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The REproductive Meristem (REM) gene family encodes for transcription factors belonging to the B3 DNA binding domain superfamily. In Arabidopsis thaliana , the REM gene family is composed of 45 members, preferentially expressed during flower, ovule, and seed developments. Only a few members of this family have been functionally characterized: VERNALIZATION1 (VRN1) and, most recently, TARGET OF FLC AND SVP1 (TFS1) regulate flowering time and VERDANDI (VDD), together with VALKYRIE (VAL) that control the death of the receptive synergid cell in the female gametophyte. We investigated the role of REM34 , REM35 , and REM36 , three closely related and linked genes similarly expressed in both female and male gametophytes. Simultaneous silencing by RNA interference (RNAi) caused about 50% of the ovules to remain unfertilized. Careful evaluation of both ovule and pollen developments showed that this partial sterility of the transgenic RNAi lines was due to a postmeiotic block in both female and male gametophytes. Furthermore, protein interaction assays revealed that REM34 and REM35 interact, which suggests that they work together during the first stages of gametogenesis. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The CRISPR/Cas9 system enables precision editing of the genome of the model plant Arabidopsis thaliana and likely of any other organism. Tools and methods for further developing and optimizing this widespread and versatile system in Arabidopsis would hence be welcomed. Here, we designed a generic vector system that can be used to clone any sgRNA sequence in a plant T-DNA vector containing an ubiquitously expressed Cas9 gene. With this vector, we explored two alternative marker systems for tracking Cas9-mediated gene-editing in vivo: BIALAPHOS RESISTANCE (BAR) and GLABROUS1 (GL1). BAR confers resistance to glufosinate and is widely used as a positive selection marker; GL1 is required for the formation of trichomes. Reversion of a frameshift null BAR allele to a functional one by Cas9-mediated gene editing yielded a higher than expected number of plants that are resistant to glufosinate. Surprisingly, many of those plants did not display reversion of the BAR gene through the germline.We hypothesize that few BAR revertant cells in a highly chimeric plant likely provide systemwide resistance to glufosinate and thus we suggest that BAR is not suitable as marker for tracking Cas9-mediated gene-editing. Targeting the GL1 gene for disruption with Cas9 provided clearly visible phenotypes of partially and completely glabrous plants. 50% of the analyzed T1 plants produced descendants with a chimeric phenotype and we could recover fully homozygous plants in the T3 generation with high efficiency. We propose that targeting of GL1 is suitable for assessing and optimizing Cas9-mediated gene-editing in Arabidopsis. [ABSTRACT FROM AUTHOR]

C4 photosynthetic plants outperform C3 plants in hot and arid climates. By concentrating carbon dioxide around Rubisco C4 plants drastically reduce photorespiration. The frequency with which plants evolved C4 photosynthesis independently challenges researchers to unravel the genetic mechanisms underlying this convergent evolutionary switch. The conversion of C3 crops, such as rice, towards C4 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 C4 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 C4 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 C4 pathway, we propose a simplified rational engineering model exclusively built with known C4 metabolic components. Moreover, we discuss an alternative approach to the progressing international engineering attempts that would combine targeted mutagenesis and directed evolution. [ABSTRACT FROM AUTHOR]

The CRISPR/Cas9 system has emerged as a powerful tool for gene editing in plants and beyond. We have developed a plant vector system for targeted Cas9-dependent mutagenesis of genes in up to two different target sites in Arabidopsis thaliana . This protocol describes a simple 1-week cloning procedure for a single T-DNA vector containing the genes for Cas9 and sgRNAs, as well as the detection of induced mutations in planta . The procedure can likely be adapted for other transformable plant species., (Copyright © 2017 The Authors; exclusive licensee Bio-protocol LLC.)