Biology subjects

Benhamed, M.

Publications and source records attributed to Benhamed, M..

At least 19 recordsLinked to original sources

bioRxiv2026

Brassinosteroid receptors reveal conserved features of exon-embedded gene regulation

Precise quantitative and spatial control of plant gene expression is typically attributed to cis-regulatory elements in promoters and other non-coding sequences. The extent to which exon-encoded regulatory information shapes plant gene expression and how remains unclear. Here we used BRASSINOSTEROID INSENSITIVE 1 (BRI1) as a model locus and found that it contains an exonic enhancer (EE), which is essential to establish the spatial BRI1 expression pattern. Its disruption restricts BRI1 expression and impairs BRI1-dependent root growth, linking exon-encoded regulatory information and development. The BRI1 EE acts in concert with its promoter, and related receptor kinase genes likewise harbor regulatory information within their coding sequences. Genome-wide surveys show that candidate EEs are associated with highly connected chromatin interaction networks and transcriptionally active loci. Moreover, species comparisons suggest that common features of exon-encoded regulatory information extend across diverse plant lineages. Thus, exon-encoded regulatory information is a vital component of plant gene regulation.

plant biology↗

bioRxiv2026

A molecular framework of chromatin extrusion in plants

Three-dimensional genome organization is a fundamental regulator of gene expression, yet the mechanisms shaping higher-order chromatin folding in plants remain poorly defined. Here, we identify the Arabidopsis cohesin regulator PDS5A as a central modulator of chromatin loop extrusion and topologically associating domain (TAD)-like architecture. Using Hi-C and Micro-C, we show that loss of PDS5A strongly enhances TAD-like domains and nucleosome-scale stripes and loops across chromosome arms, while leaving large-scale compartmentalization largely intact and revealing conserved TAD-like patterns across tissues and ploidy levels. Genetic analyses indicate that Arabidopsis TAD-like domain formation relies on the plant-specific kleisin SYN4 and is antagonized by WAPL1/2, with PDS5A acting as the dominant cohesin-unloading factor that limits loop extension. Biochemical experiments demonstrate that PDS5A physically associates with cohesin and that its function in suppressing TAD-like domains is independent of direct Tudor domain-mediated binding to H3K4me1-marked chromatin. Micro-C further resolves that chromatin loop and stripe anchors formed in pds5a are highly enriched at accessible, strongly transcribed promoters and are marked by the plant site II motif TGGGCC/T, implicating site II-binding transcription factors as plant-specific boundary elements analogous to CTCF in animals. Finally, mutational disruption of distal chromatin anchors shows that newly formed chromatin contacts in pds5a can act as cis-regulatory modules that influence target gene expression. Together, these findings identify a PDS5A-regulated chromatin extrusion module that shapes plant 3D genome architecture and uncover a promoter- and motif-based logic for loop anchoring and transcriptional control in plants.

genomics↗

bioRxiv2025

Transcriptional control of meiotic recombination by the DREAM complex

Meiotic chromosome segregation and recombination, essential for sexual reproduction and evolution, require a specialized gene set. However, how meiotic gene expression is controlled remains largely unclear in multicellular eukaryotes. Here, we identify the DREAM complex as a transcriptional regulator of meiotic recombination in Arabidopsis. We show that the DREAM complex localizes to meiotic chromosomes, and that reduced DREAM function disrupts recombination and causes sterility. Meiocyte-specific transcriptomics combined with genome-wide binding analyses revealed that recombination genes, including the crossover-promoting helicase MER3, are direct DREAM targets. Notably, bypassing DREAM regulation of MER3 partially restores recombination in DREAM-deficient plants. Our findings establish the DREAM complex as a major meiotic regulator. Given that reduced DREAM function causes reproductive defects in species such as Drosophila and mice, this role may be conserved across multicellular eukaryotes.

cell biology↗

bioRxiv2025

Replication-independent eviction of H2B.8 reveals chromatin reprogramming during seed imbibition

