Plant Cell Physiol. 42(12): 1303–1310 (2001)
JSPP © 2001
Separate Localization of Light Signal Perception for Sun or Shade Type
Chloroplast and Palisade Tissue Differentiation in Chenopodium album
Satoshi Yano 1 and Ichiro Terashima
Department of Biology, Graduate School of Science, Osaka University, 1-16 Machikaneyama-cho, Toyonaka, Osaka, 560-0043 Japan
;
Physiological and ecological characteristics of sun and
shade leaves have been compared in detail, but their developmental processes, in particular their light sensory mechanisms, are still unknown. This study compares the development of sun and shade leaves of Chenopodium album L.,
paying special attention to the light sensory site. We
hypothesized that mature leaves sense the light environment, and that this information determines anatomy of new
leaves. To examine this hypothesis, we shaded plants partially. In the low-light apex treatment (LA), the shoot apex
with developing leaves was covered by a cap made of a
shading screen and received photosynthetically active photon flux density (PPFD) of 60 mmol m–2 s–1, while the
remaining mature leaves were exposed to 360 mmol m–2 s–1.
In the high-light apex treatment (HA), the apex was
exposed while the mature leaves were covered by a shade
screen. After these treatments for 6 d, we analyzed leaf
anatomy and chloroplast ultrastructure. The anatomy of
LA leaves with a two-layered palisade tissue was similar to
that of sun leaves, while their chloroplasts were shade-type
with thick grana. The anatomy of HA leaves and shade
leaves was similar and both had one-layered palisade tissue, while chloroplasts of HA leaves were sun-type having
thin grana. These results clearly demonstrate that new
leaves differentiate depending on the light environment of
mature leaves, while chloroplasts differentiate depending
on the local light environment.
Key words: Anatomy — Chenopodium album — Chloroplast
— Light sensory mechanism — Palisade tissue — Sun and
shade leaf.
Abbreviations: HA, high-light apex; HH, high-light to high-light;
HL, high-light to low-light; LA, low-light apex; PPFD, photosynthetically active photon flux density; PCL, average number of cell layers of
the palisade tissue; TNP, index of total number of cells in the palisade
tissue.
Introduction
Plants develop ‘sun’ or ‘shade’ leaves when acclimating to
1
different irradiance levels. The anatomical and physiological
differences between sun and shade leaves have been studied
extensively. Anatomically, sun leaves have more developed
palisade tissue and larger mesophyll surface area per unit leaf
area, and are thicker than shade leaves (Haberlandt 1914, Esau
1965). Sun leaves have sun-type chloroplasts with less
appressed thylakoid membranes or grana stacking, while shade
leaves have shade-type chloroplasts having more appressed
thylakoid membranes (Anderson 1986, Anderson and Osmond
1987, Terashima and Hikosaka 1995). Functionally, the sun
leaves have higher rate of photosynthesis per unit leaf area,
higher Chl a/b ratio, higher amounts of ribulose bisphosphate
carboxylase/oxygenase, cytochromes, and PSI and PSII core
complexes than the shade leaves, on leaf area basis (Boardman
1977, Björkman 1981, Anderson 1986, Anderson and Osmond
1987, Terashima and Hikosaka 1995).
To acclimate to the light environment, plants need to have
light perception (sensory) mechanisms. So far, involvement of
several light sensory mechanisms such as phytochromes, bluelight receptors, redox state of the plastoquinone pool in thylakoid membranes, photosystems and concentration of photosynthates, have been proposed for various acclimation processes
(Anderson et al. 1995, Batschauer 1998, Ono et al. 2001).
However, the light sensory mechanism(s) regulating differentiation of sun and shade leaves have not been identified.
With the advent of a focus on global climatic change, a
number of studies has been conducted on the effect of CO2
enrichment on plants. Plants grown under high concentrations
of atmospheric CO2 form leaves with well-developed palisade
tissue (Thomas and Harvey 1983, Leadley et al. 1987, Leadley
and Reynolds 1989, Radoglou and Jarvis 1990). Therefore, the
irradiance level per se would not be directly responsible for
regulating the differentiation of sun or shade leaves. Plants
grown under high atmospheric CO2 concentration accumulate
more photosynthates, like those of high-light grown plants.
Such parallelism suggests that the concentration of photosynthates is a probable signal for leaf development. Further,
depending on the species, the developing leaves in the shoot
apex may be covered by the older leaves and are not photosynthetically competent. Thus it is unlikely that newly developing
leaves either sense the irradiance level outside of the bud or
synthesize enough photosynthates for their own development.
