Identification and Characterization of Maize floury4 as a Novel Semidominant Opaque Mutant That Disrupts Protein Body Assembly

Abstract

Zeins are the major seed storage proteins in maize (Zea mays). They are synthesized on the endoplasmic reticulum (ER) and deposited into protein bodies. Failure of signal peptide cleavage from zeins can cause an opaque endosperm in the mature kernel; however, the cellular and molecular mechanisms responsible for this phenotype are not fully understood. In this study, we report the cloning and characterization of a novel, semidominant opaque mutant, floury4 (fl4). fl4 is caused by a mutated z1A 19-kD α-zein with defective signal peptide cleavage. Zein protein bodies in fl4 endosperm are misshapen and aggregated. Immunolabeling analysis indicated that fl4 participates in the assembly of zeins into protein bodies, disrupting their proper spatial distribution. ER stress is stimulated in fl4 endosperm, as illustrated by dilated rough ER and markedly up-regulated binding protein content. Further analysis confirmed that several ER stress pathways are induced in fl4 endosperm, including ER-associated degradation, the unfolded protein response, and translational suppression by the phosphorylation of eukaryotic translational initiation factor2 α-subunit. Programmed cell death is also elevated, corroborating the intensity of ER stress in fl4. These results provide new insights into cellular responses caused by storage proteins with defective signal peptides.

Figure 1.

Phenotypic features of maize fl4 mutants. A, Light transmission by mature kernels. The homozygous mutant kernels (fl4/fl4), heterozygous kernels (fl4/wild type [WT]), and homozygous wild-type kernels (wild type/wild type) were randomly selected from segregating F2 population and viewed on a light box. B, Scanning electron microscopy analysis of the peripheral regions of mature wild-type and fl4 endosperm. PB, Protein body; SG, starch granules. Bars = 10 μm. C, Microstructure of developing endosperms of the wild type and fl4 (20 DAP). Protein bodies were adjoined into clumps in fl4 (right). PB, Protein body; SG, starch granules. Bars = 5 μm. D, Ultrastructure of developing endosperms of the wild type and fl4 (20 DAP). Small, misshapen, and aggregated protein bodies were observed in fl4 (right). Top, Low magnification. Bottom, High magnification. PB, Protein body; SG, starch granules. Bars = 500 nm (top) and 200 nm (bottom).

Figure 2.

Biochemical analysis of wild-type and fl4 endosperm. A, Comparison of zein, nonzein, and total proteins from wild-type (WT) and fl4 mature endosperm. The measurements were done on per milligram of dried endosperm. Values are the mean values with se (n = three individuals; *P < 0.05, **P < 0.01, Student’s t test). B, SDS-PAGE analysis of total proteins from wild-type and fl4 mature endosperm. C, The soluble amino acids with different content in wild-type and fl4 mature endosperm. Values are the mean values with se (n = three individuals; *P < 0.05, **P < 0.01, ***P < 0.001, Student’s t test). D, Comparison of total starch content and the percentage of amylose in wild-type and fl4 mature endosperm. The measurements were done on per milligram of dried endosperm. Values are the mean values with se (n = three individuals).

Figure 3.

Map-based cloning and identification of Fl4. A, The Fl4 locus was mapped to a 60-kb region between molecular markers SNP5.51 and InDel5.57 on chromosome 4 containing 19-kD α-zein z1A-1 subfamily. See Supplemental Table S2 for primer information. B, A novel 19-kD α-zein protein band was detected with 19-kD α-zein-specific antibody and signal peptide-specific antibody in fl4. C, Three of the 42 clones for sequencing exhibited a single amino acid replacement in the highly conserved carboxy-terminal region of the signal peptide containing the cleavage site. WT, Wild type.

Figure 4.

Immunolocalization of the 19-kD α-zein, 27-kD γ-zein, and fl4 protein in wild-type (WT) and fl4 endosperm cells (20 DAP). A, The distribution of gold particles indicating 19-kD α-zeins. Evenly distributed within the protein body core in the wild type (top). Inserted into the peripheral region in fl4 (bottom, red arrowhead). Bars = 200 nm. B, The distribution of gold particles indicating 27-kD γ-zeins. Located in the peripheral region in the wild type (top). Delocalized into the central region in fl4 (bottom, red arrowhead). Bars = 200 nm. C, The distribution of gold particles indicating fl4 protein with the antibodies recognizing the signal peptide. No signal in wild-type sample (top). Around the region connects two aggregated, misshapen protein bodies in fl4 (bottom, red arrowhead). Bars = 200 nm.

Figure 5.

ER stress evidence was observed in fl4. A, Ultrastructure of developing endosperms of the wild type (WT) and fl4 (20 DAP). Normal ER configuration in the wild type (left, red arrowhead). Dilated ER in fl4 (right, red arrowhead). Bars = 500 nm. B, Immunoblot comparing BIP accumulation in wild-type and fl4 mature kernels. Anti-Tubulin was used as a sample loading control.

Figure 6.

Effects on ERAD, UPR, and the phosphorylation of eIF2α in fl4. A, Expressions of ERAD-associated genes ERO1 (GRMZM2G108115), PDIs (GRMZM2G389173), ZmDerlin1-1 (GRMZM2G117388), and Sec61 (GRMZM2G130987) were examined in the wild type (WT) and fl4 by real-time PCR analysis (19 DAP). Values are the mean values with se (n = six individuals; *P < 0.05, **P < 0.01, ***P < 0.001, Student’s t test). B, RNA samples from wild-type and fl4 kernels (19 DAP) were analyzed for the presence of unspliced and spliced ZmbZIP60 mRNA by reverse transcription-PCR using the flanking primers (FP) assay. The level of ZmbZIP60 expression and splicing were analyzed by real-time PCR using primers for unspliced bZIP60 mRNA (SPU) or for spliced mRNA (SPS). Values are the mean values with se (n = six individuals; ***P < 0.001, Student’s t test). C, Immunoblot comparing the phosphorylated eIF2α accumulation in wild-type and fl4 kernels (18 DAP). Anti-eIF2α was used as control.

Figure 7.

Enhancement of PCD in fl4. A, Expressions of PCD-associated genes BI1 protein family members (GRMZM2G465430, GRMZM2G029087, and GRMZM2G074404) and BAGs (GRMZM2G079956 and GRMZM2G017013) were examined in the wild type (WT) and fl4 by real-time PCR analysis (19 DAP). Values are the mean values with se (n = six individuals; *P < 0.05, **P < 0.01, ***P < 0.001, Student’s t test). B, TUNEL assay in situ detection of DNA fragmentation in wild-type and fl4 endosperm (20 DAP). Left, TUNEL-detected nuclei (green); middle, PI-detected nuclei (red); right, merge of TUNEL-detected nuclei and PI-detected nuclei (yellow); histogram, statistical analysis of TUNEL-detected nuclei/PI-detected nuclei in different section samples. Values are the mean values with se (n = two individuals; *P < 0.05, Student’s t test). Bars = 50 μm.

Figure 8.

An explanation for the phenotypic consequences of zeins with uncleaved signal peptides in maize endosperm.