Control of ribosome-inactivating protein (RIP) RNA levels during maize seed development

Abstract

The maize RIP1 gene encodes proRIP, the zymogen form of a ribosome-inactivating protein that accumulates to high levels in endosperm. ProRIP is synthesized coordinately with the major storage proteins and requires a functional allele of the transcriptional activator, Opaque-2 (O2) 3 for maximal accumulation. RIP1 gene expression was characterized by developmental RNA gel blot analysis. RIP1 RNA was first detected at 10 days after pollination (DAP), and increased in abundance to reach maximal levels at mid-maturation of the kernel. In four W64A maize lines with independently-derived opaque-2 ( o2) alleles, RIP1 RNA still accumulated in a developmentally specific pattern, but showed qualitative and quantitative changes from normal. RNA gel blots reprobed with an O2-specific probe provided evidence for coordinate accumulation of RIP1 and O2 RNAs throughout normal kernel development. The pleiotropic endosperm mutations brittle-1 (bt1), brittle-2 (bt2), shrunken-2 (sh2), sugary-2 (su2), Mucronate (Mc), Defective endosperm-B30 (De ∗-B30) , and floury-2 ( fl2), affected RIP1 and O2 RNA accumulation to varying degrees. None of these mutations, however, resulted in RIP1 RNA reductions comparable to that seen in W64A o2. When coupled with o2, bt1 and bt2 mutations showed synergistic interactions in their effects of reducing RIP1 RNA levels. Thus, RIP1 gene expression and relative RNA levels are controlled by multiple factors including O2, and o2-independent component(s), genetic background, and the presence of other pleiotropic mutations.