Opaque2 modifiers alter transcription of the 27-kDa gamma-zein genes in maize

Opaque2 modifier genes cause a two- to three-fold increase in the amount of gamma-zein RNA and protein in maize kernels, and can convert the soft, starchy endosperm of an opaque2 mutant to a hard, vitreous phenotype. We analyzed several aspects of transcriptional and post-transcriptional regulation of gamma-zein gene expression in wild-type, opaque2 and modified opaque2 genotypes to investigate the molecular mechanisms by which opaque2 modifiers influence the expression of gamma-zein genes. We found that the poly(A) tails of the gamma-zein RNAs A and B were of similar length in normal, opaque2 and modified opaque2 genotypes. Multiple poly(A) addition sites were detected for the gamma-zein A and B RNAs, but no evidence was obtained that o2 modifiers influence the selection of these sites. Nucleotide sequence analysis of gamma-zein A and B cDNAs derived from 18-DAP endosperm from normal, opaque2, and modified opaque2 kernels confirmed the use of eight different poly(A) addition sites for gamma-zein A transcripts and six different sites for the gamma-zein B transcripts. It also revealed that the A/B gamma-zein RNA ratio in modified opaque2 was at least 40:1, compared to 1:1 in wild type and 3:1 in opaque2. Nuclear run-on transcription assays showed a dramatic shift in the transcription rate of the gamma-zein A gene relative to the B gene in the modified opaque2 genotype. These results are consistent with a model in which the two opaque2 modifier loci influence gamma-zein gene expression through different mechanisms: one affects transcription of the gamma-zein locus and the other influences the steady state level of gamma-zein RNA.