Botanical Studies (2006) 47: 119-127.
*
Corresponding author: E-mail: linglong@ntu.edu.tw; Tel:
886-2-33662510; Fax: 886-2-23673374.
Influence of calcium availability on deposition of calcium
carbonate and calcium oxalate crystals in the idioblasts
of Morus australis Poir leaves
Chi-Chih WU
1
, Shiang-Jiuun CHEN
2
, Tsair-Bor YEN
3
, and Ling-Long KUO-HUANG
2,
*
1
Institute of Plant and Microbial Biology, Academia Sinica, Nankang, Taipei, Taiwan
2
Department of Life Science; Institute of Ecology and Evolutionary Biology, National Taiwan University, Taipei, Taiwan
3
Institute of Tropical Agriculture and International Cooperation, National Pingtung University of Science and Technol-
ogy, Pingtung, Taiwan
(Received June 29, 2005; Accepted November 15, 2005)
ABSTRACT.
In the leaves of Morus australis, calcium carbonate formed only in lithocysts of epidermal
tissue while calcium oxalate crystals were found mostly in crystal idioblasts of bundle sheath. In order to
identify a possible influence of calcium nutrition on the formations of these two kinds of calcium depositions,
plants were grown with varying calcium supply. The results showed that the sizes of both lithocysts and cal-
cium carbonate increased as the calcium concentration increased, but the distribution density of lithocysts was
not affected. In addition, the average distribution density of calcium oxalate crystals was higher in the leaves
grown in high ca lcium solution (3750 £gmol Ca/l), and no calcium oxalate crystal was found in the leaves
grown in low calcium solution (94 £gmol Ca/l). Twenty-four days after plants were transferred from high to
low calcium solutions, the size of lithocysts in the previously formed leaves remained the same, and that of
calcium carbonate decreased slightly, but the density and size of calcium oxalate crystals decreased signifi-
cantly. After transfer from low to high calcium, the size of existing lithocysts did not respond to the change
of calcium concentration while the size of calcium carbonate and both the distribution density and size of cal-
cium oxalate crystals changed.
Keywords: Calcium carbonate; Calcium nutrition; Calcium oxalate; Morus australis.
INTRODUCTION
Calcium is known to have influences on many bio-
chemical and physiological processes in plant tissues and
cells (Bush, 1995). In higher plants, calcium oxalate crys-
tals are the most prominently deposited calcium salt and
have been found in the cells of various tissues and organs
(Arnott and Pautard, 1970; Franceschi and Horner, 1980;
Borchert, 1985; Kuo-Huang and Zindler-Frank, 1998).
The occurrence, the specific crystal shapes, and the loca-
tions of these crystals are useful taxonomic characters
(Genua and Hillson, 1985; Wu and Kuo-Huang, 1997;
Prychid and Rudall, 1999). The accumulation of calcium
oxalate crystals in plant bodies has been studied for many
years. However their function in normal plant growth and
development is still unclear. They may represent a form of
calcium and oxalate acid storage. They may act as deposi-
tories for regulation of cytosolic calcium concentration
(Franceschi and Horner, 1980; Webb, 1999). The forma-
tion of calcium oxalate crystals in the crystal idioblasts is
affected by the availability of calcium ions (Frank, 1972;
Franceschi and Horner, 1979; Borchert, 1985; Kuo-Huang
and Zindler-Frank, 1998). Calcium re-dissolved has been
observed in times of calcium depletion (Franceschi and
Horner, 1979; Borowitzka, 1984; Franceschi, 1989).
Amorphous calcium carbonate is found in several unre-
lated dicotyledonous families and is abundant in members
of the order Urticales, such as Moraceae, Urticaceae, and
Ulmaceae (Dickison, 2000). This calcium carbonate is in
the form of cystolith and mostly occurs in the epidermal
lithocysts of the leaves (Setoguchi et al., 1989; Okazaki et
al., 1986, 1991; Taylor et al., 1993). The influence of the
calcium nutrition on the precipitation of calcium carbon-
ate has been discussed (Freisleben, 1933; Rabiger, 1951;
Sugimura et al., 1999). However the bio-mineralization
mechanisms giving rise to the production of amorphous
calcium carbonate in plant cystoliths are not clear. Be-
sides, the physiological function of these deposits is un-
certain. There is no evidence of cystolith dissolution in
either intact plants or detached leaves (Taylor et al., 1993).
Nevertheless, amorphous deposits of calcium carbonate
are found in gastropod mollusks, where they provide an
ion source that can be mobilized for shell repair or acid-
base balance (Mason and Nott, 1981).
