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.

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.

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.

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.

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.

A pathogenesis-related protein GmPR08-Bet VI promotes a molecular interaction between the GmSHMT08 and GmSNAP18 in resistance to Heterodera glycines.

Soybean cyst nematode (SCN, Heterodera glycines) is the most devastating pest affecting soybean production worldwide. SCN resistance requires both the GmSHMT08 and the GmSNAP18 in 'Peking'-type resistance. Here, we describe the molecular interaction between GmSHMT08 and GmSNAP18, which is potentiated by a pathogenesis-related protein GmPR08-Bet VI. Like GmSNAP18 and GmSHMT08, GmPR08-Bet VI expression was induced in response to SCN and its overexpression decreased SCN cysts by 65% in infected transgenic soybean roots. Overexpression of GmPR08-Bet VI did not have an effect on SCN resistance when the two cytokinin-binding sites in GmPR08-Bet VI were mutated, indicating a new role of GmPR08-Bet VI in SCN resistance. GmPR08-Bet VI was mapped to a QTL for resistance to SCN using different mapping populations. GmSHMT08, GmSNAP18 and GmPR08-Bet VI localize to the cytosol and plasma membrane. GmSNAP18 expression and localization hyper-accumulated at the plasma membrane and was specific to the root cells surrounding the nematode in SCN-resistant soybeans. Genes encoding key components of the salicylic acid signalling pathway were induced under SCN infection. GmSNAP18 and GmPR08-Bet VI were also induced under salicylic acid and cytokinin exogenous treatments, while GmSHMT08 was induced only when the resistant GmSNAP18 was present, pointing to the presence of a molecular crosstalk between SCN-resistant genes and defence genes. Expression analysis of GmSHMT08 and GmSNAP18 identified the need of a minimum expression requirement to trigger the SCN resistance reaction. These results provide insight into a new response mechanism towards plant nematode resistance involving haplotype compatibility, gene dosage and hormone signalling.

Tubulin acetylation accompanies autophagy development induced by different abiotic stimuli in Arabidopsis thaliana.

Microtubules (MTs) play an important role in the regulation of autophagy development in yeast and animal as well as in plant cells. MTs participate in maturation and traffic of autophagosomes through their dynamic state changes and post-translational modifications of tubulin, namely acetylation. We subjected Arabidopsis thaliana seedlings to metabolic-, salt-, osmotic stresses as well as irradiation of ultraviolet B and investigated the involvement of plant MTs in the development of stress-induced autophagy via tubulin acetylation. For this purpose Arabidopsis thaliana line expressing autophagy-related protein 8 h (atg8h)-GFP was generated to investigate autophagy, applying the level of free GFP as an indicator of autophagy development. Using autophagosome confocal imaging and Western blot analysis of Atg8 post-translational lipidation and synchronous GFP release it was shown that all examined stressful stimuli led to pronounced development of autophagy, particularly in different root tissues. Moreover, autophagy development was accompanied by α-tubulin acetylation under all stressful conditions. Presented data indicate the possible role of the post-translational acetylation of α-tubulin in the mediation of plant stress-induced autophagy.

The Arabidopsis SWI/SNF protein BAF60 mediates seedling growth control by modulating DNA accessibility.

BACKGROUND: Plant adaptive responses to changing environments involve complex molecular interplays between intrinsic and external signals. Whilst much is known on the signaling components mediating diurnal, light, and temperature controls on plant development, their influence on chromatin-based transcriptional controls remains poorly explored. RESULTS: In this study we show that a SWI/SNF chromatin remodeler subunit, BAF60, represses seedling growth by modulating DNA accessibility of hypocotyl cell size regulatory genes. BAF60 binds nucleosome-free regions of multiple G box-containing genes, opposing in cis the promoting effect of the photomorphogenic and thermomorphogenic regulator Phytochrome Interacting Factor 4 (PIF4) on hypocotyl elongation. Furthermore, BAF60 expression level is regulated in response to light and daily rhythms. CONCLUSIONS: These results unveil a short path between a chromatin remodeler and a signaling component to fine-tune plant morphogenesis in response to environmental conditions.

Function of the Plant DNA Polymerase Epsilon in Replicative Stress Sensing, a Genetic Analysis.

Faithful transmission of the genetic information is essential in all living organisms. DNA replication is therefore a critical step of cell proliferation, because of the potential occurrence of replication errors or DNA damage when progression of a replication fork is hampered causing replicative stress. Like other types of DNA damage, replicative stress activates the DNA damage response, a signaling cascade allowing cell cycle arrest and repair of lesions. The replicative DNA polymerase ε (Pol ε) was shown to activate the S-phase checkpoint in yeast in response to replicative stress, but whether this mechanism functions in multicellular eukaryotes remains unclear. Here, we explored the genetic interaction between Pol ε and the main elements of the DNA damage response in Arabidopsis (Arabidopsis thaliana). We found that mutations affecting the polymerase domain of Pol ε trigger ATR-dependent signaling leading to SOG1 activation, WEE1-dependent cell cycle inhibition, and tolerance to replicative stress induced by hydroxyurea, but result in enhanced sensitivity to a wide range of DNA damaging agents. Using knock-down lines, we also provide evidence for the direct role of Pol ε in replicative stress sensing. Together, our results demonstrate that the role of Pol ε in replicative stress sensing is conserved in plants, and provide, to our knowledge, the first genetic dissection of the downstream signaling events in a multicellular eukaryote.

