Identifying essential components of a plant LINC complex

The higher plant NE contains a functional Linker of Nucleoskeleton and Cytoskeleton (LINC) complex based on conserved Sad1-Unc84 (SUN) domain proteins and plant specific Klarsicht/Anc1/Syne homology (KASH) domain proteins (Figure 1). Recent evidence suggests the presence of a plant lamina underneath the inner membrane and various coiled-coil proteins have been hypothesised to be associated with it including Crowded Nuclei (CRWN), Nuclear Envelope Associated Protein (NEAP) protein families as well as the CRWN binding protein KAKU4. In this study, we explore the presence of proteins of these nuclear envelope (NE) proteins from the most ancestral plant species to advanced angiosperms

Untangling chromatin interactions

International audienceUntangling chromatin interactions The plant nucleus contains myriad subdomains that define and are defined by the composition of their chromatin. On a whole nucleus scale, this includes regions of active euchromatin and inactive heterochromatin, although the boundaries between these states are blurred. Different loci constantly enter and exit nuclear structures including the nucleolus and transient regulatory nuclear bodies. Chromatin is defined by its complex combinations of epigenetic modifications and variation in its constituent histone proteins. Here we preview articles that describe many of the mechanisms that regulate this dynamic chromatin

Meeting report - INDEPTH kick-off meeting

The precise location of chromatin domains within the cell nucleus has seen growing recognition in the past decade as an additional mechanism of controlling gene expression in both plants and animals (Dekker et al., 2017). Consequently, international efforts are devoted to understanding the organising principle of this organelle in plants, and notably the nature and the role of functional compartments on gene expression (Graumann et al., 2013; Sotelo-Silveira et al., 2018). The European cooperation ‘Impact of Nuclear Domains on Gene Expression and Plant Traits’ (INDEPTH) brings together molecular cell biologists, plant physiologists, bioinformaticians, image analysts and computer scientists. They aim to address the question of how nuclear architecture, chromatin organisation and gene expression are connected in plants, particularly in relation to traits of interest such as biomass, reproduction and resistance to pathogens (https:// www.brookes.ac.uk/indepth/). The kick-off meeting of the INDEPTH consortium took place in Clermont-Ferrand, France, on 12–14thMarch 2018, where more than 80 researchers set the agenda for the coming four years of research and collaboration

Different mutations in the ZmCAD2 gene underlie the maize brown-midrib1 (bm1) phenotype with similar effects on lignin characteristics and have potential interest for bioenergy production

The maize ZmCAD2 gene has been fully sequenced in several normal and bm1 maize lines, highlighting a large diversity of mutations underlying the bm1 phenotype. Mutations in three bm1 lines (F2bm1, A619bm1, and 511Jbm1) were found corresponding to short InDels inducing premature stop codons and truncated proteins. In two lines (511Kbm1 and 5803Cbm1), mutations were limited to an only SNP or to a few SNP, modifying the catalytic sites, and likely inactivating the proteins. Results also established that the 5803Ibm7 mutant was in fact a bm1 mutant, with a sequence fully identical to the 5803Cbm1 sequence. The two new F7803bm1 (natural mutant) and Ev2210bm1 (transposon tagging Mutator investigations) both had a transposon insertion in the ZmCAD2 DNA, resulting in a truncated protein, even if the mRNA was produced. The biochemical characteristics of the Ev2210bm1 lignins corroborated the signature of CAD2 deficiency in plants, with the presence of aldehydes and atypical compounds and linkages. Considering lignin structure and content, CAD2 is likely a good target for the improvement of energy production based on maize and grass lignocellulosic biomass, including a greater susceptibility to environmentally friendly pretreatments, as it was shown in bmr sorghum. The interest in maize bm1 hybrids for cattle feeding also should be considered because there seem to be little or limited negative effects of CAD2 mutations on other agronomical traits

Exploring the evolution of the proteins of the plant nuclear envelope

In this study, we explore the plasticity during evolution of proteins of the higher plant nuclear envelope (NE) from the most ancestral plant species to advanced angiosperms. The higher plant NE contains a functional Linker of Nucleoskeleton and Cytoskeleton (LINC) complex based on conserved Sad1-Unc84 (SUN) domain proteins and plant specific Klarsicht/Anc1/Syne homology (KASH) domain proteins. Recent evidence suggests the presence of a plant lamina underneath the inner membrane and various coiled-coil proteins have been hypothesised to be associated with it including Crowded Nuclei (CRWN; also termed LINC and NMCP), Nuclear Envelope Associated Protein (NEAP) protein families as well as the CRWN binding protein KAKU4. SUN domain proteins appear throughout with a key role for mid-SUN proteins suggested. Evolution of KASH domain proteins has resulted in increasing complexity, with some appearing in all species considered, while other KASH proteins are progressively gained during evolution. Failure to identify CRWN homologs in unicellular organisms included in the study and their presence in plants leads us to speculate that convergent evolution may have occurred in the formation of the lamina with each kingdom having new proteins such as the Lamin B receptor (LBR) and Lamin-Emerin-Man1 (LEM) domain proteins (animals) or NEAPs and KAKU4 (plants). Our data support a model in which increasing complexity at the nuclear envelope occurred through the plant lineage and suggest a key role for mid-SUN proteins as an early and essential component of the nuclear envelope

Different mutations in the ZmCAD2 gene underlie the maize brown-midrib1 (bm1) phenotype with similar effects on lignin characteristics and have potential interest for bioenergy production

