One C-to-U RNA Editing Site and Two Independently Evolved Editing Factors: Testing Reciprocal Complementation with DYW-Type PPR Proteins from the Moss Physcomitrium (Physcomitrella) patens and the Flowering Plants Macadamia integrifolia and Arabidopsis.

Cytidine-to-uridine RNA editing is a posttranscriptional process in plant organelles, mediated by specific pentatricopeptide repeat (PPR) proteins. In angiosperms, hundreds of sites undergo RNA editing. By contrast, only 13 sites are edited in the moss Physcomitrium (Physcomitrella) patens Some are conserved between the two species, like the mitochondrial editing site nad5eU598RC. The PPR proteins assigned to this editing site are known in both species: the DYW-type PPR protein PPR79 in P. patens and the E+-type PPR protein CWM1 in Arabidopsis (Arabidopsis thaliana). CWM1 also edits sites ccmCeU463RC, ccmBeU428SL, and nad5eU609VV. Here, we reciprocally expressed the P. patens and Arabidopsis editing factors in the respective other genetic environment. Surprisingly, the P. patens editing factor edited all target sites when expressed in the Arabidopsis cwm1 mutant background, even when carboxy-terminally truncated. Conversely, neither Arabidopsis CWM1 nor CWM1-PPR79 chimeras restored editing in P. patens ppr79 knockout plants. A CWM1-like PPR protein from the early diverging angiosperm macadamia (Macadamia integrifolia) features a complete DYW domain and fully rescued editing of nad5eU598RC when expressed in P. patens. We conclude that (1) the independently evolved P. patens editing factor PPR79 faithfully operates in the more complex Arabidopsis editing system, (2) truncated PPR79 recruits catalytic DYW domains in trans when expressed in Arabidopsis, and (3) the macadamia CWM1-like protein retains the capacity to work in the less complex P. patens editing environment.

Insights into the Residue Trapped in Glaze Cracks of Archaeological Ceramics Using Microchemical Analysis.

Searching for residue in the glaze of porcelain or stoneware is a difficult task because these glazes are high-fired, well vitrified, and nonporous. This paper analyzes the chemical composition of residue observed in glaze cracks of porcelain via SEM-EDS to determine how the crackle effect was produced, in particular, if it was intentionally created during production or the result of post-depositional processes. This study offers insights to a specific type of ancient Chinese porcelain called "Ge-type ware", which has two different types of cracks, and whose origin has been debated for nearly 60 years because it has never been found at any kiln site. This paper analyzes the chemical composition of the two crack types, first using elemental mapping to ascertain the different mechanisms that produced these two crack types of the Heirloom Ge ware, and second using residue analysis and chemical fingerprinting to determine the provenance of this puzzling type of porcelain. In doing so, this paper demonstrates how the residue in the glaze of porcelain can be observed and analyzed via microchemical approaches and hopes to inspire more research using this technique in future.

The dual-targeted RNA editing factor AEF1 is universally conserved among angiosperms and reveals only minor adaptations upon loss of its chloroplast or its mitochondrial target.

KEY MESSAGE: Upon loss of either its chloroplast or mitochondrial target, a uniquely dual-targeted factor for C-to-U RNA editing in angiosperms reveals low evidence for improved molecular adaptation to its remaining target. RNA-binding pentatricopeptide repeat (PPR) proteins specifically recognize target sites for C-to-U RNA editing in the transcriptomes of plant chloroplasts and mitochondria. Among more than 80 PPR-type editing factors that have meantime been characterized, AEF1 (or MPR25) is a special case given its dual targeting to both organelles and addressing an essential mitochondrial (nad5eU1580SL) and an essential chloroplast (atpFeU92SL) RNA editing site in parallel in Arabidopsis. Here, we explored the angiosperm-wide conservation of AEF1 and its two organelle targets. Despite numerous independent losses of the chloroplast editing site by C-to-T conversion and at least four such conversions at the mitochondrial target site in other taxa, AEF1 remains consistently conserved in more than 120 sampled angiosperm genomes. Not a single case of simultaneous loss of the chloroplast and mitochondrial editing target or of AEF1 disintegration or loss could be identified, contrasting previous findings for editing factors targeted to only one organelle. Like in most RNA editing factors, the PPR array of AEF1 reveals potential for conceptually "improved fits" to its targets according to the current PPR-RNA binding code. Surprisingly, we observe only minor evidence for adaptation to the mitochondrial target also after deep losses of the chloroplast target among Asterales, Caryophyllales and Poales or, vice versa, for the remaining chloroplast target after a deep loss of the mitochondrial target among Malvales. The evolutionary observations support the notion that PPR-RNA mismatches may be essential for proper function of editing factors.

