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Plant, Cell and Environment
(2004)
27
, 781–793
© 2004 Blackwell Publishing Ltd
781
Blackwell Science, LtdOxford, UKPCEPlant, Cell and Environment0016-8025Blackwell Science Ltd 2004? 2004
276781793Original Article
Development of sun and shade leaves
S. Yano & I. Terashima
Correspondence: Satoshi Yano. Fax: + 81 564 55 7512; e-mail:[email protected]
*
Present address: Centre for Integrative Bioscience, Institute forBasic Biology, Myodaiji-cho, Okazaki, 444-8585, Japan.
Developmental process of sun and shade leaves in
Chenopodium album
L.
S. YANO* & I. TERASHIMA
Department of Biology, Graduate School of Science, Osaka University, Machikaneyama-cho 1–16, Toyonaka, Osaka 560–0043, Japan
ABSTRACT
The authors’ previous study of
Chenopodium album
L.revealed that the light signal for anatomical differentiationof sun and shade leaves is sensed by mature leaves, not bydeveloping leaves. They suggested that the two-cell-layeredpalisade tissue of the sun leaves would be formed withouta change in the total palisade tissue cell number. To verifythat suggestion, a detailed study was made of the develop-mental processes of the sun and shade leaves of
C. album
with respect to the division of palisade tissue cells (PCs)and the data was expressed against developmental time(leaf plastochron index, LPI). The total number of PCs perleaf did not differ between the sun and shade leavesthroughout leaf development (from LPI
----
1 to 10). In bothsun and shade leaves, anticlinal cell division of PCsoccurred most frequently from LPI
----
1 to 2. In sun leaves,periclinal division of PCs occurred synchronously with anti-clinal division. The constancy of the total number of PCsindicates that periclinal divisions occur at the expense ofanticlinal divisions. These results support the above sugges-tion that two-cell-layered palisade tissue is formed by achange of cell division direction without a change in thetotal number of PCs. PCs would be able to recognize thepolarity or axis that is perpendicular to the leaf plane andthereby change the direction of their cell divisions inresponse to the light signal from mature leaves.
Key-words
:
Chenopodium album
L.; cell division; cellpolarity; light; leaf development; leaf plastochron index;sun and shade leaves.
INTRODUCTION
Sun and shade leaves are formed in high and low lightenvironments, respectively. It has long been known that sunleaves show higher rates of photosynthesis and dark respi-ration per unit leaf area than shade leaves (Boysen-Jensen1932). Detailed comparative studies have attributed suchdifferences to differences in the amounts of components
such as ribulose bisphosphate carboxylase/oxygenase, cyto-chromes, photosystem I and II core complexes, and respi-ratory enzymes, all expressed per unit leaf area (Björkman1981; Anderson 1986; Anderson & Osmond 1987; Terash-ima & Hikosaka 1995; Noguchi, Sonoike & Terashima1996). Furthermore, sun leaves have thicker laminae,thicker palisade tissue, and a larger cumulative mesophyllsurface area per unit leaf area than shade leaves (Haber-landt 1914; Esau 1965; Björkman 1981). The greater meso-phyll surface area per unit leaf area in sun leaves facilitatesCO
2
dissolution into cell wall water and thereby decreasesresistance to CO
2
diffusion from the intercellular spaces tothe chloroplast stroma (Nobel 1977; Evans & Loreto 2000;Terashima, Miyazawa & Hanba 2001). All these studieshave revealed that sun leaves are advantageous in highlight, whereas shade leaves perform better in the shade.Thus, the differentiation of sun and shade leaves is of eco-logical importance. However, differences in developmentalprocesses between sun and shade leaves have not beenstudied.
Avery (1933) reported development of
Nicotianatabacum
leaves. In particular, early developmental eventssuch as protrusion of leaf primordia and their apical andmarginal growth were closely described. Maksymowych(1973) described leaf development of
Xanthium pennsyl-vanicum
with special reference to leaf expansion processes.However, in these classical studies, the authors did not payattention to the plasticity of leaf development. If there wereno plasticity in leaf development, the leaf would not be ableto acclimatize to its light environment. Thus, it is importantto clarify the plastic processes that are altered in responseto light signals. Sims & Pearcy (1992) reported develop-ment of
Alocasia macrorrhiza
leaves transferred from highto low light. They found that
A. macrorrhiza
leaves accli-matize to the new light environment and that young leavesdo it better than older leaves. However, the developmentalprocesses
per se
were not analysed in their study.We previously reported the effects of the light environ-
ment of mature leaves on the development of youngleaves in
Chenopodium album
L. plants (Yano & Terash-ima 2001). We grew plants in high light and then appliedthe following four treatments for 6 d, and examined theanatomy of the leaves that had been developing duringthe treatments. The treatments included: (1) low-lightapex treatment (young leaves were shaded but mature
782
S. Yano & I. Terashima
© 2004 Blackwell Publishing Ltd,
Plant, Cell and Environment,
27,
781–793
leaves were exposed to high light); (2) high-light apextreatment (young leaves were exposed but mature leaveswere shaded); (3) high–high light treatment (whole plantswere exposed to high light); and (4) high–low light treat-ment (whole plants were shaded). The young leaves ofthe plants whose mature leaves were exposed to stronglight (treatments 1 and 3) developed two-cell-layeredthick palisade tissue (sun-type leaves), but those of plantswhose mature leaves were shaded (treatments 2 and 4)developed one-cell-layered thin palisade tissue (shade-type leaves). The results indicated that the light signal issensed by mature leaves. This light information would betransferred to developing young leaves and determinetheir developmental fate.
