Plant Physiology and Biochemistry 42 (2004) 485–492

www.elsevier.com/locate/plaphy

Original article

Inhibition of cell-wall autolysis and pectin degradation by cations

J. Bernhard Wehr *, Neal W. Menzies, F. Pax C. Blamey
School of Land and Food Sciences, The University of Queensland, Brisbane, Qld. 4072, Australia
Received 27 January 2004; accepted 6 May 2004

Available online 11 June 2004

Abstract

Modification of cell wall components such as cellulose, hemicellulose and pectin plays an important role in cell expansion. Cell expansion
is known to be diminished by cations but it is unknown if this results from cations reacting with pectin or other cell wall components. Autolysis
of cell wall material purified from bean root (Phaseolus vulgaris L.) occurred optimally at pH 5.0 and released mainly neutral sugars but very
little uronic acid. Autolytic release of neutral sugars and uronic acid was decreased when cell wall material was loaded with Ca, Cu, Sr, Zn, Al
or La cations. Results were also extended to a metal–pectate model system, which behaved similarly to cell walls and these cations also
inhibited the enzymatic degradation by added polygalacturonase (EC 3.2.1.15). The extent of sugar release from cation-loaded cell wall
material and pectate gels was related to the degree of cation saturation of the substrate, but not to the type of cation. The binding strength of the
cations was assessed by their influence on the buffer capacity of the cell wall and pectate. The strongly bound cations (Cu, Al or La) resulted
in higher cation saturation of the substrate and decreased enzymatic degradability than the weakly held cations (Ca, Sr and Zn). The results
indicate that the junction zones between pectin molecules can peel open with weakly held cations, allowing polygalacturonase to cleave the
hairy region of pectin, while strongly bound cations or high concentrations of cations force the junction zone closed, minimising enzymatic
attack on the pectin backbone.
© 2004 Elsevier SAS. All rights reserved.

Keywords: Al; Autolysis; Ca; Cell wall; Cu; La; Pectin; Polygalacturonase; Sr; Zn

1. Introduction
The primary plant cell wall consists of cellulose, hemicel-
lulose, pectin and proteins [6]. Cellulose and hemicellulose
are the main load-bearing polysaccharides in the cell wall,
maintaining the cell shape and turgor pressure. Cell elonga-
tion relies on the selective enzymatic modification of the
load-bearing hemicellulose molecules [8,9]. Pectin is not
load bearing, but may control the mobility and access of
enzymes to load-bearing hemicellulose molecules
[1,2,13,15,22,26], thereby modulating cell elongation. Pectin
contains negatively charged galacturonic acid residues,
which contribute to the cell wall cation exchange capacity.
Cation binding to pectin therefore affects the charge, pH,
Abbreviations: DE, degree of esterification; DM, dry mass; GalA, galac-
turonic acid; ICPAES, inductively coupled plasma atomic emissions spec-
troscopy; PGase, polygalacturonase.
* Corresponding author.
E-mail address: b.wehr@uq.edu.au (J.B. Wehr).
© 2004 Elsevier SAS. All rights reserved.
doi:10.1016/j.plaphy.2004.05.006
swelling behaviour (pore size) and buffer capacity of the cell
wall. This can have direct effects on the mobility of cell
wall-degrading enzymes and cell elongation [2,8].
