Distant sequences determine 5' end formation of cox3 transcripts in Arabidopsis thaliana ecotype C24

Abstract

The genomic environments and the transcripts of the mitochondrial cox3 gene are investigated in three Arabidopsis thaliana ecotypes. While the proximate 5' sequences up to nucleotide position -584, the coding regions and the 3' flanking regions are identical in Columbia (Col), C24 and Landsberg erecta (Ler), genomic variation is detected in regions further upstream. In the mitochondrial DNA of Col, a 1790 bp fragment flanked by a nonanucleotide direct repeat is present beyond position -584 with respect to the ATG. While in Ler only part of this insertion is conserved, this sequence is completely absent in C24, except for a single copy of the nonanucleotide direct repeat. Northern hybridization reveals identical major transcripts in the three ecotypes, but identifies an additional abundant 60 nt larger mRNA species in C24. The extremities of the most abundant mRNA species are identical in the three ecotypes. In C24, an extra major 5' end is abundant. This terminus and the other major 5' ends are located in identical sequence regions. Inspection of Atcox3 transcripts in C24/Col hybrids revealed a female inheritance of the mRNA species with the extra 5' terminus. Thus, a mitochondrially encoded factor determines the generation of an extra 5' mRNA end.

Figure 1

Scheme of cox3 gene arrangements in the mitochondrial DNA of A.thaliana ecotype C24 (C24 mtDNA) and in the chromosome 2 mtDNA copy in the nuclear DNA of ecotype Col (Col chr. 2). A 1790 bp insertion flanked by nonanucleotide direct repeats (bold arrows) is present in Col. A putative cox3 promoter of the CNM-type (bent arrow) is found ∼640 (C24) and 2430 bp (Col) upstream of the cox3 reading frame (gray box), which overlaps with a Ψsdh4 gene (white box) in C24 and Col, respectively. Parts of this insertion are found in other genomic regions of the mtDNA of C24 (indicated by checkered boxes). An orf275 sequence in reverse orientation is given as rhomboid box with an arrow. rpl16/rps3 sequences found in the mtDNA of a maternal distorted leaf mutant are given by hatched boxes with the respective accession numbers (38). ΨAth-59 refers to a sequence with high similarity to a small non-coding RNA (44). The hybridization probe used in the Southern blot analysis of total DNA is given beneath the cox3 gene.

Figure 2

A cox3 probe detects different DNA fragments in A.thaliana ecotypes C24, Col and Ler. Total DNA isolated from green seedlings of ecotypes Col (lane 1), C24 (lane 2) and Ler (lane 3) as well as total DNA from two different cell suspension culture lines each (A and B) from Col (lanes 4 and 5) and C24 (lanes 6 and 7) were digested with HindIII (H) and hybridized with an Atcox3 probe.

Figure 3

Southern blot analysis of mtDNA isolated from cell suspension cultures of ecotypes Col and C24. mtDNA was digested with HindIII and EcoRV, respectively, and hybridized with oligonucleotide probes specific for the 1790 bp insertion in Col (ins-5′PS, left panel), for the 5′ part of the cox3 reading frame (cox3-3PS, middle panel) and for the region downstream of the cox3 gene (cox3-6, right panel).

Figure 4

Northern blot analysis of Atcox3 mRNA in total RNAs isolated from green seedlings of A.thaliana ecotypes C24, Col and Ler. A DNA probe representing the complete cox3 reading frame as well as 341 and 306 nt 5′ and 3′ flanking sequences detects transcript of ∼1500 nt (1) in all three ecotypes. A second prominent transcript of ∼1560 nt (2) is solely detectable in C24. In this ecotype, an ∼2.2 kb prominent (3) and an ∼2.8 kb low abundant (5) potential precursor RNA, respectively, are also observed. Such potential primary transcripts (4 and 6) with slightly different sizes are also present in Col. The individual RNAs are indicted by arrows and numbered in the right margin.

Figure 5

CR–RT–PCR mapping of the 5′ and 3′ extremities of Atcox3 transcripts from ecotypes C24, Col and Ler. Analyses were performed with total RNA from green seedlings (A–C) and mtRNA from cell suspension cultures (D and E). The number of individual clones analyzed is given in the right margin. Prominent ends found in several clones are indicated by pins with number of clones above. The continuous line represents sequences identical in all investigated ecotypes; dashed lines are identical in Col and Ler; the dotted line is specific for C24 in this position. The locations of the ends are given in respect to the ATG or the stop codon of the Atcox3 gene. Primers (open arrows) used in the CR–RT–PCR are a: Atcox3-1; b: Atcox3-2; c: Atcox3-3PS; d: Atcox3-4; e: Atcox3-5; f: ATB2174-2; g: Atcox3-3.

Figure 6

Transcript analysis of C24 × Col and Col × C24 hybrids. (A) Northern hybridization of total RNA from ecotypes C24, Col, Ler (lanes 1–3) and each two plants (A and B) of C24 × Col and Col × C24 hybrids (lanes 4–7). The C24 specific RNAs of ∼1560 and 2800 nt are detected in the C24 parental line (lane 1) and in hybrids with C24 female parents (lanes 4 and 5). (B) Primer extension analysis of the same RNAs as described in (A) with primer Atcox3-3. Products of ∼160 nt (1) were generated on RNAs of C24 (lane 1) and of plants A and B with C24 as female parental line (lanes 4 and 5). These products correspond to 5′ ends at positions around −440, which are exclusively detected in the CR–RT–PCR of this ecotype (Figure 5A and D). A second prominent product (2) of ∼100 nt is detected in almost all plants, except in C24 in lane 1. This ends corresponds to 5′ termini around −380, which was found in the CR–RT–PCR in all ecotypes (Figure 5).

Figure 7

Genomic arrangements and transcripts of the cox3 genes in three different A.thaliana ecotypes. The localization of the atp9, cox3 (gray boxes) and the Ψsdh4 genes (transparent box) are indicated in the mtDNAs of the investigated A.thaliana ecotypes here. The major 5′ and 3′ termini of the predominant mRNAs (dashed lines) are given including their position in respect to the ATG (5′ ends) and stop codon (3′ ends). Genomic regions that differ between the ecotypes are highlighted by a gray background. Mitochondrial promoters are given as bent arrows. Bold arrows represent the nonanucleotide direct repeat.