Biology subjects

Hartwig, T.

Publications and source records attributed to Hartwig, T..

14 recordsLinked to original sources

bioRxiv2026

A composited genome approach to analyse cistrome diversity in barley

Motivation: Functional regulatory variants are important determinants of heritable diversity, yet their systematic identification requires both genome-wide maps of transcription factor (TF) occupancy and suitable genome sequences for mapping. While chromosome-scale assemblies remain costly for large association panels, short-read sequencing data are widely available. We investigated whether draft genomes reconstructed from short reads are sufficient for comparative MOA-seq analyses of TF occupancy. Results: We generated genome-wide TF footprint maps for the barley cultivars Bowman, Morex, and Golden Promise, identifying approximately 240,000 loci per genotype. Roughly half of all footprints occurred in intergenic regions. Footprints conserved between genotypes were depleted for sequence variation and located closer to genes. Meanwhile, genotype-specific footprints were enriched for SNP-containing and gene-distal regions, highlighting the extensive diversity of regulatory landscapes among barley cultivars. We further compared two short-read-based draft genome assembly strategies: i) a SNP-replacement and ii) a Practical Haplotype Graph (PHG)-based composite genome approach, to enable MOA-seq analyses without chromosome-scale genome assemblies. Compared to the SNP-replacement strategy, the composite genome displayed 2.3-fold higher local sequence concordance for SNPs/INDELs relative to the published reference. Both draft genomes accurately reproduced MOA-seq profiles, recovering approximately 80 % of reference peak bases with per-base coverage correlations of 0.79-0.90 relative to the assembled reference. While the composite genome performed best across the genome overall, the simpler SNP-replacement strategy more accurately recovered TF occupancy within regulatory peaks due to fewer structural alignment artefacts. Availability: Pipelines consist of existing open-source tools, Bash, GAWK and Java, and are available at [zenodo; tbd].

genomics↗

bioRxiv2026

Cell identities along the proximal-distal and micropylar-chalazal axes in the Arabidopsis heart-stage seed

* Seeds are complex reproductive organs consisting of diverse maternal and filial tissues. During development, the embryo and specialized tissues for nutrient storage required for seed germination and early seedling establishment emerge. * To explore the cellular diversity and differentiation of seeds, we performed single cell RNA-sequencing on heart stage Arabidopsis seeds and identified 20,097 cells that were grouped into [≥]21 distinct cell clusters. 20 of the 21 clusters were spatially assigned by combining bioinformatic analysis, imaging reporter fusion marker lines, and spatial transcriptomics. * Our analysis revealed a high degree of differentiation of epidermal cell and inner cell layers along the rotational and axial seed axes, highlighting the importance of cell position and ontogenesis. We identified unexpected spatial domains, including a cluster marked by abscission zone-specific transcripts, and a nucellar cluster shaped by developmentally programmed cell death. Surprisingly, embryo and endosperm showed similarities in transcript profiles despite distinct and complementary functions. * In summary, our findings establish seeds as a transcriptionally complex organ with high cell type heterogeneity and provide a basis for investigating the differentiation of diverse cell layers and spatial transcript profiles.

cell biology↗

bioRxiv2025

Cis-regulatory architecture downstream of FLOWERING LOCUS T underlies quantitative control of flowering

The FLOWERING LOCUS T (FT) gene is a central integrator of floral induction in Arabidopsis thaliana, with expression tightly regulated by complex transcriptional networks. Using CRISPR/Cas9 genome editing, we dissected the functional architecture of the FT downstream region and reveal that a 2.3-kb region immediately downstream of the FT coding sequence containing the Block E enhancer is essential for proper FT expression and flowering. Fine-scale deletions revealed a 63-bp core module with adjacent CCAAT- and G-boxes, whereas other conserved motifs had minor, context-dependent effects. We also uncovered a cryptic CCAAT-box module that becomes active when repositioned, coinciding with increased transcription factor binding and local chromatin accessibility, indicating that enhancer function is governed by local chromatin and motif context. The cis-regulatory logic revealed here provides insights into manipulating gene expression through the architecture and spatial arrangement of enhancer elements, potentially applicable beyond flowering genes or plant species.

