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Notes on identification and genetics of high methionine Zein-D (10kDa)

--C. M. Wilson

D-zein is of interest because it contains 22% methionine, an essential amino acid. It has 129 amino acids (true molecular mass is 14kDa). The inbred BSSS-53 contains high levels of D-zein (and thus of methionine), with over-expression being regulated by a gene termed Zpr10/(22). Zpr10/(22) is located on chromosome 4, while the structural gene Zps10/(22) is located on chromosome 9. See Kirihara et al. (MGG 211:477-484, 1988) and Benner et al. (TAG 78:761-767, 1989) for details. I prefer the name D-zein rather than 10kDa zein because SDS-PAGE does not provide the correct molecular mass for any zein. My map of zeins on agarose IEF gels places D-zein at position 55, thus giving the term D/55 to identify this zein by position on SDS-PAGE and IEF gels, respectively (Wilson, Plant Physiol. 82:196-202, 1986).

Although D-zein is apparently identified by its position on SDS-PAGE, errors are possible because its mobility varies with different gel conditions and position identification depends upon its mobility relative to molecular mass standard proteins, which also vary considerably in mobility. Other zeins may move to the same position on IEF, another cause of misidentification. IEF followed by SDS-PAGE may provide identification of D-zein, though I have noted an alcohol-soluble protein with an apparent mass of 12kDa which is probably not D-zein (unpublished work). Because D-zein is lacking in tyrosine, D-zein can be distinguished from other zeins after separation by HPLC by a very low absorbance at 280nm relative to 210nm (Wilson, Plant Physiol. 95:777-786, 1991). HPLC also provides a quantitative assay, and preliminary results suggested that inbreds other than BSSS-53 may have above average contents of D-zein.

Hartings et al. (Genet. Agrar. 38:447-464, 1984) found two classes of D-zein which differ slightly by both SDS-PAGE and IEF. I reported what appears to be a variant D-zein (by SDS-PAGE only) in Z. diploperennis (Plant Physiol. 82:196-202, 1986). Recently I found a variant D-zein in an experimental version of B73, provided by R. P. Bergquist, RP (Pfister Hybrid Corn Co., El Paso, IL), which also had a lower mobility by SDS-PAGE. However, agarose IEF did not show a different position. Elution from an HPLC gradient was delayed by 2 min. Thus this variant can be easily detected and confirmed to be D-zein by HPLC. I made crosses to normal B73, to W64A, and to A619. Reciprocal hybrids revealed the expected two peaks, with relative peak heights suggesting that they are determined by co-dominant genes.

This program was terminated and I subsequently retired before I could complete a genetic analysis of these crosses. F1, F2, and backcross seeds (but not the experimental B73) are available for interested researchers. An HPLC analysis of A619 x ExB73 backcross seeds would confirm or eliminate, without using a translocation waxy marker inbred, the suggestion that the structural gene for D-zein is on chromosome 7 (Wilson et al., TAG 77:217-226, 1989) rather than on chromosome 9 (Benner et al., TAG 78:761-767, 1989).

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