Determination of ACC-induced cell-programmed death in roots of Vicia faba ssp. minor seedlings by acridine orange and ethidium bromide staining.

Fluorescence staining with acridine orange (AO) and ethidium bromide (EB) showed that nuclei of cortex root cells of 1-aminocyclopropane-1-carboxylic acid (ACC)-treated Vicia faba ssp. minor seedlings differed in color. Measurement of resultant fluorescence intensity (RFI) showed that it increased when the color of nuclear chromatin was changed from green to red, indicating that EB moved to the nuclei via the cell membrane which lost its integrity and stained nuclei red. AO/EB staining showed that changes in color of the nuclear chromatin were accompanied by DNA condensation, nuclei fragmentation, and chromatin degradation which were also shown after 4,6-diamidino-2-phenylindol staining. These results indicate that ACC induced programmed cell death. The increasing values of RFI together with the corresponding morphological changes of nuclear chromatin were the basis to prepare the standard curve; cells with green unchanged nuclear chromatin were alive while those with dark orange and bright red nuclei were dead. The cells with nuclei with green-yellow, yellow-orange, and bright orange chromatin with or without their condensation and fragmentation chromatin were dying. The prepared curve has became the basis to draw up the digital method for detection and determination of the number of living, dying, and dead cells in an in planta system and revealed that ACC induced death in about 20% of root cortex cells. This process was accompanied by increase in ion leakage, shortening of cells and whole roots, as well as by increase in weight and width of the apical part of roots and appearance of few aerenchymatic spaces while not by internucleosomal DNA degradation.

Kinetin induces cell death in root cortex cells of Vicia faba ssp. minor seedlings.

The double fluorescence staining with acridine orange and ethidium bromide (AO/EB) revealed that treatment of Vicia faba ssp. minor seedlings with kinetin-induced programmed cell death (PCD) in root cortex cells. Kinetin-induced cell death reflected by the morphological changes of nuclei including their invagination, volume increase, chromatin condensation and degradation as well as formation of micronuclei showed by AO/EB and 4,6-diamidino-2-phenylindol staining was accompanied by changes including increase in conductivity of cell electrolytes secreted to culture media, decrease in the number of the G1- and G2-phase cells and appearance of fraction of hypoploid cells as the effect of DNA degradation without ladder formation. Decrease in the number of mitochondria and in the activity of cellular dehydrogenases, production of reactive oxygen species (ROS), appearance of small and then large lytic vacuoles and increase in the amount of cytosolic calcium ions were also observed. The PCD was also manifested by increased width and weight of apical fragments of roots as well as decreased length of cortex cells which led to shortening of the whole roots. The kinetin-induced PCD process was almost completely inhibited by adenine, an inhibitor of phosphoribosyl transferase, and mannitol, an inhibitor of ROS production. These cell-death hallmarks and pathway of this process suggested that the induction of kinetin-specific vacuolar type of death, expressed itself with similar intensity on both morphological and metabolic levels, was a transient protecting whole roots and whole seedlings against elimination.