 2nd d). Under these conditions the transition
to the stationary phase did not occur because the cells were
diluted with fresh medium before the nutrients became
limiting and cell number reached an inhibiting level (Fig.
1a, curve 1). This high-frequency subcultivation ensured
that the culture was continuously maintained (.180 subcultivations) in the growth phase and was used to grow
cells in the absence of added boron and at different boron
concentrations. The final biomass concentration (ct) at subcultivation time and the initial biomass concentration (co)
were constant (see Fig. 1b) and, therefore, the mean
specific-growth rate (m 5 1/2 d [ln ct 2 ln 2]) equals the
mean dilution rate (r 5 1/2 d [ln 5 2 ln 2] 5 0.46 d21). This
specific-growth rate is only slightly below the maximum
growth rate of C. album.
Transition to the Stationary Phase
A sterile solution of Suc (5 mL, 400 g L21) was added to
cells (100 mL) that had been subcultured every 2nd d in
propagation culture for more than 20 cycles, and the cells
were then maintained axenically without the addition of
fresh medium. These conditions initially generated cells in
transition from the growing phase to the stationary phase
and then cells in the stationary phase. The cells were considered to be in the stationary phase when their fresh and
dry weights did not increase (Fig. 1a, curve 2) and there
was no mitosis. The cells were maintained for long periods
in the stationary phase by the addition of Suc when the
concentration of solubles decreased to less than 0.5% on the
Suc scale of the refractometer. Cell viability remained high
even though the growth rate decreased rapidly when the
cells were maintained on standard medium containing 100
mm boron. The pH of the medium changed from 5.2 (autoclaved medium) to approximately 3.8 during the 1st d,
increased to approximately 4.9 by the 2nd d, and reached
approximately 6.0 in the stationary phase.
Figure 1. Changes in the dry weights of C. album cells during their
growth phase and during the transition from the growth phase to the
stationary phase. a, Changes in the dry weights of growing cells that
were diluted with fresh medium every 2 d (curve 1). Dry weight
increase of the cells in transition from the growth phase to the
stationary phase (curve 2); before the transition to the stationary
phase, the cells had been maintained for 60 passages in the growth
phase (100 mM boric acid). The dilution of the cells with fresh
medium was at time 0, and at d 2 Suc (2 g/100 mL) was added. b,
Maximum dry weights of cells grown continuously with a high
frequency of subcultivation (every 2 d) at different boron concentrations. Dry weights (Dry Wt.) were determined before subcultivation.
The time in days is the length of time that the cells had been grown
continuously at the given boron concentration. The upper x axis is
the time for cells grown with 100 and 7 mM boron and the lower x
axis is the time for cells grown in the absence of added boron.
Cell Viability and Release of Organelles into the Medium
Cell viability was determined qualitatively by mixing
small volumes of the cells with Evans blue (Gaff and
Okong’o-Ogola, 1971) to give a dye concentration of 0.05%.
Dead cells stained deeply and were readily distinguished
from viable cells after an incubation time of 10 min. The
portion of dead cells was quantified by determining the
FAV. This is a measure of the volume of dead cells and cell
walls in the cell clusters and was determined, by a polarimetric method, as the difference between the partition
volumes of methyl b-d-arabinopyranoside (Sigma) and
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Boron Requirement of Growing and Stationary Chenopodium album L. Cells
Dextran T250 (Pharmacia) in filtered cells and expressed on
a fresh-weight basis (Fleischer and Ehwald, 1995). The
release of organelles from dead cells caused the turbidity of
the medium to increase and was estimated from the A490 of
the filtered medium.
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(Fleischer and Ehwald, 1995) and the cells have a spherical
shape. The single cells obtained by the disintegrating treatment were more or less spherical.
RESULTS
Determination of the Fresh and Dry Weights of Cells
At the designated sampling times cell cultures (10 mL)
were filtered through a G2 glass frit and the turbidity and
Suc concentration (refractive index) of the filtrate were
determined. The cells were washed with deionized water
(2 3 10 mL) and then drained with a flow of air before
determination of their fresh weights. The cells were dried for
24 h at 105°C and their dry weights were then determined.
