The effect of the nucleus on mitochondrial transcripts --Carol Leja, Susan Gabay-Laughnan and J. R. Laughnan The nucleus has been demonstrated to have an effect on mitochondrial DNA (mtDNA). The relative amounts of S1 and S2 -- two self-replicating, linear DNA molecules present in the mitochondria of cytoplasmic male-sterile maize of the S-type (cms-S) -- are determined by the nuclear background. The nucleus also affects what changes occur in the mtDNA when male-sterile plants undergo cytoplasmic reversion to male fertility. Even in the absence of cytoplasmic reversion, however, the mtDNA can be altered by changing the nuclear background.

The effect of nuclear genotype on mitochondrial gene expression in maize is less well characterized. Studies have focused on the region of the mitochondrial genome implicated in cytoplasmic male sterility and toxin sensitivity in T cytoplasm. Urf13-T transcripts have been found to be altered in cms-T (T-type cytoplasmic male-sterile) plants into which the nuclear restorer gene, Rf1, had been introduced (Cell 44:439, 1986; PNAS 84:5374, 1987). Nuclear background, apart from the presence of fertility restoration genes, has been shown to affect the abundance of urf13-T transcripts and even to direct the synthesis of an ORF25-specific transcript (Mol. Gen. Genet. 210:399, 1987). The mRNA processing event that generates a 1.1kb transcript from ORF25 may involve more than one nuclear locus (Genome 30 (Suppl. 1):316, 1988).

We report here on the effect of nuclear background on mitochondrial transcripts of a gene that has not been implicated in cytoplasmic male sterility in S cytoplasm. A Northern blot of mitochondrial RNA from WF9-N x M82510, WF9-RD (sterile; subtype of S), WF9-RD cytoplasmic revertant (cyto-rev; fertile) 81-47-15, 81-47-15 x M8257 (fertile) and 81-47-15 x M8258 (sterile; phenotype identified after first backcross to M825) was hybridized with clone TA22, a 4.2kb HindIII DNA fragment containing the atp-alpha gene and its flanking sequences as isolated from cms-T (Plt. Physiol. 79: 571, 1985).

Figure 1(a) shows the hybridization signal obtained after 19.5 hours of exposure of the hybridized blot to film. Two main transcripts are detected in the M825 nuclear background whereas the larger transcript predominates in the WF9 nuclear background. The sizes of the two transcripts are approximately the same as those described by Braun and Levings for cms-T (Plt. Physiol. 79:571, 1985). Also consistent with their data are the relative intensities of the two bands, the smaller transcript producing a more intense hybridization signal. Comparison of the TA22 hybridization signals with those obtained for a clone specific for the coding region of atp-alpha led Braun and Levings to suggest that the larger band (5,000 nts) may represent the primary transcript and the smaller band (2,600 nts) may represent the mature transcript. A more complex transcriptional pattern was noted in B37-N (Curr. Genet. 10:321-328, 1985). Three major transcripts, two of which approximate the two described by Braun and Levings, and several minor transcripts were detected. However, identical transcript sizes identical to those of B37-N were found in all male-sterile lines examined. The data for the M825 nuclear background shown in Figure 1a (lanes 1, 4 and 5) are consistent with the above findings. The two major transcripts detected in normal cytoplasm (lane 1) are also detected in S cytoplasm (lanes 4 and 5).

Figure 1. Hybridization of TA22 to Northern blot. (a) 19.5 hr. exposure. (b) 11 day exposure (same blot). Lanes: (1)WF9-N x M82510 (2)WF9-RD (sterile) (3)WF9-RD cyto-rev 81-47-15 (fertile) (4)81-47-15 x M8257 (fertile) (5)81-47-15 x M8258 (sterile). (*) indicates transcripts which appear to be present in sterile plants only. (-) indicates transcripts which appear to be present in WF9 only (not all are indicated). Positions of rRNAs on gel are indicated with arrows. The approximate sizes of the nuclear (n) and mitochondrial (m) rRNAs are given in Svedberg units.

The fact that normal and sterile cytoplasms exhibit identical transcript sizes is more significant when one considers the existence of different genomic arrangements and varying copy number of the atp-alpha gene. Small et al. have characterized four arrangements based on variability in the 3' flanking sequence (EMBO J. 6:865, 1987). T cytoplasm lines contain predominantly one gene having the type 4 organization. C cytoplasm lines also contain predominantly one gene but it has the type 1 organization. On the other hand, most normal (N) and S cytoplasms contain two atp-alpha arrangements in equal abundance. Both WF9-N and M825-N mtDNA contains type 1 and 2 arrangements, whereas WF9-RD contains type 2 and 3 arrangements. Variability in the region 3' to the atp-alpha gene does not result in different RNA transcripts. Therefore, it appears that this 3' region is not transcribed.

The fact that the two major transcripts are much larger than the coding region of the gene (1,524 nts) suggests that these transcripts contain extensive 5' and 3' untranslated regions. Although the nuclear background does not appear to affect which of the atp-alpha genomic arrangements are predominant in any one cytoplasm, the nucleus does seem to exert an influence on atp-alpha transcripts. "Mature" atp-alpha transcripts are abundantly present in the M825 nuclear background. However, in the WF9 nuclear background (Figure 1a, lanes 2 and 3), very few, if any, "mature" transcripts are present. Interestingly, this lack of "mature" transcripts is not associated with an increased level of "primary" transcript. Instead, additional transcripts of various sizes are present (Figure 1b, lanes 2 and 3). Although an 11-day exposure of the Northern hybridized with atp-alpha reveals the presence of several minor transcripts in both the M825 and WF9 nuclear backgrounds, the pattern of transcripts is inherently more complex in the WF9 nuclear background. Also, some of the minor, high molecular weight transcripts present in both nuclear backgrounds appear to be more abundant in WF9. These transcripts may result from multiple initiation/termination events, post-transcriptional processing or recombination between transcribed regions of different mitochondrial genes. The two minor transcripts of high molecular weight, which appear to be present only in sterile plants, may represent substoichiometric, sterile-specific, genomic arrangements of the atp-alpha gene. Although some of the detected transcripts may represent other genes which have flanking sequences in common with the atp-alpha gene, the almost complete lack of "mature" atp-alpha transcripts in the WF9 nuclear background (Figure 1a, lanes 2 and 3) as compared with the M825 nuclear background (Figure 1a, lanes 1, 4 and 5) provides additional evidence for nuclear-cytoplasmic interaction. The WF9 nucleus appears to affect the expression of the atp-alpha gene in RD cytoplasm primarily at the level of post-transcriptional processing.

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