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 alpha-1,4 linkage and attaching the reducing end of the new chain to a glucan chain by an alpha 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 etal. (1998), agenomic fragment (Sbe1b) of this sequence was cloned. Northern hybridizations of mutant leaf, root, tassel, endosperm and embryo tissues with non-specific Sbelb 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 sbel mutation in the presence of other genetic mutations may help to understand the role of SBEI in determining starch structure in leaves and endosperm.
Blauth
Susan L
SL
The Biotechnology Institute, Department of Horticulture, Pennsylvania State University, University Park 16802, USA.
Kim
Kyung-Nam
KN
Klucinec
Jeffery
J
Shannon
Jack C
JC
Thompson
Donald
D
Guiltinan
Mark
M
D015061:1,4-alpha-Glucan Branching Enzyme / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D004251:DNA Transposable Elements / Q000235:genetics*; D018744:DNA, Plant / Q000235:genetics / Q000302:isolation & purification; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D006720:Homozygote; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009154:Mutation; D018749:RNA, Plant / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013213:Starch / Q000378:metabolism; D003313:Zea mays / Q000201:enzymology / Q000235:genetics*
D004251:DNA Transposable Elements; D018744:DNA, Plant; D018749:RNA, Plant; D013213:Starch; D015061:1,4-alpha-Glucan Branching Enzyme