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IN THIS ISSUE

Genomic Hopscotch: Gene Transfer from Plastid to Nucleus

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The widely accepted endosymbiont theory of eukaryotic evolution holds that organelles arose from free-living prokaryotes (a proteobacterium and a cyanobacterium giving rise to mitochondria and chloroplasts, respectively) that were engulfed by an ancestral archaeal host cell (Embley and Martin, 2006; Margulis et al., 2006). Endosymbiosis was followed by massive loss of gene sequence from the endosymbiont (i.e., organellar) genomes. For example, higher plant chloroplast genomes encode 80 proteins, which is 2% of the number of protein coding genes found in the free-living cyanobacterium Synechocystis. A large number of the genes that disappeared from the endosymbiont genome were not lost to the evolving eukaryotic cell; apparently, they merely hopped over to the host cell nuclear genome. However, functional gene transfer from an organelle to the nucleus is not a mere hop but more akin to a genomic game of hopscotch, involving a number of more complicated steps. A coding sequence for a functional organellar protein that is lost from the endosymbiont genome not only must be transferred to the nuclear genome, it must also acquire gene promoter and terminator sequences necessary for transcription in the nucleus and a transit sequence necessary for targeting the protein back into the organelle (although proteins encoded by some transferred genes may acquire functions outside of the organelle). Functional transfer of mitochondrial genes to the nucleus has ceased in animals (Boore, 1999) but appears to be an ongoing process for mitochondrial and plastid genes in higher plants (reviewed in Leister, 2005).

Experimental studies in yeast and plants suggest that DNA is transferred from organelles to the nucleus at an astonishingly high rate (reviewed in Timmis et al., 2004). Huang et al. (2003a) measured the frequency of DNA transfer from chloroplast to nucleus during pollen development at one escape event per 16,000 pollen grains. In most angiosperms, paternal plastids are selectively degraded during pollen maturation, and chloroplasts are inherited maternally. Thus, degradation of paternal plastids might promote a high rate of DNA transfer relative to other cell types due to a high rate of double-strand breaks occurring in degrading pollen plastids. Stegemann et al. (2003) used a similar experimental system to measure chloroplast-to-nucleus DNA transfer in somatic leaf cells at a rate of one transfer event in 5 million cells. Both groups pointed out that the actual rate of chloroplast DNA transfer could be much higher because the experimental systems only allow for detection of the transfer of complete gene sequences (i.e., selectable antibiotic resistance genes that function only when transferred to the nuclear genome).

Acquisition of gene function for a native organellar gene transferred to the nucleus is another matter. The transcription of mitochondrial and chloroplast genes is controlled by prokaryotic-type regulatory sequences (pointing to their prokaryotic origin), which do not function in the nucleus. In addition to a difference in promoter sequences, nuclear genes contain specific sequences for faithful mRNA cleavage and polyadenylation required for transcript stability and translation of the gene product. Furthermore, nuclear gene products that function within organelles must acquire transit sequences to enable transport back into the organelle. The integration of organellar DNA to the nucleus is believed to occur via illegitimate repair (nonhomologous end-joining) of double-strand breaks (Leister, 2005). Huang et al. (2004) and Matsuo et al. (2005) have shown that a surprising amount of reshuffling takes place around integration sites. In this issue of The Plant Cell, Stegemann and Bock (pages 2869–2878) reconstruct functional gene transfer from the plastid genome to the nucleus in tobacco and show that DNA-mediated gene transfer can give rise to functional nuclear genes when followed by suitable rearrangements in the nuclear genome.

Stegemann et al. (2003) previously designed a genetic screen to isolate plants having a nuclear genome carrying DNA segments newly transferred from the chloroplast genome based on transgenic chloroplasts carrying a nuclear selectable marker gene. In their follow-up study, Stegemann and Bock employ a second genetic screen designed to select for activation of transferred genes in the nuclear genome (see figure). The acquisition of gene function in this experimental system was associated with the capture of a promoter of an upstream nuclear gene and use of AT-rich noncoding sequences downstream of the plastid gene as RNA cleavage and polyadenylation sites.

