Journal of Experimental Botany, Vol. 48, No. 307, pp. 337-344, February 1997
Journal of
Experimental
Botany
Free Ca 2+ in tissue saps of calciotrophic CAM plants as
determined with Ca2+-selective electrodes
Andreas J. Meyer1'2'4 and Marianne Popp1'3
1
1nstitut fur Okologie der Pflanzen (formerly Institut fur Angewandte Botanik), Westfalische WilhelmsUniversitat Munster, D-48143 Munster, Germany
2
Botanisches Institut, Lehrstuhl I, Universitat Karlsruhe (TH), POB 6980, D-76128 Karlsruhe, Germany
3
Institut fur Pflanzenphysiologie, Universitat Wien, POB 285, A-1091 Wien, Austria
Received 14 March 1996; Accepted 11 October 1996
Abstract
Introduction
2
Calcium was measured with Ca -selective electrodes
in calciotrophic CAM plants. Several organic acid
anions (citrate, isocitrate, malate, and malonate) were
tested for their capacity to chelate Ca 2 + in solutions
at pH 4.8. Free Ca 2 + was also calculated from the
stability constants of the chelates at pH 4.0 and pH 6.0.
The strongest chelator at pH 4.8 was citrate, reducing
free Ca 2 + from 10 to 0.5 mol m" 3 , while isocitrate and
malate decreased ionized Ca 2 + to 25% and 50%,
respectively. At pH 4.0 isocitrate is somewhat more
effective than citrate. Malonate has only slight effects
on free Ca 2 + at pH 6.0. In tissue saps from field-grown
species of Crassula sp., Crassula expansa, Aloe ramosissima, and Aloe pillansii, concentrations of total
water-soluble Ca 2 + ranged from 25 to 196 mol m~ 3 .
Measurements with Ca 2 + -selective electrodes showed
that ionized Ca 2 + was reduced to 6 2 - 8 8 % in the presence of isocitrate. Diurnal fluctuations in malate were
less important for Ca 2 + chelation, which was also true
for the situation in greenhouse-grown plants of
Kalanchoe daigremontiana, which were cultivated at
different Ca 2 + levels. Comparing the osmotic potentials measured in the tissue saps with those calculated
from the concentrations of the different solutes gave
further evidence for the chelation of Ca 2 + by organic
acid anions, since values for >//„ using only the free
Ca 2 + were much closer to the measured values of i//K
than those calculated with total water-soluble Ca 2 + .
The term 'calciotrophic' was coined by Iljin (cf. Kinzel,
1983) for plants whose tissue saps contained high concentrations of water-soluble Ca 2 + . Horak and Kinzel (1971)
defined calciotrophic plants as those with a K + /Ca 2 +
(water-soluble) ratio below 1. Plants belonging to the
family of Crassulaceae or the genera Aloe, Plectrcmthus
and Clusici may contain more than 100 mol m~ 3 plant
water of water-soluble Ca 2 + (Horak and Kinzel, 1971;
von Willert et al., 1992; Herppich, 1989; Ball et al., 1991).
These high concentrations on a plant water or tissue sap
basis are in great contrast to the submicromolar concentrations of free Ca 2 + observed in the cytoplasm by
measurements with fluorescent dyes (Williams et al.,
1990) or with Ca2+-selective microelectrodes (Sanders
and Miller, 1986; Miller and Sanders, 1987; Felle, 1988,
1989). Moreover, cytoplasmic Ca 2 + concentrations are
thought to exhibit regulatory functions (Brauer et al.,
1990; Muto, 1992; Bush, 1993). Most of the Ca 2 +
absorbed by plants is found in the cell wall (Bush and
McColl, 1987; Cleland et al., 1990) and the vacuole
(Clarkson and Hanson, 1980; Gilroy et al., 1993) which
is the most important compartment for storage of Ca 2 +
in calciotrophic plants. The balance between cations and
anions in the vacuoles of these plants is attained by the
synthesis of large quantities of malate, citrate and other
organic anions (Kinzel, 1989). Previous work has shown
that malate and citrate are effective Ca 2 + chelating agents,
and that in Kalanchoe daigremontiana only 20% of the
100 mol Ca 2 + m~ 3 plant water are in the ionized form
(Kinzel, 1989).
Key words: Calciotrophic plants, CAM, organic acids, free
Ca 2 + , Ca 2 + chelation.
Since some of the other calciotrophic CAM species
mentioned above also contained high amounts of
4
To whom correspondence should be addressed at Universitat Karlsruhe. Fax: +49 721 608 4193. E-mail: [email protected]
© Oxford University Press 1997
338
Meyer and Popp
isocitrate (Aloe sp.) and malonate (Plectranthus) it was
considered important to test these two organic acid anions
for their ability to chelate with Ca2 + . In the case of
isocitrate this was feasible because of its similarity to citrate
and the significant correlation between Ca2+ and isocitrate
content in several Crassulaceae (Rossner and Popp, 1986;
von Willert et al., 1992) and Liliaceae (von Willert et al.,
1992). On the other hand, malonate was reported to
chelate Ni2+ in two Alyssum species (Lee et al., 1978).
