Correction of frameshift mutations in the atpB gene by translational recoding in chloroplasts of Oenothera and tobacco.

Translational recoding, also known as ribosomal frameshifting, is a process that causes ribosome slippage along the messenger RNA, thereby changing the amino acid sequence of the synthesized protein. Whether the chloroplast employs recoding is unknown. I-iota, a plastome mutant of Oenothera (evening primrose), carries a single adenine insertion in an oligoA stretch [11A] of the atpB coding region (encoding the ß-subunit of the ATP synthase). The mutation is expected to cause synthesis of a truncated, nonfunctional protein. We report that a full-length AtpB protein is detectable in I-iota leaves, suggesting operation of a recoding mechanism. To characterize the phenomenon, we generated transplastomic tobacco lines in which the atpB reading frame was altered by insertions or deletions in the oligoA motif. We observed that insertion of two adenines was more efficiently corrected than insertion of a single adenine, or deletion of one or two adenines. We further show that homopolymeric composition of the oligoA stretch is essential for recoding, as an additional replacement of AAA lysine codon by AAG resulted in an albino phenotype. Our work provides evidence for the operation of translational recoding in chloroplasts. Recoding enables correction of frameshift mutations and can restore photoautotrophic growth in the presence of a mutation that otherwise would be lethal.

Multi-gene metabolic engineering of tomato plants results in increased fruit yield up to 23%.

The capacity to assimilate carbon and nitrogen, to transport the resultant sugars and amino acids to sink tissues, and to convert the incoming sugars and amino acids into storage compounds in the sink tissues, are key determinants of crop yield. Given that all of these processes have the potential to co-limit growth, multiple genetic interventions in source and sink tissues, plus transport processes may be necessary to reach the full yield potential of a crop. We used biolistic combinatorial co-transformation (up to 20 transgenes) for increasing C and N flows with the purpose of increasing tomato fruit yield. We observed an increased fruit yield of up to 23%. To better explore the reconfiguration of metabolic networks in these transformants, we generated a dataset encompassing physiological parameters, gene expression and metabolite profiling on plants grown under glasshouse or polytunnel conditions. A Sparse Partial Least Squares regression model was able to explain the combination of genes that contributed to increased fruit yield. This combinatorial study of multiple transgenes targeting primary metabolism thus offers opportunities to probe the genetic basis of metabolic and phenotypic variation, providing insight into the difficulties in choosing the correct combination of targets for engineering increased fruit yield.