Plant Science 164 (2003) 901
/909

www.elsevier.com/locate/plantsci

Review

Advances in our understanding of calcium oxalate crystal formation

and function in plants
Paul A. Nakata *
Department of Pediatrics, USDA/ARS Childrens Nutrition Research Center, Baylor College of Medicine, 1100 Bates St., Houston, TX 77030-2600,
USA
Received 6 January 2003; received in revised form 14 February 2003; accepted 19 February 2003

Abstract
Calcium oxalate crystal formation in plants appears to play a central role in a variety of important functions, including tissue
calcium regulation, protection from herbivory, and metal detoxification. Evidence is mounting to support ascorbic acid as the
primary precursor to oxalate biosynthesis. The ascorbic acid utilized in oxalate biosynthesis is synthesized directly within the
calcium oxalate crystal-accumulating cell, called the crystal idioblast. Several unique features of the crystal idioblast have been
proposed as factors that influence calcium oxalate formation. These features include an abundance of endoplasmic reticulum (ER),
acidic proteins, cytoskeletal components, and the intravacuolar matrix. A number of mutants defective in different aspects of
calcium oxalate crystal formation have been isolated. Cellular and biochemical characterizations of the various mutants have
revealed mutations affecting crystal nucleation, morphology, distribution, and/or amount. Such mutants will be useful tools in
continued efforts to decipher the pathways of crystal formation and function in plants.
Published by Elsevier Science Ireland Ltd.
Keywords: Calcium; Oxalate; Crystal formation

1. Introduction
The plant calcium oxalate crystal is considered one of
the first reported objects to have been seen under a light
microscope [1]. Since this initial report, oxalate crystals
have been found throughout nature. They have been
observed [2] in rocks, soil, and among multiple members
of all of the five kingdoms (Monera, Protista, Fungi,
Plantae, and Animalia). In all cases, the crystals are
formed from environmentally derived calcium and from
biologically synthesized oxalate.
In plants, calcium oxalate deposition is common.
Members of more than 215 plant families accumulate
crystals within their tissues [3]. Oxalate-producing

The contents of this publication do not necessarily reflect the

views or policies of the U.S. Department of Agriculture, nor does
mention of trade names, commercial products, or organizations imply
endorsement by the U.S. Government.
* Tel.: /1-713-798-7013; fax: /1-713-798-7078.
E-mail address: pnakata@bcm.tmc.edu (P.A. Nakata).

plants, which include many crop plants, accumulate

oxalate in the range of 3
/80% (w/w) of their dry weight.
As much as 90% of the total calcium of a plant can be
found as the oxalate salt [4
/7]. Crystals have been
observed in virtually all the tissues of a plant. In
whatever tissue the crystals are found, they most often
accumulate within the vacuoles of specialized cells called
crystal idioblasts [7].
Plants produce a variety of calcium oxalate crystal
shapes and sizes. Most crystals can be classified into one
of five categories, based on their morphology: crystal
sand, raphide, druse, styloid, and prismatic [8]. Usually,
the morphology of a crystal, as well as its spatial
distribution, is conserved within specific taxa. Several
functions for crystal formation in plants have been
proposed, based on such properties [8].
This review will delineate the ways in which the strides
made in recent studies contribute to our current understanding of calcium oxalate formation and its functions
in plants. Additional information is available to obtain a
more comprehensive understanding of calcium oxalate
in plants from earlier reviews [5,7
/12].

0168-9452/03/$ - see front matter. Published by Elsevier Science Ireland Ltd.

doi:10.1016/S0168-9452(03)00120-1

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2. Proposed functions of calcium oxalate

The diversity of crystal shapes and sizes, as well as
their prevalence and spatial distribution, have led to a
number of hypotheses regarding crystal function in
plants. The proposed functions include calcium regulation, ion balance (e.g. sodium and potassium), plant
protection, tissue support (plant rigidity), detoxification
(e.g. heavy metals or oxalic acid), and light gathering
and reflection [8]. Although evidence in support of many
of these hypotheses is still lacking, support for a role in
tissue calcium regulation, plant protection, and metal
detoxification has been mounting.
2.1. Calcium regulation
It has been proposed that the primary function of the
crystal idioblast is to serve as a localized calcium sink
(Fig. 1) to reduce the apoplastic calcium concentration
around adjacent cells [11,13
/15]. Studies in support of
this hypothesis have shown, using a variety of plants,
that the size and number of calcium oxalate crystals are
responsive to changes in the concentration of calcium in
the plant environment [13
/17]. Several recent reports
[18
/24] support a role for calcium oxalate formation in
calcium regulation. Two of these reports examined
whether crystals of different morphologies had varying
sensitivities to fluctuations in calcium concentrations
[20,23]. Using the raphide and druse accumulating
aquatic plant, Pistia stratiotes , Volk and colleagues
proposed that druse and raphide crystal formation serve
different but related functions. The authors presented
evidence indicating that raphide (needle-shaped) crystal
formation may serve a dual function of calcium regulation and plant defense, while druse (globular shaped)

