Opaque7 encodes an acyl-activating enzyme-like protein that affects storage protein synthesis in maize endosperm

Abstract

In maize, a series of seed mutants with starchy endosperm could increase the lysine content by decreased amount of zeins, the main storage proteins in endosperm. Cloning and characterization of these mutants could reveal regulatory mechanisms for zeins accumulation in maize endosperm. Opaque7 (o7) is a classic maize starchy endosperm mutant with large effects on zeins accumulation and high lysine content. In this study, the O7 gene was cloned by map-based cloning and confirmed by transgenic functional complementation and RNAi. The o7-ref allele has a 12-bp in-frame deletion. The four-amino-acid deletion caused low accumulation of o7 protein in vivo. The O7 gene encodes an acyl-activating enzyme with high similarity to AAE3. The opaque phenotype of the o7 mutant was produced by the reduction of protein body size and number caused by a decrease in the α-zeins concentrations. Analysis of amino acids and metabolites suggested that the O7 gene might affect amino acid biosynthesis by affecting α-ketoglutaric acid and oxaloacetic acid. Transgenic rice seeds containing RNAi constructs targeting the rice ortholog of maize O7 also produced lower amounts of seed proteins and displayed an opaque endosperm phenotype, indicating a conserved biological function of O7 in cereal crops. The cloning of O7 revealed a novel regulatory mechanism for storage protein synthesis and highlighted an effective target for the genetic manipulation of storage protein contents in cereal seeds.

Figure 1

Phenotype of the BC ear, o7, and wt kernels.

Figure 2

Scanning electron microscopy of wt and o7 endosperm. (A) o7 kernel at 21 DAP (×5000). (B) Wt kernel at 21 DAP (×5000). (C) Mature o7 kernel (×3000). (D) Mature wt kernel (×3000). st, starch; pb, protein body.

Figure 3

Transmission electron microscopy of wt and o7 endosperm. (A) o7 kernel at 21 DAP (×2500). (B) Wt kernel at 21 DAP (×2500). (C) o7 kernel at 21 DAP (×6000). (D) Wt kernels at 21 DAP (×6000). cw, cell wall; pb, protein body; st, starch; rER, rough endoplasmic reticulum; m, mitochondrion. (E) Comparison of protein body numbers per 100 μm² in o7 and wt 21-DAP endosperms (o7, protein body counts = 36.87 ± 6.30; wt, protein body count = 26.20 ± 4.21. *P < 0.05, Student’s t-test). (F) Comparison of protein body area in o7 and wt 21-DAP endosperms (o7, protein body area = 0.547 ± 0.345; n = 203. wt, protein body area = 0.624 ± 0.246; n = 270. **P < 0.01, Student’s t-test).

Figure 4

Protein analysis of wt and o7 endosperm. (A) SDS–PAGE analysis of total proteins from wt and o7 endosperm. (B) Comparison of zein, nonzein, and total proteins from wt and o7 endosperm. Values are the mean values with standard errors (*P < 0.05; **P < 0.01, Student’s t-test).

Figure 5

Positional cloning of the O7 gene. (A) Fine mapping of the O7 gene on chromosome 10. Names of the molecular markers, contig, BAC, and the recombinants are indicated (n = 13,933). ZMMBBb0166J07 and ZMMBBb0342E21 (abbreviated as b0166J07 and b0342E21) are the names of BAC clones covering this locus. The O7 locus was mapped to a ∼23-kb region containing gene1 and gene2 between the molecular markers LM4 and RM1. (B) Schematic representation of the gene structure of O7. The mutant sequence has a 12-nucleotide in-frame deletion in the second exon. ATG and TGA represent the start and stop codons, respectively.

Figure 6

Phenotypic and molecular characterization of transgenic kernels from functional complementation experiment. (A) The phenotype of 20 vitreous kernels and 10 opaque kernels, randomly selected from a transgenic line with homozygous o7 locus on chromosome 10. (B) Genotyping the 30 kernels displayed in A for o7 locus on chromosome 10 using the molecular marker Indel6819. All 30 kernels are homozygous at the o7 locus on chromosome 10. (C) Characterization of transgenic status of the 30 kernels displayed in A using the molecular marker Trans6837F3R3. The vitreous kernels (lanes 1–20) are transgenic, and the opaque kernels (lanes 21–30) are nontransformed. −, H₂O control; +, pTF102–O7Gene vector. (D) SDS–PAGE analysis of total proteins from different kernels. T+, transgenic and o7/o7 homozygous kernel; T−, nontransformed and o7/o7 homozygous kernel. (E) Comparison of zein protein content in wt, o7, pApB, transgenic homozygote o7/o7 (T+), and nontransformed homozygote o7/o7 kernels (T−).

Figure 7

Phenotypic and molecular characterization of transgenic kernels from maize RNAi transgenic experiment. (A) The vitreous and opaque phenotype of the 20 randomly selected kernels from an RNAi transgenic line. (B) Characterization of the transgenic status of the 20 kernels displayed in A using the molecular marker defined by the primer pair rice intron F/maize P3 R. The opaque kernels (lanes 1–10) are transgenic, and the vitreous kernels (lanes 11–20) are nontransformed. −, H₂O control; +, pHB–O7RNAi vector. (C) SDS–PAGE analysis of total proteins from different kernels. R1−CK and R3−CK, nontransformed vitreous kernels from transgenic lines R1 and R3; R1+ and R3+, transgenic opaque kernels from transgenic lines R1 and R3. (D) Comparison of zein protein content in pApB, R1−CK, R3−CK, R1+, and R3+ kernels.

