Chromatin dynamics and RNA metabolism are double-edged swords for the maintenance of plant genome integrity.

Maintenance of genome integrity is an essential process in all organisms. Mechanisms avoiding the formation of DNA lesions or mutations are well described in animals because of their relevance to human health and cancer. In plants, they are of growing interest because DNA damage accumulation is increasingly recognized as one of the consequences of stress. Although the cellular response to DNA damage is mostly studied in response to genotoxic treatments, the main source of DNA lesions is cellular activity itself. This can occur through the production of reactive oxygen species as well as DNA processing mechanisms such as DNA replication or transcription and chromatin dynamics. In addition, how lesions are formed and repaired is greatly influenced by chromatin features and dynamics and by DNA and RNA metabolism. Notably, actively transcribed regions or replicating DNA, because they are less condensed and are sites of DNA processing, are more exposed to DNA damage. However, at the same time, a wealth of cellular mechanisms cooperate to favour DNA repair at these genomic loci. These intricate relationships that shape the distribution of mutations along the genome have been studied extensively in animals but much less in plants. In this Review, we summarize how chromatin dynamics influence lesion formation and DNA repair in plants, providing a comprehensive view of current knowledge and highlighting open questions with regard to what is known in other organisms.

CRUMPLED LEAF supports plastid OUTER ENVELOPE PROTEIN OF 80 KDA complex formation in Arabidopsis.

Embedded ß-barrel proteins in the outer envelope membrane mediate most cellular trafficking between the cytoplasm and plastids. Although the TRANSLOCON AT THE OUTER ENVELOPE MEMBRANE OF CHLOROPLASTS 75-V (TOC75-V)/OUTER ENVELOPE PROTEIN OF 80 KDA (OEP80) complex has been implicated in the insertion and assembly of ß-barrel proteins in the outer envelope membrane of Arabidopsis (Arabidopsis thaliana) chloroplasts, relatively little is known about this process. CRUMPLED LEAF (CRL) encodes a chloroplast outer envelope membrane-localized protein, and its loss-of-function mutation 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 revealed that a missense mutation in OEP80 suppresses the pleiotropic defects of crl. Furthermore, we found that OEP80 complex formation is compromised in crl. Additionally, we demonstrated that CRL interacts with OEP80 in vivo and that a portion of CRL is present at the same molecular weight as the OEP80 complex. Our results suggest that CRL interacts with OEP80 to facilitate its complex formation. CRL is involved in plastid protein import; therefore, the pleiotropic defects in crl are likely due to the combined effects of decreased plastid protein import and altered membrane integration of ß-barrel proteins in the outer envelope membrane. This study sheds light on the mechanisms that allow ß-barrel protein integration into the plastid outer envelope membrane and the importance of this finding for plant cellular processes.

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 hinder the cell cycle, but similarly to RBR silencing, result in hyperplasia. Contrary to the growth arrest that occurs when exit from proliferation to differentiation is inhibited upon RBR silencing, the e2fabc mutant develops enlarged organs with supernumerary stem and differentiated cells as 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.

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

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 replication defects, accumulating evidence points to other pathways functioning independent of SOG1. Here, we report the roles of the Arabidopsis E2FA and EF2B transcription factors, two well-characterized regulators of DNA replication, in plant 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 plant growth in the presence of replication defects, either operating antagonistically or synergistically with SOG1. Conversely, SOG1 aids in overcoming the replication defects of E2FA/E2FB-deficient plants. Collectively, our data reveal a complex transcriptional network controlling the replication stress response in which E2Fs and SOG1 act as key regulatory factors.

HSFA1a modulates plant heat stress responses and alters the 3D chromatin organization of enhancer-promoter interactions.

The complex and dynamic three-dimensional organization of chromatin within the nucleus makes understanding the control of gene expression challenging, but also opens up possible ways to epigenetically modulate gene expression. Because plants are sessile, they evolved sophisticated ways to rapidly modulate gene expression in response to environmental stress, that are thought to be coordinated by changes in chromatin conformation to mediate specific cellular and physiological responses. However, to what extent and how stress induces dynamic changes in chromatin reorganization remains poorly understood. Here, we comprehensively investigated genome-wide chromatin changes associated with transcriptional reprogramming response to heat stress in tomato. Our data show that heat stress induces rapid changes in chromatin architecture, leading to the transient formation of promoter-enhancer contacts, likely driving the expression of heat-stress responsive genes. Furthermore, we demonstrate that chromatin spatial reorganization requires HSFA1a, a transcription factor (TF) essential for heat stress tolerance in tomato. In light of our findings, we propose that TFs play a key role in controlling dynamic transcriptional responses through 3D reconfiguration of promoter-enhancer contacts.

