Journal of Experimental Botany, Vol. 47, No. 304, pp. 1717-1724, November 1996
Journal of
Experimental
Botany
In vitro salt tolerance of cell wall enzymes from
halophytes and glycophytes
Meera Thiyagarajah1, S.C. Fry2 and A.R. Yeo1'3
1
School of Biological Sciences, The University of Sussex, Brighton BN19QG, UK
2
ICMB, Division of Biological Sciences, The University of Edinburgh, The King's Buildings, Mayfield Road,
Edinburgh EH93JH, UK
Received 12 February 1996; Accepted 20 June 1996
Abstract
The halophyte Suaeda maritima grows optimally in
high concentrations (40-60% seawater) of salt. In
these conditions the concentration of salt in the apoplast of the leaves is at least 500 mM, a concentration
which severely inhibits the activity of cytoplasmic
enzymes of both glycophytes and halophytes. The in
vitro salt tolerance of a number of cell wall enzymes
was assayed in the presence of a range of concentrations of NaCI. There was no significant inhibition of
the activity of galactosidase, glucosidase, peroxidase
or xyloglucan endo-transglycosylase extracted from
Suaeda maritima by in vitro concentrations of NaCI up
to at least 1 M. In vitro salt tolerance of cell wall
enzymes was not restricted to the halophyte, similar
enzymes from the non-halophilic relative Kochia tricophylla, and from the glycophytes Vigna radiata and
Cicer arietinum, were inhibited little, or not at all, by
the same concentrations of salt. Pectin esterase was
somewhat less tolerant, but activity at 500 mM NaCI
was still greater than at 0 mM NaCI in both Suaeda
and Vigna. It is concluded that these enzymes of the
cell wall compartment are much more salt-tolerant
than cytoplasmic enzymes of higher plants. The results
are discussed in relation to conditions thought to pertain in the apoplast.
Key words: Apoplast, cell wall enzymes, halophyte, salt
tolerance, Suaeda maritima.
Introduction
Enzymes from halobacteria have an obligate requirement
for a high concentration (one to several molar) of ions
for their maximal activity (Larson, 1967; Hochstein and
Dalton, 1968; Libel et al, 1969). However, this general
adaptation appears to be unique to the halobacteria and
enzymes from higher plants have generally been found to
be salt-sensitive, irrespective of whether they come from
salt-tolerant or from salt-sensitive species. Flowers
(1972a) showed that glucose-6-phosphate dehydrogenase
and malate dehydrogenase extracted from Suaeda maritima were inhibited by 50-70% by 333 mM NaCI while
the plants contained in excess of 800 mM sodium on a
cell water content basis. These in vitro effects of NaCI
were indistinguishable from those on the corresponding
enzymes from the salt-sensitive Pisum sativum (Flowers,
1972a) and a range of other halophytes (Flowers, 1972Z>).
Greenway and Osmond (1972) showed a similar pattern
of salt sensitivity in a number of cytoplasmic enzymes
whether extracted from the salt-tolerant A triplex spongiosa and Salicornia australis or from the salt-sensitive
Phaseolus vulgaris. Flowers et al. (1977, Table 3) were
able to list some 35 cases, covering 11 cytoplasmic
enzymes and 20 different salt-tolerant species, in which
activity was inhibited at least 50% by the average salt
concentration in the tissue from which the enzymes were
extracted. In vitro salt sensitivity has been found not only
for single enzymes, but whole cellular processes: state 3
respiration in Suaeda was inhibited 60% by 500 mM NaCI
(Flowers, 1974) and leucine incorporation into protein
was inhibited two-thirds by the addition of 200 mM
sodium to the optimal (lOOmM) potassium (Hall and
Flowers, 1973).
