The origin of lysine-containing proteins in opaque-2 maize endosperm

Abstract

The reduction of zein synthesis in the maize (Zea mays L.) opaque-2 mutant is associated with an increased percentage of lysine in the endosperm protein. When expressed on an endosperm basis, we found that W64A opaque-2 contains 490 micrograms of lysine compared with 350 micrograms in W64A normal. SDS-PAGE analysis of endosperm proteins indicated that several non-zein proteins are more abundant in the mutant than in normal genotype. To determine the subcellular origin of these proteins, we separated an endosperm homogenate from developing kernels by sucrose density gradient centrifugation and used marker enzyme assays and immunoblot analyses to identify cellular components. Amino acid analysis of proteins in the gradient fractions showed that the majority of the lysine occurs in soluble proteins at the top of the gradient. To identify these proteins, we prepared a complex antiserum against the entire soluble protein fraction and used it to immunoscreen an endosperm cDNA expression library. Sequence analysis of clones identified mRNAs involved in carbohydrate metabolism, amino acid biosynthesis, and protein synthesis. RNA dot blot hybridization analysis with these clones revealed significant variation in the levels of transcripts between normal and opaque-2 endosperm, but we identified several mRNAs that are elevated in opaque-2 and that may encode proteins responsible for the enhanced lysine content.

Elongation factor 1 alpha concentration is highly correlated with the lysine content of maize endosperm.

“…Because zeins do not contain lysine (16), all the protein-bound lysine comes from the nonzein fraction. Hence, the reduction in zein synthesis and the increased synthesis of nonzein proteins both contribute to the enhanced percentage of lysine in grain protein (17).…”

“…To investigate the basis for the pleiotropic increase in lysine-containing proteins in o2 maize, we identified genes whose expression is increased in this mutant (17). Elongation factor 1a (EF-la) was found to be one of several mRNAs whose level is significantly increased in the endosperm of W64Ao2 plants compared with its normal wild-type counterpart.…”

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

“…eEF1A. For example, the levels of a-zeins, pyruvate orthophosphate dikinase, and acetolactate synthase were decreased in o2 mutants compared to wild type (Damerval and Le Guilloux, 1998;Hunter et al, 2002), while that of eEF1A was increased (Habben et al, 1993;Sun et al, 1997). For some enzymes involved in carbohydrate metabolism, such as glyceraldehyde-3-phosphate dehydrogenase and sorbitol dehydrogenase, earlier studies showed these proteins are increased in o2 mutants (Damerval and Le Guilloux, 1998).…”

“…Both of these effects contribute to the increased Lys content of the endosperm (Moro et al, 1996), but the molecular mechanisms responsible for the larger amount of Lys-rich proteins are unknown. Habben et al (1993) investigated the nature of the Lys-containing proteins that are increased in o2 and found that elongation factor 1A (eEF1A), a protein synthesis factor, is highly increased compared to wild type. Analysis of a large number of normal and o2 inbreds with extensive variability in Lys and eEF1A content showed this protein is highly correlated (r 5 0.9) with the protein-bound Lys of the endosperm (Habben et al, 1995;Moro et al, 1996).…”

“…O2 encodes a basic domain/Leu zipper transcription factor that regulates the expression of a number of genes, including the highly abundant a-zeins (Kodrzycki et al, 1989;Schmidt et al, 1990). Defects in O2 lead to a significant reduction in a-zein synthesis and a pleiotropic increase in the synthesis of several Lys-rich, nonzein proteins (Damerval and Devienne, 1993;Habben et al, 1993). Both of these effects contribute to the increased Lys content of the endosperm (Moro et al, 1996), but the molecular mechanisms responsible for the larger amount of Lys-rich proteins are unknown.…”