Nonredundant function of zeins and their correct stoichiometric ratio drive protein body formation in maize endosperm

Abstract

Zeins, the maize (Zea mays) prolamin storage proteins, accumulate at very high levels in developing endosperm in endoplasmic reticulum membrane-bound protein bodies. Products of the multigene α-zein families and the single-gene γ-zein family are arranged in the central hydrophobic core and the cross-linked protein body periphery, respectively, but little is known of the specific roles of family members in protein body formation. Here, we used RNA interference suppression of different zein subclasses to abolish vitreous endosperm formation through a variety of effects on protein body density, size, and morphology. We showed that the 27-kilodalton (kD) γ-zein controls protein body initiation but is not involved in protein body filling. Conversely, other γ-zein family members function more in protein body expansion and not in protein body initiation. Reduction in both 19- and 22-kD α-zein subfamilies severely restricted protein body expansion but did not induce morphological abnormalities, which result from reduction of only the 22-kD α-zein class. Concomitant reduction of all zein classes resulted in severe reduction in protein body number but normal protein body size and morphology.

Figure 1.

Current model for zein protein body architecture at early (left), mid- (middle), and mature stages.

Figure 2.

Scheme of zein RNAi constructs with 27-kD γ-zein promoter (p27γ) and Cauliflower mosaic virus terminator (T35S).

Figure 3.

qRT-PCR expression analysis of α- and γ-zein genes in RNAi lines relative to wild-type nontransgenic (HiII). Each graph represents the measurement of a different zein gene in all the RNAi lines, which are shown on x axes. Values and sd (shown in insert) are the average of three biological replicate kernels and also incorporated three technical replicates of each measurement.

Figure 4.

Kernel phenotypes of zein RNAi lines. A, Half kernels showing vitreous or opaque endosperm. B, Kernels on a light box showing vitreousness or opacity. C, RNAi transgenes present in A, B, D, and E. D, Embryo genomic PCR showing genotype of kernels shown in E, which were used for TEM analysis. PCRs for double and triple construct lines were conducted separately and combined for agarose gel electrophoresis. Minus symbol refers to nontransgenic HiII control kernel, and plus symbol refers to the transgenic kernel. Size for PCR product in base pair is indicated. E, SDS-PAGE of purified zeins from developing endosperms of genotypes shown in D.

Figure 5.

TEM analysis showing protein body density in fourth subaleurone starchy layer of 18-DAP endosperm in zein RNAi lines and their crosses. Bar = 5 μm (A; refers to all panels). RNAi transgenes present are shown in bottom left of each section.

Figure 6.

TEM analysis showing protein size and morphology in fourth subaleurone starchy layer of 18-DAP endosperm in zein RNAi lines and their crosses. Bar = 1 μm (A; refers to all panels). RNAi transgenes present are shown in bottom left of each section.

Figure 7.

Immunogold TEM analysis showing α- and γ-zein distribution in protein bodies of fourth subaleurone cell layer of 18-DAP endosperm in zein RNAi lines and their crosses. Bar = 1 μm (A; refers to all panels). RNAi transgenes present are shown in bottom left of each section. α-Zein antibody was raised to total α-zeins, and γ-zein antibody was raised to 27-kD γ-zein.