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

Abstract

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.

Impact of the antifungal protein PgAFP from Penicillium chrysogenum on the protein profile in Aspergillus flavus

“…7). Untreated hyphae displayed a green color, meaning that they remained viable (Byczkowska et al 2012). The intense orange staining revealed a high penetration of EB in the PgAFP-treated hyphae through a highly compromised cell membrane.…”

The crucial elements of the ‘last step’ of programmed cell death induced by kinetin in root cortex of V. faba ssp. minor seedlings

“…The colour of chromatin of cells undergoing PCD is green-yellow, yellow or yellow-orange, reflected in greater RFI values, then in green (living) cells. The colour of chromatin of dead cells is dark orange and red and in these nuclei RFI values reach the highest levels (Byczkowska et al 2013). It allows us to distinguish living, dying and dead cells.…”

“…minor seedling roots after kinetin treatment, the ''double staining method'' described in Byczkowska et al (2013) was applied. In situ analyses of the cell death were carried out only in the cortex cells between the 4th and the 20th mm of 2 cm long apical parts of two or three roots from three independent experiments.…”

“…It allows us to distinguish living, dying and dead cells. Detection of cell death was carried out according to the procedure described in Byczkowska et al (2013), in particular, by staining the roots with the mixture of 100 lg ml -1 acridine orange (AO) and 100 lg ml -1 EB in PHB (0.01 M phosphate buffer, pH 7.4) for 4 min and fixing them with 1.0 % glutardialdehyde (POCH, Poland) in PHB for 15 min. Then the roots were cut with a razor blade along the long axis and thin sections, washed two times for 2 min with PHB, were put on glass slides with a drop of PHB, covered with coverslip and analysed (Byczkowska et al 2013).…”

“…Detection of cell death was carried out according to the procedure described in Byczkowska et al (2013), in particular, by staining the roots with the mixture of 100 lg ml -1 acridine orange (AO) and 100 lg ml -1 EB in PHB (0.01 M phosphate buffer, pH 7.4) for 4 min and fixing them with 1.0 % glutardialdehyde (POCH, Poland) in PHB for 15 min. Then the roots were cut with a razor blade along the long axis and thin sections, washed two times for 2 min with PHB, were put on glass slides with a drop of PHB, covered with coverslip and analysed (Byczkowska et al 2013). To detect cell death, the fluorescence micrographs from the specimens placed on slides were taken under blue (400-440 nm of B-2A excitation filter) light using the Optiphot-2 epifluorescence microscope (Nikon) equipped with DDX camera and Act1-software (Precoptic, Poland).…”