The transition from seed to seedling involves major changes in nuclear organization and gene expression, yet the extent to which this developmental transition requires chromatin reprogramming remains largely unexplored. Here, we report that Arabidopsis dry seed embryos accumulate the histone variant H2B.8, which contributes to higher-order chromatin organization by forming spatial clusters that structure the 3D nuclear space. H2B.8 forms heterotypic nucleosomes at euchromatic transposons and lowly expressed genes and, during imbibition, modulates the transcriptional activation of a subset of these genes. Imbibition triggers a decrease in H2B.8 transcripts and the eviction of H2B.8 proteins in a process that operates independently of DNA replication but requires protein translation and de novo transcription. Histone eviction is not limited to H2B.8 as imbibition also induces the turnover of the H3.3 histone variant, thereby initiating a broad, replication-independent chromatin reprogramming process. These findings highlight a fundamental mechanism of epigenetic regulation during early plant development.

molecular biology↗

bioRxiv2025

Role of the tomato MARS1/ROUGH gene encoding a LYSINE-SPECIFIC HISTONE DEMETHYLASE 1 in adventitious root and fruit skin formation

In contrast to animals, plants have a high regenerative capacity, and they can form new organs and even complete individuals from a few cells present in adult tissues, either in response to injury or to the alteration of their environment. In this study, we describe the isolation and characterization of the more adventitious roots1-1 (mars1-1) mutant, which exhibits enhanced regenerative potential upon wounding in tomato hypocotyl explants. Additionally, the mars1-1 fruits exhibited a rough surface due to the ectopic proliferation of subepidermal cells, which formed callus-like structures on the cuticle. The MARS1/ROUGH gene encodes a conserved lysine-specific histone demethylase, SlLSD1, which regulates a variety of processes in metazoans, including cell proliferation, stem cell pluripotency, and embryogenesis. Two CRISPR/Cas9 null alleles, mars1-2 and mars1-3, were generated and their pleiotropic phenotype was characterized. We found elevated levels of H3K4me1 in mars1/rough seedlings, which suggests that SlLSD1 is required for the demethylation of this histone mark. To ascertain the impact of altered epigenetic marks in the mars1/rough mutants on gene expression regulation, we conducted a transcriptome analysis using a variety of RNA-Seq studies on tomato hypocotyls. By employing specific bioinformatic workflows and leveraging on the resolution of directional RNA-Seq data, we have identified over several dozen distinct genomic regions that exhibit de novo expression in the mars1/rough mutants. One such region includes a novel B-type cyclin gene, which is upregulated in the mars1/rough mutants and may account for the observed phenotypes. Our findings indicate that SlLSD1 plays a role in the establishment and maintenance of silencing in specific genomic regions that are essential for tissue-specific reprogramming.

plant biology↗

bioRxiv2024

A plant-specific clade of serine/arginine-rich proteins regulates RNA splicing homeostasis and thermotolerance in tomato

High temperatures cause heat stress (HS), which has negative effects on plant growth and development and affects many cellular processes including pre-mRNA splicing. In tomato plants the splicing profile of many of genes is altered under HS, including that of HSFA2, a central transcriptional regulator of thermotolerance. To identify the core splicing regulators of HS-sensitive alternative splicing, we used HSFA2 as bait and identified two plant-specific members of the serine/arginine-rich family of splicing factors, namely RS2Z35 and RS2Z36, that inhibit HSFA2 intron splicing. Single and double CRISPR mutants of these proteins show dysregulated splicing of many genes and exhibit lower basal and acquired thermotolerance. Individual-nucleotide resolution UV cross-linking and immunoprecipitation (iCLIP) of tomato leaves revealed that the majority of HS-sensitive alternatively spliced RNAs are bound by RS2Z35 and RS2Z36 and this interaction occurs at purine-rich RNA motifs. Phenotypic and transcriptome analyses revealed that RS2Z35 and RS2Z36 are important players in the stress response and thermotolerance in plants that mitigate the negative effects of HS on RNA splicing homeostasis.

