JXB Advance Access published online on March 28, 2008
Journal of Experimental Botany, doi:10.1093/jxb/ern055
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© 2008 The Author(s).
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RESEARCH PAPER |
Difference in light-induced increase in ploidy level and cell size between adaxial and abaxial epidermal pavement cells of Phaseolus vulgaris primary leaves
1Department of Molecular and Cell Biology, Forestry and Forest Products Research Institute, Matsunosato, Tsukuba, Ibaraki 305-8687, Japan
2Department of Biology, Faculty of Science, Nara Women's University, Nara 630-8506, Japan
* To whom correspondence should be addressed. E-mail: ikinos{at}ffpri.affrc.go.jp
Received 26 October 2007; Revised 1 February 2008 Accepted 4 February 2008
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Abstract |
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Changes in nuclear DNA content and cell size of adaxial and abaxial epidermal pavement cells were investigated using bright light-induced leaf expansion of Phaseolus vulgaris plants. In primary leaves of bean plants grown under high (sunlight) or moderate (ML; photon flux density, 163 µmol m–2 s–1) light, most adaxial epidermal pavement cells had a nucleus with the 4C amount of DNA, whereas most abaxial pavement cells had a 2C nucleus. In contrast, plants grown under low intensity white light (LL; 15 µmol m–2 s–1) for 13 d, when cell proliferation of epidermal pavement cells had already finished, had a 2C nuclear DNA content in most adaxial pavement cells. When these LL-grown plants were transferred to ML, the increase in irradiance raised the frequency of 4C nuclei in adaxial but not in abaxial pavement cells within 4 d. On the other hand, the size of abaxial pavement cells increased by 53% within 4 d of transfer to ML and remained unchanged thereafter, whereas adaxial pavement cells continuously enlarged for 12 d. This suggests that the increase in adaxial cell size after 4 d is supported by the nuclear DNA doubling. The different responses between adaxial and abaxial epidermal cells were not induced by the different light intensity at both surfaces. It was shown that adaxial epidermal cells have a different property than abaxial ones.
Key words: Cell enlargement, endopolyploidization, epidermal pavement cells, incident light intensity, leaf expansion, nuclear DNA content, Phaseolus vulgaris
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Introduction |
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The adaxial (upper) side and abaxial (lower) side of a dicotyledonous leaf are morphologically asymmetrical. Corresponding with this asymmetry, the upper side of the leaf is specialized for the efficient capture of sunlight, whereas the lower side is specialized for gas exchange. Previously, it was shown that adaxial and abaxial epidermal pavement cells of bean (Phaseolus vulgaris L.) primary leaves differ from each other in nuclear DNA content (Kinoshita et al., 1991). In primary leaves of bean plants grown in moderate intensity (163 µmol m–2 s–1) white light, most adaxial epidermal pavement cells had a nucleus with the 4C amount of DNA, whereas most abaxial epidermal pavement cells had a nucleus with the 2C amount of DNA. In the present study, the influence of incident light intensity on the DNA content of adaxial and abaxial epidermal pavement cells and mesophyll cells was investigated.
The Phaseolus primary leaf is useful material to investigate leaf cell enlargement (Dale, 1988; Van Volkenburgh, 1999). Both cell division and cell enlargement contribute to leaf growth. Cell proliferation occurs in the early phase of dicotyledonous leaf development, and then the contribution of cell enlargement increases in the late phase (Van Volkenburgh and Cleland, 1979; Beemster et al., 2005). Phaseolus plants grown in low intensity red light (4 µmol m–2 s–1) for 10 d (cell proliferation in the primary leaves had already completed) showed greatly elongated stems, but the primary leaves, although green, remained unexpanded (Dale, 1988). Bringing these primary leaves into white light (250–400 µmol m–2 s–1) led to rapid leaf enlargement without cell division (Van Volkenburgh and Cleland, 1979, 1980). Using this system, Van Volkenburgh et al. (1990) showed that maximum expansion of bean leaves required at least 100 µmol m–2 s–1 white light. Light-stimulated growth of green leaves has the typical characteristics of a high irradiance response. This leaf enlargement (cell enlargement) is regulated by non-photosynthetic photosystems including phytochrome (Van Volkenburgh and Cleland, 1990; Van Volkenburgh et al., 1990). When light stimulates leaf enlargement, the proton pump in the plasma membrane is stimulated and, as a result of this event, proton efflux from the cells increases (Stahlberg and Van Volkenburgh, 1999). Acidification of the apoplast loosens the cell wall, making it more responsive to stress imposed by turgor (Rayle and Cleland, 1992). This is the early response in light-stimulated leaf cell enlargement. However, it has not been investigated whether nuclear DNA content in epidermal and mesophyll cells changes under these conditions. In the present study, these changes were investigated using primary leaves of bean plants grown under a condition similar to that described by Van Volkenburgh and Cleland (1979), but low intensity white light was used instead of low intensity red light. Bean plants were grown under the low light condition (LL, 15 µmol m–2 s–1). At 10–13 d, when cell proliferation in the primary leaves had finished, the plants were transferred to the moderate light condition (ML, 163 µmol m–2 s–1).