Generation of Denatured Cells for Analysis of Particle
Size and Wall Pore Size
Growth of C. album Cells on Media with Different
Boron Concentrations
C. album cells have a mean specific-growth rate of approximately 0.5 d21 during the first 2 d of batch culture in
standard medium and the stationary phase is reached by
the 5th d (compare Titel et al., 1997). When a borondeficient medium is used, the cells die after the third or
fourth transfer at the end of the growth phase, although
there is no marked inhibition in dry-weight increase before
loss of cell viability. Cell death in the batch-culture regime
is preceded by an increase in cell size and a slight decrease
Cells for particle and wall pore size measurements were
denatured by treating packed cells on a filter with a solution (1 bed volume) containing ethanol (80%), water (19%),
and acetic acid (1%), and then with an excess of 80%
ethanol. The denatured cells were suspended in 96% ethanol and stored at 6°C.
Determination of the Pore Size of Cell Walls Using
Dextran-Permeation Analysis
Denatured and ethanol-saturated cells (from approximately 2 g fresh weight of cells) were rehydrated and
thoroughly washed on a polypropylene filter (thickness,
0.5 mm; pore size, 35 mm) with 1 mm CaCl2 and then with
potassium phosphate, pH 7.0, containing 100 mm NaCl and
0.05% NaN3. The excess water was removed by gentle
suction and the material was then treated for 30 min with
a polydisperse dextran-probing solution (1.5–2.0 mL). The
size dependence of dextran partitioning was analyzed by
size-exclusion chromatography, as described by Woehlecke
and Ehwald (1995) and Titel et al. (1997).
Particle-Size Analysis of C. album Cells and Cell Clusters
Ethanol-saturated cells were washed with 1 mm CaCl2 to
remove the ethanol. The cell clusters were disaggregated to
single cells by sonicating (240 W, 35 kHz, 30°C) a suspension of cells (1 volume) for 1 h in 5% chromic acid (10
volumes) in a Sonorex Super sonicator (Bandelin GmbH,
Berlin, Germany). The suspension was then sheared by two
passages through a fine needle (0.8 mm in diameter and 40
mm in length). Light-microscopic analysis showed that the
cell clumps were completely disintegrated to single cells.
The volume fractions of different particle size classes in
disintegrated and nondisintegrated cell clusters were determined with a laser analyzer (Analysette 22, Fritsch
GmbH, Idar-Oberstein, Germany).
Cell numbers in the suspension were calculated from the
fresh weight and the size distribution of the cells in the
disintegrated samples. The following assumptions were
used: the cell volume per gram fresh weight is 0.7 mL
Figure 2. Particle-size distribution curves of disaggregated and untreated clusters of growth-phase C. album cells. a, Particle-size distribution curves of suspension-cultured C. album cells after disaggregating treatment without boron (2B) or with 100 mM boron (1B). The
particle-size distribution after disaggregating treatment is equivalent
to the cell-size distribution. b, Particle-size distribution curves of
untreated suspension-cultured C. album cell clusters grown with or
without boron. The peak at approximately 45 mm (2) corresponds to
single cells that have separated from cell clusters during their cultivation. Particle size is equivalent to the diameter of a spherical
particle and was determined with a laser particle-size analyzer.
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1404
Fleischer et al.
Figure 3. Particle-size distribution profiles of the polydisperse
dextran-probing solutions after equilibration with denatured growthphase cells. Denatured cells derived from cells grown in the presence
of 100 mM boron (top) and in the absence of added boron (bottom)
were equilibrated for 30 min with the polydisperse dextran-probing
solution. The particle-size distribution profile of the dextrans was
modified by their partial diffusion into the cell lumina. Untreated (or.)
and treated dextran solutions were fractionated by size-exclusion
chromatography on a Superdex 200 HR 10/30 column. The eluate
was monitored with a polarimetric detector. The dextran concentration (c) is given in arbitrary units. The Stokes’ radii of the dextrans
were derived from their elution times using a calibration function
(Woehlecke and Ehwald, 1995).
boron was done four times and in all cases a specific
growth rate of 0.46 d21 was maintained.
