Nucellain, a barley homolog of the dicot vacuolar-processing protease, is localized in nucellar cell walls

The nucellus is a complex maternal grain tissue that embeds and feeds the d eveloping cereal endosperm and embryo. Differential screening of a barley ( Hordeum vulgare) cDNA library from 5-d-old ovaries resulted in the isolatio n of two cDNA clones encoding nucellus-specific homologs of the vacuolar-pr ocessing enzyme of castor bean (Ricinus communis). Based on the sequence of these barley clones, which are called nucellains, a homolog from developin g corn (Zea mays) grains was also identified. In dicots the vacuolar-proces sing enzyme is believed to be involved in the processing of vacuolar storag e proteins. RNA-blot and in situ-hybridization analyses detected nucellain transcripts in autolysing nucellus parenchyma cells, in the nucellar projec tion, and in the nucellar epidermis. No nucellain transcripts were detected in the highly vacuolate endosperm or in the other maternal tissues of deve loping grains such as the testa or the pericarp. Using an antibody raised a gainst castor bean vacuolar-processing protease, a single polypeptide was r ecognized in protein extracts from barley grains. Immunogold-labeling exper iments with this antibody localized the nucellain epitope not in the vacuol es, but in the cell walls of all nucellar cell types. We propose that nucel lain plays a role in processing and/or turnover of cell wall proteins in de veloping cereal grains.