title: 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.
authors.affiliation: Department of Cytophysiology, University of Łódź, Pomorska 141/143, PL-90236 Łódź, Poland.
authors.forename: Anna
authors.initials: A
authors.lastname: Byczkowska
authors.forename: Anita
authors.initials: A
authors.lastname: Kunikowska
authors.forename: Andrzej
authors.initials: A
authors.lastname: Kaźmierczak
mesh_terms: D000165:Acridine Orange / Q000737:chemistry*; D000598:Amino Acids, Cyclic / Q000494:pharmacology*; D016923:Cell Death / Q000187:drug effects; D002462:Cell Membrane / Q000187:drug effects; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D004996:Ethidium / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry; D007202:Indicators and Reagents / Q000737:chemistry; D008856:Microscopy, Fluorescence / Q000379:methods; D018517:Plant Roots / Q000187:drug effects / Q000378:metabolism; D031307:Vicia faba / Q000187:drug effects* / Q000378:metabolism
chemical_list: D000598:Amino Acids, Cyclic; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; C023863:1-aminocyclopropane-1-carboxylic acid; D004996:Ethidium; D000165:Acridine Orange