Cells growing without added boron and those growing
with 7 and 100 mm boron differed visually even though
their growth rates were similar and all cultures did not
contain a significant fraction of dead cells. Cultures without added boron contained increased numbers of single
cells or cell pairs and, most notably, highly enlarged cells
(Figs. 2 and 4). Because the cells are larger under boron
deficiency, the smaller number of cells per gram fresh
weight (4.23 3 107 in the boron-deficient culture and 5.91 3
107 in the control culture) may be correlated with fewer
chromoplasts on a volume and dry-weight basis. This may
explain the observed reduction in flavonol content (to 75%
of the control). Growing cells without boron consistently
caused a discernible increase in the turbidity of the medium. This turbidity increase was less pronounced in the
growing cultures than in cultures at the stationary phase,
but in both cases was caused by the release of organelles
through cell bursting.
The size limits of the cell wall for permeation by dextrans
were determined by treating the denatured cells with a
polydisperse dextran-probing solution (Woehlecke and Ehwald, 1995). Large dextran molecules are excluded from
the cells and remain in the probing solution, whereas
smaller dextrans penetrate the wall into the cell lumina and
are partially removed from the probing solution. The dextrans that did not diffuse into the cell lumina were analyzed by high-performance size-exclusion chromatography
(Fig. 3). The elution profiles of the cell-exposed dextran
solutions contained “steps,” more gradual changes attributable to the size-dependent diffusion of the dextrans into
the cell lumina (Fig. 3). The curves were analyzed as de-
Table I. The wall-pore size limits of growing and stationary C.
album cells
Cells were grown in media containing different boric acid concentrations. Samples were taken 2 d after their last subcultivation
(growing cells) or after a subsequent 4 d of cultivation in the presence
of Suc (stationary cells).
Phase and Size Limit
in flavonol content, and is accompanied by a strong increase in the turbidity of the liquid medium.
C. album cells can be kept continuously in their growth
phase by subculturing every 2nd d at a dilution rate of 2:5.
Therefore, the effect of boron deficiency on cell division
and growth was determined under these conditions. Cells
were first grown on standard medium and on a medium
containing 7 mm boron. After approximately 50 transfers
the cells’ ability to grow in the absence of added boron
(,0.1 mm) was determined. Somewhat unexpectedly, the
cells grew in the absence of added boron and their dry
weights did not differ significantly from those of cells
grown in the presence of 7 and 100 mm boron (Fig. 1b). The
growth rate of the boron-deficient cells remained constant
for more than 180 transfers. The transfer of cells from the
boron-containing medium to the medium without added
Plant Physiol. Vol. 117, 1998
Boric Acid Concentration
0 mMa
7 mM
100 mM
nm
Growthb
LSLc
MSLd
USLe
Stationaryf
LSL
MSL
USL
4.19 6 0.32
5.62 6 0.20
8.34 6 0.28
2.73 6 0.14
3.60 6 0.13
5.66 6 0.20
2.62 6 0.12
3.41 6 0.05
5.16 6 0.44
Viability lost
2.75 6 0.30
n.d.g
n.d.
2.26 6 0.10
2.85 6 0.18
4.12 6 0.13
b
,0.1 mM boric acid.
Mean 6 SD of 10 independent replic
cates.
LSL, Lower size limit of permeation; critical Stokes’ rad
dius for equilibration of all cell lumina.
MSL, Mean size limit;
Stokes’ radius for equilibration in one-half of the cell vole
ume.
USL, Upper size limit of permeation; critical Stokes’ raf
dius for complete exclusion from cell lumina.
Mean 6 SD of at
g
least six independent replicates.
n.d., Not determinable; upper
size limit beyond the range of analysis (.9 nm).
a
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Boron Requirement of Growing and Stationary Chenopodium album L. Cells
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breakdown (turbidity) and to decrease the number of single cells (Table II).