It has been known since the early 20th century that a higher ploidy nucleus is often associated with an increase in cell size (Sugimoto-Shirasu and Roberts, 2003). Increase in cell size is often paralleled by an increase in ploidy level brought about by endoreduplication, which is successive rounds of DNA replication without mitosis (Gendreau et al., 1997, 1998). Although many studies have been done on the molecular mechanism that induces endoreduplication (Grafi and Larkins, 1995; Gendreau et al., 1997, 1998; Larkins et al., 2001; Imai et al., 2006; Yoshizumi et al., 2006), the biological significance of endopolyploidization is not well understood.
Many environmental and internal signals (e.g. light, temperature, plant growth regulators) induce endoreduplication in a variety of plant cells (Nagl, 1978). In leaf epidermal cells, environmental factors also affect the nuclear DNA content. Schlayer (1971) showed that the DNA content per cell increased 2–4-fold in epidermal cells of sugar beet cotyledons on applying stronger light or adding more moisture to the soil. Griffiths et al. (1994) showed that high temperature reduced the endopolyploidy level in adaxial epidermal bulliform cells of Lolium multiflorum. Cavallini et al. (1995) reported that endoreduplication occurred in the dark in epidermal bulliform cells of Triticum durum and that lower endopolyploidy levels were induced by light treatment. In the last two reports, light had a reductive effect on endoreduplication cycles. A similar effect of light on endoreduplication was reported in Arabidopsis hypocotyls (Gendreau et al., 1997, 1998).
In Arabidopsis leaves, the timing of endoreduplication cycles coincides with that of post-meristematic cell enlargement (Beemster et al., 2005). It has been shown that shading reduced the number of endoreduplication cycles, but increased cell area in Arabidopsis leaves (Cookson et al., 2006). However, many studies on the effect of environmental and internal signals on endoreduplication have considered the ploidy level in an organ as a whole without making a distinction between the different tissues. Moreover, it is unknown how endopolyploidization and cell enlargement are orchestrated in light-induced leaf expansion. In this study, the changes in cell size of adaxial and abaxial epidermal pavement cells that occurred when bean plants were transferred from LL to ML were investigated, and correlated with the change in nuclear DNA content of adaxial pavement cells.
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Materials and methods |
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Plant material
Bean seeds (Phaseolus vulgaris L. cv. Yamashiro-kurosando-saito) were purchased from Takii Co., Kyoto, Japan. About 30 selected seeds were surface-sterilized with NaOCl solution (1% Cl) for 10 min, then soaked in running tap water for 30 min. Next, the seeds were kept in about 3000 cm3 of water (about 3 cm depth) at 25 °C overnight. The seeds were then germinated on wet filter paper in a plastic tray at 25 °C in the dark. After 24 h, three of the germinated seeds were transplanted to a plastic beaker (diameter 10 cm, height 11 cm) containing 500 cm3 of vermiculite wetted with 250 cm3 of Hoagland's solution. In a few experiments, seedlings were grown in a glass room at 15–30 °C under sunlight. Bean plants were grown from 12 May 2000 (2-d-old) to 28 May 2000 (18-d-old). The intensity of sunlight was about 1800 µmol m–2 s–1 at midday. In most experiments, beakers were placed in a growth chamber at 25 °C under a light regimen of 16 h light and 8 h dark. Two light intensities (moderate, ML: 163 µmol m–2 s–1 and low, LL: 15 µmol m–2 s–1) were used in the growth chamber experiments. The light source was white fluorescent lamps (Toshiba FLR40S-W/M/36). To produce the LL condition, the number of fluorescent lamps was reduced and the light was shaded with filter paper (No. 2, Advantec, Tokyo, Japan). Deionized water was added to maintain about 50 cm2 of gravitational water in a plastic beaker. Primary leaves completed unfolding when the plants were 7-d-old. At this stage, seedlings were selected for uniformity.
