## 4 Discussion  
According to the results obtained, the vacuolization phenomenon was observed as early as the first treatment with 100 mM NaCl (V2). The rapid and significant accumulation of NaCl in the plant cell leads to the induction and formation of provacuoles, the main factor in vacuole formation [43]. The longer the exposure time and the higher the salt concentration, the more evident the vacuolization phenomenon. Furthermore, the results showed a significant increase in the provacuolar index (PVI), synchronized with the increase in the vacuolization index (VI). This marked increase in the provacuolar index and vacuolization index is due to the accumulation of salt in the vacuole, which also deregulates normal osmotic processes [44] and cell division by decreasing the mitotic index. Reversible vacuoles in _Helianthus annulus_ cells show normal activity under control treatments, as they are in continuous homeostatic equilibrium motion [45]. Vacuolization is also known to improve cell survival by reducing stress [23]. With the increase in the vacuolization index, normally dividing cells were found to be surrounded by vacuolized cells responsible for salt accumulation in vacuoles, highlighting the adaptive role of vacuolization in the plant tolerance mechanism. This can be seen in Figure 4D, where a circular movement of the chromosomes is observed in the vacuolized cells. Conversely, in Figure 4E, we see a normal metaphase surrounded by vacuolized cells, which reflects the importance of vacuolization in the adaptive process by accumulating NaCl in the cells and sustaining the normal mitotic division by reducing the salt concentration around it. On the other hand, our results indicate that the increase in the vacuolization index (VI) is inversely proportional to the mitotic index (MI). Studies have indicated the cellular mechanism of the effect of salt stress on plants [46]. The negative influence of salt stress on mitotic division was observed at the cellular level, with the cell division spindle blocked by circularly moving chromosomes (Figure 4D). Studies on _Oryza sativa_ indicate that vacuole destruction correlates with programmed cell death (PCD) [47]. The correlation between time and the level of exposure to different salt concentrations resulted in a synchronized response with an increase in the vacuolization index and a decrease in the mitotic index, highlighting the close link between them. A close link between vacuolization and chromosomal aberrations has also been observed. Research indicates that the vacuolization phenomenon is influenced by the endoplasmic reticulum in the sunflower, where the presence of salt stress tolerance expression genes is also localized [48]. The increase in the chromosomal aberration index at different NaCl concentrations recorded in our studies has been confirmed by numerous cytological investigations carried out on plant tissues [49; 50; 51]. The effect of salt can also be seen in the investigation carried out by [PERSON] et al. (2009) [52] on the percentage of cell viability in Microsterias denticulate after treatment with 200 mM NaCl, where they observed that after 6 h of exposure, around 80% of the cells were viable, but after 48 h of NaCl exposure, cell viability decreased to 40%. Morphological changes were observed in cell organelles: after 24 h of NaCl treatment, there was an increase in the number of mitochondria, disintegration of the Golgi apparatus into numerous small vesicles with swelling and dilatation of endoplasmic reticulum compartments, highlighting the effect of salt on the appearance of autophagy during programmed cell death (PCD) [53; 54]. The same phenomenon has been found in the results of H\({}_{2}\)O\({}_{2}\) exposure inducing PCD in cells [55; 56]. Studies show that PCD is accompanied by tonoplast cleavage (vacuole collapse) and leakage of vacuole contents into the cytoplasm. Thus, hydrolytic enzymes present in the vacuole reach the cytoplasm and begin organelle degradation, i.e., the start of the PCD process [57].