The D1 polypeptide of the photosystem II (PSII) reaction center complex contains domains that regulate primary photochemical yield and charge recombination rate. Many prokaryotic oxygenic phototrophs express two or more D1 isoforms differentially in response to environmental light needs, a capability absent in flowering plants and algae. We report that tobacco (Nicotiana tabacum) plants carrying the Synechococcus (Synechococcus elongatus PCC 7942) low-light mutation (LL-E130Qin the D1 polypeptide (Nt^LL) acquire the cyanobacterial photochemical phenotype: faster photodamage in high light and significantly more charge separations in productive linear electron flow in low light. This flux increase produces 16.5% more (dry) biomass under continuous low-light illumination (100 μE m⁻² s⁻¹, 24 h). This gain is offset by the predicted lower photoprotection at high light. By contrast, the introduction of the Synechococcus high-light mutation (HL-A152S) into tobacco D1 (Nt^HL) has slightly increased photoprotection, achieved by photochemical quenching, but no apparent impact on biomass yield compared to wild type under the tested conditions. The universal design principle of all PSII reaction centers trades off energy conversion for photoprotection in different proportions across all phototrophs and provides a useful guidance for testing in crop plants. The observed biomass advantage under continuous low light can be transferred between evolutionarily isolated lineages to benefit growth under artificial lighting conditions. However, removal of the selective marker gene was essential to observe the growth phenotype, indicating growth penalty imposed by use of the particular spectinomycin resistance gene.

Engineering the plastid genome based on homologous recombination is well developed in a few model species. Homologous recombination is also the rule in mitochondria, but transformation of the mitochondrial genome has not been realized in the absence of selective markers. The application of transcription activator-like (TAL) effector-based tools brought about a dramatic change because they can be deployed from nuclear genes and targeted to plastids or mitochondria by an N-terminal targeting sequence. Recognition of the target site in the organellar genomes is ensured by the modular assembly of TALE repeats. In this paper, I review the applications of TAL effector nucleases and TAL effector cytidine deaminases for gene deletion, base editing and mutagenesis in plastids and mitochondria. I also review emerging technologies such as post-transcriptional RNmodification to regulate gene expression, Agrobacterium- and nanoparticle-mediated organellar genome transformation, and self-replicating organellar vectors as production platforms.

Efficient plastid transformation in Arabidopsis thaliana requires genetic lines that are hypersensitive to spectinomycin due to the absence of a chloroplast ACCase encoded in the ACC2 nuclear gene. To obtain plastid transformation-competent Brassica napus, we decided to inactivate all nuclear encoded, chloroplast targeted ACCase copies using CRISPR-Cas9. B. napus (2n= 38, AACC) is a recent interspecific hybrid of B. rapa (2n=20, AA) and B. oleracea (2n=18, CC) and is expected to have at least two ACC2 copies, one from each parent. The sequenced genome has two ACC2 copies, one that is B. rapa-like and one that is B. oleracea-like. We designed single guide RNAs (sgRNAs) that could simultaneously inactivate both nuclear ACC2 copies. We expressed Cas9 from a chimeric EC1.2p promoter known to yield homozygous or biallelic mutants in Arabidopsis in the T1 generation. To maximize the probability of functionally inactivating both orthologues in a single step, each of the two vectors carried four sgRNAs. Four T₀ transgenic lines were obtained by Agrobacterium-mediated hypocotyl transformation. Amplicon sequencing confirmed mutations in ACC2 genes in 10 T₁ progeny, in seven of which no wild-type copy remained. The B. napus T2 seedlings lacking wild-type ACC2 gene copies exhibit a spectinomycin hypersensitive phenotype suggesting that they will be a useful resource for chloroplast genome transformation.

We designed a dicistronic plastid marker system that relies on the plastid's ability to translate polycistronic mRNAs. The identification of transplastomic clones is based on selection for antibiotic resistance encoded in the first open reading frame (ORF) and accumulation of the reporter gene product in tobacco chloroplasts encoded in the second ORF. The antibiotic resistance gene may encode spectinomycin or kanamycin resistance based on the expression of aadA or neo genes, respectively. The reporter gene used in the study is the green fluorescent protein (GFP). The mRNA level depends on the 5′‐untranslated region of the first ORF. The protein output depends on the strengths of the ribosome binding, and is proportional with the level of translatable mRNA. Because the dicistronic mRNA is not processed, we could show that protein output from the second ORF is independent from the first ORF. High‐level GFP accumulation from the second ORF facilitates identification of transplastomic events under ultraviolet light. Expression of multiple proteins from an unprocessed mRNA is an experimental design that enables predictable protein output from polycistronic mRNAs, expanding the toolkit of plant synthetic biology.