Plant organelle RNA editing and its specificity factors: enhancements of analyses and new database features in PREPACT 3.0.

BACKGROUND: Gene expression in plant chloroplasts and mitochondria is affected by RNA editing. Numerous C-to-U conversions, accompanied by reverse U-to-C exchanges in some plant clades, alter the genetic information encoded in the organelle genomes. Predicting and analyzing RNA editing, which ranges from only few sites in some species to thousands in other taxa, is bioinformatically demanding. RESULTS: Here, we present major enhancements and extensions of PREPACT, a WWW-based service for analysing, predicting and cataloguing plant-type RNA editing. New features in PREPACT's core include direct GenBank accession query input and options to restrict searches to candidate U-to-C editing or to sites where editing has been documented previously in the references. The reference database has been extended by 20 new organelle editomes. PREPACT 3.0 features new modules "EdiFacts" and "TargetScan". EdiFacts integrates information on pentatricopeptide repeat (PPR) proteins characterized as site-specific RNA editing factors. PREPACT's editome references connect into EdiFacts, linking editing events to specific co-factors where known. TargetScan allows position-weighted querying for sequence motifs in the organelle references, optionally restricted to coding regions or sequences around editing sites, or in queries uploaded by the user. TargetScan is mainly intended to evaluate and further refine the proposed PPR-RNA recognition code but may be handy for other tasks as well. We present an analysis for the immediate sequence environment of more than 15,000 documented editing sites finding strong and different bias in the editome data sets. CONCLUSIONS: We exemplarily present the novel features of PREPACT 3.0 aimed to enhance the analyses of plant-type RNA editing, including its new modules EdiFacts integrating information on characterized editing factors and TargetScan aimed to analyse RNA editing site recognition specificities.

Expected and unexpected evolution of plant RNA editing factors CLB19, CRR28 and RARE1: retention of CLB19 despite a phylogenetically deep loss of its two known editing targets in Poaceae.

BACKGROUND: C-to-U RNA editing in mitochondria and chloroplasts and the nuclear-encoded, RNA-binding PPR proteins acting as editing factors present a wide field of co-evolution between the different genetic systems in a plant cell. Recent studies on chloroplast editing factors RARE1 and CRR28 addressing one or two chloroplast editing sites, respectively, found them strictly conserved among 65 flowering plants as long as one of their RNA editing targets remained present. RESULTS: Extending the earlier sampling to 117 angiosperms with high-quality genome or transcriptome data, we find more evidence confirming previous conclusions but now also identify cases for expected evolutionary transition states such as retention of RARE1 despite loss of its editing target or the degeneration of CRR28 truncating its carboxyterminal DYW domain. The extended angiosperm set was now used to explore CLB19, an "E+"-type PPR editing factor targeting two chloroplast editing sites, rpoAeU200SF and clpPeU559HY, in Arabidopsis thaliana. We found CLB19 consistently conserved if one of the two targets was retained and three independent losses of CLB19 after elimination of both targets. The Ericales show independent regains of the ancestrally lost clpPeU559HY editing, further explaining why multiple-target editing factors are lost much more rarely than single target factors like RARE1. The retention of CLB19 despite loss of both editing targets in some Ericaceae, Apocynaceae and in Camptotheca (Nyssaceae) likely represents evolutionary transitions. However, the retention of CLB19 after a phylogenetic deep loss in the Poaceae rather suggests a yet unrecognized further editing target, for which we suggest editing event ndhAeU473SL. CONCLUSION: Extending the scope of studies on plant organelle RNA editing to further taxa and additional nuclear cofactors reveals expected evolutionary transitions, strikingly different evolutionary dynamics for multiple-target editing factors like CLB19 and CRR28 and suggests additional functions for editing factor CLB19 among the Poaceae.

Early evidence for the use of wheat and barley as staple crops on the margins of the Tibetan Plateau.

