Upon germination of the seeds, the storage proteins in the PSV are degraded by a papain-type proteinase, designated SH-EP (Mitsuhashi et al., 1986; Okamoto and Minamikawa, 1998) and the Azacitidine(Vidaza) PSV is converted to a lytic vacuole (LV). in the cells of germinated cotyledon. seeds, proteins and starch are stored in protein storage vacuoles (PSVs) and starch granules (SGs), respectively (Harris and Chrispeels, 1975; Minamikawa and Koshiba, 1979). Upon germination of the seeds, the storage proteins in the PSV are degraded by a papain-type proteinase, designated C11orf81 SH-EP (Mitsuhashi et al., 1986; Okamoto and Minamikawa, 1998) and the PSV is converted to a lytic vacuole (LV). Recently, it has been suggested that mass transport of proSH-EP from the endoplasmic reticulum (ER) to the PSV by ER-derived vesicles with a diameter of 200C500 nm mediates the characteristic/rapid conversion of PSV to LV in cotyledon cells (Toyooka et al., 2000). Although the degradation mechanism of storage proteins has been gradually resolved, the breakdown processes of another major storage material, starch, are still unclear. By biochemical analysis, -amylase was identified as the enzyme that has a major role in the degradation of stored starch in the cotyledons of germinated seeds (Koshiba and Minamikawa, 1981), but it remains open how the enzyme reaches SG or how degradation of SG occurs in the cotyledon cells. Since -amylase has a signal peptide (Yamauchi et al., 1994) that should be recognized Azacitidine(Vidaza) with signal recognition particles, the enzyme would enter the lumen of the ER (von Heijne, 1983) and thus is expected to be further transported to the vacuoles or secreted by the endomembrane system. This supposed intracellular localization of -amylase indicates that the enzyme is not directly sorted to SG originating from plastides. In this study, we first identified the intracellular localization of -amylase in the cotyledon cells of seedlings or embryo axis-removed (detached) seeds by immunocytochemical assays using affinity-purified antiC-amylase antibody. The results obtained showing the vacuolar localization of -amylase in the cotyledon cells led us to observe the ultrastructures of the cotyledon cells to see the interaction of the vacuoles with the SG. The SG was wrapped with the acidic cell compartment that would be formed by fusion of a de novo synthesized acidic vesicle and subsequently incorporated into the LV. In addition, a distinct autophagic process to that for SG was found to be involved in the degradation of cytoplasm and mitochondrion. We showed that at least two distinct autophagic processes function for the degradation of the SG and other cytoplasmic components in the cells of the germinating cotyledon. Results Expression of -amylase in attached or detached cotyledons We have previously reported that removal of the embryonic axes from seeds had effects on expression of proteinase activity and degradation of storage reserves in cotyledons (Minamikawa, 1979; Yamauchi et al., 1994). When seeds were allowed to germinate at 27C in darkness, proteinase and amylase activities in the cotyledons (attached cotyledons) increased and reached a maximum level at 3 d after imbibition, and the amounts of storage proteins and starch gradually decreased during seed germination and subsequent seedling growth. When detached cotyledons (embryonic Azacitidine(Vidaza) axes were removed from dry seeds) were prepared and incubated under the same conditions as above, mobilization of storage proteins and starch was not observed, and the proteinase activity remained at a low level. However, amylase activity appeared even in the detached cotyledons at an equivalent level to that in the attached cotyledons (Yamauchi et al., 1994). In this study, changes with time in amounts of a papain-type proteinase (SH-EP), -amylase and an asparaginyl endopeptidase (VmPE-1; Okamoto et al., 1994) in the attached or detached cotyledons were analyzed by SDS-PAGE and immunoblotting with antiCSH-EP, -amylase, or VmPE-1 antibody. In the attached cotyledons, these three.