Northern hybridization analysis of mitochondria gene expression in maize cytoplasm with varied nuclear backgrounds

Abstract

Type T cytoplasmic male sterility in Zea mays is associated with mitochondrial RNA (mtRNA) coding sequences found on a 1.5 kb AvaI mitochondrial DNA (mtDNA) fragment not found in other cytoplasms (N, C, or S) (Abbott and Fauron 1986). Three probes (pH3.2N, pH2.7N, and ORF 13) specific to different parts of the 1.5 AvaI T region were used in a Northern blot analysis of N mtDNAs from lines with diverse nuclear backgrounds (Rf1, Rf2 included). The N mtDNA clone pH 3.2N shows homology with the right-hand boundary of the 1.5 AvaI T region and includes a portion of an open reading frame (ORF 25). Southern blots of AvaI and BamH1 digestions of N, T, S, and C mtDNA, probed with pH3.2N demonstrate that sequences in or adjacent to this region are highly active in recombination. The clone pH 2.7N is homologous to an untranscribed region of ATPase 6 and the structural gene; and ORF 13 is a portion of the 1.5 AvaI region derived predominantly from 26S 3' flanking sequences. pH3.2N and 1.5 AvaI sequences showed identical hybridization patterns on Northern blots of N, T, Trev and Tres mtRNAs. Transcript sizes of mtRNAs homologous to the pH 3.2N probe in all of these lines were different, however, there was no variation in transcript sizes when pH 2.7N was used as a probe. Northern blots of mtRNA from N cytoplasms with various nuclear restored backgrounds showed no difference in expression when probed with pH 3.2N or pH 2.7N; however, transcripts homologous to an ORF 13 specific probe can be detected in N cytoplasm with a particular nuclear background. This may suggest activity of nuclear restorer alleles in N cytoplasm.