Corresponding author: E-mail, [email protected]; Fax, +81-6-6850-5808.
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Can developing leaves sense the light environment?
Fig. 1 Light micrographs of cross-sections of matured sun (a) and shade (b) leaves of C. album. Sun leaves grew in 360 mmol m–2 s–1, shade
leaves grew in 60 mmol m–2 s–1. Bar, 50 mm.
Recently, Lake et al. (2001) reported that during leaf development in Arabidopsis thaliana, stomatal density was affected by
both light intensity and CO2 concentration around the mature
leaves. They postulated a long-distance signaling system
between older (mature) leaves and younger developing leaves.
However, there is no report on whether such a long-distance
signal regulates chloroplast development and leaf anatomy.
Interestingly, a gradient of sun- and shade-type chloroplasts exists from the adaxial to the abaxial side of the leaf
(Skene 1974, Terashima et al. 1986). This gradient of chloroplasts can be reversed by illuminating the leaf from the abaxial
side (Terashima et al. 1986). These studies showed that chloroplast properties are also affected by local light environment
within the leaf. These findings pose questions such as (1) how
does light regulate the differentiation of sun- and shade-type
chloroplasts? and (2) is the sun or shade leaf anatomy always
accompanied by sun- or shade-type chloroplasts?
In the present study, we examined how the plants sense
the light environment for the development of young leaves in
Chenopodium album L. The sun leaves of C. album have twolayered palisade tissue and while that of shade leaves is onelayered. This criterion was available to distinguish the anatomy between the sun and shade leaves. The high-light grown
plants were shaded in various ways to identify the site(s) for
light environmental perception for differentiation of sun and
shade leaves. The leaf anatomy and ultrastructure of chloro-
plasts were studied after 6 d of the shading treatments to verify
whether (1) the light signal is perceived by mature leaves and
the information (signal) is transmitted to a developing young
leaf for differentiation into sun or shade leaf, and whether (2)
the differentiation of chloroplasts into sun- or shade-type is
coupled with the anatomical differentiation.
Results and Discussion
Preliminary studies of sun and shade leaves, grown under
continuous high light (360 mmol m–2 s–1) and low light
(60 mmol m–2 s–1), showed that these light intensities induced
sun and shade leaves (Fig. 1), respectively, and that periclinal
cell divisions of palisade tissue cells occurred at 8 mm lamina
length in sun leaves in C. album.
Young leaves with less than 8 mm lamina length were
subjected to high-light to high-light (HH), low-light apex (LA),
high-light apex (HA), and high-light to low-light (HL) treatments and the leaves were sampled after these treatments for
6 d (Fig. 2, see Materials and Methods). All the data in this
study is expressed as a function of lamina length at sampling.
Leaves with different leaf length were pooled into four separate sub-groups with a class interval of 5 mm. Thus, the lamina
lengths of sub-groups were 3–7.9 mm, 8–12.9 mm, 13–17.9 mm,
and 18–22.9 mm. Within the 6 d experimental period, the apical
leaves were still in the growing stage and even the longest
Can developing leaves sense the light environment?
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Fig. 3 Changes in PCL of LA (closed circle), HA (closed square),
HH (open triangle), and HL (open circle) leaves. Plants were grown
for about 1 month in high-light conditions, and then treated for 6 d (see
Materials and Methods). The data of the leaves with lamina lengths
ranging 3–7.9, 8–12.9, 13–17.9, and 18–22.9 mm, respectively, were
pooled. At each point, 1 to 8 leaves were examined. The vertical and
horizontal bars indicate the standard deviations of PCL and lamina
length, respectively.
Fig. 2 Partial shading treatment design. Low-light apex (LA) treatment and high-light apex (HA) treatment are left and right, respectively (a). In HA treatment, the shoot apex was illuminated with a
halogen lamp through fiber optics. Shading screens for LA treatment
(left) and for HA treatment (right) are shown (b).
leaves of about 25 mm sampled for the analysis were not fully
matured. The leaf expansion rate is not analyzed in this report.
However, the expansion growth of HL and HA leaves were
slower than that of HH and LA leaves. Long-term shading
experiments were not carried out because shade caps for LA
treatment would also shade the mature leaves if they were
greater than 20 mm.
