Developmental process of sun and shade leaves in

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1 Blackwell Science, LtdOxford, UKPCEPlant, Cell and Environment Blackwell Science Ltd 2004? Original Article Development of sun and shade leaves S. Yano & I. Terashima Plant, Cell and Environment (2004) 27, 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 , Japan ABSTRACT The authors previous study of Chenopodium album L. revealed that the light signal for anatomical differentiation of sun and shade leaves is sensed by mature leaves, not by developing leaves. They suggested that the two-cell-layered palisade tissue of the sun leaves would be formed without a change in the total palisade tissue cell number. To verify that suggestion, a detailed study was made of the developmental 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 per leaf did not differ between the sun and shade leaves throughout leaf development (from LPI -1 to 10). In both sun and shade leaves, anticlinal cell division of PCs occurred most frequently from LPI -1 to 2. In sun leaves, periclinal division of PCs occurred synchronously with anticlinal division. The constancy of the total number of PCs indicates that periclinal divisions occur at the expense of anticlinal divisions. These results support the above suggestion that two-cell-layered palisade tissue is formed by a change of cell division direction without a change in the total number of PCs. PCs would be able to recognize the polarity or axis that is perpendicular to the leaf plane and thereby change the direction of their cell divisions in response to the light signal from mature leaves. Key-words: Chenopodium album L.; cell division; cell polarity; light; leaf development; leaf plastochron index; sun and shade leaves. INTRODUCTION Sun and shade leaves are formed in high and low light environments, respectively. It has long been known that sun leaves show higher rates of photosynthesis and dark respiration per unit leaf area than shade leaves (Boysen-Jensen 1932). Detailed comparative studies have attributed such differences to differences in the amounts of components Correspondence: Satoshi Yano. Fax: ; syano8@bio.sci.osaka-u.ac.jp *Present address: Centre for Integrative Bioscience, Institute for Basic Biology, Myodaiji-cho, Okazaki, , Japan. such as ribulose bisphosphate carboxylase/oxygenase, cytochromes, photosystem I and II core complexes, and respiratory enzymes, all expressed per unit leaf area (Björkman 1981; Anderson 1986; Anderson & Osmond 1987; Terashima & Hikosaka 1995; Noguchi, Sonoike & Terashima 1996). Furthermore, sun leaves have thicker laminae, thicker palisade tissue, and a larger cumulative mesophyll surface area per unit leaf area than shade leaves (Haberlandt 1914; Esau 1965; Björkman 1981). The greater mesophyll surface area per unit leaf area in sun leaves facilitates CO 2 dissolution into cell wall water and thereby decreases resistance to CO 2 diffusion from the intercellular spaces to the chloroplast stroma (Nobel 1977; Evans & Loreto 2000; Terashima, Miyazawa & Hanba 2001). All these studies have revealed that sun leaves are advantageous in high light, whereas shade leaves perform better in the shade. Thus, the differentiation of sun and shade leaves is of ecological importance. However, differences in developmental processes between sun and shade leaves have not been studied. Avery (1933) reported development of Nicotiana tabacum leaves. In particular, early developmental events such as protrusion of leaf primordia and their apical and marginal growth were closely described. Maksymowych (1973) described leaf development of Xanthium pennsylvanicum with special reference to leaf expansion processes. However, in these classical studies, the authors did not pay attention to the plasticity of leaf development. If there were no plasticity in leaf development, the leaf would not be able to acclimatize to its light environment. Thus, it is important to clarify the plastic processes that are altered in response to light signals. Sims & Pearcy (1992) reported development of Alocasia macrorrhiza leaves transferred from high to low light. They found that A. macrorrhiza leaves acclimatize to the new light environment and that young leaves do it better than older leaves. However, the developmental processes per se were not analysed in their study. We previously reported the effects of the light environment of mature leaves on the development of young leaves in Chenopodium album L. plants (Yano & Terashima 2001). We grew plants in high light and then applied the following four treatments for 6 d, and examined the anatomy of the leaves that had been developing during the treatments. The treatments included: (1) low-light apex treatment (young leaves were shaded but mature 2004 Blackwell Publishing Ltd 781

