Transplastomics – a convergence of biotechnology and evolution

One of the going concerns in plant biotechnology is the matter of containment of transgenes in the field.  This concern arises from the inescapable fact that genetically-modified crop plants may, depending on the specific species involved, “disseminate” transgenes via hybridization with nearby plants (of the same species or closely-related ones).  A number of strategies have been devised to reduce or eliminate this possibility.  Among these is the approach of placing transgenes in the chloroplast genome of a recipient crop plant.  The rationale behind this approach lies in the fact that the chloroplast genome is inherited in a maternal fashion (much as are mitochondrial genomes in animals).  Consequently, pollen shed by a transplastomic plant (the jargon shorthand term for the plant that has one or more transgenes resident in the plastid genome, as opposed to the nuclear genome) should not carry or transmit the transgene, since transmission is through the female gamete.

Expressing foreign genes in the chloroplast comes with some other advantages.  Since the chloroplast is a prokaryotic genetic system, it is not “encumbered” by the presence of an elaborate and hard-to-control gene silencing system, one that affects nuclear-sited transgenes in a haphazard fashion.  This means that expression of chloroplast-situated transgenes is more consistent (and often attains higher levels) that that of similar nuclear transgenes.  The chloroplast is as well the location for some very highly-expressed proteins (plant physiology students learn early on that the chloroplast enzyme rubisco aka ribulose-1,5-bisphosphate carboxylase is the most abundant protein on earth), which means that it is feasible to attain higher protein levels in such systems than from nuclear transgenes.  (Of course, there are controls on mRNA and protein accumulation in the chloroplast, so that it is necessary to test and manipulate the specific transgene and its protein product to achieve the desired results.)

These considerations aside, the possibility of transmission of chloroplast-sited transgenes remains something of an open issue.  One matter should be familiar to readers who follow the field of human ancestry; work in this field has been complicated by the observation that mitochondrial genomes, typically assumed to be maternally-transmitted, may on occasion be inherited through the paternal gamete.  A similar concern applies to those plant species that are assumed to transmit chloroplast genomes maternally; for example, recent studies (5, 8 ) show that paternal inheritance of chloroplast-localized transgenes does occur.

There is yet another consideration or concern that is more fascinating, since it brings to the discussion the matter of the origins and evolution of chloroplasts and their genomes.   Chloroplasts are derived, via an endosymbiotic process, from a photosynthetic bacterium; in the course of evolution, the vast majority of the bacterial genome has migrated into the nuclear genome, along the way picking up the appropriate gene expression and organellar targeting signals.  In principle, this migration of genes could be a concern apart from the matter of paternal inheritance of plastid genomes, since it is an obvious way by which chloroplast-situated transgenes might “escape” the constraints of maternal inheritance by relocating to the nuclear genome, where the more typical Mendelian mechanisms would be in operation.  However, given that the migration of genes from plastid to nucleus is something one would expect to be in operation over evolutionary time scales, it would not seem to be a problem for chloroplast transgenes in “real time”, as the frequency of migration would seem to be too low to be an issue.

But is this really so?  Over the past five years or so, studies that indicated that this migration is much more frequent than many had supposed, and in fact can be observed in real time.  A recent review by Bock and Timmis (1) discusses these studies, and puts them in a fascinating evolutionary light.  Briefly, the relatively recent development of methods to transform chloroplasts afforded researches the opportunity to set up screens for plastid -> nucleus migration.  For this, genes that would only be active in a nuclear environment (driven by a nuclear promoter, and interrupted by a spliceosomal intron) were introduced into chloroplasts, and subsequent “generations” of plant cells (either leaf explants or seed derived from selfing of the transplastomic plants) assessed for the activation of the plastid-localized nuclear gene.  Plants or cells with putative migratory events were further analyzed to confirm that the activation of gene expression was due to migration, and not unanticipated rearrangements of the plastid-situated transgenes. Startlingly, the frequency of migration was high in both cases – more than 10 of about 250,000 seed had migrations, and more than 10 events in 1200 leaf explants (corresponding to more than 5 million somatic cells) (3, 7).  These results reveal plastid->nucleus migration to be relatively frequent, enough to easily account for both the large fraction of “bacterial” genes that now reside in the nucleus and for the numerous other chloroplast DNA fragments that litter plant nuclear genomes (4).

