Proteolytic enzymes in storage protein mobilization and cell death of the megagametophyte of Araucaria bidwillii Hook. post-germinated seeds.

In the present manuscript, we report on the proteolytic enzymes acting in the Araucaria bidwillii megagametophyte throughout seed germination. At seed maturity the megagametophyte contains a bulk of reserves for the growing embryo, thus representing the major storage tissue of the bunya pine seed. Soon after seed germination the megagametophyte undergoes storage protein mobilization, degenerating as a no longer needed tissue by the late germinative stages. By using in-solution and in-gel assays, and mass spectrometric analyses we detected exopeptidases and proteinases differently active in this tissue at selected germinative stages, and obtained preliminary data on the nature of an endopeptidase active at the late stages. Early germination stages were characterized by aminopeptidase and aspartic, metallo and cysteine proteinase activities; carboxypeptidases and serine proteinases became highly active by the late stages. Partial sequencing of a protein responsible for late stage serine peptidase activity sensitive to the caspase-6 inhibitor, showed a set of amino acid sequences with various degrees of identity with various plant subtilisin-like serine proteinases. The participation of the early stage proteases in the storage protein mobilization and the involvement of the late stage proteases in the megagametophyte cell death are proposed and discussed.

Investigation on cell death in the megagametophyte of Araucaria bidwillii Hook. post-germinated seeds.

The megagametophyte of the Araucaria bidwillii seed is a storage tissue that surrounds and feeds the embryo. When all its reserves are mobilized, the megagametophyte degenerates as a no longer needed tissue. In this work we present a biochemical and a cytological characterization of the megagametophyte cell death. The TUNEL assay showed progressive DNA fragmentation throughout the post-germinative stages, while DNA electrophoretic analysis highlighted a smear as the predominant pattern of DNA degradation and internucleosomal DNA cleavage only for a minority of cells at late post-germinative stages. Cytological investigations at these stages detected profound changes in the size and morphology of the megagametophyte nuclei. By using in vitro assays, we were able to show a substantial increase in proteolytic activities, including caspase-like protease activities during the megagametophyte degeneration. Among the caspase-like enzymes, caspase 6- and 1-like proteases appeared to be the most active in the megagametophyte with a preference for acidic pH. On the basis of our results, we propose that the major pathway of cell death in the Araucaria bidwillii megagametophyte is necrosis; however, we do not exclude that some cells undergo developmental programmed cell death.

Programmed cell death of the nucellus during Sechium edule Sw. seed development is associated with activation of caspase-like proteases.

The nucellus is a maternal tissue that embeds and feeds the developing embryo and secondary endosperm. During seed development, the cells of the nucellus suffer a degenerative process soon after fertilization as the cellular endosperm expands and accumulates reserves. Nucellar cell degeneration has been considered to be a form of developmentally programmed cell death (PCD). It was investigated whether or not this degenerative process is characterized by apoptotic hallmarks. Evidence showed that cell death is mostly localized in the border region of the tissue adjacent to the expanding endosperm. Cell death is accompanied by profound changes in the morphology of the nuclei and by a huge degradation of nuclear DNA. Moreover, an increase of activity of different classes of proteinases is reported, and the induction of caspase-like proteases sensitive to specific inhibitors was detected. Nucellar caspase-like proteases are characterized by an acid pH optimum suggesting a possible localization in the vacuole.