Biology subjects

Eekhout, T.

Publications and source records attributed to Eekhout, T..

14 recordsLinked to original sources

bioRxiv2026

A Myb-dominated gene regulatory network universally controls sexual cell fate transitions in diatoms

Diatoms are the foundation of aquatic food webs and contribute about 40% of the total marine primary productivity. Yet, the regulation of their complex size-dependent life cycles remains obscure. Here, we leveraged single-cell transcriptomics and transgenic reporter lines to uncover the molecular mechanisms behind partner recognition, nuclear fusion, and the remarkable 15-fold size expansion of auxospores. Gene regulatory network inference revealed that the irreversible commitment to differentiate into gametes is controlled by Myb transcription factors, whose specific activity in the global ocean underscores their significance for ploidy transitions across diatom clades. These findings reinforce microalgae as powerful models to study cell fate transitions and provide a mechanistic framework for the life cycle dynamics that underpin the functioning of aquatic systems worldwide. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=67 SRC="FIGDIR/small/714157v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@162f523org.highwire.dtl.DTLVardef@1cbe836org.highwire.dtl.DTLVardef@1fa6c9eorg.highwire.dtl.DTLVardef@1f12217_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2026

Decoding stage-specific symbiotic programs in the Rhizophagus irregularis-tomato interaction using single-nucleus transcriptomics

Arbuscular mycorrhizal fungi (AMF) establish a dynamic and asynchronous symbiosis with a wide range of land plants, involving distinct stages of root colonization and associated cellular responses that co-occur within the same root. Whilst decades of research have significantly advanced our understanding of the plants symbiotic gene repertoire, this spatial and temporal complexity has hindered a detailed dissection of the molecular mechanisms underlying fungal accommodation. Here, we present the first single-nucleus RNA-sequencing (snRNA-seq) dataset of Solanum lycopersicum roots colonized by Rhizophagus irregularis. Unsupervised subclustering of an AM-specific cell population resolves AM-responsive root epidermal cells as well as a developmental gradient of cortical cells across distinct stages of arbuscule formation, unveiling stage-specific transcriptional signatures during AMF colonization. Moreover, using Motif-Informed Network Inference based on single-cell EXpression data (MINI-EX), we put forward candidate transcription factors orchestrating these stage-specific transcriptional programs. Together, our data support novel hypotheses on how diverse plant developmental and physiological processes - including localized cell cycle reactivation and the integration of multiple nutritional cues - are coordinated to facilitate the establishment of a functional symbiosis. As such, this high-resolution dataset serves as a valuable resource for candidate gene prioritization and future reverse genetic studies.

plant biology↗

bioRxiv2025

Conserved transcriptional reprogramming in nematode infected root cells

Plant-parasitic nematodes are responsible for important annual losses in crop productivity worldwide1,2. Although the formation of feeding organs within the roots is essential for successful sedentary parasitism3, the molecular mechanisms underlying their development are poorly understood. This is partly because these organs originate from a limited number of root cells4-7, making difficult to capture the transcriptional reprogramming that occurs during the early stages of the infection. Here, we first developed a comparative host-pathogen framework to study the nematode infection process in Arabidopsis and rice. Using a cross-species single-cell transcriptomics approach, we identified a unique molecular signature in infected root cells and show that the cellular reprogramming during these early stages is highly conserved across both host-pathogen interactions. This transcriptional cell reprogramming is associated with stemness acquisition related to de novo organogenesis process. By cell-type specific gene regulatory network analysis, we identified AtATHB2/OsHOX28 as an evolutionary conserved and key regulator of the nematode infection process. Loss-of-function of this regulator in both species results in nematode resistance without affecting root growth. This discovery opens up new avenues for the development of sustainable nematode control strategies that could be translated across crop species.

plant biology↗

bioRxiv2025

Gene regulatory network analysis of somatic embryogenesis identifies morphogenic genes that increase maize transformation frequency

Somatic embryogenesis allows a somatic plant cell to develop into an embryo, and potentially into a fertile plant. Transcription factors such as BABY BOOM can induce somatic embryogenesis when ectopically expressed and are widely used for aiding regeneration in tissue culture for transformation and gene editing of crops. Nevertheless, regeneration remains a bottleneck and alternative morphogenic genes are highly desired. Here, we co-expressed BABY BOOM and WUSCHEL2 in zygotic maize (Zea mays L.) embryos and studied gene regulatory networks in induced somatic embryos at the single-cell level. By inferring cell-type-specific regulons, we prioritized candidate regulators and confirmed functionality of four transcription factors, bHLH48, EREB152, GRF4, and HB77, for enhanced maize transformation frequency, leading to fertile, transgenic plants. Interestingly, the basic helix-loop-helix and homeodomain-leucine zipper families had previously not been associated with induced somatic embryogenesis. Our work will contribute to more efficient transformation, much needed to deliver on the promise of gene editing for agriculture.

