Biochemical characterization of “high-lysine” endosperm mutants
--Bosio, D, Balconi, C, Motto, M

The storage proteins of maize are a subject of interest from the standpoints of grain quality and nutrition. Of these proteins, the zeins of the endosperm have received the greatest attention from biochemists, molecular biologists, and geneticists. These attentions are primarily due to the high proportion of zeins in the total protein complement and their deficiency in certain essential amino acids, the most limiting of which is lysine. Zein synthesis is affected by a number of genes, some of which are defined by mutant alleles. All mutants confer an opaque phenotype to the endosperm and the development of "high lysine" maize was predicted on the use of these mutations, because they inhibit zein synthesis and therefore, elevate the percentage of lysine in the grain. Some of these mutant genes have been mapped and their effect on zein synthesis described (Motto et al., Oxford Survey Plant Mol. Cell Biol. 6:87-114, 1989). Our research is devoted to a biochemical analysis of the opaque endosperm mutants, opaque1 (o1), opaque5 (o5), opaque9 (o9), opaque11 (o11), floury1 (fl1) and floury3 (fl3) through a characterization by SDS and IEF electrophoresis analysis of zein accumulation in the endosperm during development.

The inbred lines A69y +, A69y o1, A69y o2-m(r)+Bg, A69y o5, A69y o9, A69y o11, A69y fl1 and A69y fl3 were grown at the Institute of Cereal Research, Section of Bergamo, Italy. Ear samples were harvested at 15 and 25 days after pollination (DAP), and at maturity. The seed samples harvested at 15 and 25 DAP were frozen in liquid nitrogen and the seeds stored at -20 C and an aliquot at -80 C. For the chemical analyses endosperms without embryos were used after freeze drying for 48 h.

The procedures used for analyses are described in Balconi et al. (Plant J. 3(2):325-334, 1993). Samples of endosperm, freeze-dried and ground in a mortar, were analyzed for total nitrogen (N) content on an automated N analyzer (NA-1500 Carlo Erba). Proteins were fractionated and total protein percentage (percent N*6.4) was calculated by subtracting from the value of total N content the value of non-protein-N (NPN). Albumin plus globulin and zein fractions were analyzed by SDS gel electrophoresis. Quantification of the proportion of zein components within the total zein fraction was accoplished by scanning the SDS gels with a Personal Densitometer (Molecular DynamicsTM). Data were analyzed by analysis of variance procedures and means were compared using the protected least significant difference (LSD) test, at the O.05 probability level.

The endosperm mutants o2-m(r)+Bg, o11, and fl3, in comparison to wild-type endosperms, show a higher dry weight content throughout the sampling stages (mg/endosperm), and in particular at maturity. The results of total protein content (mg/endosperm) in wild-type and mutant endosperms during development and at maturity indicated that o2-m(r)+Bg and fl3 mutant endosperms showed a lower endosperm total protein content, with respect to the wild-type endosperms, and followed the same trend as observed for the dry weight accumulation.

The values of protein fractions as a percentage of total protein, in wild-type and mutant endosperms, showed that for all genotypes the zein fraction increases during development, whereas, albumin plus globulin and glutelin fractions decrease. For all genotypes at all developmental stages, SDS-PAGE analysis of alcohol-soluble proteins showed the presence of polypeptides with a molecular weight corresponding to all known alcohol-soluble polypeptides that are commonly referred to as zeins.

Zein partitioning into zein families, derived by densitometer tracings of the SDS-PAGE showed, as expected, that the o2 mutation causes a suppression of a specific zein family, the 22 kDa zein component; in addition at 15 DAP the 25 kDa zein component was very abundant in comparison with the wild-type. On the other hand, in the o5 endosperm mutant, at both developmental stages, a reduced 28 kDa zein component was observed. In all genotypes the 20 kDa zein component (which can be resolved into two bands) was the most abundant. For the other mutants, no specific effect on the accumulation of zein components was observed.

Interestingly, in addition to the o2-m(r)+Bg, the SDS-PAGE analysis of albumin plus globulin protein fraction of wild-type and mutant at the various sampling stages showed the absence of the b32 protein in fl3 mutant at maturity stage. In the fl3 mutant the absence, at all stages of development, of a protein band with molecular weight of 14 kDa, was also noted, which was present in the wild-type and other endosperm mutants at 25 DAP and at maturity. Studies are in progress to analyze in more detail these and other protein bands with different expression in wild-type and mutant endosperm kernels.

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