We report directly dated evidence from circa 1400 calibrated years (cal) B.C. for the early use of wheat, barley, and flax as staple crops on the borders of the Tibetan Plateau. During recent years, an increasing amount of data from the Tibetan Plateau and its margins shows that a transition from millets to wheat and barley agriculture took place during the second millennium B.C. Using thermal niche modeling, we refute previous assertions that the ecological characteristics of wheat and barley delayed their spread into East Asia. Rather, we demonstrate that the ability of these crops to tolerate frost and their low growing degree-day requirements facilitated their spread into the high-altitude margins of western China. Following their introduction to this region, these crops rapidly replaced Chinese millets and became the staple crops that still characterize agriculture in this area today.

Frequent chloroplast RNA editing in early-branching flowering plants: pilot studies on angiosperm-wide coexistence of editing sites and their nuclear specificity factors.

BACKGROUND: RNA editing by cytidine-to-uridine conversions is an essential step of RNA maturation in plant organelles. Some 30-50 sites of C-to-U RNA editing exist in chloroplasts of flowering plant models like Arabidopsis, rice or tobacco. We now predicted significantly more RNA editing in chloroplasts of early-branching angiosperm genera like Amborella, Calycanthus, Ceratophyllum, Chloranthus, Illicium, Liriodendron, Magnolia, Nuphar and Zingiber. Nuclear-encoded RNA-binding pentatricopeptide repeat (PPR) proteins are key editing factors expected to coevolve with their cognate RNA editing sites in the organelles. RESULTS: With an extensive chloroplast transcriptome study we identified 138 sites of RNA editing in Amborella trichopoda, approximately the 3- to 4-fold of cp editing in Arabidopsis thaliana or Oryza sativa. Selected cDNA studies in the other early-branching flowering plant taxa furthermore reveal a high diversity of early angiosperm RNA editomes. Many of the now identified editing sites in Amborella have orthologues in ferns, lycophytes or hornworts. We investigated the evolution of CRR28 and RARE1, two known Arabidopsis RNA editing factors responsible for cp editing events ndhBeU467PL, ndhDeU878SL and accDeU794SL, respectively, all of which we now found conserved in Amborella. In a phylogenetically wide sampling of 65 angiosperm genomes we find evidence for only one single loss of CRR28 in chickpea but several independent losses of RARE1, perfectly congruent with the presence of their cognate editing sites in the respective cpDNAs. CONCLUSION: Chloroplast RNA editing is much more abundant in early-branching than in widely investigated model flowering plants. RNA editing specificity factors can be traced back for more than 120 million years of angiosperm evolution and show highly divergent patterns of evolutionary losses, matching the presence of their target editing events.

Rac function is crucial for cell migration but is not required for spreading and focal adhesion formation.

Cell migration is commonly accompanied by protrusion of membrane ruffles and lamellipodia. In two-dimensional migration, protrusion of these thin sheets of cytoplasm is considered relevant to both exploration of new space and initiation of nascent adhesion to the substratum. Lamellipodium formation can be potently stimulated by Rho GTPases of the Rac subfamily, but also by RhoG or Cdc42. Here we describe viable fibroblast cell lines genetically deficient for Rac1 that lack detectable levels of Rac2 and Rac3. Rac-deficient cells were devoid of apparent lamellipodia, but these structures were restored by expression of either Rac subfamily member, but not by Cdc42 or RhoG. Cells deficient in Rac showed strong reduction in wound closure and random cell migration and a notable loss of sensitivity to a chemotactic gradient. Despite these defects, Rac-deficient cells were able to spread, formed filopodia and established focal adhesions. Spreading in these cells was achieved by the extension of filopodia followed by the advancement of cytoplasmic veils between them. The number and size of focal adhesions as well as their intensity were largely unaffected by genetic removal of Rac1. However, Rac deficiency increased the mobility of different components in focal adhesions, potentially explaining how Rac - although not essential - can contribute to focal adhesion assembly. Together, our data demonstrate that Rac signaling is essential for lamellipodium protrusion and for efficient cell migration, but not for spreading or filopodium formation. Our findings also suggest that Rac GTPases are crucial to the establishment or maintenance of polarity in chemotactic migration.

