Soybean seed storage protein mobilization: proteolytic activity profiling & role of membrane proton pumps.

摘要

Plants employ various proteolytic enzymes to mobilize seed storage proteins to sustain seedling development. This process is vital in ensuring plant propagation until the establishment of photosynthetic autotrophy. Study of the intrinsic proteolytic apparatus along with information about regulatory mechanisms governing such activities are fundamental to understanding plant protein metabolism. Such reports would also be valuable to ongoing efforts to develop seed-based expression systems for production of recombinant proteins, as well as in the applicability of plant proteases to combat seed protein allergenicity. Characterization of the soybean (Glycine max [L.] Merrill) cv. Williams 82 proteolytic system was undertaken to establish a foundation for future investigations. Temporal patterns of mobilization of major storage protein polypeptides, as well as trends in various proteolytic activities in soybean cotyledons during the first 14 days of germination and seedling growth are reported. Also, a more comprehensive comparative survey involving quantification of proteolytic activities separated by ion exchange chromatography is reported. Purification of cysteine proteases by various chromatographic modalities: ion exchange, thiol-disulfide exchange, and organomercurial, was evaluated.;Protein storage vacuole (PSV) acidification following germination was previously reported to be a crucial regulatory mechanism in soybean storage protein mobilization (He et al., 2007). The molecular apparatus responsible for this acidification was investigated. Soybean seed storage protein mobilization was significantly inhibited in the presence of imidodiphosphate (IDP), a specific inhibitor of vacuolar H+-PPase. Treatment with H+-ATPase inhibitor concanamycin A had no such effect on mobilization activity. Confocal microscopy of soybean cotyledon sections revealed that treatment with IDP also resulted in a substantial decrease in fluorescence intensity associated with acridine orange accumulation, indicating a marked inhibition of acidification of the PSV by IDP. The involvement of vacuolar H+-PPase in the induction of PSV acidification during early seedling growth is suggested and discussed.