plant biology↗

bioRxiv2024

Dual and spatially resolved drought responses in the Arabidopsis leaf mesophyll revealed by single-cell transcriptomics

Drought stress imposes severe challenges on agriculture by impacting crop performance. Understanding drought responses in plants at a cellular level is a crucial first step towards engineering improved drought resilience. However, the molecular responses to drought are complex as they depend on multiple factors including the severity of drought, the profiled organ, its developmental stage or even the cell types therein. Thus, deciphering the transcriptional responses to drought is specially challenging. Here, we investigated tissue-specific responses to mild drought in young Arabidopsis thaliana (Arabidopsis) leaves using single-cell RNA sequencing (scRNA-seq). To preserve transcriptional integrity during cell isolation, we inhibited RNA synthesis using the transcription inhibitor actinomycin D, demonstrating the benefits of transcriptome fixation for studying mild stress responses at single-cell level. We present a curated and validated single-cell atlas comprising 50,797 high-quality cells from almost all known cell types present in the leaf. We show that the mesophyll contains two spatially separated cell populations with distinct responses to drought: one enriched in canonical abscisic acid-related drought-responsive genes, and another one depicting iron starvation responses. Our study thus reveals a dual adaptive mechanism of the leaf mesophyll in response to mild drought and provides a valuable resource for future research on stress responses.

plant biology↗

bioRxiv2024

The dynamic and diverse nature of parenchyma cells in the Arabidopsis root during secondary growth

During the process of secondary growth, the vascular cambium produces the conductive xylem and phloem cells, while the phellogen (cork cambium) deposit phellem (cork) as the outermost protective barrier. Although most of the secondary tissues is made up by parenchyma cells which are also produced by both cambia, their diversity and function are poorly understood. Here we combined single-cell RNA sequencing analysis with lineage tracing to recreate developmental trajectories of the cell types in the Arabidopsis root undergoing secondary growth. By analysing 93 reporter lines, we were able to identify 20 different cell types or cell states, many of which have not been described before. We additionally observed distinct transcriptome signatures of parenchyma cells depending on their maturation state and proximity to the conductive cell types. Our data shows that both xylem and phloem parenchyma tissues are required for normal formation of conductive tissue cell types. Furthermore, we showed that mature phloem parenchyma gradually obtains periderm identity, and this transition can be accelerated by jasmonate or wounding. Thus, our study reveals the remarkable dynamic and diverse nature of parenchyma cells during secondary growth.

plant biology↗

bioRxiv2024

Scywalker: scalable end-to-end data analysis workflow for nanopore single-cell transcriptome sequencing

We introduce scywalker, an innovative and scalable package developed to comprehensively analyze long-read nanopore sequencing data of full-length single-cell or single-nuclei cDNA. Existing nanopore single-cell data analysis tools showed severe limitations in handling current data sizes. We developed novel scalable methods for cell barcode demultiplexing and single-cell isoform calling and quantification and incorporated these in an easily deployable package. Scywalker streamlines the entire analysis process, from sequenced fragments in FASTQ format to demultiplexed pseudobulk isoform counts, into a single command suitable for execution on either server or cluster. Scywalker includes data quality control, cell type identification, and an interactive report. Assessment of datasets from the human brain, Arabidopsis leaves, and previously benchmarked data from mixed cell lines, demonstrate excellent correlation with short-read analyses at both the cell-barcoding and gene quantification levels. At the isoform level, we show that scywalker facilitates the direct identification of cell-type-specific expression of novel isoforms.

bioinformatics↗

bioRxiv2023

Distinctive and complementary roles of E2F transcription factors during plant replication stress response

