Planta (1985)165:120-125
P l a n t a 9 Springer-Verlag1985
Cell size and chloroplast size in relation to chloroplast replication in light-grown wheat leaves J.R. Ellis 1 and R.M. Leech Department of Biology, University of York, Heslington, York Y01 5DD, UK
Abstract. As part of an investigation into the control of chloroplast replication the number and size of chloroplasts in mesophyll cells was examined in relation to the size of the cells. In first leaves of Triticum aestivum L. and T. monocoecum L. the number of chloroplasts in fully expanded mesophyll cells is positively correlated with the plan area of the cells. The linear relationship between chloroplast number per cell and cell plan area is also consistent over a fivefold range of cell size in isogenic diploid and tetraploid T. monococcum. In T. aestivum the chloroplast number per unit cell plan area varies among cells in relation to the size of the chloroplasts. Those cells containing chloroplasts with a relatively small face area have a correspondingly higher density of chloroplasts, and consequently, the total chloroplast area per unit cell plan area is very similar in all the cells. The results indicate that the proportion of the cell surface area covered by chloroplasts is precisely regulated, and that this is achieved during cell development by growth and replication of the chloroplasts. Key words: Cell size - Chloroplast replication Mesophyll (development) - Polyploidy - Triticum (chloroplast replication).
Introduction The chloroplast population of developing leaf tissues increases by divisions of the young chloroplasts, and a variety of factors have been suggested which may control this replication process (for reviews, see Leech 1976; Possingham 1980; Possingham and Lawrence 1983). Light is an important factor promoting chloroplast replication, but for plants growing in a normally illuminated environment other factors are more important in control1 Present address and address f o r correspondence: Department
of Botany, University of Durham, Science Laboratories, South Road, Durham DH1 3LE, UK
ling replication. There is some evidence that cell size might influence chloroplast replication; for example, during development of leaf tissues the average number of chloroplasts in the cells increases in parallel with the average size of the cells (Boasson et al. 1972; Kameya 1972; Tsuji et al. 1979; Scott and Possingham 1980; Whatley 1980; Wild and Wolf 1980; Olszewska etal. 1983). The number of chloroplasts per cell has also been reported to be correlated with individual cell size in " l e a v e s ' o f some mosses (MacNutt and yon Maltzahn 1960; Asahi and Toyama 1982) and in callus tissue of tobacco (Kameya 1972). These observations have been interpreted as indicating that cell expansion is a prerequisite for chloroplast replication (see discussion in Paolillo and Kass 1977), an interpretation which is consistent with the earlier proposal by Granick (1955) that chloroplasts multiply only when there is space available in the cell. Honda et al. (1971) suggested that the size of the chloroplasts is related to the number of chloroplasts on a cell face based on the observation that in Nicotiana glutinosa, Spinacia oleracea and Beta vulgaris the product of chloroplast number and chloroplast area on individual cell faces was closely related to the area of the cell faces, and so the proportion of each cell face area occupied by chloroplasts was nearly constant in a given species, e.g. 25% in N. glutinosa and 73% in S. oleracea. Using cultured fronds of Wolffia arrhiza Honda (1983) found that the chloroplast cover of the mature cells was dependent on the extent of chloroplast growth and replication. The aim of the current work was to make a detailed investigation of chloroplast size and number in mature mesophyll cells, in order to assess the influence of cell size and chloroplast size on controlling chloroplast replication. The material selected for this investigation was the mesophyll tissue of the first leaf of Triticum aestivum (hexaploid bread wheat) because development of this tissue can be examined in a precise and reproduc-
J.R. Ellis and R.M. Leech: Chloroplast replication in wheat leaves
