Background: Pentatricopeptide repeat (PPR) proteins that are required for RNA editing frequently include a C-terminal DYW deaminase domain. Results: Mutagenesis of a glutamate residue in the conserved deaminase HXE motif results in loss of editing activity. Conclusion:The glutamate residue is required for editing. Significance: The DYW deaminase domain of PPR proteins has the molecular characteristics of a deaminase.Many transcripts expressed from plant organelle genomes are modified by C-to-U RNA editing. Nuclear encoded pentatricopeptide repeat (PPR) proteins include an RNA binding domain that provides site specificity. In addition, many PPR proteins include a C-terminal DYW deaminase domain with characteristic zinc binding motifs (CXXC, HXE) and has recently been shown to bind zinc ions. The glutamate residue of the HXE motif is catalytically required in the reaction catalyzed by cytidine deaminase. In this work, we examine the activity of the DYW deaminase domain through truncation or mutagenesis of the HXE motif. OTP84 is required for editing three chloroplast sites, and transgenes expressing OTP84 with C-terminal truncations were capable of editing only one of the three cognate sites at high efficiency. These results suggest that the deaminase domain of OTP84 is required for editing two of the sites, but another deaminase is able to supply the deamination activity for the third site. OTP84 and CREF7 transgenes were mutagenized to replace the glutamate residue of the HXE motif, and transgenic plants expressing OTP84-E824A and CREF7-E554A were unable to efficiently edit the cognate editing sites for these genes. In addition, plants expressing CREF7-E554A exhibited substantially reduced capacity to edit a non-cognate site, rpoA C200. These results indicate that the DYW deaminase domains of PPR proteins are involved in editing their cognate editing sites, and in some cases may participate in editing additional sites in the chloroplast.RNA editing takes place in most land plant chloroplasts and mitochondria (1, 2). In flowering plants, the transcripts of chloroplasts and mitochondria are modified post-transcriptionally by C-to-U editing with about 35 C-to-U editing events in chloroplasts and hundreds of editing sites in the mitochondria (3).Editing in higher plants and in Physcomitrella patens is known to require nuclear proteins (4 -8).Pentatricopeptide repeat (PPR) 2 genes have been shown to be required for RNA editing (9), and form a large family of protein-coding genes in higher plants with over 400 members in Arabidopsis (10). The known editing factors are members of the PLS subfamily of PPR proteins, which are composed of characteristic P, L (long), and S (short) repeats (11). Amino acid residues located in specific locations within the repeats have been shown to specify the base recognized in the cis-element (12)(13)(14), and the PLS repeat domain interacts with specific nucleotides within the cis-element to provide site specificity for RNA editing (12,15).The PLS subfamily of PPR proteins also includes ...

An RNA-seq approach was used to investigate the role of a PLS-subfamily pentatricopeptide repeat protein, Mitochondrial Editing Factor 8 (MEF8), on editing in Arabidopsis mitochondria and plastids. MEF8 has an intact DYW domain, but possesses an unusually short PLS repeat region of only five repeats. The MEF8 T-DNA insertion (mef8) line exhibited reduced editing at 38 mitochondrial editing sites and increased editing at 24 sites; therefore the absence of MEF8 affects 11% of the mitochondrial editome. Notably, 60% of the matR transcripts' sites showed a decrease of editing extent in the mef8 mutant. An E549A substitution in the MEF8 protein replaced the putatively catalytic glutamate of the HXE motif in the DYW domain. Complementation with MEF8-E549A failed to restore editing at the main target sites but was able to restore editing at the matR transcript; it also decreased the editing extent of most of the C targets exhibiting an increase of editing extent in the mef8 mutant plant. Thus, MEF8 has two antagonistic effects on mitochondrial editing: stimulatory, which requires a catalytic glutamate for most of the targets except for the matR transcript, and inhibitory, for which glutamate is dispensable.

Arabidopsis thaliana is a small plant of significant economic and agronomic importance. While Arabidopsis is not a crop plant, it is not so different in its fundamental properties. Arabidopsis is a member of the Brassicaceae family, which constitutes one of the world's most economically important plant groups. According to the United Nations, globally Brassicaceae crops are worth $31 billion, and the number is likely to increase since a number of related species within this family are underutilized edible varieties. Its small size is an advantage to researchers with limited space and funding; simply put, a smaller plant requires fewer resources. Arabidopsis has been studied most intensely for the last 40 years and officially became a model plant in the late 1990s. Since then, the community has developed genetic and genomic resources so numerous that the barrier to entry to studying Arabidopsis is relatively low. This article provides a primer to how Arabidopsis came to be a model organism and highlights essential techniques every Arabidopsis researcher should be aware of to advance the pace of discovery. © 2019 by John Wiley & Sons, Inc.