A defective signal peptide in a 19-kD alpha-zein protein causes the unfolded protein response and an opaque endosperm phenotype in the maize De*-B30 mutant

Abstract

Defective endosperm* (De*)-B30 is a dominant maize (Zea mays) mutation that depresses zein synthesis in the developing endosperm. The mutant kernels have an opaque, starchy phenotype, malformed zein protein bodies, and highly increased levels of binding protein and other chaperone proteins in the endosperm. Immunoblotting revealed a novel alpha-zein protein in De*-B30 that migrates between the 22- and 19-kD alpha-zein bands. Because the De*-B30 mutation maps in a cluster of 19-kD alpha-zein genes, we characterized cDNA clones encoding these proteins from a developing endosperm library. This led to the identification of a 19-kD alpha-zein cDNA in which proline replaces serine at the 15th position of the signal peptide. Although the corresponding gene does not appear to be highly expressed in De*-B30, it was found to be tightly linked with the mutant phenotype in a segregating F2 population. Furthermore, when the protein was synthesized in yeast cells, the signal peptide appeared to be less efficiently processed than when serine replaced proline. To test whether this gene is responsible for the De*-B30 mutation, transgenic maize plants expressing this sequence were created. T1 seeds originating from the transformants manifested an opaque kernel phenotype with enhanced levels of binding protein in the endosperm, similar to De*-B30. These results are consistent with the hypothesis that the De*-B30 mutation causes a defective signal peptide in a 19-kD alpha-zein protein.

Figure 1.

Immunoblotting identifies a novel α-zein linked with the De^*-B30 mutation. Protein body proteins (20 μg lane^-1) from B37+ and B37De^*-B30 were separated by 10% (w/v) SDS-PAGE and blotted onto nitrocellulose. The membrane was probed with antiserum that recognized 19- and 22-kD α-zeins; their positions are denoted on the left and the identification of the novel α-zein protein is shown by the arrow on the right.

Figure 2.

Signal peptide amino acid sequences and frequency of 19-kD α-zeins isolated from a W64ADe^*-B30 cDNA library. A, The amino acid sequences of signal peptides for 19-kD α-zein mRNAs were deduced from cDNAs obtained from a 16 DAP De^*-B30 endosperm library. B, The number in parenthesis refers to the frequency of the sequence among 76 independent 19-kD α-zein cDNA clones that were analyzed (GenBank accession nos. CF752004-CF752078).

Figure 3.

Nucleotide and amino acid sequence of the 19-kD^15P α-zein deduced from a full-length cDNA clone (GenBank accession no. AY434688). The signal peptide is underlined and Pro 15 is enclosed in a box. Amino acids that differ from those in the closely related A-20 19-kD α-zein (GenBank accession no. V01476) are contrasted in gray.

Figure 4.

The gene encoding the 19-kD^S15P α-zein cosegregates with the De^*-B30 mutation. Genomic DNA was isolated from homozygous wild-type and De^*-B30 seedlings, and a 150-bp sequence spanning the 5′-UTR and signal peptide sequence was amplified by PCR using the 19 upstream and 19 downstream primers described in “Materials and Methods.” The wild-type (A) and De^*-B30 (B) PCR products were digested with the Ear I restriction enzyme and were separated by electrophoresis in 3% (w/v) agarose gel. Ear I cleaves the 19-kD^S15P PCR product into two fragments of 91 bp (arrow) and 59 bp, the latter of which is not visible in this image.

Figure 5.

The mutant 19-kD^15P α-zein is inefficiently processed in yeast cells. Proteins were extracted from wild-type and De^*-B30 maize endosperms, and from yeast cells expressing genes encoding the 19-kD^15P and 19-kD^S15P α-zeins. After separation by 12% (w/v) SDS-PAGE and transfer to a nylon membrane, the proteins were reacted with antiserum recognizing the 19-kD α-zeins. Lane 1, W64A wild type; lane 2, De^*-B30; lane 3, yeast expressing the 19-kD^S15P α-zein; lane 4, yeast expressing the 19-kD^15P α-zein.

Figure 6.

Expression of the 19-kD^15P α-zein in transgenic maize plants results in an opaque kernel phenotype. The top panel shows a representative sample of segregating opaque (o) and vitreous (v) kernels from a transgenic gz::19kD^15P α-zein ear (line 1532.231.2.9.1, genetic background Hi-II/PHR03). The bottom panel shows an immunoblot analysis of endosperm proteins from these kernels. The proteins were separated by 10% to 20% (w/v) SDS-PAGE and probed using a 19-kD B α-zein antibody. The positions of 22- and 19-kD α-zein proteins that crossreacted with the antibody are denoted at the left of the figure and the novel 19-kD^15P α-zein is shown by the asterisk.

Figure 7.

The ER chaperone BIP is highly induced in gz::19kD^15P α-zein-expressing endosperms, similar to W64ADe^*-B30 and transgenic fl2-expressing endosperm. Total endosperm protein extracts from opaque (o) and vitreous (v) seed from the transgenic gz::19kD^15P α-zein line 1532.231.2.9.1 (left), an opaque (o) and vitreous (v) kernel of a W64ADe^*-B30 F₂ ear (panel), and an opaque (o) and vitreous (v) kernel from a transgenic ear segregating for the fl2 α-zein (line 236300; right) were separated by SDS-PAGE and immunoblotted with a plant BiP-specific antibody. Equal amounts of samples were loaded into each lane based on an endosperm dry weight basis.