A Study in Scaffold Hopping: Discovery and Optimization of Thiazolopyridines as Potent Herbicides That Inhibit Acyl-ACP Thioesterase.

In the search for new chemical entities that can control resistant weeds by addressing novel modes of action (MoAs), we were interested in further exploring a compound class that contained a 1,8-naphthyridine core. By leveraging scaffold hopping methodologies, we were able to discover the new thiazolopyridine compound class that act as potent herbicidal molecules. Further biochemical investigations allowed us to identify that the thiazolopyridines inhibit acyl-acyl carrier protein (ACP) thioesterase (FAT), with this being further confirmed via an X-ray cocrystal structure. Greenhouse trials revealed that the thiazolopyridines display excellent control of grass weed species in pre-emergence application coupled with dose response windows that enable partial selectivity in certain crops.

Expression levels of inositol phosphorylceramide synthase modulate plant responses to biotic and abiotic stress in Arabidopsis thaliana.

This research was undertaken to investigate the global role of the plant inositol phosphorylceramide synthase (IPCS), a non-mammalian enzyme previously shown to be associated with the pathogen response. RNA-Seq analyses demonstrated that over-expression of inositol phosphorylceramide synthase isoforms AtIPCS1, 2 or 3 in Arabidopsis thaliana resulted in the down-regulation of genes involved in plant response to pathogens. In addition, genes associated with the abiotic stress response to salinity, cold and drought were found to be similarly down-regulated. Detailed analyses of transgenic lines over-expressing AtIPCS1-3 at various levels revealed that the degree of down-regulation is specifically correlated with the level of IPCS expression. Singular enrichment analysis of these down-regulated genes showed that AtIPCS1-3 expression affects biological signaling pathways involved in plant response to biotic and abiotic stress. The up-regulation of genes involved in photosynthesis and lipid localization was also observed in the over-expressing lines.

PALE CRESS binds to plastid RNAs and facilitates the biogenesis of the 50S ribosomal subunit.

The plant-specific PALE CRESS (PAC) protein has previously been shown to be essential for photoautotrophic growth. Here we further investigated the molecular function of the PAC protein. PAC localizes to plastid nucleoids and forms large proteinaceous and RNA-containing megadalton complexes. It co-immunoprecipitates with a specific subset of chloroplast RNAs including psbK-psbI, ndhF, ndhD, and 23S ribosomal RNA (rRNA), as demonstrated by RNA immunoprecipitation in combination with high throughput RNA sequencing (RIP-seq) analyses. Furthermore, it co-migrates with premature 50S ribosomal particles and specifically binds to 23S rRNA in vitro. This coincides with severely reduced levels of 23S rRNA in pac leading to translational deficiencies and related alterations of plastid transcript patterns and abundance similar to plants treated with the translation inhibitor lincomycin. Thus, we conclude that deficiency in plastid ribosomes accounts for the pac phenotype. Moreover, the absence or reduction of PAC levels in the corresponding mutants induces structural changes of the 23S rRNA, as demonstrated by in vivo RNA structure probing. Our results indicate that PAC binds to the 23S rRNA to promote the biogenesis of the 50S subunit.

A gene stacking approach leads to engineered plants with highly increased galactan levels in Arabidopsis.

BACKGROUND: Engineering of plants with a composition of lignocellulosic biomass that is more suitable for downstream processing is of high interest for next-generation biofuel production. Lignocellulosic biomass contains a high proportion of pentose residues, which are more difficult to convert into fuels than hexoses. Therefore, increasing the hexose/pentose ratio in biomass is one approach for biomass improvement. A genetic engineering approach was used to investigate whether the amount of pectic galactan can be specifically increased in cell walls of Arabidopsis fiber cells, which in turn could provide a potential source of readily fermentable galactose. RESULTS: First it was tested if overexpression of various plant UDP-glucose 4-epimerases (UGEs) could increase the availability of UDP-galactose and thereby increase the biosynthesis of galactan. Constitutive and tissue-specific expression of a poplar UGE and three Arabidopsis UGEs in Arabidopsis plants could not significantly increase the amount of cell wall bound galactose. We then investigated co-overexpression of AtUGE2 together with the ß-1,4-galactan synthase GalS1. Co-overexpression of AtUGE2 and GalS1 led to over 80% increase in cell wall galactose levels in Arabidopsis stems, providing evidence that these proteins work synergistically. Furthermore, AtUGE2 and GalS1 overexpression in combination with overexpression of the NST1 master regulator for secondary cell wall biosynthesis resulted in increased thickness of fiber cell walls in addition to the high cell wall galactose levels. Immunofluorescence microscopy confirmed that the increased galactose was present as ß-1,4-galactan in secondary cell walls. CONCLUSIONS: This approach clearly indicates that simultaneous overexpression of AtUGE2 and GalS1 increases the cell wall galactose to much higher levels than can be achieved by overexpressing either one of these proteins alone. Moreover, the increased galactan content in fiber cells while improving the biomass composition had no impact on plant growth and development and hence on the overall biomass amount. Thus, we could show that the gene stacking approach described here is a promising method to engineer advanced feedstocks for biofuel production.

Complex RNA metabolism in the chloroplast: an update on the psbB operon.

