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

Anna Byczkowska,Anita Kunikowska,Andrzej Kaźmierczak

doi:10.1007/s00709-012-0383-9

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.

Highlights

Programmed cell death (PCD) is an active process controlling proper development of unicellular as well as multicellular organisms by elimination of physiologically redundant, damaged, or abnormal cells (Cacas 2010; Taraphdar et al 2001).In animals, PCD may take the form of apoptosis, microand macroautophagy, or non-lysosomal as well as mitotic catastrophe, in which cells can die via apoptosis or necrosis (Hübner et al 2009)
DNA fluorescence staining with the mixture of acridine orange and ethidium bromide is one of such methods
The well-known ethylene-dependent cell death model (Drew et al 2000; Palavan-Unsal et al 2005) with ACC, ethylene precursor was prepared with roots of V. faba ssp. minor seedlings

Summary

Introduction

Programmed cell death (PCD) is an active process controlling proper development of unicellular as well as multicellular organisms (van Doorn and Woltering 2005) by elimination of physiologically redundant, damaged, or abnormal cells (Cacas 2010; Taraphdar et al 2001).In animals, PCD may take the form of apoptosis, microand macroautophagy, or non-lysosomal (van Doorn and Woltering 2005) as well as mitotic catastrophe, in which cells can die via apoptosis or necrosis (Hübner et al 2009). The latest classification described by van Doorn et al (2011) rightly confirms that apoptosis does not exist in plants (van Doorn and Woltering 2005), and death-dependent development of plants, ultrastructurally defined, is controlled via vacuolar, necrotic, as well as mixed types of plant cell death (van Doorn et al 2011). Vacuolar cell death is connected with the formation of lytic vacuoles, which increase in volume; gradual decrease of the cytoplasm; formation of actin cables; nuclear envelope disassembly; as well as nuclei segmentation and chromatin condensation (van Doorn 2011; van Doorn et al 2011). Necrotic plant cell death is defined by the absence of growing lytic vacuoles, swelling of mitochondria, early rupture of plasma membrane, and shrinkage of the protoplast. The third type of plant cell death is connected with hypersensitive response, characterized by a variety of morphological hallmarks (van Doorn et al 2011)

Methods

Results

Conclusion