Survival of living organisms is fully dependent on their maintenance of genome integrity, being permanently threatened by replication stress in proliferating cells. Although the plant DNA damage response (DDR) regulator SOG1 has been demonstrated to cope with replicative defects, accumulating evidence points to other pathways functioning independently of SOG1. Here, we have studied the role of the Arabidopsis E2FA and EF2B transcription factors, two well-characterized regulators of DNA replication, in the response to replication stress. Through a combination of reverse genetics and chromatin-immunoprecipitation approaches, we show that E2FA and E2FB share many target genes with SOG1, providing evidence for their involvement in the DDR. Analysis of double and triple mutant combinations revealed that E2FB, rather than E2FA, plays the most prominent role in sustaining growth in the presence of replicative defects, either operating antagonistically or synergistically with SOG1. Reversely, SOG1 aids in overcoming the replication defects of E2FA/E2FB-deficient plants. Our data reveal a complex transcriptional network controlling the replication stress response, in which both E2Fs and SOG1 act as key regulatory factors. ONE-SENTENCE SUMMARYThe Arabidopsis E2FA and EF2B transcription factors differently contribute to the plants response to DNA replication defects in a cooperative way with the DNA damage response regulator SOG1.

genetics↗

bioRxiv2022

Cell type-specific attenuation of brassinosteroid signalling drives stomatal asymmetric cell division

In Arabidopsis thaliana, the negative brassinosteroid (BR) signalling regulator, BR INSENSITIVE2 (BIN2) promotes and restricts stomatal asymmetric cell division (ACD) depending on its subcellular localization, which is regulated by the stomatal lineage-specific scaffolding protein POLAR. BRs inactivate BIN2, but how they govern stomatal development remains unclear. Mapping the single-cell transcriptome of stomatal lineages with either exogenous BRs or the specific BIN2 inhibitor revealed that the two modes of BR activation triggered spatiotemporally distinct transcriptional responses. We established that when in a complex with POLAR and its closest homolog POLAR-LIKE1, BIN2 is insulated from BR-mediated inactivation, nevertheless, it remains sensitive to the inhibitor. Subsequently, BR signalling is attenuated in ACD precursors, whereas it remains active in epidermal cells that would undergo differentiation. Our study demonstrates how scaffold proteins contribute to cellular signal specificity of hormonal responses in plants.

plant biology↗

bioRxiv2021

The APC/CCCS52A2 E3-Ligase Complex targeted PKN1 Protein is a Plant Lineage-Specific Driver of Cell Division

The anaphase-promoting complex/cyclosome (APC/C) marks key cell cycle proteins for proteasomal breakdown, thereby ensuring unidirectional progression through the cell cycle. Its target recognition is temporally regulated by activating subunits, one of which is called CELL CYCLE SWITCH 52 A2 (CCS52A2). We sought to expand the knowledge of identified APC/C targets by using the severe growth phenotypes of CCS52A2-deficient Arabidopsis thaliana plants as a readout in a suppressor mutagenesis screen, resulting in the identification of the previously undescribed gene called PIKMIN1 (PKN1). PKN1 deficiency rescues the disorganized root stem cell phenotype of the ccs52a2-1 mutant, whereas an excess of PKN1 inhibits growth of ccs52a2-1 plants, indicating the importance of PKN1 abundance for proper development. Accordingly, the lack of PKN1 in a wild-type background negatively impacts cell division, while its ectopic expression promotes proliferation. PKN1 shows a cell cycle phase-dependent accumulation pattern, localizing to microtubular structures, including the preprophase band, the mitotic spindle, and phragmoplast. PKN1 is conserved throughout the plant kingdom, with its function in cell division being evolutionary conserved in the liverwort Marchantia polymorpha. Our data thus demonstrate that PKN1 represents a novel, plant-specific gene with a rate-limiting role in cell division, which is proteolytically controlled by the CCS52A2-activated APC/C. One-Sentence SummaryPKN1 is a conserved plant-specific protein that is rate-limiting for cell division, likely through its interaction with microtubuli, and is proteolytically controlled by APC/CCCS52A2.

plant biology↗

bioRxiv2021

A morpho-transcriptomic map of brassinosteroid action in the Arabidopsis root

The effects of brassinosteroid signaling on shoot and root development have been characterized in great detail but did not identify a simple consistent positive or negative impact on a basic cellular parameter that would comprehensively explain the phenotype of brassinosteroid-related mutants. Here we combined digital 3D single-cell shape analysis and single-cell mRNA sequencing to characterize root meristems and mature root segments of brassinosteroid-blind mutants and wildtype. These data demonstrate that brassinosteroid signaling neither affects cell volume nor cell proliferation capacity. Instead, brassinosteroid signaling is essential for the precise orientation of cell division planes and the extent and timing of anisotropic cell expansion. Moreover, we found that the cell-aligning effects of brassinosteroid signaling can propagate to normalize the anatomy of both adjacent and distant brassinosteroid-blind cells through non-cell-autonomous functions, which are sufficient to restore overall root growth vigor. Finally, single-cell transcriptome data discern directly brassinosteroid-responsive genes from genes that can react to non-cell-autonomous brassinosteroid-dependent signals and highlight arabinogalactans as sentinels of brassinosteroid-dependent anisotropic cell expansion.