The enzymatic catabolism of cell wall polysaccharides is
difficult to study in situ due to the complexity of the cell wall,
incorporation of cell wall components into other polysaccha-
rides, uptake of carbohydrate monomers and the large num-
ber of enzymes involved (e.g. hydrolases, lyases, trans-
ferases, esterases, kinases, expansin) [8,14]. The use of a
cell-free system permits focus on the reactions occurring in
the cell wall without the confounding effects of cellular
processes. Such autolytic studies have shown that both the
uncharged arabinogalactan fraction and the rhamnogalactur-
onan “hairy region” of pectin undergo degradation during
autolysis in legumes [10,34] and it was observed that the age
and composition of the cell wall also influences the type of
carbohydrate released [33]. Pectin degradation during au-
tolysis has been observed in tomato (Lycopersicon esculen-
tum Miller) [7] while in members of the Poaceae, autolysis of
hemicelluloses predominates [16]. A non-enzymatic release
486 J.B. Wehr et al. / Plant Physiology and Biochemistry 42 (2004) 485–492

Table 1
Release of total sugars and uronic acid from control and cation-loaded cell wall material. Samples were incubated with and without added polygalacturonase
(5.7 nkat) at 35 °C for 17 h. Values are means of triplicate determinations; means followed by the same letter are not significantly different within a column
(Tukey, P = 0.05)
Treatment Autolytic sugar release With added polygalacturonase
Cation saturation µmole g–1 dry mass Cation saturation µmole g–1 dry mass
(%) Total sugars Uronic acid (%) Total sugars Uronic acid
Control 62 ± 2 82 a 15 a 31 ± 1 424 a,b 5 a,b
Cu 100 ± 3 28 c 7a 87 ± 1 206 c 1b
Zn 69 ± 2 90 a 11 a 61 ± 2 487 a 6 a,b
Ca 67 ± 3 82 a 5a 69 ± 3 436 a,b 8a
Sr 64 ± 2 84 a 1a 69 ± 4 480 a 9a
Al 107 ± 6 41 b,c 0a 101 ± 8 440 a,b 8a
La 100 ± 5 57 b 4a 100 ± 2 307 b,c 7a

of polygalacturonan was observed for potato cell wall
(Solanum tuberosum L.) [35] and bean callus [33].
Divalent cations, Ca in particular, contribute about 60% of
cations bound to the cell wall, and Ca deficiency can lead to
necrosis of leaves, internal disorders in fruit crops and in-
creased susceptibility to pathogen attack [17,18,22,37]. On
the other hand, a high Ca concentration in the cell wall
inhibits autolysis, cell expansion and fruit ripening
[5,12,26,38–40] while having no effect on the chemical (non-
enzymatic) hydrolysis of pectin [24]. The mechanism by
which Ca inhibits autolysis is not well understood but it has
been suggested that Ca inhibits the activity of pectin-
degrading enzymes such as endo-acting polygalacturonase
(PGase, EC 3.2.1.15) [23,26,29,37] directly, or by modifying
the pectin substrate [19]. Another possible explanation is that
endo-PGase becomes trapped in gelled pectin, thereby inhib-
iting movement [19,31,39]. In order to better understand the
effect of cations on cell wall metabolism and, ultimately, cell
expansion, it is necessary to focus on the interaction of
cations with structural cell wall components and cell wall
modifying enzymes.
The aim of this study was to determine the effect of several
divalent and trivalent cations on cell wall autolysis of bean
roots (Phaseolus vulgaris L.) and to determine if the effects
are due to inhibition of autolytic enzymes by cations or due to
substrate effects. Furthermore, it was hypothesised that the
effect of cations on autolysis is due to the binding strength of
cations to the cell wall material as estimated by the buffer
capacity of cell wall material and cation-neutralised pectate.
To determine if the effect of cations on cell wall material is
related to pectin, results obtained with plant root cell wall
material were then extended to study the effect of cations on
the enzymatic degradation of metal–pectate gels.
2. Results
2.1. Autolysis and PGase digestion of cell wall material
Autolysis of bean root cell wall material occurred opti-
mally at pH 5.0, releasing 185 µmole total sugars g–1 dry cell
wall material in 17 h at 35 °C. Uronic acid release was at or
below the detection limit (approximately 25 µM) over the pH
range 3.46–5.72. Addition of PGase to cell wall material
increased release of uronic acids more than of total sugar,
thus changing the ratio of autolytically released total sugar to
uronic acid from 55 (range 32–85) in the control (no PGase
added) to 11 (range 6–16) with added PGase (data not
shown).
Root cell wall material loaded with Cu, Al or La ions
released the least sugars (Table 1), whereas the autolytic
release of total sugars from Ca-, Sr- or Zn-loaded cell wall
material was similar to that of the control material, viz.
82 µmole g–1. The cation saturation of the cell wall material
was complete for Cu, Al and La but lower for Ca, Zn and Sr
(approx 64%). The control material had a cation saturation of
62%, consisting of Ca (48%), Cu (42%) and Zn (10%).
During PGase digestion of Ca-, Sr-, Zn- and Al-loaded cell
wall material, the release of total sugars was similar to that of
the control (purified cell wall material), viz. 424 µmole g–1
(Table 1). However, addition of Cu and La to cell wall
material markedly decreased the PGase-mediated release of
total sugars.