Figure 8

Phylogenetic relationships of O7 (ZmAAE3) and its homologs. The O7 gene product (shown as Zm AAE3) was aligned with AAE3 proteins from Capsicum annuum (AF354454.1), Vitis vinifera (XP 002267459), Populus trichocarpa (XP 002322473), Ricinus communis (XP 002509782), Arabidopsis thaliana (NP 190468), A. lyrata subsp. lyrata (XP 002877640), Oryza sativa Japonica (NP 001054304), Hordeum vulgare subsp. vulgare (BAK00674), Zea mays (NP 001152269.1), Sorghum bicolor (XP 002448800), A. thaliana AAE13 (At AAE13, NM 112487), and A. thaliana AAE14 (At AAE14, NM 102789).

Figure 9

Protein sequence alignment of plant AAE3 proteins. ClustalW amino acid alignment of Zm AAE3 protein and its homologs. The black shading with white lettering indicates residues conserved in all 10 members, whereas gray shading indicates conservation between two or more of the family members. The 10 sequences listed are from Zea mays (NP 001152269.1), Oryza sativa (NP 001054304), Arabidopsis thaliana (NP 190468), Sorghum bicolor (XP 002448800), Hordeum vulgare subsp. vulgare (BAK00674), Ricinus communis (XP 002509782), Capsicum annuum (AF354454.1), Populus trichocarpa (XP 002322473), Vitis vinifera (XP 002267459) and A. lyrata subsp. lyrata (XP 002877640).

Figure 10

Expression analysis of the O7 gene. (A) Real-time qRT–PCR analysis of O7 gene expression in root, stem, leaf, silk, tassel, ear, and kernel (7 DAP). (B) Real-time qRT–PCR analysis of O7 gene expression from 3-DAP to 36-DAP kernels in the W22 background. (C) Real-time qRT–PCR analysis of O7 gene expression in 12-DAP, 18-DAP, and 24-DAP samples from wt and o7 mutant kernels endosperm.

Figure 11

Immunoblot analysis of O7 protein from endosperms of wt, o7, functional complementation transgenic line, and RNAi transgenic line. (A) Immunoblot comparing the accumulation of O7 protein in 12-DAP, 15-DAP, 18-DAP, and 21-DAP kernels from wt and o7 plants with an antibody against O7 protein. (B) Immunoblot analysis using an α-tubulin antibody. (C) Immunoblot comparing the accumulation of O7 protein in 18-DAP kernels with an antibody against O7 protein. T+, transgenic homozygote o7/o7; T−, nontransformed homozygote o7/o7. (D) Immunoblot analysis using an α-tubulin antibody. T+, transgenic homozygote o7/o7; T−, nontransformed homozygote o7/o7. (E) Immunoblot comparing the accumulation of O7 protein in the 18-DAP kernels with an antibody against O7 protein. R1−CK and R3−CK, nontransformed kernels from transgenic lines R1 and R3; R1+ and R3+, transgenic kernels from transgenic lines R1 and R3. (F) Immunoblot analysis using an α-tubulin antibody. R1−CK and R3−CK:, nontransformed kernels from transgenic lines R1 and R3; R1+ and R3+, transgenic kernels from transgenic lines R1 and R3.

Figure 12

Immunoblot analysis of BiP from wt and o7 endosperms. (A) Immunoblot comparing the accumulation of BiP protein in 12-DAP, 15-DAP, and 18-DAP wt and o7/o7 kernels by using BiP antibody. (B) Immunoblot analysis using α-tubulin antibody.

Figure 13

The analysis of soluble Asx, Lys, Glx, α-ketoglutaric acid, and oxaloacetate from wt and o7 endosperms. Values are the means and standard errors (n = 3; *P < 0.05; **P < 0.01, Student’s t-test). (A and B) Soluble Asp + Asn, Lys and Glu + Gln in mature and 21-DAP kernels of wt and o7. (C and D) α-Ketoglutaric acid and oxaloacetate analysis of 21-DAP and 24-DAP kernels of wt and o7.

Figure 14

Phenotypic and molecular characterization of transgenic kernels from rice RNAi transgenic experiment. (A) The vitreous and opaque phenotype of the 20 randomly selected kernels from the RNAi transgenics. (B) Transverse sections of vitreous (top) and opaque (bottom) rice kernels. (C) Characterization of the transgenic status of the 20 kernels displayed in A using the molecular marker defined by the primer pair rice intron F/rice P3 R. The opaque kernels (lanes 1–10) are transgenic, and the vitreous kernels (lanes 11–20) are nontransformed. −, H₂O control; +, pTCK303–OsAAE3 RNAi vector. (D) SDS–PAGE analysis of total proteins from different kernels. Lane 1, Kitaake kernel; lanes 2–4, nontransformed kernels; lanes 5–7, transgenic kernels. (E and F) SEM (×350) of a nontransformed vitreous rice kernel (E) and a transgenic opaque rice kernel (F).