Botanical Studies (2007) 48: 155-164.
*
Corresponding author: E-mail: linglong@ntu.edu.tw; Tel:
886-2-33662510; Fax: 886-2-23673374.
INTRODUCTION
Peperomia is a large genus of plants primarily occur-
ring in the understory of tropical rainforests. The genus
includes species adapted to low light and with a mix of
photosynthetic mechanisms (C
3
, CAM, and CAM-cycling)
(Virzo et al., 1983; Sipes and Ting, 1985; Patel and Ting,
1987; Holthe et al., 1992; Ting et al., 1994; Helliker and
Martin, 1997). Some species have a "leaf window" which
is a colorless multiple epidermis that interfaces with a
chlorophyll-rich palisade parenchyma layer (Gibeaut and
Thomson, 1989a, b). The window tissue stores water, and
in CAM species organic acids, but also is thought to func-
tion in enhancing photosynthesis by permitting light to
penetrate to the underlying chlorenchyma (Kaul, 1977).
The palisade parenchyma cells have been shown to have
most of the C
3
photosynthetic machinery (Nishio and Ting,
1987).
A number of Peperomia species contain druse crys-
tals within the underlying palisade parenchyma cells
(Schurhoff, 1908; Horner, 1976; Franceschi and Horner,
1980; Gibeaut and Thomson, 1989a,b). Our observations
of six different species indicate the crystals are restricted
to the palisade cells and they are not related to photosyn-
thesis type as both C
3
and CAM species contain crystals
(Kuo-Huang and Franceschi, unpublished results). Cal-
cium oxalate crystals are conspicuous in many plant spe-
cies and tissues (Arnott and Pautard, 1970; Gallagher,
1975; Franceschi and Horner, 1980; Horner and Wagner,
1995; Ku-Huang, 1990; Kuo-Huang et al., 1994; Webb,
1999; Wu and Kuo-Huang, 1997), commonly formed in
cells specialized for crystal production, and their wide-
spread occurrence has raised the question of their func-
tional significance. Most studies have focused on their
role in calcium regulation as high capacity calcium sinks
to remove excess calcium (Frank, 1972; Zindler-Frank,
1975; Franceschi and Horner, 1979; Borchert, 1985, 1986;
Franceschi, 1989; Kuo-Huang and Zindler-Frank, 1998;
Kostman and Franceschi, 2000; Volk et al., 2002; Wu et
al., 2006). The observation that calcium oxalate crystals in
Peperomia are specifically produced in the photosynthetic
palisade cells rather than in cells specialized for crystal
formation indicates a function other than calcium regula-
tion in these species. Schurhoff (1908), noting that crystals
were associated with the thin layer of photosynthetic tissue
phySIOlOgy
Correlations between calcium oxalate crystals and
photosynthetic activities in palisade cells of shade-
adapted Peperomia glabella
Ling-Long KUO-HUANG
1,
*, Maurice S. B. KU
2, 3
, and Vincent R. FRANCESCHi
3
1
Department of Life Science, Institute of Ecology and Evolutionary Biology, National Taiwan University, Taipei 106,
Taiwan
2
School of Biological Sciences, Washington State University, Pullman, WA 99164-4236, USA
3
Center of Agricultural Biotechnology, National Chiayi University, Chiayi 600, Taiwan
(Received July 3, 2006; Accepted October 18, 2006)
ABSTRACT.
Each photosynthetic palisade cell in the leaves of shade-adapted Peperomia glabella contains
a druse calcium oxalate crystal which we hypothesize is involved in dispersing light to the chloroplasts.
The effect of light intensity on druse size, number and position, relative to growth and phototsynthesis
was determined. Peperomia glabella grew best at 50-100 ŁgE m
-2
s
-1
, and at 300-400 ŁgE m
-2
s
-1
had smaller
leaves with considerable yellowing. Plants grown under lower light had well developed chloroplasts while at
300-400 ŁgE m
-2
s
-1
the chloroplasts accumulated plastoglobuli and showed thylakoid swelling. Chlorophyll
content, chlorophyll a/b, and photosynthetic rate decreased with increasing light intensity. Druse crystals were
produced in palisade cells under all light conditions but crystal diameter changed, being greatest at 100 ŁgE m
-2
s
-1
and decreasing with higher light. The position of the crystals also changed with light intensity. Under 50
and 100 ŁgE m
-2
s
-1
the crystals were predominantly located at the bottom or middle of cells while at 300 and
400 ŁgE m
-2
s
-1
they were at the top of cells. The data indicate an adaptive role of calcium oxalate crystals in
photosynthesis in Peperomia.