Another interesting result of our previous paper was thatthe total number of palisade tissue cells (PCs) per leaf(
N
total
) appeared to be unchanged by these four treatments.Dale (1965) estimated the cell number of
Phaseolus vul-garis
leaves grown under various light conditions. Hisresults indicated that there were no significant differencesin total cell number of the leaf. On the other hand, Newton(1963) reported that the cell number changed with the lightenvironment in
Cucumis sativus
. Thus, the question ofwhether the cell number per leaf is constant irrespective ofthe light environment is not yet answered.
If
N
total
is constant, then the increase in the number ofcell layers in the palisade tissue can be attributed to changesin the direction of cell divisions rather than to additionalpericlinal cell divisions. In addition, this direction of celldivision may be regulated by information from the matureleaves. Moreover, to change the direction of cell division,the cells need to sense their polarities. However, we knowalmost nothing concerning the regulation of the directionof mesophyll cell division.
Our aim here was to describe quantitatively the develop-mental processes of sun and shade leaves and to clarifywhether two-cell-layered palisade tissue is formed withouta change in total number of PCs. In the previous study, weexamined the effects of the various shading treatments onthe development of palisade tissue and PCs of high-light-grown
C. album
plants. Thus, the description was imperfect
from the viewpoint of comparative development of sun andshade leaves. Moreover,
N
total
was estimated only fromtransverse sections. In the present study, we used leavesfrom plants grown under high- and low-light conditions,and analysed paradermal sections in addition to transversesections for better resolution. We used the leaf plastochronindex (LPI, Erickson & Michelini 1957) instead of chrono-logical time, because there were marked differences inexpansion rate and period, and final lamina size betweenthe sun and shade leaves.
MATERIALS AND METHODS
Plant materials and growth conditions
Seeds of
Chenopodium album
L. were germinated onmoist filter paper in a Petri dish. The germinated seedswere planted in pots (105 mm diameter, 175 mm depth;one plant per pot) containing vermiculite. Four potswere placed in a container (340 mm length
¥
195 mmwidth
¥
155 mm depth) filled with half-strength Hoag-land’s nutrient solution (6 m
M
NO
3–
). The nutrient solu-tion level was kept below about 50 mm beneath thevermiculite surface. The nutrient solution was aeratedcontinuously with an air pump and was renewed every 2weeks. The plants were grown in a phytotron (KG-50;Koito, Yokohama, Japan) with a 14 h photoperiod at 60%relative humidity. The air temperature was 25
∞
C duringthe day and 18
∞
C at night. Light was supplied by a bankof fluorescent tubes (FPR 96EX-N/A; Matsushita,Kadoma, Japan). Irradiance was measured with an LI-190Quantum Sensor (Li-Cor Inc., Lincoln, NE, USA) at theplant level; the values were 350 (sun) and 50(shade)
m
mol quanta m
-
2
s
-
1
PPFD (photosyntheticallyactive photon flux density).
On each plant, lamina lengths of all the leaves that wereyounger than the eighth leaf (counted from the base) weremeasured every other day to calculate leaf plastochronindex (LPI, Erickson & Michelini 1957). The referencelength was defined as 10 mm, since lamina growth curveswere mostly parallel with each other at this length (Fig. 1).
Figure 1.
Changes in lamina length of sun (a) and shade plants (b) plotted against actual time (days after measure-ment). Each line indicates the expansion of one leaf. For details, see Materials and methods.
Development of sun and shade leaves
783
© 2004 Blackwell Publishing Ltd,
Plant, Cell and Environment,
27,
781–793
The LPI for a given leaf, whose serial number is
a
, isexpressed as
where
n
is the serial number of the leaf that is just longerthan 10 mm (the reference length), and
L
n
is the laminalength of the
n
th leaf. When the lamina length is below10 mm, the LPI value is negative.
Fixation, sectioning, and microscopy analyses
We sampled 34 leaves from three 1.5-month-old-sun plantsand 36 leaves from three 2.5-month-old shade plants. Leafsegments (1 mm
¥
2 mm) were taken with razor bladesfrom these leaves. For structural uniformity, segments with-out major veins were taken from near the midrib. The seg-ments were fixed in 2.5% glutaraldehyde in 12.5 m
M
cacodylate buffer (pH 7.2) overnight at 4
∞
C and then in 2%osmium tetroxide for 3 h. After fixation, they were dehy-drated in an acetone series and embedded in Spurr’s resin(Spurr 1969).
