Gamma-zeins are essential for endosperm modification in quality protein maize

Abstract

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; gammaRNAi/+ 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.

Fig. 1.

Zein accumulation in normal, o2, QPM, RNAi, and their stacked mutant seeds at 18 DAP detected by SDS/PAGE. (A) CM105+, CM105o2, and CM105Mo2. The slice of protein markers run in a different gel was composited with the samples, as indicated by a white line. (B) BA (nontransgenic hybrid seed of B and A lines as a control), βRNAi, γRNAi, and the stack of the two RNAis. (C) Zein accumulation patterns for 26 kernels dissected from the cross of CM105Mo2 × O2/o2; γRNAi/+. M, protein markers from top to bottom being 25, 20, 15, and 10 kDa. Total zein loaded in each lane was equal to 500 μg of fresh endosperm at 18 DAP. The size for each band is indicated by the numbers in the “kDa” columns.

Fig. 2.

Transmission electron micrographs of protein bodies in different genotypes. (A) CM105+. (B) γRNAi. (C) CM105o2. (D) CM105Mo2. (E) Mo2/+; o2/o2; γRNAi/+. (F) Mo2/+; o2/o2; γRNAi/+; βRNAi/+. (Scale bars, 500 nm.) PB, protein body; RER, rough endoplasmic reticulum.

Fig. 3.

Scanning electron micrographs of protein bodies in different genotypes. (A) CM105+. (B) CM105o2. (C) CM105Mo2. (D) Mo2/+; o2/o2; γRNAi/+. (Scale Bars, 10 μm.) PB, protein body; SG, starch granules.

Fig. 6.

Model for vitreous endosperm formation in which midmaturation stage (18 DAP) starchy endosperm cells are depicted. PBs are represented with gray spheres, starch grains with white spheres, and proteinaceous matrix with blue lines. In wild-type and QPM, compact stable matrices give rise to glass-like, vitreous endosperm at maturity. In opaque mutants and dominant RNAi low-zein lines, small, sparse, or lobed unseparated PBs produce loose, unstable matrices, which shatter during desiccation, producing an opaque texture at maturity.

Fig. 4.

Kernel phenotype for CM105+, CM105o2, CM105Mo2, and new mutant Mo2/+; o2/o2; γRNAi/+. Photographs for intact or decapped kernels were taken under incandescent light (Top and Bottom) or with transmitted light (Middle).

Fig. 5.

Protein accumulation analysis of the γ/β RNAi, z1CRNAi and their triple-stack (z1CRNAi used as pollen) and transmission electron micrographs of their protein bodies. (A) Eight kernels dissected from the cross of γ/β RNAi × z1CRNAi at 18 DAP were analyzed. C, nontransgenic hybrid seed of B and A lines as a control. M, protein markers from top to bottom being 25, 20, 15, and 10 kDa. The slice of protein markers and control run in a different gel was composited with the samples, as indicated by a white line. Total zein loaded in each lane was equal to 500 μg of fresh endosperm at 18 DAP. The size for each band is indicated in the “kDa” column. (B–E) Transmission electron micrographs of protein bodies in γ/β RNAi stack (B), z1CRNAi (C), and the stack of γ/β RNAi and z1CRNAi (D and E). (Scale bars, 500 nm.)