Poster: Seed Biology

Abs # 555: A newly-discovered protein embedded in the phytate crystals of seeds?

Presenter:	Gillespie, Jane E., jeagley@wsu.edu
Authors	Gillespie, Jane E. (A)   Rogers, Sally W. (A)   Dupree, Paul  (B)   Rogers, John C. (A)
Affiliations:	(A): Institute of Biological Chemistry, Washington State University, USA (B): Department of Biochemistry, University of Cambridge, UK

Plant seeds contain a complex multivesicular organelle known as the protein storage vacuole (PSV). Within this PSV is a membrane-bound internal vesicle, the globoid, which stores phytate (inositol hexakisphosphate) crystals. Phytate is important as a source of phosphorus and cations for use during germination. It also acts as an antinutrient in the human diet, and is an environmental pollutant in animal waste. We want to know how phytate crystals are formed and deposited within the globoid compartment. To address this question, we isolated a protein embedded in phytate crystals. Purified B. napus PSVs were lysed and globoids purified on a sucrose step gradient. Adherent protein was removed from the crystals by heating in SDS. The phytate was then solublized in a glycine solution at pH 2.5. The protein released by this step was analyzed by SDS-PAGE; two predominant ~45 kDa proteins were present. We have sequenced four tryptic peptides belonging to these proteins by mass spectroscopy analysis. They closely match an Arabidopsis homologue, which is encoded by AT3g20370 and predicted to be similar in size to the two B. napus proteins. The sequence predicts an N-terminal signal peptide, several Asn-linked glycosylation sites, and two tandem MATH domains. The latter are known to interact to from multimers. Efforts are currently underway to determine the cellular localization of this protein in developing seeds, as well as to express the protein in tobacco protoplasts and compare its localization in these cells to that of other known PSV and secretory system markers.