Transverse and paradermal sections cut 1
m
m thick withglass knives on an ultramicrotome (Reichert Ultracut S;Leica, Vienna, Austria) and stained with 0.5% toluidineblue were viewed under a light microscope (BX-50; Olym-pus, Tokyo, Japan). Light micrographs (2048
¥
1536 pixels)were taken with a digital camera (C-3030 Zoom; Olympus,Tokyo, Japan).
Anatomical analyses
Leaf thickness and palisade tissue thickness were measuredat 10 positions on every transverse section (about 300
m
min width). Esau (1977) wrote: ‘The palisade parenchymaconsists of cells elongated perpendicular to the surface ofthe blade’. This somewhat subjective definition has causedconfusion. Instead, we defined the palisade tissue as thetissue that lies above cell layers containing vascular bundlesand consists of cylindrical mesophyll cells.
The height, width, and cross-sectional area (
C
tr
) of thePCs were measured on 19–114 cells in one transverse sec-tion. The maximum and minimum cell diameters and cross-sectional area (
C
pd
) of PCs were measured on about 200cells in one paradermal section. Thicknesses of the adaxialand abaxial epidermes were separately estimated: Thethickness of the epidermis was estimated as the cross-sec-tional area of the epidermis divided by the length of theepidermis in each transverse section. The number of celllayers in the palisade tissue (
N
layer
) was estimated for eachtransverse section (for details, see Yano & Terashima 2001).For the paradermal sections, PC density per unit area of thesection (
D
pd
) was calculated. All these parameters werequantified with image analysis software (NIH Image, publicdomain software, developed at US National Institutes ofHealth, available at http://rsb.info.nih.gov/nih-image/) aftertracing of the micrograph images. Because of their irregular
LPI = - +-
- +n a
LL L
n
n n
log loglog log
10
1
shapes, spongy tissue cells were not quantified. The spongytissue thickness was estimated as lamina thickness minusthe sum of the epidermal and palisade tissue thicknesses.
The density of PCs per unit leaf area (
D
leaf
), estimatedleaf area (
S
(
l
)), and total number of PCs per leaf (
N
total
)were calculated as follows from the above data:
D
leaf
=
D
pd
¥
N
layer
, (1)
(2)
N
total
=
S
(
l
)
¥
D
leaf
. (3)
The
l
in Eqns 2 and 3 is the LPI value. In Eqn 2, we useda leaf proportional function (
P
(
l
)). We fitted hyperbolicfunctions to the relationship between the ratio of leaflength to width and LPI.
P
(
l
) was 55.9/(
l
+ 26.6) [
R
2
= 0.879]for sun leaves and 79.4/(
l
+ 36.3) [
R
2
= 0.761] for shadeleaves.
Cell division rate
As mentioned above, we obtained
N
layer
and
N
total
for theleaves at various developmental stages. The cell divisionrate per unit LPI can be obtained as follows. First,
N
total
and
N
layer
plotted against LPI were fitted with sigmoidal func-tions,
T
(
l
) and
L
(
l
), respectively. Because the changes in
T(l
) and
L
(
l
) are attributed to cell divisions, these functionsare written as:
T
(
l
) =
a
· 2
D
(
l
)
, (4)
L
(
l
) = 1 · 2
P
(
l
)
, (5)
From these we also obtain
(6)
We analysed cell divisions from LPI =
-
1. The parameter
a
in Eqns 4 and 6 is the
N
total
value at LPI =
-
1. Factor 1 inEqn 5 is the number of cell layers at LPI =
-
1 (see Fig. 2a& b).
D
(
l
),
P
(
l
), and
A
(
l
) are total, periclinal, and anticlinalcell division frequencies, respectively, and express howmany times one PC is divided in total, periclinally, andanticlinally during the period from LPI =
-
1 to
l
. By defini-tion,
D
(
-
1) =
P(-1) = A(-1) = 0. The total, periclinal, andanticlinal cell division rates (in times per LPI), RD(l), RP(l),and RA(l), respectively, are given by differential equations:
(7)
(8)
(9)
RESULTS
Transverse and paradermal sections of the sun and shadeleaves are shown in Figs 2 and 3. At negative LPI (Figs 2a
S lP l
( )( )
,= ¥¥
¥lamina length
2lamina length
2p
T lL l
A l( )( )
.= ◊ ( )a 2
RD l
l lT l
D l( ) =( )
= ◊( )Ê
ˈ¯
dd
dd
12log
log ,a
RP l
l lL lP l( ) =
( )= ◊ ( )Ê
ˈ¯
dd
dd
12log
log ,
RA l
l lT l
L lA l( ) =
( )= ◊
( )( ) ◊
ÊËÁ
ˆ¯̃
dd
dd
12log
log .a
784 S. Yano & I. Terashima
© 2004 Blackwell Publishing Ltd, Plant, Cell and Environment, 27, 781–793
& b, 3a & b), there were no detectable differences betweenthe sun and shade leaves. As LPI increased, the sun leaveshad denser palisade tissues than the shade leaves. Severalidioblasts were identified in the paradermal sections. Inter-cellular spaces in the sun and shade leaves after full laminaexpansion (FLE) decreased to some extent (Fig. 3g–j).
