DIFFERENTIAL LOCALIZATION OF 2 ACID PROTEINASES IN GERMINATING BARLEY(HORDEUM-VULGARE) SEED

A cathepsin D-like aspartic proteinase (EC 3.4.23) is abundant in unge rminated barley (Hordeum vulgare) seed while a 30 kDa cysteine endopro teinase (EC 3.4.22) is one of the proteinases synthesized de novo in t he germinating seed. In this work, the localization of these two acid proteinases was studied at both the tissue and subcellular levels by i mmunomicroscopy. The results confirm that they have completely differe nt functions. The aspartic proteinase was present in the ungerminated seed and, during germination, it appeared in all the living tissues of the grain, including the shoot and root. Contrary to previous suggest ions, it was not observed in the starchy endosperm. By immunoblotting, the high molecular mass form of the enzyme (32+16 kDa) was found in a ll the living tissues, whereas the low molecular mass form (29+11 kDa) was not present in the shoot or root, indicating that the two enzyme forms have different physiological roles. The aspartic proteinase was localized first in the scutellar protein bodies of germinating seed, a nd later in the vacuoles which are formed by fusion of the protein bod ies. In contrast to the aspartic proteinase, the expression of the 30 kDa cysteine proteinase began during the first germination day, and it was secreted into the starchy endosperm; first from the scutellum and later from the aleurone layer. It was not found in either shoots or r oots. The 30 kDa cysteine proteinase was detected in the Golgi apparat us and in the putative secretory vesicles of the scutellar epithelium. These results suggest that the aspartic proteinase functions only in the living tissues of the grain, as opposed to the 30 kDa cysteine pro teinase which is apparently one of the proteases initiating the hydrol ysis of storage proteins in the starchy endosperm.