Cell division and subsequent radicle protrusion in tomato seeds are inhibited by osmotic stress but DNA synthesis and formation of microtubular cytoskeleton are not

Abstract

We studied cell cycle events in embryos of tomato (Lycopersicon esculentum Mill. cv Moneymaker) seeds during imbibition in water and during osmoconditioning ("priming") using both quantitative and cytological analysis of DNA synthesis and beta-tubulin accumulation. Most embryonic nuclei of dry, untreated control seeds were arrested in the G(1) phase of the cell cycle. This indicated the absence of DNA synthesis (the S-phase), as confirmed by the absence of bromodeoxyuridine incorporation. In addition, beta-tubulin was not detected on western blots and microtubules were not present. During imbibition in water, DNA synthesis was activated in the radicle tip and then spread toward the cotyledons, resulting in an increase in the number of nuclei in G(2). Concomitantly, beta-tubulin accumulated and was assembled into microtubular cytoskeleton networks. Both of these cell cycle events preceded cell expansion and division and subsequent growth of the radicle through the seed coat. The activation of DNA synthesis and the formation of microtubular cytoskeleton networks were also observed throughout the embryo when seeds were osmoconditioned. However, this pre-activation of the cell cycle appeared to become arrested in the G(2) phase since no mitosis was observed. The pre-activation of cell cycle events in osmoconditioned seeds appeared to be correlated with enhanced germination performance during re-imbibition in water.

Figure 1

Germination of control (▵) and osmoconditioned (○) tomato seeds (±se), cv Moneymaker, upon imbibition in water. During osmoconditioning in −1 MPa PEG no germination occurred.

Figure 2

Frequency of nuclei with 4C DNA contents (±se) expressed as percentage of the total number of nuclei (2C + 4C) from embryos of control (triangles) or osmoconditioned (circles) seeds during imbibition (white symbols) and from seedlings after completion of germination (black symbols) (i.e. radicle protrusion of approximately 1 mm).

Figure 3

Development of DNA synthesis in tomato embryos during seed germination. Shown are fluorescent micrographs of longitudinal sections of embryos from untreated control seeds during germination (a–g) and embryos from dried osmoconditioned seeds (h). Nuclei show red fluorescence as a result of staining with propidium iodide. Nuclei showing green fluorescence are labeled with FITC, which indicates BrdU incorporation into actively replicating DNA (S-phase). Bars indicate 100 μm (a–e, g, and h) or 25 μm (f). a, Radicle tip region of dry control seeds showing the absence of BrdU incorporation after a 3-h pulse labeling, indicating the absence of DNA synthesis. b, Radicle tip of control seeds showing nuclei labeled with BrdU after a 3-h pulse labeling at 12 h of imbibition, indicating the initiation of nDNA synthesis. c, BrdU labeling in the radicle tip of control seeds imbibed for 24 h. Note that there are more nuclei labeled with BrdU than at 12 h (b), indicating higher DNA synthesis activity at this stage. d to g, BrdU labeling in the radicle tip (d), shoot meristem (e and f), and cotyledons (g) of germinated control seeds at 48 h of imbibition. At this stage, DNA synthesis activity in the radicle tip was highest and had also started in the shoot meristem and cotyledons. In the close-up view of the shoot meristem (f), unsynchronized cells containing nuclei with various levels of BrdU labeling showing early and late stages of S-phase can be seen. h, Radicle tip region of re-dried osmoconditioned seeds.

Figure 4

β-Tubulin accumulation in embryos of tomato seed during germination. β-Tubulin levels are shown for embryos of untreated control seeds during imbibition in water (12–48 h, lanes 5–9), as well as for those of seeds after 7 d of osmoconditioning, after re-drying, and during subsequent imbibition in water (lanes 10–12). Total protein loaded per lane was 30 μg. Lanes 1 to 3 were loaded with 1, 10, and 30 ng of pure bovine brain tubulin, respectively. The films were exposed for a maximum of 1 min. ^g, Embryos of seeds that had germinated (i.e. with 1-mm radicle protrusion).