The autolytic release of uronic acids was very low from
cation-loaded cell wall materials (0–11 µmole g–1), irrespec-
tive of the bound cation, and addition of PGase did not
significantly increase the amount of uronic acid liberated
(Table 1).
2.2. Degradation of pectin and cation–pectate gels by
PGase
Extending the work with cell wall material to pectate gels
showed that Ca–, Sr– and Zn–pectate gels (DE = 0%) were
degraded readily by PGase, releasing between 1.8 and
2.3 µM min–1 reducing sugars (Table 2). Incubation of Cu–
and La–pectate gels with PGase resulted in the release of
0.3 and 0.5 µM min–1 reducing sugars, respectively, and
Al–pectate gel was least degraded by PGase (0.1 µM min–1).
This activity was not significantly different from zero. There
was, however, no statistical difference in the amount of
reducing groups released between Cu–, Al– or La–pectate
 J.B. Wehr et al. / Plant Physiology and Biochemistry 42 (2004) 485–492 487

Table 2
Reducing sugars released by polygalacturonase from cation–pectate gels
and cation–pectate gels plus added pectin. Values are means of triplicate
determinations and means in a column followed by the same letter are not
significantly different (Tukey, P = 0.05). Cation–pectate gels (degree of
esterification 0%), with and without added pectin (degree of esterification
49%) were incubated with 11.4 nkat polygalacturonase at 21.5 °C and the
amount of reducing sugars released measured. Pectin alone released 2.0 µM
reducing sugars min–1
Reducing sugars (µM min–1)
Cation Cation Pectate gel Pectate gel Difference
saturation only + pectin
(%)
Cu 54 ± 2 0.3 c 0.6 c 0.3
Fig. 1. Viscosity of apple pectin either measured alone (") or mixed with Ca
Zn 28 ± 1 2.3 a 3.5 a,b 1.2
(.) to give a final concentration of 0.45 mM Ca. Pectin (degree of esterifi-
Ca 16 ± 1 1.8 b 3.6 a,b 1.8
cation 49%) was also incubated for 15 min with either PGase alone
Sr 33 ± 1 2.1 a,b 3.9 a 1.8 (56.7 nkat) (m) or with PGase (56.7 nkat) and Ca (●). Values are the average
Al 100 ± 1 0.1 c 2.4 a,b 2.3 of two determinations and standard error bars are shown if not obscured by
La 86 ± 2 0.5 c 2.4 b 1.9 the symbols. Identical lines to those shown were obtained for Sr, Zn, Al or
La.

2.3. Influence of cation saturation of pectate gels

gels when results were corrected for differences in pectin
concentration. Likewise, there were no statistical differences
in the amount of reducing sugars released between Sr– and
Zn–pectate gels, while Ca–pectate released slightly less sug-
ars (Table 2). The cation saturation of the metal–pectate gels
ranged between 16% for Ca–pectate gel to 100% for Al–pec-
tate (Table 2), the result of cation losses during dialysis of the
gels.
Addition of pectin solution (DE = 49%) to Ca–, Sr–, Zn–,
Al– or La–pectate gels, followed by incubation with PGase
led to the release of 2.4–3.9 µM min–1 reducing sugars
(Table 2). When the amount of reducing sugars released from
the pectate gels (Table 2, column 3) was subtracted from the
amount released from the pectin–metal–pectate mixture
(Table 2, column 4), values were obtained for Ca, Sr and La
(Table 2, column 5) which were nearly identical to the
amount of reducing sugars released from pectin alone, viz.
2.0 µM min–1. Incubation of pectin–Cu–pectate and pectin–
Zn–pectate mixtures with PGase released only 0.3 and
1.2 µM min–1 reducing sugars, respectively, which is less
than the calculated amount. The pectin–Al–pectate mixture
released 2.3 µM min–1 reducing sugars, which was slightly
more than the amount released from pectin alone.
