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Flow cytometry (FCM) can be used to study cell cycle activity in developing, mature and germinating seeds. It provides information about a seed's physiological state and therefore can be used by seed growers for assessing optimal harvest times and presowing treatments. Because an augmented proportion of 4C nuclei usually is indicative of high mitotic activity, the 4C/2C ratio is commonly used to follow the progress of seed development and germination. However, its usefulness for polysomatic (i.e., containing cells with different DNA content) seeds is questioned. Changes in cell cycle/endoreduplication activity in developing seeds of five members of the Fabaceae were studied to determine a more suitable marker of seed developmental stages for polysomatic species based on FCM measurements. Seeds of Phaseolus vulgaris, Medicago sativa, Pisum sativum, Vicia sativa, and Vicia faba var. minor were collected 20, 30, 40, 50, and 60 days after flowering (DAF), embryos were isolated and the proportion of nuclei with different DNA contents in the embryo axis and cotyledon was established. The ratios 4C/2C and (Σ>2C)/2C were calculated. Dried seeds were subjected to laboratory germination tests following international seed testing association (ISTA) rules. Additionally, the absolute nuclear DNA content was estimated in the leaves of the studied species. During seed development nuclei with DNA contents from 2C to 128C were detected; the endopolyploidy pattern depended on the species, seed organ and developmental stage. The cell cycle/endoreduplication parameters correlated negatively with genome size. The (Σ>2C)/2C ratio in the cotyledons reflected the seed developmental stage and corresponded with seed germinability. Therefore, this ratio is recommended as a marker in polysomatic seed research and production instead of the 4C/2C ratio, which does not consider the occurrence of endopolyploid cells. © 2012 International Society for Advancement of Cytometry
In seeds, cell cycle activity corresponds with their physiological state and therefore can be used to follow seed development, maturation, and germination, as well as to control presowing priming treatments (1). Cell cycle progression is marked by changes in nuclear DNA content. At the G1 phase nuclei possess a 2C DNA content; some cells exit the cell cycle at this phase and enter a quiescent stage called G0, where they remain metabolically active but no longer proliferate (2). During the S phase doubling of DNA occurs, from 2C to 4C. When DNA synthesis is completed there is a second interval, called G2, during which the cell grows as synthesis of proteins that are necessary for mitosis takes place. During mitosis (the M phase) DNA is distributed to two daughter cells which consequently, after cellular division, possess 2C DNA. Because flow cytometry (FCM) is a fast and accurate method for estimation of DNA content in different plant tissues, it can be applied to the detection of nuclei at particular stages of the cell cycle in intact seeds and/or in different seed parts (e.g., embryo, endosperm; 1). Analysis of cell cycle activity by FCM can be an alternative to the determination of seed developmental stage by histological observations of embryo structure and cell division patterns in the embryo and the endosperm.
One of the most important issues in seed production is to establish an optimal harvest time. At that time the majority of a seed population should have attained physiological maturity, i.e., reached maximum germinability, viability, and vigor (2). Usually, seeds require some additional period on the mother plant (maturation drying) because at physiological maturity their moisture content is too high for safe handling and storage. Traditionally, harvest maturity is established visually (imprecisely) or by using germination tests. However, such tests usually take about 7–14 days; therefore, developmental markers that give faster information on the physiological state of a seed are desirable. FCM seems to be a suitable method to provide such a marker.
At the beginning of seed development cell cycle activity in the embryo increases, while it declines at the end of embryogenesis and becomes gradually arrested at maturation (3). While in developing embryos the proportion of 4C nuclei (being at the G2 phase of the cell cycle) is relatively high, mature embryos of desiccation-tolerant (orthodox) seeds possess predominantly nuclei with a 2C DNA content, indicating cell cycle arrest at the G0/G1 phase; in some species a small proportion of nuclei with a 4C DNA content is also present (1, 4–7). During germination or pre-sowing priming of seeds the proportion of the 4C nuclei increases again, marking the entrance of the cells into the G2 phase that precedes mitotic divisions, which typically occurs during seedling growth. Therefore, the 4C/2C ratio has been proposed as a marker for seed maturation and the advancement of seed germination (8, 9). However, there are seeds that contain 2C and 4C nuclei and also ones with a higher DNA content, which is the result of endoreduplication, a process during which the nuclei undergo repeated rounds of DNA replication without mitosis (endocycles; 10–14). Such seeds possess polysomatic organs, i.e., composed of somatic cells with different DNA contents, including endopolyploid ones. Here, it is hypothesized that for polysomatic seeds the 4C/2C ratio is not fully suitable as a marker of seed maturation, since it does not consider the presence of cells with a DNA content higher than 4C. To verify this hypothesis and to suggest another FCM marker, which reflects better the physiological state of the seed, five polysomatic species belonging to the Fabaceae family were analyzed using flow cytometry. The changes in DNA replication patterns in the embryo from 20 days after flowering (DAF) to harvest maturity, as well as after 4 months of storage, were followed. There are only a few reports on endoreduplication in the embryo during seed development and the significance of this process is still not clear (3, 14–18). The present study provides information on a relationship between endoreduplication intensity in different seed parts and genome size, and type of seedling establishment (epigeal and hypogeal). This is the first report containing FCM comparative data on DNA synthesis patterns in different polysomatic species of one family during seed development.
