Identification of Mutator insertional mutants of starch-branching enzyme 1 (sbe1) in Zea mays L.

Starch-branching enzymes (SBE) alter starch structure by breaking an α-1,4 linkage and attaching the reducing end of the new chain to a glucan chain by an α 1,6 bond. In maize, three isoforms of SBE have been identified. In order to examine the function of the SBEI isoform, a reverse-genetics PCR-based screen was used to identify a mutant line segregating for a Mutator transposon within Sbe1. Compared to wild-type controls, Sbe1 transcripts accumulate at extremely low levels in leaves of the homozygous mutant. Antibodies failed to detect SBEI in leaf tissue of mutants or wild-type controls. In contrast, the level of SBEI in endosperm is undetectable in homozygous mutants while easily detected in wild-type controls. Starches extracted from mutant leaves and endosperm have structures indistinguishable from starches of wild-type controls as determined by size-exclusion chromatography (SEC) of intact starch and high-performance SEC of debranched starch. To investigate the possibility of compensation for the lack of SBEI by expression of the homologous sequence reported by Kim et al. (1998), a genomic fragment (Sbe1b) of this sequence was cloned. Northern hybridizations of mutant leaf, root, tassel, endosperm and embryo tissues with non-specific Sbe1b probes failed to reveal expression of the homologous sequence. These results suggest that the homologous sequence is not compensating for a lack of SBEI in sbe1::Mu mutants. Further study of this sbe1 mutation in the presence of other genetic mutations may help to understand the role of SBEI in determining starch structure in leaves and endosperm.