RADA-dependent branch migration has a predominant role in plant mitochondria and its defect leads to mtDNA instability and cell cycle arrest.

Mitochondria of flowering plants have large genomes whose structure and segregation are modulated by recombination activities. The post-synaptic late steps of mitochondrial DNA (mtDNA) recombination are still poorly characterized. Here we show that RADA, a plant ortholog of bacterial RadA/Sms, is an organellar protein that drives the major branch-migration pathway of plant mitochondria. While RadA/Sms is dispensable in bacteria, RADA-deficient Arabidopsis plants are severely impacted in their development and fertility, correlating with increased mtDNA recombination across intermediate-size repeats and accumulation of recombination-generated mitochondrial subgenomes. The radA mutation is epistatic to recG1 that affects the additional branch migration activity. In contrast, the double mutation radA recA3 is lethal, underlining the importance of an alternative RECA3-dependent pathway. The physical interaction of RADA with RECA2 but not with RECA3 further indicated that RADA is required for the processing of recombination intermediates in the RECA2-depedent recombination pathway of plant mitochondria. Although RADA is dually targeted to mitochondria and chloroplasts we found little to no effects of the radA mutation on the stability of the plastidial genome. Finally, we found that the deficient maintenance of the mtDNA in radA apparently triggers a retrograde signal that activates nuclear genes repressing cell cycle progression.

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.

CmLHP1 proteins play a key role in plant development and sex determination in melon (Cucumis melo).

In monoecious melon (Cucumis melo), sex is determined by the differential expression of sex determination genes (SDGs) and adoption of sex-specific transcriptional programs. Histone modifications such as H3K27me3 have been previously shown to be a hallmark associated to unisexual flower development in melon; yet, no genetic approaches have been conducted for elucidating the roles of H3K27me3 writers, readers, and erasers in this process. Here we show that melon homologs to Arabidopsis LHP1, CmLHP1A and B, redundantly control several aspects of plant development, including sex expression. Cmlhp1ab double mutants displayed an overall loss and redistribution of H3K27me3, leading to a deregulation of genes involved in hormone responses, plant architecture, and flower development. Consequently, double mutants display pleiotropic phenotypes and, interestingly, a general increase of the male:female ratio. We associated this phenomenon with a general deregulation of some hormonal response genes and a local activation of male-promoting SDGs and MADS-box transcription factors. Altogether, these results reveal a novel function for CmLHP1 proteins in maintenance of monoecy and provide novel insights into the polycomb-mediated epigenomic regulation of sex lability in plants.

Maize ATR safeguards genome stability during kernel development to prevent early endosperm endocycle onset and cell death.

The ataxia-telangiectasia mutated (ATM) and ATM and Rad3-related (ATR) kinases coordinate the DNA damage response. The roles described for Arabidopsis thaliana ATR and ATM are assumed to be conserved over other plant species, but molecular evidence is scarce. Here, we demonstrate that the functions of ATR and ATM are only partially conserved between Arabidopsis and maize (Zea mays). In both species, ATR and ATM play a key role in DNA repair and cell cycle checkpoint activation, but whereas Arabidopsis plants do not suffer from the absence of ATR under control growth conditions, maize mutant plants accumulate replication defects, likely due to their large genome size. Moreover, contrarily to Arabidopsis, maize ATM deficiency does not trigger meiotic defects, whereas the ATR kinase appears to be crucial for the maternal fertility. Strikingly, ATR is required to repress premature endocycle onset and cell death in the maize endosperm. Its absence results in a reduction of kernel size, protein and starch content, and a stochastic death of kernels, a process being counteracted by ATM. Additionally, while Arabidopsis atr atm double mutants are viable, no such mutants could be obtained for maize. Therefore, our data highlight that the mechanisms maintaining genome integrity may be more important for vegetative and reproductive development than previously anticipated.

Polycomb-dependent differential chromatin compartmentalization determines gene coregulation in Arabidopsis.