Observations that the in vitro behaviour of cytoplasmic
enzymes from halophilic higher plants was as salt-sensitive
as corresponding enzymes from glycophytes, meaning
that enzymes from both groups would be severely inhib-
*To whom correspondence should be addressed. Fax: + 44 1273 678433, E-mail: [email protected]
Oxford University Press 1996
1718
Thiyagarajah et al.
ited at the average salt concentration in the halophyte
tissue, provided the first circumstantial evidence that, in
the protoplast of halophytes, salt is compartmentalized
within the vacuole. This model has been reinforced by
data from efflux and X-ray microanalysis and by the
elaboration of the role of compatible cytosolutes (Flowers
etai, 1977, 1986). The very specific, and highly conserved,
ionic requirements for protein synthesis (Leigh and Wyn
Jones, 1984) will have obviated any general advantage in
ionic adaptation by cytosolic enzymes. Although there is
some evidence (Flowers and Dalmond, 1992) that the
ionic requirements for protein synthesis may be a little
less stringent in the halophyte than glycophyte, synthesis
was still drastically inhibited by salt. In the presence of
optimal (100 mM) potassium, the incorporation of 35Smethionine was maximal in the presence of 100 mM NaCl
and was inhibited, relative to this, by three-quarters when
the NaCl concentration was increased to 200 mM
(Flowers and Dalmond, 1992). The general consensus is
that cytoplasmic enzymes are not exposed to more than
150—200 mM NaCl even when plants are growing in
seawater and even when plants have seawater concentrations of salt in their tissues as a whole.
The vacuoles of halophytes growing at high salinity
may contain 500 to 1000 mM NaCl. No difference from
glycophytes could be found, however, in the protontranslocating phosphatases or ion channels that span the
tonoplast (Leach et al., 1990; Maathuis et al., 1992). The
conclusion must be that any salt-sensitive domains are
directed into the cytoplasm and not into the vacuole.
Little attention has yet been paid, however, to the salt
tolerance of enzymes present in the cell wall compartment.
It is now known from studies with the pressure probe
(Clipson et al., 1985) and by X-ray microanalysis
(Hajibagheri and Flowers, 1989) that the apoplast of
Suaeda maritima has an ion concentration equivalent to
that in the vacuole: 554 mM on a water content basis at
the growth optimum (Flowers, 1985). As with the tonoplast, enzymes which span the plamalemma may have
their salt-sensitive domains directed into the cytoplasm.
However, there is also a group of enzymes which are
located in the cell wall compartment. These include
enzymes which participate directly or indirectly in cell
wall loosening or tightening. The plant grows optimally,
becomes succulent and so shows the most cell expansion
in conditions where apoplastic enzymes will be exposed
to salt concentrations sufficient to inhibit the activity of
most cytoplasmic enzymes. This implies either that the
cell wall-located enzymes are much more tolerant of salt
than the cytoplasmic enzymes, or else that they are
protected in some way within the wall. If the apoplastic
enzymes are more tolerant of salt than cytoplasmic
enzymes, then the question arises as to whether this is a
general property of this group of enzymes, irrespective of
the salt tolerance of the species of origin, or whether this
is a particular adaptation of halophytes.
The experiments reported were to investigate the in
vitro response to NaCl of xyloglucan endotransglycosylase
(XET: an enzyme proposed to be involved in cell wall
loosening) and a number of other enzymes considered to
be located in the apoplast, which were extracted from
Suaeda maritima, from a non-halophilic member of the
Chenopodiaceae {Kochia tricophylla which is grown as an
ornamental) and from two salt-sensitive glycophytes
(Vigna radiata and Cicer arietinum).
Materials and methods
Growth of plants
Seeds of Suaeda maritima (L.) Dum. were collected from the
Cuckmere estuary in East Sussex and were germinated in
washed silver sand in a growth chamber (Saxcil). The growth
conditions were a 16 h photoperiod of 450 ^mol m~ 2 s" 1
(photosynthetically active radiation) at 22 °C and 60% relative
humidity and a dark period of 18 °C and 70% relative humidity.
Seedlings were transplanted and grown in culture solution
(Stout and Arnon, 1939) supplemented with 4%, 40% or 100%
artificial sea water (ASW: Harvey, 1966). The solutions were
aerated and the air line filtered through water. Enzyme
preparations of Suaeda were made using leaves from apical
regions (1-2 cm) of at least 10 plants.