Cytofluorometry
The most serious obstacle to getting reliable nuclear DNA content data of leaf mesophyll cells is high and irregular background fluorescence emitted by chloroplast DNA (Fig. 1A). To reduce this background level, mesophyll protoplasts, flattened between the microscope slide and coverslip (Kinoshita et al., 1991) (Fig. 1B) were used. Pavement cells in the peeled epidermal tissue are very good material for cytofluorometric measurement of nuclear DNA content because of their low background level (Fig. 1C). In the present study, peeled epidermal tissues and flattened mesophyll protoplasts were stained with 4',6-diamidino-2-phenylindole (DAPI) and the intensity of fluorescence emitted from the nuclei was measured.
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Epidermal tissue was peeled from the central half of the leaf between the tip and the base. At least five epidermal fragments from the adaxial or abaxial surface of 2–3 leaves were placed on a gelatin-coated microscope slide with their external face in contact with the gelatin, and stained with DAPI as described by Kinoshita et al. (1991). Mesophyll protoplasts were isolated from the leaves, flattened between the microscope slide and cover slip, and then stained with DAPI as described by Kinoshita et al. (1991). Experiments were repeated several times and the data were accumulated.
The fluorescence intensity of each nucleus was measured by cytofluorometry. Measurements were made under an epifluorescence microscope, a Nikon Optiphot (Nikon, Tokyo, Japan) equipped with a microphotometer Nikon P1 (Nikon, Tokyo, Japan). UV excitation (excitation filter, EX 330–380) was used. Emissions shorter than 470 nm were cut by an IF470 filter. Fluorescence intensity was measured earlier than 10 s after beginning excitation. Values were converted to arbitrary units by dividing them by the mean value of 20 measurements of chick red blood cells on the same slide.
Detection of DNA synthesis
5-bromo-2'-deoxyuridine (BrdU), an analogue of thymidine, was used. A primary leaf attached to a bean plant was submerged in 50 cm3 of BrdU solution (10 µM) in a 9 cm Petri dish. The Petri dish was then placed in a plastic bag, and the open end of the bag was sealed by wrapping cellophane tape around the petiole, after which the outside of the plastic bag was completely covered with aluminium foil because BrdU solution is light-sensitive (Darzynkiewicz and Juan, 1997). The primary leaves were incubated in BrdU solution for 19 h.
Epidermal tissues were peeled from the adaxial and abaxial surfaces, then guided by a dissecting microscope spread on the surface of a water droplet placed on a gelatin-coated microscope slide. The external face of the epidermis was pasted to the microscope slide by air-drying and then fixed with 100% methanol. This procedure was the same as the microscope slide preparation for cytofluorometry.
BrdU incorporated into the nuclear DNA was made visible by the method of Levi et al. (1987). Slides were hydrolysed in 1.5 N HCl for 30 min at 25 °C, washed thoroughly in TRIS buffer [10 mM tris (hydroxy-methyl)-aminomethane, 10 mM EDTA-2Na, 100 mM NaCl, pH 7.2 containing 0.5% (w/v) Triton X-100], and dried. Next, the samples were treated with anti-BrdU mouse monoclonal antibody (Becton Dickinson Immunocytometry Systems, San Jose, CA, USA) and goat anti-mouse IgG conjugated with FITC (Becton Dickinson Immunocytometry Systems, San Jose, CA, USA), after which they were counterstained for 15 min with DAPI, as described by Kinoshita et al. (1991), and observed under an epifluorescence microscope with appropriate filter combinations.
Leaf area and cell area determination
At least six primary leaves were harvested. They were photocopied, and the copied leaf shapes were cut out and weighed. Based on a paper weight of 10x10 cm (100 cm2), the weights of the copied leaf shapes were converted to leaf area.
Epidermal tissues were peeled from adaxial and abaxial surfaces of the leaves and pasted on to a microscope slide as described above. Next, photographs were taken under a microscope with a digital camera. For each sample, areas of 200 pavement cells were measured with the computer software Image-Pro Express (Media Cybernetics, Inc., Silver Spring, MD, USA). At least six leaves from separate plants were used in each measurement.
Statistical analysis
The data of leaf area and epidermal cell area were analysed, respectively, by the t test and the Mann–Whitney test.
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Results |
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Nuclear DNA content distribution in primary leaves of sunlight-grown bean plants
Using primary leaves of bean plants grown in ML, it has been shown that the nuclear DNA contents of adaxial and abaxial epidermal pavement cells differ from each other (Kinoshita et al., 1991). However, the nuclear DNA content of bean plants grown in sunlight has not been measured, although the intensity of sunlight is much higher. Figure 2 shows the nuclear DNA content distributions in adaxial and abaxial epidermal pavement cells. This indicates that most adaxial pavement cells have a 4C DNA nucleus and that most abaxial pavement cells have a 2C nucleus. This is similar to our previous reported findings about the plants grown under ML (Kinoshita et al., 1991). This indicates that ML is sufficient to produce the difference in nuclear DNA content between adaxial and abaxial pavement cells.
