Expression of a mutant alpha-zein creates the floury2 phenotype in transgenic maize

Abstract

The maize floury2 mutation results in the formation of a soft, starchy endosperm with a reduced amount of prolamin (zein) proteins and twice the lysine content of the wild type. The mutation is semidominant and is associated with small, irregularly shaped protein bodies, elevated levels of a 70-kDa chaperone in the endoplasmic reticulum, and a novel 24-kDa polypeptide in the zein fraction. The 24-kDa polypeptide is a precursor of a 22-kDa alpha-zein protein that is not properly processed. The defect is due to an alanine-to-valine substitution at the C-terminal position of the signal peptide, which causes the protein to be anchored to the endoplasmic reticulum. We postulated that the phenotype associated with the floury2 mutation is caused by the accumulation of the 24-kDa alpha-zein protein. To test this hypothesis, we created transgenic maize plants that produce the mutant protein. We found that endosperm in seeds of these plants manifests the floury2 phenotype, thereby confirming that the mutant alpha-zein is the molecular basis of this mutation.

Figure 1

Transformation of the 24-kDa α-zein gene into transgenic maize plants leads to synthesis of the encoded protein and overexpression of BiP. Analysis of 3 of 17 independent transgenic lines is shown. One normal and one floury kernel from transgenic lines 236300 (lanes 3 and 4), 236133 (lanes 5 and 6), and 236294 (lanes 7 and 8) were analyzed and compared with kernels from the untransformed (lane 1, P1), the untransformed parent outcrossed to a normal inbred (lane 2, P1 × P2), and W64Afl2 (lane 9) plants. (A) Using PCR primers specific to the 24-kDa α-zein gene, a 560-bp DNA fragment was amplified from genomic DNA of seedlings germinated from floury kernels, but not wild-type kernels. (B) An immunoblot of α-zeins shows a 24-kDa protein band (indicated by arrowheads) in samples from floury kernels, but not wild-type kernels. (C) An immunoblot of the 70-kDa maize homolog of BiP.

Figure 2

Comparison of protein bodies from wild-type and transgenic maize endosperm. Protein bodies in the endosperm of wild-type seeds are round (A and C) compared with the misshapen protein bodies in the endosperm of seeds expressing the 24-kDa α-zein gene (B and D). Comparison of these protein bodies at lower magnification revealed that they appear as discreet spheres (circles in cross-section) in cells not expressing the 24-kDa α-zein gene (A), whereas they have a convoluted shape and are aggregated in cells expressing this gene (B). Comparison of these protein bodies at higher magnification highlights their differences in the internal structure. In cells not expressing the gene, the darkly staining zein proteins are found primarily on the periphery of the protein bodies (C, arrowheads), whereas in cells expressing the gene, the protein bodies have many locules of darkly staining zein in their interior (D, arrowheads). The irregularities seen in B and D are similar to those found in protein bodies of W64Afl2. PB, protein body; RER, rough endoplasmic reticulum; S, starch grain. Bars = 0.5 μm.