The relationship between germination and melatonin applied during osmo- and hydropriming was studied in cucumber seeds. The proportion of nuclei with different DNA contents, the mean ploidy and the (2C + 4C = 8C)/2C ratio in unprimed and primed, dry and imbibed at 10 degrees C seeds were established by flow cytometry. Thiobarbituric acid reactive substances and protein oxidation were also estimated. Melatonin and indole-3-acetic acid (IAA) concentrations in the seeds were determined using high-performance liquid chromatography with electrochemical detection. Being sensitive to chilling stress, seeds that germinated well (99%) at 25 degrees C showed only 30% germination at 15 degrees C, and almost no germination (4%) at 10 degrees C. Hydropriming in water improved seed germination to 50-60% at 15 degrees C and the addition of melatonin (25-100 M) also increased the rate of germination. Osmopriming in polyethylene glycol increased germination at 15 degrees C to 78%, and 98% when combined with 50 M melatonin. Osmoprimed seeds germinated even at 10 degrees C and reached 43%, and 83% when 50 M melatonin was applied. None of the treatments induced DNA synthesis, although during the first 24 hr of imbibition at 10 degrees C the mean ploidy and the (2C + 4C = 8C)/2C ratio increased, which is indicative of the advanced Phase II of germination. Hydro- and osmopriming slightly decreased IAA content in the seeds in most of the cases; only hydropriming with 100 and 500 M melatonin increased it. Melatonin protected membrane structure against peroxidation during chilling, but excessive melatonin levels in cucumber seeds (approximately 4 microg/g fresh weight) provoked oxidative changes in proteins. There is still lack of information explained clearly the role of melatonin in plant physiology. This molecule acts multidirectionally and usually is alliged to other compounds.

The completion of germination of seeds of Arabidopsis thaliana is marked by the appearance of the radicle through the surrounding endosperm and testa. Using confocal microscopy and green fluorescent protein (GFP)-transformed embryos to highlight the epidermal cell walls it has been possible to conduct time-lapse photography of individual embryos during their germination. This reveals that the elongation of embryo cells to effect completion of germination does not occur within the radicle itself, but rather within a discrete region that is immediately proximal to the radicle. This region, identifiable as the lower hypocotyl and hypocotyl-radicle transition zone, is also definable by accumulation of carbohydrate-containing bodies during germination, and distinct GFP expression of GAL4-GFP in enhancer trap lines. Flow cytometric studies show that there is an increase in the proportion of 4C nuclei in the axis which coincides with a considerable increase in length of the hypocotyl, and the occurrence of endopolyploid (8C and 16C) nuclei accompanies the 2-fold increase in mean cell size in the region of elongation, the lower hypocotyl, and hypocotyl-radicle transition zone. Thus the observed cell elongation during germination is accompanied by an increase in nuclear DNA content, and the resultant elongation of the axis to effect radicle emergence is due to cell expansion, not to cell division. When studying the molecular events involved in the completion of germination, therefore, it may be prudent to focus on this region of elongation.

The nucleus is a definitive feature of eukaryotic cells, comprising twin bilamellar membranes, the inner and outer nuclear membranes, which separate the nucleoplasmic and cytoplasmic compartments. Nuclear pores, complex macromolecular assemblies that connect the two membranes, mediate communication between these compartments. To explore the morphology, topology, and dynamics of nuclei within living plant cells, we have developed a novel method of confocal laser scanning fluorescence microscopy under time-lapse conditions. This is used for the examination of the transgenic expression in Arabidopsis thaliana of a chimeric protein, comprising the GFP (Green-Fluorescent Protein of Aequorea victoria) translationally fused to an effective nuclear localization signal (NLS) and to ␤-glucuronidase (GUS) from E. coli. This large protein is targeted to the nucleus and accumulates exclusively within the nucleoplasm.This article provides online access to movies that illustrate the remarkable and unusual properties displayed by the nuclei, including polymorphic shape changes and rapid, longdistance, intracellular movement. Movement is mediated by actin but not by tubulin; it therefore appears distinct from mechanisms of nuclear positioning and migration that have been reported for eukaryotes. The GFP-based assay is simple and of general applicability. It will be interesting to establish whether the novel type of dynamic behavior reported here, for higher plants, is observed in other eukaryotic organisms. INTRODUCTIONA definitive feature of eukaryotic cells is the nucleus, first described in stamen cells of Tradescantia by Robert Brown in 1831, which is found in all cell types at some stage of their development. The nucleus comprises twin bilamellar membranes, the inner and outer nuclear membranes, which serve to separate the nuclear contents, the nucleoplasm, from the cytoplasm. Communication between these compartments is mediated by nuclear pores, complex macromolecular assemblies that connect the two membranes.All of our information concerning nuclear morphology, topology, and dynamics comes from the use of various forms of microscopy. The major components of the nucleoplasm, including proteins, RNA, and DNA, do not absorb in the visible part of the spectrum. Thus, nuclei are nearly translucent and nonfluorescent. This impedes the observation of nuclei within living cells with standard bright-field or fluorescence microscopy. Although other forms of light microscopy can be used to examine nuclei, particularly phase contrast and differential interference contrast microscopy, which rely on optical properties other than absorbance and fluorescence, this is practical only in nonpigmented cells and in tissues having simple three-dimensional organization.We have developed an alternative method for observation of nuclei in living plant cells that uses confocal laser scanning fluorescence microscopy (Chytilova et al., 1999). Based on the transgenic expression of the Green Fluorescent Protein (GFP) of Aequorea victoria, it involves ...

Background: Nuclear DNA content in plants is commonly estimated using flow cytometry (FCM). Plant material suitable for FCM measurement should contain the majority of its cells arrested in the G 0 /G 1 phase of the cell cycle. Usually young, rapidly growing leaves are used for analysis. However, in some cases seeds would be more convenient because they can be easily transported and analyzed without the delays and additional costs required to raise seedlings. Using seeds would be particularly suitable for species that contain leaf cytosol compounds affecting fluorochrome accessibility to the DNA. Therefore, the usefulness of seeds or their specific tissues for FCM genome size estimation was investigated, and the results are presented here. Methods: The genome size of six plant species was determined by FCM using intercalating fluorochrome propidium iodide for staining isolated nuclei. Young leaves and different seed tissues were used as experimental material. Pisum sativum cv. Set (2C ϭ 9.11 pg) was used as an internal standard. For isolation of nuclei from species containing compounds that interfere with propidium iodide intercalation and/or fluorescence, buffers were used supplemented with reductants.

The 2C nuclear DNA content has been estimated by flow cytometry in 18 species and botanical forms of the genus Lupinus (family Fabaceae), using propidium iodide as a fluorescent dye. They represented distinct infrageneric taxonomic groups and differed in somatic chromosome numbers. Estimated 2C DNA values ranged from 0.97 pg in L. princei to 2.44 pg in L. luteus, which gives a more than 2.5-fold variation. Statistical analysis of the data obtained resulted in a grouping that supports the generally accepted taxonomic classification of the Old World lupins. The rough-seeded L. princei turned out to be an interesting exception, getting closer to smooth-seeded species. Results of DNA content analyses are discussed with regards to the phylogenetic relationships among the Old World lupins and some aspects of the evolution of the genus.