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Induction of rapid leaf expansion
When bean plants were grown under the LL condition, stems elongated, and LL-grown plants were more than twice the height of ML-grown ones at 13 d. When 13-d-old LL-grown plants (Fig. 3A) were transferred to ML, cells in the primary leaves enlarged rapidly (Fig. 3B). Both the thickness of the leaves and the thickness of epidermal cells increased markedly during the ML treatment (Fig. 3B). However, the thickness of epidermal cells was not measured as it was difficult to make accurate transverse sections suitable for quantitative analysis. Leaf area had increased by 58% 5 d after transfer to ML (Fig. 3C). Leaves of the plants exposed to ML for 5 d were significantly larger than those of LL-grown plants of the same age (P <0.001) (Fig. 3C). However, leaves of 18-d-old LL-grown plants were not significantly larger than those of 13-d-old LL-grown plants (P >0.05) (Fig. 3C). This shows that the increase in the leaf area of LL-grown plants had almost reached a plateau at 13 d. However, when 13-d-old plants were transferred to ML, leaf area increased rapidly.
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Changes in nuclear DNA content distribution after transfer to ML
When bean plants were grown under the LL condition, most adaxial epidermal pavement cells had a 2C DNA nucleus, and the 4C peak area did not increase even when plants reached 21-d-old (Fig. 4A–D). Moreover, abaxial pavement cells and mesophyll cells showed the same nuclear DNA content distributions as did those cells of ML-grown plants (Kinoshita et al., 1991); most of those cells had a 2C DNA nucleus (Fig. 4E–G).
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Next, 13-d-old plants grown under the LL condition were transferred to ML, and the nuclear DNA contents of their adaxial pavement cells were measured at 2, 4, 6, and 8 d after exposure (Fig. 5A–D). Two days after transfer to the ML condition, the 4C peak area had increased slightly and became higher at 4 d after transfer. Subsequently, the proportion of 4C nuclei did not change significantly. On the other hand, nuclear DNA content distribution in the abaxial epidermal pavement cells was not affected by exposure to ML (Fig. 5E, F). This was also the case with mesophyll cells (Fig. 5G). These results indicate that abaxial pavement cells and mesophyll cells had been differentiated from adaxial pavement cells in their reactivity to ML. Similar results were obtained when 12-d-old LL-grown plants were transferred to ML, but nuclear DNA content of most adaxial pavement cells doubled within two days (data not shown). Also, similar results were obtained when low intensity red light was used instead of LL, which is the same condition as that used by Van Volkenburgh and Cleland (1979) (data not shown).
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When 21-d-old LL-grown plants were transferred to ML, nuclear DNA content distributions of the leaves did not change (Fig. 6).
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Investigation of DNA synthesis
In nuclei whose DNA content increased from 2C to 4C, one round of DNA synthesis must have occurred. DNA synthesis in epidermal pavement cells was therefore investigated by a non-radioactive labelling method that used BrdU. BrdU was incorporated into replicating DNA in bean primary leaves and bound with anti-BrdU mouse monoclonal antibody. The antibody was then bound with anti-mouse goat IgG conjugated with FITC for visualization under a fluorescence microscope. Nuclei made visible with FITC fluorescence under blue excitation (Fig. 7A, C) had progressed through the S-phase. All the nuclei present were made visible with DAPI fluorescence under UV excitation (Fig. 7B, D), but only a small number were labelled with BrdU (arrows, Fig. 7B, D). Both the total and labelled nuclei were counted in the same field by fluorescence microscopy, respectively, under UV and blue excitation. The ratios of labelled nuclei were calculated (Fig. 8). No DNA synthesis occurred in guard cells and trichomes (Fig. 7).
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Cell division continued during the early phase of leaf development in leaves of dicotyledonous plants, division ceasing earlier in the tip than in the base (Granier and Tardieu, 1998, 1999; Tardieu and Granier, 2000). Epidermal tissue, peeled from the central half of the leaf between the tip and base, was used. To determine the age at which the cell cycle progression of adaxial and abaxial pavement cells stops, DNA synthesis was assayed in nuclei of pavement cells of primary leaves of bean plants grown under the ML or LL condition. In the adaxial epidermis of 10-d-old and 12-d-old ML-grown plants, respectively, 1.03% and 0.1% of the nuclei synthesized DNA in the period of BrdU uptake (19 h), whereas no synthesis was detected in nuclei in the abaxial epidermis (Fig. 8). However, in the primary leaves of LL-grown plants of the same ages, nuclear DNA synthesis was not detected in the adaxial or abaxial epidermal pavement cells. When plants were 13-d-old, no nuclear DNA synthesis was detected in either type of epidermal pavement cell, irrespective of the growth irradiance used (Fig. 8). This shows that mitotic cell cycle progression ceased before 10 d of age in both adaxial and abaxial epidermal pavement cells of LL-grown plants, as it did in abaxial pavement cells of ML-grown plants. In the case of adaxial pavement cells of ML-grown plants, DNA synthesis finished between 12 d and 13 d. This result agrees with the report of Van Volkenburgh and Cleland (1979) showing that primary leaves of bean plants grown in low intensity red light complete cell proliferation before 10 d.