Pectin (DE = 49%) had an intrinsic viscosity of 0.23 l g–1,
which did not change significantly upon addition of Ca
(Fig. 1), Sr, Zn, Al or La (not shown). Only Cu yielded a hazy
solution with greatly increased viscosity (not shown), al-
though the intrinsic viscosity remained unchanged. Incuba-
tion of pectin with PGase for 15 min, in the absence of added
cations, resulted in a drastic loss of viscosity (Fig. 1), the
viscosity ranging between 0–0.08 l g–1 with 5.7 nkat PGase
and 0–0.03 l g–1 with 56.7 nkat PGase. Addition of Ca, Cu,
Sr, Zn, Al or La (0.45 mM) to pectin, followed by incubation
for 15 min with 5.7 nkat PGase resulted in the intrinsic
viscosity ranging between 0.08–0.09 l g–1, while incubation
with 56.7 nkat PGase yielded an intrinsic viscosity ranging
between and 0.01–0.03 l g–1 (Fig. 1).
on PGase activity
The degradation of Ca– and Al–pectate gels by PGase,
assessed by the amount of reducing sugars liberated, de-
creased with increasing cation saturation (Fig. 2). There was
an essentially linear decrease in the quantity of reducing
sugars released as the degree of saturation increased from
35% to 75% (Fig. 2). Above 75%, less reducing sugars were
released by PGase per unit increase in saturation. It is note-
worthy that no difference was observed in the amount of
sugars released from Ca–pectate and Al–pectate gels for a
given cation saturation.
The magnitude of reducing sugar release during PGase
digestion of cation–pectate gels, as shown in Table 2, was
highly negatively correlated (r = –0.86) with the degree of
cation saturation of these gels: sugar release declined at a rate
of 0.025 µmole min–1 for every percentage point increase in
cation saturation. Reducing sugar release was not signifi-
cantly correlated (r = –0.29) with the cation binding strength
as estimated by the Langmuir adsorption constant (data not
Fig. 2. The release of reducing sugars as a function of the Ca (C) and Al (●)
saturation of pectic acid. Pectic acid (degree of esterification 0%) was mixed
with varying volumes of CaCl2 or AlCl3 to give 40–100% charge neutrali-
sation of pectic acid. Assays contained 5.7 nkat PGase. Each data point is the
average of two or three determinations and standard errors are shown if not
obscured by the symbols.
488 J.B. Wehr et al. / Plant Physiology and Biochemistry 42 (2004) 485–492
Fig. 3. Buffer capacity of cell wall material from bean roots (A) and pectic
acid (B) without added cation (●) or with Ca (D), Cu (h), Sr (m), Zn ("),
Al (.) or La (∇) added. No correction has been made for the buffer capacity
of water, which is shown separately (C). The curves are means of 2–3 deter-
minations and standard error bars are shown if not obscured by the symbols.
shown). Similar results were obtained with cation-loaded cell
wall material: the autolytic release of total sugars was highly
negatively correlated (r = –0.91) to the cation saturation of
the material.
2.4. Buffer capacity of cell wall material and pectic acid
The buffer capacity of bean root cell wall material peaked
at pH 5, which is due to the pKa of galacturonic acid (GalA)
(Fig. 3A). Pectic acid showed a pronounced peak in buffer
capacity at pH 4.1 (Fig. 3B) [32]. The difference in buffer
capacity between cell wall material and pectic acid is a result
of local concentration effects (polyelectrolyte effect) in the
cell wall [28,32] and the presence of other charged groups in
cell wall material. The buffer capacity of cell wall material
and pectic acid increased below pH 3.5, probably due to
protonation of hydroxyl groups of GalA.
Addition of cations to cell wall material decreased the
buffer capacity between pH 4.5 and 6 due to binding of
cations to negatively charged groups such as GalA. Addition
of Zn, Ca, and Sr to cell wall material increased the buffer
capacity at pH < 4.3, while the buffer capacity increased at
pH < 4.0 for Cu and La (Al was not included in this experi-
ment) (Fig. 3A). Addition of cations to pectic acid led to
marked reduction in buffer capacity at pH 4.1 but increased
the buffer capacity at pH < 3.7 for Zn, Ca and Sr and at
pH < 3.3 for Cu, La and Al (Fig. 3B). These differences are
related to the ease with which the bound cations can be
displaced or exchanged by protons in the solution phase.
Hence, Ca, Sr and Zn are displaced more readily than are Cu,
Al, and La.
3. Discussion
3.1. Degradation of bean cell wall material releases
mainly neutral sugars
Cell wall material isolated from bean root tips released no
detectable uronic acid, indicating that autolysis of polygalac-
turonan in the root cell wall is negligible (Table 1). Since
little uronic acid was released, the total sugar fraction can be
equated to neutral sugars. Based on the predominant release
of neutral sugars, it appears that autolysis of bean root tips
follows the same characteristics as in the pea epicotyl (Pisum
sativum L.) [10] and the chickpea (Cicer arietinum L.) hypo-
cotyl [34,36], i.e. degradation of the “hairy region” of pectin.