plant biology↗

bioRxiv2023

Genome-wide evidence of the role of Paf1C in transcription elongation and histone H2B monoubiquitination in Arabidopsis

The evolutionarily conserved Paf1 complex (Paf1C) participates in transcription, and research in animals and fungi suggests that it facilitates RNAPII progression through chromatin remodeling. To obtain evidence that Paf1C acts in transcription elongation in Arabidopsis, we examined the genomic distribution of the ELF7 and VIP3 subunits of Paf1C. The occupancy of both subunits was confined to thousands of gene bodies and positively correlated with RNAPII occupancy and the level of gene expression, supporting a role as a transcription elongation factor. We found that monoubiquitinated histone H2B, which marks most transcribed genes, was strongly reduced genome-wide in elf7 seedlings. Genome-wide profiling of RNAPII revealed that in elf7 mutants, RNAPII occupancy was reduced throughout the gene body and at the transcription end site of Paf1C-targeted genes, suggesting a direct role for the complex in transcription elongation. Overall, our observations suggest that there is a direct functional link between Paf1C activity, mono-ubiquitination of histone H2B, and the transition of RNPII to productive elongation. However, for several genes, Paf1C may also act independently of H2Bub deposition or occupy these genes more stably than H2Bub marking, possibly reflecting the dynamic nature of Paf1C association and H2Bub turnover during transcription.

plant biology↗

bioRxiv2023

RTEL1 is required for silencing and epigenome stability

Transcriptional silencing is an essential mechanism for controlling the expression of genes, transgenes and heterochromatic repeats through specific epigenetic marks on chromatin that are maintained during DNA replication. In Arabidopsis, silenced transgenes and heterochromatic sequences are typically associated with high levels of DNA methylation, while silenced genes are enriched in H3K27me3. Reactivation of these loci is often correlated with decreased levels of these repressive epigenetic marks. Here, we report that the DNA helicase REGULATOR OF TELOMERE ELONGATION 1 (RTEL1) is required for transcriptional silencing. RTEL1 deficiency causes upregulation of many genes enriched in H3K27me3 accompanied by a moderate decrease in this mark, but no loss of DNA methylation at reactivated heterochromatic loci. Instead, heterochromatin exhibits DNA hypermethylation and increased H3K27me3 in rtel1. We further find that loss of RTEL1 suppresses the release of heterochromatin silencing caused by the absence of the MOM1 silencing factor. RTEL1 is conserved among eukaryotes and plays a key role in resolving DNA secondary structures during DNA replication. Inducing such aberrant DNA structures using DNA cross-linking agents also results in a loss of transcriptional silencing. These findings uncover unappreciated roles for RTEL1 in transcriptional silencing and in stabilizing DNA methylation and H3K27me3 patterns.

genomics↗

bioRxiv2023

The CRL plastid outer envelope protein supports TOC75-V / OEP80 complex formation in Arabidopsis

Embedded {beta}-barrel proteins in the outer envelope membrane mediate most cellular traffic between the cytoplasm and the plastids. Although TOC75-V/OEP80 has been implicated in the insertion and assembly of {beta}-barrel proteins in the outer envelope membrane of Arabidopsis thaliana, relatively little is known about this process. CRUMPLED LEAF (CRL) encodes a protein localizing in the outer envelope membrane, and its loss of function results in pleiotropic defects, including altered plant morphogenesis, growth retardation, suppression of plastid division, and spontaneous light intensity-dependent localized cell death. A suppressor screen conducted on mutagenized crl mutants with ethyl methanesulfonate revealed that a missense mutation in OEP80 suppresses crls pleiotropic defects. Furthermore, we found that the complex formation of OEP80 was compromised in crl. Furthermore, we demonstrated that CRL interacts with OEP80 in vivo and that a portion of CRL is present in protein complexes with the same molecular weight as the OEP80-associated complex. Our results suggest that CRL interacts with OEP80 to regulate its complex formation. CRL has been shown to be involved in plastid protein import; therefore, pleiotropic defects in crl are likely due to the combined effects of decreased plastid protein import and altered membrane integration of {beta}-barrel proteins in the outer envelope membrane. This study sheds light on the mechanisms that allow the integration of {beta}-barrel proteins into the outer envelope membrane of plastids and the significance of this finding for plant cellular processes.