To investigate the effect of elevated light intensity on nuclear DNA synthesis in leaf epidermal cells, 13-d-old LL-grown plants were transferred to ML. These LL-grown plants were exposed to ML for 1–3 d, after which their primary leaves were submerged in BrdU solution for 19 h to take it up. After exposure to ML for 1 d, 2.59% of the nuclei in adaxial pavement cells, but no nuclei in abaxial pavement cells, synthesized DNA (Fig. 8). After exposure to ML for 3 d also, 2.16% of the nuclei in the adaxial pavement cells and 0.06% (almost zero) of those in the abaxial pavement cells synthesized DNA. Similar results were obtained when both 10-d-old and 12-d-old LL-grown plants were transferred to the ML condition (data not shown). The frequencies of the nuclei that synthesized DNA during 19 h of BrdU uptake ranged from about 2% to 3% in adaxial pavement cells (Fig. 8) clearly showing that ML induced nuclear DNA synthesis in these cells, although the efficiency of BrdU uptake might be low.
In every experiment done in this study, the light source was positioned higher than the plants. Therefore, light intensity at the upper surface of the leaves was higher than that at the lower surface. Was this the cause of the different responses between adaxial and abaxial epidermal pavement cells? To answer this question, primary leaves of 13-d-old LL-grown plants were fixed on a glass plate with the abaxial surface facing fluorescent lamps and kept for 1 d, after which the leaves were submerged in BrdU solution. Although the abaxial surface received higher irradiance than the adaxial one, DNA synthesis frequencies of the nuclei were 2.09% for the adaxial and 0% for the abaxial epidermal pavement cells (Fig. 8). These results indicate that adaxial pavement cells have a different property than abaxial ones.
Cell enlargement after transfer to ML
Photographs of peeled epidermal tissues (Fig. 9A–F) show that adaxial pavement cells are much larger than abaxial ones at 12 d after transfer to ML (Fig. 9C, F). To compare cell sizes quantitatively, areas of pavement cells were measured. In 13-d-old LL-grown plants, median cell area of abaxial pavement cells was 673 µm2. The size increased by 53% in 4 d after transfer to ML (1027 µm2) and remained unchanged thereafter (951 µm2 at 12 d after transfer to ML) (Fig. 9H). The size at 4 d after transfer to ML was significantly different from the size of abaxial cells in 13-d-old LL-grown plants (P=0.008). However, abaxial cell size was not significantly different between 4 d and 12 d after transfer (P=0.912). In contrast to abaxial cells, adaxial pavement cells continuously enlarged for 12 d after transfer to ML (Fig. 9G). The median size of adaxial cells in 13-d-old LL-grown plants was 958 µm2, the size was 1817 µm2 at 4 d after transfer and 2294 µm2 at 12 d after transfer. The size at 4 d after transfer was significantly different from the size of adaxial cells in 13-d-old LL-grown plants (P <0.001). The size of adaxial cells at 12 d after transfer was also significantly different from the size of adaxial cells at 4 d after transfer (P=0.005). These results show that both nuclear DNA doubling (Fig. 5) and continuous cell enlargement occurred in adaxial pavement cells after transfer to ML. At 12 d after transfer to ML, adaxial pavement cells were 2.4 times the size of abaxial ones, although this ratio had been 1.4 times in 13-d-old LL-grown plants. This seems to be a cause of bulging (Fig. 9I) of the adaxial side of the primary leaves. The leaves had been flat before transfer to ML.
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Discussion |
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When bean plants grown in low intensity red light were transferred to bright white light, leaf expansion began within 10–20 min (Dale, 1988). This leaf expansion is brought about by activation of the proton pump in the plasma membrane and loosening of acidified cell wall (Van Volkenburgh and Cleland, 1980; Cleland et al., 1983; Staal et al. 1994; Linnemeyer et al., 1990; Stahlberg and Van Volkenburgh, 1999). In contrast to this short-term response, it is shown here that the nuclear DNA content of adaxial epidermal pavement cells of 13-d-old LL-grown plants increased 4 d after transfer to ML (Fig. 5). This is a long-term response. This increase in the proportion of 4C nuclei is paralleled by cell enlargement (Fig. 9). However, when 21-d-old LL-grown plants were transferred to ML, the increase in the proportion of 4C nuclei did not occur (Fig. 6). This indicates that the bright white light-induced nuclear DNA doubling does not occur after the beginning of leaf senescence.
