Intracellular gene transfer in action: Dual transcription and multiple silencings of nuclear and mitochondrial cox2 genes in legumes

Abstract

The respiratory gene cox2, normally present in the mitochondrion, was previously shown to have been functionally transferred to the nucleus during flowering plant evolution, possibly during the diversification of legumes. To search for novel intermediate stages in the process of intracellular gene transfer and to assess the evolutionary timing and frequency of cox2 transfer, activation, and inactivation, we examined nuclear and mitochondrial (mt) cox2 presence and expression in over 25 legume genera and mt cox2 presence in 392 genera. Transfer and activation of cox2 appear to have occurred during recent legume evolution, more recently than previously inferred. Many intermediate stages of the gene transfer process are represented by cox2 genes in the studied legumes. Nine legumes contain intact copies of both nuclear and mt cox2, although transcripts could not be detected for some of these genes. Both cox2 genes are transcribed in seven legumes that are phylogenetically interspersed with species displaying only nuclear or mt cox2 expression. Inactivation of cox2 in each genome has taken place multiple times and in a variety of ways, including loss of detectable transcripts or transcript editing and partial to complete gene loss. Phylogenetic evidence shows about the same number (3-5) of separate inactivations of nuclear and mt cox2, suggesting that there is no selective advantage for a mt vs. nuclear location of cox2 in plants. The current distribution of cox2 presence and expression between the nucleus and mitochondrion in the studied legumes is probably the result of chance mutations silencing either cox2 gene. Most of these recent evolutionary transfers involve mt genes in plants (e.g., refs. 3-9); in contrast, the numerous sequenced mt genomes of animals all contain the same set of 13 (or occasionally 12) protein genes (10).Previously characterized cases of gene transfer have revealed several intermediate stages in the process of intracellular gene transfer. In most cases, physical transfer of plant mt genes has been inferred to occur by an RNA intermediate because the nuclear copy resembles an edited mt mRNA rather than an unedited mt gene (e.g., refs. 3-5). Acquisition of regulatory elements and a mt targeting sequence is critical for nuclear gene activation. This has been inferred to have occurred by recombination with a duplicated targeting sequence from a preexisting nuclear gene encoding a mt protein (6), by alternative splicing of a targeting sequence shared with a preexisting mitochondrialtargeted gene (8, 9), and by exon shuffling from a nuclear gene encoding a cytosolic protein and gain of mt targeting function (11). After nuclear gene activation, both the nuclear and mt copies should be expressed, at least transiently, but no such examples have been reported previously in plants. Inactivation (silencing) and loss have been reported only for the organellar copy but not the nuclear copy, suggesting that inactivation of the organellar gene is favored.The respiratory gene cox2, encoding subunit 2 of cy...

Intracellular gene transfer: Reduced hydrophobicity facilitates gene transfer for subunit 2 of cytochromecoxidase

“…Gene transfer of cox2 from the mitochondrion to the nucleus has only been reported twice, once in Chlamydomonad algae and once in a group of legumes (12,15). Because cox2 is present in the mitochondrion of most completely sequenced mitochondrial genomes (3), transfer of the cox2 gene to the nucleus appears to be rare.…”

“…3A). As mtAbCox2 represents a functional protein in a species that is closely related to soybean (15), it provides a control that is not dependent on predicting C to U editing patterns.…”

“…Plant mitochondrialencoded genes undergo extensive post-transcriptional C to U editing, at sites that are well conserved between species. As the cox2 transcript is not edited in soybean mitochondria, it is a pseudogene (15). For the purposes of this study, we changed the soybean cox2 gene at C to U editing sites by using site-directed mutagenesis based on the conserved editing patterns from pea and cowpea (17,18,27) so that the gene product should reflect an authentic Cox2.…”

“…Any changes that occurred after gene transfer are evident, and the effect of such changes can be tested experimentally. Such a situation exists for cox2 in legumes, where active nuclear and mitochondrialencoded copies exist in some species (15)(16)(17)(18). This situation is open to experimental manipulation to determine whether changes in hydrophobicity that have occurred after gene transfer to the nucleus played a role in facilitating import of the protein into the mitochondrion, and thus successful gene transfer.…”

“…Some of these events have involved hydrophobic inner membrane proteins (10)(11)(12)(13)(14)(15)(16)(17), suggesting that the proposed hydrophobicity problem can be overcome. In the cases where the gene for a hydrophobic protein has been transferred to the nucleus, it was noted that the product of the nuclear gene was less hydrophobic than its mitochondrial counterpart.…”