plant biology↗

bioRxiv2023

Long noncoding RNA-mediated epigenetic regulation of auxin-related genes controlling shade avoidance syndrome in Arabidopsis thaliana

The long noncoding RNA (lncRNA) AUXIN-REGULATED PROMOTER LOOP (APOLO) recognizes a subset of target loci across the Arabidopsis thaliana genome by forming RNA-DNA hybrids (R-loop) and modulating local three-dimensional chromatin conformation. Here we show that APOLO is involved in regulating the shade avoidance syndrome (SAS) by dynamically modulating the expression of key factors. In response to far-red (FR) light, the expression of APOLO anticorrelates with its target BRANCHED1 (BRC1), a master regulator of shoot branching in Arabidopsis thaliana. APOLO deregulation results in BRC1 transcriptional repression and an increase in the number of branches. APOLO transcriptional accumulation fine-tunes the formation of a repressive chromatin loop encompassing the BRC1 promoter, which normally occurs only in leaves as well as in a late response to FR treatment in axillary buds. In addition, our data reveal that APOLO participates in leaf hyponasty, in agreement with its previously reported role in the control of auxin homeostasis through direct modulation of YUCCA2 (auxin synthesis), PID and WAG2 (auxin efflux). We found that direct application of APOLO RNA to leaves results in a rapid increase in auxin accumulation that is associated with changes in the response of the plants to FR light. Collectively, our data support the view that lncRNAs coordinate the shade avoidance syndrome in Arabidopsis thaliana and shed light on the potential of lncRNAs as bioactive exogenous molecules. Deploying exogenous RNAs that modulate plant-environment interactions are important new tools for sustainable agriculture.

plant biology↗

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

The canonical E2Fs together with RETINOBLASTOMA-RELATED are required to establish quiescence during plant development

Maintaining stable and transient quiescence in differentiated and stem cells, respectively, requires repression of the cell cycle. The plant RETINOBLASTOMA-RELATED (RBR) has been implicated in stem cell maintenance, presumably by forming repressor complexes with E2F transcription factors. Surprisingly we find that mutations in all three canonical E2Fs do not compromise the cell cycle, but similarly to RBR silencing, result in overproliferation. Contrary to the growth arrest upon RBR silencing, when exit from proliferation to differentiation is inhibited, the e2fabc mutant develops enlarged organs with supernumerary stem and differentiated cells as the quiescence is compromised. While E2F, RBR and the M-phase regulatory MYB3Rs are part of the DREAM repressor complexes, and recruited to overlapping groups of targets, they regulate distinct sets of genes. Only the loss of E2Fs but not the MYB3Rs interferes with quiescence, which might be due to the ability of E2Fs to control both G1-S and some key G2-M targets. We conclude that collectively the three canonical E2Fs in complex with RBR have central roles in establishing cellular quiescence during organ development, leading to enhanced plant growth.

plant biology↗

bioRxiv2022

Predictable and stable epimutations induced during clonal propagation with embryonic transcription factors

