Plant Science 128 (1997) 217 223

Cell wall proteins of in vitro cultured chili pepper lines differing

in water stress tolerance
Quintero-Higuera Mara Fernanda, Santos-Daz Mara del Socorro *,
Garca-de la Cruz Ramon Fernando
Laboratorio de Bioqumica, Facultad de Ciencias Qumicas de la UASLP, A6. Manuel Na6a No. 6, CP 78210,
San Luis Potos, Mexico
Received 21 January 1997; received in revised form 29 May 1997; accepted 17 June 1997

Abstract
As a strategy to understand cellular mechanisms of drought tolerance, we analyzed the electrophoretic pattern of
cell wall proteins from non adapted cells (ST) and a 15% polyethylenglycol (PEG)-tolerant clone (T7) of chili pepper
(Capsicum annuum). To separate proteins bound to structural polymers by non-covalent links or disulfide bonds, cell
walls were treated with sodium dodecyl sulphate (SDS), urea and 2-mercaptoethanol. After treatment three major
proteins with apparent molecular masses of 9, 11 and 14 kDa accumulated in higher quantities in clone T7 than in
ST cells. Cell walls were also solubilized with commercial lytic enzymes, such as lyticase and cellulase, to separate
proteins joined by covalent bonds. The main difference was the presence of a new band with an apparent molecular
mass of 10 kDa (p10) in clone T7 but not in ST cells. This band was also evident after autodigestion of walls by
hydrolytic enzymes. Since p10 was liberated by cellulase and lyticase we suggest that this protein is covalently
attached to the cellulose and glucan skeleton in clone T7. The potential role of p10 on the mechanism of drought
tolerance is discussed. 1997 Elsevier Science Ireland Ltd.
Keywords: Capsicum annuum; Cell wall proteins; Drought tolerance

1. Introduction

Abbre6iations: ABA, abscisic acid; PEG, polyethylenglycol;

PMSF, phenyl-methylsulfonyl fluoride; SDS, sodium dodecyl
sulphate; cw, water potential.
* Corresponding author.

In vitro selection of cells exhibiting increased

tolerance to water stress is considered a biotechnological alternative to conventional plant breeding and a model to study physiological and
biochemical processes involved in the mechanisms
of resistance. Tolerant lines of different species

0168-9452/97/$17.00 1997 Elsevier Science Ireland Ltd. All rights reserved.

PII S 0 1 6 8 - 9 4 5 2 ( 9 7 ) 0 0 1 5 5 - 6

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Q.-H.M. Fernanda et al. / Plant Science 128 (1997) 217223

have been isolated using polyethylenglycol (PEG)

and mannitol as osmotic compounds to simulate
drought conditions [1 4]. Santos-Daz and
Ochoa-Alejo [5] described the isolation and characterization of PEG-tolerant cell clones of chili
pepper (Capsicum annuum). Growth of nonselected cell suspension of chili pepper was inhibited
to 50% when cells were exposed to 5% or higher
PEG, whereas clone T7 was capable of growing
on 1520% PEG. The tolerant clone exhibited a
three-fold decrease in the osmotic potential as
compared to that of the nonselected cells suggesting that osmotic adjustment occurred. However,
additional work is required to better understand
the nature of adaptation of the PEG-tolerant
clone to water deficit.
There are many reports indicating that cell wall
constituents are altered in response to biotic and
abiotic stresses [68]. In relation to water stress,
Bozarth et al. [9] described the accumulation of a
28 kDa protein in the cell wall fraction of soybean
seedlings exposed to low water potentials. The
level of the 28 kDa protein increased in the area
of the dividing region soon after the initial growth
inhibition, suggesting a possible role in the regulation of growth and adaptation of seedling to
water deficit. Similar results were reported by
Covarrubias et al. [10]. These authors described
the accumulation of two cell wall glycoproteins
with apparent molecular masses of 36 and 33 kDa
which are accumulated in bean seedlings in response to water deficit. The proteins were induced
by abscisic acid (ABA) treatment and were
present mostly in the elongation region of
hypocotyls. It has also been demonstrated that
cultured cells of tomato and tobacco, which do
not enlarge after adaptation to water stress, undergo physiological and biochemical changes of
cell wall components [11,12]. On the other hand,
the cellular response to water stress involved factors related to cell wall metabolism, particularly
those process associated to cell extensibility and
hydraulic conductivity [13,14].
Based on the assumption that water deficit may
promote changes in cell wall composition, we
performed a comparative electrophoretic analysis
of cell wall proteins obtained from clone T7
adapted to growth in 15% PEG and non selected

cells of chili pepper (ST). To extract cell wall

proteins associated with structural polymers by
non covalent links, we used sodium dodecyl sulphate (SDS), urea and 2-mercaptoethanol,
whereas cellulase and lyticase digestion were used
to isolate covalently joined proteins. We propose
that modifications of structural proteins, enzymatic activities and/or synthesis of specific peptides might play a role in drought tolerance at the
cellular level.