Leaf growth
Changes in lamina length with LPI are shown in Fig. 4a.Both sun and shade leaves ceased expansion by LPI = 7 or8, and the final lengths were about 60 and 72 mm, respec-tively. Maximum lamina elongation rates were 8 and14 mm LPI-1, respectively. These values correspond to 6.4and 4 mm d-1, respectively. Changes in leaf thickness areshown in Fig. 4b. The sun leaves were thicker (300 mm) thanthe shade leaves (250 mm). The maximum thickening rateswere 25 and 20 mm LPI-1, respectively. Leaf thickness con-tinued to increase after FLE in both types of leaf.
At the same LPI, the difference in leaf thickness betweenthe sun and shade leaves was not very large (Fig. 4b). How-ever, in Fig. 4c, in which leaf thickness is plotted againstlamina length, the difference is marked. For example, thedifferences at lamina lengths of 30 and 50 mm were 30and 80 mm, respectively. On the other hand, the differencebecame ambiguous when the lamina length of the sunleaves reached the maximum (60 mm). This resulted froma considerable increase in leaf thickness after FLE.
Changes in thickness of the adaxial and abaxial epider-mes are shown in Fig. 5. The thickness of both somewhatdecreased between LPI -1 and 2, and then increased. The
Figure 2. Transverse sections of the sun (a, c, e, and g) and shade leaves (b, d, f, and h). The LPI values are also shown. The lamina lengths are 6.9, 7.5, 29.2, 46.8, 50.0, 70.1, 59.6, and 76.8 mm (alpha-betical order). Bar = 100 mm.
Figure 3. Paradermal sections of the sun (a, c, e, g, and i) and shade leaves (b, d, f, h, and j). The LPI values are also shown. The lamina lengths are 8.8, 7.5, 19.6, 20.2, 34.9, 46.8, 50.0, 70.1, 59.6, and 76.8 mm (alphabetical order). Asterisks indicate idioblasts. Bar = 100 mm.
Development of sun and shade leaves 785
© 2004 Blackwell Publishing Ltd, Plant, Cell and Environment, 27, 781–793
adaxial epidermis attained a final thickness of 16 mm inboth sun and shade leaves. The adaxial epidermis of theshade leaves was always thicker than that of the sunleaves, although the differences were not statistically sig-nificant. For the abaxial epidermis, the difference was sig-nificant around LPI 7, but not significant for otherperiods. Both epidermes continued to increase theirthickness after FLE.
Figure 4. Lamina length (a) and thickness (b) plotted against LPI, and thickness plotted against lamina length (c). The data for the sun and shade leaves are indicated as open circles (solid line) and squares (dotted line), respectively. Bar = SD.
Figure 5. Thicknesses of adaxial (a) and abaxial (b) epidermes plotted against LPI. The data for the sun and shade leaves are indicated as open circles (solid line) and squares (dotted line), respectively.
�
786 S. Yano & I. Terashima
© 2004 Blackwell Publishing Ltd, Plant, Cell and Environment, 27, 781–793
Development of palisade tissue cells
Changes in height and width of the PCs measured in trans-verse sections are shown in Fig. 6. The average height ofPCs did not differ between sun and shade leaves at any LPI,and increased exponentially to 70 mm by LPI 10 (Fig. 6a).The height continued to increase after FLE, in a similarmanner to the leaf thickness (Fig. 4b). The width of PCsdecreased between LPI -1 and 1, and then increased inboth sun and shade leaves (Fig. 6b). The increment in thePC width in the sun leaves was less than that in the shadeleaves, and the PC width reached 20 and 24 mm in the sunand shade leaves, respectively. The PC width also continuedto increase after FLE in both types of leaf.
The diameter of PCs measured in the paradermal sec-tions (Fig. 7) was almost identical to the width of PCs mea-
Figure 6. Height (a) and width (b) of PCs measured in the trans-verse sections plotted against LPI. The data for the sun and shade leaves are indicated as open circles (solid line) and squares (dotted line), respectively. Bar = SD
Figure 7. Maximum (a) and minimum (b) diameters of PCs mea-sured in the paradermal sections plotted against LPI. Maximum diameter re-plotted against lamina length (c). The data for the sun and shade leaves are indicated as open circles (solid line) and squares (dotted line), respectively. Bar = SD.