Chloroplasts around the plant cell cycle.

Plastids arose from an endosymbiosis between a host cell and free-living bacteria. One key step during this evolutionary process has been the establishment of coordinated cell and symbiont division to allow the maintenance of organelles during proliferation of the host. However, surprisingly little is known about the underlying mechanisms. In addition, due to their central role in the cell's energetic metabolism and to their sensitivity to various environmental cues such as light or temperature, plastids are ideally fitted to be the source of signals allowing plants to adapt their development according to external conditions. Consistently, there is accumulating evidence that plastid-derived signals can impinge on cell cycle regulation. In this review, we summarize current knowledge of the dialogue between chloroplasts and the nucleus in the context of the cell cycle.

LHP1 Regulates H3K27me3 Spreading and Shapes the Three-Dimensional Conformation of the Arabidopsis Genome.

Precise expression patterns of genes in time and space are essential for proper development of multicellular organisms. Dynamic chromatin conformation and spatial organization of the genome constitute a major step in this regulation to modulate developmental outputs. Polycomb repressive complexes (PRCs) mediate stable or flexible gene repression in response to internal and environmental cues. In Arabidopsis thaliana, LHP1 co-localizes with H3K27me3 epigenetic marks throughout the genome and interacts with PRC1 and PRC2 members as well as with a long noncoding RNA. Here, we show that LHP1 is responsible for the spreading of H3K27me3 towards the 3' end of the gene body. We also identified a subset of LHP1-activated genes and demonstrated that LHP1 shapes local chromatin topology in order to control transcriptional co-regulation. Our work reveals a general role of LHP1 from local to higher conformation levels of chromatin configuration to determine its accessibility to define gene expression patterns.

Involvement of Inositol Biosynthesis and Nitric Oxide in the Mediation of UV-B Induced Oxidative Stress.

The involvement of NO-signaling in ultraviolet B (UV-B) induced oxidative stress (OS) in plants is an open question. Inositol biosynthesis contributes to numerous cellular functions, including the regulation of plants tolerance to stress. This work reveals the involvement of inositol-3-phosphate synthase 1 (IPS1), a key enzyme for biosynthesis of myo-inositol and its derivatives, in the response to NO-dependent OS in Arabidopsis. Homozygous mutants deficient for IPS1 (atips1) and wild-type plants were transformed with a reduction- grx1-rogfp2 and used for the dynamic measurement of UV-B-induced and SNP (sodium nitroprusside)-mediated oxidative stresses by confocal microscopy. atips1 mutants displayed greater tissue-specific resistance to the action of UV-B than the wild type. SNP can act both as an oxidant or repairer depending on the applied concentration, but mutant plants were more tolerant than the wild type to nitrosative effects of high concentration of SNP. Additionally, pretreatment with low concentrations of SNP (10, 100 µM) before UV-B irradiation resulted in a tissue-specific protective effect that was enhanced in atips1. We conclude that the interplay between nitric oxide and inositol signaling can be involved in the mediation of UV-B-initiated oxidative stress in the plant cell.

Role of the Polymerase ϵ sub-unit DPB2 in DNA replication, cell cycle regulation and DNA damage response in Arabidopsis.

Faithful DNA replication maintains genome stability in dividing cells and from one generation to the next. This is particularly important in plants because the whole plant body and reproductive cells originate from meristematic cells that retain their proliferative capacity throughout the life cycle of the organism. DNA replication involves large sets of proteins whose activity is strictly regulated, and is tightly linked to the DNA damage response to detect and respond to replication errors or defects. Central to this interconnection is the replicative polymerase DNA Polymerase ϵ (Pol ϵ) which participates in DNA replication per se, as well as replication stress response in animals and in yeast. Surprisingly, its function has to date been little explored in plants, and notably its relationship with DNA Damage Response (DDR) has not been investigated. Here, we have studied the role of the largest regulatory sub-unit of Arabidopsis DNA Pol ϵ: DPB2, using an over-expression strategy. We demonstrate that excess accumulation of the protein impairs DNA replication and causes endogenous DNA stress. Furthermore, we show that Pol ϵ dysfunction has contrasting outcomes in vegetative and reproductive cells and leads to the activation of distinct DDR pathways in the two cell types.