Keywords: Calcium oxalate; Crystals; Peperomia; Photosynthesis; Shade plants.
pg_0002
156
Botanical Studies, Vol. 48, 2007
in Peperomia leaf, suggested that they might be involved
with the photosynthetic process. This possibility has also
been mentioned in reviews on calcium oxalate function
(Arnott and Pautard, 1970; Franceschi and Horner, 1980),
but no one has ever examined this further.
As an understory plant Peperomia is likely to be ex-
posed to primarily low light but also sunflecks of varying
intensity and duration. The window tissue will maximize
transmission of light into the photosynthetic tissue layer.
The palisade cell chloroplasts are distributed to the anticli-
nal surface and bottom of the cells while the adaxial end is
clear of chloroplasts. We hypothesize that the calcium oxa-
late crystals can act to distribute light entering the palisade
cell towards the chloroplasts along the wall of the cell. The
crystals are all of the druse type, which is a spherical con-
glomerate of multiple facets (see Franceschi and Horner,
1980). This crystal structure would be ideal for diffrac-
tion or reflection of light around the entire cell, and under
high light conditions (prolonged sunflecks) the crystals
could scatter some of the light back into the window tissue
to avoid photodamage to the low light-adapted palisade
chloroplasts. The purpose of this study was to examine the
relationship of druse crystals to photosynthesis in Pepero-
mia. We determined if crystal size and number, or cellular
features changed when plants were grown under a range of
light conditions. After screening a number of species we
chose Peperomea glabella, a C
3
species, for our studies.
The results support a role of Peperomia druse crystals in
photosynthesis.
MATERIAlS AND METhODS
plant material
Peperomia glabella L. plants were obtained from a
commercial nursery and acclimated for two weeks in a
growth chamber at 26
˘X
C with 16 h day length under wide
spectrum fluorescent lights (approximately 100 ŁgE m
-2
s
-1
).
The plants were randomly divided into five groups which
were placed in the growth chamber at different heights
to give light intensities at the top of the plants of 50, 100,
200, 300 and 400 ŁgE m
-2
s
-1
. Leaves that developed during
growth under each light intensity were used for analysis.
immunolabeling of six different Peperomia species for
Rubisco and PEP carboxylase (see protocols in Voznesen-
skaya et al., 1999) indicated that P. glabella had very little
PEP carboxylase and was a C
3
species.
light microscopy
Fresh free-hand sections were used for initial compari-
son of the leaf anatomical features. Sections were viewed
with partially crossed polarizing filters for visualization of
the birefringent crystals. Resin embedded samples were
used for higher resolution observation. Small pieces (1
mm
2
) from the same region of leaves from each treatment
were fixed overnight at 4
˘X
C in 2.5% (v/v) glutaraldehyde
and 2% (v/v) paraformaldehyde in 50 mM Pipes buffer (pH
7.2). The specimens were post-fixed overnight in 1% (w/
v) osmium tetroxide in sodium phosphate buffer (50 mM,
pH 7.2), rinsed with buffer, dehydrated in an acetone se-
ries, and embedded in Spurr resin (Spurr, 1969). For light
microscopy, sections 1 Łgm thick were cut on glass knives,
dried onto gelatin coated slides and stained with Stevenelˇ¦s
blue (Del Cerro et al., 1980). A drop of immersion oil was
placed on the section, a cover slip applied, and images of
the sections captured on an Olympus BM2 microscope.
For determining crystal densities and sizes, leaves were
cleared of chlorophyll and other pigments by soaking in
70% acetone for 2-3 days. The leaves were rehydrated,
infiltrated with glycerol, and examined with an Olympus
BM2 microscope by using crossed polarizing filters. The
crystals were counted within two 0.15 mm
2
areas of three
leaves of each treatment. The diameter of the crystals was
measured on 30 crystals along a line on the image of each
leaf. A total of 90 crystals were measured per treatment.
