Efficient Plastid Transformation in Arabidopsis1[OPEN]

Plastid transformation is routine in tobacco (Nicotiana tabacum) but 100-fold less frequent in Arabidopsis (Arabidopsis thaliana), preventing its use in plastid biology. A recent study revealed that null mutations in ACC2, encoding a plastid-targeted acetylcoenzyme A carboxylase, cause hypersensitivity to spectinomycin. We hypothesized that plastid transformation efficiency should increase in the acc2 background, because when ACC2 is absent, fatty acid biosynthesis becomes dependent on translation of the plastid-encoded ACC b-carboxylase subunit. We bombarded ACC2-defective Arabidopsis leaves with a vector carrying a selectable spectinomycin resistance (aadA) gene and gfp, encoding the green fluorescence protein GFP. Spectinomycin-resistant clones were identified as green cell clusters on a spectinomycin medium. Plastid transformation was confirmed by GFP accumulation from the second open reading frame of a polycistronic messenger RNA, which would not be translated in the cytoplasm. We obtained one to two plastid transformation events per bombarded sample in spectinomycin-hypersensitive Slavice and Columbia acc2 knockout backgrounds, an approximately 100-fold enhanced plastid transformation frequency. Slavice and Columbia are accessions in which plant regeneration is uncharacterized or difficult to obtain. A practical system for Arabidopsis plastid transformation will be obtained by creating an ACC2 null background in a regenerable Arabidopsis accession. The recognition that the duplicated ACCase in Arabidopsis is an impediment to plastid transformation provides a rational template to implement plastid transformation in related recalcitrant crops.