Iwasaki el al. (1992) reported two types of channels
involved in malate ion transport across the tonoplast of
Graptopetalum paraguayense. One of these channels is
probably involved in the efflux of malate out of the
vacuoles. Since it could not be observed at cytoplasmic
concentrations of free Ca2+ lower than 10~3 mol m" 3 a
high cytoplasmic Ca 2+ concentration could be a necessary
prerequisite for malate release from the vacuoles during
the diurnal CAM rhythm (Iwasaki et al., 1992). From
the high vacuolar Ca 2+ content it is evident that the
vacuole serves as a reservoir for regulating the concentration of free Ca 2+ in the cytoplasm (Evans et al., 1991;
Arata et al., 1992; Muto, 1992). In tonoplast membranes
at least three types of selective Ca 2+ channels have been
found (Bush, 1993). The open-state probability of one of
these channels in sugar beet tap roots, increases markedly
with positive voltage changes in the physiological range.
Also, elevation of vacuolar Ca 2+ shifts the threshold for
voltage activation to less positive tonoplast potentials
(Johannes et al., 1992). This raises the possibility that
the concentration of vacuolar free Ca 2+ is involved in
regulation of cytoplasmic free Ca2 + . The difference in
concentrations of the divalently dissociated form of malic
acid across the tonoplast might be another factor that
regulates both the uptake and the release of malate since
it determines the probability of the responsible ion channels being open (Iwasaki et al., 1992; Arata et al., 1992).
The chelation of Ca2+ by organic acid anions should,
therefore, affect both the vacuolar concentration of free
Ca 2+ and the concentration of divalently dissociated
malic acid. This study attempts to determine whether
there are changes in the content of vacuolar free Ca2 +
and to which degree Ca2+ chelation depends on fluctuations of malic acid during the diurnal CAM rhythm.
In addition, chelation of Ca 2+ should clearly affect the
osmolality of the cell sap especially in calciotrophic plants.
This assumption was tested by a comparison of the
osmolality measured by the freezing point depression of
cell saps and by summing the detected solutes.
in their natural habitat at Numees (Richtersveld, Republic of
South Africa) during October.
Plants of Kalanchoe daigremontiana Hamet and Perrier de la
Bathie (Crassulaceae) were cultivated under controlled conditions in a greenhouse in two nutrient solutions of different
Ca 2 + concentrations. In experiments referred to as 'high Ca 2 + '
plants were grown on solutions with 0.5 mol m " 3 KN0 3 , 1.25
mol m" 3 Ca(NO 3 ) 2 , 0.4 mol m " 3 MgSO4, and 10 mol m " 3
CaCl2. In 'low Ca 2 + ' experiments plants were grown on
solutions containing 2.8 mol m~ 3 KNO 3 , 0 1 mol m~ 3
Ca(NO 3 ) 2 , 0.4 mol m " 3 MgSO 4 , and 10 mol m " 3 K.C1.
Micronutrients were added in the following concentrations to
both solutions: Na 2 B 4 O 7 7.2, Fe-EDTA 3.7, MnCl 2 3.6, ZnSO 4 ,
CuS0 4 0.3 mmol m" 3 . pH of the solutions was always adjusted
to 6.0 with 8 ml of 200 mol m " 3 ammonium phosphate buffer
((NH 4 ) 2 HPO 4 :(NH 4 )H 2 PO 4 = 8:92) per litre nutrient solution.
The light regime consisted of 12 h light and 12 h dark. Light
intensities at the top of the plants were about 275 /Mmol photons
m" 2 s" 1 . During the light period the temperature was 30 °C
with a relative humidity of 65%. In the dark the temperature
was 17 °C and relative humidity between 35% and 40%.
Tissue sap preparation
Under field as well as under laboratory conditions the harvested
leaves were put into tight scintillation vials and immediately
heated in a microwave oven for 3 x 15 s with a power output
of 500 W. After cooling the material was centrifuged to extract
the tissue sap as described by Smith and Lttttge (1985).
Preliminary experiments with samples of Ananas comosus (L.)
Merr. had proven that the described microwave treatment is
sufficient to eliminate any further enzymatic activities. Solute
concentration in tissue saps prepared by the microwave method
and those in freeze-dried material, which was hot-water
extracted and data calculated on a tissue water basis gave the
same results within SD-margins (Popp et al., 1996).
Chemical analysis of tissue saps
Concentrations of water-soluble cations (Na + , Mg2 + , K + ,
Ca 2+ ) were determined by atomic absorption spectroscopy.
Measurements of free acid were performed by automatic
titration with 50 mol irT 3 NaOH (DL 21 Titrator, Mettler,
Greifensee, Switzerland). Chloride was determined by electrochemical titration with 10 mol m~ 3 AgNO 3 . Sulphate was
measured by automatic titration with 5 mol m~ 3 Ba(C10 4 ) 2
and thorine as an indicator for free Ba 2+ ions following Albert
and Kinzel (1973) (DL 21 Titrator with Phototrode DP550,
Mettler). Nitrate was measured enzymatically in tissue saps
(Beutlerefa/., 1986).
The organic acid anions malate, citrate and isocitrate were
determined enzymatically (Mollering. 1974; Mollering and
Gruber, 1966; Siebert, 1974) as well as by gas chromatography.
Inorganic phosphate was also determined by gas chromatography (Popp et al., 1996). The components of the neutral
fraction were also analysed by two different GC-methods (Ball
el al., 1991).
Osmolality of tissue saps was measured by a cryoscopic
method (Osmomat 030, Gonotec, Berlin, FRG).
