Molecular organization of a gene in barley which encodes a protein similar to aspartic protease and its specific expression in nucellar cells during degeneration

The nucellar cells of barley undergo progressive degeneration after ovule fertilization. This degeneration is a characteristic of programmed cell death. Increasing evidence has indicated that proteases are important regulators of programmed cell death in animals. We have cloned and characterized a barley gene which encodes an aspartic protease-like protein and is specifically expressed in nucellar cells during degeneration. The gene contains eight exons and seven introns and encodes a polypeptide of 410 amino acid residues. The deduced polypeptide is characterized by having two aspartic protease catalytic site motifs, the Asp-Thr-Gly-Ser in the N-terminal and Asp-Ser-Gly-Ser in the C-terminal region, and two other regions nearly identical to two regions of plant aspartic proteases. However, it shares <20% overall sequence identity with the known plant aspartic proteases, and does not contain a 'prosequence' or a 'plant-specific insert' which are characteristics of plant aspartic proteases. We have named this aspartic protease-like protein 'nucellin'. In northern analyses nucellin transcripts were most abundant in ovaries 3-4 days after pollination, but only marginally detectable before pollination or 10 days after pollination. RNA in situ hybridization showed that before pollination the nucellin gene was expressed at a very low level only in a cluster of nucellar cells close to the embryo sac at the chalazal end, but after pollination it was highly expressed in most nucellar cells surrounding the entire embryo sac. Furthermore, no nucellin transcripts were detectable in anther, leaf, or root tissue. The temporal and spatial pattern of the nucellin gene expression is synchronal with nucellar cell degeneration and thus, nucellin may be involved with nucellar cell death.