Average number of cell layers of the palisade tissue (PCL)
There was only one cell-layer in the palisade tissue from
all the leaves less than 8 mm, grown either at high-light or at
low-light conditions (Fig. 3). Palisade tissue cells divided periclinally as the lamina length exceeded 8 mm and PCL
increased to 2 (average values ³1.9) in 20 mm lamina in HH
and LA leaves. In contrast, the palisade tissue remained onelayered with an average value of <1.35 in HL and HA leaves
(Fig. 3). PCL in HL and HA leaves were comparable to that of
the shade leaves from plants grown continuously under lowlight conditions (data not shown). These results clearly demonstrate that the perception of light environment by mature leaves
and not by developing leaves regulates the sun or shade leaf
anatomy in C. album. The periclinal division of the palisade
tissue cells was less when the mature leaves were under low
light conditions.
Index of total number of cells in the palisade tissue (TNP)
TNP for all the treatments were similar within the same
leaf length groups (Fig. 4). TNP increased linearly from 1,000
at 5.5 mm lamina length to 3,000 at 15.5 mm and 4,000 at
20.5 mm (no data for HL treatments). Although HH and LA
treatments induced two-layered palisade tissue and HL and HA
treatments produced only one-layered palisade tissue, the TNP
was the same in all these leaves. These results indicate that no
additional divisions are required to attain two-layered palisade
tissue. If the periclinal divisions did not occur additionally but
just in place of anticlinal divisions in HH and LA leaves, the
constant increase in cell number is possible. Then, as shown in
a diagram (Fig. 4, inset), the cells in HL and HA treatments
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Can developing leaves sense the light environment?
growth is regulated by the light environment of the mature
leaves.
Fig. 4 Changes in TNP of LA (closed circle), HA (closed square),
HH (open triangle), and HL (open circle) leaves. Plants were grown
for about 1 month in high-light, and then treated for 6 d (see Materials
and Methods). Inset indicates a palisade tissue model of HH, HL, LA,
and HA leaves. The data of the leaves with lamina lengths ranging 3–
7.9, 8–12.9, 13–17.9, and 18–22.9 mm, respectively, were pooled. At
each point, 1 to 8 leaves were examined. The vertical and horizontal
bars indicate the standard deviations of TNP and lamina length,
respectively.
would be slender. In fact, the column widths of HL and HA
were less than those of HH and LA leaves (see Materials and
Methods for the definition of ‘column’). Average width was
about 8 mm in HL and HA leaves and 12 mm in HH and LA
leaves, respectively, when their lamina length was about
20 mm (data not shown).
Leaf thickness and palisade tissue thickness, and crosssectional column area of the palisade tissue
The leaf thickness (Fig. 5a), the palisade tissue thickness
(Fig. 5b) and cross-sectional column area of the palisade tissue
(Fig. 5c) increased gradually with that of lamina length, but the
rate of increase (measured as the slope) for HL and HA leaves
was less than that of HH and LA leaves. All these parameters
were the same for the leaves of 5.5 mm length. The leaf thickness of 20 mm long leaves was 130 mm for HH and LA leaves
and less than 90 mm in HL and HA leaves (Fig. 5a). Similarly,
the palisade tissue thickness was 35–48 mm in HH (no data for
18–22.9 mm) and LA leaves and 17–22 mm in HL (no data for
18–22.9 mm) and HA leaves (Fig. 5b). The cross-sectional column area of the palisade tissue also increased faster for HH (no
data for 18–22.9 mm) and LA leaves than HL (no data for 18–
22.9 mm) and HA leaves (Fig. 5c). These results suggest that
not only the direction of cell division but also the cell and leaf
Light perception
Lake et al. (2001) reported that stomatal development in
young developing leaves is affected by the light intensity or
by the CO2 concentration around the mature leaves. A longdistance signaling from mature leaves to young developing
leaves was proposed. Our present study revealed that the lightinduced signals from mature leaves to young leaves are also
responsible for the development of sun and shade type palisade tissue. Although it is difficult to predict the nature of light
signal in the present processes, possible involvement of phytochromes, blue-light receptors, redox signaling and photosynthates are briefly considered.
