Nuclear genes that encode mitochondrial proteins for DNA and RNA metabolism are clustered in the Arabidopsis genome

Abstract

The plant mitochondrial genome is complex in structure, owing to a high degree of recombination activity that subdivides the genome and increases genetic variation. The replication activity of various portions of the mitochondrial genome appears to be nonuniform, providing the plant with an ability to modulate its mitochondrial genotype during development. These and other interesting features of the plant mitochondrial genome suggest that adaptive changes have occurred in DNA maintenance and transmission that will provide insight into unique aspects of plant mitochondrial biology and mitochondrial-chloroplast coevolution. A search in the Arabidopsis genome for genes involved in the regulation of mitochondrial DNA metabolism revealed a region of chromosome III that is unusually rich in genes for mitochondrial DNA and RNA maintenance. An apparently similar genetic linkage was observed in the rice genome. Several of the genes identified within the chromosome III interval appear to target the plastid or to be targeted dually to the mitochondria and the plastid, suggesting that the process of endosymbiosis likely is accompanied by an intimate coevolution of these two organelles for their genome maintenance functions.

Figure 1.

Arabidopsis Gene Duplications Involving the Organelle Gene-Rich Interval of Chromosome III. (A) Extensive interchromosomal and intrachromosomal duplications involving genes on chromosome III. Members of larger gene families were excluded from the figure. Genes involved in DNA metabolism (red), RNA metabolism (blue), and mismatch repair (green) are designated. Predicted targeting is indicated by m (mitochondrial) or cp (chloroplast), and asterisks indicate ambiguous target predictions. Duplicated genomic regions, as described by Blanc et al. (2000), are indicated with like-colored stripes. (B) Rice chromosome I homologous gene alignment with Arabidopsis chromosome III. Organellar genes with sequence homology were identified and aligned on rice chromosome I and Arabidopsis chromosome III to assess the conservation of gene order. cM, centimorgan.

Figure 2.

Sequence Comparison of Two Arabidopsis Genes Predicted to Encode SSB-Like Proteins. (A) Alignment of SSB-like sequences. The N-terminal portions of the sequences, which encode the mitochondrion-targeting presequence, were truncated before alignment using CLUSTAL X. Identical residues are shown by reverse contrast and similar residues with shading, with dark gray indicating a block of similar and light gray weakly similar amino acid residues. In the interest of space, only a portion of the identified genes are shown. (B) Phylogenetic tree developed from SSB-like sequences reveals two plant forms of the gene. The phylogenetic tree was constructed using the NJ method (Saitou and Nei, 1987) based on a distance matrix computed using the PROTDIST program in the PHYLIP software package version 3.6(α3) (Felsenstein, 2002). Estimated statistical confidence was generated from 100 bootstrapped data sets using the SEQBOOT program, and the consensus tree was generated using the CONSENSE program in the PHYLIP package (Felsenstein, 2002). EST sequences were derived from BLAST searches. Two distinct ESTs were identified for nearly every plant species surveyed, providing evidence of SSB gene duplication.

Figure 3.

Evidence of Targeting of Presequence Exchange during the Evolution of Mitochondrial Genes in the Chromosome III Organellar Gene-Rich Interval in Arabidopsis. (A) Amino acid sequence alignment of the Arabidopsis proteins sharing common N-terminal amino acid sequence with the DNA polymerase-like protein (At3g20540), the RNA helicase-like protein (At3g22310), or the RNA binding protein (At3g28830). Identical residues are shown by reverse contrast and similar residues with shading, with dark gray indicating a block of similar and light gray weakly similar amino acid residues. (B) The predicted mitochondrial presequence from the RNA helicase (At3g22310; underlined) was used in a BLAST search to identify homologous sequences in the database. The three most significant hits were obtained for mitochondrial presequences of three neighboring loci within the same chromosomal interval, with amino acid identities ranging from 50 to 66%.

Figure 4.

Organellar Targeting of a Putative γ-Like DNA Polymerase. (A) Amino acid sequence alignment reveals conserved motifs within exonuclease and polymerase domains of the Pol1-like DNA polymerase in both chromosome I and III versions of the Arabidopsis genes. The alignment presented here incorporates data taken directly from Lewis et al. (1996) for comparison. (B) Arabidopsis leaf cell bombardment was used to test for organellar targeting of the putative DNA polymerase I–like gene products encoded on chromosome III (AtPolγ1; At3g20540) and chromosome I (AtPolγ2; At1g50840). Plastids are shown red as a result of autofluorescence. Enhanced GFP was used as the reporter gene. Although the product from AtPolγ2 appeared to target plastids, the product from AtPolγ1 targeted both plastids and mitochondria (the white arrow indicates mitochondria, and the green arrows indicate plastids).