Download calcium requirement for the secretion of peroxidases by plant cell

J. Cell Set. 48, 34S-353 (1981)
Printed in Great Britain © Company of Biologists Limited 1981
CALCIUM REQUIREMENT FOR THE
SECRETION OF PEROXIDASES BY
PLANT CELL SUSPENSIONS
LILIANE STICHER, CLAUDE PENEL AND HUBERT GREPPIN
Laboratoire de Physiologic vigitale, University de Geneve,
3 place de V University, 1211 Geneve 4, Switzerland
SUMMARY
Spinach {Spinacia oleracca, L.) cells in liquid culture release peroxidases. This release is
reduced by EGTA and promoted by calcium ions. In a medium deprived of calcium the rate
of peroxidase release is low, but immediately after addition of 1 mM calcium there is a sudden
increase of the extracellular peroxidase activity. Extracellular calcium apparently penetrates
into the cultured cells rather freely and, as a consequence, the rate of peroxidase secretion by
these cells is directly correlated with the concentration of calcium in the medium. Magnesium,
at twice the concentration used for calcium, has no effect on the release of peroxidases. Cells
treated with Na azide, Na hydrogenarsenate or fluphenazine secrete less peroxidase upon
addition of calcium.
INTRODUCTION
The enzyme peroxidase (EC 1.11.1.7) is widely distributed in higher plants but,
in spite of the large number of papers devoted to its study, little is known about the
control of its biosynthesis and its actual function in the plant. One of the best
established facts concerning this enzyme is its presence in the plant cell wall (De Jong,
1967; Birecka & Miller, 1975). Moreover, it is known that cells in culture (Olson,
Evans, Frederick & Jansen, 1969; van Huystee & Turcon, 1973; Fry, 1979) and even
whole organs such as roots (Gaspar & Xhaufflaire, 1967) release peroxidases into
their surrounding medium. The presence of this enzyme outside the cells may be
explained either by passive leakage through the plasma membrane, or by active
secretion. A study of peroxidase localization in the onion root tip showed that the
enzyme was present not only in the cell wall but also on the plasmalemma, in the
Golgi apparatus cisternae and vesicles, in the endoplasmic reticulum and on
membrane-bound ribosomes (Goff, 1975). These localizations characterize a secretory
protein. In animals, several tissues have been shown to secrete peroxidases in response
to a precise stimulus (Herzog, Sies & Miller, 1976). It is likely that plant cells also
control the release of their peroxidases in order to regulate their elongation through
the rigidification of their wall (Fry, 1979) or through the degradation of the plant
growth regulator auxin (Ricard & Nari, 1966). In addition, wall peroxidases are
Address for correspondence: Dr C. Penel, Laboratoire de Physiologie v6g£tale, 3 place de
l'Universite, 1211 Geneve 4, Switzerland.
12
CEL 4R
346
L. Sticher, C. Penel and H. Greppin
responsible for hydrogen peroxide biosynthesis (Elstner & Heupel, 1976) and
incorporation of hydroxyproline-containing proteins into lignin monomers (Withmore,
1978).
The process of secretion generally requires the presence of calcium which is
needed for the secretory vesicles to be discharged from the cells (Dahl, Ekerdt &
Gratzl, 1979). This mechanism is widespread in animals. In plants, however, although
several aspects of the secretion of macromolecules are well documented (Chrispeels,
1976), there is little literature available on the role of Ca2+ in this process. Morris &
Northcote (1977) have shown that Ca2+, as well as other ions, trigger the secretion
of polysaccharides in sycamore suspension cultures. Reiss & Herth (1979 a), on the
other hand, have recently observed in the tip region of pollen tubes of Lilium
longiflorum a perturbation of an exocytosis mechanism following treatment with the
ionophore A23187. The aim of this work is to look for a possible requirement of
Ca2+ for the release of peroxidase in plant cell cultures.