Transition from the Growth Phase to the Stationary
Phase for Cells Grown in the Presence of Different
Boron Concentrations
The growth rate of C. album cells was significantly reduced on the 4th d of cultivation if the cultures were fed
with Suc and not diluted with fresh medium (Fig. 1a). Cell
wall expansion in stationary control suspensions was
strongly reduced, although no secondary walls were
formed (cells resume growth after subcultivation). At the
transition to the stationary phase intercellular spaces increased, meristematic spheric or oval cell complexes with
plane-segmenting walls disappeared, and cells became
nearly sphere shaped. Stationary cells were smaller and
more uniform when obtained from cultures grown with
100 rather than with 7 mm boron (Fig. 5). The stationary
cells (100 mm boron) remained viable for more than 3 weeks
without a significant increase in cell size. The cell diameter
increased at the transition phase and remained constant
during the stationary phase. Stationary control cells did not
release turbid material and their FAV remained low if Suc
depletion was prevented (Fig. 6, a and b). When cells
grown in the absence of boron or at 7 mm boron were fed
with Suc and not diluted, their dry-weight increase was
similar to that of control cells (Fig. 6a). However, the turbidity of the medium, an indication of cell bursting, increased at the onset of the stationary phase, as did the
number of dead cells and the FAV (Fig. 6, a and b). The
FAV is a measure of the liquid-volume fraction of cell walls
and dead cells. A FAV of 50% corresponds to the death of
most cells (see Fig. 6a), since the FAV includes neither
liquid volumes within intercellular spaces and surface film
nor the volumes of solid materials (Fleischer and Ehwald,
1995). Almost complete cell damage was confirmed by
staining with Evans blue. In addition, the size of the cells
grown without boron or with 7 mm boron increased during
the transition to the stationary phase (Fig. 6c).
Stationary cells pregrown with 7 mm boron were characterized by a broadening of the dextran-permeation region
due to selective increase of the upper size limit to values
beyond the range of analysis (Fig. 7; Table I). The strong
increase in the upper size limit of boron-deficient stationary cultures may be explained at least partly by ruptured
Figure 4. Effect of boron concentrations on the cell size and the
mean size limits (MSL) of the walls of C. album cells. , Mean size
limits 6 SD (n 5 10). F, Cell size (mode of the cell size distribution)
of mechanically disaggregated clusters 6 SD (n 5 8). Cells were
grown continuously in media containing different boric acid concentrations. With the exception of the control, cells were previously
grown without boron and, before analysis, subcultivated at least five
times in media of the given boron concentrations.
scribed by Woehlecke and Ehwald (1995) to determine the
size limits for approximate equilibration with 95%, 50%,
and 5% of the inner cell volume, which correspond to the
lower size limit, the mean size limit, and the upper size
limit of cell wall permeation, respectively. In cells growing
in boron-deficient medium, all of these size limits were
greatly increased (Table I).
To estimate the boron concentrations that are required to
decrease wall pore size, reduce medium turbidity, and
decrease the number of single cells, cultures grown continuously without boron were transferred to media with boron concentrations between 1 and 100 mm. The mean size
limit of cell wall permeation was comparable to that of
control cells when the boron concentration was 7 mm (boron content of the cells at harvest time was approximately
8 mg g21 dry weight). No change in the pore size of the cell
wall was obtained with 1 or 2 mm boron (Fig. 4), whereas 4
mm boron resulted in an intermediate pore size. The apparent half-effect concentration was 4.5 mm. The dependence
of cell size on boron concentration was similar to that of the
wall pore size, although 2 mm boron did have a discernible
effect (Fig. 4). In contrast, the lowest boron concentrations
(1 and 2 mm) were sufficient to reduce the mechanical cell
Table II. The effect of the addition of boric acid on cell rupture and the frequency of single cells and small clusters of C. album cells
Cells were grown continuously in media containing different boric acid concentrations. Cells with 1 and 2 mM boron were previously grown
without boron and, before analysis, subcultivated at least five times in media of the given boron concentrations. Cell rupture was estimated by
measuring the A490 of the medium, and particle size was determined with a laser analyzer.
Boric Acid Concentration
Parameter
0 mM
A490b
Frequency of particles (% of total particle volume)c
,50 mM (mainly single cells)
,100 mM (includes small cluster)
a
,0.1 mM boric acid.
b
1 mM
2 mM
100 mM
0.077 6 0.023
0.039 6 0.012
0.039 6 0.011
0.017 6 0.006
11.50 6 2.46
27.68 6 4.82
6.63 6 0.74
16.67 6 1.85
4.60 6 0.48
10.64 6 0.98
4.22 6 0.64
12.68 6 2.12
a
Mean 6 SD of at least 10 independent replicates.
c
Compare Figure 2 (mean 6 SD of 10 independent replicates).
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Fleischer et al.
Plant Physiol. Vol. 117, 1998
size of these cells and control cells were comparable (Table
I; Fig. 8).