plant biology↗

bioRxiv2021

Loss of FAS1 function reveals rescue of an aberrant intra-S-phase checkpoint by the G2/M checkpoint regulator SOG1

The WEE1 and ATR kinases represent important regulators of the plant intra-S-phase checkpoint, as evidenced by the hypersensitivity of WEE1KO and ATRKO roots to replication inhibitory drugs. Here, we report on the identification of a defective allele of the FASCIATA1 (FAS1) subunit of the chromatin assembly factor 1 (CAF-1) complex as a suppressor of WEE1- or ATR-deficient plants. We demonstrate that lack of FAS1 activity results in the activation of an ATM- and SOG1-mediated G2/M-arrest that makes the ATR and WEE1 checkpoint regulators redundant. This ATM activation accounts for telomere erosion and loss of ribosomal DNA described for the fas1 plants. Knocking out SOG1 in the fas1 wee1 background restores replication stress sensitivity, demonstrating that SOG1 plays a prominent role as secondary checkpoint regulator in plants that fail to activate the intra-S-phase checkpoint. One-Sentence SummaryLack of the chromatin assembly factor-1 subunit FAS1 results in a DNA damage response that overrules the need for replication checkpoint activators.

plant biology↗

bioRxiv2020

Arabidopsis Casein Kinase 2 triggers Stem Cell Exhaustion under Al Toxicity and Phosphate Deficiency Through activation of the DNA Damage Response pathway

Aluminum (Al) toxicity and inorganic phosphate (Pi) limitation are widespread chronic abiotic and mutually enhancing stresses that profoundly affect crop yield. Both stresses cause a strong inhibition of root growth, resulting from a progressive exhaustion of the stem cell niche. Here, we report on a casein kinase 2 (CK2) inhibitor identified by its capability to maintain a functional root stem cell niche under Al toxic conditions. CK2 operates through phosphorylation of the cell cycle checkpoint activator SUPPRESSOR OF GAMMA RADIATION1 (SOG1), priming its activity under DNA-damaging conditions. In addition to yielding Al tolerance, CK2 and SOG1 inactivation prevents meristem exhaustion under Pi starvation, revealing the existence of a low Pi-induced cell cycle checkpoint that depends on the DNA damage activator ATAXIA-TELANGIECTASIA MUTATED. Overall, our data reveal an important physiological role for the plant DNA damage response pathway under agriculturally limiting growth conditions, opening new avenues to cope with Pi limitation. ONE-SENTENCE SUMMARYCasein kinase 2 and DNA damage response regulators play a pivotal role in the control of Arabidopsis root growth in response to Al toxicity and phosphate limitation.

plant biology↗

bioRxiv2020

CYCA3;4 Is a Post-Prophase Target of the APC/CCCS52A2 E3 Ligase Controlling Formative Cell Divisions in Arabidopsis

The Anaphase Promoting Complex/Cyclosome (APC/C) controls unidirectional progression through the cell cycle by marking key cell cycle proteins for proteasomal turnover. Its activity is temporally regulated by the docking of different activating subunits, known in plants as CDC20 and CCS52. Despite the importance of the APC/C during cell proliferation, the number of identified targets in the plant cell cycle is limited. Here, we used the growth and meristem phenotypes of Arabidopsis CCS52A2-deficient plants in a suppressor mutagenesis screen to identify APC/CCCS52A2 substrates or regulators, resulting in the identification of a mutant cyclin CYCA3;4 allele. CYCA3;4 deficiency partially rescues the early ccs52a2-1 phenotypes, whereas increased CYCA3;4 levels enhances them. Furthermore, whereas CYCA3;4 proteins are promptly broken down after prophase in wild-type plants, they remain present in later stages of mitosis in ccs52a2-1 mutant plants, marking them as APC/CCCS52A2 substrates. Strikingly, CYCA3;4 overexpression results in aberrant root meristem and stomatal divisions, mimicking phenotypes of plants with reduced RBR1 activity. Correspondingly, RBR1 hyperphosphorylation was observed in CYCA3;4-overproducing plants. Our data thus demonstrate that an inability to timely destroy CYCA3;4 attributes to disorganized formative divisions, likely in part caused by the inactivation of RBR1. ONE-SENTENCE SUMMARYTimely post-prophase breakdown of the Arabidopsis cyclin CYCA3;4 by the Anaphase Promoting Complex/Cyclosome is essential for meristem organization and development.

plant biology↗