Arginine-specific cleavage is the primary method used to prepare lysine-rich histone proteins in bottom-up proteomics. As the Arg-C enzyme has demonstrated suboptimal specificity, cleavage at the carboxyl side of arginine residues is typically achieved through the chemical derivatization of lysines followed by trypsin digestion. Recent improvements in proteolytic enzymes are reflected in the introduction of Arg-C Ultra, a recombinant proteinase with a substantially improved digestion specificity. Here, using mammalian histone extract, we demonstrate that Arg-C Ultra facilitates histone preparation for LC-MS/MS. We show the performance of Arg-C Ultra in terms of digestion specificity, number of modified forms identified, and yield of quantitative information compared with Arg-C and trypsin digestion combined with chemical derivatization with trimethylacetic anhydride. Importantly, we show that chemical derivatization at the peptide level, i.e., after Arg-C Ultra digestion, is still necessary to improve the quantification of short histone peptidoforms as well as positional isomers.

• MeSH
• Arginine metabolism   chemistry   MeSH
• Chromatography, Liquid methods   MeSH
• Histones * chemistry   metabolism   isolation & purification   MeSH
• Liquid Chromatography-Mass Spectrometry MeSH
• Humans MeSH
• Tandem Mass Spectrometry * methods   MeSH
• Trypsin metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Arginine MeSH
• Histones * MeSH
• Trypsin MeSH

At the molecular scale, adaptive advantages during plant growth and development rely on modulation of gene expression, primarily provided by epigenetic machinery. One crucial part of this machinery is histone posttranslational modifications, which form a flexible system, driving transient changes in chromatin, and defining particular epigenetic states. Posttranslational modifications work in concert with replication-independent histone variants further adapted for transcriptional regulation and chromatin repair. However, little is known about how such complex regulatory pathways are orchestrated and interconnected in cells. In this work, we demonstrate the utility of mass spectrometry-based approaches to explore how different epigenetic layers interact in Arabidopsis mutants lacking certain histone chaperones. We show that defects in histone chaperone function (e.g., chromatin assembly factor-1 or nucleosome assembly protein 1 mutations) translate into an altered epigenetic landscape, which aids the plant in mitigating internal instability. We observe changes in both the levels and distribution of H2A.W.7, altogether with partial repurposing of H3.3 and changes in the key repressive (H3K27me1/2) or euchromatic marks (H3K36me1/2). These shifts in the epigenetic profile serve as a compensatory mechanism in response to impaired integration of the H3.1 histone in the fas1 mutants. Altogether, our findings suggest that maintaining genome stability involves a two-tiered approach. The first relies on flexible adjustments in histone marks, while the second level requires the assistance of chaperones for histone variant replacement.

• Keywords
• Arabidopsis, chromatin remodeling, histone chaperone complex, histone variants, immunochemistry, mass spectrometry, post-translational modifications,
• MeSH
• Arabidopsis * genetics   metabolism   MeSH
• Epigenesis, Genetic * MeSH
• Chromatin Assembly Factor-1 metabolism   genetics   MeSH
• Histone Chaperones * metabolism   genetics   MeSH
• Histones * metabolism   MeSH
• Mutation MeSH
• Protein Processing, Post-Translational MeSH
• Arabidopsis Proteins * metabolism   genetics   MeSH
• Gene Expression Regulation, Plant MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Chromatin Assembly Factor-1 MeSH
• Histone Chaperones * MeSH
• Histones * MeSH
• Arabidopsis Proteins * MeSH

Correlative light and electron microscopy (CLEM) is an important tool for the localisation of target molecule(s) and their spatial correlation with the ultrastructural map of subcellular features at the nanometre scale. Adoption of these advanced imaging methods has been limited in plant biology, due to challenges with plant tissue permeability, fluorescence labelling efficiency, indexing of features of interest throughout the complex 3D volume and their re-localization on micrographs of ultrathin cross-sections. Here, we demonstrate an imaging approach based on tissue processing and embedding into methacrylate resin followed by imaging of sections by both, single-molecule localization microscopy and transmission electron microscopy using consecutive CLEM and same-section CLEM correlative workflow. Importantly, we demonstrate that the use of a particular type of embedding resin is not only compatible with single-molecule localization microscopy but shows improvements in the fluorophore blinking behavior relative to the whole-mount approaches. Here, we use a commercially available Click-iT ethynyl-deoxyuridine cell proliferation kit to visualize the DNA replication sites of wild-type Arabidopsis thaliana seedlings, as well as fasciata1 and nucleolin1 plants and apply our in-section CLEM imaging workflow for the analysis of S-phase progression and nucleolar organization in mutant plants with aberrant nucleolar phenotypes.