A reduction in water potential by Suc (10 g L21, 0.8
MPa), which is sufficient to compensate the osmotic potential of the cell sap in C. album (Fleischer and Ehwald, 1995),
caused a transient (3–4 d) halt in the increase in the fresh
weight and cell size that typically occurs during the transition of boron-deficient cells to the stationary phase. The
reduction in turgor also caused a marked reduction in the
increase of turbidity and reduced the percentage of dead
cells (stained with Evans blue; data not shown), but had no
significant effect on the size limits of cell wall pores
(Table III).
Figure 5. Light micrographs of clusters of stationary-phase C. album
cells obtained from cultures grown with 100 or 7 mM boric acid.
Before the transition to the stationary phase the cells were grown
continuously in medium containing 100 mM boron (top) or 7 mM
boron (bottom). Scale bar, 50 mm. Cells were photographed 9 d after
their last subcultivation.
cells. In stationary boron-deficient cells only the lower size
limit could be easily compared with that of the control.
Whereas the control showed a significant decrease in all
size limits at the transition to the stationary phase, there
was no decrease in the lower size limit of boron-deficient
cells (Table I).
The progression of cell necrosis in the stationary phase
was interrupted by supplementing the media with boron
(Fig. 8). The addition of boron (100 mm) to boron-deficient
cells during the transition phase stopped further cell enlargement and organelle release, and after 8 d the wall-pore
Figure 6. Effect of boron concentrations on the dry weight, FAV,
turbidity, and cell size during the transition of cells from the growth
to the stationary phase. Cells grown continuously for 125 passages
in the absence of added boron (E), 7 mM boron (ƒ), or 100 mM
boron (F) were not diluted with fresh medium but supplied with Suc
(2%, w/v) 2 d after the last subcultivation. a, Increase in dry weight
(DWt.) and FAV. b, Turbidity of the filtered medium (A490). c, Cell
size (mode of the size distribution of the mechanically disaggregated cell clusters). The data are means 6 SD obtained from three
independent experiments.
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Boron Requirement of Growing and Stationary Chenopodium album L. Cells
Figure 7. Pore-size distribution profiles of the polydisperse dextranprobing solutions after equilibration with denatured stationary cells.
Denatured stationary cells derived from cells grown in medium
containing 100 mM boron (top) or 7 mM boron (bottom). The pore-size
distribution profile of the untreated dextran-probing solution is
shown in Figure 3. c, Dextran concentration.
1407
deficiency are stable and are reversed by transferring
growing cells from a medium without boron to the standard medium. Finally, the sensitivity of stationary cells to
boron deficiency is not reduced by long-term growth without boron.
The rupture of a small number of cells in the borondeficient growth medium may be attributable to a subpopulation of cells that have left the cell-division cycle and
entered the stationary phase. These cells, like cells in the
early stationary phase, would then be considerably more
sensitive to boron deficiency (Fig. 6). The presence of a
subpopulation of stationary cells may not be completely
prevented even by the high frequency of subcultivation.
Although in the whole plant it is difficult to separate the
direct and indirect effects of boron deficiency on early
meristematic growth because meristems are under correlative control by other plant parts, cell enlargement is believed to require higher boron levels than cell division
(Torssell, 1956; Slack and Whittington, 1964; Birnbaum et
al., 1974; Kouchi and Kumazawa, 1976; Dell and Huang,
1997). Our findings are consistent with a lower boron requirement for cell division growth, but are in apparent
contrast to studies reporting growth reduction of
suspension-cultured cells at suboptimal boron concentrations (Seresinhe and Oertli, 1991; Matoh et al., 1992; Hu and
Brown, 1994). Those studies used batch cultures that were
made boron deficient by washing the cells with a borondeficient medium and thus are not comparable to our
growth conditions. By this treatment, conditioning factors
are removed and a pronounced lag phase is induced.
Suspension-cultured plant cells may be more sensitive to
boron deficiency during the lag phase than in the growth
phase. However, suspension cultures appear to differ in
their ability to maintain high rates of propagation growth at
low boron concentrations. For example, a culture derived
DISCUSSION
The Boron Requirement for Plant Cell
Division and Growth
All vascular plants need boron (Augsten and Eichhorn,
1976; Dugger, 1983; Loomis and Durst, 1992), but this does
not necessarily imply that boron is essential for the viability of all suspension-cultured higher plant cells. Our results
show a striking difference in the boron requirement of
growing and stationary C. album cells and provide evidence that these cells continue to divide and grow even in
the absence of added boron. However, the presence of
nanomolar concentrations of boron in the growth medium
cannot be excluded, nor can the possibility that submicromolar levels of boron are essential for cell growth.