The maize ZmCAD2 gene has been fully sequenced in several normal and bm1 maize lines, highlighting a large diversity of mutations underlying the bm1 phenotype. Mutations in three bm1 lines (F2bm1, A619bm1, and 511Jbm1) were found corresponding to short InDels inducing premature stop codons and truncated proteins. In two lines (511Kbm1 and 5803Cbm1), mutations were limited to an only SNP or to a few SNP, modifying the catalytic sites, and likely inactivating the proteins. Results also established that the 5803Ibm7 mutant was in fact a bm1 mutant, with a sequence fully identical to the 5803Cbm1 sequence. The two new F7803bm1 (natural mutant) and Ev2210bm1 (transposon tagging Mutator investigations) both had a transposon insertion in the ZmCAD2 DNA, resulting in a truncated protein, even if the mRNA was produced. The biochemical characteristics of the Ev2210bm1 lignins corroborated the signature of CAD2 deficiency in plants, with the presence of aldehydes and atypical compounds and linkages. Considering lignin structure and content, CAD2 is likely a good target for the improvement of energy production based on maize and grass lignocellulosic biomass, including a greater susceptibility to environmentally friendly pretreatments, as it was shown in bmr sorghum. The interest in maize bm1 hybrids for cattle feeding also should be considered because there seem to be little or limited negative effects of CAD2 mutations on other agronomical traits

Image analysis workflows to reveal the spatial organization of cell nuclei and chromosomes

Nucleus, chromatin, and chromosome organization studies heavily rely on fluorescence microscopy imaging to elucidate the distribution and abundance of structural and regulatory components. Three-dimensional (3D) image stacks are a source of quantitative data on signal intensity level and distribution and on the type and shape of distribution patterns in space. Their analysis can lead to novel insights that are otherwise missed in qualitative-only analyses. Quantitative image analysis requires specific software and workflows for image rendering, processing, segmentation, setting measurement points and reference frames and exporting target data before further numerical processing and plotting. These tasks often call for the development of customized computational scripts and require an expertise that is not broadly available to the community of experimental biologists. Yet, the increasing accessibility of high- and super-resolution imaging methods fuels the demand for user-friendly image analysis workflows. Here, we provide a compendium of strategies developed by participants of a training school from the COST action INDEPTH to analyze the spatial distribution of nuclear and chromosomal signals from 3D image stacks, acquired by diffraction-limited confocal microscopy and super-resolution microscopy methods (SIM and STED). While the examples make use of one specific commercial software package, the workflows can easily be adapted to concurrent commercial and open-source software. The aim is to encourage biologists lacking custom-script-based expertise to venture into quantitative image analysis and to better exploit the discovery potential of their images

NucleusJ: an ImageJ plugin for quantifying 3D images of inter- phase nuclei

ABSTRACT Summary: NucleusJ is a simple and user-friendly ImageJ plugin dedicated to the characterization of nuclear morphology and chromatin organization in 3D. Starting from image stacks, the nuclear boundary is delimited by combining the Otsu segmentation method with optimization of nuclear sphericity. Chromatin domains are segmented by partitioning the nucleus using a 3D watershed algorithm and by thresholding a contrast measure over the resulting regions. As output, NucleusJ quantifies 15 parameters including shape and size of nuclei as well as intra-nuclear objects and their position within the nucleus. A step-by-step documentation is available for selftraining, together with data sets of nuclei with different nuclear organization. Availability: Data set of nuclei is available at https://www.gredclermont.fr/media/WorkDirectory.zip. NucleusJ is available at http://imagejdocu.tudor.lu/doku.php?id=plugin:stacks:nuclear_analysis_plugin:start

The INDEPTH (Impact of Nuclear Domains On Gene Expression and Plant Traits) Academy: A community resource for plant science

This community resource paper introduces the range of materials developed by the INDEPTH (Impact of Nuclear Domains on Gene Expression and Plant Traits) COST Action made available through the INDEPTH Academy. Recent rapid growth in understanding of the significance of epigenetic controls in plant and crop science has led to a need for shared, high-quality resources, standardisation of protocols and repositories for open access data. The INDEPTH Academy provides a range of masterclass tutorials, standardised protocols and teaching webinars together with a rapidly developing repository to support imaging and spatial analysis of the nucleus and deep-learning for automated analysis. These resources were developed partly as a response to the COVID-19 pandemic, but also driven by needs and opportunities identified by the INDEPTH community of around 200 researchers in 80 laboratories from 32 countries. This community report outlines the resources produced and how they will be extended beyond the INDEPTH project, but also aims to encourage the wider community to engage with epigenetics and nuclear structure by accessing these resources

The LINC complex contributes to heterochromatin organisation and transcriptional gene silencing in plants

​The LInker of Nucleoskeleton and Cytoskeleton (LINC) complex is an evolutionary well-conserved protein bridge connecting the cytoplasmic and nuclear compartments across the nuclear membrane. While recent data support its function in nuclear morphology and meiosis, its implication in chromatin organisation has not been studied in plants. Here 3D imaging methods have been used to investigate nuclear morphology and chromatin organisation in interphase nuclei of the model plant Arabidopsis thaliana, in which heterochromatin cluster in conspicuous chromatin domains called chromocentres. Chromocentres form a repressive chromatin environment contributing to transcriptional silencing of repeated sequences, a general mechanism needed for genome stability. Quantitative measurements of 3D position of chromocentres indicate their close proximity to the nuclear periphery but that their position varies with nuclear volume and can be altered in specific mutants affecting the LINC complex. Finally we propose that the plant LINC complex contributes to proper heterochromatin organisation and positioning at the nuclear periphery, since its alteration is associated with the release of transcriptional silencing as well as decompaction of heterochromatic sequences