2 782 S. Yano & I. Terashima leaves were exposed to high light); (2) high-light apex treatment (young leaves were exposed but mature leaves were shaded); (3) high high light treatment (whole plants were exposed to high light); and (4) high low light treatment (whole plants were shaded). The young leaves of the plants whose mature leaves were exposed to strong light (treatments 1 and 3) developed two-cell-layered thick palisade tissue (sun-type leaves), but those of plants whose mature leaves were shaded (treatments 2 and 4) developed one-cell-layered thin palisade tissue (shadetype leaves). The results indicated that the light signal is sensed by mature leaves. This light information would be transferred to developing young leaves and determine their developmental fate. Another interesting result of our previous paper was that the 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 vulgaris leaves grown under various light conditions. His results indicated that there were no significant differences in total cell number of the leaf. On the other hand, Newton (1963) reported that the cell number changed with the light environment in Cucumis sativus. Thus, the question of whether the cell number per leaf is constant irrespective of the light environment is not yet answered. If N total is constant, then the increase in the number of cell layers in the palisade tissue can be attributed to changes in the direction of cell divisions rather than to additional periclinal cell divisions. In addition, this direction of cell division may be regulated by information from the mature leaves. Moreover, to change the direction of cell division, the cells need to sense their polarities. However, we know almost nothing concerning the regulation of the direction of mesophyll cell division. Our aim here was to describe quantitatively the developmental processes of sun and shade leaves and to clarify whether two-cell-layered palisade tissue is formed without a change in total number of PCs. In the previous study, we examined the effects of the various shading treatments on the development of palisade tissue and PCs of high-lightgrown C. album plants. Thus, the description was imperfect from the viewpoint of comparative development of sun and shade leaves. Moreover, N total was estimated only from transverse sections. In the present study, we used leaves from plants grown under high- and low-light conditions, and analysed paradermal sections in addition to transverse sections for better resolution. We used the leaf plastochron index (LPI, Erickson & Michelini 1957) instead of chronological time, because there were marked differences in expansion rate and period, and final lamina size between the sun and shade leaves. MATERIALS AND METHODS Plant materials and growth conditions Seeds of Chenopodium album L. were germinated on moist filter paper in a Petri dish. The germinated seeds were planted in pots (105 mm diameter, 175 mm depth; one plant per pot) containing vermiculite. Four pots were placed in a container (340 mm length 195 mm width 155 mm depth) filled with half-strength Hoagland s nutrient solution (6 mm NO 3 ). The nutrient solution level was kept below about 50 mm beneath the vermiculite surface. The nutrient solution was aerated continuously with an air pump and was renewed every 2 weeks. 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 during the day and 18 C at night. Light was supplied by a bank of fluorescent tubes (FPR 96EX-N/A; Matsushita, Kadoma, Japan). Irradiance was measured with an LI-190 Quantum Sensor (Li-Cor Inc., Lincoln, NE, USA) at the plant level; the values were 350 (sun) and 50 (shade) mmol quanta m -2 s -1 PPFD (photosynthetically active photon flux density). On each plant, lamina lengths of all the leaves that were younger than the eighth leaf (counted from the base) were measured every other day to calculate leaf plastochron index (LPI, Erickson & Michelini 1957). The reference length was defined as 10 mm, since lamina growth curves were 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 measurement). Each line indicates the expansion of one leaf. For details, see Materials and methods.