Phytochrome regulation of photomorphogenic processes
in plants is well documented (Mancinelli 1994, Quail 1994,
Batschauer 1998). The red/far-red ratio is the major determinant for the phytochrome action in plants (Smith 1994). The
red/far-red ratio for daylight ranges from 1.05 to 1.25, and that
for canopy shade ranges from 0.05 to 1.15 (Smith 1994). The
red/far-red ratio in the present study was 2.5 or 4.4 (see
Materials and Methods), which was two- or four-times that of
the daylight. The ratio did not decrease in either the HL or HA
treatments and the leaves of these treatments were shade type,
which excludes the role of phytochrome in the differentiation
of sun and shade leaves.
Blue-light receptors CRY1, CRY2, PHOT1 (NPH1) and
PHOT2 (NPL1) are also involved in photomorphogenesis (Batschauer 1998, Kagawa et al. 2001), and blue-light-induced leaf
thickening was reported (Schuerger et al. 1997). However,
Weston et al. (2000) clearly showed that there was no anatomical difference between the wild type, CRY1, CRY2, and NPH1
mutants of A. thaliana under low or high light intensity conditions. Thus, the involvement of blue-light receptors, CRY1,
CRY2, and PHOT1 (with the exception of PHOT2, not yet
analyzed) in the differentiation of sun and shade leaves is
unlikely. Since there still remains a possibility that blue-light
receptors (including unknown receptors) relate to sun and
shade leaf development, further studies are required.
The redox state of the quinone pool might be a sensor for
leaf development, since it can control expression levels of
some genes (Anderson et al. 1995, Escoubas et al. 1995, Huner
et al. 1998). The photosynthates may also act as light signals in
plants (Ono et al. 2001). However, it should be noted that these
photosensory signal(s) should be transferred from the mature
leaves to the developing apical leaves. This makes photosynthates the most likely candidates as light signals from mature
leaves to the developing leaves (in the palisade tissue) differentiation, although other signals cannot be excluded.
Besides the anatomical differences, sun and shade leaves
differ in their chloroplast ultrastructure. Sun-type chloroplasts
have more non-appressed thylakoids and shade-type chloroplasts have more appressed thylakoids (Anderson 1986). In
Can developing leaves sense the light environment?
order to test whether the signal(s) from mature leaves to developing leaves also regulate sun- or shade-type chloroplast
ultrastructure, we examined chloroplast electron micrographs
for the relative abundance of appressed and non-appressed
regions of thylakoid membrane.
Chloroplast ultrastructure
Electron micrographs of chloroplasts from young leaves
about 20 mm in lamina length treated under different light conditions for 6 d are shown in Fig. 6. Chloroplasts from HH
leaves were sun-type with less appressed thylakoids or more
stroma lamellae (Fig. 6a). Chloroplasts in HL leaves were
shade-type with well-appressed thylakoids or grana stackings
(Fig. 6b). Similar to that of HH leaves, the chloroplasts of HA
leaves had more non-appressed thylakoids (Fig. 6c). On the
other hand, the chloroplasts of LA leaves were shade-type with
more appressed thylakoids (Fig. 6d). These results clearly indicate that the local light environment of the developing leaf
plays a determinant role in the differentiation of chloroplasts to
sun- and shade-types. The present study, thus, also provides
clear-cut evidence that sun- or shade-type chloroplast development is fairly independent of the anatomical differentiation of
the tissue in the developing leaves. The local light environment of the developing leaf plays a determinant role in the
differentiation of chloroplasts to sun- and shade-types.
However, under the extreme experimental conditions
these processes may not be completely independent, because it
has been shown that chloroplast development is a prerequisite
for the development of palisade tissue cells (Chatterjee et al.
1996, Keddie et al. 1996). In these studies, through examination of roles of DAG (Antirrhinum majus) and DCL (tomato)
genes, which act at an early step of chloroplast development,
they showed that elongation and division of the palisade tissue
cells require cell-specific chloroplast development. Probably
because the present study was conducted with the wild plants,
and even the low irradiance level used was sufficient for
chloroplast development, we did not observe this effect. When
the shoot apices were placed in the extreme shade or in the
complete darkness, the effect of plastid development on the
development of palisade tissue cells could be observed. Thus,
the present conclusions may not apply to such extreme experimental conditions.
Fig. 5 Changes in leaf thickness (a), the palisade tissue thickness (b),
and cross-sectional column area (c) of LA (closed circle), HA (closed
square), HH (open triangle), and HL (open circle) leaves. Plants were
grown for about 1 month in high light condition, and then treated for
6 d (see Materials and Methods). The data of the leaves with lamina
lengths ranging 3–7.9, 8–12.9, 13–17.9, and 18–22.9 mm, respectively,
were pooled. At each point, 1 to 8 leaves were examined for leaf thickness, 32 to 71 cells from 1 to 8 leaves were examined for the palisade
tissue thickness and cross-sectional column area of the palisade tissue.
