New Methods for Extraction and Quantitation of Zeins Reveal a High Content of γ-Zein in Modified <i>opaque-2</i> Maize

Wallace, John C.; Lopes, M. A.; Paiva, Edílson; Larkins, Brian A.

doi:10.1104/pp.92.1.191

Cited by 240 publications

(205 citation statements)

References 12 publications

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“…Prolamins were extracted according to a previously described method 34 and separated on 15% polyacrylamide SDS-PAGE gels, transferred to nitrocellulose and probed with total rabbit a-zein antiserum at 1/10,000 dilution. Secondary antibody was horse radish peroxidase-conjugated goat anti-rabbit (Pierce) used at 1/5,000 dilution, and the detection was done with the West Pico chemiluminescent detection system (Pierce).…”

Section: Methodsmentioning

confidence: 99%

Mutation in the seed storage protein kafirin creates a high-value food trait in sorghum

Yuan

Guo

et al. 2013

Nat Commun

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Sustainable food production for the earth's fast-growing population is a major challenge for breeding new high-yielding crops, but enhancing the nutritional quality of staple crops can potentially offset limitations associated with yield increases. Sorghum has immense value as a staple food item for humans in Africa, but it is poorly digested. Although a mutant exhibiting high-protein digestibility and lysine content has market potential, the molecular nature of the mutation is previously unknown. Here, building on knowledge from maize mutants, we take a direct approach and find that the high-digestible sorghum phenotype is tightly linked to a single-point mutation, rendering the signal peptide of a seed storage protein kafirin resistant to processing, indirectly reducing lysine-poor kafirins and thereby increasing lysine-rich proteins in the seeds. These findings indicate that a molecular marker can be used to accelerate introduction of this high nutrition and digestibility trait into different sorghum varieties.

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Section: Methodsmentioning

confidence: 99%

Mutation in the seed storage protein kafirin creates a high-value food trait in sorghum

Yuan

Guo

et al. 2013

Nat Commun

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“…Zein and nonzein proteins from maize endosperm were prepared as described previously (Wallace et al, 1990). Five-microliter aliquots of the zein and nonzein protein extracts were separated by 12.5% and 7.5% SDS-PAGE, respectively.…”

Section: Analysis Of Proteinsmentioning

confidence: 99%

The Maize Zmsmu2 Gene Encodes a Putative RNA-Splicing Factor That Affects Protein Synthesis and RNA Processing during Endosperm Development

et al. 2007

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We characterized two maize (Zea mays) mutants, zmsmu2-1 and zmsmu2-3, that result from insertion of a Mutator (Mu) transposable element in the first exon of a gene homologous to the nematode gene, smu-2, which is involved in RNA splicing. In addition to having a starchy endosperm with reduced levels of zein storage proteins, homozygous zmsmu2-1 mutants manifest a number of phenotypes, including defective meristem development. The zmsmu2 mutants have poor seedling viability and surviving plants are sterile. The gene encoding ZmSMU2 is expressed in the endosperm, embryo, and shoot apex, which explains the pleiotropic nature of the mutation. We found that proper expression of Zmsmu2 is required for efficient ribosomal RNA processing, ribosome biogenesis, and protein synthesis in developing endosperm. Based on the pleiotropic nature of the mutations and the known function of animal Zmsmu2 homologs, we propose a possible role for ZmSMU2 in the development of maize endosperm, as well as a mechanism by which misregulation of zmsmu2 causes the mutant phenotypes.

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“…Protein extracts from equal amounts of endosperm flour of each genotype were partitioned into zein and nonzein fractions (Wallace et al, 1990). Figure 1A illustrates a Coomassie Blue-stained gel showing the nature and relative abundance of various zein proteins in the parental inbreds and the RILs.…”

Section: Development Of Recombinant Inbred Lines With High and Low Eementioning

confidence: 99%

“…In addition, we analyzed four RILs that differ in eEF1A content. Protein extracts from equal amounts of lyophilized endosperm of each genotype were partitioned into zein and nonzein fractions (Wallace et al, 1990) and analyzed using SDS-PAGE. Figure 5A illustrates a Coomassie Blue-stained gel showing the relative abundance of zein proteins in 18-DAP endosperm of the parental inbreds and six RILs.…”