3 Development of sun and shade leaves 783 The LPI for a given leaf, whose serial number is a, is expressed as logln - log10 LPI = n- a+ logl logl n - n+ 1 where n is the serial number of the leaf that is just longer than 10 mm (the reference length), and L n is the lamina length of the nth leaf. When the lamina length is below 10 mm, the LPI value is negative. Fixation, sectioning, and microscopy analyses We sampled 34 leaves from three 1.5-month-old-sun plants and 36 leaves from three 2.5-month-old shade plants. Leaf segments (1 mm 2 mm) were taken with razor blades from these leaves. For structural uniformity, segments without major veins were taken from near the midrib. The segments were fixed in 2.5% glutaraldehyde in 12.5 mm cacodylate buffer (ph 7.2) overnight at 4 C and then in 2% osmium tetroxide for 3 h. After fixation, they were dehydrated in an acetone series and embedded in Spurr s resin (Spurr 1969). Transverse and paradermal sections cut 1 mm thick with glass knives on an ultramicrotome (Reichert Ultracut S; Leica, Vienna, Austria) and stained with 0.5% toluidine blue were viewed under a light microscope (BX-50; Olympus, Tokyo, Japan). Light micrographs ( pixels) were taken with a digital camera (C-3030 Zoom; Olympus, Tokyo, Japan). Anatomical analyses Leaf thickness and palisade tissue thickness were measured at 10 positions on every transverse section (about 300 mm in width). Esau (1977) wrote: The palisade parenchyma consists of cells elongated perpendicular to the surface of the blade. This somewhat subjective definition has caused confusion. Instead, we defined the palisade tissue as the tissue that lies above cell layers containing vascular bundles and consists of cylindrical mesophyll cells. The height, width, and cross-sectional area (C tr ) of the PCs were measured on cells in one transverse section. The maximum and minimum cell diameters and crosssectional area (C pd ) of PCs were measured on about 200 cells in one paradermal section. Thicknesses of the adaxial and abaxial epidermes were separately estimated: The thickness of the epidermis was estimated as the cross-sectional area of the epidermis divided by the length of the epidermis in each transverse section. The number of cell layers in the palisade tissue (N layer ) was estimated for each transverse section (for details, see Yano & Terashima 2001). For the paradermal sections, PC density per unit area of the section (D pd ) was calculated. All these parameters were quantified with image analysis software (NIH Image, public domain software, developed at US National Institutes of Health, available at after tracing of the micrograph images. Because of their irregular shapes, spongy tissue cells were not quantified. The spongy tissue thickness was estimated as lamina thickness minus the sum of the epidermal and palisade tissue thicknesses. The density of PCs per unit leaf area (D leaf ), estimated leaf 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) lamina length lamina length Sl () = p, 2 2 Pl () N total = S(l) D leaf. (3) The l in Eqns 2 and 3 is the LPI value. In Eqn 2, we used a leaf proportional function (P(l)). We fitted hyperbolic functions to the relationship between the ratio of leaf length to width and LPI. P(l) was 55.9/(l ) [R 2 = 0.879] for sun leaves and 79.4/(l ) [R 2 = 0.761] for shade leaves. Cell division rate As mentioned above, we obtained N layer and N total for the leaves at various developmental stages. The cell division rate per unit LPI can be obtained as follows. First, N total and N layer plotted against LPI were fitted with sigmoidal functions, T(l) and L(l), respectively. Because the changes in T (l) and L(l) are attributed to cell divisions, these functions are written as: T(l) = a 2 D(l), (4) L(l) = 1 2 P(l), (5) From these we also obtain Tl () Al () = a 2. (6) Ll () 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 in Eqn 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 anticlinal cell division frequencies, respectively, and express how many times one PC is divided in total, periclinally, and anticlinally during the period from LPI = -1 to l. By definition, D(-1) = P(-1) = A(-1) = 0. The total, periclinal, and anticlinal cell division rates (in times per LPI), R D(l), R P(l), and R A(l), respectively, are given by differential equations: R R R Dl ()= Pl ()= Al ()= RESULTS ddl () d Ê 1 Tl () ˆ = log, dl dlë log2 a dpl () d Ê 1 = Ll () ˆ log, dl dlë log2 dal () d Ê 1 Tl () ˆ = Á log. dl dlë log2 Ll () a Transverse and paradermal sections of the sun and shade leaves are shown in Figs 2 and 3. At negative LPI (Figs 2a (2) (7) (8) (9)

4 784 S. Yano & I. Terashima At the same LPI, the difference in leaf thickness between the sun and shade leaves was not very large (Fig. 4b). However, in Fig. 4c, in which leaf thickness is plotted against lamina length, the difference is marked. For example, the differences at lamina lengths of 30 and 50 mm were 30 and 80 mm, respectively. On the other hand, the difference became ambiguous when the lamina length of the sun leaves reached the maximum (60 mm). This resulted from a considerable increase in leaf thickness after FLE. Changes in thickness of the adaxial and abaxial epidermes are shown in Fig. 5. The thickness of both somewhat decreased 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 (alphabetical order). Bar = 100 mm. & b, 3a & b), there were no detectable differences between the sun and shade leaves. As LPI increased, the sun leaves had denser palisade tissues than the shade leaves. Several idioblasts were identified in the paradermal sections. Intercellular spaces in the sun and shade leaves after full lamina expansion (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 or 8, and the final lengths were about 60 and 72 mm, respectively. Maximum lamina elongation rates were 8 and 14 mm LPI -1, respectively. These values correspond to 6.4 and 4 mm d -1, respectively. Changes in leaf thickness are shown in Fig. 4b. The sun leaves were thicker (300 mm) than the shade leaves (250 mm). The maximum thickening rates were 25 and 20 mm LPI -1, respectively. Leaf thickness continued to increase after FLE in both types of leaf. 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.

