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Dep. of Agronomy and Plant Genetics, Univ. of Minnesota, 1991 Upper Buford Circle, St. Paul, MN 55108
* Corresponding author (jones012{at}tc.umn.edu)
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Abbreviations: C, the DNA content of a haploid nucleus in maize • DAP, d after pollination • FCM, flow cytometry • FW, fresh weight • HTT, high temperature treatment • LSD, least significant difference
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Maize endosperm development consists of several different phases. The endosperm nucleus results from a fusion of one sperm with two polar nuclei. Mitotic divisions are observed within 3 to 5 h of fertilization (Kiesselbach, 1949). At 3 d after fertilization, cell walls are developed, and 2 d later the endosperm is completely celluarized (Kiesselbach, 1949). Most of the endosperm cells are produced between 4 and 12 DAP, with the mitotic index peaking between 6 and 8 DAP (Kowles and Phillips, 1988; Engelen-Eigles et al., 2000). DNA endoreduplication and starch synthesis start between 10 and 12 DAP (Jones et al., 1985; Kowles and Phillips, 1985).
DNA endoreduplication leads to enlarged nuclei with elevated DNA contents. DNA endoreduplication has been observed in the endosperm of Z. mays L. (Kowles and Phillips, 1985) and Lycopersicon esculentum Mill. (Bino et al., 1992), as well as in other tissues in plants, for example Abies balsamea (L.) Mill. (Mellerowicz and Riding, 1992) and Arabidopsis thaliana (L.) Heynh. (Galbraith et al., 1991). During maize endosperm development, the cell cycle takes two discrete forms: a mitotic cycle and an endoreduplication cycle. The mitotic cell cycle consists of G1, S, G2, and M; thus the nuclear DNA value of an endosperm mitotic cell is expected to be 3 or 6 C. However, during DNA endoreduplication, the cell cycle consists of G and S only; the cells do not divide, and no mitosis-like structural changes can be seen in the nucleus (Nagl, 1982, 1990). Recent research shows that endoreduplicating tobacco (Nicotiana tabacum L.) cells can revert back to cell division when supplied with auxin and cytokinin, resulting in a reduction of the DNA content per cell (Valente et al., 1998). The intricate details of the regulation of the endoreduplication cycle are beginning to be unraveled. Recent molecular studies suggest that the inactivation of p34cdc2/cyclin B kinase, a mitotic kinase, and the activation of S-phase related protein kinases are required for DNA endoreduplication to occur (Grafi and Larkins, 1995; Nagl, 1995; Grafi, 1998).
Maize DNA endoreduplication starts at 
10 DAP and peaks at 16 DAP, with the presence of at least four DNA content classes (12, 24, 48, 96 C or higher). By 18 DAP, the resolution of the individual C-classes becomes unclear, which is thought to be due to DNA degradation and/or starch granule interference, or it may be an artifact of the DNA preparation protocol (Kowles and Phillips, 1985). The onset of DNA endoreduplication and the number of C-classes depend on the maize genotype (Kowles et al., 1997) and environment (Meyers et al., 1992; Engelen-Eigles et al., 2000). The magnitude of maize endosperm DNA endoreduplication appears to be maternally inherited (Kowles et al., 1997), and the extra DNA synthesized by the endoreduplication process is likely important in maize kernel development (Kowles and Phillips, 1985; Kowles et al., 1992). It has been suggested that the increased DNA content during endoreduplication may provide for increased gene expression during endosperm development and kernel filling, since it coincides with increased enzyme activity and protein accumulation at this time (Kowles et al., 1992). DNA content and endosperm FW are highly correlated r = 0.93) between 4 to 18 DAP, indicating the importance of DNA endoreduplication in determining kernel mass (Engelen-Eigles et al., 2000).