plant biology↗

bioRxiv2025

From lab to field: analyses of genome-edited bacterial blight resistant rice

Bacterial blight (BB) of rice, caused by Xanthomonas oryzae pv. oryzae (Xoo) is one of the major drivers of yield losses in Africa and Asia. Xoo secretes TAL-effectors (TALe) that induce host SWEET sucrose uniporter by binding to the effector binding element (EBE) of SWEET promoters, likely required for Xoo reproduction and virulence. We had multiplex edited the EBEs of three SWEET genes to prevent TALe binding, producing genome-edited (GEd) rice mega-varieties (IR64, Ciherang-Sub1 for Asia, and Komboka for Africa) that were resistant to a wide spectrum of Xoo strains. Here, we report comprehensive analyses of the GEd lines, including evaluation of agronomic performance in multi-location multi-season experimental field plots under different fertilization regimes, and tests for the presence/absence of foreign DNA/transgene in the offspring of GEd lines (IR64-BC1T6, Ciherang-Sub1-BC1T5, Komboka-T3). Various strategies were evaluated, including herbicide tolerance, PCR, DNA gel blotting, whole genome sequencing (WGS), and specific tests stipulated by country-specific biosafety guidelines. Different WGS technologies were evaluated and also used to identify heritability of the edits, single nucleotide polymorphisms (SNPs), and insertions/deletions (indels) that might have resulted from somaclonal variation and potential GE-induced off-target mutations. Complete genome reference sequences for the parental lines IR64, Ciherang-Sub1, and Komboka are provided. In the field experiments, the GEd lines did not show performance defects. Together, the results indicate that select GE lines do not contain foreign DNA or transgene fragments and fulfill the requirements for treatment equivalent to classical breeding lines in countries such as India and Kenya.

plant biology↗

bioRxiv2024

Investigating the barley methylome, its variation and association with genomic, transcriptomic, and phenotypic variation

Epigenetic variation contributes to explaining the missing heritability of complex traits. In order to understand the genome-wide methylation variation in spring barley, our objectives were to gain fundamental insight into the barley methylome through whole genome bisulfite sequencing, characterizing methylation variation among 23 parental inbreds of a community resource for genetic mapping of phenotypic traits, and assessing the association of differentially methylated regions (DMRs) with single nucleotide polymorphisms (SNPs) and gene expression variation. Compared to other angiosperms, barley was found to have a highly methylated genome with an average genome wide methylation level of 88.6%, 58.1%, and 1.4% in the CpG, CHG, and CHH sequence context, respectively. We identified just below 500 000 differentially methylated regions (DMRs) among the inbreds. About 64%, 64%, and 83% of the DMRs were not associated with genomic variation in the CpG, CHG, and CHH context, respectively. The methylation level of around 6% of all DMRs was significantly associated with gene expression, where the directionality of the correlation was depended on the relative location of the DMR to the respective gene with a recognizable pattern. Notably, this pattern was much more specific and spatially confined than the association of methylation with gene expression across genes in a singular inbred line. We exemplified this association between DNA methylation and gene expression on the known flowering promoting gene VRN-H1 and identified a highly methylated epiallele associated with earlier flowering time. Finally, methylation was shown to improve the prediction abilities of genomic prediction models for a variety of traits over models using solely SNPs and gene expression as predictors. These observations highlight the independence of DNA methylation to sequence variation and their difference in information content. Our discoveries suggest that epigenetic variation provides a layer of information likely not predictable by other means and is therefore a valuable addition to genomic prediction models.

plant biology↗

bioRxiv2024

The role of methylation and structural variants in shaping the recombination landscape of barley