Resin sections were used to determine location of crys-
tals within the palisade cells. Crystal location was catego-
rized into upper, middle and lower positions (thirds) within
the palisade cells. Crystals within 30 randomly selected
cells were examined in each leaf. A total of 90 cells were
examined per treatment.
Scanning electron microscopy
For scanning electron microscopy (SEM) analysis
of crystal structure, leaf samples were fixed as for light
microscopy. After washing and dehydration with an
alcohol series, samples were freeze fractured in liquid
nitrogen, critical point dried from CO
2
, and sputter coated
with gold prior to examination on a Hitachi S-570 SEM.
Transmission electron microscopy
Leaf samples prepared for light microscopy were also
used for transmission electron microscopy (TEM). Thin
sections were made using a diamond knife on a Reichard
Ultramicrotome and picked up onto 200 mesh formvar
coated nickel grids. Sections were stained with saturated
uranyl acetate and 1% (w/v) KMnO
4
and observed with a
JEOL 1200EX TEM.
photosynthetic rate and chlorophyll contents
Five mature leaves from plants under each light
intensity were analyzed for O
2
evolution using a Hansatech
CB1D oxygen electrode apparatus. For each leaf sample
the O
2
evolution rate was measured at five levels of light
intensity: 50, 100, 200, 300 and 400 ŁgE m
-2
s
-1
. The O
2
evolution of mature leaves of rice (Oryza sativa) under
1200 ŁgE m
-2
s
-1
was also measured as a reference and to
ensure that the O
2
apparatus was operating properly.
For chlorophyll analysis, leaves were ground and
extracted in 95% ethanol. The total chlorophyll content
and chlorophyll a/b ratio were determined by absorbance
at 665 and 649 nm using a Perkin-Elmer 552A UV/ViS
spectrophotometer. Chlorophyll a and b levels were
calculated from the absorbance following the protocol of
Wintermans and De Mots (1965).
pg_0003
KUO-HUANG et al. ˇX Calcium oxalate crystals in
Peperomia
157
RESUlTS
Effect of light intensity on growth
Peperomia glabella is well adapted to grow under low
light (see Figure 1A). Optimum growth in terms of leaf
size, chlorophyll content and branching was observed with
a light intensity of 100
Łg
E m
-2
s
-1
, or approximately 5% of
full sunlight. The plants also grew well at 50
Łg
E m
-2
s
-1
,
although they had fewer branches and their leaves were
thinner than leaves grown under 100
Łg
E m
-2
s
-1
. At 200
Łg
E
m
-2
s
-1
the leaves were smaller and some slight yellowing
was apparent, and plants grown at 300 and 400
Łg
E m
-2
s
-1
had leaves that showed considerable or complete yellow-
ing, respectively (Figure 1A).
general leaf anatomy and light intensity
The leaves of P. glabella are differentiated into four
distinct tissue layers: upper multiple epidermis ("window
tissue" or water storage tissue), a one-cell layered palisade
parenchyma, spongy mesophyll, and lower epidermis
(Figure 1B). The palisade cells are much darker green
than the spongy mesophyll (Figure 1B). Each palisade cell
generally contains a single druse crystal (Figure 1B and
1C) in the vacuole, although occasionally more than one
crystal is present. The druse is a roughly spherical crystal
conglomerate of multiple facets (Figure 1D). Crystals were
only found in the palisade cells.
Light level had several anatomical effects. The thick-
ness of the window tissue as a percent of total leaf cross
sectional thickness was 43
ˇÓ
4% for plants grown at 50
Łg
E m
-2
s
-1
and increased to 54
ˇÓ
3% in the leaves of plants
grown under 400
Łg
E m
-2
s
-1
. There were also some slight
changes in the size of the palisade and spongy mesophyll
cells, but were not quantified in this study (see Figure 2).
The morphology and distribution of chloroplasts in the
chlorenchyma were strongly influenced by light intensity.
