Fig 8. Scheme depicting experimental procedures used to reveal and to support the fact that the activation of vacuolar/autolytic type of plant-specific, apoptosis-like programmed cell death [(V/A) AL-PCD] is a secondary result of the caffeine-induced premature chromosome condensation (PCC) in hydroxyurea (HU) synchronized Vicia faba root meristem cells.

The scheme shows that PCC leads to the formation of cells with different phenotypes (phenotype A–typical of cells not diverging from the morphology of regular cells; phenotype B–characteristic of cells with the morphology typical of PCC induction from G2 phase; and phenotype C–cells with strongly fragmented chromosomes, induced to PCC, probably in early or middle sub-periods of S phase). The scheme shows that the induction of PCC is associated with the generation of DNA damage (mainly single-strain breaks [SSB] but also to a lesser extent, double-strain breaks [DSBs]). The occurrence of various types of DNA damage during PCC induction has been proven following the results of many experimental procedures (immunocytochemistry, with the use of antibodies against specific marker proteins, tissue printing methods, Western blot and comet tests–both alkaline and neutral). In turn, double staining with acridine orange and ethidium bromide allowed us to distinguish in planta, the sub-fractions of living, dying and dead cells. Preliminary histochemical analyses (classic staining with Schiff’s reagent in Feulgen’s method) showed the presence of such changes in the structure of cellular nuclei that could indicate the occurrence of apoptosis-like programmed cell death (AL-PCD). On the other hand, more detailed analyses, i.e. DNA electrophoresis and TUNEL reaction, confirmed by ultrastructural tests (performed at a level of transmission electron microscopy) proved the existence of vacuolar/autolytic type of AL-PCD (V/A-type AL-PCD) in stressed V. faba roots.