5 Development of sun and shade leaves 785 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. adaxial epidermis attained a final thickness of 16 mm in both sun and shade leaves. The adaxial epidermis of the shade leaves was always thicker than that of the sun leaves, although the differences were not statistically significant. For the abaxial epidermis, the difference was significant around LPI 7, but not significant for other periods. Both epidermes continued to increase their thickness after FLE. 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.

6 786 S. Yano & I. Terashima Figure 6. Height (a) and width (b) of PCs measured in the transverse 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 Development of palisade tissue cells Changes in height and width of the PCs measured in transverse sections are shown in Fig. 6. The average height of PCs 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 similar manner to the leaf thickness (Fig. 4b). The width of PCs decreased between LPI -1 and 1, and then increased in both sun and shade leaves (Fig. 6b). The increment in the PC width in the sun leaves was less than that in the shade leaves, and the PC width reached 20 and 24 mm in the sun and shade leaves, respectively. The PC width also continued to increase after FLE in both types of leaf. The diameter of PCs measured in the paradermal sections (Fig. 7) was almost identical to the width of PCs mea- Figure 7. Maximum (a) and minimum (b) diameters of PCs measured 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.

7 Development of sun and shade leaves 787 sured in the transverse sections (Fig. 6). Interestingly, the difference between the maximum and minimum diameters remained unchanged, being 2 3 mm at any LPI. Thus, the difference relative to the diameter decreased with LPI. This is due to changes in PC shape with time: the paradermal sectional view of PCs changed from square or polygonal in the early periods (Fig. 3a d) to circular in later periods (Fig. 3e j). The maximum diameter of the PCs was plotted against lamina length (Fig. 7c). Being similar to the pattern of lamina thickness (Fig. 4c), the diameter also increased and the data points spread vertically after FLE. When plotted against lamina length, the difference of the diameter between the sun and shade leaves became ambiguous. The cross-sectional areas of PCs measured in the transverse sections (C tr ) and in the paradermal sections (C pd ) are shown in Fig. 8. C tr was 100 mm 2 at LPI -1 in both the sun and shade leaves (Fig. 8a). The value of C tr increased to 1000 mm 2 in the sun leaves and to 1300 mm 2 in the shade leaves at LPI 10. At LPI -1 C pd was about 60 mm 2 in both the sun and shade leaves (Fig. 8b). The value of C pd decreased from LPI -1 to 1 in both types of leaf (about 35 mm 2 ), as was observed for the cell width and diameters (Figs 6b & 7). After the decrease, C pd increased to 270 and 500 mm 2 at LPI 10 in the sun and shade leaves, respectively. The C pd of the shade leaves was greater than that of the sun leaves from LPI 4. Both C tr and C pd also continued to increase after FLE. From the data of C pd (Fig. 8b) and cell height (Fig. 6a), the volume of PCs was calculated (Fig. 8c). At LPI -1, the volumes in both types of leaf were 800 mm 3. The cell volume exponentially increased with age to and mm 3 in the sun and shade leaves, respectively. Development of palisade and spongy tissues Figure 8. Cross-sectional area of PCs for the transverse (a, C tr ) and paradermal sections (b, C pd ) 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. Changes in the palisade tissue thickness are shown in Fig. 9a. The thickness in both types of leaf was similar from LPI -1 to 2, but from LPI 3 the thickness in the sun leaves increased more than that in the shade leaves. At the end of the observation, the palisade tissue thicknesses reached 120 and 90 mm in the sun and shade leaves, respectively. Changes in the number of cell layers in the palisade tissue (N layer ) are shown in Fig. 9b. The value of N layer of the sun leaves increased rapidly in the early period to 1.7 and N layer also increased later to some extent in the shade leaves. The N layer for one cell lineage in the vertical direction varied from 1 to 3, even in adjacent cells. The average PC heights of the sun and shade leaves were very similar at all LPIs (Fig. 6a). Thus, the difference in the palisade tissue thickness was caused by a change in cell layer number. The thicknesses of the palisade and spongy tissues and of the epidermes are plotted against lamina thickness (Fig. 10a, b & c, respectively). The palisade tissue thickness increased linearly as the lamina thickness increased. The regression lines (y = x [R 2 = 0.976] for sun leaves; y = x [R 2 = 0.982] for shade leaves) were statistically different [analysis of covariance (ANCOVA), P = ]. The spongy tissue thickness

