JXB Advance Access originally published online on January 17, 2007
Journal of Experimental Botany 2007 58(5):1059-1070; doi:10.1093/jxb/erl267
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RESEARCH PAPER |
Protein storage vacuole acidification as a control of storage protein mobilization in soybeans
Department of Biological Sciences, State University of New York at Binghamton, Binghamton, NY 13902-6000, USA
To whom correspondence should be addressed. E-mail: annatan{at}binghamton.edu
Soybean protease C1 (EC 3.4.21.25 [EC] ), the subtilisin-like serine protease that initiates the proteolysis of seed storage proteins in germinating soybean [Glycine max (L.) Merrill], was localized to the protein storage vacuoles of parenchyma cells in the cotyledons by immunoelectron microscopy. This was demonstrated not only in germination and early seedling growth as expected, but also in two stages of protein storage vacuole development during seed maturation. Thus, the plant places the proteolytic enzyme in the same compartment as the storage proteins, but is still able to accumulate those protein reserves. Since soybean protease C1 activity requires acidic conditions for activity, the hypothesis that the pH condition in the protein storage vacuole would support protease C1 activity in germination, but not in seed maturation, was tested. As hypothesized, acridine orange accumulation in the protein storage vacuole of storage parenchyma cells was detected by fluorescence confocal microscopy in seedlings before the onset of mobilization of reserve proteins as noted by SDS-PAGE. Accumulation of the dye was reversed by inclusion of the weak base methylamine to dissipate the pH gradient across the vacuolar membrane. Also as hypothesized, acridine orange did not accumulate in the protein storage vacuole of those parenchyma cells during seed maturation. These results were obtained using cells separated by pectolyase treatment and also using cotyledon slices.
Key words: Acidity, cotyledon, enzyme regulation, protease C1, protein storage vacuole, proteolytic enzyme, seed maturation, soybean [Glycine max (L.) Merrill], storage protein mobilization, subcellular localization
* These authors contributed equally to this work.
Present address: Department of Biology, University of Pennsylvania, Philadelphia, PA 19104, USA.
Present address: Proteomics Core, COBRE Center for Cancer Research Development, Rhode Island Hospital, Providence, RI 02903, USA.
Received 28 July 2006; Revised 24 October 2006 Accepted 13 November 2006
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