Journal of Experimental Botany, Vol. 54, No. 384, pp. 913-922,
March 1, 2003
© 2003 Oxford University Press
Uncleaved legumin in developing maize endosperm: identification, accumulation and putative subcellular localization
Received 5 June 2002; Accepted 28 October 2002
1 Laboratory of Molecular Cell Biology, Department of Biological Resources, Faculty of Agriculture, Ehime University, Matsuyama 790-8566, Japan
2 Botany Department, Box 7612, North Carolina State University, Raleigh, NC 27695, USA
3 To whom correspondence should be addressed. Fax: +81 89 946 9853. e-mail: abe{at}mcb.agr.ehime-u.ac.jp
Abbreviations: CDB, cytoskeleton depolymerizing buffer; CSB, cytoskeleton stabilizing buffer; DTT, dithiothreitol; EGTA, ethylenebis(oxyethylenenitrilo)tetraacetic acid; HEPES, N-2-hydroxyethylpiperazine- N'-2-ethanesulphonic acid; NP-40, Nonidet P-40; PMSF, phenylmethylsulphonyl fluoride; PTE, polyoxyethylene-10-tridecyl ether; SDS, sodium dodecyl sulphate; SDS-PAGE, SDS-polyacrylamide gel electrophoresis; TritonX-100, polyethylene glycol mono-p-isooctylphenyl ether; Tris, tris-(hydroxymethyl) aminomethane.
While identifying proteins present in the cytoskeleton and protein body fractions from maize (Zea mays L.) endosperm, a 51 kDa protein was discovered in a fraction containing small (
200 nm in diameter) protein bodies. Based on partial amino acid sequences of V8 protease fragments, degenerate primers were made and fragments of cDNA encoding these partial sequences were cloned. Using 3' and 5' PCR, a full-length cDNA encoding this 51 kDa protein was obtained, which was identified as legumin-1. In other plants, this protein is generally cleaved into 20 and 35 kDa subunits after synthesis. However, SDS-PAGE of both the native and denatured protein indicates that cleavage does not occur in corn endosperm, even though the cleavage site (asparagine) is conserved. The lack of cleavage is presumably because the canonical cleavage sequence downstream from the cleavage site is almost totally absent. levels of transcript and encoded protein were compared in all three varieties and it was shown that both are more abundant in wild-type maize than in opaque-2 or sweet corn. Finally, using TEM, it was shown that the protein apparently occurs in morphologically distinct protein bodies, very similar to the protein bodies in legumes.
Key words: Cytoskeleton, gene expression, legumin, peptide mapping, protein body, zein.
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