Recio et al. [33] observed that bean epicotyl cell wall under-
went enzymatic hydrolysis (i.e. autolysis), releasing mainly
neutral sugars, in agreement with our results, whereas bean
callus cell wall material showed a non-enzymatic release of
uronic acids. These differences can probably be attributed to
changes in cell wall composition between callus and normal
cells.
To determine whether the minimal release of uronic acids
was due to unavailability of uronic acid containing cell wall
polymers or due to lack of endo-PGase activity, cell wall
material was also incubated with PGase. Since PGase incu-
bation increased uronic acid release more than total sugar
release (Table 1), it appears that the observed lack of degra-
dation of uronic acid containing polymers is due to low
endogenous endo-PGase activity of the cell wall material.
While it can be argued that PGase (pectinase) is a crude
polygalacturonase preparation, containing several enzyme
activities apart from endo-PGase (we found pectin esterase
but no lyase activity), the predominant activity is endo-
PGase. Furthermore, the native plant cell wall also contains
several enzymes acting in concert to degrade cell wall poly-
mers (e.g. PGase, pectin esterase and other glycanases).
Therefore, we believe that PGase (pectinase) is a suitable
model enzyme preparation to digest plant cell walls in au-
tolysis studies focussing on pectin.
While the autolysis model has its shortcomings in under-
standing the control of cell expansion in vivo, the turnover of
cell wall carbohydrates during cell elongation is very difficult
to study in vivo. Most carbohydrates are not released in vivo
but remain in the cell wall and are either incorporated into
other polymers or play a role as signal molecules. In our
study, we used the apical part of roots (0–5 mm section)
which is the most extensible and hence, the native enzymes
present in the cell wall should be involved in cell wall
metabolism and cell expansion. Thus, the released sugars in a
cell-free system may be indicative of the sugars released in
vivo during cell expansion.
 J.B. Wehr et al. / Plant Physiology and Biochemistry 42 (2004) 485–492
3.2. Cations inhibit degradation of bean root cell wall
material
It is known that Ca inhibits autolysis [19,38,39] but it is
less well understood if other cations have a similar effect on
autolysis. Therefore, cell wall material was loaded with Ca,
Cu, Sr, Zn, Al and La and the autolytic release of sugars
determined.
Autolytic release of total sugars from cell wall material
loaded with Cu, Al or La ions was inhibited, whereas Ca, Sr
or Zn ions had little effect (Table 1). This may be due to the
relatively low cation saturation of the Ca-, Sr- or Zn-loaded
cell wall material, the autolytic release of total sugars being
highly negatively correlated (r = –0.91) with the cation satu-
ration of the cell wall material. Also, the differential effect of
cations on autolysis makes it unlikely that the observed sugar
release can be attributed to non-enzymatic degradation of the
cell wall, e.g. via b-elimination of pectin, since non-
enzymatic degradation of pectin is unaffected by cations
[24].
Release of uronic acid from cation loaded cell wall mate-
rial was low during autolysis and addition of PGase did not
significantly increase uronic acids (Table 1). This suggests
that the uronic acid containing polysaccharides in metal-
loaded cell wall material were unavailable for enzymatic
degradation. Otherwise, the added PGase should have greatly
increased the amount of uronic acid liberated. The non-
availability could have been due to cation binding, making
the cell wall (probably pectin) resistant to enzymatic degra-
dation. It is less likely that the uronic acid-containing
polysaccharides had a high DE, preventing PGase action,
since this would have resulted in a low cation binding capac-
ity, which was not observed. Several researchers have ob-
served a decrease in autolytic sugar release, especially uronic
acid, in response to higher Ca concentrations, whereas addi-
tion of chelators increases autolysis [7,19,24,37] and the
consensus view is that Ca binding to pectin decreases its
degradability by enzymes.
