The aspartic proteinase is expressed in Arabidopsis thaliana seeds and localized in the protein bodies

We have been studying a seed aspartic proteinase, termed AtAP, from Arabido psis thaliana. In previous work, we purified the proteinase, analysed its a ctivity and isolated the cDNA sequence. In this paper, the expression of th e mRNA for the aspartic proteinase was analysed in seed tissues both by Nor thern blots for overall regulation and by in situ hybridization to follow c ell-specific localization of message. We found a 1.9 kb aspartic proteinase message in dry seeds and seed pods. This message was expressed in many dif ferent cell types of the mature dry seed. The localization of the protein w ithin these cells was also determined. Antibodies were raised against the A tAP and purified using affinity chromatography on an AtAP-immobilized-pepst atin A-agarose column. This purified antibody recognized several AtAP pepti des in seeds. To localize the enzyme in cells, we isolated protein bodies f rom the dry seeds of Arabidopsis using a non-aqueous isolation method. The AtAP activity and antigenic peptides were found to be highest in the protei n body fraction and colocalized with the seed storage protein 2S albumin an d the vacuolar marker enzyme ol-mannosidase. This protein body localization of the AtAP was confirmed with immunocytochemical localization by electron microscopy and shows that the protein is not secreted by these cells.