It is unlikely that long-term propagation of C. album cells
at a low boron concentration resulted in the selection of
cells with both an irreversibly altered wall structure and a
low boron demand, because these cells have been transferred several times from standard medium to borondeficient medium without a reduction in their mean
specific-growth rate. Furthermore, the symptoms of boron
Figure 8. The effect of 100 mM boron on cells grown at 7 mM boron
during their transition to the stationary phase. Cells were grown in
medium containing 7 mM boron and at time 0 kept without dilution
and allowed to reach the stationary phase. Boric acid (100 mM) was
added at time 0 or at the times indicated by the arrows. Suc was
added at d 2. Cell rupture was followed by measuring the turbidity
(A490) of the filtered medium. Cell size (mode of the size distribution
of the mechanically disaggregated cell clusters) and the mean size
limit (MSL) of the walls were measured at d 8. n.d., Not determinable; upper size limit beyond the range of analysis (.9 nm).
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1408
Fleischer et al.
Plant Physiol. Vol. 117, 1998
Table III. The effect of reduced osmotic potential on the enlargement, rupture, and wall-pore size of stationary-phase C. album cells
maintained at low boric acid concentration
Cells grown continuously in the presence of 7 or 100 mM boric acid were cultivated without dilution to reach their stationary phase. The media
were supplemented with Suc (20 or 100 g L21) at d 2 to determine the effect of reduced turgor pressure on the development of boron-deficiency
symptoms at the onset of the stationary phase.
Boric Acid Concentration
Parametera
7 mM
Reduction in osmotic potential of the medium (MPa)
Fresh wt (g L21)
d2
d5
Cell size (mm)
d2
d5
MSLb (nm)
d2
d5
A490
d2
d5
0.16
100 mM
0.80
0.16
0.80
187.79 6 8.21
276.42 6 3.86
187.79 6 8.21
189.78 6 2.26
171.38 6 5.20
264.63 6 13.27
171.38 6 5.20
195.78 6 4.16
35.68 6 0.33
43.03 6 1.36
35.68 6 0.33
35.24 6 0.85
33.66 6 0.96
35.22 6 0.39
33.66 6 0.96
33.90 6 0.76
3.68 6 0.08
n.d.c
3.68 6 0.08
n.d.
3.30 6 0.10
2.75 6 0.05
3.30 6 0.10
2.77 6 0.11
0.055 6 0.011
1.050 6 0.118
0.055 6 0.011
0.420 6 0.058
0.019 6 0.003
0.042 6 0.013
0.019 6 0.003
0.026 6 0.007
a
b
Each value represents the mean 6 SD of three independent replicates.
MSL, Mean size limit; Stokes’ radius for equilibration in one-half
c
of the cell volume.
n.d., Not determinable, upper size limit beyond the range of analysis (.9 nm).
from carrot grows in the absence of added boron, whereas a
culture of Dioscorea deltoidea Wall. requires boron to maintain continuous growth (A. Fleischer, unpublished results).
The Effect of Boron on Cell Wall Structure
C. album cells maintain a high specific-growth rate for an
apparently unlimited period of time, even at extremely low
boron concentrations in the growth medium. Nevertheless,
symptoms of boron deficiency that indicate an altered cell
wall structure are observed in these cells. These symptoms,
which include decreased cell wall resistance to mechanical
stress (cell-cluster disintegration, uncontrolled cell expansion, and the bursting of cells), are comparable to the
effects of boron deficiency in other plants (Loomis and
Durst, 1992). The high frequency of single cells and small
clusters of cells (see Fig. 2b) is consistent with the weakening of wall-to-wall contact between cells; a weakened
middle lamella has been reported to be a symptom of
boron deficiency in plant organs (Loomis and Durst, 1992;
Marschner, 1995). We have also shown that C. album cells
grown in the absence of boron are larger than the cells
grown with boron. Again, such a result is consistent with the
reported uncontrolled enlargement of cells in the growing
regions of boron-deficient plants (Loomis and Durst, 1992).