According to the results obtained in this study, changes of ploidy level and cell size after transfer to ML are schematically shown in Fig. 10 for adaxial and abaxial pavement cells of 13-d-old LL-grown plants. In abaxial pavement cells, ploidy level remained unchanged during the experimental period, whereas cell size increased in the early phase. This cell enlargement is brought about by activation of the plasma membrane proton pump and loosening of acidified wall. However, cell enlargement finished 4 d after transfer to ML and cell size remained unchanged thereafter. On the other hand, ploidy level increased to 4C in most adaxial pavement cells within 4 d and cell enlargement continued after the increase in ploidy level, suggesting that cell enlargement occurring after 4 d is supported by nuclear polyploidization.
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When 13-d-old LL-grown plants were transferred to ML, leaf area increased by 58% in 5 d (Fig. 3) and cell area of abaxial and adaxial pavement cells increased, respectively, by 53% and 89% in 4 d (Fig. 9G, H). The reason why the rate of increase of adaxial pavement cell area exceeded that of leaf area is probably that the leaf surfaces were not flat. These results suggest that few cell divisions occurred in the epidermal pavement cells after transfer to ML.
In our previous work (Kinoshita et al., 1991), it was shown that benzyladenine treatment did not induce cell division but induced an increase in nuclear DNA content from 4C to 8C in most adaxial epidermal pavement cells of 13-d-old ML-grown bean primary leaves. This indicates that these cells had switched from mitotic cell cycle to endocycle when cell proliferation had finished. Therefore, the ML-induced increase in nuclear DNA content from 2C to 4C in adaxial epidermal cells of the leaves of LL-grown plants (Fig. 5A-D) is shown to be an endopolyploidization.
It is known that there is proportionality between nuclear DNA level and cell size in epidermal pavement cells of Arabidopsis leaves (Melaragno et al., 1993). This suggests that cell ploidy has some impact on cell size determination (Sugimoto-Shirasu and Roberts, 2003). However, phenomena contradicting this relationship are often observed. For example, Beemster et al. (2002) showed that root cells from different ecotypes of Arabidopsis are of various sizes but that there was no correlation between mature cell size and endoreduplication. In this study, nuclear DNA content in abaxial pavement cells of LL-grown plants did not change after transfer to ML (Fig. 5E, F), although the cell area of those cells increased by 53% (Fig. 9H). Consequently, it has been claimed that there is a positive correlation between nuclear DNA content and amount of cytoplasm and that some control mechanism ensures that the amount of cytoplasm a cell can make and sustain is proportional to the amount of DNA in its nucleus (Sugimoto-Shirasu and Roberts, 2003). Both cell growth (i.e. increase in cytoplasmic macromolecular mass) and cell expansion (i.e. increase in cell volume through vacuolation) contribute independently to an increase in cell size in plants (Sugimoto-Shirasu and Roberts, 2003). Endoreduplication could allow greater cell growth, but cell expansion (vacuolation) occurs independently of endoreduplication. Furthermore, the correlation between ploidy level and cell growth is explained as follows: an increase in nuclear DNA content could induce an increase in protein synthesis activity of the cells through activation of ribosome regeneration and gene expression (Nagl, 1976; Barow and Meister, 2003; Sugimoto-Shirasu and Roberts, 2003). In this study, it is shown that nuclear DNA doubling only occurred in adaxial pavement cells. This suggests that not only vacuolation but also cell growth contributed to the enlargement of adaxial epidermal cells (Fig. 9).
Another possible significance of the increase in nuclear DNA content in adaxial epidermal pavement cells could be protection of cells from irradiation damage (Nagl, 1978), because solar radiation is absorbed first by the adaxial epidermal cells in field-grown plants.
It is well known that endopolyploidy levels differ largely among plant species. For example, Arabidopsis leaf cells have 2C, 4C, 8C, and 16C nuclei (Galbraith et al., 1991). In woody plants, in contrast, the mean number of endoreduplication cycles per nucleus (cycle value) is very low (Barow and Meister, 2003), namely, endoreduplication rarely occurs in woody plants. In the leaves of poplar (Populus sieboldii Miq.), all adaxial and abaxial epidermal pavement cells had a 2C nucleus, irrespective of light intensity during their growth (I Kinoshita, unpublished data).
