Gene transfer from organelles to the nucleus: frequent and in big chunks.

C hloroplasts arose 1.2 billion years ago (1) when a freeliving cyanobacterium became an endosymbiont in a eukaryotic host. Since that time, chloroplast genomes have undergone severe reduction, because chloroplast genomes encode between 50 and 200 proteins, whereas cyanobacterial genomes encode several thousand. Accordingly, endosymbiotic theories have always assumed that the cyanobacterial ancestor of plastids relinquished much of its genetic autonomy: ‘‘it is not surprising that chloroplasts lost their ability to live independently long ago,’’ as Mereschkowsky put it in 1905 (2). In today’s terms, that means that during the course of evolution, genes must have been transferred from the ancestral chloroplast to the nucleus, where they acquired the proper expression and targeting signals to allow the encoded proteins to be synthesized on cytosolic ribosomes and reimported into the organelle with the help of a transit peptide. This process, a special kind of lateral gene transfer called endosymbiotic gene transfer (3), appears to be very widespread in nature: 18% of the nuclear genes in Arabidopsis seem to come from cyanobacteria (4), and obvious remnants of the chloroplast DNA have been found in higher plant nuclear chromosomes (5). Evolutionary biologists have long been able to infer endosymbiotic gene transfer from evolutionary sequence comparisons but have not been able to watch it happen in the lab until now. In this issue of PNAS, Stegemann et al. (6) report gene transfer from the tobacco chloroplast genome to nuclear chromosomes under laboratory conditions. Their findings, together with other recent developments, open up new chapters in our understanding of organelle–nuclear DNA dynamics and have far-reaching evolutionary implications. The experimental design used by Stegemann et al. (6) was simple and effective. Using a technology called chloroplast transformation (7), they introduced a cassette containing two foreign genes into tobacco chloroplast DNA. The first one encoded spectinomycin resistance (aad) under the control of a chloroplast-specific promoter; the second one encoded kanamycin resistance (npt) under the control of a nuclearspecific promoter. They took advantage of the fact that whole tobacco plants can be regenerated from single cells. By subjecting transformed tobacco tissues to several rounds of selection on medium containing spectinomycin, they were able to obtain tobacco plants that were homoplastomic for aad and npt; that is, all copies of the chloroplast DNA in all plastids in those plants contained the new cassette. By placing small sections of leaves from those aad npt homoplastomic lines on kanamycincontaining medium, they initiated selection for strong expression of the npt gene under the control of the nuclearspecific promoter. That was the key step, because on kanamycin medium, only such tobacco cells will survive whose nuclear DNA has incorporated a segment of the genetically modified chloroplast DNA containing the new npt gene. With that simple scheme, they obtained 12 independent kanamycin-resistant regenerant plants, and using some rough calculations, they estimate that 1 in every 5,000,000 tobacco leaf cells that they assayed contained a highly expressed, independently transferred, chloroplast-derived npt gene in the nucleus. But was the npt gene in those 12 plants in the nucleus? Simple genetics says yes: the resistance gene behaved as a normal Mendelian dominant marker. Using their resistant plants as pollen donors in crosses to wild-type tobacco, they obtained a 1:1 ratio of kanamycin-resistant to kanamycin-sensitive progeny. Because chloroplast DNA is not transmitted through the pollen in tobacco (8), this ratio means but one thing: the npt gene, which they had originally inserted into chloroplast DNA, had found its way via a natural mechanism from the chloroplast to the nucleus and was being expressed there. On an evolutionary or environmental scale, 1 in 5,000,000 cells is a whopping number; to some it will be unbelievable (9) and by no means will it make everybody happy. Some biotechnologists are adamant that foreign genes introduced into the chloroplast can be sequestered there and thus will not escape via pollen (introgress) from cultivated fields into wild species like nuclear genes can (9). So for many the first question will be: Are these surprising findings reproducible? The answer is yes (10). In independent work, Huang et al. (11) reported chloroplast-to-nucleus gene transfer also by using the npt gene. They transformed a different region of the chloroplast genome, placed a typical nuclear intron in the npt gene, and used a different approach to look for kanamycin resistance (11). Whereas Stegemann et al. (6) took the low road, assaying leaf tissue on a few convenient Petri dishes and finding 12 independent transfers among some 60,000,000 vegetative cells, Huang et al. (11) took the high road, assaying seeds from pollen outcrosses of homoplastomic plants and finding 16 independent transfers among 250,000 male gametes so tested. Of course, it is one thing to get a transferred gene expressed when it brings along its own nuclear promoter, as in the present findings (6, 11), but quite another to acquire a good promoter through recombination (9, 10). But with two independent laboratories reporting massive rates of chloroplast DNA escape to the nucleus in laboratory regimens, it is time to consider the mechanistic and evolutionary implications of such findings. In what physical form are these chloroplast genes making their way to the nucleus? In principle, there are three simple possibilities: as bulk chloroplast DNA, mRNA, or cDNA (possibly virusmediated). Stegemann et al. (6) checked the pollen-outcrossed progeny that possessed only the expressed nuclear copy of the npt gene to see whether the aad gene was still physically linked to npt. It was, suggesting that a contiguous piece of bulk chloroplast DNA had escaped from the plastid and had recombined into a nuclear chromosome. Huang et al. (11) made the same observation, and because they had furthermore inserted a nuclear-specific intron GT–AG into their npt gene, the involvement of an mRNA or cDNA intermediate in their 16 organelle-to-nucleus transfer events can reasonably be excluded. That raises the question of whether bulk DNA recombination is also involved in chloroplast-to-nucleus gene transfer events in nature.