Johannes Thiel

Programmed chromosome elimination is a highly controlled developmental process in which specific chromosomes are selectively lost from defined cell types during development. Despite its broad occurrence across plants and animals, the molecular mechanisms driving the tissue-specific elimination of chromosomes remain largely unresolved. Here, we exploit the root-specific elimination of supernumerary B chromosomes in Aegilops speltoides as a tractable model to identify the genetic basis of programmed chromosome loss. A high-quality, chromosome-scale genome assembly was generated, assigning 398 Mb of sequence to the Ae. speltoides B chromosome. Transcriptome profiling across seven tissue types representing chromosome elimination-active, elimination-negative, and B chromosome nondisjunction conditions identified 3,262 genes consistently upregulated in elimination-associated tissues, including 1,035 B genes. Stepwise subtraction of genes expressed in post-elimination and B chromosome-retaining reference tissues, followed by intersection with genes expressed during B nondisjunction in anthers, identified a candidate gene set enriched for chromosome segregation functions. From this set, we prioritized SYN2-B, a B chromosome-encoded cohesin α-kleisin subunit whose Arabidopsis thaliana ortholog AtSYN2 induces chromosome bridges and micronucleus formation when misregulated. CENH3-B, an α-type centromeric histone variant identified through GO enrichment analysis of B genes expressed in elimination-associated tissues, was shown to be incorporated in centromeres of both A and B chromosomes by transient gene expression assays using protoplasts and 3D structured illumination microscopy. These findings support a model in which B chromosome-encoded perturbations of cohesin activity and centromere composition contribute to selective B chromosome nondisjunction and their elimination in root tissues. Moreover, the SYN2-B promoter is enriched for ethylene response factor-binding sites compared to its A-encoded paralog, suggesting that ethylene is implicated in the root identity pathway driving root-specific B chromosome elimination.

Background: Selective DNA elimination occurs across diverse species and plays a crucial role in evolution and development. This process encompasses small deletions, complete removal of chromosomes, or even the elimination of entire parental genomes. Despite its importance, the molecular mechanisms governing selective DNA elimination remain poorly understood. Our study focuses on the tissue-specific elimination of Sorghum purpureosericeum B chromosomes during embryo development. Results: In situ B chromosome visualisation, complemented by transcriptomic profiling and gene-enrichment analysis, allows us to identify 28 candidate genes potentially linked to chromosome elimination. We show that elimination is a developmentally programmed process, peaking during mid-embryogenesis and nearly completed at later stages, leaving B chromosomes only in restricted meristematic regions. Genome sequencing reveals that the sorghum B chromosome is of multi-A chromosomal origin, has reduced gene density, is enriched in repetitive sequences, and carries a novel centromeric repeat, SpuCL166. Transcriptome analyses identify B-specific variants of kinetochore, cohesion, and checkpoint genes that are expressed during active elimination, while structural modeling of CENH3 and CENP-C indicates functional divergence at the kinetochore interface. Conclusions: Here, we provide the first comprehensive genomic and transcriptomic characterization of B chromosome and its elimination in Sorghum purpureosericeum . Our findings suggest that B chromosomes express modified mitotic machinery to control their own fate. By establishing a framework of candidate genes, this study opens new avenues for dissecting the molecular mechanisms of chromosome elimination and provides a critical foundation for understanding how genomes evolve to regulate and tolerate supernumerary chromosomal elements.

