These authors contributed equally to this work. SUMMARYC 4 photosynthetic plants outperform C 3 plants in hot and arid climates. By concentrating carbon dioxide around Rubisco C 4 plants drastically reduce photorespiration. The frequency with which plants evolved C 4 photosynthesis independently challenges researchers to unravel the genetic mechanisms underlying this convergent evolutionary switch. The conversion of C 3 crops, such as rice, towards C 4 photosynthesis is a long-standing goal. Nevertheless, at the present time, in the age of synthetic biology, this still remains a monumental task, partially because the C 4 carbon-concentrating biochemical cycle spans two cell types and thus requires specialized anatomy. Here we review the advances in understanding the molecular basis and the evolution of the C 4 trait, advances in the last decades that were driven by systems biology methods. In this review we emphasise essential genetic engineering tools needed to translate our theoretical knowledge into engineering approaches. With our current molecular understanding of the biochemical C 4 pathway, we propose a simplified rational engineering model exclusively built with known C 4 metabolic components. Moreover, we discuss an alternative approach to the progressing international engineering attempts that would combine targeted mutagenesis and directed evolution.
The data suggest that the REM34, REM35 and REM36 group is the most interesting one, as REM34 is co-expressed with the floral meristem identity (FMI) genes, they are bound by AP1, SVP, AP3 and PI, and they are expressed in the floral meristem and during the earliest stages of flower development. However, it appears that high levels of functional redundancy may conceal the exact function of these transcription factor genes.
The CRISPR/Cas9 system has emerged as a powerful tool for targeted genome editing in plants and beyond. Double-strand breaks induced by the Cas9 enzyme are repaired by the cell’s own repair machinery either by the non-homologous end joining pathway or by homologous recombination (HR). While the first repair mechanism results in random mutations at the double-strand break site, HR uses the genetic information from a highly homologous repair template as blueprint for repair of the break. By offering an artificial repair template, this pathway can be exploited to introduce specific changes at a site of choice in the genome. However, frequencies of double-strand break repair by HR are very low. In this study, we compared two methods that have been reported to enhance frequencies of HR in plants. The first method boosts the repair template availability through the formation of viral replicons, the second method makes use of an in planta gene targeting (IPGT) approach. Additionally, we comparatively applied a nickase instead of a nuclease for target strand priming. To allow easy, visual detection of HR events, we aimed at restoring trichome formation in a glabrous Arabidopsis mutant by repairing a defective glabrous1 gene. Using this efficient visual marker, we were able to regenerate plants repaired by HR at frequencies of 0.12% using the IPGT approach, while both approaches using viral replicons did not yield any trichome-bearing plants.
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