Although clonal propagation is frequently used in commercial plant breeding and plant biotechnology programs because it minimizes genetic variation, it is not uncommon to observe clonal plants with stable phenotypic changes, a phenomenon known as somaclonal variation. Several studies have shown that epigenetic modifications induced during regeneration are associated with this newly acquired phenotypic variation. However, the factors that determine the extent of somaclonal variation and the molecular changes associated with it remain poorly understood. To address this gap in our knowledge, we compared clonally propagated Arabidopsis thaliana plants derived from somatic embryogenesis using two different embryonic transcription factors-RWP-RK DOMAIN-CONTAINING 4 (RKD4) or LEAFY COTYLEDON2 (LEC2) and from two epigenetically distinct tissues. We found that both the epi(genetic) status of explant and the regeneration protocol employed play critical roles in shaping the molecular and phenotypic state of clonal plants. Phenotypic variation of regenerated plants can be largely explained by the inheritance of tissue-specific DNA methylation imprints, which are associated with specific transcriptional and metabolic changes in sexual progeny of clonal plants. Moreover, regenerants from roots were particularly affected by the inheritance of epigenetic imprints, which resulted in increased accumulation of salicylic acid in leaves and accelerated plant senescence. Collectively, our data reveal pathways for targeted manipulation of phenotypic variation in clonal plants.

plant biology↗

bioRxiv2021

A hierarchical transcriptional network activates specific CDK inhibitors that regulate G2 to control cell size and number in Arabidopsis

How cell size and number are determined during organ development remains a fundamental question in cell biology. Here, we identified a GRAS family transcription factor, called SCARECROW-LIKE28 (SCL28), with a critical role in determining cell size in Arabidopsis. SCL28 is part of a transcriptional regulatory network downstream of the central MYB3Rs that regulate G2 to M phase cell cycle transition. We show that SCL28 forms a dimer with the AP2-type transcription factor, AtSMOS1, which defines the specificity for promoter binding and directly activates transcription of a specific set of SIAMESE-RELATED (SMR) family genes, encoding plant-specific inhibitors of cyclin-dependent kinases and thus inhibiting cell cycle progression at G2 and promoting the onset of endoreplication. Through this dose-dependent regulation of SMR transcription, SCL28 quantitatively sets the balance between cell size and number without dramatically changing final organ size. We propose that this hierarchical transcriptional network constitutes a cell cycle regulatory mechanism that allows to adjust cell size and number to attain robust organ growth.

plant biology↗

bioRxiv2021

Arabidopsis Topless-related 1 mitigates physiological damage and growth penalties of induced immunity

Transcriptional corepressors of the Topless family are important regulators of plant hormone and immunity signaling. The lack of a genome-wide profile of their chromatin associations limits understanding of transcriptional regulation in plant immune responses. Chromatin immunoprecipitation with sequencing (ChIP-seq) was performed on GFP-tagged Topless-related 1 (TPR1) expressed in Arabidopsis thaliana lines with and without constitutive immunity dependent on Enhanced Disease Susceptibility 1 (EDS1). RNA-seq profiling of pathogen-infected tpl/tpr mutants and assessments of growth and physiological parameters were employed to determine TPL/TPR roles in transcriptional immunity and defense homeostasis. TPR1 bound to promoter regions of ~1,400 genes and ~10% of the detected binding required EDS1 immunity signaling. A tpr1 tpl tpr4 (t3) mutant displayed mildly enhanced defense-related transcriptional reprogramming upon bacterial infection but not increased bacterial resistance. Bacteria or pep1 phytocytokine-challenged t3 plants exhibited, respectively, photosystem II dysfunction and exacerbated root growth inhibition. Transgenic expression of TPR1 restored the t3 physiological defects. We propose that TPR1 and TPL-family proteins function in Arabidopsis to reduce detrimental effects associated with activated transcriptional immunity.

plant biology↗

bioRxiv2021

Sequence-unrelated long noncoding RNAs converged to modulate the activity of conserved epigenetic machineries across kingdoms