ible manner, as a consequence of the organised pattern of leaf growth (Boffey et al. 1979). We have previously shown that extensive growth and replication of the chloroplasts occur during the development of these mesophyll cells (Leech et al. 1981 ; Ellis et al. 1983). To investigate a wider range of mesophyll cell size within a single genotype we also examined isogenic diploid and tetraploid lines of Triticum monococcum (einkorn wheat), since increased cell size is generally associated with polyploidy (Stebbins 1950, 1971). The study of the autopolyploid allowed us to examine nucleotypic effects (Bennett 1971) independently of genotypic variation. Materials and methods Plant material. Seedlings of Triticum aestivum L. cv. Maris Dove and of T. monococcum L. were grown under carefully controlled environmental conditions. Surface-sterilized grains were sown in Levington Universal compost (Fisons, UK) at a depth of 1.3 cm, and the seedlings grown under a photoperiod of 16 h light (45-50 W m -2) at 20 ~ C, 8 h darkness at 15 ~ C, and 70% relative humidity. For studies of leaf (lamina) tissue development, specific regions of the first leaves, defined by distance from the leaf base, were sampled from seedlings 7 d after sowing (Boffey et al. 1979). Fully expanded tissue was routinely ' sampled 8.0 to 8.5 cm from the leaf base. Measurement of cell size, chloroplast number, and chloroplast size in separated cells. To measure cell size, chloroplast number and chloroplast area per cell, the mesophyll tissue was separated into its component cells by a gentle maceration procedure. Transverse leaf slices (1 mm wide) were fixed in 2.5% (v/v) glutaraldehyde in 0.1 M K-phosphate buffer (pH 7.2) for 1 h. The leaf slices were washed in the phosphate buffer, and incubated in 2% (w/v) Onozuka eellulase R-10 and 1% (w/v) Macerozyme R-10 (Yakult Biochemicals, Nishinomiya, Japan) in 0.05 M K-citrate/0.1 M K-phosphate buffer (pH 5.0) at 30 ~ C. After 2.0 to 2.5 h the leaf tissue was teased apart on a slide and the suspension of cells examined under a Zeiss (Oberkochert, FRG) photomieroscope. The plan area of a cell and the face areas of individual chloroplasts were measured from photomicrographs (bright-field optics and Nomarski interference optics, respectively) using a 9864A Hewlett-Packard (Bracknell, Berks., UK) digitizer linked to a Model 30 calculator. Only those chloroplasts exhibiting a face view were measured, i.e. those lying with their face against the upper or lower surfaces of the cell (approx. 40% of the chloroplasts in each cell). The number of chloroplasts per cell was counted directly from the microscope under Nomarski interference optics. The proportion of the cell surface area covered by chloroplasts was obtained by planimetry of cell faces. Measurement of the chloroplast compartment in cell sections. Quantitative information on the chloroplast compartment as a component of the mesophyll cell was obtained from thin sections of the leaf tissue using high-resolution light and electron microscopy. Leaf specimens (1 ram2), taken between the midvein and margin of the leaf, were fixed and resin-embedded as previously described (Boffey et al. 1979). Transverse sections were cut at a thickness of I pm and stained with toluidine blue 0 for light microscopy, or at a thickness of 60-120 nm for electron microscopy. The latter ultrathin sections were spread with
121 chloroform vapour, stained on coated grids with uranyl acetate (saturated solution in 50% (v/v) ethanol) followed by lead citrate solution (Reynolds 1963) and were examined in a Corinth 500 electron microscope (Kratos, Manchester, UK). Morphometric values were determined using the stereological procedures of Weibel (1969). The proportion of the cell volume occupied by the chloroplast compartment, measured as the area fraction of the chloroplast compartment, was obtained directly from the electron microscope by superimposing an array of test points on the specimen image. The porportion of the cell surface area covered by the chloroplast compartment was obtained by using a test lattice of parallel straight lines, and was recorded from electron-microscope images for younger tissue (1.0-2.5 cm from the leaf base) and from light-microscope images for older tissue (3.0-8.5 cm from the leaf base). Each result presented from this morphometry was the mean value from six sections, each derived from a different seedling. Measurement of the size of isolated chloroplasts. A suspension of isolated chloroplasts was prepared from the leaf tissue to allow adequate sampling of the chloroplast population. Leaf pieces (5 mm wide) sampled from six seedlings were diced with a razor blade on a microscope slide in 0.4 M sorbitol plus 0.75 mM MgC12 in 0.1 M K-phosphate buffer (pH 7.6), and the plan area of the isolated chloroplasts measured from photomicrographs using the digitizer. Only intact chloroplasts as judged by their refractive appearance under phase-contrast optics (Kahn and von Wettstein 1961) were measured. The proportion of intact chloroplasts was 60-80%.
