br Materials and methods br Results In the leaves of

Materials and methods
Results In the leaves of the studied varieties, one to three nucleoli per nucleus were observed (Fig. 1a), except for the variety Bastardo were a maximum of two nucleoli per nucleus were detected. In the root-tip cells, most of the varieties presented one to three nucleoli per interphase nucleus apart from the variety Pinot Noir that showed one to four nucleoli per nucleus (Fig. 1b). The average frequencies of the nucleoli scored in the leaves and root-tips of different V. vinifera varieties are presented in Table 1. In all varieties, independently of the tissue, one single nucleolus per nucleus appeared more frequently, justifying the fact of all average values of nucleoli per cell were lower than 2 (Table 1). For the average number of nucleoli per cell, the Tukey test distributed the 7 varieties into 5 groups with statistically significant differences (p < 0.001) among them. When comparing the different varieties, independently of the tissue, there are statistically significant differences (p < 0.001) for the nucleoli average frequencies. The interaction leaf × variety showed statistically significant differences (p < 0.001) among the studied varieties for all the parameters, with the obvious exception of the average frequency of four nucleoli. Once again, in the average number of nucleoli per cell data, the Tukey test distributed the 7 varieties into 5 groups. This did not happen for the interaction root × variety. Here, only Pinot Noir stands out because it is the only variety that presented four nucleoli. Due to the small size of the chromosomes that hampers the accurate identification of Ag-NORs and to the reduced mitotic index, particularly in the leaf tissue, it was not possible to score the number of Ag-NORs in a high number of metaphase Sulfaphenazole of the 7 varieties to achieve valuable statistical data. Nevertheless, in the root-tip cells of most of the varieties, one to three Ag-NORs per metaphase were observed (Fig. 2a to c; Table 1). In fact, the Ag-NORs identified after silver nitrate staining were further confirmed by sequential FISH performed with the 45S rDNA probe, pTa71 (Fig. 2c and d; Table 1). Except for Tinto Cão that showed three rDNA loci after FISH (Fig. 2d), the remaining varieties presented four rDNA loci independently of the maximum number of Ag-NORs previously detected (Fig. 2e; Table 1). 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 cell cycle (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.