Flow cytometric estimation of nuclear DNA content was performed in six plant species employing three fluorochromes showing different DNA base preferences: propidium iodide (no base preference), 4′,6‐diamidino‐2‐phenylindole (DAPI; AT preference), and mithramycin (GC preference). Nuclei isolated from human leukocytes were used as a primary reference standard. While nuclear DNA contents estimated using propidium iodide were in agreement with published data obtained using other techniques, the values obtained using fluorochromes showing base preference were significantly different. It was found that the differences were caused by the differences in overall AT/GC ratios, and by the species‐specific differences in binding of these fluorochromes to DNA. It was concluded that nuclear DNA content estimations performed with fluorochromes showing base preference should be interpreted with caution even when AT/GC ratios of the reference and the sample are equal. The use of intercalting dyes (e.g. propidium iodide) is recommended for this purpose. On the other hand, comparison of the staining behaviour of intercalating dyes with that of dyes showing base preference may give additional information on chromatin structural differences and arrangement of molecule pairs in DNA.

The membrane integrity of a cell is a well-accepted criterion for characterizing viable (active or inactive) cells and distinguishing them from damaged and membrane-compromised cells. This information is of major importance in studies of the function of microbial assemblages in natural environments, in order to assign bulk activities measured by various methods to the very active cells that are effectively responsible for the observations. To achieve this task for bacteria in freshwater and marine waters, we propose a nucleic acid doublestaining assay based on analytical flow cytometry, which allows us to distinguish viable from damaged and membrane-compromised bacteria and to sort out noise and detritus. This method is derived from the work of S. Barbesti et al. (Cytometry 40:214-218, 2000) which was conducted on cultured bacteria. The principle of this approach is to use simultaneously a permeant (SYBR Green; Molecular Probes) and an impermeant (propidium iodide) probe and to take advantage of the energy transfer which occurs between them when both probes are staining nucleic acids. A full quenching of the permeant probe fluorescence by the impermeant probe will point to cells with a compromised membrane, a partial quenching will indicate cells with a slightly damaged membrane, and a lack of quenching will characterize intact membrane cells identified as viable. In the present study, this approach has been adapted to bacteria in freshwater and marine waters of the Mediterranean region. It is fast and easy to use and shows that a large fraction of bacteria with low DNA content can be composed of viable cells. Admittedly, limitations stem from the unknown behavior of unidentified species present in natural environments which may depart from the established permeability properties with respect to the fluorescing dyes.

S. 1992. Comparison of three DNA fluorochromes for flow cytometric estimation of nuclear DNA content in plants. -Physiol. Plant. 85:'625-631.Flow cytometric estimation of nuclear DNA content was performed in sis plant species employing three fluorochromes showing different DNA base preferences: propidium iodide (no base preference), 4',6-diamidino-2-phenylindole (DAPI; AT preference), and mithramycin (GC preference). Nuclei isolated from human leukocytes were used as a primary reference standard. While nuclear DNA contents estimated using propidium iodide were in agreement with published data obtained using other techniques, the values obtained using fluorochromes showing base preference were significantly different. It was found that the differences were caused by the differences in overall AT/GC ratios, and by the spedes-specific differences in binding of these fluorochromes to DNA. !t was coneluded that nuclear DNA content estimations performed with fluorochromes showing base preference should be interpreted with caution even when AT/GC ratios of the reference and the sample are equal. The use of intercalating dyes (e.g. propidium iodide) is recommended for this porpose. On the other hand, comparison of the staining behaviour of intercalating dyes with that of dyes showing base preference may give additional information on chromatin structural differences and arrangement of molecule pairs in DNA.

The vegetation in a high alpine site of the European Alps experienced changes in area between 1953 and 2003 as a result of climate change. Shrubs showed rapid expansion rates of 5.6% per decade at altitudes between 2400 m and 2500 m. Above 2500 m, vegetation coverage exhibited unexpected patterns of regression associated with increased precipitation and permafrost degradation. As these changes follow a sharp increase in both summer and annual temperatures after 1980, we suggest that vegetation of the alpine (2400–2800 m) and nival (above 2800 m) belts respond in a fast and flexible way, contradicting previous hypotheses that alpine and nival species appear to have a natural inertia and are able to tolerate an increase of 1–2°C in mean air temperature.

The present study was to assess the effect of heavy metal stress on the DNA methylation of a metal-sensitive plant, Trifolium repens L. and of a metal-tolerant plant, Cannabis sativa L. The changes in the level of 5-methylcytosine (5mC) in the root DNA of plants grown on soils contaminated with different concentrations of Ni 21 , Cd 21 and Cr 61 compared with that of untreated plants, were determined by immunolabelling with a monoclonal antibody, using the Slot-Blot technique. Results showed that DNA of hemp control plants was about three times more methylated than clover DNA, for the same amount of root DNA. Heavy metal treatments induced a global dose-dependent decrease of 5mC content, both in hemp and clover, ranging from 20 to 40%. Changes in methylation pattern of 5 0 -CCGG-3 0 containing sequences were investigated by methylation-sensitive amplification polymorphism (MSAP) technique. Control plants of the same species showed a very similar pattern, suggesting that, in normal condition, methylation involves precise sites. Heavy metals induced DNA methylation changes mainly related to hypomethylation events. These variations were not randomly directed but involved specific DNA sequences, since the detected polymorphisms were the same in all the plants analysed for each treatment.Abbreviations -5mC, 5-methylcytosine; AFLP, amplified fragment length polymorphism; MSAP, methylation-sensitive amplification polymorphism; oxo8dG, 8-oxo-2 0 -deoxyguanosine; ROS, reactive oxygen species.

No abstract

The origin of the grapevine was investigated with archaeobotanical, cultural and historical data. A primary domestication centre was located in the Near East region but there is no agreement on the existence or role of secondary domestication centres. In this work, PCR-based microsatellite analysis has been applied to study the origin of some Italian cultivated grapevines from in situ direct domestication of the wild autoctonous grapevine. Three different Italian locations in Grosseto, Cosenza and Nuoro were identified for this study, and domesticated grapevine as well as wild local accessions growing in these location, were analysed by SSR markers. Cluster analysis performed on Cosenza and Grosseto samples showed a high value of genetic distance between domesticated and wild accessions. On the contrary two cultivars (Bovale Murru and Bovale Muristellu) recovered in Nuoro (in the Sardinia island) were very close to some wild varieties. This suggests that the latter two cultivars may have originated from wild grapevines and consequently that in this location a secondary grapevine domestication event occurred. Six Lambrusco varieties were also included in this analysis as ancient putative ancestors of the cultivated grapevines. The molecular analysis excluded this hypothesis and suggest Lambrusco as an independent Vitis taxon.

The correlation between environmental stress and DNA methylation has been studied by following the methylation status of cytosine residues in the DNA of pea root tips exposed to water deficit. DNA methylation was evaluated by two complementary approaches: (i) immunolabelling by means of a monoclonal antibody against 5‐methylcytosine; (ii) MSAP (Methylation‐Sensitive Amplified Polymorphism) to verify if methylation and de‐methylation in response to water deficit may be related to specific DNA sequences. Immunolabelling showed that water stress induces cytosine hypermethylation in the pea genome. Regarding the CCGG target sequence, an increase in methylation specifically in the second cytosine (about 40 % of total site investigated) was revealed by MSAP analyses. In addition, MSAP band profile detected in three independent repetitions was highly reproducible suggesting that, at least for the CCGG target sequence, methylation was addressed to specific DNA sequences.