8 788 S. Yano & I. Terashima Figure 9. Changes of palisade tissue thickness (a) and N layer (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. also increased linearly, but the slopes and y-intercepts were not statistically different (ANCOVA, P = and P = 0.743, respectively). When leaves of the same lamina thickness were compared therefore the sun leaves had thicker palisade tissue than the shade leaves, whereas the spongy tissue thicknesses were very similar. The sums of the adaxial and abaxial epidermes were statistically different between the sun and shade leaves (ANCOVA, P = ; Fig. 10c). The regression lines were y = x (R 2 = 0.943) for the sun leaves and y = x (R 2 = 0.910) for the shade leaves. Thus, the difference in the palisade tissue thickness was compensated for by the difference in epidermal thicknesses. PC density in the paradermal sections (D pd ) is shown in Fig. 11a. At LPI -1, D pd 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.

9 Development of sun and shade leaves 789 shade leaves ( ). The value of D pd increased as the PC diameter decreased (that is, as the palisade cell division increased) until LPI 1 to 2 and then D pd decreased gradually with the expansion of the lamina (LPI < 7 or 8). The D pd of the sun leaves was greater than that of the shade leaves. PC densities per unit leaf area (D leaf ) were calculated as the product of D pd and N layer (Fig. 11b). The D leaf of the sun leaves was always greater than that of the shade leaves except at LPI < 0. The total number of PCs per leaf (N total ) was calculated from D leaf and the lamina length and width (see Materials and methods). The value of N total of both types of leaf increased exponentially from LPI -1 to 2 and stabilized at about It is noteworthy that N total of the sun and shade leaves did not differ markedly at any LPI. Cell division rates The total (R D(l) ), periclinal (R P(l) ), and anticlinal (R A(l) ) cell division rates were calculated. Both L(l) and T(l) were sigmoidal functions of N layer (Fig. 9b) and N total (Fig. 11c), respectively (Table 1). These functions were substituted into Eqns 4 6, and total (D(l)), periclinal (P(l)), and anticlinal (A(l)) cell division frequencies were obtained (Table 1). These three functions were differentiated with respect to LPI (Eqns 7 9, see Materials and methods) and plotted against LPI. Changes in the anticlinal cell division rate (R A, LPI -1 ) are shown in Fig. 12a. The maximum R A values were 1.4 LPI -1 in the sun leaves and 3.1 LPI -1 in the shade leaves. Although it seemed that R A of the sun leaves was smaller than that of the shade leaves, integrated values of R A, which say how many times PCs divided anticlinally from LPI = -1 to 12, were not very different (4.0 and 4.6 in the sun and shade leaves, respectively). The periods in which the anticlinal cell divisions occurred most frequently almost agreed in the sun and shade leaves. The periclinal cell division rates (R P ) are shown in Fig. 12c. The R P values of the sun leaves were greater than those of the shade leaves for LPI -1 to 3. Interestingly, the maximum cell division rate of the shade leaves occurred much later than that of the sun leaves. The maximum R P and integrated value of R P for the sun leaves were 0.35 LPI -1 at LPI = 1.7 and 0.7, and those for the shade leaves were 0.09 LPI -1 at LPI = 3.9 and 0.4, respectively. The maximum total cell division rate (R D ) of the sun leaves was 1.6 LPI -1 at LPI = 0.9 and that of the shade leaves was 3.0 LPI -1 at LPI = 1.0 (Fig. 12b). The integrated values of R P were 4.8 and 5.0 for the sun and shade leaves, respectively. The small differences between R D and R A + R P were errors due to the curve fitting. Figure 11. D pd (a), D leaf (b), and N total (c) plotted against LPI. The data for the sun and shade leaves are indicated as open circles (solid line) and squares (dotted line), respectively. DISCUSSION Developmental processes Early event, LPI = -1 to 1 In this period, PCs started to divide vigorously (Fig. 12), and PC diameter and width (Figs 7 & 6b, respectively)