Production of the Ca 2+ -selective electrodes
Materials and methods
Plant material
Cmssula expansa Dryand, Crassula sp., Aloe ramosissima Pillans
and Aloe pillansii (L.) Guthrie were collected at dawn and dusk
Free Ca 2 + concentrations were measured with laboratory built
Ca2+-selective electrodes. The solvent polymeric membranes
were prepared using 1 wt% ETH 129 carrier (N,N,N~,Pftetracyclohexyl-3-oxapentanediamide; Fluka 21193, Fluka AG,
Buchs, Switzerland), 66 wt% o-NPOE (o-nitrophenylether;
puriss. p.a., Fluka 73732) and 33 wt% PVC (polyvinylchloride.
Ca2+ in calciotrophic CAM plants
high molecular, purum p.a., Fluka 81392) (Schefer el al., 1986).
A solution of PVC and o-NPOE in THF (tetrahydrofuran,
Fluka 87369) was made first and then the ionophore dissolved
in 2 ml of this base solution. The clear solution was carefully
poured into a 40 mm diameter glass ring which was tightly
fixed to a clean glass plate. During evaporation of the solvent
for 48 h the membrane solution was protected from dust and
currents of air. The homogenous membrane was carefully taken
from the glass plate and 6 mm discs fixed to PVC-tubing (4 mm
in diameter) with a few drops of the base solution. The
electrodes were filled with 100 mol m~ 3 CaCl 2 . Care was taken
to exclude air bubbles from the electrode body. For electrical
connection an Ag/AgCl-wire was inserted into the tubing. The
electrode potential readings were taken on a pH meter
(Microprocessor-pH-Meter 763, Knick, Berlin, FRG) using an
Ag/AgCl-microelectrode as reference electrode (Metrohm
6.0712.100, Metrohm AG, Herisau, Switzerland). Stirred sample
and standard solution of 1.5-2 ml volume were thermostatted
at 25 °C. Before calibration the Ca 2 +-selective electrodes were
equilibrated in 100 mol m~ 3 CaCl 2 for at least 1 h.
Ion-selective electrodes normally measure the activity of an
ion. Therefore, the electrodes were calibrated against solutions
with defined Ca 2 + activity. In order to ensure that the activity
coefficients in both solutions for electrode calibration and
samples are the same, solutions for calibration were mixed with
a solution which had the same concentration of inorganic ions
other than Ca 2 + as the sample. To adjust the ionic strength
NH4C1 was added.
The separate solution method (Ammann, 1986) was used to
determine the selectivity factors (lg ATCaM). These factors were
obtained in 10 mol m~ 3 unbuffered solutions of the corresponding chloride salts. Electrodes showed good selectivity for Ca2 +
over N a + (lg KCaNa = -3.0), Mg 2 + (lg KCaMg = -4.5), and K +
(lg ^caK= -3.2) with a linear response over a range from 0.01
mol m " 3 to 100 mol m " 3 Ca2 + . The selectivity for Ca 2 + over
H + was lg A"CaH = -1.5. Preceding experiments with test
solutions of millimolar Ca 2 + solutions had shown that no
relevant H + interference was present at pH values between 3
and 6. Calibration curves had a slope between 23 mV and 27
mV per 10-fold increase in Ca 2 + concentration. The emf drift
was lower than 1.5 mVh" 1 .
The ability of different organic acid anions to chelate Ca 2 +
was determined in standard solutions with a basic concentration
of 10 mol m~ 3 CaCl 2 . The decrease in the ionized portion of
total Ca 2 + , in the presence of different amounts of the respective
organic acid anion, was measured with the Ca 2 + -electrode. The
pH of the solution was always adjusted to 4.8 with diethanolamine. In view of the high ionic strength in most of the
cell saps the ionic strength in the standard solutions was always
adjusted to 1000 mol m " 3 with KC1. Concentrations of free
Ca 2 + in standard solutions at pH 4.0 and pH 6.0 were calculated
by using the relevant stability constants given by Martell and
Smith (1977, 1982).
Results
Ca2+ chelation by organic acid anions in vitro
The four organic acid anions used in the procedure
described above differed markedly in their ability to
chelate Ca 2 + . While with 50 mol m " 3 citrate at pH 4.8
only 5% of the original 10 mol m~ 3 Ca 2 + remained in
the free state, the same amount of isocitrate only reduced
the free Ca 2 + to 25% (Fig. IB). In comparison to the
I
'
i
'
i
'
339
r
10
20
30
40
50
Organic Acid Anion [mol nr 3 ]
60
i
' i i i ' i ' r
10
20
30
40
50
Organic Acid Anion [mol rrv3]
I ' I ' I
' T ' T
10
20
30
40
50
Organic Acid Anion [mol nr 3 ]
Fig. 1. Ca2+ chelation by different organic acid anions in standard
solutions at different pH; (A) pH 4.0, (B) pH 4.8, (C) pH 6.0. Values
in (B) were measured with Ca2 + -selective electrodes and given as means
from five measurements. Standard deviations are not shown because
they were always smaller then 10% of the mean and therefore often
within the size of the symbols. Values in (A) and (C) were calculated.
