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 theproteolytic enzymes acting in the Araucaria bidwilliimegagametophyte throughout seed germination. At seedmaturity the megagametophyte contains a bulk of reservesfor the growing embryo, thus representing the major storagetissue of the bunya pine seed. Soon after seed germinationthe megagametophyte undergoes storage proteinmobilization, degenerating as a no longer needed tissue bythe late germinative stages. By using in-solution and in-gelassays, and mass spectrometric analyses we detected exopeptidasesand proteinases differently active in this tissueat selected germinative stages, and obtained preliminarydata on the nature of an endopeptidase active at the latestages. Early germination stages were characterized byaminopeptidase and aspartic, metallo and cysteine proteinaseactivities; carboxypeptidases and serine proteinasesbecame highly active by the late stages. Partial sequencingof a protein responsible for late stage serine peptidaseactivity sensitive to the caspase-6 inhibitor, showed a set ofamino acid sequences with various degrees of identity withvarious plant subtilisin-like serine proteinases. The participationof the early stage proteases in the storage proteinmobilization and the involvement of the late stage proteasesin the megagametophyte cell death are proposed anddiscussed.