Plastid transformation is routine in tobacco, but 100-fold less frequent in Arabidopsis, the most important model species of plant research. A recent study revealed that null mutations in ACC2, encoding a plastid-targeted acetyl-CoA-carboxylase, causes hypersensitivity to spectinomycin. We hypothesized that plastid transformation efficiency should increase in the acc2 background, because fatty acid biosynthesis becomes dependent on the translation of a plastid-encoded subunit of a redundant acetyl-CoA-carboxylase. Plastid transformation in the ACC2 knockout background resulted in 100-fold increase in the plastid transformation efficiency, as predicted. We are now refining the protocol to regularly obtain fertile transplastomic Arabidopsis plants. We are also working on extending high-efficiency plastid transformation to Brassica napus, an oilseed crop, which also has a duplicated ACCase system in the chloroplasts.

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The plastid has been proposed as the chassis for synthetic biology applications due to the opportunity to express multiple genes in operons and the ease of genome manipulation.  Identification of regulatory elements that provide a predictable output in a dynamic expression range are essential for the balanced expression of multiple genes in synthetic operons. We design synthetic operons to tune chloroplast transgene expression with dynamic range based on the nuclear-encoded PPR10 RNA binding protein and its cognate binding site upstream of plastid genes. We have found that choosing variant binding sites and incorporating tRNA cis-elements tune reporter gene expression over a 60-fold range (0.4%-25%). We utilize this system for the expression of therapeutic proteins in chloroplasts for biomedical applications.

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