3.3. Cation binding to pectin may inhibit degradation
of pectin and cell walls
Our results show that cell wall autolysis is decreased by
cations but it is not clear if this effect is due to cation binding
to pectin in the cell wall. Therefore, we employed a metal–
pectate model system to test the effect of cations on the
enzymatic degradation of the gels. Results indicate that metal
pectate gels, formed by the addition of Cu, Al or La to pectic
acid (DE = 0%), were poorly degraded by PGase whereas
Ca–, Sr– and Zn–pectate gels were degraded more rapidly
(Table 2). The low activity of PGase on cation–pectate gels
was not due to enzyme inhibition since addition of pectin
solution (DE = 49%) to the gels increased the release of
reducing sugars to rates nearly identical to those measured
for pectin alone. Similarly, the viscometry analysis of PGase
action on pectin in the presence and absence of cations
489
(Fig. 1) indicated that PGase was not inhibited by the added
cations and the effects of cations must be related to cation–
pectin steric effects. Only Cu– and Zn–pectate gels appeared
to partially inhibit pectin hydrolysis by PGase, while Al–pec-
tate appeared to slightly enhance PGase activity (Table 2).
The studies with cell wall material and metal–pectate gels
indicated that the latter is a good model for the study of cation
effects on bean cell walls. Our results suggest that cation
saturation is more important to controlling enzymatic pectin
or cell wall degradation than the type of cation. Hence, we
investigated the degree of cation saturation in more detail and
focussed on Ca and Al cations, which differ vastly in binding
strength [3].
3.4. Cation saturation of pectin controls enzymatic
degradation
The Ca- or Al-saturation of the pectate gels varied be-
tween 35% and 100% and the degradability by PGase was
only related to the cation saturation and not the type of cation
(Fig. 2). It seems likely that galacturonic acid residues not
contributing to the junction zones or neutralised with cations
can be liberated by PGase. This is similar to our earlier work
showing that the number of non-methoxylated carboxyl
groups determines the rates of pectin degradation [41]. Thus,
only carboxyl groups that are undissociated can bind PGase
and are sites that can be cleaved by the enzyme.
It is worthy of note that the cation saturation at the inflec-
tion point in Fig. 2 corresponds to the normally observed
cation saturation of cell wall material (60–80%) and may be
relevant for the normal functioning of the cell wall in terms of
buffer capacity [17,18]. The buffer capacity of cell wall
material and pectin shifted to lower pH due to cation binding
and indicates that Al, La and Cu bind stronger to cell wall
material and pectic acid than does Ca, Zn and Sr. This may
partly explain the toxic effect of Cu, Al and La, especially on
root growth, inhibiting the degradation of pectin which in
turns shields hemicellulose [1] and ultimately leads to a
reduction in cell expansion. Obviously, a strongly bound
cation may lead to a higher cation saturation of the cell wall
since it cannot be displaced easily, thereby having a more
detrimental effect on pectin degradation.
In conclusion, the results of these cell wall and pectate-gel
studies may be explained by considering that an endogenous
enzyme (probably PGase) splits the pectin backbone, releas-
ing the “hairy regions” which contain large branches com-
prising neutral sugars but little uronic acid [30] (Fig. 4). The
terminal GalA groups in a junction zone do not bind Ca, Sr or
Zn strongly [21,27]. Therefore, at low concentrations of
these cations, the junction zone can “peel open” from the
end, allowing endo-PGase to cleave the polygalacturonan
backbone and liberate the “hairy region”. On the other hand,
strongly binding cations (Cu, Al and La [20]), or high con-
centrations of cations, bind to terminal GalA groups of the
junction zone, forcing it closed and leading to a change in
pectin structure (Fig. 4). The sterical constraint prevents
490 J.B. Wehr et al. / Plant Physiology and Biochemistry 42 (2004) 485–492
Fig. 4. Schematic representation of rhamnogalacturonan I in the cell wall.
Unbranched polygalacturonan regions (“smooth region”) are terminated by
rhamnose sugars, which contain branches (“hairy region”) of galactan,
arabinogalactan or arabinan pectin. Polygalacturonan regions can form
junction zones (egg-boxes) with divalent cations. A, Proposed structure with
weakly bound cations (e.g. Ca) leading to a “peeling open” of the junction
zone and subsequent cleavage by PGase (endo-PGase); B, strongly bound
cations (e.g. Cu) limit “peeling open” and prevent PGase (endo-PGase)
action.
endo-PGase action, thereby reducing the liberation of sugars.
Such a model would describe the observed effects of cations
on the autolytic release of sugars from cell walls. It should be
kept in mind, however, that other enzymes with a different
mode of action are also involved in autolysis of pectin and
cell walls. Further detailed studies with these enzymes are
warranted.