Plants grown under lower light (50 and 100
Łg
E m
-2
s
-1
)
had rounder and larger chloroplasts compared to those
grown under higher light (Figure 2, see insets). Starch
was abundant at 50 and 100
Łg
E m
-2
s
-1
, but was essentially
absent at 300 and 400
Łg
E m
-2
s
-1
(see Figure 2, but also
examined with PAS staining; data not shown). At the two
lowest light levels the chloroplasts of the palisade tissue
are distributed along the anticlinal walls and lower part of
the cells, with the grana stacks oriented with their surfaces
facing the vacuole or cell wall (Figure 3), while in spongy
mesophyll, the chloroplasts are located mostly along upper
and lower sides of the cells (Figure 2A and 2B). For plants
grown above 200
Łg
E m
-2
s
-1
, the chloroplasts are located
predominantly along the anticlinal periphery of both pali-
sade and spongy mesophyll cells (Figure 2C and 2D), and
the grana stacks have less strict orientation (Figure 3H).
Calcium oxalate crystals were produced under all light
conditions (Figure 2), although there were changes in size
and distribution. The overall structure of the crystals did
not appear to be affected by light intensity (Figure 3). The
crystals always appeared as multifaceted conglomerates,
although the size of the individual facets became smaller
at light levels of 200
Łg
E m
-2
s
-1
and above (Figure 3). The
crystals facets were always surrounded by a membrane.
While there was material associated with the crystal sur-
faces (Figure 3), no connection between the crystal and the
tonoplast was observed.
Figure 1. General features of Peperomia
glabella leaf anatomy. A, Effect of light
intens ity on le af siz e and chlorophyll
content. Leaves are of the same age and
taken from plants grown at the light level
indicated on the leaf (in ŁgE m
-2
s
-1
). Plants
grown at 300 and 400 ŁgE m
-2
s
-1
show se-
vere photobleaching; B, A free-hand cross
section taken from a fresh leaf. Four dis-
tinct tissues are present: a multiple epider-
mis called window tissue and involved in
water storage, a palisade cell layer of dark
green cells, a spongy mesophyll contain-
ing less chlorophyll, and the lower epi-
dermis. Calcium oxalate crystals (bright
spots) are only found in the palisade cells.
Bar = 200 Łgm; C, A sca nning electron
microscope view of two palis ade cells
that have been cleaved open to reveal the
drus e calcium oxalate crys ta ls . Gener-
ally, only one crystal is formed in each
palis ade cell. Bar = 10 Łgm ; D, A hi gh
magnification image of a druse crystal.
The crystal is a conglomerate of multiple
facets that radiate out from a central core.
Bar = 3 Łgm.
pg_0004
158
Botanical Studies, Vol. 48, 2007
in contrast to the stability of crystal structure, chlo-
roplasts in the palisade cells changed considerably in
response to changing light intensity. At 50
Łg
E m
-2
s
-1
, the
chloroplasts had numerous small grana stacks, multiple
starch grains, and only a few very small plastoglobuli (Fig-
ure 3B). At 100
Łg
E m
-2
s
-1
, the grana stacks were fewer but
much larger (Figure 3D), while other features were similar.
At 200
Łg
E m
-2
s
-1
, the grana stacks show some disruption
and there are numerous large plastoglobuli (Figure 3F). At
300 and 400
Łg
E m
-2
s
-1
, the chloroplasts showed consider-
able thylakoid swelling and grana stacks were poorly de-
fined (Figure 3H). These observations are consistent with
the observation of whole leaf coloration indicating loss of
chlorophyll at higher light intensities.
Effect of light on chlorophyll and photosynthetic
rate
in P. glabella, both the chlorophyll content and chloro-
phyll a/b ratio were sensitive to light intensity. The total
chlorophyll content of leaves grown under lower light (50
and 100
Łg
E m
-2
s
-1
) was about 41.5
Łg
g cm
-2
(compared to
62.9
ˇÓ
16.8
Łg
g cm
-2
for rice leaf, plant grown under 200-
300
Łg
E m
-2
s
-1
) and decreased significantly with increas-
ing light intensity (Figure 4A). The chlorophyll a/b ratio
in leaves grown under lower light intensities was about
2.2 (compared to 3.8
ˇÓ
0.4 for rice leaf) and decreased as
the light intensity increased (Figure 4B). This change in
chlorophyll a/b ratio was due to a decrease in chlorophyll
a with increasing light intensity (data not shown). it is
possible related to different decay rates of chlorophyll a
and b.