MATERIALS AND METHODS
Spinach (Spinacia oleracea L., cv. Nobel) cell cultures were derived from calluses obtained
from hypocotyls of 14-day-old seedlings grown in sterile conditions. Cell suspensions were
cultured in 250-ml flasks in 90 ml of the liquid medium (MS) of Murashige & Skoog (1962)
supplemented with 2 % sucrose, 5 x io~' M indolylbutyric acid and 5 x io~7 M benzyladenine.
The flasks were continuously shaken under continuous illumination with fluorescent white
light at 20 °C. After a 14-day culture, the cells were transferred twice (48 and 24 h before
the assay) to fresh MS Ca-free medium containing o-i min ethylene-glycol-6u-(amino-ethyl
ether) iV.W-tetracetic acid (EGTA). They were kept in darkness after the second transfer.
The release of peroxidases by plant cell suspensions was followed under dim green light
at room temperature. The cells were harvested and divided into aliquots of 250 mg fresh
weight. Each aliquot was resuspended in 1-5 ml of buffer containing 50 DIM N-morpholino3-propane-sulphonic acid adjusted to pH 7-5 with tris-methylhydroxyaminomethane and
20 mM KC1 (MTK). Each aliquot was left for 2 h without stirring.
Calcium chloride and the other chemicals were added to cell suspensions at the required
concentrations using small volumes of stock solutions.
During the assay, the cell suspensions were gently stirred at fixed intervals with a glass rod.
Peroxidase activity present in the medium was followed by taking 5o-/il samples. The
peroxidase assay was performed in 2'5 ml of phosphate buffer containing 8 mM guaiacol and
2 mM hydrogen peroxide at pH 6-i and 20 °C. The increase in absorbence at 470 nm was read
after 10 min. Each experiment was repeated at least 3 times.
RESULTS
The release of peroxidases by plant cells in culture can be easily followed by taking
a sample of the medium at fixed intervals and assaying it for peroxidase activity.
Using this procedure, we observe (Fig. 1) that the release of peroxidases in the medium
occurs faster in MTK buffer alone than in MTK containing o-1 mM EGTA. In both
cases, the addition of 1 mM Ca2+ promptly enhances the level of activity measured
in the medium. The effect of Ca2+ is stronger in the EGTA-containing medium. The
response of the cells can be detected as soon as 30 s after the addition of Ca2+.
As the peroxidases are mainly bound to the wall constituents through ionic
interactions, it is useful to study the pH-dependence of peroxidase release, in order
Calcium and secretion of plant peroxidases
347
to choose the optimal conditions. This was done by using MTK buffers between
pH 6-o and 8-o (Fig. 2). It appears that changing the pH of the medium has little
effect on the weak release observed in the absence of Ca2+. On the other hand, the
Ca2+-dependent release of peroxidases is somewhat promoted by slightly alkaline
pH, with an optimum at pH 7-8. At this pH, either retention of peroxidases by the
walls is minimal, or the secretory process is promoted.
Q-
0-6
Peroxidaseactivir
1
1
D
/
•V""
,/
0-4
I;
0-2
00
120
150
180
210
240
270
Time after resuspension, mm
Fig. 1. Peroxidase activity released by spinach cells resuspended in MTK without and
with 01 M EGTA. Ca l+ (1 IHM) is added 225 min (arrow) after resuspension; # , O,
MTK without and with Ca, respectively; • , Q, MTK + EGTA without and with Ca,
respectively.
The presence of extracellular Ca2+ is required for a substantial release of peroxidase
to occur. This means that under the conditions of the experiment, some Ca2+ can
penetrate into the cells. Fig. 3 shows what happens when the Ca2+ ionophore A23187
is added to the medium. Before addition of Ca2+, the level of external peroxidase
activity is higher in the presence of the ionophore. This could be explained by the
fact that the ionophore facilitates the penetration of the few Ca2+ still present in the
wall and in the medium in spite of the presence of EGTA. Upon addition of Ca2+,
increase in the extracellular activity is observed in both cases and is little greater
in the presence of the ionophore.