RNA-DNA hybrid (R-loop)-associated long noncoding RNAs (lncRNAs), including the Arabidopsis lncRNA AUXIN-REGULATED PROMOTER LOOP (APOLO), are emerging as important regulators of three-dimensional chromatin conformation and gene transcriptional activity. Here, we showed that in addition to the PRC1-component LIKE-HETEROCHROMATIN PROTEIN 1 (LHP1), APOLO interacts with the methylcytosine-binding protein VARIANT IN METHYLATION 1 (VIM1), a conserved homolog of the mammalian DNA methylation regulator UBIQUITIN-LIKE CONTAINING PHD AND RING FINGER DOMAINS 1 (UHRF1). The APOLO-VIM1-LHP1 complex directly regulates the transcription of the auxin biosynthesis gene YUCCA2 by dynamically determining DNA methylation and H3K27me3 deposition over its promoter during the plant thermomorphogenic response. Strikingly, we demonstrated that the lncRNA UHRF1 Protein Associated Transcript (UPAT), a direct interactor of UHRF1 in humans, can be recognized by VIM1 and LHP1 in plant cells, despite the lack of sequence homology between UPAT and APOLO. In addition, we showed that increased levels of APOLO or UPAT hamper VIM1 and LHP1 binding to YUCCA2 promoter. Collectively, our results uncover a new mechanism in which a plant lncRNA coordinates Polycomb action and DNA methylation, and reveal that evolutionary unrelated lncRNAs may exert similar functions across kingdoms.

plant biology↗

bioRxiv2021

The LEAFY floral regulator displays pioneer transcription factor properties

Pioneer transcription factors (TFs) are a special category of TFs with the capacity to bind to closed chromatin regions in which DNA is wrapped around histones and often highly methylated. Subsequently, they are able to modify the chromatin state to initiate gene expression. In plants, LEAFY (LFY) is a master floral regulator and has been suggested to act as a pioneer TF in Arabidopsis. Here, we demonstrate that LFY is able to bind both methylated and non-methylated DNA using a combination of in vitro genome-wide binding experiments and structural modeling. Comparisons between regions bound by LFY in vivo and chromatin accessibility data suggest that LFY binds a subset of regions occupied by nucleosomes. We confirm that LFY is able to bind nucleosomal DNA in vitro using reconstituted nucleosomes. Finally, we show that constitutive LFY expression in seedling tissues is sufficient to induce chromatin accessibility in the LFY direct target genes, APETALA1 and AGAMOUS. Taken together, our study suggests that LFY possesses key pioneer TF features that contribute to launch the floral gene expression program.

plant biology↗

bioRxiv2020

New insights into homoeologous copy number variations in the hexaploid wheat genome

Bread wheat is an allohexaploid species originating from two successive and recent rounds of hybridization between three diploid species that were very similar in terms of chromosome number, genome size, TE content, gene content and synteny. As a result, it has long been considered that most of the genes were in three pairs of homoeologous copies. However, these so-called triads represent only one half of wheat genes, while the remaining half belong to homoeologous groups with various number of copies across subgenomes. In this study, we examined and compared the distribution, conservation, function, expression and epigenetic profiles of triads with homoeologous groups having undergone a deletion (dyads) or a duplication (tetrads) in one subgenome. We show that dyads and tetrads are mostly located in distal regions and have lower expression level and breadth than triads. Moreover, they are enriched in functions related to adaptation and more associated with the repressive H3K27me3 modification. Altogether, these results suggest that triads mainly correspond to housekeeping genes and are part of the core genome, while dyads and tetrads belong to the Triticeae dispensable genome. In addition, by comparing the different categories of dyads and tetrads, we hypothesize that, unlike most of the allopolyploid species, subgenome dominance and biased fractionation are absent in hexaploid wheat. Differences observed between the three subgenomes are more likely related to two successive and ongoing waves of post-polyploid diploidization, that had impacted A and B more significantly than D, as a result of the evolutionary history of hexaploid wheat. Core ideasOnly one half of hexaploid wheat genes are in triads, i.e. in a 1:1:1 ratio across subgenomes Triads are likely part of the core genome; dyads and tetrads belong to the dispensable genome Subgenome dominance and biased fractionation are absent in hexaploid wheat Subgenome differences are related to two successive waves of post-polyploid diploidization

genomics↗