The vertical and horizontal bars indicate the standard deviations.
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Conclusion
1. The differences in number of cell-layers in the palisade tissue
between HH/LA leaves, and HL/HA leaves clearly demonstrate
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Can developing leaves sense the light environment?
Fig. 6 Electron micrographs of ultra-thin sections of chloroplasts in upper most side of HH (a), HL (b), HA (c), and LA (d) leaves having 20,
17, 19, and 21 mm lamina length, respectively. Arrow-heads indicate well stacked grana. Bar, 200 nm.
Fig. 7 A model of light sensory mechanisms for developing leaves. Anatomical characteristics of developing leaves are regulated by longdistance signal, which bears light environment information sensed in mature leaves. Chloroplast characteristics of developing leaves are regulated by local light environment.
that the light environment of mature leaves and not developing
leaves regulates the sun or shade leaf anatomy in C. album. The
signal for periclinal division of palisade tissue cells less
occurred when the mature leaves were transferred to low light.
2. The cells in the palisade tissue of HH and LA leaves divided
both in periclinal and anticlinal directions, but that in HL and
Can developing leaves sense the light environment?
HA leaves underwent only anticlinal division.
3. The results of leaf thickness, palisade tissue thickness, and
cross-sectional column area of the palisade tissue suggest that
not only the direction of cell division but also the cell and leaf
growth is largely regulated by the light environment of the
mature leaves.
4. Chloroplast development appears to be independent of the
anatomical differentiation of the developing leaves. The local
light environment of the leaf lamina regulates the chloroplast
differentiation to ‘sun-type’ and ‘shade-type’ chloroplasts.
These clearly showed the separate localization of light signal perception for sun- or shade-type chloroplast and palisade
tissue differentiation in C. alubum (diagrammatically shown in
Fig. 7).
Materials and Methods
Plant material and growth conditions
Chenopodium album L. seeds were collected from a native population of the Tokyo area (a gift from Dr. Hisae Nagashima, Botanical
Gardens, The University of Tokyo, Nikko, Japan). Seeds were germinated on moist filter paper in a Petri dish. One germinated seed was
planted in a pot (105 mm diameter, 175 mm height) containing vermiculite. Eight pots were placed in a container (560 mm length,
260 mm width, 180 mm height) filled with the nutrient medium containing 2 mM KNO3, 2 mM Ca(NO3)2, 0.75 mM MgSO4, 0.665 mM
NaHPO4, 25 mM Fe-EDTA, 0.5 mM ZnSO4, 0.5 mM CuSO4, 25 mM
H3BO4, 0.25 mM Na2MoO4, 50 mM NaCl, and 0.1 mM CoSO4. The
nutrient medium was aerated continuously with an air pump. The solution was renewed twice a month. Plants were grown in a phytotron
(KG–50HLA–S, Koito, Yokohama, Japan) with 14 h photoperiod. The
day/night air temperatures were 25/18°C, and the relative humidity
was 60%. Light was supplied by a bank of fluorescent tubes (FPR
96EX-N/A, Matsushita, Kadoma, Japan), and the photosynthetically
active photon flux density (PPFD, 400–700 nm) at the plant level
measured with a quantum sensor (LI-190SB, Li-COR, Lincoln, NE,
U.S.A.) was 360 mmol m–2 s–1. Under this PPFD, plants formed sun
leaves with two-layered palisade tissue (Fig. 1a). The red/far-red
(660 nm/730 nm) ratio of the light, measured with a home-made sensor with two photodiodes (S1227-BR, Hamamatsu Photonics, Hamamatsu, Japan) and two interference filters (maximal transmittance are
660 nm with half band width 7 nm and 730 nm with half band width
9 nm, Vacuum Optics Corporation of Japan, Gotenba, Japan) was 2.5
under this growth light condition. Under these growth conditions, 1month-old plants had leaves showing a similar lamina growth rate and
uniform final lamina length (about 30 mm).
Shading treatment
One-month-old high-light grown plants were shaded (low-light,
L) in various ways with black shade screen, such that in each case
shaded leaves received PPFD at 60 mmol m–2 s–1. The LA treatment
was made by covering the developing leaves in the shoot apex with a
cylindrical shape screen of 20 mm diameter and 20 mm height (Fig. 2).