Development of sun and shade leaves 787
© 2004 Blackwell Publishing Ltd, Plant, Cell and Environment, 27, 781–793
sured in the transverse sections (Fig. 6). Interestingly, thedifference between the maximum and minimum diametersremained unchanged, being 2–3 mm at any LPI. Thus, thedifference relative to the diameter decreased with LPI. Thisis due to changes in PC shape with time: the paradermalsectional view of PCs changed from square or polygonal inthe early periods (Fig. 3a–d) to circular in later periods(Fig. 3e–j). The maximum diameter of the PCs was plottedagainst lamina length (Fig. 7c). Being similar to the patternof lamina thickness (Fig. 4c), the diameter also increasedand the data points spread vertically after FLE. When plot-ted against lamina length, the difference of the diameterbetween the sun and shade leaves became ambiguous.
The cross-sectional areas of PCs measured in the trans-verse sections (Ctr) and in the paradermal sections (Cpd) areshown in Fig. 8. Ctr was 100 mm2 at LPI -1 in both the sunand shade leaves (Fig. 8a). The value of Ctr increased to1000 mm2 in the sun leaves and to 1300 mm2 in the shadeleaves at LPI 10. At LPI -1 Cpd was about 60 mm2 in boththe sun and shade leaves (Fig. 8b). The value of Cpd
decreased from LPI -1 to 1 in both types of leaf (about35 mm2), as was observed for the cell width and diameters(Figs 6b & 7). After the decrease, Cpd increased to 270 and500 mm2 at LPI 10 in the sun and shade leaves, respectively.The Cpd of the shade leaves was greater than that of the sunleaves from LPI 4. Both Ctr and Cpd also continued toincrease after FLE.
From the data of Cpd (Fig. 8b) and cell height (Fig. 6a),the volume of PCs was calculated (Fig. 8c). At LPI -1, thevolumes in both types of leaf were 800 mm3. The cell volumeexponentially increased with age to 1.7 ¥ 104 and3.3 ¥ 104 mm3 in the sun and shade leaves, respectively.
Development of palisade and spongy tissues
Changes in the palisade tissue thickness are shown inFig. 9a. The thickness in both types of leaf was similar fromLPI -1 to 2, but from LPI 3 the thickness in the sun leavesincreased more than that in the shade leaves. At the end ofthe observation, the palisade tissue thicknesses reached 120and 90 mm in the sun and shade leaves, respectively.Changes in the number of cell layers in the palisade tissue(Nlayer) are shown in Fig. 9b. The value of Nlayer of the sunleaves increased rapidly in the early period to 1.7 and Nlayer
also increased later to some extent in the shade leaves. TheNlayer for one cell lineage in the vertical direction variedfrom 1 to 3, even in adjacent cells. The average PC heightsof the sun and shade leaves were very similar at all LPIs(Fig. 6a). Thus, the difference in the palisade tissue thick-ness was caused by a change in cell layer number.
The thicknesses of the palisade and spongy tissues andof the epidermes are plotted against lamina thickness(Fig. 10a, b & c, respectively). The palisade tissue thicknessincreased linearly as the lamina thickness increased. Theregression lines (y = -22.5 + 0.499x [R2 = 0.976] for sunleaves; y = -17.3 + 0.425x [R2 = 0.982] for shade leaves)were statistically different [analysis of covariance(ANCOVA), P = 1.60 ¥ 10-4]. The spongy tissue thickness
Figure 8. Cross-sectional area of PCs for the transverse (a, Ctr) and paradermal sections (b, Cpd) and calculated PC volume (c) plotted against LPI. The data for the sun and shade leaves are indicated as open circles (solid line) and squares (dotted line), respectively. Bar = SD.
788 S. Yano & I. Terashima
© 2004 Blackwell Publishing Ltd, Plant, Cell and Environment, 27, 781–793
also increased linearly, but the slopes and y-intercepts werenot statistically different (ANCOVA, P = 0.0853 andP = 0.743, respectively). When leaves of the same laminathickness were compared therefore the sun leaves hadthicker palisade tissue than the shade leaves, whereas thespongy tissue thicknesses were very similar. The sums of theadaxial and abaxial epidermes were statistically differentbetween the sun and shade leaves (ANCOVA, P = 7.86 ¥10-9; Fig. 10c). The regression lines were y = 12.4 + 0.0654x(R2 = 0.943) for the sun leaves and y = 11.7 + 0.107x(R2 = 0.910) for the shade leaves. Thus, the difference in thepalisade tissue thickness was compensated for by the dif-ference in epidermal thicknesses.
PC density in the paradermal sections (Dpd) is shown inFig. 11a. At LPI -1, Dpd was almost the same in the sun and
Figure 10. Thickness of palisade tissue (a), spongy tissue (b), and epidermes (c) plotted against lamina thickness. The sun and shade leaves are indicated as open circles (line) and squares (dotted line), respectively. Bar = SD.