4. Methods
4.1. Pectic acid and cell wall preparation
Apple pectin with a degree of esterification (DE) of 49%
and a GalA content of 73% (w/w) was obtained from Sigma
Chemical Co. (St. Louis, USA). Pectic acid (DE = 0%) was
prepared from apple pectin by alkaline de-esterification with
0.5 M NaOH under N2 gas for 15 min before desalting with
Amberlite IR-120 (H+) cation exchange resin. Purified cell
wall material was obtained from the apical 0–5 mm of bean
(P. vulgaris L. cv. Sinatra) roots grown in tap water at
ambient temperature and light. Approximately 6000 root tips
were homogenised in 10 mM sodium acetate buffer (pH
4.65) with a pestle and mortar on ice. The homogenate was
washed on stainless steel screens (38–125 µm) sequentially
with 2 l ice cold (0–4 °C) sodium acetate buffer, 250 ml 70%
acetone, 350 ml ice-cold deionised water and 100 ml sodium
acetate buffer. The cell wall material was stored at 0–4 °C as
a slurry in sodium acetate buffer and used within 10 d. At this
temperature, no autolysis of stored cell wall material was
observed within 3 d.
4.2. Enzyme assays
The activity of crude endo-PGase (PGase; EC 3.2.1.15)
(pectinase, Cat. No. P9179, Sigma Chemical Co., St. Louis,
USA) was assayed in “PGase buffer” (25 mM sodium acetate
in 125 mM NaCl, pH 4.5) [41]. The enzyme preparation
contained a low activity of pectin esterase (reducing the DE
from 49% to 45% within 30 min under the assay conditions),
while no pectin lyase or pectate lyase activity was detected
under the assay conditions. Viscometric and colorimetric
tests indicate that the preparation contained negligible exo-
PGase activity.
4.3. Quantitation
Total sugars were measured with the phenol-sulphuric
acid method [11] using galactose as a standard. Reducing
sugars were quantified with the alkaline ferricyanide method
using GalA as standard [41]. Uronic acids were determined
with a modification of the method of Blumenkrantz and
Asboe-Hansen [4], using 2-phenylphenol (98% pure, Fluka)
instead of 3-phenylphenol. Statistical analyses were per-
formed with the REG and GLM procedures of SAS Release
6.03 (SAS Institute Inc., Cary, NC, USA). Significance was
tested at the 5% probability level and treatment means were
compared with Tukey’s t-test.
4.4. Autolysis and PGase digestion of cell wall material
Cell wall material (0.4 ml of slurry) was incubated in
1.6 ml autolysis buffer (50 mM sodium acetate buffer (pH
5.0) containing 150 mM NaCl and 2 mM sodium azide) with
or without added PGase (5.7 nkat). Cell wall material (1.5 ml
of the slurry) was also loaded with cations by mixing with
1.5 ml of 5 mM Cu, Ca, Sr, Zn, Al or La chloride solutions for
2 h at 0–4 °C. After centrifugation, the pellet was suspended
in 3 ml of 5 mM metal chloride solution for 1 h at 0–4 °C.
This step was repeated a third time with standing for 30 min
at 0–4 °C. Thereafter, the material was washed three times in
deionised water and centrifuged. The pellet was mixed with
autolysis buffer and incubated for 17 h at 35 °C or 0–4 °C
(control) with or without added PGase (5.7 nkat). After
incubation, samples were centrifuged and the concentrations
of total sugar and uronic acid in the supernatant determined
and expressed on a dry mass (DM) basis of cell wall material.
The experiment was replicated three times. The cation satu-
ration of cation-loaded cell wall material was expressed
relative to the amount of La bound, after wet-ashing and
determination of the cation concentration by inductively
coupled plasma atomic emission spectroscopy (ICPAES).
 J.B. Wehr et al. / Plant Physiology and Biochemistry 42 (2004) 485–492
4.5. Degradation of cation–pectate gels and pectin by
PGase
Cation–pectate gels were prepared by adding an excess of
5 mM metal chloride (Ca, Cu, Sr, Zn, Al or La) to pectic acid.
The pH was increased to 4.5 (4.0 for Al) with 0.1 M NaOH
and the gels allowed to stand overnight. Gels were washed
five times with deionised water by centrifugation and finally
by dialysis. After dialysis, gels (3 ml) were mixed with 50 ml
PGase buffer. Aliquots (5 ml) were mixed with either 1 ml
0.1% pectin (DE = 49%, to assess possible enzyme inhibition
of PGase) or deionised water (control) and incubated with
11.4 nkat PGase at 21.5–22 °C. Aliquots were withdrawn
after various time intervals and assayed for reducing sugars
[41]. Blanks employed heat-inactivated PGase (boiled for
10 min). The experiment was replicated three times.
