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Essential amino acids like lysine and tryptophan are deficient in corn meal because of the abundance of zein storage proteins that lack these amino acids. A natural mutant, opaque 2 (o2) causes reduction of zeins, an increase of nonzein proteins, and as a consequence, a doubling of lysine levels. However, o2's soft inferior kernels precluded its commercial use. Breeders subsequently overcame kernel softness, selecting several quantitative loci (QTLs), called o2 modifiers, without losing the high-lysine trait. These maize lines are known as \"quality protein maize\" (QPM). One of the QTLs is linked to the 27-kDa γ-zein locus on chromosome 7S. Moreover, QPM lines have 2- to 3-fold higher levels of the 27-kDa γ-zein, but the physiological significance of this increase is not known. Because the 27- and 16-kDa γ-zein genes are highly conserved in DNA sequence, we introduced a dominant RNAi transgene into a QPM line (CM105Mo2) to eliminate expression of them both. Elimination of γ-zeins disrupts endosperm modification by o2 modifiers, indicating their hypostatic action to γ-zeins. Abnormalities in protein body structure and their interaction with starch granules in the F1 with Mo2/+; o2/o2; γRNAi/+ genotype suggests that γ-zeins are essential for restoring protein body density and starch grain interaction in QPM. To eliminate pleiotropic effects caused by o2, the 22-kDa α-zein, γ-zein, and β-zein RNAis were stacked, resulting in protein bodies forming as honeycomb-like structures. We are unique in presenting clear demonstration that γ-zeins play a mechanistic role in QPM, providing a previously unexplored rationale for molecular breeding.

Maize ( Zea mays L.) grain is an important feedstock for the ethanol-producing industry. However, little is known about the optimum grain quality for optimizing ethanol yielding efficiencies. We specifically investigated the response of ethanol yields (L Mg −1 ) to kernel hardness, and its physiological determinant endosperm zein protein profiles, as affected by genotype selection, field nitrogen (N) fertilization, and crop growth environment. We measured ethanol yield and related this to different kernel hardness indicators, kernel composition, and zein profiles. We also described changes in field ethanol yield (L ha −1 ), by taking into account the crop yield (Mg ha −1 ). Hard endosperm genotypes always yielded less ethanol than softer endosperm ones per grain mass (L Mg −1 ). Higher N fertilization rates increased kernel hardness and decreased ethanol yield (L Mg −1 ) on soft endosperm dented genotypes but had no effect on hard endosperm ones. Ethanol yield was negatively correlated with kernel density, kernel protein concentration, and Z1 and Z2 zein fractions. Within Z2, 15 kDa β-zein explained the largest ethanol yield variation generated by genotypes, N fertilizations, and growth environments. However, and although these differences were as large as 10%, ethanol field yield (L ha −1 ) was mainly driven by crop yields ( r 2 0.98) due to the large crop yield (Mg ha −1 ) differences observed across treatments. Together, our results helped describe the magnitude that changes in maize kernel hardness can have over ethanol yield, both through genotype selection or crop management. A particular Z2 zein protein rises as relevant for future genetic manipulations of maize ethanol yield determination.