BIOCHEMISTRY
pg_0002
120
Botanical Studies, Vol. 47, 2006
Deposits of calcium carbonate generally dissolve in
acid while forming CO
2
bubbles (Nultsch and Grahle,
1988). Calcium oxalate crystals are insoluble in acetic
acid, but they dissolve in hydrochloric and sulfuric acid
without forming bubbles (Kuo-Huang, 1990). Most stud-
ies of calcium deposits in higher plants focus on the calci-
um oxalate crystals (Franceschi and Horner, 1980; Webb,
1999). Noticeably fewer reports concern the formation of
calcium carbonate (Scott, 1946; Watt et al., 1987; Yu and
Li, 1990; Taylor et al., 1993), and studies comparing both
kinds of calcium deposits in a given species are rare (Wu
and Kuo-Huang, 1997).
The calcium carbonate in the lithocyst idioblasts of
mulberry (Morus alba) leaves was studied by Sugimura
et al. (1999). However, their work made no mention of
the occurrence of calcium oxalate crystals. In the leaves
of Morus australis conspicuous calcium carbonate and
calcium oxalate depositions were found (Wu and Kuo-
Huang, 1997). The calcium carbonate depositions are
formed mostly in the adaxial epidermal lithocysts while
the prismatic or druse-shaped calcium oxalate crystals are
frequently located in the bundle sheath cells. The purpose
of this study was to examine the influence of calcium con-
centration on the development of plants and on the forma-
tions of calcium oxalate and carbonate depositions in the
leaves of hydroponically grown plants of Morus australis.
MATERIALS AND METHODS
Mature fruits of mulberry plants (Morus australis Poir)
were collected from the mature healthy plants grown in
the experimental farm of National Taiwan University. The
seeds were washed out, dried in the oven at 40ĘXC for 12
h, and then stored in plastic bags at 4ĘXC. The prepared
seeds were imbibed in distilled water for 2-4 h and then
planted in vermiculite and watered with distilled water.
After 12-14 days, as the hypocotyls hook was stretching,
the seedlings were carefully transferred to modified
Hoagland solutions in a growth chamber under conditions
as described by Horner and Zindler-Frank (1982). The
nutrient solutions used in this study contained one of the
four calcium concentrations as Ca(NO
3
)
2
: 3750 £gmol
Ca/l (i.e. 5 Ca; high Ca); 750 £gmol Ca/l (1 Ca; normal
Ca), 94 £gmol Ca/l (1/8 Ca; low Ca), and 47 £gmol Ca/l
(1/16 Ca; deficient). The nitrate contents of the low and
deficient calcium solutions were adjusted to the high
calcium solution by the addition of NaNO
3
. During the
hydroponical culture process, the growing length of the
5
th
to 10
th
leaves of the seedlings was measured every 2
days. After two months, five high Ca cultured plants were
transferred to low Ca solutions, and another five low Ca
cultured plants were transferred to high Ca solutions for
another 24 days. Investigations were made with the 5
th
to
7
th
fully-expanded leaves of the plants grown in the four
levels of calcium supply as well as with the latest and
newly formed fully-expanded leaves of the transferred
plants.
For light (LM) and scanning electron microscopy
(SEM), the investigated leaves were fixed at 2 h after the
beginning of the 12-h light period. Each experiment was
done with leaves from at least two different plants from
different sowing dates. Small squares were sliced out of
the middle of the half leaf, placed in 2.5% glutaraldehyde
in a 0.1 M phosphate buffer at pH 7.2 at room tempera-
ture for 2-3 h, washed three times with buffer, and post-
fixed in 1% osmium tetroxide in the same buffer for 4 h.
The specimens were then rinsed three times with buffer,
dehydrated through an acetone series (each step 20-30
min), and then either prepared for SEM observation or
embedded in Spurr's resin (Spurr, 1969) for sectioning.
Some one-£gm thick sections were made and stained with
0.1% toluidine blue. Observations and photographs were
made with a Leica Diaplan photomicroscope. The materi-
als for SEM were then dried with a Hitachi Critical Point
Dryer (HCP-1). A coating of about 30 nm was made with
IB-2 ion coater and examined by a Hitachi S-2400 SEM.
For clearing, the segments of leaves were hardened and
decolorized in 95% ethanol. Then they were either cleared
with 50% lactic acid solution or stored in 95% ethanol.
