Cell-to-cell movement of mitochondria in plants

We report cell-to-cell movement of mitochondria through a graft junction. Mitochondrial movement was discovered in an experiment designed to select for chloroplast transfer from Nicotiana sylvestris into Nicotiana tabacum cells. The alloplasmic N. tabacum line we used carries Nicotiana undulata cytoplasmic genomes, and its flowers are male sterile due to the foreign mitochondrial genome. Thus, rare mitochondrial DNA transfer from N. sylvestris to N. tabacum could be recognized by restoration of fertile flower anatomy. Analyses of the mitochondrial genomes revealed extensive recombination, tentatively linking male sterility to orf293, a mitochondrial gene causing homeotic conversion of anthers into petals. Demonstrating cell-to-cell movement of mitochondria reconstructs the evolutionary process of horizontal mitochondrial DNA transfer and enables modification of the mitochondrial genome by DNA transmitted from a sexually incompatible species. Conversion of anthers into petals is a visual marker that can be useful for mitochondrial transformation.Horizontal gene transfer (HGT), the acquisition of gene(s) across species mating boundaries, results in a phylogeny of the transferred gene(s) that is incongruent with the phylogeny of the organism. In flowering plants, HGT is relatively rare in the nucleus, but frequently involves mitochondrial DNA (mtDNA); for reviews see refs. 1–3. Pioneering papers described HGT of several mitochondrial genes (4–6) and, in an extreme case, incorporation of six genome equivalents in the 3.9-Mb Amborella trichopoda mtDNA (7). These findings imply that mechanisms exist for DNA delivery between unrelated species. Parasitic plants are frequent participants in HGT, either as donors (8, 9) or recipients (5) of foreign DNA; the DNA exchange between the host and parasite is probably facilitated by the physical connection (for review, see ref. 3). HGT between nonparasites, however, necessitates alternative modes of DNA transfer. Transfer via vectoring agents such as viruses, bacteria, fungi, insects, and pollen; transformational uptake of plant DNA released into the soil; and occasional grafting of unrelated species were proposed (6). The first experimental evidence in support of grafting as a potential mechanism of HGT came from demonstrating exchange of plant DNA in tobacco tissue grafts (10). Movement of entire chloroplast genomes was subsequently demonstrated through tobacco graft junctions, interpreted as evidence for cell-to-cell movement of the organelles (11, 12). However, evidence for cell-to-cell movement of mitochondrial DNA is missing in plants despite the fact that the majority of horizontal gene transfer events involve mitochondrial sequences.We report here an experimental system for the successful identification of a rare mitochondrial HGT event. Replacing the cytoplasm of Nicotiana tabacum with the cytoplasm of Nicotiana undulata makes the flowers of N. tabacum male sterile due to conversion of anthers to stigmatoid petals (Fig. 1 D–G). Such N. tabacum plants are called alloplasmic substitution lines for carrying an alien cytoplasm and are cytoplasmic male sterile (CMS) because they inherit male sterility only from the maternal parent (13). We reasoned that movement of Nicotiana sylvestris mitochondria into CMS cells should restore anther morphology and pollen production, a change that is easy to detect in plants even if restricted to a few flowers.Open in a separate windowFig. 1.Restoration of fertile flower anatomy facilitates identification of mitochondrial graft transmission event. (A) N. tabacum Nt-CMS and fertile N. sylvestris Ns-F graft partners and GT19-C seed progeny. (B) Grafting tobacco in culture. The scion is Nt-CMS, which carries the nuclear gentamycin resistance marker; and the rootstock is Ns-F, which carries the plastid spectinomycin resistance (aadA) and aurea bar^au genes. Arrow points to graft junction. (C) Selection of gentamycin and spectinomycin double-resistant clones. (Right) Stem slices from the graft region; (Left) from above and below. Arrow points to double-resistant clone. (D) One isolated anther from a wild-type N. tabacum flower (above) and the anther after homeotic conversion of the N. tabacum alloplasmic substitution line (below). (E) Flower morphology of the graft partners and mixed flower anatomy on the GT19-C graft transmission plant. (Right) Flowers are shown with corolla; (Left) with corolla removed. Note homeotic transformation of anthers into stigmatoid petals in Nt-CMS graft partner and the GT-CMS flowers. GT-F and N. sylvestris Ns-F flowers are fertile. The flowers of Nt-CMS graft partner and GT19-C plant (GT-CMS and GT-F) are pink, a nuclear trait; those of the N. sylvestris graft partner are white. A close-up of (F) GT-CMS, (G) GT-intermediate, and (H) GT-F flowers. (Scale bars in Lower Right corners, 10 mm.)We looked for cell-to-cell movement of mitochondria in stem grafts of two species, N. tabacum and N. sylvestris. We first selected for the nuclear marker from N. tabacum and the chloroplast marker in N. sylvestris and regenerated plants from double-resistant tissue derived from the graft junction. We identified branches with fertile flowers on one of the regenerated plants, indicating presence of fertile mtDNA in the otherwise CMS plant, and analyzed the mtDNA of its fertile and CMS seed progeny. Recombination at alternative sites in the mitochondrial genome facilitated the identification of a candidate mitochondrial gene responsible for homeotic transformation of anthers resulting in CMS.