The results of our study provide evidence that in a
boron-deficient medium cell death is caused primarily by
the weakening of the cell wall. The death of resting cells is
accompanied by the release of cell organelles, which is
indicative of cell wall rupture. The rupture of borondeficient cell walls results from plasmoptysis, a phenomenon in which localized bursting of the cell wall causes
damage to the plasma membrane (Küster, 1958). Plasmoptysis may be induced by local weakening of cell walls
(defect plasmoptysis) or by an increase in turgor pressure
(osmotic plasmoptysis). Plasmoptysis may account for the
tip bursting that occurs at low boron concentrations during
pollen-tube growth of some plants (Schmucker, 1934) and
may also account for the increased solute efflux and cell
damage in boron-deficient plants that has been reported
(Cakmak et al., 1995). Solute leakage may be misinterpreted as indicating a high permeability of lipid membranes if plasmoptysis is not taken into account (compare
e.g. Simon, 1977; Ehwald et al., 1980, 1984).
To our knowledge, our data are the first to show that
pore size in the cell wall is markedly affected by the presence or absence of boron. The pore size of the cell walls, i.e.
the size of pores limiting unrestricted diffusion of polymers
through the wall, has been shown to depend on the concentration and/or conformation of the pectic polymers
(Baron-Epel et al., 1988; Ehwald et al., 1992; Carpita and
Gibeaut, 1993). The increased pore size limits of the wall in
the boron-deficient cells is indicative of a disorganized
pectic network and may result from the absence of borate
ester cross-linked RG-II.
We suggest that in the cell wall, borate ester cross-linked
RG-II regulates the macroscopic conformation of the pectin
network and that the formation of borate ester cross-linked
RG-II is required for normal cell growth. We also suggest
that the boron-dependent pectin conformation, which itself
may control the permeation of macromolecules through the
wall, will affect the accessibility of load-bearing structures
to wall-loosening protein molecules, since the size limits of
cell wall permeation and wall extensibility are reduced at
the transition to the stationary phase and both changes
require high boron concentrations. Another possibility to
explain the observed parallel changes of wall extensibility
and pore size might be the influence of pore size on the
retention of polymer compounds (Bonilla et al., 1997) that
are necessary for wall stiffening, e.g. extensins and Pro-rich
proteins (Carpita and Gibeaut, 1993).
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Boron Requirement of Growing and Stationary Chenopodium album L. Cells
The increase in the mean size limit of cell wall permeation of growing cells was the most sensitive of the borondeficiency symptoms observed. The restoration of the
mean size limit of cell wall permeation to normal values
required higher boron concentrations than were required
to reduce the turbidity of the growth medium and to decrease the number of single cells (compare Fig. 4 and Table
II). The ability of boron to alter cell wall pore size was not a
secondary effect resulting from increased wall expansion,
since it also occurred at reduced water potential, under
which cell enlargement was completely prevented (Table
III). Moreover, the wall pore size of boron-deficient enlarged
cells was reduced by adding boron to the cells (Fig. 8).
Stationary-phase cells had a much higher boron requirement than growing cells. For example, 7 mm boron was
sufficient to keep the porosity values of propagating cells
comparable to those of control cells (Fig. 4; Table I), but
was not sufficient for the decrease of the wall pore size at
the transition from the growth phase to the stationary
phase (Fig. 7; Table I). A decrease of all wall size limits at
the transition to the stationary phase is characteristic of
control cells (Table I) (Titel et al., 1997).
The disappearance of boron-deficiency symptoms in
stationary-phase cells after the addition of boron (Fig. 8)
suggests that boron affects wall structure even in nongrowing cells, in which an increase in cell wall dry weight is
negligible. In C. album cells, boron appears to be essential
for wall stiffening at growth termination. This may allow
the cells to maintain their size within certain limits and
over long periods of time, when genes for the synthesis of
wall polymers are not expressed and the deposition of new
wall polymers does not occur. The boron-deficiency symptoms most likely result from insufficient boron in the cell
wall rather than from a boron deficiency in the cytoplasm,
because at low boron concentrations the cell wall boron is
the predominant boron pool in suspension-cultured cells
and higher plant tissues (Loomis and Durst, 1991; Matoh et
al., 1992; Hu and Brown, 1994; Hu et al., 1996).