Each cleared leaf segment was mounted on a slide in
50% aquatic glycerin (Sporne, 1948) and observed under
a bright field or polarized light microscope. Counts and
observations were made in the square areas (0.4 mm
2
)
distributed on the vein areas (for calcium oxalate crystals)
and intercostals areas (for lithocysts) of the specimen (Fig-
ure 1). The results given are average values.
RESULTS
The shoot apexes of all the plants grown in the 47 £gM
Ca/l (1/16 Ca; deficient Ca) solutions withered after the
formation of the first foliage leaf. The lengths of inter-
nodes and leaves of plants in 94 £gM Ca/l (1/8 Ca; low Ca)
solution were shorter than those grown in 750 £gM Ca/l (1
Ca; normal Ca) or 3750 £gM Ca/l (5 Ca; high Ca) solutions
(Figure 2). The morphology of the plants grown in normal
calcium and those grown in high calcium were similar.
Figure 1. Localization of the test squares on the blade of Morus
australis. °Ĺ, for lithocysts; °ľ, for calcium oxalate crystals.
pg_0003
WU et al. °X Calcium depositions in
Morus
leaves
121
Figure 2. Young plants of Morus grown for 2 months with 5 Ca (high calcium), 1 Ca (normal calcium), and 1/8 Ca (low calcium) of
calcium concentrations in the nutrient solution. Plant grown in low calcium solution showed signs of calcium deficiency. Bar=1 cm.
Figu re 3. Growth curves of leaves of the plants grown with
different calcium concentrations in the nutrient solutions.
Table 1. The anatomical characteristics in the cross sections (C.S.) of the leaves of Morus australis grown with different concen-
trations of calcium in the nutrient solution.
Characters
1/8 Ca
1 Ca
5 C a
P
Thickness of leaf (£gm) (n=6)
a
104.7°”5.2
b
87.9°”4.7
c
68.9°”5.0
0.01
Area % of palisade tissue in the C. S. of leaf (n=6)
a
29.46°”3.73
a
33.10°”3.00
a
33.35°”2.30 0.05
Area % of chloroplasts in the C. S. of palisade cell (n=6)
a
80.39°”3.24
b
88.03°”2.51
c
92.22°”3.35 0.05
Area % of chloroplasts in the C. S. of spongy cell (n=6)
a
61.05°”3.55
b
78.36°”5.25
b
77.63°”6.66 0.05
Density of lithocyst (No./mm
2
) (n=20)
a
768.5°”175.24
a
680.4°”189.28
a
816.9°”178.63 0.20
Area of C.S. of lithocyst (£gm
2
) (n=25)
a
2851.1°”918.78
a
2554.3°”959.24
b
3569.9°”1170.6 0.001
Area of C.S. of Ca carbonate deposition (£gm
2
) (n=25)
---
a
341.41°”314.95
b
1403.1°”766.20 0.005
The values with different letters in the same row are significantly different by LSD.
The average lengths of fully expanded leaves of the plants
grown in low, normal, and high calcium solutions were
27.3 mm, 47.3 mm, and 45.3 mm, respectively (Figure
3). The thickness of leaf decreased as the calcium sup-
ply increased (Figures 4A, C, and E; Table 1). However
the percentage of the areas of chloroplasts in palisade and
spongy parenchyma were higher for the plants grown with
high calcium supply than those grown in normal or low
calcium supplies (Figures 4 A-F; Table 1).
The formation of calcium carbonate deposition was
affected by the calcium concentrations. The size of both
lithocyst and calcium carbonate deposition increased
as the calcium concentration increased (Table 1). In the
leaves of the plants grown in the low calcium solution,
most lithocysts contained only the stalk (Figures 4A and
B), but most plants grown in normal calcium solution had
both the stalk and the cystolith body structures (Figures
4C and D). In the plants grown in the high calcium solu-
pg_0004
122
Botanical Studies, Vol. 47, 2006
Figure 5. Paradermal sections of leaves from the plants grown with low (A) and normal (B) calcium concentrations in the nutrient
solution. No calcium oxalate crystals were found in the low calcium supply, but many druses (arrows) occurred in the cells of bundle
sheath. (Bar=50 £gm)
Figure 4. Light and scanning electron photographs of cros s sections of leaves from the plants grown with different calcium
concentrations in the nutrient solutions. The sizes of the chloroplasts in palisade parenchyma and the calcium carbonate depositions in
the lithocysts were smaller in the plants grown with low calcium supply (A and B) than those with normal (C and D) or with high (E
and F) calcium supplies. Lithocyst in the plants grown with low calcium contains only the structure of cystolith stalk (CS), but in the
plants grown in normal or high calcium solutions, the calcium carbonate depositions formed both the structures of stalk and cystolith
body (Cy). In the plants grown in the high calcium solution the lithocyst was almost filled with the cystolith body (Cy). (Bar=25
£g
m)
pg_0005
WU et al. °X Calcium depositions in
Morus
leaves
123
tion, the lithocyst was always filled with calcium carbon-
ate deposition (Figures 4E and F). In contrast, the calcium
oxalate crystal idioblasts, the density of lithocysts was not
affected by the different concentrations of calcium (Table
1). The formation of calcium oxalate crystals in the cells
of fully-expanded leaves was also related to the calcium
concentration of the solution. No oxalate crystal was
found in the leaf of the plants grown in low calcium solu-
tion (Figure 5A). In the bundle sheath cells, many druses
and prismatic crystals were observed in the leaves for the
plants grown in high and normal calcium solutions (Figure
5B). The distribution density of calcium oxalate crystals
was higher in the high calcium solution group. There were
about 9,200 and 29,100 crystal idioblasts per mm
2
leaf
area in the mature leaf of the plants grown in the normal
calcium and high calcium solutions, respectively.
