Mutations in regulatory loci alter maize zein gene transcription
Abstract
Zeins, the storage proteins of maize seed, are encoded by a large multigene family that is developmentally regulated and expressed in a tissue-specific manner during endosperm development. Zeins can be divided into several classes based on their solubility and amino acid composition. Alpha-zeins, the most abundant class, proteins of M$\sb{\rm r}$ 19,000 and 22,000, are extracted from maize endosperm by 70% ethanol. When a reducing agent is included in the aqueous ethanol, the other zein classes are solubilized. Beta-zein consists of a M$\sb{\rm r}$ 14,000 polypeptide, gamma-zeins consist of M$\sb{\rm r}$ 16,000 and 27,000 proteins, and delta-zein consists of a M$\sb{\rm r}$ 10,000 polypeptide. The accumulation of zeins is reduced by several mutations such as opaque-2, floury-2, brittle-1, and brittle-1/opaque-2. These mutations affect primarily the alpha-zeins. The opaque-2 and floury-2 mutations also cause a reduction in zein mRNA levels and therefore may act through lowering the transcription rate of the affected gene products. There is no data concerning the effects of brittle-1 or brittle-1/opaque-2 on zein mRNA accumulation. To better understand the mechanisms controlling gene expression, the normal genotype of maize and the above-mentioned mutations were analyzed at the protein, mRNA, and transcriptional levels. Zein protein accumulations was assayed by SDS-polyacrylamide gel electrophoresis. The amount of zein mRNA was determined by quantitative hybridization and the rate of zein gene transcription was measured by nuclear run-on transcription assays. Analysis of the normal genotype of maize showed that zein transcription is developmentally regulated and tissue specific. Furthermore, comparison of the rate of zein gene transcription with the level of zein mRNA in polysomes showed evidence for post-transcriptional regulation of the M$\sb{\rm r}$ 22,000 alpha-zein genes. Transcription of genes encoding alpha-zeins is significantly inhibited in opaque-2 mutants, with expression of those encoding the M$\sb{\rm r}$ 22,000 proteins being almost totally blocked. Other gene subfamilies were affected but to a lesser extent. The floury-2 mutation also causes a reduction in the rate of zein gene transcription. While this effect is greatest on the alpha-zein genes, the transcription rate of other zein classes were also reduced. Analysis of brittle-1 and brittle-1/opaque-2 showed that zein mRNA was decreased in the kernels of these mutants. Subsequent analysis of by nuclear run-on transcription assays showed that brittle-1 and brittle-1/opaque-2 reduce the rate of zein gene transcription. The level of zein mRNA and transcription was drastically reduced in brittle-1/opaque-2. The degree to which the double mutation affects mRNA and transcription is greater than the additive effects of both mutants alone. Comparison of zein mRNA levels with the rate of zein gene transcription in brittle-1 showed evidence for post-transcriptional regulation of alpha-zein genes.
Degree
Ph.D.
Advisors
Larkins, Purdue University.
Subject Area
Molecular biology
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