In summary, we have shown that C. album cells grow in
the absence of added boron and that several cell wallrelated symptoms of boron deficiency in growing and stationary cells can be examined. These deficiency symptoms
are reversed by the addition of boron to the cells. For
preventing boron-deficiency symptoms much higher boron
concentrations are necessary in the stationary phase than in
the growth phase. It is not known if the failure to form a
borate ester cross-linked RG-II dimer is directly responsible
for the effects of boron deficiency on wall structure. Determining the monomeric RG-II content of boron-deficient C.
album cell walls and determining if the amount of RG-II in
their walls increases at the onset of the stationary phase
may provide evidence for the physiological role of this
boron-binding pectic polysaccharide.
ACKNOWLEDGMENT
The authors thank Dr. M.A. O’Neill (Complex Carbohydrate
Research Center, The University of Georgia, Athens) for critical
review and improvement of the manuscript.
1409
Received February 9, 1998; accepted May 8, 1998.
Copyright Clearance Center: 0032–0889/98/117/1401/10.
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Plant Physiol. (1998) 118: 1101–1103
The Electronic Plant Gene Register
Plant Gene Register titles for PGR 98–175 to PGR 98–189 appear below. The sequences have been
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Weers B, Thornburg R (1998) Characterization of the cDNA and gene for the Arabidopsis
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Plant Gene Register PGR 98–175
Molecular-Mass Heat-Shock Proteins (Accession Nos.
U83669, U83670, and U83671).
Characterization of the cDNA and Gene for the Arabidopsis GDP-Mannose Pyrophosphorylase (Accession
No. AF076484).
Jiahn-Chou Guan, Fa-Cheng Chang, Tong-Shun Tseng, PiFang L. Chang, Kai-Wun Yeh, Yih-Ming Chen, and ChuYung Lin*.
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Nucleotide Sequences of cDNAs (Accession Nos.
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Cloning and Characterization of a Cellulose Synthase
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Molecular Characterization of a cDNA Encoding a
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The Genomic Organization of the Arabidopsis 6–4 Photolyase Gene (Accession No. AB017331).
Ayako Sakamoto*, Atsushi Tanaka, Shigemitsu Tano, Saatoshi Nakajima, Kazuo Yamamoto, and Hiroshi Watanabe.
Plant Resources Laboratory, Japan Atomic Energy Research Institute, Watanuki-machi 1233, Takasaki 3701292, Japan (A.S., A.T., S.T., H.W.); and Biological Institute, Graduate School of Science, Tohoku University,
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Nucleotide Sequence of a Probable Aminotransferase
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CORRECTION
Volume 117: 1401–1410, 1998
Fleischer, A., Titel, C., and Ehwald, R. The Boron Requirement and Cell Wall Properties
of Growing and Stationary Suspension-Cultured Chenopodium album L. Cells.
Several inaccuracies were printed in this article and they are corrected below.
On page 1401, the correct e-mail address for Rudolf Ehwald is:
[email protected].
On page 1402 under the heading Propagation Culture, the second to last sentence should
read: The final biomass concentration (ct) at subcultivation time and the initial biomass
concentration (co) were constant (see Fig. 1b) and, therefore, the mean specific-growth
rate (m 5 1⁄2 d [ln ct 2 ln co]) equals the mean dilution rate (r 5 1⁄2 d [ln 5 2 ln 2] 5 0.46
d21).
On page 1403, the second and third sentences in the legend to Figure 2 should read:
a, Particle-size distribution curves of suspension-cultured C. album cells after disaggregating treatment. Cells grown without boron (2B) or with 100 mm boron (1B).
On page 1404, the first sentence in the legend to Figure 3 should read: Size distribution
profiles of the polydisperse dextran-probing solutions after equilibration with denatured
growth-phase cells.
On page 1404, the last complete sentence in the right column should read: The elution
profiles of the cell-exposed dextran solutions contained “steps,” or more gradual changes
attributable to the size-dependent diffusion of the dextrans into the cell lumina (Fig. 3).
On page 1407, the entire legend to Figure 7 should read: Size distribution profiles of the
polydisperse dextran-probing solutions after equilibration with denatured stationary
cells. Denatured stationary cells derived from cells grown in medium containing 100 mm
boron (top) or 7 mm boron (bottom). The size distribution profile of the untreated
dextran-probing solution is shown in Figure 3. c, Dextran concentration.
1103