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

Published online by Cambridge University Press:  22 February 2007

Asuman Mutlu ,
Xia Chen ,
Sridhar M. Reddy  and
Susannah Gal
Asuman Mutlu
Affiliation:
Department of Biological Sciences, The State University of New York at Binghamton, Binghamton, NY 13902–6000, USA
Xia Chen
Affiliation:
Department of Biological Sciences, The State University of New York at Binghamton, Binghamton, NY 13902–6000, USA
Sridhar M. Reddy
Affiliation:
Department of Biological Sciences, The State University of New York at Binghamton, Binghamton, NY 13902–6000, USA
Susannah Gal*
Affiliation:
Department of Biological Sciences, The State University of New York at Binghamton, Binghamton, NY 13902–6000, USA
*
*Correspondence Fax: 607 777 6521 Email: sgal@binghamton.edu
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Abstract

We have been studying a seed aspartic proteinase, termed AtAP, from Arabidopsis thaliana. In previous work, we purified the proteinase, analysed its activity and isolated the cDNA sequence. In this paper, the expression of the mRNA for the aspartic proteinase was analysed in seed tissues both by Northern blots for overall regulation and by in situ hybridization to follow cell-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 different cell types of the mature dry seed. The localization of the protein within these cells was also determined. Antibodies were raised against the AtAP and purified using affinity chromatography on an AtAP–immobilized-pepstatin A–agarose column. This purified antibody recognized several AtAP peptides in seeds. To localize the enzyme in cells, we isolated protein bodies from the dry seeds of Arabidopsis using a non-aqueous isolation method. The AtAP activity and antigenic peptides were found to be highest in the protein body fraction and co-localized with the seed storage protein 2S albumin and the vacuolar marker enzyme α-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.

Keywords

2S albuminin situ hybridizationprobarley lectinprocessing proteaseprotein precursorprotein storage vacuolesstorage protein
Type
Research Article
Information
Seed Science Research , Volume 9 , Issue 1 , January 1999 , pp. 75 - 84
Copyright
Copyright © Cambridge University Press 1999

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