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

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 40¢XC for 12

h, and then stored in plastic bags at 4¢XC. 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. ¡½, for lithocysts; ¡¼, for calcium oxalate crystals.

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¡Ó5.2

b

87.9¡Ó4.7

c

68.9¡Ó5.0

0.01

Area % of palisade tissue in the C. S. of leaf (n=6)

a

29.46¡Ó3.73

a

33.10¡Ó3.00

a

33.35¡Ó2.30 0.05

Area % of chloroplasts in the C. S. of palisade cell (n=6)

a

80.39¡Ó3.24

b

88.03¡Ó2.51

c

92.22¡Ó3.35 0.05

Area % of chloroplasts in the C. S. of spongy cell (n=6)

a

61.05¡Ó3.55

b

78.36¡Ó5.25

b

77.63¡Ó6.66 0.05

Density of lithocyst (No./mm

2

) (n=20)

a

768.5¡Ó175.24

a

680.4¡Ó189.28

a

816.9¡Ó178.63 0.20

Area of C.S. of lithocyst (£gm

2

) (n=25)

a

2851.1¡Ó918.78

a

2554.3¡Ó959.24

b

3569.9¡Ó1170.6 0.001

Area of C.S. of Ca carbonate deposition (£gm

2

) (n=25)

---

a

341.41¡Ó314.95

b

1403.1¡Ó766.20 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-

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)

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)

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).

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

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.

LITERATURE CITED

Arnott, H.J. and F.G.E. Pautard. 1970. Calcification in plants.

In H. Schraer (ed.), Biological Calcification. New York:

Appleton-Centry Crofts, pp. 375-446.

Borchert, R. 1985. Calcium-induced patterns of calcium-oxalate

crystals in isolated leaflets of Gleditsia triacanthos L. and

Albizia julibrissin Durazz. Planta 165: 301-310.

Borowitzka, M.A. 1984. Calcification in aquatic plants. Plant

Cell & Eviron. 7: 457-466.

Bush, D.S. 1995. Calcium regulation in plant cells and its role

in signaling. Ann. Rev. Plant Physiol. Plant Mol. Bio. 46:

95-122.

Dickis on, W.C. 2000. Integrative P lant Anatomy. Academic

Press, A Harcourt Science and Technology Company, USA,

pp. 40-44.

Franceschi, V.R. 1989. Calcium oxalate formation is a rapid and

reversible proces s in Lemna minor L. Protoplasma 148:

130-137.

F ranceschi, V.R. and H.T. Horner. 1979. Use of Psychotr ia

punctata callus in study of calcium oxalate crystal idioblast

formation. Z. Pflanzenphysiol. 92: 61-75.

Franceschi, V.R. and H.T. Horner. 1980. Calcium oxalate crys-

tals in plants. Bot. Rev. 46: 361-427.

Frank, E. 1972. The formation of crystal idioblasts in Canavalia

ensiformis DC. at different levels of calcium s upply. Z.

Pflanzenphysiol. 67: 350-358.

Freisleben, R. 1933. Untersuchungen uber Bildung und Auflo-

sung von Cystolithen bei den Urticales. Flora 27: 1-45.

Guena, J.M. and C. J. Hillson. 1985. The occurrence, type and

location of calcium oxalate crystals in the leaves of fourteen

species of Araceae. Ann. Bot. 56: 351-361.

Horner, H.T. and E. Zindler-Frank. 1982. Calcium oxalate

cryst als a nd crystal cells in the le aves of R hynchosi a

caribaea (Leguminosae: Papilionoideae). Protoplasma 111:

10-18.

Jian, L.C., J .H. Li, and P.H. Li. 2000. Seas onal alteration in

amount of Ca

2+

in apical bud cells of mulberry (Morus bom -

bciz Koidz): an electron microscopy-cytochemical study.

Tree Physiol. 20: 623-628.

Kuo-Huang, L.L. 1990. Calcium oxalate crystals in the leaves of

Nelumbo nucifera and Nymphaea tetragona. Taiwania 35:

178-190.

Kuo-Huang, L.L. and E. Zindler-Frank. 1998. Structure of crys -

tal cells and influences of leaf development on crystal cell

development and vice versa in Phaseolus vulgaris (Legu-

minosae). Bot. Acta 111(5): 337-345.

Kuo-Huang, L.L. and M.L. Lin. 1997. Morphological and X-ray

microanalytical study on the calcium carbonate crystals in

the leaves of Justicia procumbens L. Sup Electron Micros-

copy (Japan), 32, 22-IV-1030, pp. 157.

K u o- Hu a n g , L . L ., S . H. C h e n , a n d S . J . C h e n . 2 00 0 .