2. Materials and methods

2.1. Plant material

Cell suspensions of Serrano tampiqueno pepper
were grown and maintained as described previously [5]. Non-selected cells are referred to as ST
cells and 15% PEG-tolerant cells as clone T7.
Briefly, clone T7 was isolated by plating 70 000
cells on filter paper discs supported by
polyurethane foam bathed with liquid medium
MS containing 15% PEG (PEG 8000, water potential (cw)= 0.98 MPa). Cell colonies obtained after 12 months were established as
suspension cultures in MS medium supplemented
with 15% PEG. Clone T7 has been maintained in
selective medium for more than 3 years [5].

2.2. Cell wall preparation

Cells at the stationary growth phase were collected in conical tubes and centrifuged to 1000
g, 20 min. To remove culture medium, ST cells
and clone T7 were washed with 50 ml deionized
water three and six times, respectively. Cells were
broken by sonication (Braun-Sonic U) at 165 W,
for 1 min at 4C and this operation was repeated
three times or until all cells were broken as judged
by microscope examination. To eliminate cytoplasmic contaminants the ST and clone T7 preparations were centrifuged at 1000g for 15 min,
five and ten times, respectively, with 50 ml of
TrisHCl 10 mM, pH 6.8 containing 1 mM
phenyl-methylsulfonyl fluoride (PMSF) (buffer
A). Additionally, cell walls from clone T7 were
dialyzed 48 h against buffer A. Finally, to obtain

Q.-H.M. Fernanda et al. / Plant Science 128 (1997) 217223

a fine preparation, cells walls were homogenized

with a borosilicate glass tissue grinder, washed
five times with buffer A and lyophilized. All procedures for isolating cell walls were carried out at
4C. Cell wall purity was assessed by examination
of the preparation under phase-contrast microscope.

2.3. Protein extraction

Purified walls, essentially free of contamination
(30 mg) were hydrated with 400 ml of deionized
water and treated separately with 1 ml of either:
(a) 5 min with 2% SDS in boiling water; (b) 1 hr
with 6 M urea at room temperature; (c) 30 min
with 2-mercaptoethanol at 30C. Following these
treatments, samples were centrifuged at 8800 g,
10 min and the supernatant was collected.
Enzyme solutions were prepared by dissolving 2
mg of cellulase in 1 ml of 10 mM acetate pH 5.5
containing 1 mM PMSF, or 2 mg of lyticase in 1
ml of 10 mM phosphate pH 7.5 containing 1 mM
PMSF. About 30 mg of dry cell walls were hydrated with 400 ml of deionized water and mixed
with 1 ml of enzymatic solution of lyticase or
cellulase. Samples were incubated for 2.5 h at
25C (lyticase) or at 37C for 2.5 h (cellulase). In
each case, controls with only enzymatic preparation were included.
To measure autolysis, 30 mg of lyophilized cell
walls were hydrated with 400 ml of deionized
water and incubated in 1 ml 50 mM acetate pH
5.5 containing 1 mM PMSF for 24 h at 25C and
constant stirring. Samples were centrifuged at
8800g, for 10 min and the supernatant was
collected.
The supernatants obtained after chemical (SDS,
urea, 2-mercaptoethanol) and enzymatic solubilization (cellulase, lyticase, autolysis) were treated
with cold 75% ethanol (v/v) to precipitate
proteins. Samples were incubated for 18 h at 4C
and centrifuged at 8800 g for 10 min. The pellet
was collected and resuspended (v/v) in sample
buffer (0.125 M Tris HCl pH 6.8, 4% SDS, 20%
glycerol, 10% 2-mercaptoethanol and 0.02% bromophenol blue) and analyzed by SDS-PAGE.

219

2.4. Gel electrophoresis

SDS-PAGE was carried out according to the
method of Laemmli [15] in 12% polyacrylamide
gels. The following molecular weight standards
were run in parallel: phosphorylase B (106 kDa),
bovine albumin (80 kDa), egg albumin (49.5
kDa), carbonic anhydrase (32.5 kDa), trypsin
(27.5 kDa) and lysozyme (18.5 kDa). Gels were
stained with silver nitrate as described by Wayne
et al. [16].

