Genomics analysis of genes expressed in maize endosperm identifies novel seed proteins and clarifies patterns of zein gene expression

Abstract

We analyzed cDNA libraries from developing endosperm of the B73 maize inbred line to evaluate the expression of storage protein genes. This study showed that zeins are by far the most highly expressed genes in the endosperm, but we found an inverse relationship between the number of zein genes and the relative amount of specific mRNAs. Although alpha-zeins are encoded by large multigene families, only a few of these genes are transcribed at high or detectable levels. In contrast, relatively small gene families encode the gamma- and delta-zeins, and members of these gene families, especially the gamma-zeins, are highly expressed. Knowledge of expressed storage protein genes allowed the development of DNA and antibody probes that distinguish between closely related gene family members. Using in situ hybridization, we found differences in the temporal and spatial expression of the alpha-, gamma-, and delta-zein gene families, which provides evidence that gamma-zeins are synthesized throughout the endosperm before alpha- and delta-zeins. This observation is consistent with earlier studies that suggested that gamma-zeins play an important role in prolamin protein body assembly. Analysis of endosperm cDNAs also revealed several previously unidentified proteins, including a 50-kD gamma-zein, an 18-kD alpha-globulin, and a legumin-related protein. Immunolocalization of the 50-kD gamma-zein showed this protein to be located at the surface of prolamin-containing protein bodies, similar to other gamma-zeins. The 18-kD alpha-globulin, however, is deposited in novel, vacuole-like organelles that were not described previously in maize endosperm.

Figure 1.

Storage Protein Transcript Levels in B73 Endosperm as Reflected by Their Percentage among Randomly Chosen Clones from 15- to 40-DAP Endosperm-Specific cDNA Libraries. For abbreviations, see Table 1.

Figure 2.

Amino Acid Sequence Alignment of Members of the Maize α-Zein Protein Family. (A) Polypeptides correspond to alleles of the 22-kD α-zeins and the B and D subfamilies of 19-kD α-zeins in the B73 inbred line (see Table 1 for GenBank accession numbers and abbreviations). Identical residues are shown in boldface, and similar amino acids and conservative changes are shaded. Underlined residues designate peptide sequences that were expressed in bacteria and used to develop subfamily-specific antibodies (see Methods). (B) Dendrogram showing an alignment of α-zein polypeptides that correspond to W64A alleles (GenBank accession numbers M12143 [A1], M12146 [C1], M12144 [D1], and M12141 [22C]) and to B73 alleles (Table 1). The bootstrapping values of all internal branches exceeded 95% (data not shown), strongly supporting the correct topology of the dendrogram. The branches of the three α-zein subfamilies are indicated. The nomenclatures of Heidecker and Messing (1986) and Rubenstein and Geraghty (1986) are shown in parentheses. Family members within the three subgroups share 75 to 95% identical amino acid residues, and there is 40 to 55% identity between residues in subgroups.

Figure 2.

Amino Acid Sequence Alignment of Members of the Maize α-Zein Protein Family. (A) Polypeptides correspond to alleles of the 22-kD α-zeins and the B and D subfamilies of 19-kD α-zeins in the B73 inbred line (see Table 1 for GenBank accession numbers and abbreviations). Identical residues are shown in boldface, and similar amino acids and conservative changes are shaded. Underlined residues designate peptide sequences that were expressed in bacteria and used to develop subfamily-specific antibodies (see Methods). (B) Dendrogram showing an alignment of α-zein polypeptides that correspond to W64A alleles (GenBank accession numbers M12143 [A1], M12146 [C1], M12144 [D1], and M12141 [22C]) and to B73 alleles (Table 1). The bootstrapping values of all internal branches exceeded 95% (data not shown), strongly supporting the correct topology of the dendrogram. The branches of the three α-zein subfamilies are indicated. The nomenclatures of Heidecker and Messing (1986) and Rubenstein and Geraghty (1986) are shown in parentheses. Family members within the three subgroups share 75 to 95% identical amino acid residues, and there is 40 to 55% identity between residues in subgroups.

Figure 3.

Amino Acid Sequence Alignment of Members of the Maize γ-Zein Protein Family. (A) The polypeptides correspond to alleles of the 16-, 27-, and 50-kD γ-zeins and the 15-kD β-zein in the B73 inbred line (for GenBank accession numbers and abbreviations, see Table 1). Conserved Cys residues are indicated with asterisks. Identical residues are shown in boldface, and similar amino acids and conservative changes are shaded. Underlined residues indicate peptide sequences that were either expressed in bacteria or synthesized chemically and used to develop monospecific antibodies (see Methods). (B) Dendrogram of the alignment shown in (A). The bootstrapping values of all internal branches exceeded 95% (data not shown).