Photosynthesis as measured by O
2
evolution was higher
(6-10
Łg
mol m
-2
s
-1
) in leaves of plants grown under lower
light intensities (50 and 100
Łg
E m
-2
s
-1
) and was reduced
(less than 6
Łg
mol m
-2
s
-1
) in leaves grown under light in-
tensities of 200
Łg
E m
-2
s
-1
and greater (Figure 5). For plants
grown at a given light intensity, the photosynthetic rates
of the leaves increased with increasing light intensity. But
Figure 2. Light microscopy of the anatomy of leaves from plants grown at different light intensities (P-50, etc.). insets show higher
magnification of the palisade cells. A, 50 ŁgE m
-2
s
-1
. The cells of the palisade layer (large arrow) contain relatively large chloroplasts
and the crystals are displaced towards the middle or bottom of the palisade cells (arrows in inset); B, 100 ŁgE m
-2
s
-1
. Palisade cells and
crystal position are similar to that in plants grown at 50 ŁgE m
-2
s
-1
; C, 300 ŁgE m
-2
s
-1
. Palisade cells are longer and the chloroplasts are
smaller. The crystals (arrows in inset) are now displaced to the top of the palisade cells; D, 400 ŁgE m
-2
s
-1
. Chloroplasts are very re-
duced in the palisade layer and spongy mesophyll. The crystals are still displaced to the top of the cells but they are small. Bars are 100
Łgm on low magnification pictures and 20 Łgm on insets.
pg_0005
KUO-HUANG et al. ˇX Calcium oxalate crystals in
Peperomia
159
Figure 3. Transmission electron microscopy of the crystals and chloroplasts of plants grown at different light intensities (P-50 etc). All
bars = 1 Łgm. A and B, 50 ŁgE m
-2
s
-1
. The sections demonstrate the druse is made of a mass of individual facets (clear spaces in section).
The chloroplasts are well developed with many small grana stacks (arrows) and some starch; C and D, 100 ŁgE m
-2
s
-1
. Crystal morphol-
ogy has not changed but the chloroplasts have larger grana stacks (arrows); E and F, 200 ŁgE m
-2
s
-1
. This crystal has a slightly modified
structure with rings of facets. The chloroplasts have disrupted thylakoids as well as some distinct grana stacks, and there is accumula-
tion of lipid drops (arrows); G and H, 400 ŁgE m
-2
s
-1
. The crystal is smaller and has multiple small facets. The chloroplasts have slightly
swollen thylakoids and the grana stacks are poorly defined. Note that starch is absent.
pg_0006
160
Botanical Studies, Vol. 48, 2007
the increase in photosynthesis was greater (steeper slope)
in the leaves of plants grown under 50 and 100
Łg
E m
-2
s
-1
than that of plants grown under higher light intensities
(Figure 5). For comparison, the photosynthetic O
2
evolu-
tion rate for rice leaves under a light intensity of 1200
Łg
E
m
-2
s
-1
was about 14
ˇÓ
3
Łg
mol m
-2
s
-1
.
Effect of light on palisade crystals
Under all light conditions used for growth, crystals
were only formed in palisade cells. There was not a sta-
tistical difference in the number of crystals formed under
the different light conditions (Figure 6A). Crystal density
(number per unit area) in the leaves of plants grown under
different light intensities increased slightly from 3,800 to
4,280 mm
-2
leaf area (Figure 6A). While crystal number
did not change significantly, there was a difference in
the size of the crystals within the palisade cells in plants
grown at different light levels, which ranged from 5.2 to
Figure 4. Effect of light intensity (ŁgE m
-2
s
-1
) during growth on
chlorophyll in P. glabella leaves. A, Total chlorophyll levels de-
cline significantly at light above 100 ŁgE m
-2
s
-1
; B, Chlorophyll
a/b ratio decreases with increasing light intensity. Bars give the
mean ˇÓ SD.
Figure 5. Effect of light intensity (ŁgE m
-2
s
-1
) during growth on
photosynthesis rate (mean ˇÓ SD) as measured by O
2
evolution.