Kochia trichophylla L. is a member of the same family
(Chenopodiaceae), but is not a native of salt marshes. The
seeds used were of a selection used for ornamental horticulture
(Suttons Seeds Ltd., UK). Seeds of Kochia were germinated in
fresh water and grown in a pot of sand irrigated with Stout
and Arnon culture solution without added ASW in the same
growth conditions as Suaeda. Enzyme preparations were made
from young leaves.
Seeds of Vigna radiata (L.) Wilczek (mung bean, Suttons
Seeds Ltd., UK) and Cicer arietinum L. (chickpea, Central Soil
Salinity Research Institute, India) were soaked in distilled water
overnight and germinated in darkness at 25 °C and 75% relative
humidity. One-week-old etiolated hypocotyls were used for cell
wall preparations.
Ceil wall isolation
Leaf material was frozen in liquid nitrogen and ground in a
pre-cooled mortar and pestle. The tissue was further ground in
TRIS buffer (10 mM, pH 9.2) with 1% Triton X-100 and 0.1%
mercaptoethanol. The resulting extract was centrifuged at 27
000 g for 10 min and the pellet was reground in the same buffer
and re-centrifuged. The pellet was resuspended again in the
same buffer and centrifuged at 27 000 g for 15 min (modified
from Birecka et al., 1973).
Extraction of hydrolases
The cell wall preparation (the pellet from above) was suspended
in 2 M NaCl and allowed to stand on ice for 1 h. The extract
was then centrifuged at 27 000 g for 15 min and the supernatant
was collected for enzyme assays. The supernatant was desalted
by dialysing against phosphate buffer (5mM, pH 6.5) at 4°C
overnight. The desalted extract was then concentrated by
dialysis against (25% w/v) PEG (MW 20000). This extract was
used to assay hydrolases, pectin esterase and peroxidase
(modified from Seara et al., 1988).
Salt tolerance of cell wall enzymes
Protein content determination
Protein content was determined according to Bradford (1976)
using Coomassie brilliant blue G (Biorad) and bovine serum
albumin (Sigma) as standard.
1719
of the assay mix was adjusted to be constant. An NaCl-induced
pH shift (Flowers, 1972a; Munns et al., 1983) can cause
apparent stimulation or inhibition of enzyme activity. In the
other enzyme assays employed there was no appreciable effect
of NaCl on the pH.
XET extraction
XET was extracted directly from fresh tissue according to Fry
et al. (1992). Individual leaves ( l i d after salinization, at least
3 replicates ) were weighed and ground with acid-washed sand
in a mortar and pestle in an ice-cold extractant consisting of
CaCl 2 (10 mM), ascorbic acid (10 raM), succinic acid (50 mM),
DTT (1 mM) at pH 5.5 using 1 ml extractant per gram fresh
weight. The homogenate was centrifuged at 2000 g for 5 min
and the resulting supernatant was used for the XET assay.
XET assay
Tamarind xyloglucan with pH]XLLGol was used as the
substrate for the XET enzyme (Fry et al., 1992). The reaction
mixture contained xyloglucan (62 ^g), 1 kBq [ 3 H]XLLGol, 10
^1 enzyme extract and NaCl +water to give a range of NaCl
concentrations up to 2.0 M in a final volume of 30 y\ in an
Eppendorf vial. The reaction was allowed to proceed for 1 h at
25 °C and the reaction was stopped by the addition of 20%
(w/v) formic acid (100 fA). The total reaction mixture was
loaded and dried on to chromatography paper divided into
squares of 40 mm. The paper was washed at least 1 h in running
tap water to remove unreacted [ 3 H]XLLGol. Each square of
paper was then dried at 60 °C, rolled into a cylinder with the
loaded side outermost, placed in a 22 ml scintillation bottle and
soaked with approximately 2 ml of scintillant (Optiphase Safe).
Radioactive polymeric product, which had remained on the
paper, was assayed by liquid scintillation spectrometry (LKB)
and the enzyme activity expressed as: Bq product formed kBq"'
substrate h " 1 g" 1 fresh weight.