(O ) Citrate, ( • ) isocitrate, ( • ) malate, ( • ) malonate.
tricarboxylic acids malate was less effective in chelating
Ca 2 + , but still the proportion of free Ca 2 + was reduced
to less than half (Fig. IB). Malonate had hardly any
influence on the concentration of free Ca 2 + at pH 4.8
(Fig. IB). At pH 6.0 the degree of chelation is always
higher than at pH 4.8 (Fig. 1C). Small amounts of anions,
particularly of tricarboxylic acids caused a large reduction
of free Ca 2 + . In more acidic solutions the degree of
chelation is generally reduced (pH 4.0, Fig. 1A). At
pH 4.0 isocitrate is a stronger chelator of Ca 2 + than
citrate while at higher pH the reverse is true.
Ca2+ chelation in tissue saps of calciotrophic CAM plants
Examination of the tissue saps of four species of South
African succulents revealed a very high degree of chelation
340
Meyer and Popp
of Ca 2 + (Fig. 2). Between 88% (A. ramosissima) and 62%
(C. sp.) of total water-soluble Ca 2 + were chelated, but
when the ionic strength of the saps was taken into account
values for active Ca 2 + between 4.1 to 22.5 mol m " 3 were
obtained (Fig. 2). Isocitrate was the most effective of the
four organic acid anions examined (Table 1). Its concentration alone explained most of the observed Ca 2 + chelation (Fig. 2).
In dawn samples 84% (A. pillansii) to 88% (A. ramosissima) of total water-soluble Ca 2 + were chelated. In contrast to these values, the degree of chelation in dusk
samples was only 77% in A. pillansii and 70% in A.
ramosissima. In the case of dawn samples, it may be
assumed that malate also contributed to the chelation of
Ca 2 + . These two species were the only field-grown species
in which high diurnal variations in malate (Table 1) as
well as changes in the degree of Ca 2 + chelation (Fig. 2)
were observed. While an increase in the degree of Ca 2 +
chelation in A. ramosissima led to a decreasing concentration of free Ca 2 + , in A. pillansii a corresponding change
was not found. The differing degrees of chelate formation
might be explained by alterations of total water-soluble
Ca 2 + in dawn and dusk samples. The reason for this
observation remains unclear.
A more detailed study of diurnal changes in the degree
of Ca 2 + chelation was carried out in greenhouse experiments on K. daigremontiana grown at two Ca 2 + levels.
£.O\J
Addition of 10 mol m 3 CaCl 2 and KC1, respectively, to
the nutrient solutions resulted in high concentrations of
both cations in the tissue saps (Fig. 3). In the same way
the high external Cl~ concentrations led to high internal
Cl" contents. High Ca 2 + plants in this experiment showed
a significantly smaller increase in malate during the night
than low Ca 2 + plants (Figs 3, 4). This inhibition might
be caused by the adverse effect of the higher Cl~ concentration in the tissue saps of high Ca 2 + plants. The degree
of fluctuation in malate in low Ca 2 + plants was similar
to those previously reported (Kinzel, 1989; Arata et al.,
1992).
Different amounts of stored cations and inorganic
anions require compensation of electrical charges within
the vacuoles. In K. daigremontiana this was achieved by
stoichiometric synthesis of organic acid anions of which
isocitrate is the main component (Fig. 3).
Measurements of free Ca 2 + with a Ca2+-selective electrode revealed much higher degrees of Ca 2+ chelation in
low Ca 2 + plants than in plants grown on high Ca 2 + .
Contrary to expectations in low Ca 2 + plants, Ca 2 + chelation did not increase with increasing malate concentration
during the night. The nocturnal increase in malate was
accompanied by a decrease in Ca 2 + chelation from 80%
to 45% of total soluble Ca 2 + (Fig. 4). This surprising
result is probably due to the degree of the nocturnal
increase in malate in proportion to the amount of organic
200-
A
200-
150l
l 1501100-
88%
70%
50n_
84%
-t
m 1
dawn
dusk
Aloe ramosissima
•
*
77%
50-
-£-
0
—
dawn
Uii_
dusk
Aloe pillansii
200150-
r+i
o
73%
dawn
dusk
Crassula sp.
chelated Ca2*
72%
dawn
dusk
Crassula expansa
free Ca2*
^ • i
active Ca2*
Fig. 2. Chelation of C a J i and Ca 2 * activity in cell saps from four different species grown in the southern Namib Desert collected at dawn and
dusk. Total water-soluble calcium, i.e. the sum of free and chelated calcium, was measured by atomic spectroscopy. Data were taken from Table 2.
Free Ca 2 * and the resulting activity were measured with Ca2*-selective electrodes. Values are based on at least three measurements and standard
deviations are given by vertical bars. The numbers next to the columns give the percentage of total water-soluble Ca 2 * that is chelated by organic
acid anions.
Ca2 + in calciotrophic CAM plants
341
Osmotic considerations
300
High Low
Ca 2+ Ca 2+
9.00
High
Ca 2+
Low
Ca 2+
To show that the strong Ca 2 + chelation that was found
in the field-grown CAM plants is feasible the osmolality
of tissue saps was compared with the sum of all the
osmolytes either including total water-soluble Ca 2 +
(Table 1) or including only free Ca 2 + (Fig. 2). The results
using only free Ca 2 + agreed much better with the measured osmolality than those including total water-soluble
Ca 2 + (Table 2). C. expcmsa was an exception, however,
even in this case the sums including total water-soluble
Ca 2 + exceeded the measured osmolality, while correction
for chelated Ca 2 + resulted in values 12-16% lower than
measured osmolality. It is evident, that this way of
calculation can only lead to estimates, since these tissue
saps were far beyond the concentration of an ideal
solution. Nevertheless, this approach confirmed the evaluation of free Ca 2 + , because the sum of the other solutes
was an underestimate rather than an overestimate.
