Expression of a mutant α-zein creates the floury 2 phenotype in transgenic maize

Abstract

The maize floury2 mutation results in the formation of a soft, starchy endosperm with a reduced amount of prolamin (zein) proteins and twice the lysine content of the wild type. The mutation is semidominant and is associated with small, irregularly shaped protein bodies, elevated levels of a 70-kDa chaperone in the endoplasmic reticulum, and a novel 24-kDa polypeptide in the zein fraction. The 24-kDa polypeptide is a precursor of a 22-kDa ␣-zein protein that is not properly processed. The defect is due to an alanine-tovaline substitution at the C-terminal position of the signal peptide, which causes the protein to be anchored to the endoplasmic reticulum. We postulated that the phenotype associated with the floury2 mutation is caused by the accumulation of the 24-kDa ␣-zein protein. To test this hypothesis, we created transgenic maize plants that produce the mutant protein. We found that endosperm in seeds of these plants manifests the floury2 phenotype, thereby confirming that the mutant ␣-zein is the molecular basis of this mutation.Zeins are prolamin storage proteins that accumulate in the endosperm of maize (Zea mays L.) seeds. They are composed of four different types of polypeptides, classified as ␣-, ␤-, ␥-, and ␦-zeins (1). Accretions of zein proteins form spherical protein bodies within the lumen of the endoplasmic reticulum (ER), and there is a distinct spatial arrangement of these proteins within a protein body: ␤-and ␥-zeins are located on the periphery, whereas ␣-and ␦-zeins are found in the interior (2, 3). Collectively, the zein proteins are rich in glutamine and proline, but they lack lysine and tryptophan. Because zeins constitute such a large proportion of the total seed protein (60-70%), the amino acid composition of these proteins causes the grain to be of inferior nutritional quality for monogastric animals.Efforts to improve the protein quality of maize seed have focused on mutants in which zein synthesis is reduced and the lysine content is increased. The first ''high-lysine'' mutants to be identified were opaque2 (o2) and floury2 ( fl2) (4, 5). Unfortunately, the favorable nutritional quality of these mutants is offset by the inferior physical properties of their soft, starchy endosperms. It appears that the starchy endosperm of the o2 and fl2 mutants is caused by changes in the nature of their protein bodies. The o2 mutation affects a transcriptional activator of a subset of ␣-zein genes, leading to a reduction in ␣-zein protein synthesis and the formation of protein bodies that are significantly smaller than normal (6-9). The fl2 mutation, which is semidominant, causes a decrease in synthesis of all classes of zeins, and the resultant protein bodies are not only smaller than normal, but they are also asymmetrical and misshapen (10, 11). Another feature of fl2 endosperm is the overexpression of the ER-resident binding protein (BiP), which becomes deposited at the periphery of the mutant protein bodies (12-15).We have postulated that the phenotype associated with fl2 is caused by ...

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

“…Therefore, we showed that the coding region of the mutant gene, which is tightly linked to the PB phenotype in sorghum P721Q, produces the same phenotype in maize as a gain of function. This also confirmed that as in floury2 in maize 20 , the compartmentalization of storage proteins in PBs is conserved between the two species.…”

“…Analogous to the sorghum phenotype, similar irregular and clumped PB morphologies in endosperm cells have previously been observed in two maize mutants with semi-dominant and dominant floury kernels [19][20][21] . These mutants resulted from point mutations at different sites in the ER signal peptides of a 22-kDa a-zein (floury2) and a 19-kDa a-zein (De-*B30).…”

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

“…Several opaque mutants, e.g. floury2 (fl2) and Defective endosperm B30 (De-B30; Coleman et al, 1997;Kim et al, 2004a), are caused by defective signal peptides in a-zein proteins that disrupt protein body assembly (Zhang and Boston, 1992), leading to increased expression of genes involved in the unfolded protein response (Hunter et al, 2002). These observations are consistent with the hypothesis that the size, number, and structure of zein protein bodies influence the texture and vitreous phenotype of the mature endosperm (Coleman and Larkins, 1999).…”

The Difference of Protein Composition of Central Region between White-core Grains and Non-white-core Grains in the Same Rice Cultivar for Sake Brewery.

“…In maize mutants, the increase in the precursor of storage protein, 22 kDa-alpha-zein, was observed in flo-2 (Coleman et al 1997) and the quantitative increase of a protein (34 kDa, pI = 5.57) and decrease of some proteins were shown in o2 (Damerval and Guilloux 1998). The quantitative difference of proteins observed between wild and mutant kernels of o2 and flo-2 were not detected between whitecore and non-white-core grains of rice.…”

“…In maize (Zea mays), it is reported that flo-2 mutant bore soft kernels with floury endosperm, and the kernels contained some proteins that were not detected in those of the wild type (Coleman et al 1995(Coleman et al , 1997. Damerval and Guilloux (1998) reported that novel proteins were synthesized in developing endosperm of opaque kernels of o2 mutant.…”