Pectin esterase assay
Pectin esterase was detected by spectrometry using the change
in colour of a pH-sensitive indicator dye (bromothymol blue)
to indicate the acidification of the reaction medium as hydrolysis
of the pectin substrate releases protons. The reaction mixture
in a final volume of 1.2 ml contained; 0.5 ml of apple pectin
(0.1% (w/v) Sigma), 0.1 ml bromothymol blue (0.2% w/v),
0.5 ml NaCl + water (to provide a final concentration up to 1
M) and 0.1 ml enzyme extract (5-10 /ig protein). Before adding
the enzyme the reaction mix was alkalinized by adding a few
microlitres of 200 mM NaOH using a syringe to bring the
initial absorbance to 0.8 at 590 nm. The reaction was started
by adding the enzyme extract. The change in absorbance was
monitored at 590 nm for 5 min and the rate of reaction was
calculated using the Biochrom enzyme kinetics programme
(LKB) and expressed as AAS9O min" 1 mg" 1 protein (Warrilow
et al., 1994). The rate of reaction without enzyme was negligible.
Peroxidase assay
1.0 ml of guaiacol (Sigma: 25 mM in 10 mM phosphate buffer,
pH 6.5) was mixed with 0.1 ml of hydrogen peroxide (10 mM)
adjusted to pH 7.0 using TRIS buffer (10 mM, pH 9.2).
NaCl + water was added to give five final concentrations of
NaCl of 0-2 M in a final volume of 2.6 ml. The reaction was
started by the addition of 0.1 ml of enzyme extract and
monitored for 2 min at 470 nm. The rate of reaction was
calculated using the Biochrom programme and expressed as
AA410 min" 1 ^ g " 1 protein. Addition of NaCl produced substantial changes in pH in this assay medium, consequently the pH
Galactosidase and glucosidase assay
The enzyme activity of hydrolases was assayed by monitoring
the release of p-nitrophenol from ^-nitrophenol glycoside
substrates (pnp a- and j3-galactoside, pnp a- and (3-glucoside,
Sigma). The reaction mixture in a final volume of 0.5 ml
comprised 0.3 ml substrate (3 mM in acetate buffer 10 mM,
pH 5.2) and 0.1 ml NaCl + water (to give a final concentration
of NaCl from 0-1 M) and 0.1 ml cell wall extract. The reaction
was initiated by adding the enzyme extract and incubating at
25 °C for 20 min. The reactions were terminated by adding
0.5 ml glycine buffer (pH 10.4) and /I400 was measured. The
activity of the enzymes are expressed as AA^ min" 1 mg" 1
protein.
Results
Galactosidase and glucosidase
The activities of all the hydrolases assayed were found to
be unaffected by concentrations of NaCl up to 1 M. The
absolute activities of the glucosidases were higher in Vigna
than in Suaeda, but there was no significant difference at
^ = 0.05 in the effect of NaCl according to whether the
enzymes were extracted from Suaeda leaves or from Vigna
hypocotyls (Fig. 1). Enzymes from both halophyte and
glycophyte showed tolerance in vitro to a range of salt
concentrations much higher than reported for cytoplasmic
enzymes of higher plants (Flowers 1972a, b; Flowers
et al., 1976a; Greenway and Osmond, 1972; Beer et al.,
1975).
Pectin esterase
The activity of pectin esterase was maximal in the range
50-200 mM NaCl for Suaeda and 80-500 mM for Vigna
(Fig. 2). Enzymes from both glycophyte and halophyte
were initially activated by salt and above an optimum
activity decreased at higher salt concentrations. The
degree of salt activation (with respect to 0 NaCl) was
greater in Suaeda (20-fold) than the Vigna enzyme
(2.3-fold). Even though the activity was reduced at higher
concentrations of NaCl, it was still higher in the presence
of 500 mM NaCl than in 0 mM NaCl in both species.
The absolute activity (per unit protein) was, at its maximum, about 4-fold higher in Suaeda than in Vigna.
