Does and asparaginyl-specific cysteine endopeptidase trigger phaseolin degradation in cotyledons of kidney bean seedlings?

An asparaginyl‐specific cysteine endopeptidase which was named ’legumain‐like proteinase' (LLP) and has an apparent molecular mass of 38.1 kDa was isolated from cotyledons of kidney bean (Phaseolus vulgaris L.) seedlings and partially characterized. It is, to our knowledge, the first known proteinase which in vitro extensively degrades native phaseolin, the major storage globulin of this grain legume. Phaseolin that in vitro had been partially degraded by LLP (Pvitro) and phaseolin that was isolated after partial in vivo breakdown 6 days after the start of seed imbibition (Pvivo) showed similar fragment patterns on SDS/polyacrylamide gels. The fragments had identical cleavage sites in Pvitro and Pvivo as determined by partial amino acid sequencing. In both types of partially degraded phaseolin, these cleavage sites have asparagine in the P1 position. Two of the cleavage sites are located in the β‐barrel domain of the C‐terminal module and only one cleavage site was found in the β‐barrel domain of the N‐terminal module according to the consensus structural model of phaseolin subunits. These results suggest that very likely LLP could in vivo be responsible for the initiation of phaseolin proteolysis. Two different legumain‐specific clones named cp6b and p21b were isolated from a cDNA library of germinated bean cotyledons. Cp6b encodes LLP, while p21b encodes a VPE‐like enzyme. Southern‐blot analysis revealed a single gene copy for Pv‐VPE and, presumably, at least two gene copies for LLP in the kidney bean genome. Northern‐blot analysis indicated that mRNAs for both clones appear de novo during seed germination. However, the developmental patterns of the transcript levels corresponding to the two clones differed significantly. The temporal pattern of phaseolin degradation and of LLP polypeptide levels agreed well with the suggestion that LLP plays a key role in the mobilization of phaseolin during and after kidney bean germination.