Results
The first leaf of the wheat seedling grows from a basal intercalary meristem and thus different stages of mesophyll development can be examined by sampling specific regions of the leaf (Boffey et al. 1979). Differentiation of these mesophyll cells is characterized by the loss of mitotic activity, expansion of the cells, and expansion and replication of the chloroplasts (Ellis et al. /983). The results from the present investigation are conveniently divided into observations on (i) the fully expanded cells, when the final chloroplast number has been attained, and on (ii) the developing cells during the phase of cell expansion and chloroplast replication. Fully expanded cells. The size of the expanded mesophyll cells of T. aestivum, measured as plan area,
varied over a threefold range, and there was a positive linear relationship between the number of chloroplasts in a cell and the plan area of the cell (Fig. 1). The number of chloroplasts per unit cell plan area, which is a measure of the numerical density of the chloroplasts, varied little between the cells (coefficient of variation= 0.14). Fully expanded mesophyll cells of isogenic diploid and tetraploid T. monococcum were generally smaller and contained fewer chloroplasts than those of T. aestivum. The mean plan area of the
122
J.R. Ellis and R.M. Leech: Chloroplast replication in wheat leaves
25
250
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u
21. v
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20
r=-0.87 p
P l a n t a 9 Springer-Verlag1985
Cell size and chloroplast size in relation to chloroplast replication in light-grown wheat leaves J.R. Ellis 1 and R.M. Leech Department of Biology, University of York, Heslington, York Y01 5DD, UK
Abstract. As part of an investigation into the control of chloroplast replication the number and size of chloroplasts in mesophyll cells was examined in relation to the size of the cells. In first leaves of Triticum aestivum L. and T. monocoecum L. the number of chloroplasts in fully expanded mesophyll cells is positively correlated with the plan area of the cells. The linear relationship between chloroplast number per cell and cell plan area is also consistent over a fivefold range of cell size in isogenic diploid and tetraploid T. monococcum. In T. aestivum the chloroplast number per unit cell plan area varies among cells in relation to the size of the chloroplasts. Those cells containing chloroplasts with a relatively small face area have a correspondingly higher density of chloroplasts, and consequently, the total chloroplast area per unit cell plan area is very similar in all the cells. The results indicate that the proportion of the cell surface area covered by chloroplasts is precisely regulated, and that this is achieved during cell development by growth and replication of the chloroplasts. Key words: Cell size - Chloroplast replication Mesophyll (development) - Polyploidy - Triticum (chloroplast replication).
Introduction The chloroplast population of developing leaf tissues increases by divisions of the young chloroplasts, and a variety of factors have been suggested which may control this replication process (for reviews, see Leech 1976; Possingham 1980; Possingham and Lawrence 1983). Light is an important factor promoting chloroplast replication, but for plants growing in a normally illuminated environment other factors are more important in control1 Present address and address f o r correspondence: Department
of Botany, University of Durham, Science Laboratories, South Road, Durham DH1 3LE, UK
ling replication. There is some evidence that cell size might influence chloroplast replication; for example, during development of leaf tissues the average number of chloroplasts in the cells increases in parallel with the average size of the cells (Boasson et al. 1972; Kameya 1972; Tsuji et al. 1979; Scott and Possingham 1980; Whatley 1980; Wild and Wolf 1980; Olszewska etal. 1983). The number of chloroplasts per cell has also been reported to be correlated with individual cell size in " l e a v e s ' o f some mosses (MacNutt and yon Maltzahn 1960; Asahi and Toyama 1982) and in callus tissue of tobacco (Kameya 1972). These observations have been interpreted as indicating that cell expansion is a prerequisite for chloroplast replication (see discussion in Paolillo and Kass 1977), an interpretation which is consistent with the earlier proposal by Granick (1955) that chloroplasts multiply only when there is space available in the cell. Honda et al. (1971) suggested that the size of the chloroplasts is related to the number of chloroplasts on a cell face based on the observation that in Nicotiana glutinosa, Spinacia oleracea and Beta vulgaris the product of chloroplast number and chloroplast area on individual cell faces was closely related to the area of the cell faces, and so the proportion of each cell face area occupied by chloroplasts was nearly constant in a given species, e.g. 25% in N. glutinosa and 73% in S. oleracea. Using cultured fronds of Wolffia arrhiza Honda (1983) found that the chloroplast cover of the mature cells was dependent on the extent of chloroplast growth and replication. The aim of the current work was to make a detailed investigation of chloroplast size and number in mature mesophyll cells, in order to assess the influence of cell size and chloroplast size on controlling chloroplast replication. The material selected for this investigation was the mesophyll tissue of the first leaf of Triticum aestivum (hexaploid bread wheat) because development of this tissue can be examined in a precise and reproduc-
J.R. Ellis and R.M. Leech: Chloroplast replication in wheat leaves
