Exogenous glutathione induces sister chromatid exchanges,clastogenicity and endoreduplication in V79-E Chinese hamster cells

Glutathione (GSH) dissolved in Eagle's MEM and added to cultures o of V79-E cells in concentrations between 2.5 × 10^–4 and 10^–3 moles/l for 1 h induces a dose-dependent cell cycle delay, sister chromatid exchanges and clastogenic damage. 7–8% of the metaphases showed endoreduplication at a recovery phase of 25 and 30 h after treatment with 10^–3 molesll GSH. Higher concentrations were lethal. The highest tolerated dose corresponds to the intracellular GSH level in V79-E cells. In the same range of concentrations, glutathione disulfide was inactive. Endoreduplication induction by GSH is G2-phase specific and endoreduplication metaphases show a reduced occurrence of single SCEs when extrapolated to the diploid complement. The adverse effects of GSH are independent of the presence of serum in the culture fluid but completely abolished when the treatment is performed in Hank's solution instead of MEM. The mechanism of genotoxicity of exogenous GSH is discussed but, at present, no pertinent explanation can be given.Abbreviations BUdR 5-bromodeoxyuridine - GSH glutathione - GSSG glutathione disulfide - SCE sister chromatid exchange     

Nuclear DNA content in differentiated tissues of sunflower (Helianthus annuus L.)

Summary Scanning cytophotometry following Feulgen-staining was used to determine nuclear DNA content in many differentiated tissues of nine cultivars, hybrids or selfed lines ofHelianthus annuus. Apart from such ephemeral tissues as endosperm and anther tapetum, it was found that tissue differentiation in sunflower occurs in the diploid condition, cells being arrested in the DNA presynthetic phase (G₁). In certain cases, however, the nuclear DNA content of differentiated G₁ cells does not exactly match the 2C DNA content found in meristematic cells, but may be either higher or lower. In endosperm and anther tapetum cells, nuclear DNA content may be as high as 24 C and 32 C, respectively. Cytological and autoradiographic analyses after³H-thymidine incorporation reveal that polyploidy in the tapetal cells is due to chromosome endoreduplication. No detectable difference between male-fertile and male-sterile plants exists as far as occurrence and level of cell polyploidy are concerned. The results are discussed in the context of previous investigations on the nuclear condition of differentiatedHelianthus annuus tissue.     

Effect of sucrose on polyploidization in early callus cultures of Solanum tuberosum

Leaf segments of a monohaploid, dihaploid and tetraploid genotype of the potato (Solanum tuberosum; x = 12) were cultured on callus-inducing medium with 10, 20, 30 or 40 gl^–1 sucrose. After 5 and 7 days of culture, metaphases contained the somatic or polyploidized number of mono- or diplochromosomes. The percentages of polyploidized metaphases were inversely correlated with the number of chromosome sets of the genotypes. In monohaploid leaf segments the percentages of polyploidized metaphases and of metaphases with diplochromosomes increased when the sucrose concentration was raised from 10 or 20 to 30 gl^–1 and remained constant or decreased from 30 to 40 gl^–1. Higher concentrations of sucrose but not higher osmolalities of the medium due to mannitol induced endoreduplication in more cells. The frequency of polyploidized metaphases and metaphases with diplochromosomes in dihaploid and tetraploid leaf segments remained constant through increases in sucrose concentrations.     

Chromosome endoreduplication in endosperm cells of two maize genotypes and their progenies

Summary Chromosome endoreduplication is a very common process in higher plants but its function and genetic control are still to be clarified. In our experiments we analyzed, by Feulgen cytophotometry, chromosome endoreduplication in endosperm cells of two maize genotypes, IHP and ILP, having high and low protein content in their seed, respectively. Chromosome endoreduplication occurs in both lines within 24 days after pollination, attaining a maximum ploidy level of 384C (7 DNA replication rounds) in IHP and of 192C (6 replication rounds) in ILP. In the mature seed, endosperms of the two lines show different mean ploidy level. In reciprocal crosses between IHP and ILP the f₁ endosperms have mean ploidy levels analogous to that of the maternal parent, showing that the difference in ploidy level between the two genotypes is maintained. After selfing of the f₁ plants, the difference in ploidy level between the two F₂ populations is reduced. In F₂ the mean ploidy level is as variable as in f₁, indicating the absence of genetic segregation. From our data, it is apparent that both the genetic constitution (cytoplasmic and nuclear) of the maternal parent and the genotype of the individual endosperms influence the ploidy level. An analysis of the protein content in endosperms carried out on the same seed sample as analyzed cytophotometrically showed that the protein content increases, during seed development, parallel to chromosome endoreduplication and varies, in the two lines, in reciprocal crosses and their progeny, according to the same trend as mean ploidy level, suggesting a correlation between the two parameters.     