Fig. 1. Calcium-accumulating crystal idioblast. Reflected-transmitted

image of a microautoradiograph of a Pistia stratiotes leaf section after
the plantlet was placed on high-calcium growth medium (containing
45
Ca) overnight. The image shows that the crystal idioblasts accumulate large amounts of calcium (red color) in comparison to surrounding
mesophyll cells. Bar/20 mm.

crystal formation is strictly involved in calcium regulation [23]. Druse crystal formation was found to be
dynamic and responsive to fluctuations in calcium
levels. When calcium levels were high, druse crystal
size and number rapidly increased. When calcium was
limiting, druse crystal size and number decreased,
presumably freeing up the calcium for utilization by
the plant. The disappearance of crystals in tissues under
conditions of calcium deficiency and active growth has
been reported in a number of plants [9,15,17,25
/27]. In
addition, Volk and colleagues identified a putative
enzyme, using an oxalate oxidase antibody, that may
play a role in the degradation of liberated oxalate. This
antigen was abundant in the crystal idioblast under
plant growth conditions of calcium limitation, and was
absent under growth conditions of calcium availability
[23].
2.2. Plant protection
Recently published works [28
/32] have cited a role
for crystal formation in plant protection, based primarily on temporal, spatial and/or morphological parameters of crystal formation. Two studies have reported
that calcium oxalate crystal accumulation increased in
leaves of Sida rhombilfolia [33] and seeds of Norway
spruce [29] in response to artificial herbivory or tissue
wounding. Thus, in some plants, calcium oxalate
formation appears to be an inducible defense response.
In a gazelle herbivory study [30,34], the authors
noticed that only the leaf tips of the desert lilies were
consumed. Upon microscopic examination of the leaves,
it became apparent that the tips were the only areas of
the leaf devoid of raphide crystals [34]. Ruiz et al. [31]
compared the amount of calcium oxalate the plants
accumulated to the amount of herbivory that occurred
at a particular location. Desert lilies at three different
locations (Ardon valley, Neqarot canyon, and Machmal
valley) were studied. The authors found that the lilies
which were grown at the locations where grazing was the
highest contained the highest amount of crystals while
those that were grown in locations where grazing was
minimal accumulated few, if any, crystals. Clipping or
wounding the plant leaves, however, did not result in an
increase in calcium oxalate accumulation, indicating
that crystal formation was not inducible in this case.
Thus, it appears that the crystal formation in that study
was a developmentally programmed process, and that
the crystal formation may have been under the selective
pressure of herbivory [31].
Investigations [35
/39] into outbreaks of contact
dermatitis among field workers and processors, resulted
in the discovery of a link between the presence of
calcium oxalate crystals and the dermal rashes. The
raphide needles of calcium oxalate punctured the skin of
the workers allowing the sap of the plants to enter the

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body at the wound site. Examples of such dermal

irritations were reported to be common in the flower
growing [35,36] and distillery industries [38], where an
abundance of raphide needles is liberated during the
harvesting and processing of plant tissues. The agave
juice that is used to make tequila has been reported to
contain as many as 6000 crystals/ml [38].
2.3. Detoxification of heavy metals
Several studies have shown that plants utilize organic
acids (e.g. citrate, malate, and/or oxalate) as mechanisms enabling them to tolerate various heavy metals [40].
Some plants utilize oxalate to detoxify hazardous
metals, such as lead [41], aluminum [42], strontium
[43,44], and cadmium [45], when present in the environment. Studies have noted that the exogenous application
of such metals to the plant environment often results in
the incorporation of the heavy metal into oxalate
crystals [43
/46]. In acid soils (approximately 40% of
the worlds arable land), aluminum toxicity is a major
problem limiting crop production [47]. Micromolar
concentrations of aluminum can inhibit root growth
and affect the acquisition of nutrients and water. This
reduction in root function is a major issue in the
production of many agriculturally important crop
species [42,48]. Two mechanisms of oxalate utilization,
exclusion and internal tolerance, have evolved in plants
to enable them to resist aluminum toxicity [48,49]. The
exclusion mechanism involves the excretion of oxalate
into the environment by the roots and occurs in
response to external aluminum stress [46]. The internal
tolerance mechanism involves the sequestration of
aluminum in the non-toxic form of aluminum-oxalate
within the aerial portion of the plant [50]. Plants such as
buckwheat use both mechanisms of aluminum detoxification.