Figure 6. The morphology of lithocyst and calcium carbonate depositions before and after the transfer experiments from high to low
(A, B, and E) and from low to high (C, D, and F) calcium concentration. Mature leaves before transferring experiments (A and C).
Previously formed mature leaves after transferring experiments (B and D). Newly formed mature leaves after transferring experiments
(E and F). (Bar=20 £gm)
pg_0006
124
Botanical Studies, Vol. 47, 2006
Twenty-four days after transfer from the high to low
calcium concentrations, the plants grew equally well. In
the previously formed mature leaves, the density of litho-
cysts remained the same, and the size of calcium carbon-
ate depositions decreased slightly (Figures 6 A and C), but
the density of calcium oxalate crystals decreased (Figures
7A and C) from 29,000 to 23,300 crystal idioblasts per
mm
2
leaf area. After transfer from low calcium to high
calcium, the size of existing calcium carbonate depositions
in the previously formed mature leaves increased (Figures
6B and D). In addition, the distribution density of calcium
oxalate crystals increased (Figures 7B and D) from 0 to
2,400 crystal idioblasts per mm
2
leaf area. As expected,
the newly formed calcium carbonate and calcium oxalate
in the mature leaves that followed the lowering of the cal-
cium concentrations of the growth solutions was similar
to those in the leaves of plants grown in unaltered calcium
concentrations throughout the experiment (Figures 6E and
F; 7E and F).
Figure 7. The distribution of calcium oxalate crystals (arrows) in the cleared leaves before and after the transferring experiments from
high to low (A, C, and E) and from low to high (C, D, and F) calcium concentration. Mature leaves before transferring experiments
(A and C). Previously formed mature leaves after transferring experiments (B and D). Newly formed mature leaves after transferring
experiments (E and F). (Bar= 200
£g
m).
pg_0007
WU et al. °X Calcium depositions in
Morus
leaves
125
DISCUSSION
Calcium deficiency generally has a rapid and strongly
deleterious effect on the growth of higher plants. Morus
australis plants grown with a low calcium supply (94
£gmol Ca/l) showed retardation. The average length of
fully expanded leaves of the plants grown in low calcium
solution was only about 60% of the length of leaves grown
in normal and high calcium solutions. The thickness of
leaf also decreases at the calcium supply increased. It is
interesting to find that the percentage of areas contain-
ing chloroplasts in palisade and spongy parenchyma was
higher for plants grown with a high calcium supply than
for those grown in normal or low calcium supplies.
All the seedlings grown in the deficient solution (47
£gmol Ca/l) died out before the expansion of the first foli-
age. For Morus australis, a calcium supply below 94
£gmol Ca/l will reduce the growth, and plants will survive
only when the calcium concentration in the growth solu-
tion is higher than 47 £gmol Ca/l. The minimum required
calcium concentration differs between plant species. Plants
of Phaseolus vulgaris grow well in the solutions with 47
£gmol Ca/l (Zindler-Frank et al., 1988), but for Justicia
procumbens the minimal calcium requirement is 188 £gmol
Ca/l (Kuo-Huang and Lin, 1997).