Section: Patterns Of Protein Synthesis In Developing Endospermmentioning

confidence: 99%

“…Protein fractions were obtained from mature endosperms or lyophilized, developing endosperms as described by Wallace et al (1990). Fifty milligrams of flour was extracted overnight with 1 mL of borate buffer (12.5 mM Na 2 B 4 O 7 , 1% [w/v] SDS, 2% 2-mercaptoethanol, pH 10) at 37°C, and the soluble proteins were partitioned into zeins (supernatant) and nonzeins (pellet) by the addition of absolute ethanol to a final concentration of 70% (v/v).…”

Section: Zein and Nonzein Protein Extraction Quantification And Sdsmentioning

confidence: 99%

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Cytoskeletal Proteins Are Coordinately Increased in Maize Genotypes with High Levels of eEF1A

et al. 2004

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The opaque2 (o2) mutation increases the Lys content of maize (Zea mays) endosperm by reducing the synthesis of zein storage proteins and increasing the accumulation of other types of cellular proteins. Elongation factor 1A (eEF1A) is one of these proteins, and its concentration is highly correlated with the amount of other Lys-containing proteins in the endosperm. We investigated the basis for this relationship by comparing patterns of protein accumulation and gene expression between a high (Oh51Ao2) and a low (Oh545o2) eEF1A inbred, as well as between high and low eEF1A recombinant inbred lines obtained from their cross. The content of a-zein and several cytoskeletal proteins was measured in high and low eEF1A inbred lines, and the levels of these proteins were found to correlate with that of eEF1A. To extend this analysis, we used an endosperm expressed sequence tag microarray to examine steady-state levels of RNA transcripts in developing endosperm of these genotypes. We identified about 120 genes coordinately regulated in association with eEF1A content. These genes encode proteins involved in several biological structures and processes, including the actin cytoskeleton, the endoplasmic reticulum, and the protein synthesis apparatus. Thus, higher levels of eEF1A in o2 mutants may be related to a more extensive cytoskeletal network surrounding the rough endoplasmic reticulum and increased synthesis of cytoskeleton-associated proteins, all of which contribute significantly to the Lys content of the endosperm.The discovery that the opaque2 (o2) mutation almost doubles the Lys content of maize (Zea mays) endosperm (Mertz et al., 1964) led to extensive efforts to develop genotypes with better nutritional quality for human and animal diets. Unfortunately, the inferior agronomic traits associated with this mutation, including reduced yield and protein content, soft kernels, and pathogen susceptibility, made it difficult to develop o2 in breeding programs (Glover and Mertz, 1987). To overcome these problems, maize breeders at International Center for Development of Maize and Wheat, Mexico (Villegas et al., 1992) and the University of Natal, South Africa (Gevers and Lake, 1992) developed modified o2 mutants called Quality Protein Maize (QPM). These genotypes combine the improved nutritional quality trait of o2 with those responsible for vitreous endosperm and normal yield. However, additional improvement of protein quality is required for Quality Protein Maize to meet the minimal Lys content (5 mg/100 mg of protein) recommended for human nutrition (Young et al., 1998).Several studies have documented a broad range of variability in Lys content among normal, o2, and modified o2 maize genotypes (Moro et al., 1996;Zarkadas et al., 2000), showing that the effect of the mutation is highly dependent on the genetic background. These studies suggested the Lys content of the kernel can be improved by appropriate selection. However, identification of more nutritional maize genotypes has been limited by knowledge of the genetic...

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New Methods for Extraction and Quantitation of Zeins Reveal a High Content of γ-Zein in Modified opaque-2 Maize

Cited by 240 publications

References 12 publications

Mutation in the seed storage protein kafirin creates a high-value food trait in sorghum

Mutation in the seed storage protein kafirin creates a high-value food trait in sorghum

The Maize Zmsmu2 Gene Encodes a Putative RNA-Splicing Factor That Affects Protein Synthesis and RNA Processing during Endosperm Development

Cytoskeletal Proteins Are Coordinately Increased in Maize Genotypes with High Levels of eEF1A

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