Chloroplasts

The introduction of a transgene to alter the properties of the chloroplast raises the question of whether the transgene should be integrated into the nuclear or plastid genome (Daniell et al. 2004; Grevich and Daniell 2005; Maliga 2002, 2004). For the nucleus, we need to consider a plastid targeting sequence, gene silencing, and cell- and development-specific expression. For both locations, regulation of gene expression is a concern, but different mechanisms predominate in each location. Transcriptional regulation is the most important issue to address in the nucleus, whereas post-transcriptional regulation is primary in the plastid. Success in this endeavor may be further affected by the presence of multiple copies of the genome per plastid and multiple plastids per cell.

Another consideration is the structure of the plastid chromosome (Bendich 2004). Since we require that the transgene be present in all cells derived from the cell containing the initial transformed plastid, it is important to target a plastid DNA molecule capable of acting as a chromosome, a segregating genetic unit. Thus, we need to know what a plastid chromosome looks like and where in the plant to find such a chromosome. The concept of the circular chloroplast chromosome has impeded progress toward an understanding of the process by which chloroplast (cp) DNA is replicated and inherited. The “ploidy paradox” illustrates the problem: there is a small number of segregating genetic units, but a high level of ploidy (computed as the mass of DNA per plastid divided by its genome size) (Birky 1994; Gillham 1994). If the chromosome were comprised of a multigenomic structure of replicating cpDNA, this paradox would be resolved. Furthermore, we could then aim our transgene at cells containing bona fide plastid chromosomes and avoid cells no longer containing cpDNA able to serve as a plastid chromosome.