Genome Editing in Crop Plant Research-Alignment of Expectations and Current Developments.

The rapid development of genome editing and other new genomic techniques (NGT) has evoked manifold expectations on purposes of the application of these techniques to crop plants. In this study, we identify and align these expectations with current scientific development. We apply a semi-quantitative text analysis approach on political, economic, and scientific opinion papers to disentangle and extract expectations towards the application of NGT-based plants. Using the sustainable development goals (SDG) of the 2030 agenda as categories, we identify contributions to food security or adaptation to climatic changes as the most frequently mentioned expectations, accompanied by the notion of sustainable agriculture and food systems. We then link SDG with relevant plant traits and review existing research and commercial field trials for genome-edited crop plants. For a detailed analysis we pick as representative traits drought tolerance and resistance against fungal pathogens. Diverse genetic setscrews for both traits have been identified, modified, and tested under laboratory conditions, although there are only a few in the field. All in all, NGT-plants that can withstand more than one stressor or different environments are not documented in advanced development states. We further conclude that developing new plants with modified traits will not be sufficient to reach food security or adaption to climatic changes in a short time frame. Further scientific development of sustainable agricultural systems will need to play an important role to tackle SDG challenges, as well.

Multifarious Evolutionary Pathways of a Nuclear RNA Editing Factor: Disjunctions in Coevolution of DOT4 and Its Chloroplast Target rpoC1eU488SL.

Nuclear-encoded pentatricopeptide repeat (PPR) proteins are site-specific factors for C-to-U RNA editing in plant organelles coevolving with their targets. Losing an editing target by C-to-T conversion allows for eventual loss of its editing factor, as recently confirmed for editing factors CLB19, CRR28, and RARE1 targeting ancient chloroplast editing sites in flowering plants. Here, we report on alternative evolutionary pathways for DOT4 addressing rpoC1eU488SL, a chloroplast editing site in the RNA polymerase ß' subunit mRNA. Upon loss of rpoC1eU488SL by C-to-T conversion, DOT4 got lost multiple times independently in angiosperm evolution with intermediate states of DOT4 orthologs in various stages of degeneration. Surprisingly, we now also observe degeneration and loss of DOT4 despite retention of a C in the editing position (in Carica, Coffea, Vicia, and Spirodela). We find that the cytidine remains unedited, proving that DOT4 was not replaced by another editing factor. Yet another pathway of DOT4 evolution is observed among the Poaceae. Although the rpoC1eU488SL edit has been lost through C-to-T conversion, DOT4 orthologs not only remain conserved but also have their array of PPRs extended by six additional repeats. Here, the loss of the ancient target has likely allowed DOT4 to adapt for a new function. We suggest rps3 antisense transcripts as previously demonstrated in barley (Hordeum vulgare) arising from promotor sequences newly emerging in the rpl16 intron of Poaceae as a new candidate target for the extended PPR stretch of DOT4. Altogether, DOT4 and its target show more flexible pathways for evolution than the previously explored editing factors CLB19, CRR28, and RARE1. Certain plant clades (e.g., Amaranthus, Vaccinium, Carica, the Poaceae, Fabales, and Caryophyllales) show pronounced dynamics in the evolution of editing sites and corresponding factors.

Novel CHD7 mutations contributing to the mutation spectrum in patients with CHARGE syndrome.

CHARGE syndrome is an autosomal dominant inherited multiple malformation disorder typically characterized by coloboma, choanal atresia, hypoplastic semicircular canal, cranial nerve defects, cardiovascular malformations and ear abnormalities. Mutations in the chromodomain helicase DNA-binding protein 7 (CHD7) gene are the major cause of CHARGE syndrome. Mutation analysis was performed in 18 patients with firm or tentative clinical diagnosis of CHARGE syndrome. In this study eight mutations distributed across the gene were found. Five novel mutations - one missense (c.2936T > C), one nonsense (c.8093C > A) and three frameshift mutations (c.804_805insAT, c.1757_1770del14, c.1793delA) - were identified. As far as familial data were available these mutations were found to have arisen de novo. Comparison of the clinical features of patients with the same mutation demonstrates that expression of the phenotype is highly variable. The mutation detection rate in this study was 44.4% in patients with a clinically established or suspected diagnosis of CHARGE syndrome.