Meiotic recombination is not only a key mechanism for sexual adaptation in eukaryotes but crucial for the accumulation of beneficial alleles in breeding populations. The effective manipulation of recombination requires, however, a better understanding of the mechanisms regulating the rate and distribution of recombination events in genomes. Here, we identified the genomic features that best explain the recombination variation among a diverse set of segregating populations of barley at a resolution of 1 Mbp and investigated how methylation and structural variants determine recombination hotspots and coldspots at a high-resolution of 10 kb. Hotspots were found to be in proximity to genes and the genetic effects not assigned to methylation were found to be the most important factor explaining differences in recombination rates among populations along with the methylation and the parental sequence divergence. Interestingly, the inheritance of a highly-methylated genomic fragment from one parent only was enough to generate a coldspot, but both parents must be equally low methylated at a genomic segment to allow a hotspot. The parental sequence divergence was shown to have a sigmoidal correlation with recombination indicating an upper limit of mismatch among homologous chromosomes for CO formation. Structural variants (SVs) were shown to suppress COs, and their type and size were not found to influence that effect. Methylation and SVs act jointly determining the location of coldspots in barley and the weight of their relative effect depends on the genomic region. Our findings suggest that recombination in barley is highly predictable, occurring mostly in multiple short sections located in the proximity to genes and being modulated by local levels of methylation and SV load.

genomics↗

bioRxiv2023

Phenotypic variation in maize can be largely explained by genetic variation at transcription factor binding sites

Comprehensive maps of functional variation at transcription factor (TF) binding sites (cis-elements) are crucial for elucidating how genotype shapes phenotype. Here we report the construction of a pan-cistrome of the maize leaf under well-watered and drought conditions. We quantified haplotype-specific TF footprints across a pan-genome of 25 maize hybrids and mapped over two-hundred thousand genetic variants (termed binding-QTL) linked to cis-element occupancy. Three lines of evidence support the functional significance of binding-QTL: i) they coincide with numerous known causative loci that regulate traits, including VGT1, Trehalase1, and the MITE transposon near ZmNAC111 under drought; ii) their footprint bias is mirrored between inbred parents and by ChIP-seq; iii) partitioning genetic variation across genomic regions demonstrates that binding-QTL capture the majority of heritable trait variation across [~]70% of 143 phenotypes. Our study provides a promising approach to make previously hidden cis-variation more accessible for genetic studies and multi-target engineering of complex traits.

genomics↗

bioRxiv2023

MINI-AC: Inference of plant gene regulatory networks using bulk or single-cell accessible chromatin profiles

Gene regulatory networks (GRNs) represent the interactions between transcription factors (TF) and their target genes. GRNs control transcriptional programs involved in growth, development and stress responses, ultimately affecting diverse agricultural traits. While recent developments in accessible chromatin (AC) profiling technologies make it possible to identify context-specific regulatory DNA, learning the underlying GRNs remains a major challenge. We developed MINI-AC (Motif-Informed Network Inference based on Accessible Chromatin), a method that combines AC data from bulk or single-cell experiments with TF binding site information to learn GRNs in plants. We benchmarked MINI-AC using bulk AC datasets from different Arabidopsis thaliana tissues and showed that it outperforms other methods to identify correct TFs binding sites. In maize, a crop with a complex genome and abundant distal AC regions, MINI-AC successfully inferred leaf GRNs with experimentally confirmed, both proximal and distal, TF-target gene interactions. Furthermore, we showed that both AC regions and footprints are valid alternatives to infer AC-based GRNs with MINI-AC. Finally, we combined MINI-AC predictions from bulk and single-cell AC datasets to identify general and cell-type specific maize leaf regulators. Focusing on C4 metabolism, we identified diverse regulatory interactions in specialized cell types for this photosynthetic pathway. MINI-AC represents a powerful tool for inferring accurate AC-derived GRNs in plants and identifying known and novel candidate regulators, improving our understanding of gene regulation in plants.