Peroxidase
The activity of guaiacol peroxidase was found to be
unaffected by NaCl concentrations of up to 1.9 M in both
halophytic and glycophytic species (Fig. 3). Relative to
an assay medium without NaCl, the activity of both the
Suaeda and Vigna wall-bound peroxidase was stimulated
1720
Thiyagarajah et a).
1.2
!E
.
(A) Suaada
6 _ (B) Vigna
I
1.0 - L S L r
T-
T-
T -
5
[Ml/
4
- 1
3
-
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5" °
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Z> o>
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r1
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<
T
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B
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T
i
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B
B"
2
02
0
( T r'
\
(-
„ -
A
1
_
0
1
0.0
0.2
1
1
1
1
i
i
i
i
i
i
0.4
0.6
0.8
1.0
0.0
0.2
0.4
0.6
0.8
1.0
NaCI concentration (M)
Fig. 1. Effect of NaCI on the in vitro activities of cell wall hydrolases. Four hydrolases (a-glucosidase (O), 0-glucosidase ( • ) , a-galactosidase (d)
and /3-galactosidase (•)) were extracted from eel) walls obtained from leaves of Suaeda marittma (A) and etiolated hypocotyls of Vigna radiam (B)
and assayed in a range of concentrations of NaCI. Means and standard error (n = 3). Where error bars are not visible they are smaller than the
data marker.
wall enzymes may be exposed to much more variable
conditions. The activity of these enzymes is regulated by
the electrostatic potential between the inside and outside
of the cell wall and by the pH in the apoplast (Ricard
XET
and Noat, 1986). Ricard and Noat (1986) proposed that
the activity of the wall enzymes is regulated by the wall
XET was extracted from the halophytic Suaeda, the nonpH, the enzymes becoming active at a particular pH
halophytic chenopod Kochia and a glycophyte system
(etiolated Cicer arietinum). In neither Suaeda nor Kochia range otherwise remaining inactive.
There are many enzymes present in the cell wall comwas there any significant effect of NaG concentration up
partment,
but only some of them are likely to be directly
to 2.0 M NaCI (Fig. 4). The XET activity from Cicer
involved
in
the growth of the wall (Fry, 1988, 1995). The
was significantly (P= 0.01) reduced at 1.0 M NaCI relative
hydrolases
are
involved in cell wall autolysis which leads
to the control (at 0 NaCI), but was still 65% of the
to
changes
in
the
structure of the cell wall associated with
control in the presence of 2.0 M NaCI. The enzymes from
growth
(Huber
and
Nevins, 1979; Mufioz et ai, 1993).
both halophyte and glycophyte were highly tolerant of
Although
the
galactosidases
and glucosidases are not
NaCI in vitro with that from the halophyte being someconsistently
correlated
with
growth,
they are known to
what more tolerant.
hydrolyse wall polymers which can cause structural
changes in the cell wall (Huber and Nevins, 1979; Dopico
Discussion
et ai, 1989; Munoz et ai, 1993).
Peroxidases are haemoproteins which catalyse the
The cell wall is structurally complex and its surface charge
oxidation of phenolics and decomposition of H2O2 (Kalir
is dominated by galacturonans (Grignon and Sentenac,
1991). Cations are electrostatically attracted to these fixed et ai, 1984), which probably occur in all primary cell
walls. Their expression is sensitive to a wide range of
negative charges and also exist in solution in the water
stimuli, e.g. hormones, temperature, drought, infection,
in the apoplastic compartment. The enzymes in the cell
and Ca 2+ (Fry, 1988; Gasper et ai, 1985). Peroxidases
wall are either freely soluble or are bound to the wall
occur in many isoforms, both acidic and basic (Gasper
polymers, either covalently or ionically (Huber and
et ai, 1985). The activity of these different isoforms can
Nevins, 1979; Fry, 1988; Grigon and Sentenac, 1991).
differ according to the pH and inorganic ion concentraSalt-extractable wall enzymes are ionically bound to the
tion. The physiological importance of peroxidase may be
wall (Fry, 1988).
the tightening of cell walls through oxidative cross-linking
Unlike cytoplasmic enzymes which experience the
of phenolics, which restricts further growth of the cell.
highly regulated conditions within the protoplast, cell(though not significantly at P=0.05) in 1.9 M NaCI. The
peroxidase activities vary according to the substrate used
and assay conditions employed.