Figure 9. Changes of palisade tissue thickness (a) and Nlayer (b) plotted against LPI. The data for the sun and shade leaves are indicated as open circles (solid line) and squares (dotted line), respectively. Bar = SD.
Development of sun and shade leaves 789
© 2004 Blackwell Publishing Ltd, Plant, Cell and Environment, 27, 781–793
shade leaves (1.7 ¥ 104). The value of Dpd increased as thePC diameter decreased (that is, as the palisade cell divisionincreased) until LPI 1 to 2 and then Dpd decreased graduallywith the expansion of the lamina (LPI < 7 or 8). The Dpd ofthe sun leaves was greater than that of the shade leaves. PCdensities per unit leaf area (Dleaf) were calculated as theproduct of Dpd and Nlayer (Fig. 11b). The Dleaf of the sunleaves was always greater than that of the shade leavesexcept at LPI < 0. The total number of PCs per leaf (Ntotal)was calculated from Dleaf and the lamina length and width(see Materials and methods). The value of Ntotal of bothtypes of leaf increased exponentially from LPI -1 to 2 andstabilized at about 1.5 ¥ 107. It is noteworthy that Ntotal ofthe sun and shade leaves did not differ markedly at any LPI.
Cell division rates
The total (RD(l)), periclinal (RP(l)), and anticlinal (RA(l)) celldivision rates were calculated. Both L(l) and T(l) weresigmoidal functions of Nlayer (Fig. 9b) and Ntotal (Fig. 11c),respectively (Table 1). These functions were substitutedinto Eqns 4–6, and total (D(l)), periclinal (P(l)), and anti-clinal (A(l)) cell division frequencies were obtained(Table 1). These three functions were differentiated withrespect to LPI (Eqns 7–9, see Materials and methods) andplotted against LPI.
Changes in the anticlinal cell division rate (RA, LPI-1) areshown in Fig. 12a. The maximum RA values were 1.4 LPI-1
in the sun leaves and 3.1 LPI-1 in the shade leaves. Althoughit seemed that RA of the sun leaves was smaller than thatof the shade leaves, integrated values of RA, which say howmany times PCs divided anticlinally from LPI = -1 to 12,were not very different (4.0 and 4.6 in the sun and shadeleaves, respectively). The periods in which the anticlinal celldivisions occurred most frequently almost agreed in the sunand shade leaves.
The periclinal cell division rates (RP) are shown inFig. 12c. The RP values of the sun leaves were greater thanthose of the shade leaves for LPI -1 to 3. Interestingly, themaximum cell division rate of the shade leaves occurredmuch later than that of the sun leaves. The maximumRP and integrated value of RP for the sun leaves were 0.35LPI-1 at LPI = 1.7 and 0.7, and those for the shade leaveswere 0.09 LPI-1 at LPI = 3.9 and 0.4, respectively. The max-imum total cell division rate (RD) of the sun leaves was1.6 LPI-1 at LPI = 0.9 and that of the shade leaves was3.0 LPI-1 at LPI = 1.0 (Fig. 12b). The integrated values ofRP were 4.8 and 5.0 for the sun and shade leaves, respec-tively. The small differences between RD and RA + RP wereerrors due to the curve fitting.
DISCUSSION
Developmental processes
Early event, LPI = -1 to 1
In this period, PCs started to divide vigorously (Fig. 12), andPC diameter and width (Figs 7 & 6b, respectively)
Figure 11. Dpd (a), Dleaf (b), and Ntotal (c) plotted against LPI. The data for the sun and shade leaves are indicated as open circles (solid line) and squares (dotted line), respectively.
790 S. Yano & I. Terashima
© 2004 Blackwell Publishing Ltd, Plant, Cell and Environment, 27, 781–793
decreased. Daughter cells tended to divide before theyexpanded to the same size as their mother cells during thisperiod. This temporal decrease in PC diameter was alsoreported for Xanthium pennsylvanicum (Maksymowych1973). The PC density per unit area in the paradermal sec-tions (Dpd, Fig. 11a) and per unit leaf area, taking account ofthe number of cell layers (Dleaf, Fig. 11b), increased, since thelaminar expansion was slower than the vigorous PC produc-tion. In addition to the decrease in PC diameter, PCs elon-gated vertically (Fig. 6a). Thus, depression was observed inneither Ctr nor PC volume (Fig. 8a & c, respectively).
In this period, developmental processes of the sun andshade leaves did not differ, except for the periclinal divisionrate of PCs, which started to increase in the sun leaves(Fig. 12c).
Expansion period, LPI = 1 to FLE
Remarkable PC expansion and elongation occurred in thisperiod (Figs 6 & 7), while the cell division gradually dimin-ished and ceased by LPI = 4 (Fig. 12). A significant increasein PC diameter was observed after LPI 4 (Fig. 7). The cross-sectional area of PCs measured in the transverse (Ctr) andparadermal (Cpd) sections and the PC volume also rapidlyincreased after LPI 4 (Fig. 8). The values of Dpd and Dleaf
increased early in this period but decreased after LPI 2(Fig. 11).