View larger version (78K): [in this window] [in a new window] [as a PowerPoint slide]   Selection for Functional Activation of a Transferred Chloroplast Gene in the Nucleus. Stegemann and Bock (2006) designed a genetic screen for which the chloroplast-type aadA gene, engineered into the chloroplast genome of tobacco plants, could become functional only after transfer to the nucleus, acquisition of a nuclear promoter sequence, and the presence of appropriate terminator sequences. Primary lines with an activated aadA gene (arrow, left panel) were identified by selection on plant regeneration medium containing spectinomycin and streptomycin, resistance to which is dependent on the aadA gene product. Shoots were regenerated from tissue pieces of primary lines in the presence of spectinomycin (right). Genetic analysis of inheritance and sequence analysis indicated integration of the aadA gene into the nuclear genome.

A second screen was then performed to isolate plants that acquired resistance to spec/strep, indicating functional activation of the aadA gene in the nucleus. Eight independent lines were isolated, in which aadA was activated as a result of various short DNA deletions that removed parts of the nptII gene to bring aadA under control of the CaMV 35S promoter. In all cases, the nptII gene function was knocked out as a result of these deletions, such that acquisition of spec/strep resistance was accompanied by loss of kanamycin resistance. This suggests that acquisition of native nuclear promoters in the real world might often be accompanied by loss of function of a nuclear gene associated with the captured promoter. Thus, it might be of interest to design an experimental system that required capture of a native nuclear promoter, for example, by reversing the position of nptII and aadA genes and their promoters in the experimental construct.

The authors also investigated the downstream regions of the transferred aadA genes to determine if and how they might have acquired mRNA cleavage and polyadenylation sites. Interestingly, they did not find any incidence of rearrangements in this region. Rather, it appeared the nuclear translational machinery made use of a fortuitous native chloroplast sequence present in the 3' untranslated region. The experimental construct made use of the 3' terminator sequence of the chloroplast psbA gene (i.e., aadA was made to resemble an authentic chloroplast gene by incorporation of chloroplast promoter and terminator sequences). Further investigation of this sequence revealed that it matched the loose consensus sequence for nuclear mRNA 3' end formation in plants, which is highly AU-rich. AT richness is commonly found in chloroplast genes, particularly in untranslated and intergenic regions (Shinozaki et al., 1986; Ohyama et al., 1988), and Stegemann and Bock suggest that this might contribute significantly to the success rate of chloroplast gene transfer during evolution by eliminating the need to acquire specific sequences for faithful mRNA end processing. The work of Stegemann and Bock provides a model for the process of the activation of transferred organelle genes, suggesting that activation might frequently occur via short deletions and acquisition of the promoter of a nearby upstream gene and use of AT-rich noncoding sequences downstream of the plastid gene as RNA cleavage and polyadenylation sites.

As a final comment, the occurrence of high rates of organelle-to-nucleus DNA transfer has raised concerns about the effectiveness of chloroplast transgenes for transgene containment in genetically modified crops (Daniell and Parkinson, 2003; Huang et al., 2003b). The demonstration by Stegemann and Bock of functional gene transfer of a chloroplast gene to the nucleus is likely to exacerbate these concerns in some circles. However, it is important to realize that increased knowledge of the mechanism(s) of gene transfer, such as provided by Stegemann and Bock, will greatly enhance our ability to design transgenes that have a very low probability of functional transfer to the nucleus (see Maliga, 2003).

	Footnotes

 
www.plantcell.org/cgi/doi/10.1105/tpc.106.049031

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Related articles in Plant Cell:

Experimental Reconstruction of Functional Gene Transfer from the Tobacco Plastid Genome to the Nucleus

Sandra Stegemann and Ralph Bock
Plant Cell 2006 18: 2869-2878. [Abstract] [Full Text]  

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