3. Oxalate biosynthesis
A number of pathways for oxalate production have
been hypothesized. These pathways include the cleavage
of isocitrate, hydrolysis of oxaloacetate, glycolate/glyoxylate oxidation, and/or oxidative cleavage of L-ascorbic
acid [2]. Biochemical measurements, using radio-labeled
precursors, support a C2/C3 cleavage of ascorbic acid as
a major pathway of oxalate production [51
/57]. Recently, there has been renewed interest in ascorbate as
the precursor to oxalate biosynthesis. By incorporating
microautoradiography into the radiotracer studies, investigators have strengthened this proposition [58
/60].
In these studies, radiolabeled compounds were introduced into cultures of Pistia stratiotes plantlets [58],
Yucca torreyi roots [59], or isolated Pistia crystal
idioblasts [60]. The radiolabel was incorporated into

903

the oxalate crystals when supplied in the form of C1labeled ascorbate [58,59] or a precursor leading to C1labeled ascorbate [60]. Taken together, these studies
suggested ascorbic acid as the precursor to oxalic acid
production, and moreover, that the ascorbate precursor
is most likely produced within the crystal idioblast itself
[60] via the Wheeler-Smirnoff pathway [61,62]. The
presence of L-galactono-g-lactone dehydrogenase within
the mitochondria of the crystal idioblast, the enzyme
responsible for the conversion of L-galatonolactone to
ascorbic acid, supports these findings [63].

4. Calcium oxalate crystal formation

Crystals of calcium oxalate are usually formed in a
defined shape and spatial location. This property may
be a useful feature in plant taxonomy and systematics
[7,22,64]. Spatially, crystal formation most commonly
occurs inside the vacuoles of specialized cells called
crystal idioblasts. Crystal idioblasts exhibit characteristic features including an enlarged nucleus, specialized
plastids, increased ER, elevated levels of rRNA, and
unique vacuolar components [7
/9,65].
4.1. Intravacuolar matrix
Intravacuolar matrices (e.g. crystal chambers) are
macromolecular structures that have been suggested to
play an important role in defining crystal shape and
growth [9,66
/69]. Crystal chambers appear to form a
lipid and polysaccharide partition, separating the vacuolar compartment from the space in which the crystal
develops. The shape of the crystal can often be predicted
before it actually forms, based on the shape of these
membrane chambers [9,66
/68]. In some cases, the
crystals are even found encased in cell wall material
[70,71], or surrounded by lamellated sheaths [72].
Interestingly, the transcripts encoding proteins putatively involved in lipid, polysaccharide, and cell wall
metabolism were recently identified and found to be
expressed at higher levels during calcium oxalate crystal
accumulation [73]. Whether proteins encoded by the
identified transcripts are specifically involved in crystal
formation remains to be determined.
4.2. Crystal-associated proteins
Using isolated crystals, researchers have identified
proteins associated with the plant crystal or crystal
matrix [69,74]. Webb et al., showed that there are a
number of different polypeptides associated with raphide bundles from Vitis [69]. Recently, Bouropoulos et
al. reported their investigations into crystal-associated
matrix macromolecules and their involvement in regulating crystal nucleation and morphology [74]. Crystals

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from tobacco, tomato, and bougainvillea were shown to

contain acidic proteins [74] with compositions similar to
those found in the spicules of sponges [75] and antlers
[76]. In vitro nucleation assays revealed that the
assemblage of crystal-associated macromolecules from
tobacco promotes nucleation of calcium oxalate crystals, while various control polypeptides did not [74]. The
results support a role for crystal-associated macromolecules in the initiation and growth of a crystal.

crystal idioblast (Fig. 2). High-resolution immunolocalization revealed higher levels of calreticulin within
distended regions of the ER [80]. Such an observation
has been reported in other cell types where calreticulin
was over-expressed to 100-fold wild-type levels [81].
Interestingly, these distended regions appeared similar
to regions recently found to be enriched in calcium
(Kostman et al., unpublished). We speculate that the
high-capacity calcium binding protein, calreticulin, may
be involved in sequestering the calcium within these
distended regions.