Differences between the sun and shade leaves were sig-nificant in this period. The most significant differences werefound in the number of cell layers in the palisade tissue(Nlayer, Fig. 9b) and in PC expansion growth (Figs 6b & 7).The Nlayer of the sun leaves increased around LPI 2 andbecame stable by LPI 4. On the other hand, Nlayer in theshade leaves started to increase at around LPI 3, and thegradual increase continued until FLE. In the shade leaves,PC diameter markedly increased after LPI 4. Thus, the dif-ferences in PC volume and Cpd became marked betweenthe sun and shade leaves (Fig. 8c & b, respectively).
After FLE (LPI > 7–8)
The lamina expansion ceased in this period (Fig. 4a). How-ever, elongation and expansion of PCs (Figs 6, 7 & 8) andthickening of the palisade tissue (Fig. 9) and the lamina(Fig. 4b) continued, although the increases in this periodwere much less than those in the expansion period. Theshade leaves showed greater PC expansion growth than thesun leaves in this period as well (Fig. 7).
Cell divisions and cell axis
Our results show that the anticlinal cell division occurredin almost the same LPI period in sun and shade leaves(Fig. 12a), but that the periclinal division occurred in dif-ferent periods (Fig. 12c). In the sun leaves, the periclinaldivision occurred at almost the same time as the anticlinaldivision. However, in the shade leaves, the periclinal divi-sion occurred much later than the anticlinal division. Thisdifference between the anticlinal and periclinal divisionperiods in the shade leaves suggests that these divisions arecontrolled independently.
The two-cell-layered palisade tissue is formed as a resultof periclinal cell divisions. If periclinal divisions occurred inaddition to anticlinal divisions, the total numbers of PCs(Ntotal) should be greater in the sun leaves than in the shadeleaves. However, the Ntotal values of the sun and shadeleaves were almost identical (Fig. 11c). Moreover, asmentioned above, the periclinal and anticlinal divisionsoccurred simultaneously in the sun leaves. These resultsindicate that, in the sun leaves, periclinal division takesplace at the expense of anticlinal division, and that thisdirectional change of cell division makes two-cell-layeredpalisade tissue.
We have reported that mature leaves, but not developingleaves, sense the light signal for differentiation of sun andshade leaves (Yano & Terashima 2001). The light informa-tion is transferred to the developing leaves by some signal
Table 1. List of mathematical functions fitted to the data in Fig. 11c; T(l) and Fig. 9b, (L(l)). a is the value of T(-1). D(l), P(l), and A(l) were calculated for the sun and shade leaves
Sun leaf R2 Shade leaf R2
T(l) 0.795 0.645
L(l) 0.765 0.682
a 4.90 ¥ 105 4.80 ¥ 105
D(l) 0.999 0.999
P(l) 0.999 0.998
A(l) 0.999 0.995
See Materials and methods for details.
3 17 101 31 10
1 3 15 1 455
7
..
exp . .¥ + ¥
+ - ¥( )l4 62 10
1 51 101 4 52 2 78
57
..
exp . .¥ +
¥+ - ¥( )l
0 9720 708
1 3 04 1 68.
.exp . .
++ - ¥( )l
0 9760 402
1 2 79 0 702.
.exp . .
++ - ¥( )l
- ++ - ¥( )
5 825 36
1 1 08 1 20.
.exp . . l
- ++ - ¥( )
0 08655 11
1 2 28 2 31.
.exp . . l
0 7471 3 23 1 86
.exp . .+ - ¥( )l
0 4571 3 22 0 814
.exp . .+ - ¥( )l
0 02704 62
1 2 55 2 72.
.exp . .
++ - ¥( )l
- ++ - ¥( )
0 6964 72
1 0 781 1 14.
.exp . . l
Development of sun and shade leaves 791
© 2004 Blackwell Publishing Ltd, Plant, Cell and Environment, 27, 781–793
transduction system, and the fate of developing leaves isthus determined. The present results indicate that the signalcontrols the direction of cell division. They also indicatethat mesophyll cells, or at least palisade tissue cells, recog-nize the axis perpendicular to the leaf plane.
Axis recognition in mesophyll cells has been studied inArabidopsis thaliana. The ANGUSTIFOLIA (AN) andROTUNDIFOLIA3 genes control cell enlargement in theleaf-width and leaf-length directions, respectively (for areview, see Tsukaya 2002). Leaves of an angustifolia (an)mutant were narrower and thicker than those of wild-typeplants, although lengths were identical (Tsuge, Tsukaya &Uchiyama 1996). Interestingly, the number of cell layers inthe an mutant was greater than that of wild-type plants. Asthe number of cell layers in the sun leaves of C. album isalso greater than that in the shade leaves, there may besome relationship between AN and the formation of sunleaves. Alternatively, as the sun leaves are not narrowerthan the shade leaves, there may be genes controlling thedirection of cell division other than AN. Further studies arerequired.