Nagl (1976) demonstrated that there is a negative correlation between genome size and endopolyploidy level. To explain the significance of this phenomenon, it has been suggested that endopolyploidization occurs in the cells of species with a small genome in order to supply a minimum amount of nuclear DNA to maintain the regulatory and functional state (Nagl, 1976; De Rocher et al., 1990; Galbraith et al., 1991). On the other hand, Barow and Meister (2003) conjectured that endopolyploidization is a means to accelerate the growth of the plant species in niches, which require and support fast development. Genome sizes (2C) of P. vulgaris and Arabidopsis thaliana are 1.58 pg and 0.43 pg, respectively (Barow and Meister, 2003). The mean numbers of endoreduplication cycles per nucleus are 0.31 and 1.66 for lower leaves of P. vulgaris and A. thaliana, respectively (Barow and Meister, 2003). This agrees with Nagl's hypothesis (Nagl, 1976). In this study, the following was shown: although the average endopolyploidization level of bean primary leaves is low, endopolyploidization occurred specifically in adaxial pavement cells, which rapidly grew for a long time under moderate irradiance conditions.
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Footnotes |
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Present address: Development Laboratory 5, Lion Corporation, 13-12 Hirai 7-chome, Edogawa-ku, Tokyo 132-0035, Japan. 
Deceased in June 2006. |
References |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Barow M, Meister A. Endopolyploidy in seed plants is differently correlated to systematics, organ, life strategy and genome size. Plant, Cell and Environment (2003) 26:571–584.[CrossRef]
Beemster GTS, De Veyler L, Vercruysse S, West G, Rombaut D, Van Hummelen P, Galichet A, Gruissem W, Inzé D, Vuylsteke M. Genome-wide analysis of gene expression profiles associated with cell cycle transitions in growing organs of Arabidopsis. Plant Physiology (2005) 138:734–743.
Beemster GTS, De Vusser K, De Tavernier E, De Bock K, Inzé D. Variation in growth rate between Arabidopsis ecotypes is correlated with cell division and A-type cyclin-dependent kinase activity. Plant Physiology (2002) 129:854–864.
Cavallini A, Baroncelli S, Lercari B, Cionini G, Rocca M, D'Amato F. Effect of light and gibberellic acid on chromosome endoreduplication in leaf epidermis of Triticum durum Desf. Protoplasma (1995) 186:57–62.[CrossRef][Web of Science]
Cleland RE, Cosgrove D, Tepfer M. Characterization of the in vitro acid extension curves of Avena coleoptiles. Planta (1983) 170:379–385.[CrossRef]
Cookson SJ, Radziejwoski A, Granier C. Cell and leaf size plasticity in Arabidopsis: what is the role of endoreduplication? Plant, Cell and Environment (2006) 29:1273–1283.[CrossRef][Medline]
Dale JE. The control of leaf expansion. Annual Review of Plant Physiology and Plant Molecular Biology (1988) 39:267–295.[CrossRef][Web of Science]
Darzynkiewicz Z, Juan G. Analysis of DNA content and BrdU incorporation. In: Current protocols in cytometry—Robinson JP, Darzynkiewicz Z, Hyun W, Orfao A, Rabinovitch PS, eds. (1997) New York: John Wiley & Sons, Inc. 7.7.1–7.7.9.
De Rocher EJ, Harkins KR, Galbraith DW, Bohnert HJ. Developmentally regulated systemic endopolyploidy in succulents with small genomes. Science (1990) 250:99–101.
Galbraith DW, Harkins KR, Knapp S. Systemic endopolyploidy in Arabidopsis thaliana. Plant Physiology (1991) 96:985–989.
Gendreau E, Höfte H, Grandjean O, Brown S, Traas J. Phytochrome controls the number of endoreduplication cycles in the Arabidopsis thaliana hypocotyl. The Plant Journal (1998) 13:221–230.[CrossRef][Web of Science][Medline]
Gendreau E, Traas J, Desnos T, Grandjean O, Caboche M, Höfte H. Cellular basis of hypocotyl growth in Arabidopsis thaliana. Plant Physiology (1997) 114:295–305.[Abstract]
Grafi G, Larkins BA. Endoreduplication in maize endosperm: involvement of M phase-promoting factor inhibition and induction of S phase-related kinases. Science (1995) 269:1262–1264.
Granier C, Tardieu F. Spatial and temporal analyses of expansion and cell cycle in sunflower leaves. A common pattern of development for all zones of a leaf and different leaves of a plant. Plant Physiology (1998) 116:991–1001.
Granier C, Tardieu F. Water deficit and spatial pattern of leaf development. Variability in responses can be simulated using a simple model of leaf development. Plant Physiology (1999) 119:609–619.