3. Results
To determine whether changes in the cell wall
structure are related to the mechanisms of adaptation to water stress, we analyzed the composition
of cell wall proteins from cellular suspensions of
chili pepper with different tolerances to PEG.
Proteins associated by non-covalent links were
separated by treatment of the cell walls with SDS,
urea or 2-mercaptoethanol. In the electrophoretic
analysis of SDS-solubilized cell wall proteins from
ST cells it was not possible to distinguish individual bands (data not shown). Therefore, we used
15 mg of dry cell walls and the solubilized material was analyzed. The results showed a pattern of
bands with apparent molecular masses in the
range of 752 kDa (Fig. 1, lane a). By using the
same amount of clone T7 derived-walls, we didnt
obtain a good resolution of low molecular weigth
bands, since an over loading of the gel was evident (Fig. 1, lane b); a higher expression of three
proteins with apparent molecular masses between
40 and 52 kDa was observed. The solubilization
of 5 mg dry lyophilized walls from clone T7
showed five major bands with apparent molecular
masses of 9, 11, 14, 16 and 20 (referred to as p9,
p11, p14, p16 and p20, respectively); these bands
appeared with higher intensity in clone T7 than in
extracts of ST cells (Fig. 1, lane c).
After treatment with urea, p9, p11, p12, p14
and p20 were particularly distinguished in ST
cells. A substantial increase in p9, p11 and p14
was seen in clone T7 (Fig. 2A). Cell walls solubilized with 2-mercaptoethanol also showed differences between ST and clone T7 cells (Fig. 2B).

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The most remarkable one was an increase of p9 in

clone T7; p11 and p14 also increased but to a
lesser extent.
To separate proteins covalently attached to cellulose or glucan, cell walls were digested with
cellulase and lyticase as described in Section 2. A
blank containing the enzymatic preparation only
was included to identify the corresponding enzyme bands. As shown in Fig. 3A, after digestion
with cellulase, a similar pattern of bands were
observed in clone T7 and ST cell walls but p10
was specifically accumulated in the PEG-tolerant
clone. The treatment with lyticase (Fig. 3B) liberated more proteins in ST cells (p9, p11, p12, p14
and p20) than in clone T7, however p10 was only
present in tolerant clone.
Another strategy to analyze cell wall proteins is
to liberate proteins by cell wall autodigestion. In
ST cultures about 14 bands were observed with
apparent molecular masses in the range of 8 56
kDa. In clone T7 around 12 bands were evident,
of which p9, p10 and p34 were specifically present
in this culture (Fig. 4).

Fig. 1. Electrophoretic pattern of cell wall proteins solubilized

with SDS. (a) Extracts obtained from ST cells (15 mg); (b)
clone T7 (15 mg); and (c) clone T7 (5 mg). Molecular weights
(103) of standard proteins are listed to the right of the figure.

Fig. 2. Analysis of extracts obtained from ST cells (ST) and

clone T7 (T7) after treatment with 6 M urea (A) or 10%
2-mercaptoehtanol (B). Molecular weights (103) of standard
proteins are listed to the right of the figure.

4. Discussion
Cell walls in higher plants are made up of
complexes of cellulosic microfibrils and a non-cellulosic matrix composed of pectins, hemicelluloses
and proteins [17]. The synthesis and the crosslinking of these wall proteins are under strict control
and can be environmentally induced by a number
of factors. For example, there is evidence supporting the existence of an extensin-pectin crosslinking
[8]. The positively charged lysine and protonated
histidine residues of extensin could interact with
the negatively charged uronic acid of pectins.
Such interactions can be regulated by changes in
cell wall pH and Ca2 + and thus alter the physicochemical properties of the wall. The expression of
glycine-rich proteins is also regulated by a variety
of stress conditions, including ABA and drought
stress [8].
On the other hand, tolerance to water stress is
related to a higher elasticity of cell wall, described
by the so-called volumetric elastic modulus (o).
Low o provide cells with a higher resistance to
short-term fluctuations in the cw of the environment. A high o value insures that relative changes

Q.-H.M. Fernanda et al. / Plant Science 128 (1997) 217223

Fig. 3. Protein patterns of extracts obtained from ST cells (ST)

and clone T7 (T7) after treatment with cellulase (A) and
lyticase (B). The analysis was performed using 30 mg of
protein per lane. A blank containing only cellulase and lyticase
were included as control (BCO). Molecular weights (103) of
standard proteins are listed to the right of the figure.

in cell water content is much smaller than in those

cells with a lower o value. This may be of great
ecological relevance particularly to plants living in
regions with water restrictions [18].

