Nuclease activities and DNA fragmentation during programmed cell death of megagametophyte cells of white spruce (Picea glauca) seeds

The haploid megagametophyte of white spruce (Picea glauca) seeds undergoes programmed cell death (PCD) during post-germinative seedling growth. Death of the megagametophyte storage parenchyma cells was preceded by reserve mobilization and vacuolation. TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling)-positive nuclei indicated that the first megagametophyte cells to die were those closest to the radicle at the micropylar end of the seed as well as those that comprised the most peripheral and innermost layers at the chalazal end of the seed. The death process was accompanied by nuclear fragmentation and internucleosomal DNA cleavage and the sequential activation of several nucleases. The latter comprised at least two groups: those induced relatively early during post-germinative seedling growth, that had pH optima in the neutral range (33, 31, 17 and 15 kDa), and those induced later that had pH optima in the acidic range (73, 62, 48, 43 and 29 kDa). Activities of all of the nucleases were stimulated by Ca2+, Mg2+ and Mn2+; only the nucleases active at neutral pH were inhibited by Zn2+. The temporal pattern of induction of the neutral and acidic nucleases may suggest that the latter function after tonoplast rupture.