Light effects on PC anatomy
There is little quantitative information about the effects oflight environment on PC size. Björkman (1981) found noobvious differences in cell width on the basis of a cursoryexamination of published micrographs or camera lucidadrawings of leaf sections from sun and shade leaves ofseveral species. There are also observations that cell widthis smaller in shade leaves than in sun leaves (Wilson &Cooper 1969; Ballantine & Forde 1970). In the presentstudy, the PC diameters of the shade leaves were greaterthan those of the sun leaves when the data were plottedagainst LPI. When the data were plotted against laminalength, however, PC diameters were somewhat smaller inthe shade leaves than in the sun leaves while the leaveswere expanding. Thus, comparison of cell size between sunand shade leaves requires careful material sampling.
The sun and shade plants were grown at the same timein the same phytotron. Thus, the air temperature and rela-tive humidity around plants were almost the same for thesun and shade plants. However, owing to the different irra-diance levels, the leaf temperature and thereby vapourpressure deficit were probably greater in the sun leavesthan in the shade leaves. In other words, the sun leaves wereprobably exposed to more xeric conditions than the shadeleaves. In fact, the sun leaves contained more idioblasts,which we identified as water storage cells by their shapeand contents (Fig. 3). The more xeric conditions might alsobe responsible for the smaller PC size (Nobel & Walker1985) and thinner epidermes.
Cell elongation/expansion after full lamina expansion
After FLE, PC height, palisade tissue thickness, epidermalthicknesses, and leaf thickness increased (Figs 6a, 9a, 5 &
Figure 12. Anticlinal (a), total (b), and periclinal cell division rates (c) per LPI plotted against LPI. The data for the sun and shade leaves are indicated as open circles and squares, respectively.
792 S. Yano & I. Terashima
© 2004 Blackwell Publishing Ltd, Plant, Cell and Environment, 27, 781–793
4b, respectively). In other words, leaves that had ceasedexpanding in area were still thickening. These results weredifferent from the results of X. pennsylvanicum (Maksy-mowych 1973), in which thickness growth ceased beforeFLE. Why leaf developmental patterns differ between C.album and X. pennsylvanicum is not clear. In the presentstudy, it was difficult to define maturation of leaves, becausethe edges of leaves that were older than those used in thisstudy turned brownish, and some leaves whose LPI wasmore than 15 withered completely. Cell division and elon-gation and leaf longevity are affected by nitrogen supply(see review, Forde 2002), and the plants used in this studywere grown under a nutrient-rich condition. In such a case,leaves might continue to thicken after FLE.
The PC diameter (Figs 6b & 7) and cross-sectional areameasured in the paradermal sections (Cpd, Fig. 8b) alsoincreased after FLE. Thus, intercellular spaces shoulddecrease. We did not quantify intercellular spaces but thedecrease can be seen (compare Fig. 3g, i, h and j).
Vertical elongation and horizontal expansion growth ofPCs after FLE was reported in Castanopsis sieboldii, Quer-cus glauca, and Phaseolus vulgaris (Miyazawa & Terashima2001; Miyazawa, Makino & Terashima 2003). If such cellelongation and expansion growth after FLE is general, mat-uration of leaves as judged by area growth would bemisleading.
Factors that limit expansion growth of the leaf areunknown (see review, Van Volkenburgh 1999). Elongationof the coleoptiles, stems, and roots is regulated by thegrowth of epidermis (Masuda & Yamamoto 1972; Cosgrove1986; Kutschera, Bergfeld & Schopfer 1987). Wilson &Bruck (1999) peeled the adaxial epidermis off Pisum sati-vum leaves and compared the shapes of the leaves and PCswith those in intact leaves. They did not find detectablechanges in the expansion of the leaflets or the shape of PCs.In the present study, the elongation and expansion of PCsoccurred after FLE, which means that the cessation of epi-dermal expansion does not affect the elongation or expan-sion of PCs, supporting the results of Wilson & Bruck(1999).
CONCLUSIONS
1 Elongation and expansion growth of PCs and leaf thick-ening were observed after FLE. These results indicatethat mesophyll growth was not synchronized with laminaexpansion.
2 Analyses of Ntotal and cell division rates indicate that, inthe sun leaves, periclinal division occurred in the phaseof vigorous cell division to form two-cell-layered palisadetissue. Thus, light signals from mature leaves would con-trol the direction of cell division in this phase.
ACKNOWLEDGMENTS
We thank Dr K. Noguchi for his kind advice, especially onstatistics. This study was financially supported by the Min-istry of Education, Culture, Sports, Science and Technology
of Japan and Research Fellowships of the Japan Society forthe Promotion of Science for Young Scientists to S.Y.
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Received 15 October 2003; received in revised form 15 January 2004;accepted for publication 22 January 2004