ible manner, as a consequence of the organised pattern of leaf growth (Boffey et al. 1979). We have previously shown that extensive growth and replication of the chloroplasts occur during the development of these mesophyll cells (Leech et al. 1981 ; Ellis et al. 1983). To investigate a wider range of mesophyll cell size within a single genotype we also examined isogenic diploid and tetraploid lines of Triticum monococcum (einkorn wheat), since increased cell size is generally associated with polyploidy (Stebbins 1950, 1971). The study of the autopolyploid allowed us to examine nucleotypic effects (Bennett 1971) independently of genotypic variation. Materials and methods Plant material. Seedlings of Triticum aestivum L. cv. Maris Dove and of T. monococcum L. were grown under carefully controlled environmental conditions. Surface-sterilized grains were sown in Levington Universal compost (Fisons, UK) at a depth of 1.3 cm, and the seedlings grown under a photoperiod of 16 h light (45-50 W m -2) at 20 ~ C, 8 h darkness at 15 ~ C, and 70% relative humidity. For studies of leaf (lamina) tissue development, specific regions of the first leaves, defined by distance from the leaf base, were sampled from seedlings 7 d after sowing (Boffey et al. 1979). Fully expanded tissue was routinely ' sampled 8.0 to 8.5 cm from the leaf base. Measurement of cell size, chloroplast number, and chloroplast size in separated cells. To measure cell size, chloroplast number and chloroplast area per cell, the mesophyll tissue was separated into its component cells by a gentle maceration procedure. Transverse leaf slices (1 mm wide) were fixed in 2.5% (v/v) glutaraldehyde in 0.1 M K-phosphate buffer (pH 7.2) for 1 h. The leaf slices were washed in the phosphate buffer, and incubated in 2% (w/v) Onozuka eellulase R-10 and 1% (w/v) Macerozyme R-10 (Yakult Biochemicals, Nishinomiya, Japan) in 0.05 M K-citrate/0.1 M K-phosphate buffer (pH 5.0) at 30 ~ C. After 2.0 to 2.5 h the leaf tissue was teased apart on a slide and the suspension of cells examined under a Zeiss (Oberkochert, FRG) photomieroscope. The plan area of a cell and the face areas of individual chloroplasts were measured from photomicrographs (bright-field optics and Nomarski interference optics, respectively) using a 9864A Hewlett-Packard (Bracknell, Berks., UK) digitizer linked to a Model 30 calculator. Only those chloroplasts exhibiting a face view were measured, i.e. those lying with their face against the upper or lower surfaces of the cell (approx. 40% of the chloroplasts in each cell). The number of chloroplasts per cell was counted directly from the microscope under Nomarski interference optics. The proportion of the cell surface area covered by chloroplasts was obtained by planimetry of cell faces. Measurement of the chloroplast compartment in cell sections. Quantitative information on the chloroplast compartment as a component of the mesophyll cell was obtained from thin sections of the leaf tissue using high-resolution light and electron microscopy. Leaf specimens (1 ram2), taken between the midvein and margin of the leaf, were fixed and resin-embedded as previously described (Boffey et al. 1979). Transverse sections were cut at a thickness of I pm and stained with toluidine blue 0 for light microscopy, or at a thickness of 60-120 nm for electron microscopy. The latter ultrathin sections were spread with
121 chloroform vapour, stained on coated grids with uranyl acetate (saturated solution in 50% (v/v) ethanol) followed by lead citrate solution (Reynolds 1963) and were examined in a Corinth 500 electron microscope (Kratos, Manchester, UK). Morphometric values were determined using the stereological procedures of Weibel (1969). The proportion of the cell volume occupied by the chloroplast compartment, measured as the area fraction of the chloroplast compartment, was obtained directly from the electron microscope by superimposing an array of test points on the specimen image. The porportion of the cell surface area covered by the chloroplast compartment was obtained by using a test lattice of parallel straight lines, and was recorded from electron-microscope images for younger tissue (1.0-2.5 cm from the leaf base) and from light-microscope images for older tissue (3.0-8.5 cm from the leaf base). Each result presented from this morphometry was the mean value from six sections, each derived from a different seedling. Measurement of the size of isolated chloroplasts. A suspension of isolated chloroplasts was prepared from the leaf tissue to allow adequate sampling of the chloroplast population. Leaf pieces (5 mm wide) sampled from six seedlings were diced with a razor blade on a microscope slide in 0.4 M sorbitol plus 0.75 mM MgC12 in 0.1 M K-phosphate buffer (pH 7.6), and the plan area of the isolated chloroplasts measured from photomicrographs using the digitizer. Only intact chloroplasts as judged by their refractive appearance under phase-contrast optics (Kahn and von Wettstein 1961) were measured. The proportion of intact chloroplasts was 60-80%.
Results
The first leaf of the wheat seedling grows from a basal intercalary meristem and thus different stages of mesophyll development can be examined by sampling specific regions of the leaf (Boffey et al. 1979). Differentiation of these mesophyll cells is characterized by the loss of mitotic activity, expansion of the cells, and expansion and replication of the chloroplasts (Ellis et al. /983). The results from the present investigation are conveniently divided into observations on (i) the fully expanded cells, when the final chloroplast number has been attained, and on (ii) the developing cells during the phase of cell expansion and chloroplast replication. Fully expanded cells. The size of the expanded mesophyll cells of T. aestivum, measured as plan area,
varied over a threefold range, and there was a positive linear relationship between the number of chloroplasts in a cell and the plan area of the cell (Fig. 1). The number of chloroplasts per unit cell plan area, which is a measure of the numerical density of the chloroplasts, varied little between the cells (coefficient of variation= 0.14). Fully expanded mesophyll cells of isogenic diploid and tetraploid T. monococcum were generally smaller and contained fewer chloroplasts than those of T. aestivum. The mean plan area of the
122
J.R. Ellis and R.M. Leech: Chloroplast replication in wheat leaves
25
250
(M
E
u
21. v
~,zoo
O
ee
20
r=-0.87 p