The HA treatment was given by a cylindrical screen of 110 mm diameter (height adjusted as per the plant size) with a central cylindrical
aperture of 20 mm diameter and 20 mm height (Fig. 2). The light
intensity at the shoot apex was adjusted to 360 mmol m–2 s–1 by an
additional halogen light source (COLDSPOT PICL-NEX, NPI, Tokyo,
Japan) as shown in Fig. 2a. Under this condition, the red/far-red ratio
of the light that the shoot apex received was 4.4. The whole plant was
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grown under the high-light condition throughout the experimental
period (high-light to high-light, HH) or shifted to low light condition
(high-light to low-light, HL). The leaf anatomy and ultrastructure of
chloroplasts was studied after 6 d of the above treatments.
A preliminary study showed that palisade tissue cells started periclinal division when the lamina length was 8 mm and the palisade tissue became two-cell-layered in high-light grown plants. When the leaf
laminas were less than 8 mm, there were no differences in leaf anatomy between high-light grown and low-light grown (grown in PPFD
at 60 mmol m–2 s–1) plants. Therefore the plants having apical leaves
with lamina length less than 8 mm were subjected to the various light
treatments.
Leaf structure and chloroplast ultrastructure
Leaf segments, adjoining the mid rib, excluding major veins
(1´3 mm), were taken from the basal part of the foliage with a razor
blade (29, 21, 14 and 15 leaves of LA, HA, HH and HL, respectively).
The leaves were sampled within 2 h from the start of the light period.
The segments were fixed overnight at 4°C in 2.5% glutaraldehyde in
12.5 mM cacodylate buffer, pH 7.2, and then treated with 2% osmium
tetroxide for 3 h. The fixed segments were dehydrated in a graded acetone series and embedded in Spurr’s resin (Spurr 1969).
Light microscopy (BX-50, Olympus, Tokyo, Japan) was carried
out with 1 mm thick transverse sections of the leaf cut with a glass
knife on an ultramicrotome (Reichert Ultracut S, Leica, Vienna, Austria) and stained with 0.5% toluidine blue. Light micrographs were
taken with a digital camera (C-3030 Zoom, Olympus, Tokyo, Japan).
Transmission electron microscopy of chloroplast ultrastructure
was made with 40 nm ultra-thin sections cut with a diamond knife on
the ultramicrotome (as above) and stained with uranium acetate and
lead citrate double staining. Chloroplasts of the uppermost part of the
leaf sections were viewed under an electron microscope (JEM-1200EX
Electron Microscope, JEOL, Tokyo, Japan).
Leaf anatomy
Leaf thickness, number of cell layers in the palisade tissue, and
total number of cells in the palisade tissue were quantified. Leaf thickness of each leaf was calculated with image analysis software (NIH
Image, written by National Wayne Rasband at the National Institute of
Health and available from NTIS, 5285 Port Royal Road, Springfield,
VA, U.S.A.), from the average thickness measured at 10 different positions of each micrograph. The indices of total number of cells in the
palisade tissue (TNP) and the average number of cell layers of the palisade tissue (PCL) of the leaf were obtained as:
TNP = (Total number of cells in the palisade tissue on a section/section
width) ´ lamina length in mm,
PCL = Total number of cells in the palisade tissue on a section/number
of cell columns on a section.
The image of palisade tissue on the micrograph was traced on a
computer using photo retouch software and height, width of palisade
tissue cells and cross-sectional area of the cell column in the palisade
tissue were obtained with image analysis software. A “column”
denotes vertical cellular pillar in the palisade tissue whether it consists
of one cell or two cells. The cells with faint edges and the cells with
irregular shapes that resulted from irregular cell divisions, such as
three cells in line in a column, or those with ‘Y’ or ‘upside-down Y’shapes, were excluded from the analysis. The palisade tissue thickness
and column area were calculated by averaging the palisade column
height and area within each section. The spongy mesophyll cells were
not analyzed because of their irregular shapes.
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Can developing leaves sense the light environment?
Acknowledgements
We are grateful to Dr. Hisae Nagashima for C. album seeds and
Prof. Amarendra Narayan Misra for editing an early draft. This work
was supported by a grant from the Ministry of Education, Culture,
Sports, Science and Technology of Japan and the Research Fellowships of Japan Society for the Promotion of Science for Young Scientists to S. Y.
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(Received July 18, 2001; Accepted October 15, 2001)