Wheat mitochondrial orf256-coxl RNA binding proteins and tissue -specific cDNAs

The chimeric mitochondrial gene orf256 is associated with cytoplasmic male sterility (CMS) in Triticum aestivum/T. timopheevi hybrids. It is upstream of coxl in T. timopheevi mitochondrial DNA and is expressed with coxl in the same mRNA in T. timopheevi, fertility restored (FR), and CMS lines. The gene is not present in T. aestivum. Expression as a 7 kD protein occurs only in CMS lines. CMS and FR lines have full length transcripts and processed transcripts with 5' termini within the orf256 coding region. T. timopheevi mitochondria have major transcripts with 5' termini within the orf256 coding region, precluding any significant expression of an orf256 protein product. To determine the basis for the transcript differences, orf256-coxl mRNA was used to search for RNA binding proteins by RNA mobility shift and Northwestern blots. Results include observations on a number of proteins that bind the RNA, regions of the RNA necessary for binding, phosphorylation states, and purification studies of the proteins. Differential expression of mRNA in wheat tissues was studied using differential display of cDNAs derived from mRNA as an approach to clone tissue-specific promoters for use in targeting expression of desired genes to a particular tissue. Wheat leaves, anthers, and ovaries were used as sources of mRNA. A number of mRNAs showed differential expression and coded for proteins similar to histidine rich proteins, zinc finger protein, various enzymes, or other unknown proteins. For example, one fragment is only expressed as mRNA in anthers and is similar to the C-terminus coding region of MAPK9 of Arabidopsis thaliana.