ALTERATION OF THE STRUCTURAL-PROPERTIES OF STARCH COMPONENTS BY THE LACK OF AN ISOFORM OF STARCH BRANCHING ENZYME IN RICE SEEDS

This study describes the effect of starch-synthesizing enzymes on bios ynthesis of storage starch in rice amylose-extender mutants, which con tain branched D-glucans with abnormal structures. Western blot analysi s indicated that two out of five amylose-extender mutant lines lacked an isoform of starch branching enzyme, termed RBE3, although the level s of granule-bound starch synthase and a major form of branching enzym e, RBE1, were normal in these two mutants. Proteins corresponding to t he 87-kDa RBE3 molecule were present in the three other amylose-extend er mutants as well as in the wild type. However, the level of branchin g enzyme activity significantly decreased in all amylose-extender muta nts, suggesting that the 87-kDa proteins in these three mutants are in active forms of RBE3. Therefore, we conclude that formation of the abn ormal branched glucans in the amylose-extender mutant of rice is due t o the lack of the RBE3 activity. The cDNA clones encoding RBE3 have be en identified from a normal rice seed cDNA library in lambdagt11, usin g a synthetic oligonucleotide as a probe. The deduced amino acid seque nce of RBE3 indicates that this protein is initially synthesized as a precursor of 825 amino acids, including a 65-residue transit peptide a t the NH2 terminus. The sequences of the catalytic regions in amylolyt ic enzymes are highly conserved in the sequence of RBE3. Thus, the bra nching enzyme isoform belongs to a family of the amylolytic enzymes. R BE3 also shares a noticeable degree of sequence identity with RBE1, es pecially at the central portion of the protein molecule. However, RBE3 possesses an approximately 70-residue extra sequence at the NH2 termi nus and lacks a COOH-terminal sequence of almost 50 residues as compar ed with RBE1. The structural differences at both termini may explain t he distinct role in starch synthesis for RBE1 and RBE3.