opaque15, a soft endosperm mutation that reduces gamma-zein content and has the properties of a defective opaque2 modifier
--Joanne Dannenhoffer, Gloverson Moro, Dwight Bostwick, Etti Or, Ronald Burnett and Brian Larkins

We have recently characterized (MNL this issue) a novel, opaque mutation that reduces synthesis of gamma zein in the endosperm. The mutation causes a two- to three-fold reduction in accumulation of the 27 kDa-gamma-zein mRNA and protein, but there is no pronounced effect on the other classes of zein proteins, or on non-zein proteins. o15 has the properties of a defective opaque2 modifier. In contrast to o15, o2 modifier genes are associated with a two- to three-fold increase in the synthesis of the 27-kDa gamma zein mRNA and protein. There are two gamma-zein genes, A and B. Transcripts of the two genes were distinguished using a PCR- based assay, and the mutation alters the ratio of mRNAs encoded by the genes; o15 causes a preferential reduction in the level of the A gene transcript. o2 modifier genes are associated with an increase in the proportion of the A gene transcript. Using a combination of bulk-segregant analysis and RFLP mapping, the chromosome location of o15 was determined to be on the distal end of chromosome 7L, coincident with the location of an o2 modifier. Protein bodies in o15 endosperm are comparable in size to those in O15, however, there are fewer protein bodies per cross-sectional area.

o15 acts as a single, recessive gene and kernels that are homozygous for o15 do not germinate when planted in soil or on moist filter paper. To analyze this mutation we used kernels from heterozygous ears. In an effort to grow homozygous o15 plants to use for further work on this mutation, we found that seedlings develop if we imbibe seeds in sterile water and germinate them on MS media. This indicates that the inability to germinate is a nutritional deficiency.

Our original analysis of the population of EMS-induced opaque mutants from Dr. M.G. Neuffer identified another mutant with reduced amounts of gamma-zein protein. We conducted an allelism test between o15 and this second mutant. Detection of opaque seeds in the F1 indicates that they are allelic. We are now mapping the second mutation to confirm its identification. 

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