bioinformatics↗

bioRxiv2022

Mapping Responsive Genomic Elements to Heat Stress in a Maize Diversity Panel

Many plant species exhibit genetic variation for tolerating environmental stress. A transcriptome investigation of over 100 maize inbreds revealed many cis- and trans-acting eQTLs that influence the expression response to heat stress. The cis-acting eQTL in response to heat stress are identified in genes with differential responses to heat stress between genotypes as well as genes that are only expressed under heat stress. The cis-acting variants for heat stress responsive expression likely result from distinct promoter activities and the differential heat responses of the alleles were confirmed for selected genes using transient expression assays. Global foot-printing of transcription factor binding was performed in control and heat stress conditions to document regions with heat-enriched transcription factor binding occupancies. Footprints enriched near proximal regions of characterized heat-responsive genes in a large association panel can be utilized for prioritizing functional genomic regions that regulate genotype-specific responses under heat stress.

plant biology↗

bioRxiv2022

Conserved and variable responses of the HEAT SHOCK FACTOR transcription factor family in maize and Setaria viridis

Responding to the environment is a core aspect of plant growth and development. Mounting an effective response is important for plants to balance growth and survival. The HEAT SHOCK FACTOR (HSF) transcription factor family is a central and required component of plant heat stress responses and acquired thermotolerance. The HSF family has dramatically expanded in plant lineages, often including a repertoire of 20 or more genes. Here we assess the composition and heat responsiveness of the HSF family in Setaria viridis (Setaria), a model C4 panicoid grass, and make targeted comparisons between the HSF families of Setaria and maize. Examples of both conserved and variable expression responses to a heat stress event were observed when comparing the two species. Novel and existing data on chromatin accessibility, histone modifications, and genome-wide DNA binding profiles were utilized to assess the chromatin of HSF family members with distinct responses to heat stress. We observed significant variability for both expression and chromatin state within syntenic and orthologous sets of HSFs between Setaria and maize, as well as between syntenic pairs of maize HSFs retained following its most recent genome duplication event. These observations collectively support a complex scenario of expansion and sub-functionalization within this transcription factor family that has significant untapped potential for better understanding the evolution of large gene families. Significance StatementA comparison of the Heat Shock Factor transcription factors in maize and Setaria reveals examples of consistent and variable expression responses to heat stress and provides insights into the role of chromatin in predicting expression responses.

plant biology↗

bioRxiv2022

Structural variants in the barley gene pool: precision and sensitivity to detect them using short-read sequencing and their association with gene expression and phenotypic variation

In human genetics, several studies have shown that phenotypic variation is more likely to be caused by structural variants (SV) than by single nucleotide variants (SNV). However, accurate while cost-efficient discovery of SV in complex genomes remains challenging. The objectives of our study were to (i) facilitate SV discovery studies by benchmarking SV callers and their combinations with respect to their sensitivity and precision to detect SV in the barley genome, (ii) characterize the occurrence and distribution of SV clusters in the genomes of 23 barley inbreds that are the parents of a unique resource for mapping quantitative traits, the double round robin population, (iii) quantify the association of SV clusters with transcript abundance, and (iv) evaluate the use of SV clusters for the prediction of phenotypic traits. In our computer simulations based on a sequencing coverage of 25x, a sensitivity >70% and precision >95% was observed for all combinations of SV types and SV length categories if the best combination of SV callers was used. We observed a significant (P < 0.05) association of gene-associated SV clusters with global gene-specific gene expression. Furthermore, about 9% of all SV clusters that were within 5kb of a gene were significantly (P < 0.05) associated with the gene expression of the corresponding gene. The prediction ability of SV clusters was higher compared to that of single nucleotide polymorphisms from an array across the seven studied phenotypic traits. These findings suggest the usefulness of exploiting SV information when fine mapping and cloning the causal genes underlying quantitative traits as well as the high potential of using SV clusters for the prediction of phenotypes in diverse germplasm sets.

genomics↗

bioRxiv2020

Arabidopsis ACINUS is O-glycosylated and regulates transcription and alternative splicing of regulators of reproductive transitions