In animals, distant H3K27me3-marked Polycomb targets can establish physical interactions forming repressive chromatin hubs. In plants, growing evidence suggests that H3K27me3 acts directly or indirectly to regulate chromatin interactions, although how this histone modification modulates 3D chromatin architecture remains elusive. To decipher the impact of the dynamic deposition of H3K27me3 on the Arabidopsis thaliana nuclear interactome, we combined genetics, transcriptomics, and several 3D epigenomic approaches. By analyzing mutants defective for histone H3K27 methylation or demethylation, we uncovered the crucial role of this chromatin mark in short- and previously unnoticed long-range chromatin loop formation. We found that a reduction in H3K27me3 levels led to a decrease in the interactions within Polycomb-associated repressive domains. Regions with lower H3K27me3 levels in the H3K27 methyltransferase clf mutant established new interactions with regions marked with H3K9ac, a histone modification associated with active transcription, indicating that a reduction in H3K27me3 levels induces a global reconfiguration of chromatin architecture. Altogether, our results reveal that the 3D genome organization is tightly linked to reversible histone modifications that govern chromatin interactions. Consequently, nuclear organization dynamics shapes the transcriptional reprogramming during plant development and places H3K27me3 as a key feature in the coregulation of distant genes.

The plant DNA polymerase theta is essential for the repair of replication-associated DNA damage.

Safeguarding of genome integrity is a key process in all living organisms. Due to their sessile lifestyle, plants are particularly exposed to all kinds of stress conditions that could induce DNA damage. However, very few genes involved in the maintenance of genome integrity are indispensable to plants' viability. One remarkable exception is the POLQ gene, which encodes DNA polymerase theta (Pol θ), a non-replicative polymerase involved in trans-lesion synthesis during DNA replication and double-strand break (DSB) repair. The Arabidopsis tebichi (teb) mutants, deficient in Pol θ, have been reported to display severe developmental defects, leading to the conclusion that Pol θ is required for normal plant development. However, this essential role of Pol θ in plants is challenged by contradictory reports regarding the phenotypic defects of teb mutants and the recent finding that rice (Oryza sativa) null mutants develop normally. Here we show that the phenotype of teb mutants is highly variable. Taking advantage of hypomorphic mutants for the replicative DNA polymerase epsilon, which display constitutive replicative stress, we show that Pol θ allows maintenance of meristem activity when DNA replication is partially compromised. Furthermore, we found that the phenotype of Pol θ mutants can be aggravated by modifying their growth conditions, suggesting that environmental conditions impact the basal level of replicative stress and providing evidence for a link between plants' responses to adverse conditions and mechanisms involved in the maintenance of genome integrity.

Pharmacoresistant Epilepsy in Childhood: Think of the Cerebral Folate Deficiency, a Treatable Disease.

Cerebral folate deficiency (CFD) is a neurological disorder characterized by low levels of 5-methyltetrahydrofolate (5-MTHF) in the cerebrospinal fluid (CSF). The prevalence of this autosomal recessive disorder is estimated to be <1/1,000,000. Fifteen different pathogenic variants in the folate receptor 1 gene (FOLR1) encoding the receptor of folate α (FRα) have already been described. We present a new pathogenic variation in the FOLR1 in a childhood-stage patient. We aim to establish the core structure of the FRα protein mandatory for its activity. A three-year-old child was admitted at hospital for a first febrile convulsions episode. Recurrent seizures without fever also occurred a few months later, associated with motor and cognitive impairment. Various antiepileptic drugs failed to control seizures. Magnetic resonance imaging (MRI) showed central hypomyelination and biological analysis revealed markedly low levels of 5-MTHF in CSF. Next generation sequencing (NGS) confirmed a CFD with a FOLR1 homozygous variation (c.197 G > A, p.Cys66Tyr). This variation induces an altered folate receptor α protein and underlines the role of a disulfide bond: Cys66-Cys109, essential to transport 5-MTHF into the central nervous system. Fortunately, this severe form of CFD had remarkably responded to high doses of oral folinic acid combined with intravenous administrations.

A new role for histone demethylases in the maintenance of plant genome integrity.