But these assays reveal only that plastid DNA can migrate to the nucleus.  As mentioned above, in the course of evolution, plastid genes need to acquire appropriate transcription and protein localization signals in order to reach the status we see today in most plants.  To see how frequent this event might be, Stegemann and Bock (6) performed further screens of plants identified in the first migration studies, to identify individuals in which the (now silent) plastid-specific aadA gene (that enables resistance to spectinomycin) was subsequently activated.  Individuals were indeed identified; the activation resulted from deletions or other rearrangements that brought the appropriate transcription signals into close proximity to the aadA gene.   In this way, these authors showed how relatively frequent rearrangements (eight events in 5500 leaf explants) can bring together nuclear promoter and plastid gene to yield an active gene.  (Interestingly, the relatively A+T-rich chloroplast DNA fragment flanking the aadA gene sufficed to serve as a polyadenylation signal.  This is not surprising, given the nature of plant poly(A) signals.)

These results provide limits for estimating another way by which plastid-localized transgenes might escape the constraints of the plastid and become transmissible as an active genes by Mendelian means.  The frequency is likely very low (one needs to “stack” the migration and subsequent rearrangement that would activate the plastid gene), but the above numbers indicate that it could conceivably approach the 1 in 10^5 to 1 in 10^6 frequency for paternal transmission that has reported by several groups. These frequencies are low and safe for the most part, but, as stated by Ruf et al. (5), are high enough to require other additional approaches to attain “perfect” containment of chloroplast transgenes.

Of course, these frequencies are well within the possibilities of genetic change over evolutionary time.  Add to this both the relatively high frequency of plastid-localized nucleus-encoded proteins and the relatively non-descript nature of the transit peptide (it is highly degenerate (2), and probably occurs relatively frequently in even random peptide collections), and the feasibility of migration and activation of plastid-localized genes is plain.

References:

1.    Bock, R., and J. N. Timmis. 2008. Reconstructing evolution: gene transfer from plastids to the nucleus. Bioessays 30:556-66.
2.    Bruce, B. D. 2000. Chloroplast transit peptides: structure, function and evolution. Trends Cell Biol 10:440-7.
3.    Huang, C. Y., M. A. Ayliffe, and J. N. Timmis. 2003. Direct measurement of the transfer rate of chloroplast DNA into the nucleus. Nature 422:72-6.
4.    Martin, W., T. Rujan, E. Richly, A. Hansen, S. Cornelsen, T. Lins, D. Leister, B. Stoebe, M. Hasegawa, and D. Penny. 2002. Evolutionary analysis of Arabidopsis, cyanobacterial, and chloroplast genomes reveals plastid phylogeny and thousands of cyanobacterial genes in the nucleus. Proc Natl Acad Sci U S A 99:12246-51.
5.    Ruf, S., D. Karcher, and R. Bock. 2007. Determining the transgene containment level provided by chloroplast transformation. Proc Natl Acad Sci U S A 104:6998-7002.
6.    Stegemann, S., and R. Bock. 2006. Experimental reconstruction of functional gene transfer from the tobacco plastid genome to the nucleus. Plant Cell 18:2869-78.
7.    Stegemann, S., S. Hartmann, S. Ruf, and R. Bock. 2003. High-frequency gene transfer from the chloroplast genome to the nucleus. Proc Natl Acad Sci U S A 100:8828-33.
8.    Svab, Z., and P. Maliga. 2007. Exceptional transmission of plastids and mitochondria from the transplastomic pollen parent and its impact on transgene containment. Proc Natl Acad Sci U S A 104:7003-8.