γ-Zeins are essential for endosperm modification in quality protein maize

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 gamma-zein locus on chromosome 7S. Moreover, QPM lines have 2- to 3-fold higher levels of the 27-kDa gamma-zein, but the physiological significance of this increase is not known. Because the 27-and 16-kDa gamma-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 gamma-zeins disrupts endosperm modification by o2 modifiers, indicating their hypostatic action to gamma-zeins. Abnormalities in protein body structure and their interaction with starch granules in the F1 with Mo2/+; o2/o2;gamma RNAi/+ genotype suggests that gamma-zeins are essential for restoring protein body density and starch grain interaction in QPM. To eliminate pleiotropic effects caused by o2, the 22-kDa alpha-zein, gamma-zein, and beta-zein RNAis were stacked, resulting in protein bodies forming as honeycomb-like structures. We are unique in presenting clear demonstration that gamma-zeins play a mechanistic role in QPM, providing a previously unexplored rationale for molecular breeding.