4.6. Influence of the degree of cation saturation on PGase
activity
Pectate gels with varying degrees of Ca- or Al-saturation
were prepared by adding calculated volumes of Ca or Al
chloride to pectic acid. The gels were stood overnight before
raising the pH to 4.25 (Ca gels) or pH 4.0 (Al gels) with
20 mM NaOH. Gels were washed three times with deionised
water by centrifugation followed by dialysis. Gels (2.6 ml)
were mixed with 30 ml PGase buffer (pH 4.5 for Ca; pH
4.0 for Al), and 5 ml aliquots mixed with 5.7 nkat PGase and
incubated at 26–26.5 °C. After various time intervals, 1 ml
aliquots were withdrawn and assayed for reducing sugars
[41]. Aliquots of the gels were also dried and the results
expressed on DM basis and represent the average of two
replicates. The degree of cation saturation was determined by
removing bound Ca and Al ions with Amberlite IR 120 (H+)
resin and measuring the number of carboxyl groups of pectic
acid by titration with NaOH to pH 7–8. The cation concen-
tration of the dried gel was determined by ICPAES after
wet-ashing the sample with nitric/perchloric acid (5:1, v/v)
and hydrogen peroxide.
4.7. Buffer capacity of cell wall material and pectic acid
Solutions containing stoichiometric quantities of Ca, Cu,
Sr, Zn, Al and La were added to cell wall material or pectic
acid. The resultant cation–cell wall suspensions and cation–
pectate gels were slowly titrated to pH 6.0–6.5 with 0.02 M
NaOH using a Radiometer ABU 901 autoburette connected
to a Radiometer TIM 900 autotitrator (Radiometer Analyti-
cal, Copenhagen, Denmark). Thereafter, samples were al-
lowed to equilibrate for 2–6 d at 0–4 °C before being slowly
titrated (approx. 150 µl min–1) to pH 3 with 0.02 M HCl and
the titration curves automatically recorded. All measure-
ments were conducted in triplicate with pectate and in dupli-
cate with cell wall material. Back-titration curves were
analysed with the TableCurve 2D program (Jandel Scientific,
San Rafael) and a linear equation with the best fit (highest r2)
491
and continuous first derivative chosen. The buffer capacity
was calculated from the first derivative of the selected equa-
tion, thereby minimising noise in the buffer curve.
4.8. Viscometric assessment of cation effects on pectin
and PGase
A Cannon–Fenske type viscometer was employed for
capillary viscosity measurements. Solutions were serially
diluted with 125 mM NaCl and the reduced viscosity (here-
after referred to as viscosity) calculated from the drainage
times. The intrinsic viscosity was determined by extrapola-
tion to zero pectin concentration [25]. Four treatments were
compared: (a) “Control” 2.5 ml 1% pectin solution, 2 ml
PGase buffer and 0.5 ml deionised water. (b) “PGase” 2.5 ml
1% pectin, 2 ml PGase buffer, 0.45 ml deionised water and
50 µl PGase (5.7 nkat). (c) “Cation” 2.5 ml 1% pectin solu-
tion, 2 ml PGase buffer, 0.45 ml of 5 mM Ca, Cu, Sr, Zn, Al
or La solution (as chlorides) and 50 µl deionised water. (d)
“Cation + PGase” 2.5 ml 1% pectin solution, 2 ml PGase
buffer, 0.45 ml of 5 mM Ca, Cu, Zn, Sr, Al or La solution and
50 µl PGase (5.7 nkat). Solutions containing PGase were
incubated for 15 min at 25 °C before boiling for 4 min to
inactive the enzyme. Preliminary studies showed that boiling
for 4 min did not influence the viscosity of the solution.
Aluminium effects on viscosity were also studied at pH 4.0 to
limit possible formation of hydroxo–Al complexes; the re-
sults were the same as those obtained at pH 4.5.
Acknowledgements
One author (JBW) gratefully acknowledges receipt of a
Commonwealth of Australia Overseas Postgraduate Re-
search Scholarship and a University of Queensland Ernest-
Singer scholarship to conduct this study.
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