Selective DNA elimination occurs across diverse species and plays a crucial role in evolution and development. This process encompasses small deletions, complete removal of chromosomes, or even the elimination of entire parental genomes. Despite its importance, the molecular mechanisms governing selective DNA elimination remain poorly understood. Our study focused on the tissue-specific elimination of Sorghum purpureosericeum B chromosome(s) during embryo development. In situ B chromosome visualisation, complemented by transcriptomic profiling and gene-enrichment analysis, allowed us to identify 28 candidate genes potentially linked to chromosome elimination. We show that elimination is a developmentally programmed process, peaking during mid-embryogenesis and nearly completed at later stages, leaving B chromosomes only in restricted meristematic regions. Genome sequencing revealed that the sorghum B chromosome is of multi-A chromosomal origin, has reduced gene density, is enriched in repetitive sequences, and carries a novel centromeric repeat (SpuCL166). Transcriptome analyses identified B-specific variants of kinetochore, cohesion, and checkpoint genes that are expressed during active elimination, while structural modeling of CENH3 and CENP-C indicated functional divergence at the kinetochore interface. Here, we provide the first comprehensive genomic and transcriptomic characterization of B chromosome and its elimination in Sorghum purpureosericeum. Our findings suggest that B chromosomes express modified mitotic machinery to control their own fate. By establishing a framework of candidate genes, this study opens new avenues for dissecting the molecular mechanisms of chromosome elimination and provides a critical foundation for understanding how genomes evolve to regulate and tolerate supernumerary chromosomal elements.

Oilseed rape (Brassica napus) seeds are of major economic and nutritional value since they are rich in both oil and proteins, which accumulate predominantly in the embryonic cotyledons during the filling period. Developmental phases such as embryogenesis, seed filling, and maturation have been associated with specific changes in the transcriptional landscape and are controlled by interactions of regulatory components, particularly transcription factors and cis-regulatory elements. However, the global changes on the protein level remain largely elusive. Here, we investigated the dynamics of seed development by an integrative analysis of the seed transcriptome and proteome. Plants of the winter-type cultivar Express 617 were grown under controlled, field-like conditions in the IPK PhenoSphere, and developing seeds were collected for temporally and spatially resolved multi-omics analyses. The dataset covers five stages, from pre-storage to seed maturation, and includes spatial information on four dissected organs/tissues. It provides comprehensive insights into differentiation and developmental processes of the Brassica napus seed and may serve as starting point to select potentially important genes for detailed functional investigations.

Background: The two-component system (TCS) serves as a common intracellular signal transduction pathway implicated in various processes of plant development and response to abiotic stress. With regard to the important cereal crop barley, only partial information about the occurrence of TCS signaling elements in the genome and putative functions is available. Results: In this study, we identified a total of 67 non-redundant TCS genes from all subgroups of the phosphorelay in the latest barley reference genome. Functional annotation and phylogenetic characterization was combined with a comprehensive gene expression analysis of the signaling components. Expression profiles hint at potential functions in vegetative and reproductive organs and tissue types as well as diverse stress responses. Apparently, a distinct subset of TCS genes revealed a stringent grain-specificity not being expressed elsewhere in the plant. By using laser capture microdissection (LCM)-based transcript analysis of barley grain tissues, we refined expression profiles of selected TCS genes and attributed them to individual cell types within the grain. Distinct TCS elements are exclusively expressed in the different maternal and filial cell types, particularly in the endosperm transfer cell (ETC) region. These genes are deemed to be selected in the domestication process of modern cultivars. Moreover, barley plants transformed with a synthetic sensor (TCSn::GFP) showed a high and specific activity in the ETC region of grains monitoring transcriptional output of the signaling system. Conclusions: The results provide comprehensive insights into the TCS gene family in the temperate cereal crop barley and indicate implications in various agronomic traits. The dataset is valuable for future research in different aspects of plant development and will be indispensable not only for barley, but also for other crops of the Poaceae.

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Endosperm development in barley starts with the formation of a multinucleate syncytium, followed by cellularization in the ventral part of the syncytium generating endosperm transfer cells (ETCs) as first differentiating subdomain, whereas aleurone (AL) cells will originate from the periphery of the enclosing syncytium. Positional signaling in the syncytial stage determines cell identity in the cereal endosperm. Here, we performed a morphological analysis and employed laser capture microdissection (LCM)-based RNA-seq of the ETC region and the peripheral syncytium at the onset of cellularization to dissect developmental and regulatory programs directing cell specification in the early endosperm. Transcriptome data revealed domain-specific characteristics and identified two-component signaling (TCS) and hormone activities (auxin, ABA, ethylene) with associated transcription factors (TFs) as the main regulatory links for ETC specification. On the contrary, differential hormone signaling (canonical auxin, gibberellins, cytokinin) and interacting TFs control the duration of the syncytial phase and timing of cellularization of AL initials. Domain-specific expression of candidate genes was validated by in situ hybridization and putative protein–protein interactions were confirmed by split-YFP assays. This is the first transcriptome analysis dissecting syncytial subdomains of cereal seeds and provides an essential framework for initial endosperm differentiation in barley, which is likely also valuable for comparative studies with other cereal crops.