Environmental perturbations such as water-deficit and exposure to high temperature affect the maize endosperm cell cycle and the magnitude of endoreduplication, and lead to reduced nuclei number and mature kernel mass (Meyers et al., 1992; Engelen-Eigles et al., 2000). In vitro studies have shown that kernel water potential decreases in response to decreased media water potential (Meyers et al., 1992). Media of -1.6 or -2.0 MPa resulted in a reduction of endosperm cell number compared with the nonstressed kernels (Meyers et al., 1992). Maize endosperm DNA endoreduplication was significantly reduced when water stress treatments were given from 1 to 10 DAP (Artlip et al., 1995). While the optimum temperature for maize kernel development is between 27 and 32°C (Keeling and Greaves, 1990), Jones et al. (1984) found that nuclei number, starch granule number, and kernel FW were reduced by exposure to 35°C for 6 to 8 d during the most sensitive phase of endosperm cell division (4 to 10 DAP). Night temperatures above 30°C during the early stages of maize endosperm development are especially detrimental to maize kernel development (Teixeira, 1995). Our recent studies show that the magnitude of maize endosperm DNA endoreduplication is reduced by 4- and 6-d HTTs imposed at 4 DAP. Since such treatment causes the endosperm cells to remain predominately mitotic, we concluded that HTT restricts the entry of mitotic cells into the endoreduplication phase of the cell cycle (Engelen-Eigles et al., 2000). However, the specific interval during the early stages of maize endosperm development during which the DNA endoreduplication process is most sensitive to HTTs remains unclear. Moreover, it is not known to what extent HTT affects DNA endoreduplication per se. Thus, the specific objective of this study was to determine when during endosperm development short-term (4 d) or long-term (6 d) exposure to HTT is most detrimental to endosperm DNA endoreduplication and kernel development in maize.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Endosperm nuclei (a combination of three endosperms per petri dish) were stained with mithramycin A (a GC-binding DNA fluorochrome; Sigma, St. Louis, MO) for analysis by flow cytometry (FCM) and were prepared as follows: endosperm cells were carefully dissected from the kernels and macerated with a flattened probe by forcing them through a 150-µm mesh screen (Bellco Glass Inc., Vineland, NJ) placed on top of a small funnel in a 1.5-mL microfuge tube. The screen was washed three times with 500 µL grinding buffer [100 mM glycine, 10.0 mL L-1 hexylene glycol (v/v), 1.0 mL L-1 Triton X-100 (v/v), and 2.0 g L-1 phenylmethylsulfonylfluoride (w/v)]. The sample was then centrifuged for 1 min at 180 g. The supernatant was decanted and the pellet resuspended in 400 µL mithramycin buffer [45 mM MgCl2, 30 mM sodium citrate, 20 mM 3-(N-Morpholino) propanesulphoric acid, 1.0 mL L-1 Triton X-100 (v/v), pH adjusted to 7.0 with 1.0 M NaOH], and allowed to equilibrate for 30 min. The samples were again centrifuged for 1 min at 180 g, and the pellet was then resuspended in 200 µL mithramycin A stain solution (0.25 mg mithramycin A mL-1 mithramycin buffer). Endosperm nuclei were stained overnight in complete darkness (Kowles et al., 1994). The magnitude of DNA endoreduplication was determined with a Coulter Epics flow cytometer (Coulter Corp., Hialeah, FL). The water-cooled argon laser was aligned at 488 nm with DNA microspheres at a coefficient of variance <2.0. The laser was set to 455 nm for endosperm DNA content analysis. Up to 2000 nuclei were analyzed for each sample. The sample readings were gated to eliminate nuclear debris. Only samples up to 18 DAP were analyzed by FCM because later samples contained too much starch for accurate analysis.
Endosperm FW data were taken at the time of endosperm preparation for FCM; nine endosperms (three kernels per petri dish x three replicates) per HTT were weighed to determine endosperm FW. Nuclei number was determined by FCM by adding a known concentration of DNA microspheres (DNA Check, Coulter Corp., Hialeah, FL) to the endosperm nuclei preparation. The flow cytometer was programmed to run up to 2000 DNA microspheres while the nuclei were being counted. These data were used to calculate the number of nuclei per endosperm. The mitotic index was determined as the number of cells in prophase, anaphase, metaphase, and telophase divided by the total number of cells. For mitotic index determination, endosperm tissue was stained with acetocarmine and squashed with an iron needle. The preparation was briefly heated to clear the stain from the cytoplasm. Six endosperms (two kernels per petri dish x three replicates) were prepared, and 300 nuclei per endosperm were counted for mitotic index determination.
Statistical Design
Treatments were arranged in a complete randomized design. Each treatment consisted of 3 petri dishes with 5 kernels per petri dish. The experiment was performed in 1994 and repeated in 1995. Analysis of variance was calculated, and means were compared using the least significant difference (LSD) test at P < 0.05. There was no statistical difference between years, therefore the data were pooled. All data were analyzed with Statistix (Analytical Software, St. Paul, MN).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The 4-d HTT initiated at 10 or 12 DAP did not result in lower nuclei numbers compared with the control at 16 and 18 DAP (Fig. 2A). The mitotic index was low for these treatments and not significantly different from the control (Fig. 2B). Comparable to the 4-d HTT, the 6-d HTT initiated at 4 and 6 DAP reduced the endosperm nuclei number more dramatically than when this treatment was started at 8, 10, or 12 DAP (data not shown). At 18 DAP, nuclei numbers in all 6-d HTTs were significantly lower than in the control endosperm. Analogous to the endosperm FW data, the endosperm nuclei number and mitotic index data also support the suggestion that 4 to 10 DAP is the most sensitive period to high temperature.