Histone modifications deposited by the Polycomb repressive complex 2 (PRC2) play a critical role in the control of growth, development, and adaptation to environmental fluctuations of most multicellular eukaryotes. The catalytic activity of PRC2 is counteracted by Jumonji-type (JMJ) histone demethylases, which shapes the genomic distribution of H3K27me3. Here, we show that two JMJ histone demethylases in Arabidopsis, EARLY FLOWERING 6 (ELF6) and RELATIVE OF EARLY FLOWERING 6 (REF6), play distinct roles in H3K27me3 and H3K27me1 homeostasis. We show that failure to reset these chromatin marks during sexual reproduction results in the transgenerational inheritance of histone marks, which cause a loss of DNA methylation at heterochromatic loci and transposon activation. Thus, Jumonji-type histone demethylases play a dual role in plants by helping to maintain transcriptional states through development and safeguard genome integrity during sexual reproduction.

A novel pathogenic variant in DYNC1H1 causes various upper and lower motor neuron anomalies.

OBJECTIVE: To perform genotype-phenotype, clinical and molecular analysis in a large 3-generation family with autosomal dominant congenital spinal muscular atrophy. METHODS: Using a combined genetic approach including whole genome scanning, next generation sequencing-based multigene panel, whole genome sequencing, and targeted variant Sanger sequencing, we studied the proband and multiple affected individuals of this family who presented bilateral proximal lower limb muscle weakness and atrophy. RESULTS: We identified a novel heterozygous variant, c.1826T > C; p.Ile609Thr, in the DYNC1H1 gene localized within the common haplotype in the 14q32.3 chromosomal region which cosegregated with disease in this large family. Within the family, affected individuals were found to have a wide array of clinical variability. Although some individuals presented the typical lower motor neuron phenotype with areflexia and denervation, others presented with muscle weakness and atrophy, hyperreflexia, and absence of denervation suggesting a predominant upper motor neuron disease. In addition, some affected individuals presented with an intermediate phenotype characterized by hyperreflexia and denervation, expressing a combination of lower and upper motor neuron defects. CONCLUSION: Our study demonstrates the wide clinical variability associated with a single disease causing variant in DYNC1H1 gene and this variant demonstrated a high penetrance within this large family.

Involvement of Arabidopsis BIG protein in cell death mediated by Myo-inositol homeostasis.

Programmed cell death (PCD) is essential for several aspects of plant life. We previously identified the mips1 mutant of Arabidopsis thaliana, which is deficient for the enzyme catalysing myo-inositol synthesis, and that displays light-dependent formation of lesions on leaves due to Salicylic Acid (SA) over-accumulation. Rationale of this work was to identify novel regulators of plant PCD using a genetic approach. A screen for secondary mutations that abolish the mips1 PCD phenotype identified a mutation in the BIG gene, encoding a factor of unknown molecular function that was previously shown to play pleiotropic roles in plant development and defence. Physiological analyses showed that BIG is required for lesion formation in mips1 via SA-dependant signalling. big mutations partly rescued transcriptomic and metabolomics perturbations as stress-related phytohormones homeostasis. In addition, since loss of function of the ceramide synthase LOH2 was not able to abolish cell death induction in mips1, we show that PCD induction is not fully dependent of sphingolipid accumulation as previously suggested. Our results provide further insights into the role of the BIG protein in the control of MIPS1-dependent cell death and also into the impact of sphingolipid homeostasis in this pathway.

The matrix revolutions: towards the decoding of the plant chromatin three-dimensional reality.

In recent years, we have witnessed a significant increase in studies addressing the three-dimensional (3D) chromatin organization of the plant nucleus. Important advances in chromatin conformation capture (3C)-derived and related techniques have allowed the exploration of the nuclear topology of plants with large and complex genomes, including various crops. In addition, the increase in their resolution has permitted the depiction of chromatin compartmentalization and interactions at the gene scale. These studies have revealed the highly complex mechanisms governing plant nuclear architecture and the remarkable knowledge gaps in this field. Here we discuss the state-of-the-art in plant chromosome architecture, including our knowledge of the hierarchical organization of the genome in 3D space and regarding other nuclear components. Furthermore, we highlight the existence in plants of topologically associated domain (TAD)-like structures that display striking differences from their mammalian counterparts, proposing the concept of ICONS-intergenic condensed spacers. Similarly, we explore recent advances in the study of chromatin loops and R-loops, and their implication in the regulation of gene activity. Finally, we address the impact that polyploidization has had on the chromatin topology of modern crops, and how this is related to phenomena such as subgenome dominance and biased gene retention in these organisms.

Maize Thymidine Kinase Activity Is Present throughout Plant Development and Its Heterologous Expression Confers Tolerance to an Organellar DNA-Damaging Agent.