Figure 4.

Amino Acid Sequence Alignment of Members of the Maize δ-Zein Protein Family. The polypeptides correspond to the 18- and 10-kD δ-zein alleles of the B73 inbred line (see Table 1 for GenBank accession numbers and abbreviations) and the δ-zein allele of the Mo17 inbred line (Swarup et al., 1995; GenBank accession number U31541). Identical residues are shown in boldface, and conservative changes are shaded. Underlined residues indicate peptide sequences that were synthesized chemically and used to develop specific antibodies (see Methods).

Figure 5.

Amino Acid Sequence Alignment of the 18-kD α-Globulin and Related Protein Sequences. (A) Conserved Cys residues are indicated with asterisks. Identical residues are shown in boldface, and similar amino acids and conservative changes are shaded. The “Trp box” in wheat puroindolin and a similar Trp-rich sequence in the maize 18-kD α-globulin are underlined. The unique wheat HMW glutenin repeat domain between residues 217 and 616 was omitted for brevity. GenBank accession numbers are as follows: maize α-globulin, AF371278; wheat HMW glutenin, P08488; rice α-globulin, D50643; and wheat puroindolin b, X69912. (B) Dendrogram of the alignment shown in (A). The bootstrapping values of all internal branches exceeded 95% (data not shown).

Figure 6.

Immunodetection of Proteins in Maize Endosperm Extracts. (A) Total protein from B73 endosperm was separated by 4 to 20% gradient SDS-PAGE and stained with Coomassie blue. Positions of molecular mass markers are indicated at left. Proteins that were clearly identified with monospecific polyclonal antibodies are specified at right. The asterisk indicates the approximate position of the 19-kD α-zein D, the 18-kD δ-zein, and the 18-kD α-globulin polypeptides, all of which migrated to a similar position. (B) and (C) Immunoblots of the protein extract in (A) separated by 4 to 20% gradient SDS-PAGE (B) and by 10 to 20% SDS-PAGE (C). Identical lanes were cut from the blots and incubated with the designated monospecific antibody (for abbreviations, see Table 1).

Figure 7.

Detection of mRNAs in Maize Endosperm by in situ Hybridization of Antisense Probes. Longitudinal median sections of maize kernels at 10 ([A] to [D]), 15 ([E] to [H]), 20 ([I] to [L]), and 25 DAP ([M] to [P]) were hybridized with antisense RNA probes of 22-kD α-zein ([A], [E], [I], and [M]), 19-kD B1 α-zein ([B], [F], [J], and [N]), 27-kD γ-zein ([C], [G], [K], and [O]), and 16-kD γ-zein ([D], [H], [L], and [P]). Most sections for a particular developmental stage were derived serially from one or two kernels. Embryos are found at the lower right corners of the sections. Bars = 1 mm.

Figure 8.

Detection of mRNAs in Maize Endosperm by in situ Hybridization of Antisense Probes. Developing endosperm and tissue sections were prepared as in Figure 7. The kernel sections were hybridized with antisense RNA probes of 50-kD γ-zein ([A], [E], [I], and [M]), 15-kD β-zein ([B], [F], [J], and [N]), 10-kD δ-zein ([C], [G], [K], and [O]), and 18-kD α-globulin ([D], [H], [L], and [P]). Bars = 1 mm.

Figure 9.

Immunodetection of the 50-kD γ-Zein and the 18-kD α-Globulin in Protein Bodies of 20-DAP Endosperm. Protein bodies were labeled singly with 50-kD γ-zein antibodies (A) or 18-kD α-globulin antibodies (B) and double labeled with 50-kD γ-zein antibodies (10-nm gold beads) and 19-kD α-zein antibodies (5-nm gold beads) (C) or 18-kD α-globulin antibodies (10-nm gold beads) and 19-kD α-zein antibodies (5-nm gold beads) (D). (E) shows a representative transmission electron micrograph showing a region of the third to fifth starchy endosperm cell layers labeled with 18-kD α-globulin antibodies (10-nm gold beads) and 19-kD α-zein antibodies (5-nm gold beads). The large empty hole is the former location of a starch grain. α-Globulin–containing protein bodies are indicated by arrowheads in (B), (C), (D), and (E). Bars = 0.4 μm in (A) and (C) and 2 μm in (E).