4.3. Endoplasmic reticulum

Prolific networks of ER have been reported throughout the cytoplasm of the crystal idioblast [7,77,78]. This
prolific ER network may be necessary for the idioblast
to tolerate the high influxes of calcium that can occur
during the formation of calcium oxalate crystals [5]. In
order for the ER to function in calcium storage it must
possess a mechanism to reduce the luminal calcium
concentration to prevent precipitation with phosphate
and other anions. A recent report revealed that a
calsequestrin-like calcium binding protein was expressed
in the idioblasts of Pistia stratiotes [79]. Since this initial
report, calreticulin was shown to be enriched in the

4.4. Microtubules
Cytoskeletal elements, which include microtubules,
have been shown to function in the regulation of cell
shape during cell growth [82
/84]. Since crystal idioblasts
(e.g. raphide and styloid) become elongated during
crystal growth, researchers proposed that tubules may
be playing a role in coordinating crystal and cell growth
[7]. Careful coordination of cell and crystal growth
would be paramount, especially under circumstances of
rapid crystal formation. Using the aquatic plant model,
Pistia stratiotes , Kostman and Franceschi showed that

Fig. 2. Calreticulin expression in the crystal idioblast. (A) In situ analysis of calreticulin expression. Paraffin-embedded sections of Pistia stratiotes
leaves were hybridized with antisense calreticulin probe and visualized by colormetric development. Strong expression is shown within the developing
idioblast (indicated by arrowheads). Bar/10 mm (B) Immunolocalization of a LR white embedded Pistia leaf section. The section was
immunostained with anti-calreticulin polyclonal antiserum followed by protein A-gold and silver enhancement. The reflected and transmitted images
were merged to generate the final image. Bar/5 mm.

P.A. Nakata / Plant Science 164 (2003) 901
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raphide crystal idioblasts possess a cortical microtubule

network [65]. The microtubule network appears to limit
an increase in cell diameter, but not elongation. Thus,
the raphide idioblast elongates at its ends, generating a
football-shaped cell. Using colchicine to prevent microtubule polmerization, the authors observed a dramatic
change in idioblast shape, but no effect on the crystal
form (other than crystal length). Microtubules appear to
be involved in controlling idioblast shape, but not
crystal growth [65].

905

genome size (three to four times the size of Arabidopsis ),

ability to be transformed, autogamous and diploid
nature, and spatial pattern of calcium oxalate crystal
formation [90]. By visually inspecting an EMS-mutagenized M. truncatula population, mutants have been
identified that differ in calcium oxalate crystal content,
spatial distribution, number, and morphology [89,91].
As anticipated, genetic analysis of the different mutants
indicates that crystal formation is a complex process
involving multiple loci [89,91].

4.5. Crystal hydration state

5.1. Alterations in calcium oxalate content
Recently, several studies have analyzed the hydration
state of the crystals from a number of different plants,
which include ribbon plant [78,85], cactus [22], tobacco
[74], tomato [74], and water lettuce [23]. Hydration state
is another factor that has been shown to influence
crystal morphology in vitro [86]. Two hydration states
have been reported in plants [86]. They are the monohydrate (whewellite) and dihydrate (weddellite). The
relative concentration of calcium to oxalate has been
shown to affect the hydration of crystals formed in vitro.
Monohydrate crystals predominated when calcium was
added to a concentrated solution of oxalate, while
dihydrate crystals comprised the most abundant form
when oxalate was mixed into a concentrated solution of
calcium [86,87]. Crystals of the same morphology,
however, are found in both hydration states in plants
[8,22,87].

A number of mutants were isolated from a mutagenized M. truncatula population that showed an alteration
in calcium oxalate content. Two of the more extreme
mutants in this regard were calcium oxalate defective 4

(cod4 ) and cod5 [89]. The mutant cod4

accumulated
more calcium oxalate, while cod5 accumulated less
calcium oxalate than controls (Fig. 3A). Wild-type M.
truncatula plants accumulated prismatic crystals along
the vascular strands in all the different plant tissues with
the exception of roots, in which no crystals were
observed. Measurements of oxalate content in the
different cod4 tissues showed elevated tissue oxalate
levels as a result of druse crystal accumulation. The

4.6. Crystal twinning

Twinning represents an alteration in the orientation
of the crystal lattice along a plane within the calcium
oxalate crystal [71]. Although twinning is common in
plant crystal formation, its biological significance remains to be fully determined. In a recent study, the
nature and orientation of twinning were examined using
raphide crystals from grape [88]. In this study, Arnott
and Webb showed that the raphide crystals are twinned
along their length and that the twinning is rotational.
The authors proposed that twinning may enhance
crystal stability and strength. Such a factor could play
a role in allowing for synthesis and persistence of the
raphide shape within the idioblast [88].