In plant cells and tissues, the formation of calcium de-
positions is not a random event but occurs in a series of
biologically organized steps (Webb, 1999). The results of
this study demonstrated that the depositaries of calcium
oxalate and calcium carbonate in cells may act as Ca-
sinks. These cells enable the plant tissues to accumulate
surplus Ca absorbed under experimental conditions and,
implicitly, natural conditions either as Ca-oxalate or as Ca-
carbonate, and to maintain relatively low concentrations
of soluble calcium in the vacuoles and apoplast of the tis-
sues. If the capacity of existing Ca-sinks is insufficient to
precipitate most unnecessary Ca, the content of soluble
Ca in the tissues will rise and induce the formation of new
calcium oxalate crystal cells or of larger calcium carbon-
ate depositions in the lithocysts.
The formation of calcium oxalate crystals may involve
the induction of mechanisms for the transport of calcium
into the vacuole as well as for the crystal initiation. The
literature cited has shown that the number of crystal idio-
blasts formed is related to the amount of calcium available
to the plant (Frank, 1972; Zindler-Frank, 1975; Franceschi
and Horner, 1979; van Balen et al., 1980). Depending
upon the species and the test conditions, both the number
and size of the calcium oxalate crystals will be influenced
by calcium nutrition to a certain degree. In this study,
the distribution density of calcium oxalate crystals in the
mature leaves of plants grown in high calcium solution
was three times higher than that of the plants grown in
normal calcium solution. No oxalate crystal was found in
the leaves of the plants grown in low calcium solution, but
following transfer from low to high calcium solutions, the
formation of calcium oxalate crystal in the mature bundle
sheath cells occurred. However, the distribution density of
these crystal idioblasts was much lower than that of plants
grown either in high or normal calcium solutions in this
study. In the mature leaves of Alibizia julibrissin, a higher
concentration of calcium acetate was required to induce
the mesophyllous cells to form calcium crystals (Borchert,
1985).
The density of lithocysts in the mature leaves of
Morus
australis
was not significantly affected by the dif-
ferent concentrations of calcium. The results suggested
that extra lithocysts were not induced under the high
calcium supply, nor were fewer lithocysts formed under
a low calcium supply. Only the sizes of lithocyst and
calcium carbonate depositions increased from the low
calcium to the high calcium solutions. The initial num-
ber of lithocysts in the epidermal tissue was genetically
determined, but their differentiation was affected by
the amount of Ca available. During leaf development, a
continuous surplus of Ca
+2
influx from the high calcium
solution enables the lithocyst initially to form a larger
cell and a larger calcium carbonate deposition inside.
Otherwise, when under calcium deficiency the lithocysts
are smaller, and most calcium carbonate depositions
contain only the stalk with no obvious body structure.
In Morus alba, the increase in the Ca content of leaves
was proportional to the increase in leaf age, and it was
found to be closely related to the Ca-sink capacity of
the developing lithocysts (Sugimura et al., 1999). The
seasonal dynamics of intracellular Ca
2+
concentration
in the apical bud cells of Morus bomciz have a close
relationship to growth cessation and the development of
dormancy and cold hardiness (Jian et al., 2000).
Since crystals appear to be reabsorbed during cal-
cium deficient conditions, it was suggested that calcium
oxalate serves as a form of calcium storage in plants
(Franceschi and Horner, 1979; Borowitzka, 1984; Fran-
ceschi, 1989). The results of the transferring experi-
ments of this study showed that the number and size of
lithocysts in the previously formed mature leaves did
not change though the size of calcium carbonate deposi-
tions changed slightly. However, the density and size of
the calcium oxalate crystals changed significantly. After
transfer from high calcium to low calcium, the size of
the first 2-3 newly formed leaves was larger than those
grown only in the lower calcium throughout the experi-
ment. This suggests that in the mature leaves of Morus
australis
, both calcium oxalate and calcium carbon-
ate could serve as forms of calcium storage, and after
transferring to the low calcium conditions, the calcium
depositions were dissolved as the calcium ions could be
utilized for the growth of new leaves. This phenomenon
was revealed during the development of new leaves be-
cause the formation of lithocysts with calcium carbonate
depositions was found to occur earlier than the bundle
sheath cells with calcium oxalate crystals. However, the
calcium oxalate crystals were dissolved and transported
to the other tissues and organs earlier than the calcium
pg_0008
126
Botanical Studies, Vol. 47, 2006
carbonate depositions. It would be interesting to test the
hypothesis that the timing of dissolution is related to the
distances between the vascular tissue and the calcium
deposition cells. Further experiments are needed to un-
derstand the regulation mechanisms associated with the
formations and redistributions of calcium oxalate and
calcium carbonate.
Acknowledgments. The authors thank Dr. C. L. Huang
for helpful comments concerning this study and Ms. C.-Y.
Lin for assistance in operating the electron microscope.
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