18.00
Discussion
Fig. 3. Total concentrations of
ions in tissue saps of Kalanchoe
solutions with different Ca 2 +
Materials and methods). Values
four different plants.
water-soluble inorganic and organic
daigremontiana grown on two nutrient
concentrations (for composition see
are means of four samples taken from
background anions. In terms of the number of carboxyl
groups in low Ca 2 + plants this ratio was about 4:1 and
therefore much higher than in high Ca 2 + plants where
the ratio was only 1:1 (Fig. 3).
Using Ca2+-selective electrodes, citrate, isocitrate, malate,
and malonate were tested for their capacity to chelate
Ca2 + . The observed chelating effects of citrate and malate
at pH 4.8 were consistent with data presented by Kinzel
(1989). In solutions with pH 6.0 and pH 4.8 isocitrate
was not as effective in chelating Ca 2 + as citrate (Fig. IB,
C). In contrast to this in more acidic solutions isocitrate
seems to be the stronger chelator. This observation can
be explained with the slightly different dissociation constants (pA"a) for the acids and the stability constants (lg
K) for the respective Ca 2 + chelates given by Martell and
Smith (1977, 1982). Dissociation constants for isocitric
Low Ca2+
High C a "
20
100 E
10
50 -
ill
CO
o
0
6.0 -
Icium fmol
E
e
CO
o
0
80
60
i
40
40
20
c—-e—a-"8""^
I
I i I
18 21 24
I
3
(0
CO
®-^>
I I I I I
6 9 12 15 18
Time
i
i i
18 21 24
i
3
i i i i i
6 9 12 15 18
Time
Fig. 4. Changes in pH. malate, total water-soluble and chelated Ca.2* in tissue saps of Kalanchoe daigremontiana during a diurnal cycle grown at
two different Ca 2 + concentrations. Total water-soluble calcium is shown by open bars and chelated Ca 2 + by the filled ones. Values are the means
from four measurements Standard deviations that are within the size of the symbols are not shown. Note that there are different scales for Ca 2 ^
values (total water-soluble Ca 2 * and chelated Ca 2 + ) in high Ca 2 + plants and low Ca 2T plants.
342
Meyer and Popp
Table 1. Total concentrations of water-soluble cations, inorganic unions, organic acid unions, und carbohydrates in cell saps of different
CAM plants collected at natural sites in South Africa
Solute
+
H
Ca 2 "
K+
Mg 2 +
Na +
Cl"
Residuals
Malate
Citrate
Isocitrate
Quinate
Glucose
Fructose
Sucrose
Myo-inositol
Aloe ramosissima
Aloe pillansu
Crassula expansa
Crassula sp.
Dawn
Dusk
Dawn
Dusk
Dawn
Dusk
Dawn
Dusk
172.6+ 18 9
175.1 + 13.6
60.9 + 7.9
52.0 + 6.7
66 0 + 4.7
25.8 + 4.3
2.8±0.5
93.1+7.7
16 3±2.3
151.4+13.6
n.d.
7.9 + 0.5
5.2 + 0.5
n.d.
n.d.
23.3+1.2
196.0 ±9.8
63 7 + 4.6
70.8+10.3
67.7 + 8.1
43.2 + 4.5
1 9+1.6
20.4+1.8
9.7±0.7
156.9+11.2
n.d
5.4 + 0 5
3.5 + 0.2
4 4 + 0.3
n.d.
83.7 + 5.3
143 5+10.1
9 1 + 1.2
77.2±5.3
102.4+10 9
150 + 2.1
2.1+0.6
30.7+1.8
11 9+1 6
161.8 + 7.0
n.d.
4.3+0.8
2 7 + 1.0
n.d.
n.d.
32.8 + 2.4
105.1 + 12.8
8.4 ±1.0
77.6 + 8.6
83.5 + 9.1
22.7+1.9
4.5 + 2.6
n.d.
5.4±0.6
131.2+10.3
n.d.
11.8 + 0.8
8.5 + 1.0
0 4 + 0.1
n.d.
126.9 + 11.3
25.2 + 1.9
10.6±0.6
13.3+0.8
30.5 + 3.5
47.2 + 5.6
2.1+0.9
37.8 + 2.5
0.9 + 0.4
19.6 + 0.5
n.d.
12.3 + 1.5
6.4+1.2
n.d.
3.6 + 0.7
60.2 ±3.8
31.1+4.6
6.7±0.9
12.5 + 1.0
19.6 + 0.9
35.8 + 5.6
4.5±0.8
9.0 ±1.5
nd.
26.9 ±2.4
n.d.
20.5 + 2.3
17.3+1.1
n.d.
2.1 ±0.5
51.3 + 8.5
130.8 ±7.9
43.1+4 0
58.1+4.9
7.0 + 0.2
46.1+4.9
3.5 + 1.9
10.5+1.5
6.5 + 0.4
69.2 + 4.1
4.6 + 0.3
12.3 + 1.1
6.8 + 0 8
n.d.
