© 2001 Heron Publishing—Victoria, Canada
Characterization of proteinase activity in stratified Douglas-fir seeds
Benjamin S. Forward (1), Timothy J. Tranbarger (2) and Santosh Misra (1, 3)
1. Department of Biochemistry and Microbiology and Centre for Forest Biology, University of Victoria, Victoria, B.C., V8W 3P6,
Canada / 2. Biochimie et Physiologie Moléculaire des Plantes, INRA/ENSA-M/CNRS URA 2133, Place Viala, 34060 Montpellier Cedex 1, France / 3. Author to whom correspondence should be addressed (smisra@uvic.ca) / Received October 31, 2000
Summary
We investigated the effect of stratification on the proteinase activity involved in mobilization of the major soluble ~45
kDa storage protein during germination of Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) seeds. Complete hydrolysis of the ~45 kDa protein was observed approximately 7 days after exposure of stratified
seeds to germination conditions. Coincident with the onset of mobilization, proteinase activity was detected primarily in
microsomal extracts from stratified seeds. Microsomal-associated proteinase activity was most active at pH 8.0 and had a molecular
mass > 175 kDa as determined by gelatin SDS-PAGE gels. In vitro digestion of soluble protein extracts indicated that, following stratification, there was a significant increase in proteinase
activity and hydrolysis of the ~45 kDa storage protein. Whether this increase was a result of activation of preexisting proteinase(s)
or de novo synthesis remains unknown. In vitro digestion of soluble protein extracts in the presence of various proteinase inhibitors showed that digestion of the ~45 kDa
storage protein is mediated primarily by a metalloproteinase and to a lesser degree by a serine proteinase. The accumulation
of ~25 kDa protein products following in vitro digestion suggests that mobilization of the ~45 kDa soluble storage protein is mediated by a multi-step process involving
the action of different classes of proteinases.
Keywords:
germination, storage protein mobilization, stratification.