Kip-related protein 3 is required for control of endoreduplication in the shoot apical meristem and leaves of Arabidopsis

The cell cycle plays an important role in the development and adaptation of multicellular organisms; specifically, it allows them to optimally adjust their architecture in response to environmental changes. Kip-related proteins (KRPs) are important negative regulators of cyclin-dependent kinases (CDKs), which positively control the cell cycle during plant development. The Arabidopsis genome possesses seven KRP genes with low sequence similarity and distinct expression patterns; however, why Arabidopsis needs seven KRP genes and how these genes function in cell cycle regulation are unknown. Here, we focused on the characterization of KRP3, which was found to have unique functions in the shoot apical meristem (SAM) and leaves. KRP3 protein was localized to the SAM, including the ground meristem and vascular tissues in the ground part of the SAM and cotyledons. In addition, KRP3 protein was stabilized when treated with MG132, an inhibitor of the 26S proteasome, indicating that the protein may be regulated by 26S proteasome-mediated protein degradation. KRP3-overexpressing (KRP3 OE) transgenic plants showed reduced organ size, serrated leaves, and reduced fertility. Interestingly, the KRP3 OE transgenic plants showed a significant reduction in the size of the SAM with alterations in cell arrangement. In addition, compared to the wild type, the KRP3 OE transgenic plants had a higher DNA ploidy level in the SAM and leaves. Taken together, our data suggest that KRP3 plays important regulatory roles in the cell cycle and endoreduplication in the SAM and leaves.     

Analysis of the spatial expression pattern of seven Kip related proteins (KRPs) in the shoot apex of Arabidopsis thaliana

BACKGROUND AND AIMS: Kip-related-proteins (KRPs), negative regulators of cell division, have recently been discovered in plants but their in planta function is as yet unclear. In this study the spatial expression of all seven KRP genes in shoot apices of Arabidopsis thaliana were compared. METHODS: In situ hybridization analyses were performed on longitudinal sections of shoot apices from 2-month-old Arabidopsis plants. KEY RESULTS: The study provides evidence for different expression pattern groups. KRP1 and KRP2 expression is restricted to the endoreduplicating tissues. In contrast, KRP4 and KRP5 expression is mainly restricted to mitotically dividing cells. KRP3, KRP6 and KRP7 can be found in both mitotically dividing and endoreduplicating cells. CONCLUSION: The results suggest differential roles for the distinct KRPs. KRP1 and KRP2 might specifically be involved in the establishment of polyploidy. In contrast, KRP4 and KRP5 might be involved in regulating the progression through the mitotic cell cycle. KRP3, KRP6 and KRP7 might have a function in both types of cell cycle.     

Analysis of the tomato fruit growth response to temperature and plant fruit load in relation to cell division, cell expansion and DNA endoreduplication

BACKGROUND AND AIMS: To better understand the regulation of fruit growth in response to environmental factors, the effects of temperature and plant fruit load on cell number, cell size and DNA endoreduplication were analysed. METHODS: Plants were grown at 20/20 degrees C, 25/25 degrees C and 25/20 degrees C day/night temperatures, and inflorescences were pruned to two ('2F') or five ('5F') flowers. KEY RESULTS AND CONCLUSIONS: Despite a lower fruit growth rate at 20/20 degrees C, temperature did not affect final fruit size because of the compensation between cell number and size. The higher cell number at 20/20 degrees C (9.0 x 10(6) against 7.9 x 10(6) at 25/25 degrees C and 7.7 x 10(6) at 25/20 degrees C) resulted from an extended period of cell division, and the smaller cell size was due to a shorter period of expansion rather than a lower expansion rate. By contrast, the lower fruit growth rate and size of 5F fruits compared with 2F fruits resulted from the slow down of cell expansion, whereas the number of cells was hardly affected in the proximal fruit. However, within the inflorescence the decreasing gradient of fruit size from proximal to distal fruits was due to a decrease in cell number with similar cell size. Fruit size variations within each treatment were always positively correlated to variations in cell number, but not in cell size. Negative correlations between cell size and cell number suggested that cells of tomato pericarp can be seen as a population of competing sinks. Mean ploidy was slightly delayed and reduced in 5F fruits compared with 2F fruits. It was highest at 25/25 degrees C and lowest at 25/20 degrees C. Treatments did not affect ploidy and cell size in similar ways, but within each treatment, positive correlations existed between mean ploidy and cell size, though significant only in the 2F-25/20 treatment.