In order to determine whether the rate of peroxidase release is related to the
external Ca2+ concentration, spinach cells, previously washed twice in MTK
containing io~3 and I O ^ M EGTA, respectively, were resuspended in media containing io~6 M EGTA and increasing concentrations of Ca2+ (Fig. 4). This
experiment shows that the level of external peroxidase activity depends on the avail-
L. Sticker, C. Penel and H. Greppin
348
0-8
0-6
•3
0-4
0-2
00
L6-4
60
7-2
6-8
7-6
80
pH
Fig. 2. Peroxidase activity released by spinach cells in MTK buffers of various pH
without (O) and with (A) i mM Ca*+. The activities are measured 120 min after
resuspension of the cells.
o
0-6
-
0-4
-
0-2
-
150
180
210
240
270
Time after resuspension, min
Fig. 3. Peroxidase activity released by spinach cells resuspended in MTK without
(O) and with ( # ) o-oi mM A23187. Ca1+ (1 ITIM) is added 180 min (arrow) after
the resuspension.
Calcium and secretion of plant peroxidases
2+
349
2+
ability of external Ca . In these conditions, even a Ca concentration as low as
10-6 M slightly increases the extracellular peroxidase activity. The maximum effect
is obtained at 1 mM Ca 2+ ; a higher concentration (10 mM) does not exhibit a much
greater effect.
240
270
Time after resuspemion, min
Fig 4 Peroxidase activity released by spinach cells resuspended in MTK containing
1 fiM EGTA. Caa+ at various concentrations are added 190 min (arrow) after
resuspension. •, A, A, D, • , and O, Ca1+ concentrations i o - \ 10-3, io"4, io" 6 ,
io~* and o M, respectively.
All the data presented above show that external Ca2+ promote increase in peroxidase
activity in the cell suspension medium. Ca2+, however, when added to a suspension
medium separated from its spinach cells, also induce a weak increase in peroxidase
activity (see Fig. 6). Thus, some extracellular peroxidases can be reactivated by Ca2+.
This fact can be explained by the observation of Haschke & Friedhoff (1978), who
showed that Ca2+ contribute to the structural stability of 2 isoperoxidases of horseradish and is essential to their en2ymic activity.
The possibility that Ca2+ act in a non-specific way is tested by using another
divalent cation (Fig. 5). The release of peroxidases by cell suspension is followed
before and after the addition of 2 mM Mg2+. It is found that Mg 2+ has only a weak
effect which can be explained by the displacement of some Ca2+ bound to the walls
of the plasmalemma. On the other hand, Ca2+ added after Mg 2+ triggers a sudden
increase in extracellular peroxidase activity.
Fig. 6 shows the effect of Ca2+ under several conditions. In one set of experiments
Caa+ are added to cell suspensions which were pretreated for 1 h with one of the
following inhibitors: Na azide, Na hydrogenarsenate or fluphenazine. In other
experiments, Ca2+ are added to a medium from which the cells had just been
L. Sticker, C. Penel and H. Greppin
35°
T*
3
0-6
-
150
180
210
240
270
Time after resuspeniion, min
Fig. 5. Peroxidase activity released by spinach cells resuspended in MTK. Effect of
the addition of 2 mM Mg (downward pointing arrow) followed by I mM Ca a+ (upward
pointing arrow).
30
Time after Ca addition, min
Fig. 6. Effect of i mM Ca1+ on the release of peroxidase activity by spinach cells
pretreated for 6o min with 20 mM Na azide ( A), 10 mM Na-hydrogen arsenate ( • ) or
o-oi mMfluphenazine(O). The effect of Ca l+ on a suspension medium after removal
of the cells (MTK alone, • ) and on cells frozen during 24 h at — 20 °C and thawed
(A) is also shown. # , control, MTK.
Calcium and secretion of plant peroxidases
351
withdrawn or to a medium containing cells which were frozen for 24 h, thawed and
then resuspended in MTK. The 3 inhibitors reduce the Ca2+ effect by at least 50%.