Salt tolerance of cell wall enzymes
0
150
300
450
600
750
150
300
450
600
750
NaCI concentration (mM)
Fig. 2. Effect of NaCI on the m vitro activity of pectin esterase. The
enzyme was extracted from cell walls obtained from leaves of Suaeda
maritima (A) and etiolated hypocotyls of Vigna radiata (B) and assayed
in a range of concentraions of NaCI. Means and standard error (n = 3).
Unlike the other enzymes studied there are data for
salt tolerance of peroxidases available for comparison,
because enzymes with similar activities occur in other
compartments of the cell. Unfortunately, all the data are
for cytoplasmic peroxidases and the salt response of
peroxidases is not typical of cytosolic enzymes in general.
The cytoplasmic peroxidase (substrate /?-phenylenediamine) from Suaeda showed 50% inhibition by 330 mM
NaCI at pH 5.0, but was unaffected at pH 7.0 (Flowers,
1972a). The same enzyme from another halophyte
(Spartina) showed optimal activity at 500 mM NaCI (substrate o-dianisidine, at pH 6.0) and whilst the activity was
inhibited above 500 mM it was still higher at 3 M than
in the control (Gettys et al., 1980). The activity of the
enzyme from the halophyte Halimione with guiacol as
substrate (at pH 5.0) and in 2.5 M NaCI was double that
in the 0 NaCI treatment (Kalir et al, 1984). Peroxidase
enzymes, in general, appear to be substantially tolerant
of ionic concentrations, irrespective of the cell compartment of origin.
0.5
1.0
1.5
1721
2.0
NaCI concentration (M)
Fig. 3. Effect of NaCI on the in vitro activity of cell wall peroxidase.
Peroxidase was extracted from cell walls obtained from leaves of Suaeda
maritima (A) and etiolated hypocotyls of Vigna radiata (B) and assayed
in a range of concentrations of NaCI with guaiacol as substrate. Means
and standard error (n = 3).
Pectin esterase is responsible for de-esterifying the
methyl galacturonate residues in the pectin of the wall.
The activity of this enzyme has several consequences:
alteration of apoplastic pH, effects on charge density and
ionic environment in the wall, and modulation of the
ability of the pectin to form rigid gels (Pressey, 1984;
Jones and Warrilow, 1994). The de-esterified pectin can
bind with Ca2+ ions and form gels. The gelling properties
of cell wall pectin may modulate the wall extensibility
which is associated with plant growth (Yamaoka and
Chiba, 1983; Pressey, 1984; Jarvis, 1984; McCann et al,
1994). A similar pattern of concentration-dependence to
that observed in these studies was reported in tomato
pectin esterase (Warrilow and Jones, 1995). The enzyme
binds to the substrate by electrostatic attraction and it is
interaction with cations that is thought to account for
the concentration-dependency on salt. Initially, the
sodium ion activates the enzyme. The activation effect of
cations is to shield the negative charges of the pectin
1722
Thiyagarajah et al.
0.5
1.0
1.5
2.0
0.5
1.0
1.5
2.0
100
90
80
ICtlVlt
fresh
T
*
70
q>
50
o
E .c
N 7
^^
HI
40
30
m
20
[Bq.
C
60
10
0
0.0
NaCI concentration (M)
Fig. 4. Effect of NaCI on the in vitro activity of xylogJucan endotransglycosylase. The enzyme was extracted from leaves of Suaeda
mantima (A) and Kochia trkhophylla (B, O) and from etiolated
hypocotyls of Cicer arietinum (B, • ) , and assayed in a range of
concentrations of NaCI. Means and standard error (n = 3).
which reduces the formation of inactive enzyme-pectin
complexes and thereby increases the enzyme activity
indirectly (Warrilow and Jones, 1995). At higher concentrations, Na + inhibits enzyme activity by occupying the
initiation sites on the substrate and behaves as a competitive inhibitor (Nari et al., 1991). The optimum is the
balance point between the two opposing effects of Na + .
