• megagametophyte
• DNA ladder
• caspase-like proteases
• bunya pine
• PCD

Programmed cell death (PCD) is a genetically-controlled process necessary for the proper development of most living organisms. The megagametophyte of the bunya pine (Araucaria bidwillii Hook.) seed is a storage tissue that surrounds and feeds the embryo. At seed maturity, the megagametophyte is a living tissue and contains protein and starchy reserves. Following seed germination, when all the reserves are hydrolyzed and transferred to the embryo, the megagametophyte degenerates as a no longer needed tissue. In the current study it was investigated whether the megagametophyte degeneration could be recognized as a form of PCD, by searching typical hallmarks of apoptosis. The viability assay showed a progressive spreading of cell death over the tissue in the post-germinative stages. Biochemical data revealed a strong reduction of protein levels and a marked increase in proteolytic activities at late post-germinative stages. Among the proteases detected, some serine proteases appeared the most interesting, because of their high sensitivity towards the caspase 6 inhibitor. DNA fragmentation was demonstrated both in situ by the TUNEL assay and in gel by electrophoresis. Concurrently with DNA fragmentation, increased nuclease activities were detected by using both in vitro and in-gel assays. Finally, the measurements of auxin content revealed a possible role of this hormone in the induction of the megagametophyte PCD.