Ultras tructural s tudy on t he form ation of sc lereids in

the floating leaves of Nymphoides coreana and Nuphar

schimadai. Bot. Bull. Acad. Sin. 41: 283-291.

Mas on, A.Z . a nd J .A. Nott. 1981. The role of intracellular

biomineralized granules in the regulation and detoxification

of metals in gastropods with special reference to the marine

prosobranch Littorina littorea. Aquat. Toxicol. 1: 239-256.

Nultsch, W. and A. Grahle. 1988. Mikroskopisch-Botanisches

Praktikum. 8

th

Ed. Georg Thieme Verlag Stuttgart, pp.

53-56.

Okazaki, M., H. Setoguchi, and E. Hisanaga. 1991. Inorganic

composition of cystoliths isolated from leaves of higher

plants. In S. Suga and H. Nakahara (eds.), In Mechanisms

and Phylogeny of Mineralization in Biological Systems.

Tokyo, Springer-Verlag, pp. 173-177.

Okazaki, M., H. Setoguchi, H. Aoki, and S. Suga. 1986. Ap-

plication of soft X-ray microradiography to observation of

cystoliths in the leaves of various higher plants. Bot. Mag.

Tokyo 99: 281-287.

Prychid, C.J. and P.J. Rudall. 1999. Calcium oxalate crystals in

monocotyledons: A review of their structure and systemat-

ics. Ann Bot. 84: 725-739.

Rabiger, F.H. 1951. Untersuchungen an einigen Acanthaceen

und Urticaceen zur Function der Cystolithen. Planta 40:

121-144.

Scott, F.M. 1946. Cystoliths and plasmodesmata in Beloperone,

Ficus and Boehmeria. Bot. Gaz. 107: 372-378.

Setoguchi, H., M. Okazaki, and S. Suga. 1989. Calcification

in higher plants with special reference to cystoliths. In R.

E. Crick (ed.), Origin, Evolution, and Modern Aspects of

Biomineralization in Plants and Animals, New York, Ple-

num Press, pp. 409-418.

Sheue, C.R., Y.P. Yang, and L.L. Kuo-Huang. 2003. Altitudinal

variation of resin ducts in Pinus taiwanensis Hayata (Pina-

ceae) needles. Bot. Bull. Acad. Sin. 44: 305-313.

Sporne, K.R. 1948. A note ona rapid clearing technique of wide

application. New Phyto. 47: 290-291.

Spurr, A.R. 1969. A low-viscosity epoxy resin embedding medi-

um for electron microscopy. J. Ultrastruct. Res. 26: 31-34.

Sugimura, Y., T. Mori, I. Nitta, E. Kotani, T. Furusawa, M. Tat-

sumi, S. Kusakari, M. Wada, and Y. Morita. 1999. Calcium

deposition in idioblasts of mulberry leaves. Ann. Bot. 83:

543-550.

Taylor, M.G., K. Simkiss, G.N. Greaves, M. Okazaki, and S.

WU et al. ¡X Calcium depositions in

Morus

leaves

127

Mann. 1993. An X-ray absorption spectroscopy s tudy of

the structure and transformation of amorphous calcium car-

bonate from plant cystoliths. Proc. Royal Soc. Lond. 252:

75-80.

Van Balen, E., S.C. van de Gejn, a nd G.M. Desmet. 1980.

Autora diog raphi c e vide nce for t he i ncorp orat ion of

cadmium into calcium oxalate crystals. Z. Pflanzenphysiol.

97: 123-133.

Watt, W.M., C.K. Morrell, D.L. Smith, and M.W. Steer. 1987.

Cystolith development and structure in Pilea cadierei (Urti-

caceae). Ann. Bot. 60: 71-84.

Webb, M.A. 1999. Cell-mediated crys tallization of calcium

oxalate in plants. Plant Cell 11: 751-761.

Wu, C.C. and L.L. Kuo-Huang. 1997. Calcium crystals in the

leaves of some species of Moraceae. Bot. Bull. Acad. Sin.

38: 97-104.

Yu, F.G. and Z.I. Li. 1990. Anatomy of the lithocyst in the epi-

dermis of leaf in Broussonetia papyrifera. Acta. Bot. Sinica

33: 249-255.

Zindler-Frank, E. 1975. On the formation of the pattern of crys -

tal idioblasts in Canavalia ensiformis D.C. VII. Calcium

and oxalate content of the leaves in dependence of calcium

nutrition. Z. Pflanzenphysiol. 77: 80-85.

Zindler-Frank, E., E. Wichmann, and M. Korneli. 1988. Cells

with crystals of calcium-oxalate in the leaves of Phaseolus

vulgaris - a comparison with those in Canavalia ensiformis.

Bot. Acta 101: 246-253.