Chloroplast Activity and 3'phosphadenosine 5'phosphate Signaling Regulate Programmed Cell Death in Arabidopsis.

Programmed cell death (PCD) is a crucial process both for plant development and responses to biotic and abiotic stress. There is accumulating evidence that chloroplasts may play a central role during plant PCD as for mitochondria in animal cells, but it is still unclear whether they participate in PCD onset, execution, or both. To tackle this question, we have analyzed the contribution of chloroplast function to the cell death phenotype of the myoinositol phosphate synthase1 (mips1) mutant that forms spontaneous lesions in a light-dependent manner. We show that photosynthetically active chloroplasts are required for PCD to occur in mips1, but this process is independent of the redox state of the chloroplast. Systematic genetic analyses with retrograde signaling mutants reveal that 3'-phosphoadenosine 5'-phosphate, a chloroplast retrograde signal that modulates nuclear gene expression in response to stress, can inhibit cell death and compromises plant innate immunity via inhibition of the RNA-processing 5'-3' exoribonucleases. Our results provide evidence for the role of chloroplast-derived signal and RNA metabolism in the control of cell death and biotic stress response.

A SWI/SNF Chromatin Remodelling Protein Controls Cytokinin Production through the Regulation of Chromatin Architecture.

Chromatin architecture determines transcriptional accessibility to DNA and consequently gene expression levels in response to developmental and environmental stimuli. Recently, chromatin remodelers such as SWI/SNF complexes have been recognized as key regulators of chromatin architecture. To gain insight into the function of these complexes during root development, we have analyzed Arabidopsis knock-down lines for one sub-unit of SWI/SNF complexes: BAF60. Here, we show that BAF60 is a positive regulator of root development and cell cycle progression in the root meristem via its ability to down-regulate cytokinin production. By opposing both the deposition of active histone marks and the formation of a chromatin regulatory loop, BAF60 negatively regulates two crucial target genes for cytokinin biosynthesis (IPT3 and IPT7) and one cell cycle inhibitor (KRP7). Our results demonstrate that SWI/SNF complexes containing BAF60 are key factors governing the equilibrium between formation and dissociation of a chromatin loop controlling phytohormone production and cell cycle progression.

Involvement of Arabidopsis Hexokinase1 in Cell Death Mediated by Myo-Inositol Accumulation.

Programmed cell death (PCD) is essential for several aspects of plant life, including development and stress responses. We recently identified the mips1 mutant of Arabidopsis thaliana, which is deficient for the enzyme catalyzing the limiting step of myo-inositol (MI) synthesis. One of the most striking features of mips1 is the light-dependent formation of lesions on leaves due to salicylic acid (SA)-dependent PCD. Here, we identified a suppressor of PCD by screening for mutations that abolish the mips1 cell death phenotype. Our screen identified the hxk1 mutant, mutated in the gene encoding the hexokinase1 (HXK1) enzyme that catalyzes sugar phosphorylation and acts as a genuine glucose sensor. We show that HXK1 is required for lesion formation in mips1 due to alterations in MI content, via SA-dependant signaling. Using two catalytically inactive HXK1 mutants, we also show that hexokinase catalytic activity is necessary for the establishment of lesions in mips1. Gas chromatography-mass spectrometry analyses revealed a restoration of the MI content in mips1 hxk1 that it is due to the activity of the MIPS2 isoform, while MIPS3 is not involved. Our work defines a pathway of HXK1-mediated cell death in plants and demonstrates that two MIPS enzymes act cooperatively under a particular metabolic status, highlighting a novel checkpoint of MI homeostasis in plants.

Chloroplast dysfunction causes multiple defects in cell cycle progression in the Arabidopsis crumpled leaf mutant.

The majority of research on cell cycle regulation is focused on the nuclear events that govern the replication and segregation of the genome between the two daughter cells. However, eukaryotic cells contain several compartmentalized organelles with specialized functions, and coordination among these organelles is required for proper cell cycle progression, as evidenced by the isolation of several mutants in which both organelle function and overall plant development were affected. To investigate how chloroplast dysfunction affects the cell cycle, we analyzed the crumpled leaf (crl) mutant of Arabidopsis (Arabidopsis thaliana), which is deficient for a chloroplastic protein and displays particularly severe developmental defects. In the crl mutant, we reveal that cell cycle regulation is altered drastically and that meristematic cells prematurely enter differentiation, leading to reduced plant stature and early endoreduplication in the leaves. This response is due to the repression of several key cell cycle regulators as well as constitutive activation of stress-response genes, among them the cell cycle inhibitor SIAMESE-RELATED5. One unique feature of the crl mutant is that it produces aplastidic cells in several organs, including the root tip. By investigating the consequence of the absence of plastids on cell cycle progression, we showed that nuclear DNA replication occurs in aplastidic cells in the root tip, which opens future research prospects regarding the dialogue between plastids and the nucleus during cell cycle regulation in higher plants.