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In seeds of Fabaceae species the endosperm is almost totally resorbed during embryo development and cotyledons become the major storage organ, which supports seedling growth before it establishes photosynthetic activity. At the beginning of embryo development, the cotyledon cells actively divide, but mitotic activity ceases during maturation (18, 25, 26). At this time and in mature seeds, in such Fabaceae species as P. vulgaris, P. sativum, V. faba, Glycine max, Lupinus albus, and Vigna radiata, high endopolyploidy was detected in the cotyledons (4, 16–18, 25, 26). Endoreduplication in this organ is directly related to storage protein and carbohydrate/oil synthesis, which confirms a suggestion that one of its roles is to increase the potential of gene activity in supplying multiple templates for transcription and translation (16, 27). In the present study endopolyploid nuclei were found in the cotyledons, supporting the hypothesis on endopolyploidization of storage cells. However, in M. sativa this occurred only at the beginning of seed development and as a low proportion. Evidently, in this species another mechanism must exist for the support of early seedling growth. Lack of endopolyploid nuclei can be related to a small size of the seeds of this species, a phenomenon that often correlates with endoreduplication intensity (25) and epigeal seedling development, which is characterized by the emergence of cotyledons above the soil surface; they become photosynthetic as the stored reserves are being depleted (2). In another species with epigeal seedling establishment, large-seeded P. vulgaris, however, endopolyploid nuclei up to 128C occurred, which suggests that the type of seedling emergence is not indicative of endoreduplication intensity. The type of cotyledons could be more determinative: M. sativa produces foliar ones, which have a greater initial allocation of photosynthetic tissue than the reserve-type ones of P. vulgaris and may respond quicker to light availability. The intensity of endoreduplication in the cotyledons of V. faba observed here was the same (32C) as previously detected (18), but in P. sativum it was lower than the earlier reported endopolyploidy level of up to 64C (26). However, variability in the maximum endopolyploidy in the cotyledons of this species (from 32C to 128C) is known, depending on seed size (25).
The pattern of endoreduplication in the cotyledons established in the present experiments depends on the species; nevertheless it can be considered as a marker of seed maturity. The beginning of establishment of physiological maturity was clearly marked in four out of the five species by the change in the number of endocycles: in P. vulgaris, V. sativa, and V. faba var. minor an additional endocycle occurred, and in M. sativa the endopolyploid 8C nuclei were no longer present (Table 3). When the seeds were dried at this time (at 30 DAF for P. vulgaris and V. sativa, and at 40 DAF for V. faba var. minor and M. sativa), they already had established some, but not maximal, germination energy and capacity (Table 2). After another 10 days they attained germinability close to 100%, related to full physiological maturity. Only in P. sativum cotyledons did endoreduplication intensity fail to correspond with the seed developmental stages, although physiological maturity was marked by the occurrence of an additional endocycle in the embryo axis at 30 DAF. The (Σ>2C)/2C ratio in the cotyledons also marked well seed physiological maturity and additionally indicated harvest maturity by the decrease in their value in all species but P. vulgaris. In the latter, the marker increased.
There is very little information on the pattern of DNA synthesis in the embryo axis of Fabaceae seeds. Bino et al. (4) observed some 8C nuclei in the radicle of the mature seed of P. vulgaris; however, in the developing embryo axis of G. max no endopolyploid cells were observed (16). In the species studied here, only in P. sativum did endoreduplication in the embryo axis reach 16C, while in the other species one endocycle occurred, resulting in the presence of 8C nuclei. The proportion of these nuclei in M. sativa, V. sativa, and V. faba var. minor did not change much during development, thus it did not correspond with the developmental stage of the embryo.
According to Barow and Meister (28), taxonomic position (family affiliation) is the major factor determining the degree of endopolyploidy within a species, while life cycle, genome size and organ type have a minor but also significant effect on endopolyploidization. This was not fully the case in seeds of species belonging to Fabaceae family. Although endoreduplication was detected in all studied species, its intensity varied significantly between them, with maximal endopolyploidy from 8C in M. sativa to 128C in P. vulgaris. As found previously, no endopolyploid nuclei occur in seeds of such perennial Fabaceae species as Olneya tesota and Parkinsonia aculeata (29). This instead confirms a suggestion that endoreduplication is negatively correlated with the length of the plant life cycle. Organ-specificity of endoreduplication was also clearly visible here and since the process was more intensive in the cotyledons its functional significance can be assumed. The negative correlation between endoreduplication and genome size, suggested by Nagl (27), was also confirmed.
In conclusion, in the Fabaceae, a marker based on the intensity of endoreduplication in the cotyledons, the (Σ>2C)/2C ratio, can be applied to follow seed development. Therefore, this is recommended to seed producers for establishing seed maturity and, consequently, optimal harvest time. The 4C/2C ratio should not be used for polysomatic species, because it can be misleading. Endopolyploidy occurrence in the cotyledons confirms that DNA amplification is necessary in the highly specialized storage cells, which play an important role in seedling development, unless another mechanism for supplying nutrients is provided. Further investigations, however, on a broader spectrum of species expressing epigeal and hypogeal seedling emergence are necessary to confirm this suggestion.
The seed embryo is a suitable model in which to follow plant tissue development and therefore the results obtained here can be used as part of a basis to study in vivo as well as in vitro cell/tissue differentiation. FCM, as a unique fast method for detecting cell cycle activity and endopolyploidy, can supplement or even replace more complex and time-consuming techniques, e.g. the establishment by microscopy of mitotic index and anatomy, as used to follow such processes as somatic embryogenesis or development of treachery elements. The 4C/2C ratio is recommended for tissues/organs with dividing cells and the (Σ>2C)/2C ratio for those also with endoreduplicating ones.