The opaque-2 mutation of maize differentially reduces zein gene transcription.

Abstract

Zeins, the storage proteins of maize seed, are encoded by a large multigene family that is regulated developmentally and expressed in a tissue-specific manner during endosperm development. The synthesis of these proteins is affected by mutations, such as opaque-2, that cause a reduction in the accumulation of zein proteins and mRNAs. We used nuclear run-on transcription assays to analyze the expression of zein genes in developing normal and opaque-2 endosperms and to map the origin of these transcripts with respect to the coding and noncoding regions of the genes. These analyses demonstrate that zein gene expression is regulated transcriptionally and posttranscriptionally in developing endosperm. Transcription of genes encoding a-zeins is inhibited significantly in opaque-2 mutants, with expression of those encoding the M, 22,000 proteins being almost totally blocked. Other gene subfamilies were affected but to a lesser extent.

Quantification of dominance for proteins pleiotropically affected by opaque-2 in maize

“…Higher amounts of gene products in the wild type than in the mutant could simply be due to a direct action of the transcription activator, as shown for 22 kDa zeins (Kodrzycki et al, 1989). For six acidic and one basic polypeptides, the amount of which was increased in the mutant, such a hypothesis cannot be retained unless 02 does not act as an activator; for example oncoprotein Fos, a bZip factor, has been shown to act as a positive or negative regulator in different DNA contexts (Curran & Franza, 1988;Vogt & Bos, 1989).…”

The Differential Transcription Network between Embryo and Endosperm in the Early Developing Maize Seed

“…To further characterize the developmental stage of the maize seeds, expression profiles of storage protein genes were specifically compiled using the RNA-seq data generated in this study (Supplemental Table S1). Previous studies showed that the expression of zein genes is temporally regulated during endosperm development (Kodrzycki et al, 1989;Woo et al, 2001). In our RNA-seq data, two genes, 18-kD d-zein and 19-kD a-zein, were most abundantly expressed.…”

Phosphorylation of Opaque2 changes diurnally and impacts its DNA binding activity.

“…Although the degree of this effect appears influenced by the inbred background and stage of endosperm development, this result is consistent with the prediction that hyperphosphorylation of 0 2 during the night inhibits DNA binding and impacts target gene expression. Clearly, nuclear run-on experiments (Kodrzycki et al, 1989) are required to demonstrate an effect of 0 2 phosphorylation on target gene transcription.…”

“…Although the degree of this effect appears influenced by the inbred background and stage of endosperm development, this result is consistent with the prediction that hyperphosphorylation of 0 2 during the night inhibits DNA binding and impacts target gene expression. Clearly, nuclear run-on experiments (Kodrzycki et al, 1989) are required to demonstrate an effect of 0 2 phosphorylation on target gene transcription.We noted that the 02-676 and o21t polypeptides, both mutated within the basic region of the DNA binding domain (Aukerman et al, 1991; B. Lazzari and A. Viotti, unpublished results), do not bind the 0 2 target oligonucleotide in vitro but are phosphorylated similarly to the wild-type 0 2 in vivo. Because it is known that 02-676 is nuclearly localized in maize endosperm cells (Varagona and Raikhel, 1994), the addition of phosphates may depend on nuclear localization but is likely not dependent on zein promoter recognition.…”