Griffiths PD, Ougham HJ, Jones RN. Genotypic and environmental effects on endopolyploidy in the epidermal tissues of Lolium perenne L. and Lolium multiflorum Lam. New Phytologist (1994) 128:339–345.[CrossRef][Web of Science]
Imai KK, Ohashi Y, Tsuge T, Yoshizumi T, Matsui M, Oka A, Aoyama T. The A-type cyclin CYCA2;3 is a key regulator of ploidy levels in Arabidopsis endoreduplication. The Plant Cell (2006) 18:382–396.
Kinoshita I, Sanbe A, Yokomura E. Increases in nuclear DNA content without mitosis in benzyladenine-treated primary leaves of intact and decapitated bean plants. Journal of Experimental Botany (1991) 42:667–672.
Larkins BA, Dilkes BP, Dante RA, Coelho CM, Woo Y, Liu Y. Investigating the hows and whys of DNA endoreduplication. Journal of Experimental Botany (2001) 52:183–192.
Levi M, Sparvoli E, Sgorbati S, Chiatante D. Rapid immunofluorescent determination of cells in the S phase in pea root meristems: an alternative to autoradiography. Physiologia Plantarum (1987) 71:68–72.[CrossRef]
Linnemeyer P, Van Volkenburgh E, Cleland RE. Characterization and effect of light on the plasma membrane H+-ATPase of bean leaves. Plant Physiology (1990) 94:1671–1676.
Melaragno JE, Mehrotra B, Coleman AW. Relationship between endopolyploidy and cell size in epidermal tissue of Arabidopsis. The Plant Cell (1993) 5:1661–1668.[Abstract]
Nagl W. DNA endoreduplication and polyteny understood as evolutionary strategies. Nature (1976) 261:614–615.[CrossRef][Medline]
Nagl W. Endopolyploidy and polyteny in differentiation and evolution. Towards an understanding of quantitative and qualitative variation of nuclear DNA in ontogeny and phylogeny (1978) Amsterdam: Elsevier/North-Holland Biochemical Press.
Rayle DL, Cleland RE. The acid growth theory of auxin-induced cell elongation is alive and well. Plant Physiology (1992) 99:1271–1274.
Schlayer G. Modifikationen des DNS-Gehalts in Zuckerrübenzellen. Planta (1971) 98:294–299.[CrossRef][Web of Science]
Staal M, Elzenga JTM, Van Elk AG, Prins HBA, Van Volkenburgh E. Red and blue light-stimulated proton efflux by epidermal leaf cells of the Argenteum mutant of Pisum sativum. Journal of Experimental Botany (1994) 45:1213–1218.
Stahlberg R, Van Volkenburgh E. The effect of light on membrane potential, apoplastic pH and cell expansion in leaves of Pisum sativum L. var Argenteum. Role of the plasma-membrane H+-ATPase and photosynthesis. Planta (1999) 208:188–195.[CrossRef][Web of Science]
Sugimoto-Shirasu K, Roberts K. ‘Big it up’: endoreduplication and cell-size control in plants. Current Opinion in Plant Biology (2003) 6:544–553.[CrossRef][Web of Science][Medline]
Tardieu F, Granier C. Quantitative analysis of cell division in leaves: methods, developmental patterns and effects of environmental conditions. Plant Molecular Biology (2000) 43:555–567.[CrossRef][Web of Science][Medline]
Van Volkenburgh E. Leaf expansion: an integrating plant behaviour. Plant, Cell and Environment (1999) 22:1463–1473.[CrossRef]
Van Volkenburgh E, Cleland RE. Separation of cell enlargement and division in bean leaves. Planta (1979) 146:245–247.[CrossRef][Web of Science]
Van Volkenburgh E, Cleland RE. Proton excretion and cell expansion in bean leaves. Planta (1980) 148:273–278.[CrossRef][Web of Science]
Van Volkenburgh E, Cleland RE. Light-stimulated cell expansion in bean (Phaseolus vulgaris L.) leaves. I. Growth can occur without photosynthesis. Planta (1990) 182:72–76.[Web of Science][Medline]
Van Volkenburgh E, Cleland RE, Watanabe M. Light-stimulated cell expansion in bean (Phaseolus vulgaris L.) leaves. II. Quantity and quality of light required. Planta (1990) 182:77–80.[Web of Science][Medline]
Yoshizumi T, Tsumoto Y, Takiguchi T, Nagata N, Yamamoto YY, Kawashima M, Ichikawa T, Nakazawa M, Yamamoto N, Matsui M. INCREASED LEVEL OF POLYPLOIDY1, a conserved repressor of CYCLINA2 transcription, controls endoreduplication in Arabidopsis. The Plant Cell (2006) 18:2452–2468.
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