10 790 S. Yano & I. Terashima 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 R 2 Shade leaf R 2 7 T(l) exp ( l) 1+ exp ( l) L(l) exp ( l) 1 + exp ( l) a D(l) exp ( l ) 1 + exp ( l ) P(l) exp ( l) 1+ exp ( l) A(l) exp ( l) 1 + exp ( l ) 7 See Materials and methods for details. decreased. Daughter cells tended to divide before they expanded to the same size as their mother cells during this period. This temporal decrease in PC diameter was also reported for Xanthium pennsylvanicum (Maksymowych 1973). The PC density per unit area in the paradermal sections (D pd, Fig. 11a) and per unit leaf area, taking account of the number of cell layers (D leaf, Fig. 11b), increased, since the laminar expansion was slower than the vigorous PC production. In addition to the decrease in PC diameter, PCs elongated vertically (Fig. 6a). Thus, depression was observed in neither C tr nor PC volume (Fig. 8a & c, respectively). In this period, developmental processes of the sun and shade leaves did not differ, except for the periclinal division rate 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 this period (Figs 6 & 7), while the cell division gradually diminished and ceased by LPI = 4 (Fig. 12). A significant increase in PC diameter was observed after LPI 4 (Fig. 7). The crosssectional area of PCs measured in the transverse (C tr ) and paradermal (C pd ) sections and the PC volume also rapidly increased after LPI 4 (Fig. 8). The values of D pd and D leaf increased early in this period but decreased after LPI 2 (Fig. 11). Differences between the sun and shade leaves were significant in this period. The most significant differences were found in the number of cell layers in the palisade tissue (N layer, Fig. 9b) and in PC expansion growth (Figs 6b & 7). The N layer of the sun leaves increased around LPI 2 and became stable by LPI 4. On the other hand, N layer in the shade leaves started to increase at around LPI 3, and the gradual increase continued until FLE. In the shade leaves, PC diameter markedly increased after LPI 4. Thus, the differences in PC volume and C pd became marked between the sun and shade leaves (Fig. 8c & b, respectively). After FLE (LPI > 7 8) The lamina expansion ceased in this period (Fig. 4a). However, elongation and expansion of PCs (Figs 6, 7 & 8) and thickening of the palisade tissue (Fig. 9) and the lamina (Fig. 4b) continued, although the increases in this period were much less than those in the expansion period. The shade leaves showed greater PC expansion growth than the sun leaves in this period as well (Fig. 7). Cell divisions and cell axis Our results show that the anticlinal cell division occurred in almost the same LPI period in sun and shade leaves (Fig. 12a), but that the periclinal division occurred in different periods (Fig. 12c). In the sun leaves, the periclinal division occurred at almost the same time as the anticlinal division. However, in the shade leaves, the periclinal division occurred much later than the anticlinal division. This difference between the anticlinal and periclinal division periods in the shade leaves suggests that these divisions are controlled independently. The two-cell-layered palisade tissue is formed as a result of periclinal cell divisions. If periclinal divisions occurred in addition to anticlinal divisions, the total numbers of PCs (N total ) should be greater in the sun leaves than in the shade leaves. However, the N total values of the sun and shade leaves were almost identical (Fig. 11c). Moreover, as mentioned above, the periclinal and anticlinal divisions occurred simultaneously in the sun leaves. These results indicate that, in the sun leaves, periclinal division takes place at the expense of anticlinal division, and that this directional change of cell division makes two-cell-layered palisade tissue. We have reported that mature leaves, but not developing leaves, sense the light signal for differentiation of sun and shade leaves (Yano & Terashima 2001). The light information is transferred to the developing leaves by some signal

11 Development of sun and shade leaves 791 transduction system, and the fate of developing leaves is thus determined. The present results indicate that the signal controls the direction of cell division. They also indicate that mesophyll cells, or at least palisade tissue cells, recognize the axis perpendicular to the leaf plane. Axis recognition in mesophyll cells has been studied in Arabidopsis thaliana. The ANGUSTIFOLIA (AN) and ROTUNDIFOLIA3 genes control cell enlargement in the leaf-width and leaf-length directions, respectively (for a review, see Tsukaya 2002). Leaves of an angustifolia (an) mutant were narrower and thicker than those of wild-type plants, although lengths were identical (Tsuge, Tsukaya & Uchiyama 1996). Interestingly, the number of cell layers in the an mutant was greater than that of wild-type plants. As the number of cell layers in the sun leaves of C. album is also greater than that in the shade leaves, there may be some relationship between AN and the formation of sun leaves. Alternatively, as