γ-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 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.electron microscopy | stacking of RNAi events | storage organs | opaque phenotype | kernel hardness G rain hardness is a key agronomic trait in maize (Zea mays L.)because it provides resistance to damage during harvesting and marketing, as well as to insect and fungal damage. Kernel texture is determined by the relative amounts of hard (vitreous) and soft (opaque) endosperm and there is a positive correlation between zein storage proteins and kernel vitreousness (1). Zeins are a heterogeneous mixture of alcohol-soluble proteins, falling into four classes based on their structure (α-, β-, γ-, and δ-zeins) (2). The zeins extracted with the Osborne method (3) are classified as z1 (19-and 22-kDa α-zeins) and the cross-linked z2 group (50-, 27-, and 16-kDa γ-zeins, 15-kDa β-zein, and 18-and 10-kDa δ-zeins) (4, 5). Zeins are deposited in rough endoplasmic reticulum-delimited protein bodies (PBs) in endosperm cells from around 10 d after pollination (DAP) (6, 7). Alpha-and δ-zeins are mainly stored in the center of PBs, and γ-and β-zeins are deposited in the peripheral region (8). The Cys-rich γ-and β-zeins have redundant function in the stabilization of PB morphology (9). The translucency (vitreousness) of the mature kernel is influenced by PB composition and the spatial organization of α-, β-, γ-, and δ-zeins (10-16).Because zeins are essentially devoid of lysine and tryptophan, their high-lev...

Functions of maize genes encoding pyruvate phosphate dikinase in developing endosperm

“…Determining the functions of each target is necessary to understand fully how o2-affects grain quality and in turn how to best achieve nutritional quality traits. The effects of zein deficiencies and SSIIIa mutations have been characterized (4)(5)(6), but maize endosperm PPDK function has not been described.…”

Mutation in the seed storage protein kafirin creates a high-value food trait in sorghum

“…Therefore, a lysine-enhanced and highly digestible sorghum variety has gained broad attention. Nevertheless, similar to quality protein maize, hdhl requires additional selection to identify modifier genes that make the mutant kernels harder 29 . As shown in maize, the introduction of such different mutations into local germplasm is a complex and time-consuming process 30 .…”

The Maize High-Lysine Mutantopaque7Is Defective in an Acyl-CoA Synthetase-Like Protein

“…Thin sections were prepared and analyzed under the transmission electron microscope as described elsewhere (Wu et al 2010). For total zein protein extraction, the same method was used as in Wu et al (2010). Two individual kernels were processed for all four samples in Figure 5C.…”