Among the four rDNA loci two

Among the four rDNA loci, two hybridisation signals were more intense, but all were localized near the nucleoli detectable by the faint DAPI staining also typical of more decondensed heterochromatin, as exemplified in Fig. 2e. The same nucleus also present heterochromatic foci (dense chromatin regions) visualized by a strong DAPI staining corresponding to AT-rich repeat sequences (Fig. 2e).
Discussion The nucleolus is the most prominent nuclear sub-compartment, being the site of rRNA transcription and ribosome biosynthesis. The nucleoli are formed at the end of mitosis around NORs and their expression is regulated by different mechanisms, according to the cell need for ribosomes (Preuss and Pikaard, 2007). The higher frequencies of nuclei with a single nucleolus per nucleus detected in the grapevine varieties studied here can be due to a dosage control of the rDNA, since eukaryotes have more rRNA genes than required for ribosome biosynthesis at any given time (Grummt and Pikaard, 2003). The necessary quantity of rRNA genes could be controlled by activating/repressing the transcription of a constant subset of rDNA loci (Neves et al., 2005b). The whole mechanism it is still unknown but some components have already been identified (for detailed reviews on the matter see Neves et al. (2005b), Preuss and Pikaard (2007)). The occurrence of one nucleolus per interphase nucleus can also be explained by nucleolar fusion, where two or more nucleoli are formed in a nucleus and fuse during the GSK180 (Jordan et al., 1982). The average percentage values of interphase nuclei with a variable number of nucleoli scored in this work for the different grapevine varieties were consistent with those reported by Haas and Alleweldt (2000). These authors scored silver nitrate stained nucleoli in root-tip cells of V. vinifera variety Bacchus and detected a highest frequency of one nucleolus per interphase cell as well as a smallest frequency of nuclei with three nucleoli. Haas and Alleweldt (2000) did not report the presence of four nucleoli per nucleus in Bacchus. These authors reported the existence of secondary constrictions in the chromosome pairs 1 and 3 but reinforced that only one satellite chromosome pair (no. 1) can be positively stained with silver nitrate. However, after performing FISH with the rDNA probe pTa71, Haas and Alleweldt (2000) referred the detection of four 45S rDNA loci located on the chromosome pairs 1 and 16. Four 45S rDNA loci were also detected in other V. vinifera varieties (Houel et al., 2010; Pereira et al., 2005, Pereira et al., 2014). In the present study, four rDNA loci were detected after FISH in all varieties except in Tinto Cão. In the particular case of Pinot Noir, a maximum of four nucleoli per nucleus were previously detected after silver nitrate staining. Hence, in Pinot Noir the four 45S rDNA loci were transcriptionally active as revealed by the positive silver nitrate staining of four nucleoli per nucleus despite it was not found metaphase cells with discriminative four Ag-NORs.