Expression of most plastid genes involves multiple post-transcriptional processing events, such as splicing, editing, and intercistronic processing. The latter involves the formation of mono-, di-, and multicistronic transcripts, which can further be regulated by differential stability and expression. The plastid pentacistronic psbB transcription unit has been well characterized in vascular plants. It encodes the subunits CP47 (psbB), T (psbT), and H (psbH) of photosystem II as well as cytochrome b (6) (petB) and subunit IV (petD) of the cytochrome b (6) f complex. Each of the petB and petD genes contains a group II intron, which is spliced during post-transcriptional modification. The small subunit of photosystem II, PsbN, is encoded in the intercistronic region between psbH and psbT but is transcribed in the opposite direction. Expression of the psbB gene cluster necessitates different processing events along with numerous newly evolved specificity factors conferring stability to many of the processed RNA transcripts, and thus exemplarily shows the complexity of RNA metabolism in the chloroplast.

RHON1 is a novel ribonucleic acid-binding protein that supports RNase E function in the Arabidopsis chloroplast.

The Arabidopsis endonuclease RNase E (RNE) is localized in the chloroplast and is involved in processing of plastid ribonucleic acids (RNAs). By expression of a tandem affinity purification-tagged version of the plastid RNE in the Arabidopsis rne mutant background in combination with mass spectrometry, we identified the novel vascular plant-specific and co-regulated interaction partner of RNE, designated RHON1. RHON1 is essential for photoautotrophic growth and together with RNE forms a distinct ∼800 kDa complex. Additionally, RHON1 is part of various smaller RNA-containing complexes. RIP-chip and other association studies revealed that a helix-extended-helix-structured Rho-N motif at the C-terminus of RHON1 binds to and supports processing of specific plastid RNAs. In all respects, such as plastid RNA precursor accumulation, protein pattern, increased number and decreased size of chloroplasts and defective chloroplast development, the phenotype of rhon1 knockout mutants resembles that of rne lines. This strongly suggests that RHON1 supports RNE functions presumably by conferring sequence specificity to the endonuclease.

The cutting crew - ribonucleases are key players in the control of plastid gene expression.

Chloroplast biogenesis requires constant adjustment of RNA homeostasis under conditions of on-going developmental and environmental change and its regulation is achieved mainly by post-transcriptional control mechanisms mediated by various nucleus-encoded ribonucleases. More than 180 ribonucleases are annotated in Arabidopsis, but only 17 are predicted to localize to the chloroplast. Although different ribonucleases act at different RNA target sites in vivo, most nucleases that attack RNA are thought to lack intrinsic cleavage specificity and show non-specific activity in vitro. In vivo, specificity is thought to be imposed by auxiliary RNA-binding proteins, including members of the huge pentatricopeptide repeat family, which protect RNAs from non-specific nucleolytic attack by masking otherwise vulnerable sites. RNA stability is also influenced by secondary structure, polyadenylation, and ribosome binding. Ribonucleases may cleave at internal sites (endonucleases) or digest successively from the 5' or 3' end of the polynucleotide chain (exonucleases). In bacteria, RNases act in the maturation of rRNA and tRNA precursors, as well as in initiating the degradation of mRNAs and small non-coding RNAs. Many ribonucleases in the chloroplasts of higher plants possess homologies to their bacterial counterparts, but their precise functions have rarely been described. However, many ribonucleases present in the chloroplast process polycistronic rRNAs, tRNAs, and mRNAs. The resulting production of monocistronic, translationally competent mRNAs may represent an adaptation to the eukaryotic cellular environment. This review provides a basic overview of the current knowledge of RNases in plastids and highlights gaps to stimulate future studies.

Recruitment of a ribosomal release factor for light- and stress-dependent regulation of petB transcript stability in Arabidopsis chloroplasts.

Land plant genomes encode four functional ribosomal peptide chain release factors (Prf) of eubacterial origin, two (PrfA and PrfB homologs) for each endosymbiotic organelle. Formerly, we have shown that the Arabidopsis thaliana chloroplast-localized PrfB homolog, PrfB1, is required not only for termination of translation but also for stabilization of UGA stop codon-containing chloroplast transcripts. A previously undiscovered PrfB-like protein, PrfB3, is localized to the chloroplast stroma in a petB RNA-containing complex and found only in vascular plants. Highly conserved positions of introns unequivocally indicate that PrfB3 arose from a duplication of PrfB1. Notably, PrfB3 is lacking the two most important tripeptide motifs characteristic for all eubacterial and organellar PrfB homologs described so far: the stop codon recognition motif SPF and the catalytic center GGQ for peptidyl-tRNA hydrolysis. Complementation studies, as well as functional and molecular analyses of two allelic mutations in Arabidopsis, both of which lead to a specific deficiency of the cytochrome b6f complex, revealed that PrfB3 is essentially required for photoautotrophic growth. Plastid transcript, polysome, and translation analyses indicate that PrfB3 has been recruited in vascular plants for light- and stress-dependent regulation of stability of 3' processed petB transcripts to adjust cytochrome b6 levels.

Construction, database integration, and application of an Oenothera EST library.

Coevolution of cellular genetic compartments is a fundamental aspect in eukaryotic genome evolution that becomes apparent in serious developmental disturbances after interspecific organelle exchanges. The genus Oenothera represents a unique, at present the only available, resource to study the role of the compartmentalized plant genome in diversification of populations and speciation processes. An integrated approach involving cDNA cloning, EST sequencing, and bioinformatic data mining was chosen using Oenothera elata with the genetic constitution nuclear genome AA with plastome type I. The Gene Ontology system grouped 1621 unique gene products into 17 different functional categories. Application of arrays generated from a selected fraction of ESTs revealed significantly differing expression profiles among closely related Oenothera species possessing the potential to generate fertile and incompatible plastid/nuclear hybrids (hybrid bleaching). Furthermore, the EST library provides a valuable source of PCR-based polymorphic molecular markers that are instrumental for genotyping and molecular mapping approaches.