Apoptosis‐like cell death of Brassica napus leaf protoplasts

Abstract

Summary• The cleavage of nuclear DNA into oligonucleosomal fragments that is the hallmark of apoptosis in animal cells occurs during the culture of Brassica napus leaf protoplasts.• The changes in nuclei of cultured Brassica napus leaf protoplasts were studied by propidium iodide (PI) and 4 ′ , 6-diamino-2-phenylindole, dihydrochloride (DAPI) staining, transmission electron microscopy, flow cytometry analysis, and DNA laddering staining with ethidium bromide and Southern hybridization.• Free 3 ′ -OH termini of nuclear DNA fragments were labelled with DIG-dUTP, catalyzed by terminal deoxynucleotidyl transferase (TdT), and used as probes for Southern hybridization. This method (TUNEL on membrane ) allowed visualization of DNA fragments with 3 ′ -OH termini on a nylon membrane.• These results suggest that loss of viability of protoplast with culture time is accompanied by apoptosis-like cell death. However, the forms or processes undergoing to apoptotic cell death in B. napus leaf protoplasts appears to be different in some details to those in animal cells.

Generation of intracellular reactive oxygen species during the isolation of Brassica napus leaf protoplasts

“…Before plant regeneration can occur, protoplasts must undergo a process of dedifferentiation. Previously, we showed that Brassica napus leaf protoplasts initiate senescence during the isolation process and die via a process of apoptosis, similar to that observed in animal cells but with several morphological differences (Watanabe et al 1998;Watanabe et al 2002).…”

Iron deprivation-induced reactive oxygen species generation leads to non-autolytic PCD in Brassica napus leaves

“…Protoplasts were isolated from young expanded leaves of different iron treatments as described previously (Watanabe et al, 2002). The protoplasts were washed and re-suspended in 0.6 M sorbitol in 5 mM MES buffer (pH 5.8) and 5 mM CaCl 2 , and loaded with 100 μM H 2 DCF-DA for 30 min in darkness.…”

“…Protoplasts were isolated and fixed in 2% glutaraldehyde in sorbitol buffer (10 mM phosphate buffer, 0.6 M sorbitol, pH 7.4) for 2 h at room temperature and stained with DAPI (Watanabe et al, 2002). The protoplasts were washed and re-suspended in the sorbitol buffer.…”

Role of calcium in the mitigation of heat stress in the cyanobacterium Anabaena PCC 7120

“…Strong blue DAPI stain characterized healthy cyanobacterial cells; however, cells with features of apoptosis exhibited weaker DAPI stain (Zheng et al, 2013). Likewise, DAPI-stained Brassica napus leaf protoplasts under fluorescence microscopy clearly showed enlarged nuclei with condensed chromatin (Watanabe et al, 2002).…”