Fig. 4. Proteins liberated by cell wall autolysis from ST cells

(ST) and from clone T7 (T7). The analysis was performed
using 30 mg of proteins per lane. Molecular weights (103) of
standard proteins are listed to the right of the figure.

221

To understand cellular mechanism of PEG tolerance of in vitro cultures of chili pepper we

analyzed cell wall proteins. Results presented in
this paper showed that the electrophoretic pattern
of proteins associated by weak, non-covalent and
disulfide bonds was similar in ST cells and clone
T7. Treatment of cell walls with SDS, urea or
2-mercaptoethanol showed quantitative but not
qualitative differences. Regardless of the treatment p9, p11 and p14 were detected in higher
amounts in clone T7 in relation to ST cells. The
bands between 40 and 52 kDa and p11 observed
after solubilization of 15 mg cell wall from clone
T7 also were detected in ST cells when higher
quantities (30 mg cell walls) were used (data not
shown); the presence of p11 in ST cells was corroborated after treatment with urea and 2-mercaptoethanol (Fig. 2). These proteins are probably
soaked in cell wall polymers by ionic bonds that
can be broken without requiring enzymatic digestion. Arabinogalactan-associated proteins are
readily solubilized during tissue extraction with
low ionic strength aqueous solutions [8]. Additional work will be required to determine whether
the cell wall proteins separated by chemical treatments correspond to this type of proteins in clone
T7 and ST cells.
Degradation of cell wall fibrillar polymers by
lytic enzymes promoted the solubilization of
proteins covalently associated with its structure.
Solubilization of cell wall with cellulase and lyticase showed differences between ST cells and
clone T7. The most remarkable qualitative difference was the presence of a protein band with an
apparent molecular mass of 10 kDa in clone T7.
Incubation with cellulase released proteins
strongly associated with cellulosic microfibrils,
whereas lyticase liberated proteins cross-linked to
b-1,3 glucan. Release of p10 by both cellulase and
lyticase, suggests that p10 is attached covalently
to the cellulose and glucan skeleton probably by
forming a bridge between these polymers. The
accumulation of low molecular mass wall protein
in response to environmental stress has been described by others authors. Iraki et al. [12] observed the accumulation of 20 and 11 kDa
proteins in tobacco cells adapted to grow in 428
mM NaCl.

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Autolysis of cell wall during in vitro incubation is due to glycosylases associated to this
structure. The results corroborated that p10
specifically accumulated in clone T7 but not in
ST cells. The electrophoretic analysis also revealed the presence of p9 and p34, probably
solubilized when the fibrillar structure of cell
wall of T7 was totally digested after the long
period of incubation with its own lytic enzymes
(Fig. 4).
Since clone T7 has been mantained for several years in 15% PEG it is difficult to believe
that appearance of p10 was the result of cellular damage or just a consequence of water
stress. The possibility that some of the differences between ST cells an clone T7 was due to
contaminating proteases is low, since cell walls
were extracted at 4C in the presence of PMSF.
Besides the electrophoretic pattern obtained after 24 h of incubation (Fig. 4) was very similar
to that obtained at 5 min (Fig. 1a).
We believe that the precise localization and
spatial orientation of the fibrilar and amorphous components of cell wall are implicated
directly in its final architecture and function.
The incorporation of p10 may participate in the
supramolecular arrangement of the cell wall, either as an enzyme or as a structural protein,
acting as scaffolds for the deposition of other
molecules or changing the cell-wall-plasma
membrane continuum; p10 probably could also
affect the mechanical strength of the cell wall
by increasing the value of o and consequently
reducing the changes in cell water content.
Further studies are required to elucidate the
distribution and topology of p10 in cell wall, as
well as its contribution to mechanism of tolerance to water deficit in clone T7. The use of
specific antibodies may permit us to determine
the localization of p10 at the ultrastructural
level. Another strategy to study the role of p10
in the mechanism of tolerance to PEG, would
be to correlate its presence with water stress. If
p10 were an inducible protein, we would expect
its disappearance from the cell wall after gradually transfering clone T7 to media without
PEG.

Acknowledgements
We are indebted to Dr Everardo Lopez
Romero for critical review of the manuscript.

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