Identification, cDNA cloning and possible roles of seed-specific rice asparaginyl endopeptidase, REP-2

We previously showed that two major cysteine endopeptidases, REP-1 and REP-2, were present in germinated rice (Oryza sativa L.) seeds, and that REP-1 was the enzyme that digests seed storage proteins. The present study shows that REP-2 is an asparaginyl endopeptidase that acts as an activator of REP-1, and we separated it into two forms, REP-2α (39 kDa) and REP-2β (40 kDa), using ion-exchange chromatography and gel filtration chromatography. Although analysis of the amino terminals revealed that 10 amino acids of both forms were identical, their isoelectric points were different. SDS–PAGE/immunoblot analysis using an antiserum raised against legumain, an asparaginyl endopeptidase from jack bean, indicated that both forms were present in maturing and germinating rice seeds, and that their amounts transiently decreased in dry seeds. Northern blot analysis indicated that REP-2 mRNA was expressed in both maturing and germinating seeds. In germinating seeds, the mRNA was detected in aleurone layers but not in shoot and root tissues. Incubation of the de-embryonated seeds in 10–6 M gibberellic acid induced the production of large amounts of REP-1, whereas REP-2β levels declined rapidly. Southern blot analysis showed that there is one gene for REP-2 in the genome, indicating that both REP-2 enzymes are generated from a single gene. The structure of the gene was similar to that of β-VPE and γ-VPE isolated from Arabidopsis thaliana.