DNA Endoreduplication
The DNA content of maize endosperm cells are broadly divided into two levels: one level corresponds to cells with a mitotic DNA content of 3 or 6 C, and another level corresponds to cells with an endoreduplicating DNA content of 12 C or higher. It is important to note, however, that a cell with a 6 C content can divide and result in two 3 C cells, or it can continue into the endoreduplicating phase of the cell cycle. Unfortunately, the flow cytometric procedure employed in this study did not permit us to distinguish 6 C cells that are being committed to endoreduplication from those that remain mitotic. The mitotic DNA content (3 and 6 C) for the control nuclei declined over time from 84% at 10 DAP to 41% at 18 DAP (Fig. 3A). The mitotic DNA content for endosperms of kernels exposed to 4-d HTT imposed at 4 or 6 DAP also declined over the period sampled, but remained significantly higher compared with that of the control endosperm at 14, 16, and 18 DAP (Fig. 3A). With the exception of 16 DAP, the mitotic DNA content for the 4-d HTT imposed at 10 DAP was not significantly different from that of the control. Likewise, the mitotic DNA content for the 4-d HTT imposed at 12 DAP was not significantly different from that of the control at all sampling dates (Fig. 3A).
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To elucidate when DNA endoreduplication is most vulnerable to high temperature, both the 4- and 6-d HTTs were imposed at the time of high mitotic activity (4 to 10 DAP) and when DNA endoreduplication had been initiated (10 DAP). DNA endoreduplication was observed at 10 DAP for the 4-d HTT imposed at 4 and 6 DAP (Fig. 3B). At 10 DAP, the 4-d HTT imposed at 4 and 6 DAP showed 14% and 13% of the nuclei with an endoreduplicating DNA content (Fig. 3B). The endoreduplicating DNA content increased from 10 to 18 DAP for these two HTTs, but the increase in percentage of nuclei with an endoreduplicating DNA content was not as dramatic as that observed for the control endosperm and the HTT imposed after 8 DAP. For example, the 4-d HTT beginning at 4 DAP showed that only 23% of the nuclei left the 3- and 6-C pool for endoreduplication between 10 and 18 DAP (Fig. 3B). In contrast, the control endosperm showed 43% of the nuclei entering the DNA endoreduplication phase of the endosperm cell cycle during the same time interval.
The results from the 4-d HTT applied when DNA endoreduplication was in progress were considerably different from the results obtained when these HTTs were applied during the mitotic phase of endosperm development. The 4-d HTT imposed at 8, 10, or 12 DAP resulted in a percentage of nuclei with an endoreduplicating DNA content that was not significantly different from the control nuclei at 14, 16, and 18 DAP (Fig. 3B). The 6-d HTT yielded similar results to the 4-d HTT (data not shown). The results from the 4-d or 6-d HTT confirmed that DNA endoreduplication was most reduced when these treatments were applied during the mitotic phase of endosperm development (4–10 DAP). When these treatments were initiated at 10 or 12 DAP (i.e., during endoreduplication), the extent of DNA endoreduplication was much less affected. Therefore, the DNA endoreduplication process itself appeared to be quite resistant to either short- or long-term exposure to high temperature.
Average DNA Content
The average DNA content per endosperm was calculated by multiplying the percentage of nuclei in each C class by the C value of that class. Thus, a group of endosperm cells containing mostly endoreduplicating nuclei will yield a higher average DNA content than one with the majority of its nuclei as mitotic nuclei (3 and 6 C). The control endosperm showed a gradual increase in average DNA content to reach its maximum at 18 DAP (Fig. 4).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Maize endosperm FW was significantly reduced by the HTTs that were imposed during the mitotic phase (4–10 DAP) of the endosperm cell cycle. In contrast, the HTTs given during the endoreduplication phase of the endosperm cell cycle (10 to 16 DAP) resulted in a FW reduction that was less severe. The control endosperm FW increased steeply from 14 to 18 DAP, whereas the FW for the HTTs did not (Fig. 1). The reduction in FW by the HTT imposed during the mitotic phase of the cell cycle might have been the result of reduced cell proliferation and cell expansion growth. Indeed, a 4-d HTT imposed early in development resulted in ultra-structural changes that led to less endosperm cavity filling (Commuri and Jones, 1999). In addition, the FW data suggest that starch biosynthesis, which normally starts between 14 and 16 DAP, was optimal for the control endosperm and contributed significantly to the endosperm FW at 18 DAP. Starch biosynthesis may have been less optimal for the HTTs applied from 4 to 10 DAP, thus contributing less to the lower rate of endosperm growth. We have reported that starch granule number was reduced in maize endosperm that was exposed to 35°C during the early stages of kernel development (Jones et al., 1984 and 1985; Commuri and Jones, 1999). The 4- and 6-d HTTs applied early during kernel development (4 DAP) has been shown to reduce the activity of RNA transcript levels of the key starch synthesis enzymes such as ADPglucose pyrophosphorylase (ADPG-Ppase) and soluble and insoluble forms of starch synthase (Teixeira, 1995; Commuri, 1997). We surmise from the current data that such effects would be more severe when the HTT are imposed during the mitotic stage of endosperm development than during the endoreduplication stage.
