CHANGES IN UBIQUITIN AND UBIQUITIN-PROTEIN CONJUGATES DURING SEED FORMATION AND GERMINATION

Changes in both free ubiquitin and ubiquitin-protein conjugates were followed in cotyledons of lupin (Lupinus albus L.) during the course of seed formation, from the flower to the dry seed, and during germination and seedling growth, from the dry seed to the senescing cotyledons. The observed levels of ubiquitin conjugates, detected by immunoblotting using antiubiquitin antibodies and by autoradiography using (125)l- labelled ubiquitin, suggest an intense involvement of the ubiquitin-mediated proteolytic pathway during the highly regulated phases of seed formation and germination, High amounts of free ubiquitin are present at all stages in all tissues examined. With the exception of the dry seed, the high molecular mass ubiquitin-protein conjugates are also present at all stages. Higher amounts of these conjugates were found during the initial stages of pod development and seed germination and during the most active phases of storage protein deposition and degradation. Germination and seedling growth in total darkness not only delays the degradation of the storage proteins, but also extends the period characterized by the presence of a high amount of these conjugates. No such conjugates were detected in the dry seeds, probably reflecting the extremely low metabolic activity observed in these organs. A number of smaller molecular mass polypeptides were also detected at different stages of seed development, germination and seedling growth. Of particular interest is the abrupt accumulation of an abundant 20 kDa polypeptide in the cotyledons during the 4th day after imbibition, which is maintained in high amounts in these organs, rapidly declining after about 12-14 d. The pattern of accumulation of the 20 kDa polypeptide is controlled neither by light nor by the embryo axes, and large variations in its concentration are observed during heat shock.