1.8 + 0.2
38.6+ 11.9
141.2+11 4
38.9 + 3 9
52 2 + 7.8
6.0+1.0
44.3+4.1
1 8±0.5
6 1+0.5
n.d.
84.3 + 7.1
6 5 + 0.5
14.6 + 0.9
7.9 + 0.6
nd.
21+0 1
Values are means from at least three samples ( ± S D ) and given in molm
HPO 2 .-. SO2," and NO3"\
3
tissue water (n.d.: not detected). Residuals comprise
Table 2. Osmolality of tissue saps in mosmol kg ' sap as determined bv crvoscopy and solute concentrulions calculated from the data
of Table 1 with or without chelated Ca2+, respectively
Osmolality (mOsmol kg ' )
Solutes (mol m " 3 )
^(solutes-chclatcd Ca) ( m o 1
m
~
3
)
Aloe ramosissima
Aloe pillansii
Dawn
Dusk
Dawn
Dusk
Dawn
Dusk
Dawn
Dusk
475.7
656.5
485.5
455.5
643.6
459.9
402.8
560.7
423.4
394.7
459.1
360.6
188.2
209.5
188.8
180.1
186 0
161.3
329.9
400 3
290.5
342.1
405.9
287.2
acid (p/C,=2.29; p# 2 = 3.73; pA:3 = 5.06) indicate that it
is a slightly stronger acid than citric acid (p#,=2.80;
p# 2 = 4.08; pA"3 = 5.33). On the other hand the chelate
effect of citrate on Ca2+ is stronger than that of isocitrate
dg #caHcit = 2.10; lg #Cacit_=3.18; lg A^,HlsocU=1.21; lg
^caisocit-—2.16). The amount of free, i.e. non-protonated,
organic acid anions available for chelation of Ca2+ and
hence the extent to which chelate formation occurs, varies
accordingly with the pH and the magnitude of stability
constants. At higher pH values when most protons are
ionized from the acid anions citrate is able to bind more
Ca 2+ than isocitrate. With decreasing pH the protonation
reactions compete with the chelating action towards Ca2 + .
Due to the different dissociation constants the number of
citrate anions decrease faster than the number of isocitrate. This leads to the fact that at pH 4.0 more isocitrate
anions are present and thus more Ca 2+ is bound by
isocitrate than by citrate.
Although malonate was reported to chelate Ni 2+ (Lee
et al., 1978) and therefore was expected to chelate Ca2 +
too, it failed to reduce the proportion of ionized Ca2+ in
the test solutions at pH 4.8. This agrees with the stability
constants for Ni-malonate (lg #=3.30; Lee et al., 1978)
and Ca-malonate (lg #=1.47; Martell and Smith, 1977).
Nevertheless, in less acidic solutions malonate is able to
Crassula sp.
Crassula expansa
chelate Ca2+ to some extent (Fig. 1C). This agrees with
the observation that several calciotrophic species in the
Fabaceae (Popp and Kinzel, 1981) and Brassicaceae
(Rattenbock, 1978) store high amounts of malonate. An
attempt was made to test Ca2+ chelation by malonate in
tissue saps using Plectranthus marrubioides which is
known to accumulate malonate as counterion for cations
(Herppich, 1989). However, tissue saps of this CAM
plant had deleterious effects on the electrode membranes.
Isocitrate which was shown to be the best chelating
agent in standard solutions at pH 4.0 (Fig. 1A) was the
major chelator in tissue saps under investigation. This
confirms findings by Rossner and Popp (1986) and von
Willert et al. (1992) that there is a correlation between
Ca2+ content and isocitrate in a number of CAM plants.
Dealing with CAM plants raises the question as to how
the diurnal acidification influences Ca2+ chelation. Arata
et al. (1992) reported that the concentrations of the
divalently dissociated malic acid and Ca-malate were
relatively constant throughout the day in Graptopetalum
paraguayense and K. daigremontiana. Since the monovalently dissociated form has only slight ability to bind Ca2 +
accumulation of this anion does not affect the degree of
Ca2+ chelation directly. Nevertheless, day-night changes
in the degree of Ca2+ chelation occurred in Aloe species
Ca2+ in calciotrophic CAM plants
(Fig. 2) and in K. daigremontiana (Fig. 4). The results
indicate that the part of total water-soluble Ca 2 + that is
in the free form is influenced by the amount of malic acid
accumulated during CAM in relation to the level of
background organic acid anions. Accumulation and
release of malic acid take place with a ratio of mal 2 " to
H + of 1:2. Because of its dissociation constants (pA",=
3.11; pK2 = 4A5) at the vacuolar pH malic acid will exist
mainly in the monovalently dissociated form. Depending
on the dissociation constants the residual protons will
bind to the background organic acid anions, especially to
isocitrate. With increasing numbers of bound protons
Ca 2 + will dissociate from its chelates with these anions.
Thus it is the ratio of carboxyl groups from malate
accumulated during the night and from background carboxyl groups which led either to slightly increasing degrees
of Ca 2 + chelation in the Aloe species (Fig. 2) or to
constant levels of chelation in the field-grown Crassula
species (Fig. 2) or even to lower percentages of chelated
Ca 2 + in tissue saps from low Ca 2 + plants of K. daigremontiana (Fig. 3) at dawn. K. daigremontiana plants with
Ca 2 + concentration similar to that in our high Ca 2 +
treatments, but a three times higher citrate concentration
exhibited a Ca 2 + chelation of 75-79% without changes
between dawn and dusk samples (Kinzel, 1989). These
values agree very well with those found in field-grown
succulents in this investigation.