the sun leaves are not narrower than the shade leaves, there may be genes controlling the direction of cell division other than AN. Further studies are required. Light effects on PC anatomy There is little quantitative information about the effects of light environment on PC size. Björkman (1981) found no obvious differences in cell width on the basis of a cursory examination of published micrographs or camera lucida drawings of leaf sections from sun and shade leaves of several species. There are also observations that cell width is smaller in shade leaves than in sun leaves (Wilson & Cooper 1969; Ballantine & Forde 1970). In the present study, the PC diameters of the shade leaves were greater than those of the sun leaves when the data were plotted against LPI. When the data were plotted against lamina length, however, PC diameters were somewhat smaller in the shade leaves than in the sun leaves while the leaves were expanding. Thus, comparison of cell size between sun and shade leaves requires careful material sampling. The sun and shade plants were grown at the same time in the same phytotron. Thus, the air temperature and relative humidity around plants were almost the same for the sun and shade plants. However, owing to the different irradiance levels, the leaf temperature and thereby vapour pressure deficit were probably greater in the sun leaves than in the shade leaves. In other words, the sun leaves were probably exposed to more xeric conditions than the shade leaves. In fact, the sun leaves contained more idioblasts, which we identified as water storage cells by their shape and contents (Fig. 3). The more xeric conditions might also be responsible for the smaller PC size (Nobel & Walker 1985) and thinner epidermes. 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. Cell elongation/expansion after full lamina expansion After FLE, PC height, palisade tissue thickness, epidermal thicknesses, and leaf thickness increased (Figs 6a, 9a, 5 &

12 792 S. Yano & I. Terashima 4b, respectively). In other words, leaves that had ceased expanding in area were still thickening. These results were different from the results of X. pennsylvanicum (Maksymowych 1973), in which thickness growth ceased before FLE. Why leaf developmental patterns differ between C. album and X. pennsylvanicum is not clear. In the present study, it was difficult to define maturation of leaves, because the edges of leaves that were older than those used in this study turned brownish, and some leaves whose LPI was more than 15 withered completely. Cell division and elongation and leaf longevity are affected by nitrogen supply (see review, Forde 2002), and the plants used in this study were 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 area measured in the paradermal sections (C pd, Fig. 8b) also increased after FLE. Thus, intercellular spaces should decrease. We did not quantify intercellular spaces but the decrease can be seen (compare Fig. 3g, i, h and j). Vertical elongation and horizontal expansion growth of PCs after FLE was reported in Castanopsis sieboldii, Quercus glauca, and Phaseolus vulgaris (Miyazawa & Terashima 2001; Miyazawa, Makino & Terashima 2003). If such cell elongation and expansion growth after FLE is general, maturation of leaves as judged by area growth would be misleading. Factors that limit expansion growth of the leaf are unknown (see review, Van Volkenburgh 1999). Elongation of the coleoptiles, stems, and roots is regulated by the growth of epidermis (Masuda & Yamamoto 1972; Cosgrove 1986; Kutschera, Bergfeld & Schopfer 1987). Wilson & Bruck (1999) peeled the adaxial epidermis off Pisum sativum leaves and compared the shapes of the leaves and PCs with those in intact leaves. They did not find detectable changes in the expansion of the leaflets or the shape of PCs. In the present study, the elongation and expansion of PCs occurred after FLE, which means that the cessation of epidermal expansion does not affect the elongation or expansion of PCs, supporting the results of Wilson & Bruck (1999). CONCLUSIONS 1 Elongation and expansion growth of PCs and leaf thickening were observed after FLE. These results indicate that mesophyll growth was not synchronized with lamina expansion. 2 Analyses of N total and cell division rates indicate that, in the sun leaves, periclinal division occurred in the phase of vigorous cell division to form two-cell-layered palisade tissue. Thus, light signals from mature leaves would control the direction of cell division in this phase. ACKNOWLEDGMENTS We thank Dr K. Noguchi for his kind advice, especially on statistics. 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