In tobacco (Nicotiana tabacum
) it has been possible to observe experimentally, in real-time, the transfer of plastid DNA to the nucleus.5,9,10
These observations were made in plants containing, in their plastome, an aminoglycoside resistance gene (aadA
) used to select the transplastomic lines, together with a closely linked kanamycin resistance gene (neo
) designed for exclusive nuclear expression. As neo
was not active in the chloroplast but would be active if relocated to the nucleus, transfer of this gene could be detected simply by screening seedling progeny, or cells in tissue culture, for kanamycin resistance ( and screen 1). As neo
was already equipped for nuclear expression, these experiments did not measure the frequency of endosymbiotic gene transfer per se but merely the frequency at which plastid DNA transfers to the nucleus.
Figure 1 Consecutive screens were used to identify endosymbiotic transfer of a chloroplast transgene. Screen 1, used transplastomic tobacco plants containing a plastid specific copy of aadA and a nucleus specific copy of neo in their plastid genome. These kanamycin (more ...)
The transfer frequency was found to be remarkably high, particularly in the male germline, with one in 11,000 to 16,000 pollen grains containing a new nuclear copy of the gene.5,9
This frequency was at least 15-fold higher than that observed in the female germline9
and ~300 fold higher than that observed in somatic tissue.10
The exceptionally high rate is thought to relate to the programmed degeneration and exclusion of plastids in developing male gametophytes,9
both a result and probable cause of uni-parental inheritance. During this process plastid DNA is presumably released into the cytoplasm from where it can transfect nuclear chromosomes. It follows that other factors that compromise organelle integrity, such as environmental stress,12
may also lead to an increase in the frequency with which plastid DNA enters the nucleus.
While the rate at which plastid DNA relocates is an important question, perhaps more interesting, from both evolutionary and biotechnological perspectives, is the rate at which a cytoplasmic organelle gene may become functionally active in the nucleus. This is not a trivial process as it requires not only transfer of DNA encoding a protein to the nucleus but also acquisition of a nuclear promoter and polyadenylation signal and, if the gene product is to be targeted back to the organelle, it must also acquire a sequence specifying a transit peptide or another mechanism of protein targeting.
We addressed the question of functional gene transfer using the kanamycin resistant lines generated in the genetic screens outlined above. In these lines the chloroplast fragments transferred to the nucleus were large and so, in almost all cases, aadA
was co-transferred with neo
to the nucleus. Although these plants contained a copy of aadA
in the nucleus, they were sensitive to aminoglycosides, as the gene had a plastid promoter and terminator and was therefore inactive (these lines no longer contained aadA
in the plastome as they were the progeny of backcrosses to female wild type; ). By screening cells in tissue culture for aminoglycoside resistance we were able to regenerate plants in which aadA
had undergone nuclear activation ( and screen 2), paralleling the pathway of genes functionally transferred during endosymbiotic evolution. This approach had been used once previously in a study by Stegemann and Bock.13
We extended their work, screening a much larger number of independent lines and using a significantly different arrangement of the experimental genes which enabled us to uncover more diverse and evolutionarily applicable activation events. Further, we reported the complete sequence of a de novo nupt
and its flanking sequences, providing insight into how chloroplast DNA fragments are incorporated into nuclear chromosomes.