Thymidine kinase 1 (TK1) phosphorylates thymidine nucleosides to generate thymidine monophosphate. This reaction belongs to the pyrimidine salvage route that is phylogenetically conserved. In the model plant Arabidopsis thaliana, TK activity contributes to maintain nuclear and organellar genome integrity by providing deoxythymidine-triphosphate (dTTP) for DNA synthesis. Arabidopsis has two TK1 genes (TK1a and TK1b) and double mutants show an albino phenotype and develop poorly. In contrast, maize (Zea mays L.) has a single TK1 (ZmTK1) gene and mutant plants are albino and display reduced genome copy number in chloroplasts. We studied the role of ZmTK1 during development and genotoxic stress response by assessing its activity at different developmental stages and by complementing Arabidopsis tk1 mutants. We found that ZmTK1 transcripts and activity are present during germination and throughout maize development. We show that ZmTK1 translocation to chloroplasts depends on a 72-amino-acid N-signal and its plastid localization is consistent with its ability to complement Arabidopsis tk1b mutants which are hypersensitive to ciprofloxacin (CIP), a genotoxic agent to organellar DNA. Also, ZmTK1 partly complemented the Arabidopsis double mutant plants during development. Our results contribute to the understanding of TK1 function in monocot species as an organellar enzyme for genome replication and repair.

GCN5 modulates salicylic acid homeostasis by regulating H3K14ac levels at the 5' and 3' ends of its target genes.

The modification of histones by acetyl groups has a key role in the regulation of chromatin structure and transcription. The Arabidopsis thaliana histone acetyltransferase GCN5 regulates histone modifications as part of the Spt-Ada-Gcn5 Acetyltransferase (SAGA) transcriptional coactivator complex. GCN5 was previously shown to acetylate lysine 14 of histone 3 (H3K14ac) in the promoter regions of its target genes even though GCN5 binding did not systematically correlate with gene activation. Here, we explored the mechanism through which GCN5 controls transcription. First, we fine-mapped its GCN5 binding sites genome-wide and then used several global methodologies (ATAC-seq, ChIP-seq and RNA-seq) to assess the effect of GCN5 loss-of-function on the expression and epigenetic regulation of its target genes. These analyses provided evidence that GCN5 has a dual role in the regulation of H3K14ac levels in their 5' and 3' ends of its target genes. While the gcn5 mutation led to a genome-wide decrease of H3K14ac in the 5' end of the GCN5 down-regulated targets, it also led to an increase of H3K14ac in the 3' ends of GCN5 up-regulated targets. Furthermore, genome-wide changes in H3K14ac levels in the gcn5 mutant correlated with changes in H3K9ac at both 5' and 3' ends, providing evidence for a molecular link between the depositions of these two histone modifications. To understand the biological relevance of these regulations, we showed that GCN5 participates in the responses to biotic stress by repressing salicylic acid (SA) accumulation and SA-mediated immunity, highlighting the role of this protein in the regulation of the crosstalk between diverse developmental and stress-responsive physiological programs. Hence, our results demonstrate that GCN5, through the modulation of H3K14ac levels on its targets, controls the balance between biotic and abiotic stress responses and is a master regulator of plant-environmental interactions.

Wheat chromatin architecture is organized in genome territories and transcription factories.

BACKGROUND: Polyploidy is ubiquitous in eukaryotic plant and fungal lineages, and it leads to the co-existence of several copies of similar or related genomes in one nucleus. In plants, polyploidy is considered a major factor in successful domestication. However, polyploidy challenges chromosome folding architecture in the nucleus to establish functional structures. RESULTS: We examine the hexaploid wheat nuclear architecture by integrating RNA-seq, ChIP-seq, ATAC-seq, Hi-C, and Hi-ChIP data. Our results highlight the presence of three levels of large-scale spatial organization: the arrangement into genome territories, the diametrical separation between facultative and constitutive heterochromatin, and the organization of RNA polymerase II around transcription factories. We demonstrate the micro-compartmentalization of transcriptionally active genes determined by physical interactions between genes with specific euchromatic histone modifications. Both intra- and interchromosomal RNA polymerase-associated contacts involve multiple genes displaying similar expression levels. CONCLUSIONS: Our results provide new insights into the physical chromosome organization of a polyploid genome, as well as on the relationship between epigenetic marks and chromosome conformation to determine a 3D spatial organization of gene expression, a key factor governing gene transcription in polyploids.