Organ development in plants predominantly occurs postembryonically through combinatorial activity of meristems; therefore, meristem and organ fate are intimately connected. Inflorescence morphogenesis in grasses (Poaceae) is complex and relies on a specialized floral meristem, called spikelet meristem, that gives rise to all other floral organs and ultimately the grain. The fate of the spikelet determines reproductive success and contributes toward yield-related traits in cereal crops. Here, we examined the transcriptional landscapes of floral meristems in the temperate crop barley (Hordeum vulgare L.) using RNA-seq of laser capture microdissected tissues from immature, developing floral structures. Our unbiased, high-resolution approach revealed fundamental regulatory networks, previously unknown pathways, and key regulators of barley floral fate and will equally be indispensable for comparative transcriptional studies of grass meristems.

Some eukaryotes exhibit dramatic genome size differences between cells of different organs, resulting from programmed elimination of chromosomes. Here, we present the first transcriptome analysis of programmed chromosome elimination using laser capture microdissection (LCM)-based isolation of the central meristematic region of Aegilops speltoides embryos where B chromosome (B) elimination occurs. The comparative RNA-seq analysis of meristematic cells of embryos with (Bplus) and without Bs (B0) allowed the identification of 14,578 transcript isoforms (35% out of 41,615 analyzed transcript isoforms) that are differentially expressed during the elimination of Bs. A total of 2908 annotated unigenes were found to be up-regulated in Bplus condition. These genes are either associated with the process of B chromosome elimination or with the presence of B chromosomes themselves. GO enrichment analysis categorized up-regulated transcript isoforms into 27 overrepresented terms related to the biological process, nine terms of the molecular function aspect and three terms of the cellular component category. A total of 2726 annotated unigenes were down-regulated in Bplus condition. Based on strict filtering criteria, 341 B-unique transcript isoforms could be identified in central meristematic cells, of which 70 were functionally annotated. Beside others, genes associated with chromosome segregation, kinetochore function and spindle checkpoint activity were retrieved as promising candidates involved in the process of B chromosome elimination.

SUMMARY: Cereal endosperm represents the most important source of the world’s food; nevertheless, the molecular mechanisms underlying cell and tissue differentiation in cereal grains remain poorly understood. Endosperm cellularization commences at the maternal–filial intersection of grains and generates endosperm transfer cells (ETCs), a cell type with a prominent anatomy optimized for efficient nutrient transport. Barley HISTIDINE KINASE1 (HvHK1) was identified as a receptor component with spatially restricted expression in the syncytial endosperm where ETCs emerge. Here, we demonstrate its function in ETC fate acquisition using RNA interference‐mediated downregulation of HvHK1. Repression of HvHK1 impairs cell specification in the central ETC region and the development of transfer cell morphology, and consecutively defects differentiation of adjacent endosperm tissues. Coinciding with reduced expression of HvHK1, disturbed cell plate formation and fusion were observed at the initiation of endosperm cellularization, revealing that HvHK1 triggers initial cytokinesis of ETCs. Cell‐type‐specific RNA sequencing confirmed loss of transfer cell identity, compromised cell wall biogenesis and reduced transport capacities in aberrant cells and elucidated two‐component signaling and hormone pathways that are mediated by HvHK1. Gene regulatory network modeling was used to specify the direct targets of HvHK1; this predicted non‐canonical auxin signaling elements as the main regulatory links governing cellularization of ETCs, potentially through interaction with type‐B response regulators. This work provides clues to previously unknown molecular mechanisms directing ETC specification, a process with fundamental impact on grain yield in cereals.