It has been suggested that cytoplasmic Ca 2 + regulate
the efflux of malate from the vacuole via a slow activating
channel and that the vacuole could be functioning as a
reservoir for the regulation of cytoplasmic concentration
of Ca 2 + (Arata et ai, 1992; Iwasaki et al, 1992).
Undoubtedly, with this way of regulation it is the free
concentration of vacuolar Ca 2 + that is of interest. The
results reported here indicate that day-night changes of
vacuolar free Ca 2 + do not take place in the same way in
different CAM species and not even in different plants
from the same species as shown for K. daigremontiana
(Fig. 4). Therefore, it can be concluded that possible
diurnal fluctuations in vacuolar free Ca 2 + are not involved
in triggering CAM. Independent of the degree of chelation
the concentration of free Ca 2 + is rather high so that the
vacuole still may serve as a Ca 2 + reservoir for regulation
of the cytoplasmic Ca 2 + concentration.
The complex interrelationships between total watersoluble Ca 2 + , organic background anions and accumulated malic acid during CAM lead to the question whether
storage of Ca 2 + and organic counter anions like isocitrate
affect the capacity of the vacuoles to store malic acid
during night fixation of CO 2 . A high buffer capacity
inside the vacuole due to a high level of organic background anions causes a smaller decrease of pH during
accumulation of malic acid. Nevertheless, the amplitude
of day-night changes in low Ca 2 + plants of K. daigremontiana (Fig. 4) are consistent with data presented by Kinzel
343
(1989) though in that case tissue saps contained almost
ten times total water-soluble Ca 2 + and more organic
background anions. However, it cannot be excluded, that
the total concentration of inorganic cations increases the
amount of malic acid accumulated during the dark period.
The degree of Ca 2 + chelation between 62% and 88%
(Fig. 2) should have a clear effect on the osmolality of
the cell saps. The calculation of the osmolality from the
sum of detected solutes showed that these values were
considerably higher than osmolalities measured by freezing point depression of the cell saps. The calculation of
the sum of all osmotically active solutes minus the chelated Ca 2 + is still an estimate for two reasons. Firstly,
the cell sap is not an ideal solution and the concentrations
would have had to be corrected for the osmotic coefficients. Secondly, there are a few other solutes (e.g. amino
acids) that were not included in the present determinations. In spite of these restrictions the use of free Ca 2 +
values should be preferred for osmotic evaluations in
calciotrophic plants. In contrast to those plants which
precipitate excess Ca2 + in the form of sparingly soluble
Ca-oxalate, calciotrophic plants make use of soluble Ca 2 +
chelates as osmolytes. This is particularly important for
desert plants since they are often exposed to drought
stress.
Acknowledgements
We gratefully acknowledge the support of Professor Dr DJ von
Willert, who made research work in the Namib Desert possible
for AM and his help in taking samples. We wish to thank Mrs
Hildegard Schwitte for excellent technical assistance and for
taking care of the K. daigremontiana experiment. We also
acknowledge Professor Dr Warwick Bottomley for reading the
manuscript and correcting the English language. This research
was
supported
by
a
grant
of
the
Deutsche
Forschungsgemeinschaft to MP (PO 323/2-1).
References
Albert R, Kinzel H. 1973. Unterscheidung von Physiotypen bei
Halophyten des Neusiedlerseegebietes (Osterreich). Zeitschrift
fur Pflanzenphysiologie 70, 138-58.
Ammann D. 1986. Ion-selective microelectrodes. Principles, design
and application. Berlin: Springer Verlag.
Arata H, Iwasaki I, Kusumi K, Nishimura M. 1992.
Thermodynamics of malate transport across the tonoplast of
leaf cells of CAM plants. Plant Cell Physiology 33, 873-80.
Ball E, Hann J, Kluge M, Lee HSJ, Liittge U, Orthen B, Popp
M, Schmitt A, Ting IP. 1991. Ecophysiological comportment
of the tropical CAM-tree Clusia in the field. II. Modes of
photosynthesis in trees and seedlings. New Phytologist
117,483-91.
Beutler H-O, Wurst B, Fischer S. 1986. Eine neue Methode zur
enzymatischen Bestimmung von Nitrat in Lebensmitteln.
Deutsche Lebensmittel-Rundschau 82, 283-9.
Brauer M, Sanders D, Stitt M. 1990. Regulation of photosynthetic sucrose synthesis: a role for calcium? Planta 182,
236-43.
344
Meyer and Popp
Bush DS. 1993. Regulation of cytosolic calcium in plants. Plant
Physiology 103, 7-13.
Bush DS, McColl JD. 1987. Mass-action expressions of ion
exchange applied to Ca 2 *, H + , K + and Mg 2 + sorption on
isolated cell walls of leaves from Brassica oleracea. Plant
Physiology 85, 247-60.
Clarkson DT, Hanson JB. 1980. The mineral nutrition of plants.
Annual Review of Plant Physiology 31, 239-98.