Leaves from plants grown at each of 5 different light intensities
were measured at 5 different light levels. The results indicate
this plant is adapted to low light levels and its photosynthetic
machinery is damaged by prolonged exposure to light levels
higher than 100 ŁgE m
-2
s
-1
.
Figure 6. Druse crystal number and size in relation to light in-
tensity (indicated as ŁgE m
-2
s
-1
). Bars represent the mean ˇÓ SD
calculated from 6 areas located in 3 different leaves. A, Crystal
number remains relatively constant regardless of light intensity
the plants were grown under; B, Crystal size changes in relation
to light intensity during growth. The crystals are smaller at the
two extremes of our experimental light conditions.
(B)
3
2.5
2
1.5
1
0.5
0
P-50 P-100 P-200 P-300 P-400
(A)
50
40
30
20
10
0
P-50 P-100 P-200 P-300 P-400
14
12
10
8
6
4
2
0
-2
0 100 200 300 400 500
Light intensity (
Łg
E
-1
s
- 1
)
(A)
4000
3000
2000
1000
0
P-50 P-100 P-200 P-300 P-400
(B)
10
8
6
4
2
0
P-50 P-100 P-200 P-300 P-400
pg_0007
KUO-HUANG et al. ˇX Calcium oxalate crystals in
Peperomia
161
8.5 Łgm diameter with increasing light intensity. Crystal
diameter was greatest in the leaves of plants grown at 100
Łg
E m
-2
s
-1
and decreased dramatically in plants grown at
higher light intensities (Figure 6B). There was also a slight
decrease in the size of crystals in plants grown at the low-
est light level of 50
Łg
E m
-2
s
-1
.
The crystals are located in the vacuoles of the palisade
cells (see Figure 2) and there was a clear change in the
position of the crystals within the vacuole relative to light
intensity. Under low light levels (50 and 100
Łg
E m
-2
s
-1
)
the crystals were predominantly (two thirds) found at the
bottom or middle of the palisade cells while at higher light
levels (300 or 400
Łg
E m
-2
s
-1
) they were predominantly at
the top of the palisade cells (Figure 7). Plants grown at
200
Łg
E m
-2
s
-1
showed a crystal distribution pattern that
was intermediate to that found in plants grown at lower
and higher light levels (Figure 7). This distribution pattern
is also illustrated visually in the insets in Figure 2.
DISCUSSION
Measurement of photosynthetic rates in Peperomia
glabella under different light intensities demonstrates that
this C
3
species is adapted for growth under very low light.
The large and abundant grana stacks seen under optimal
growth are typical of shade adapted leaves (Boardman,
1977). it appears that this plant cannot effectively adapt
to light levels above about 200
Łg
E m
-2
s
-1
. At 300-400
Łg
E m
-2
s
-1
the leaves have obvious visual signs of photo-
bleaching, which is supported by analyses showing loss of
chlorophyll and greatly reduced photosynthetic capacity.
Even at 200
Łg
Em
-2
s
-1
the chloroplasts begin to show signs
of light damage, such as accumulation of lipids and thyla-
koid swelling (Bjorkman et al., 1972; Lichtenthaler et al.,
1981; Pearcy and Franceschi, 1986), and these symptoms
are severe at 300 and 400
Łg
E m
-2
s
-1
. Given these observa-
tions, it is interesting to note that plants grown at a low
light level show increased short term photosynthesis rate
at light intensities that cause severe damage during long
term growth. it is likely that this is an adaptation to the
constantly changing light conditions under the canopy and
that anatomical features of the leaves are involved in the
short term acclimation to light fluctuations. These ana-
tomical features include presence of a window tissue, hav-
ing the preponderance of chlorophyll in the palisade layer
directly beneath the window tissue, and the presence of a
refractive crystal of calcium oxalate at the center of each
primary photosynthetic cell.
The window tissue in Peperomia would satisfy a need
for water storage capacity in these mostly epiphytic plants
while maximizing collection of light and its transmission
to the photosynthetic palisade cell layer (Krulik, 1980),
which are the primary photosynthetic cells in this plant.