The endosperm nuclei number (i.e., cell number) was also significantly reduced by the HTTs when applied early in endosperm development. These data are in agreement with earlier observations (Jones et al., 1984; Commuri and Jones, 1999; Engelen-Eigles et al., 2000). Coincident with the nuclei number data, the 4-d HTT imposed at 4 or 6 DAP delayed the peak mitotic index. Generally, the mitotic index for endosperm growing at 25°C peaks between 6 and 8 DAP (Fig. 2B) (Kowles and Phillips, 1988; Engelen-Eigles et al., 2000). The reduced nuclei number observed when the 4-d HTT was imposed at 4 or 6 DAP may be due to the disruption of mitosis as was shown by the delay in the mitotic index. Indeed, when the 4-d HTT was given after the mitotic phase of the endosperm cell cycle (e.g., at 10 or 12 DAP), nuclei number and mitotic index were not significantly different from those of the control endosperm. Therefore, the data show that the imposition of HTTs during the period of active cell division in the endosperm results in a significant decrease in kernel sink capacity via a reduction in the number of endosperm cells formed.
The magnitude of DNA endoreduplication is significantly affected when 4-d HTTs are imposed during the mitotic phase of kernel development; this may further contribute to the decrease in sink potential. These HTTs led to a reduced percentage of nuclei with an endoreduplicating DNA content (Fig. 3B) and to a significantly lower average DNA content (Fig. 4). The majority of nuclei remained mitotic (3 or 6 C) when the 4-d HTTs were initiated at 4 or 6 DAP (Fig. 3a), thus supporting the hypothesis that HTT seem to restrict the entry of mitotic cells into the endoreduplication cell cycle. Commuri and Jones (1999) have shown that kernels exposed to 4- and 6-d HTTs showed irregular-shaped nuclei and altered size of the nucleolus. Clearly, such structural changes may have resulted in copious numbers of cells with nuclei that had lost the competency to move to the endoreduplication phase of the endosperm cell cycle. In addition, the disruption of the endoreduplication cycle induced by the HTTs imposed at 4 and 6 DAP may have been due to the disruption of key molecular processes such as the inactivation of p34cdc2/cyclin B kinase, believed to be part of the required molecular processes for DNA endoreduplication to occur (Grafi and Larkins, 1995; Grafi, 1998; Nagl, 1995). The prolonged cell divisions observed for these 4-d HTTs (Fig. 2A) suggest that the p34cdc2/cyclin B kinase complex was not completely deactivated. These data suggest that further research should focus on the effect of HTTs on the molecular processes that control DNA endoreduplication.
From this study, it was clear that HTTs did not have a direct effect on DNA endoreduplication, since the 4-d HTT starting at 8, 10, or 12 DAP did not affect the magnitude of DNA endoreduplication and resulted in an average DNA content at 18 DAP that was similar to that of the control (Fig. 3 and 4). Thus, these data indicate that the DNA endoreduplication process itself is quite resilient to high temperature. It is the period before the cell cycle is committed to DNA endoreduplication (e.g., during the mitotic phase of the cell cycle) that is most sensitive to high temperature, which results in reduced DNA endoreduplication later in kernel development.
The present findings are in concert with the water deficit studies on maize endosperm DNA endoreduplication by Artlip et al. (1995). They concluded that DNA endoreduplication was significantly reduced when water stress treatments were given from 1 to 10 DAP. Water stress applied later in endosperm development was less deleterious to DNA endoreduplication than when given early in endosperm development. We conclude that the period from 4 to 10 DAP, that is, the time during which endosperm cells are in a mitotic cell cycle, is the most sensitive period to environmental perturbations. The magnitudes of endosperm FW, nuclei number, and endosperm DNA endoreduplication are clearly determined during this sensitive period in maize endosperm development.
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ACKNOWLEDGMENTS |
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Received for publication May 6, 2000.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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