O-GlcNAc modification plays important roles in metabolic regulation of cellular status. Two homologs of O-GlcNAc transferase, SECRET AGENT (SEC) and SPINDLY (SPY), which have O-GlcNAc and O-fucosyl transferase activities, respectively, are essential in Arabidopsis but have largely unknown cellular targets. Here we show that AtACINUS is O-GlcNAcylated and O-fucosylated and mediates regulation of transcription, alternative splicing (AS), and developmental transitions. Knocking-out both AtACINUS and its distant paralog AtPININ causes severe growth defects including dwarfism, delayed seed germination and flowering, and abscisic acid (ABA) hypersensitivity. Transcriptomic and protein-DNA/RNA interaction analyses demonstrate that AtACINUS represses transcription of the flowering repressor FLC and mediates AS of ABH1 and HAB1, two negative regulators of ABA signaling. Proteomic analyses show AtACINUSs O-GlcNAcylation, O-fucosylation, and association with splicing factors, chromatin remodelers, and transcriptional regulators. Some AtACINUS/AtPININ-dependent AS events are altered in the sec and spy mutants, demonstrating a function of O-glycosylation in regulating alternative RNA splicing.

molecular biology↗

bioRxiv2020

Phloem loading via the abaxial bundle sheath cells in maize leaves

Leaves are asymmetric, with differential functionalization of abaxial and adaxial tissues. The bundle sheath (BS) surrounding the vasculature of the C3 crop barley is dorsoventrally differentiated into three domains: adaxial structural, lateral S-type, and abaxial L-type. S-type cells seem to transfer assimilates towards the phloem. Here we used single-cell RNA sequencing to investigate BS differentiation in C4 maize. Abaxial BS (abBS) cells of rank-2 intermediate veins specifically expressed three SWEET sucrose uniporters (SWEET13a, b, and c) and UmamiT amino acid efflux transporters. SWEET13a, b, c were also identified in the phloem parenchyma (PP). Thus maize acquired a unique mechanism for phloem loading in which abBS cells provide the main pathway for apoplasmic sucrose transfer towards the phloem. This pathway predominates in veins responsible for phloem loading (rank-2 intermediate), while rank-1 intermediate and major veins export sucrose from the phloem parenchyma (PP) adjacent to the sieve element companion cell (SE/CC) complex, as in Arabidopsis. We surmise that abBS identity is subject to dorsoventral patterning and has components of PP identity. These observations provide first insights into the unique transport-specific properties of abBS cells and support for a modification to the canonical phloem loading pathway of maize, which may be generalizable to other C4 monocots.

plant biology↗

bioRxiv2017

Impaired phloem loading in genome-edited triple knock-out mutants of SWEET13 sucrose transporters

Crop yield depends on efficient allocation of sucrose from leaves to seeds. In Arabidopsis, phloem loading is mediated by a combination of SWEET sucrose effluxers and subsequent uptake by SUT1/SUC2 sucrose/H+ symporters. ZmSUT1 is essential for carbon allocation in maize, but the relative contribution to apoplasmic phloem loading and retrieval of sucrose leaking from the translocation path is not known. We therefor tested whether SWEETs are important for phloem loading in maize.\n\nHere we identified three leaf-expressed SWEET sucrose transporters as key components of apoplasmic phloem loading in Zea mays L. Notably, ZmSWEET13 paralogs (a, b, c) are among the highest expressed genes in the leaf vasculature. Genome-edited triple knock-out mutants are severely stunted. Photosynthesis of mutants was impaired and leaves accumulated starch and soluble sugars. RNA-seq revealed profound transcriptional deregulation of genes associated with the photosynthetic apparatus and carbohydrate metabolism. GWAS analyses may indicate that variability in ZmSWEET13s is correlated with agronomical traits, specifically flowering time and leaf angle.\n\nThis work provides support for cooperation of three ZmSWEET13s with ZmSUT1 in phloem loading in Zea mays L. Our study highlights these three ZmSWEET13 sucrose transporters as possible candidates for the engineering of crop yield.\n\nOne Sentence Summary: Three SWEET sucrose transporter paralogs are necessary for phloem loading in maize.

plant biology↗