XET is a glycoprotein with high substrate specificity;
it promotes transglycosylation, lacks hydrolase activity
and is possibly involved in wall loosening (Fry et al.,
1992). Most XETs are soluble apoplastic enzymes (Fry,
1995) though a proportion can be ionically bound to the
cell wall. XET activity is moderately enhanced by various
cations such as Ca2 + , Mg 2+ and Mn2+ and inhibited by
Ag + , Hg2 + , La3 + , and Zn 2+ (Fry et al., 1992).
The extrapolation from in vitro to in vivo activities is
always beset by problems. The activity of enzymes at
different concentrations of inorganic ions depends on
several other factors such as pH, substrate concentration,
temperature and enzyme concentration which has to be
allowed for in experimental design and in interpretation.
The effect of substrate concentration on the salt sensitivity
of enzymes has often been noted (Greenway and Sims,
1974; Shomer-Ilan et al., 1985). In vitro conditions may
affect enzyme conformation (Munns et al., 1983; Flowers
et al., 1916b) and kinetic parameters, which is a characteristic mechanism of uncompetitive inhibition (Shomer-Ilan
et al., 1985). Enzymes which are bound to the cell wall
in vivo may behave differently in vitro when they are
solubilized, but it should be emphasized that the cell wall
enzyme XET, which is readily solubilized, behaved in a
similar way in vitro to those enzymes that required salt
for their solubilization from the cell wall for assay.
In this study, a range of cell wall enzymes from several
species was studied under different conditions with the
general finding that cell wall enzymes from both halophytes and glycophytes were much more tolerant of salt
in vitro than were cytoplasmic enzymes of higher plants.
There are a few cases of cytoplasmic enzymes having
tolerance (such as the peroxidases and acid phosphatases),
but this is not a general feature of the enzymes of
halophytes. The need for ionic adaptation by cytoplasmic
enzymes has been avoided in the halophytes by compartmentation. The development of a salt-tolerant cytoplasm
would require changes in a great number of enzymes and,
as discussed earlier, is precluded by the conserved ionic
requirements for protein synthesis.
Evidence from X-ray microanalysis and from cell water
relations provides strong arguments that the concentration (the osmotically active concentration, and not only
the electrostatically bound component) of inorganic ions
in the cell walls of halophytes is at least 500 mM for
plants making optimal growth. This is a concentration at
which the great majority of enzymes from both glycophytes and halophytes would be seriously inhibited. The
results of this study showed that the cell wall enzymes of
the halophyte Suaeda maritima were tolerant of NaCI up
to concentrations of 1 or 2 M. However, the results also
show that this is not a specific adaptation of halophytes
because the corresponding enzymes extracted from a nonhalophilic member of the Chenopodiaceae as well as from
a glycophyte showed substantial and in some cases equivalent tolerance. The results suggest that ionic tolerance is
a general property of enzymes of the cell wall compartment, and that this tolerance permits the enzymes of
halophytes to function in the conditions to be found in
their cell walls, rather than being an adaptation to do so.
Although the conditions likely to occur in the walls of
halophytes are extreme in terms of ionic concentration,
conditions in the apoplast will generally be much more
variable than within the membrane-bound compartments
of the cell. Acting throughout the evolution of terrestrial
plants, this may provide sufficient reason for this group
Salt tolerance of cell wall enzymes
of enzymes to have developed much wider adaptibility
than those localized in the cytoplasm.
Acknowledgements
This work was funded in part by the European Communities'
Biotechnology Programme as part of the Project of
Technological Priority, 1993-1996. MT was supported by a
Scholarship from the British Council under a link between The
University of Sussex and Eastern University, Sri Lanka. We
thank Dr George Jones, Aberystwyth, Wales, for help and
advice on the extraction and assay of peroxidase and pectin
esterase.
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