• MeSH
• Arabidopsis * MeSH
• Microscopy, Electron MeSH
• Electrons MeSH
• Microscopy, Fluorescence methods   MeSH
• Microscopy, Electron, Transmission MeSH
• Single Molecule Imaging * methods   MeSH
• Publication type
• Journal Article MeSH

Telomerase, an essential enzyme that maintains chromosome ends, is important for genome integrity and organism development. Various hypotheses have been proposed in human, ciliate and yeast systems to explain the coordination of telomerase holoenzyme assembly and the timing of telomerase performance at telomeres during DNA replication or repair. However, a general model is still unclear, especially pathways connecting telomerase with proposed non-telomeric functions. To strengthen our understanding of telomerase function during its intracellular life, we report on interactions of several groups of proteins with the Arabidopsis telomerase protein subunit (AtTERT) and/or a component of telomerase holoenzyme, POT1a protein. Among these are the nucleosome assembly proteins (NAP) and the minichromosome maintenance (MCM) system, which reveal new insights into the telomerase interaction network with links to telomere chromatin assembly and replication. A targeted investigation of 176 candidate proteins demonstrated numerous interactions with nucleolar, transport and ribosomal proteins, as well as molecular chaperones, shedding light on interactions during telomerase biogenesis. We further identified protein domains responsible for binding and analyzed the subcellular localization of these interactions. Moreover, additional interaction networks of NAP proteins and the DOMINO1 protein were identified. Our data support an image of functional telomerase contacts with multiprotein complexes including chromatin remodeling and cell differentiation pathways.

• Keywords
• Arabidopsis, chromatin, folding, mitochondria, protein–protein interaction, replication, telomerase, transport,
• MeSH
• Arabidopsis metabolism   MeSH
• Transcription, Genetic MeSH
• Golgi Apparatus metabolism   MeSH
• Telomere Homeostasis MeSH
• Protein Interaction Maps MeSH
• Mitochondria metabolism   MeSH
• Multiprotein Complexes metabolism   MeSH
• Nucleosomes metabolism   MeSH
• Peptides metabolism   MeSH
• RNA Processing, Post-Transcriptional genetics   MeSH
• Arabidopsis Proteins chemistry   metabolism   MeSH
• Telomere-Binding Proteins metabolism   MeSH
• Gene Expression Regulation, Plant MeSH
• DNA Replication MeSH
• Chromatin Assembly and Disassembly MeSH
• Ribosomes metabolism   MeSH
• Telomerase metabolism   MeSH
• Protein Binding MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Multiprotein Complexes MeSH
• Nucleosomes MeSH
• Peptides MeSH
• Arabidopsis Proteins MeSH
• Telomere-Binding Proteins MeSH
• Telomerase MeSH

The WEE1 and ATM AND RAD3-RELATED (ATR) kinases are important regulators of the plant intra-S-phase checkpoint; consequently, WEE1KO and ATRKO roots are hypersensitive to replication-inhibitory drugs. Here, we report on a loss-of-function mutant allele of the FASCIATA1 (FAS1) subunit of the chromatin assembly factor 1 (CAF-1) complex that suppresses the phenotype of WEE1- or ATR-deficient Arabidopsis (Arabidopsis thaliana) plants. We demonstrate that lack of FAS1 activity results in the activation of an ATAXIA TELANGIECTASIA MUTATED (ATM)- and SUPPRESSOR OF GAMMA-RESPONSE 1 (SOG1)-mediated G2/M-arrest that renders the ATR and WEE1 checkpoint regulators redundant. This ATM activation accounts for the telomere erosion and loss of ribosomal DNA that are described for fas1 plants. Knocking out SOG1 in the fas1 wee1 background restores replication stress sensitivity, demonstrating that SOG1 is an important secondary checkpoint regulator in plants that fail to activate the intra-S-phase checkpoint.

• MeSH
• Arabidopsis genetics   physiology   MeSH
• Ataxia Telangiectasia Mutated Proteins genetics   metabolism   MeSH
• Stress, Physiological MeSH
• Genome, Plant MeSH
• Genomic Instability MeSH
• Protein Serine-Threonine Kinases genetics   metabolism   MeSH
• Arabidopsis Proteins genetics   metabolism   MeSH
• Proto-Oncogene Proteins c-myb genetics   metabolism   MeSH
• DNA Replication * MeSH
• Signal Transduction * MeSH
• Transcription Factors genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• ATM protein, Arabidopsis MeSH Browser
• Ataxia Telangiectasia Mutated Proteins MeSH
• ATR1 protein, Arabidopsis MeSH Browser
• FAS protein, Arabidopsis MeSH Browser
• Protein Serine-Threonine Kinases MeSH
• Arabidopsis Proteins MeSH
• Proto-Oncogene Proteins c-myb MeSH
• SOG1 protein, Arabidopsis MeSH Browser
• Transcription Factors MeSH
• WEE1 protein, Arabidopsis MeSH Browser

Genes encoding ribosomal RNA (rDNA) are essential for cell survival and are particularly sensitive to factors leading to genomic instability. Their repetitive character makes them prone to inappropriate recombinational events arising from collision of transcriptional and replication machineries, resulting in unstable rDNA copy numbers. In this review, we summarize current knowledge on the structure and organization of rDNA, its role in sensing changes in the genome, and its linkage to aging. We also review recent findings on the main factors involved in chromatin assembly and DNA repair in the maintenance of rDNA stability in the model plants Arabidopsis thaliana and the moss Physcomitrella patens, providing a view across the plant evolutionary tree.

• Keywords
• CAF-1, RAD51, RTEL1, genome stability, rDNA organization, rRNA genes, ribosome,
• MeSH
• Arabidopsis genetics   MeSH
• DNA, Plant genetics   MeSH
• Transcription, Genetic MeSH
• Gene Dosage MeSH
• Humans MeSH
• Bryopsida genetics   MeSH
• Genomic Instability MeSH
• DNA Repair * MeSH
• DNA Replication MeSH
• Chromatin Assembly and Disassembly MeSH
• DNA, Ribosomal genetics   MeSH
• Aging genetics   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• DNA, Plant MeSH
• DNA, Ribosomal MeSH