5. Isolation of mutants of calcium oxalate formation

To complement existing biochemical and cellular
investigations efforts have been made to identify a
suitable genetic system to study calcium oxalate formation. A recent report [89] offered the model legume
Medicago truncatula as such a system. Key attributes of
M. truncatula include its short generation time, small

Fig. 3. Calcium oxalate defective (cod ) mutants. (A) Whole mount leaf
clearing showing a secondary vein and surrounding mesophyll cells
from wild-type, cod4 , and cod5 plants viewed between crossedpolarizers. The calcium oxalate crystals appear as bright spots.
Bar/10 mm. (B). Calcium and oxalate content of leaves from wildtype, cod4 , and cod5 plants.

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calcium content, however, did not increase in proportion to the increase in oxalate (Fig. 3B). Thus, more of
the tissue calcium was partitioned into the crystalline
form in the leaves and stems from cod4 than controls.
Biomass and chlorophyll measurements showed that the
cod4 mutation also resulted in an overall reduction in
plant growth and chlorophyll content [92].
In contrast, no prismatic crystals were detected in any
of the different tissues of the cod5 mutant. Measurements of the oxalate content in the different tissues
confirmed the cod5 crystal phenotype by exhibiting low
oxalate levels compared to those of controls. Although
compromised in its ability to accumulate crystals of
calcium oxalate, cod5 exhibited growth similar to that of
controls [93]. Moreover, cod5 and controls contained
similar amounts of calcium (Fig. 3B), sodium, and
potassium [93]. These findings suggest that calcium
oxalate crystal formation is not essential for plant
growth or development in the case of M. truncatula .

oxalate-accumulating plant halogeton was considered

one of the most economically destructive poisonous
plants [98]. Consumption of large amounts of this plant,
which can occur during grazing, resulted in large losses
of sheep and cattle. The cause of death was attributable
to calcium oxalate crystal formation in rumen walls,
arteries, and kidneys (kidney stones).
Efforts continue to screen germplasms [101] and
carefully evaluate plant growth conditions [102
/104] in
order to produce crops with lower oxalate levels in
edible tissues. Recent evidence indicates that genetic
manipulation to reduce the oxalate content in plant
foods is another feasible alternative [93]. Examples of
some of the plant foods that contain substantial
amounts of oxalate are spinach, rhubarb, and dry beans
[5,27,101,105]. A better understanding of the mechanisms regulating calcium oxalate formation in plants
could lead to strategies to improve the nutritional
composition of plant foods.

5.2. Alterations in crystal morphology

7. Conclusion
The factors affecting crystal morphology are numerous and the process is complex. Isolation of a number of
different crystal morphology defective (cmd ) mutants
provided genetic evidence supporting this complexity
[91]. The mutants revealed that a single point mutation
could drastically alter the size and morphology of the
crystal. A comparison of the different shapes present in
various legumes to those present in the cmd population
revealed striking similarities. Some of these mutations
resulted in crystal morphologies virtually identical to
those observed in other legumes. Based on these
comparisons, the authors speculate that such point
mutations over the course of evolution contributed to
the variations in crystal morphologies commonly observed in these legumes today [91,94]. Further characterization of these mutants should provide new insights
into the regulation of crystal morphology.

Although much remains to be discovered about

calcium oxalate crystal formation and function in
plants, recent reports show that progress is being
made. Studies indicate that the pathway of oxalate
biosynthesis utilizes ascorbate as the primary precursor,
and that the ascorbate utilized is produced directly
within the crystal idioblast itself. Plant oxalate production appears to serve diverse functional roles that
protect the plant from succumbing to a variety of
environmental stresses such as herbivory or excess
metals in the soil in which it grows. Recent results
suggest that manipulating the amount and spatial
distribution of calcium oxalate formation is feasible.
Improvements in production (e.g. by improving plant
defense) and enhancement of nutritional quality of crop
plants (e.g. by increasing calcium bioavailability) may
soon be possible through the manipulation of calcium
oxalate crystal formation.

6. Plant oxalates and human health

Oxalate in plant foods impacts human health in at
least two significant ways [2,5,95]. First, oxalate is an
antinutrient in that it renders calcium unavailable for
nutritional absorption by humans [96,97]. This issue of
calcium bioavailability is important when one considers
the reliance of different populations around the world
on plant foods as their main source of calcium and other
minerals, as well as the failure of many people in the
United States to meet the recommended daily allowance
(RDA) for calcium intake. Second, soluble oxalates
have been shown to pose a hazard in some cases when
ingested by grazing livestock [98] and humans
[95,99,100]. From the 1940s through the 1970s, the

Acknowledgements
Thanks goes to Dr Todd Kostman and Dr Vincent
Franceschi for contributing images to this review. This
research was supported in part by the U.S. Department
of Agriculture, Agricultural Research Service, under
Cooperative Agreement number 58-6250-6-001.

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