Cleland RE, Virk SS, Taylor D, Bjorkman T. 1990. Calcium,
cell walls and growth. In: Leonard RT, Hepler PK, eds.
Calcium in plant growth and development. Current topics in
plant physiology. An American Society of Plant Physiologists
Series, Vol.4, 9-16.
Evans DE, Briars S-A, Williams LE. 1991. Active calcium
transport by plant cell membranes. Journal of Experimental
Botany 42, 285-303.
Felle H. 1988. Cytoplasmic free calcium in Riccia fluitans L.
and Zea mays L.: interaction of Ca 2 + and pH? Planta
176, 248-55.
Felle H. 1989. Ca 2 +-selective microelectrodes and their application to plant cells and tissues. Plant Physiology 91, 1239-42.
Gilroy S, Bethke PC, Jones RL. 1993. Calcium homeostasis in
plants. Journal of Cell Science 106, 453-62.
Herppich W. 1989. CAM-Auspragung in Plectranthus marrubioides Benth.(Fam. Lamiaceae). Einflu/? der Faktoren Licht,
Blattemperatur, Luftfeuchtigkeit, Bodenwasserverfiigbarkeit
und Blattwasserzustand. PhD thesis, University of Miinster.
Horak O, Kinzel H. 1971. Typen des Mineralstoffwechsels bei
den hoheren Pfianzen. Osterreichische botanische Zeitschrift
119,475-95.
Iwasaki I, Arata H, Kijima H, Nishimura M. 1992. Two types
of channels involved in the malate ion transport across the
tonoplast of a crassulacean acid metabolism plant. Plant
Physiology 98, 1494-7.
Johannes E, Brosnan JIM, Sanders D. 1992. Parallel pathways
for intracellular Ca 2 + release from the vacuole of higher
plants. The Plant Journal 2, 97-102.
Kinzel H. 1983. Influence of limestone, silicates and soil pH on
vegation. In: Lange OL, Nobel PS, Osmond CB, Ziegler H,
eds. Encyclopedia of plant physiology. New series, Vol. 12C.
Physiological plant ecology III. Berlin: Springer Verlag,
201-44.
Kinzel H. 1989. Calcium in the vacuoles and cell walls of plant
tissue. Forms of deposition and their physiological and
ecological significance. Flora 182, 99-125.
Lee J, Reeves RD, Brooks RR, Jaffre T. 1978. The relation
between nickel and citric acid in some nickel-accumulating
plants. Phytochemistry 17, 1033-5.
Martell AE, Smith RM. 1977. Critical stability constants, Vol. 3.
Other organic ligands. New York: Plenum Press.
Martell AE, Smith RM. 1982. Critical stability constants, Vol. 5.
Supplement 1. New York: Plenum Press.
Miller AJ, Sanders D. 1987. Depletion of cytosolic free calcium
induced by photosynthesis. Nature 326, 397-400.
Mollering H. 1974. Malatbestimmung. In: Bergmeyer HU, ed.
Methoden der enzymatischen Analyse, Bd. 2. Weinheim: Verlag
Chemie, 1636-9.
Mollering H, Gruber W. 1966. Determination of citrate with
lyase. Analytical Biochemistry 17, 369-76.
Muto S. 1992. Intracellular Ca 2 + messenger system in plants.
International Review of Cytology 142, 305-45.
Popp M, Kinzel H. 1981. Changes in the organic acid content
of some cultivated plants induced by mineral ion deficiency.
Journal of Experimental Botany 32, 1-8.
Popp M, Lied W, Meyer AJ, Richter A, Schiller P, Schwitte H.
1996. Sample preservation for determination of organic
compounds: microwave versus freeze-drying. Journal of
Experimental Botany 47, 1469-73.
Rattenbock H. 1978. Chemisch-physiologische Charakterisierung der Brassicaceae. Ein Beitrag zum Physiotypen-Konzept.
PhD thesis, University of Vienna.
Rossner H, Popp M. 1986. Ion patterns in some Crassulaceae
from Austrian habitats. Flora 178, 1-10.
Sanders D, Miller AJ. 1986. Measurement of cytoplasmic
calcium activity with ion-selective microelectrodes. In:
Trewavas AJ, ed. Molecular and cellular aspects of calcium in
plant development. London: Plenum Press, 149-56
Schefer U, Ammann D, Pretsch E, Oesch U, Simon W. 1986.
Neutral carrier-based Ca 2+ -selective electrode with detection
limit in the sub-nanomolar range. Analytical Chemistry
58, 2282-5.
Siebert G. 1974. Isocitrat. UV-spektroskopische Bestimmung.
In: Bergmeyer HU, ed. Methoden der enzvmatischen analyse,
Bd. 2. Weinheim: Verlag Chemie, 1616-19.
Smith JAC, Liittge U. 1985. Day-night changes in leaf water
relations associated with the rhythm of crassulacean acid
metabolism in Kalanchoe daigremontiana. Planta 163, 272-82.
von Willert DJ, Eller BM, Werger MJA, Brinckmann E, Ihlenfeld
HD. 1992. Life strategies of succulents in deserts. With special
reference to the Namib desert. Cambridge University Press.
Williams DA, Cody SH, Gehring CA, Parish RW, Harris PJ.
1990. Confocal imaging of ionized calcium in living plant
cells. Cell Calcium 11,291-7.