Figure 5

Development of the microtubular cytoskeleton in embryos during tomato seed germination. Fluorescent micrographs of longitudinal sections of embryos from untreated control seeds during germination (a–l), and from osmoconditioned seeds before and after re-drying and during renewed imbibition in water (m–t) labeled with anti-β-tubulin/FITC are shown. The latter images (“primed”) are all in “confocal Z-series” projections to enhance the visualization of β-tubulin either in microtubules or in granules. Bars indicate 20 μm. Because the sections are relatively thin (4 μm) with respect to the diameter of the cells, only a few cells have their cortical cytoplasm with microtubules in the plane of the section. a to c, Radicle tip (a), shoot meristem (b), and cotyledon (c) of embryos from untreated dry seeds. Note the absence of microtubules. There were only remnants of microtubules in the radicle tips (arrows) and fluorescent granules (arrowheads) in the shoot meristem and cotyledons. d to f, Radicle tip (d), shoot meristem (e), and cotyledon (f) of embryos from untreated seeds imbibed for 12 h showing β-tubulin labeling in microtubules. Note that an integrated cortical microtubular cytoskeleton was formed in the radicle tip. Microtubules accumulated in the shoot meristem and meristele of the cotyledons concomitantly with the disappearance of the tubulin granules. g to i, Radicle tip (g), shoot meristem (h), and cotyledon (i) of embryos from untreated seeds imbibed for 24 h. Both early and later mitotic phragmoplasts (cytokinesis, arrows), and divided cells (arrowheads) can be observed in the radicle tip. j to l, Radicle tip (j), shoot meristem (k), and cotyledon (l) of embryos from germinated seeds imbibed for 48 h. At this stage the microtubular cytoskeleton was abundant throughout the embryo. More mitotic arrays and divisions were observed in the radicle tip, and could also be observed in the hypocotyl (not shown). A well-established cytoskeleton was then observed in the shoot meristem and in the cotyledons (l). m to o, Radicle tip (m), shoot meristem (n), and cotyledon (o) of embryos from osmoconditioned seeds before re-drying. A cortical microtubular cytoskeleton had formed during osmoconditioning throughout the radicle tip, hypocotyl (shown in “s”) and shoot meristem. In the cotyledons, microtubules were only observed in cells of the meristele, whereas the tubulin granules were still (detected also in a) present in the mesophyll. Mitotic arrays were not detected in embryos of osmoconditioned seeds. p to r, Radicle tip (p), shoot meristem (q), and cotyledon (r) of embryos from osmoconditioned seeds after re-drying. Note in the radicle tip, hypocotyl (shown in t), and shoot meristem the presence of a large number of tubulin granules resulting from degradation of the microtubular cytoskeleton accumulated during osmoconditioning. s and t, Hypocotyls of osmoconditioned seed before (s) and after re-drying (t). As in the radicle tip (m and p), the microtubular cytoskeleton, which was well formed after osmoconditioning, degraded after re-drying as a result of depolymerization of microtubules.

Figure 5

Development of the microtubular cytoskeleton in embryos during tomato seed germination. Fluorescent micrographs of longitudinal sections of embryos from untreated control seeds during germination (a–l), and from osmoconditioned seeds before and after re-drying and during renewed imbibition in water (m–t) labeled with anti-β-tubulin/FITC are shown. The latter images (“primed”) are all in “confocal Z-series” projections to enhance the visualization of β-tubulin either in microtubules or in granules. Bars indicate 20 μm. Because the sections are relatively thin (4 μm) with respect to the diameter of the cells, only a few cells have their cortical cytoplasm with microtubules in the plane of the section. a to c, Radicle tip (a), shoot meristem (b), and cotyledon (c) of embryos from untreated dry seeds. Note the absence of microtubules. There were only remnants of microtubules in the radicle tips (arrows) and fluorescent granules (arrowheads) in the shoot meristem and cotyledons. d to f, Radicle tip (d), shoot meristem (e), and cotyledon (f) of embryos from untreated seeds imbibed for 12 h showing β-tubulin labeling in microtubules. Note that an integrated cortical microtubular cytoskeleton was formed in the radicle tip. Microtubules accumulated in the shoot meristem and meristele of the cotyledons concomitantly with the disappearance of the tubulin granules. g to i, Radicle tip (g), shoot meristem (h), and cotyledon (i) of embryos from untreated seeds imbibed for 24 h. Both early and later mitotic phragmoplasts (cytokinesis, arrows), and divided cells (arrowheads) can be observed in the radicle tip. j to l, Radicle tip (j), shoot meristem (k), and cotyledon (l) of embryos from germinated seeds imbibed for 48 h. At this stage the microtubular cytoskeleton was abundant throughout the embryo. More mitotic arrays and divisions were observed in the radicle tip, and could also be observed in the hypocotyl (not shown). A well-established cytoskeleton was then observed in the shoot meristem and in the cotyledons (l). m to o, Radicle tip (m), shoot meristem (n), and cotyledon (o) of embryos from osmoconditioned seeds before re-drying. A cortical microtubular cytoskeleton had formed during osmoconditioning throughout the radicle tip, hypocotyl (shown in “s”) and shoot meristem. In the cotyledons, microtubules were only observed in cells of the meristele, whereas the tubulin granules were still (detected also in a) present in the mesophyll. Mitotic arrays were not detected in embryos of osmoconditioned seeds. p to r, Radicle tip (p), shoot meristem (q), and cotyledon (r) of embryos from osmoconditioned seeds after re-drying. Note in the radicle tip, hypocotyl (shown in t), and shoot meristem the presence of a large number of tubulin granules resulting from degradation of the microtubular cytoskeleton accumulated during osmoconditioning. s and t, Hypocotyls of osmoconditioned seed before (s) and after re-drying (t). As in the radicle tip (m and p), the microtubular cytoskeleton, which was well formed after osmoconditioning, degraded after re-drying as a result of depolymerization of microtubules.