Having most of the chlorophyll in a single cell layer re-
duces the problem of light attenuation if the light had to
move through multiple photosynthetic cell layers. The
palisade cell crystals are an interesting structural feature
that is shown here to vary in position with changing light
intensity, and may play a unique, though indirect role in
photosynthesis. We feel the primary function of the crys-
tals is to disperse the light coming into the top of the cell
so that all the chloroplasts of the cell, which are primarily
lined up above each other along the anticlinal sides, are
exposed to a roughly equal amount of light. Horner (1976)
noted the grana "are usually oriented perpendicular to
imaginary radii drawn from the center of the crystals". We
also observed that the chloroplast grana are oriented to-
wards the vacuole (or wall) of the cell. Reflection of light
off the crystal surfaces towards the grana stacks would
help to maximize light capture/utilization, especially at
lower light levels.
A role of crystals in photosynthesis in Peperomia is dif-
ficult to demonstrate directly. However, our observations
strongly support such a hypothesis. The crystals are only
formed in the palisade cells, which make up the primary
photosynthetic tissue. The only type of crystal formed is
druse, which has multiple, radial oriented facets and the
potential reflective properties that would help disperse
light to the surrounding chloroplasts. The number of crys-
tals is held constant in the palisade layer regardless of light
level, although crystal size changes. This would indicate
that the crystals are required by these primary photosyn-
thetic cells. Even at higher light levels where starch is
absent and photoassimilates are limited, resources are still
put into production of calcium oxalate, though carbon
limitation may result in reduction of size of the crystals at
light intensity above or below optimum levels for growth.
A surprising observation was that the position of the
druse crystals within the palisade cells was altered in re-
sponse to growth under different light levels. This further
supports our hypothesis of a role of the crystals in the
photosynthesis process. At low light levels the crystals
were mostly at the middle or bottom of the cells. This
would help to ensure distribution of the limited light to
the chloroplasts towards the bottom half of the cells, and
help maximize light capture. At an intermediate light level,
Figure 7. Position of druse crystals in palisade cells in rela-
tion to light intensity (indicated as ŁgE m
-2
s
-1
). Bars represent
the position of the crystal within the cell as a percent of all the
cells counted. At low light levels the crystals are primarily in the
middle to lower part of the cell while at higher light they are at
the top of the cell.
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
P-50 P-100 P-200 P-300 P-400
pg_0008
162
Botanical Studies, Vol. 48, 2007
where light would more easily penetrate deeper into the
palisade cells, the crystals were at the middle and top half
of the cell. At high light levels the crystals were primarily
at the top of the palisade cells. Under high light the crys-
tals at the upper surface of the cell may act to reflect part
of the light back into the window tissue and thus provide
some protection of the shade adapted chloroplasts from
photo-damage by high light. Changing the position of the
druse crystal within the palisade cell appears to provide a
means for roughly regulating the amount of light entering
the cell and how that light is distributed within the cell.
Calcium oxalate crystals are a common component of
many plant species, but the regulation of their formation
and the various functions they play in the plant are still
being resolved. They are clearly involved in bulk regula-
tion of calcium in some plants, defense in other plants,
and possibly mechanical properties as well (reviewed by
Arnott and Pautard, 1970; Horner and Franceschi, 1980;
Webb, 1999). We suggest that crystals originally evolved
as a means to remove excess calcium as a physiologically
and osmotically inactive precipitate, at a minimum cost
of carbon since oxalic acid has only two carbons but two
carboxylic acid groups. Over time, plants have evidently
modified crystal form and patterns of distribution for sec-
ondary functions such as defense (i.e. larger crystals with
pointed and/or barbed tips). Here we provide evidence that
in some plants calcium oxalate crystals have also been
utilized as part of the overall photosynthetic process. it
will be interesting to survey other low light and "window
plants" to see how widespread this phenomenon is.
Acknowledgements. Part of this work was conducted by
the authors in the WSU Electron Microscopy Center. We
thank V. Lynch-Holm and C. Davitt of the WSU EMC, and
N. Tarlyn for assistance with plants and specimen prepara-
tion. L.-L. Kuo-Huang was supported in part by a grant,
NSC-38018F, from the National Science Council, Taiwan.
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