This means that the action of Ca2+ depends on the metabolic energy (inhibition by
azide and arsenate) and could be related to calmodulin, a protein playing the role
of intermediate in several Ca2+-controlled cellular processes (inhibition by the
antipsychotic drug fluphenazine which specifically binds to calmodulin) (Klee,
Crouch & Richman, 1980). Thus, poisoned cells partially lose their ability to respond
to Ca2+, while cells killed by freezing are completely unable to be stimulated by Ca2+.
Table 1. Effect of 1 mM Cat+ added in vivo (on cell suspensions) or in vitro (on extracts)
fresh weight) of extracts* from spinach
on the peroxidase activity (t^Ail0.min~l.mg*1
cells in suspension culture
Extracts
Cell suspension
1+
— Ca
+ Ca 1 +
-Ca' +
+Ca'+
0-056
0-056
° - i95
0-106
• Extraction in 100 mM phosphate buffer pH 7-0. T h e activity is measured in the supernatant
after centrifugation at 10 000 g for 10 min.
The effect of Ca2+ on the release of peroxidases can also be visualized by measuring
the peroxidase activity remaining in the cells after addition of Ca2+. It appears
(Table 1) that cells deprived of Ca2+ and cells which received 1 mM Ca2+ 15 min
before the extraction contain the same level of peroxidase activity. The addition of
Ca2+ to these cell-free extracts enhances activity. The activation is much stronger
in the extract from cells which were not treated in vivo with Ca2+. Cells cultured
without Ca8+ seem to contain more peroxidases in an inactive form than cells which
were in contact with Ca2+ before the extraction.
DISCUSSION
The major difficulty encountered when studying secretion in plant cells is the
presence of a cell wall able to retain the secretion products. Peroxidases are always
found associated with the wall either in situ (Gordon & Alldridge, 1971) or after
cell fractionation (Ridge & Osborne, 1970). This association mainly results from
electrostatic interactions. In our work, we tried to minimize these interactions by
using a buffer of adequate ionic strength and pH. Nevertheless, it is likely that
a fraction of the extracellular peroxidases remains trapped or linked in the walls and
therefore, Ca2+ added even at low concentrations could act on the wall itself by
displacing wall peroxidases through a very specific mechanism. This possibility
cannot be completely ruled out, but it was shown that the wall of frozen cells which
have retained their peroxidase activity does not respond to addition of Ca2+ (Fig. 6)
and cells pretreated with metabolic poisons partially lose their ability to respond to
Ca2+.
352
L. Sticker, C. Penel and H. Greppin
Our data provide good evidence that plant cells need Ca2+ to release peroxidases
into the medium. Cells deprived of Ca2+ respond very rapidly to the addition of this
cation. This fast response probably means that Ca2+ trigger the discharge of preexisting enzyme molecules outside the cells. These molecules perhaps accumulate
within the cells in an inactive form and could be activated by Ca2+ during the
secretory process, as suggested by the effect of Ca2+ on cellular extracts (Table i).
In the experimental conditions used in the present study, some external Ca2+
apparently can move into the cells and the level of secreted peroxidases is determined
by the Ca concentration of the medium. In the plant, however, it seems likely that
the cells are able to control their permeability to Caa+. For example, such a control
is achieved by the plant pigment phytochrome in the green alga Mougeotia (Dreyer &
Weisenseel, 1979) and accumulation of Ca2+ seems to be related to growth rate in
several plant systems (Reiss & Herth, 1979 b).
It appears in conclusion that Ca is a key factor in the control of plant peroxidases,
since it is involved in the activation and secretion of these enzymes and is able to
bind them to several kinds of cell membranes (Penel & Greppin, 1979).
We thank Dr N. Bernardini for his help in obtaining spinach cells in culture. The ionophore
A23187 andfluphenazinewere gifts from E. Lilly & Company and Van Heyden GmbH,
respectively.
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{Received 22 May 1980 - Revised 19 September 1980)
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