Nuclear-mitochondrial interactions affecting transcription of mitochondrial open reading frames --Torbert R. Rocheford* and Daryl R. Pring

*Present address: Agronomy Department, University of Illinois, Urbana

The circular mitochondrial genome of maize (570 kb) consists mainly of single copy sequences. For several maize mitochondrial genes, multiple mRNA transcript size classes are present. mRNA processing or multiple transcript initiation sites can contribute to the presence of multiple mRNA transcripts for an individual mitochondrial gene. Some mt mRNA processing events have been shown to be under nuclear control. We are reporting on the effect of nuclear genotype on the transcriptional patterns of the maize mitochondrial gene T-urf13 and open reading frame 25. T-urf13 is expressed only in T cytoplasm maize and is associated with T cytoplasmic male sterility. ORF25 is transcribed in all four major maize cytoplasms (N,C,S,T), and is cotranscribed with T-urf13 in T cytoplasm.

Variation in T-urf13/ORF25 transcripts among inbred lines in T cytoplasm and ORF25 transcripts among inbred lines within N, C and S cytoplasms was detected. Northern analysis of genetic progenies developed from inbred lines associated with these transcriptional differences indicated presence or relative abundance of specific ORF25 transcripts was under nuclear control.

Transcriptional patterns of the T-urf13/ORF25 region include up to six transcripts. Our survey of diverse maize inbreds in T cytoplasm revealed absence of the generally detected 1538 nt transcript for the inbreds 33-16(T) and B14A(T). The absence of this transcript is accompanied with presence of novel transcripts of 1400 and 1475 nt, respectively. These two novel transcripts were not detected in all other inbred lines evaluated. Detection of the 1400 nt transcript and absence of the 1538 nt transcript in the F1 progeny of Wf9 (T) X 33-16(N) indicates presence of this transcript is under nuclear control.

To investigate the relationship of the T-urf13/ORF25 processing event encoded by Rf1 and the effect associated with inbred 33-16, the line R213(N) Rf1 Rf1 rf2 rf2 was crossed onto 33-16(T) rf1 rf1 Rf2 Rf2. The T-urf13/ORF25 transcriptional pattern of this F1 exhibited a 1400 nt transcript, and the Rf1-associated reduction in abundance of 2013, 1830, and 1785 nt transcripts and appearance of the 1571 nt Rf1 specific transcript. Therefore the mRNA processing associated with Rf1 appears independent of the event generating the 1400 nt transcript. Additional information was gained by evaluating fertility phenotypes and corresponding T-urf13/ORF25 transcript patterns of some stocks. Since inbred 33-16(T) (male sterile) and the F1 progeny of 33-16(T) X R213(N) (male fertile) both exhibit the novel 1400 nt transcript and not the 1538 nt transcript generally detected, T-urf13/ORF25 transcriptional patterns can therefore be variable in both sterile and fertile states.

Evaluation of ORF25 transcript patterns for the inbred W64A in N, C, and S cytoplasms revealed a difference in the size of the most highly abundant transcript detected. This transcript migrated at 2200 nt in N cytoplasm and 2100 nt in C and S cytoplasms. Since the nuclear complement was essentially the same among the three cytoplasms, the transcript size difference is probably due to a mitochondrial configuration difference, possibly a 100 bp deletion or insertion. For each of these cytoplasms, this 2200/2100 nt ORF25 transcript varied considerably in abundance when associated with different inbred lines. For example, the 2100 nt transcript is not detected or is detected in very low abundance in RDW182BNRf(C). The F1 of RDW182BNRf(C) X A619(C) exhibits the highly abundant 2100 nt transcript. The other ORF25 transcripts (3400, 3200, 1350 nt) detected in the F1 did not change in abundance in comparison to RDW182BNRf(C). Reciprocal crosses of RDW182BNRf(C) X A619(C) both exhibited a highly abundant 2100 nt transcript, suggesting dominant gene action for control of the highly abundant form of the 2100 nt transcript.

A minor 2800 nt ORF25 transcript is present in some inbred lines, e.g. Wf9(C), that do not exhibit the highly abundant 2100 nt transcript. This 2800 nt transcript is not apparent in lines that exhibit the highly abundant 2100 nt transcript, e.g. W64A(C). The F1 progeny of Wf9 (C) X W64A(N) did not exhibit the 2800 nt transcript and did exhibit the 2100 nt transcript. This indicates the W64A genotype somehow modifies transcription of the 2800 nt transcript and there is a relationship between the absence of the 2800 nt transcript and presence of the 2100 nt transcript.

The ORF25 transcript pattern associated with inbred A188(N) is different from all other lines examined in N cytoplasm (e.g. 187-2) in that the largest mRNA species detected is 3100 nt and not the 3500 nt species generally detected. To examine whether this transcript size difference was due to mitochondrial configuration or due to nuclear effects, the cross A188(N) X 187-2(N) was performed. The ORF25 transcript pattern of the F1 progeny exhibited transcripts of both 3500 and 3100 nt, indicating nuclear control of the presence of these transcripts. Since the 3500 nt transcript is the largest species detected, this transcript may be a primary initiated transcript.

The tissue culture mutant A188(T7), which has lost T-urf13, displays an ORF25 transcript pattern that is identical to A188(N) (3100, 1700 & 1300 nt). The F1 progeny of A188(T7) X 187-2(N) exhibits an additional highly abundant 2100 nt transcript. This 2100 nt transcript is not present in lines with T-urf13. The deletion of T-urf13 and the observation of the highly abundant 2100 nt transcript when 187-2(N) is crossed onto this deletion mutant, suggests that the mtDNA region associated with expression of the highly abundant 2100 nt transcript was present in T cytoplasm mt DNA. However, this region apparently was unable to contribute to presence of the highly abundant 2100 nt transcript. These data demonstrate the complex interactions of the nucleus and mitochondria during the process of mt DNA transcription. The T-urf13 data are suggestive of additional nuclear controlled processing events for this gene. Collectively, the ORF25 data are consistent with modification of transcription by nuclear encoded gene products that enhance initiation of specific ORF25 transcripts.

Sequence analysis of ORF25 and the region 5' to ORF25 in B37(N) revealed high sequence similarity with the region 160 bp 5' to the presumed wheat atp6 amino terminus leader sequence. Interestingly, comparison of predicted amino acids of the amino termini of these two open reading frames revealed 13 of the initial 18 amino acids were shared.


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