Leaves to Measure Light Intensity.

Quantitative measurement of light intensity is a key step in ensuring the reliability and the reproducibility of scientific results in many fields of physics, biology, and chemistry. The protocols presented so far use various photoactive properties of manufactured materials. Here, leaves are introduced as an easily accessible green material to calibrate light intensity. The measurement protocol consists in monitoring the chlorophyll fluorescence of a leaf while it is exposed to a jump of constant light. The inverse of the characteristic time of the initial chlorophyll fluorescence rise is shown to be proportional to the light intensity received by the leaf over a wide range of wavelengths and intensities. Moreover, the proportionality factor is stable across a wide collection of plant species, which makes the measurement protocol accessible to users without prior calibration. This favorable feature is finally harnessed to calibrate a source of white light from exploiting simple leaves collected from a garden.

The TELOMERE REPEAT BINDING proteins TRB4 and TRB5 function as transcriptional activators of PRC2-controlled genes to regulate plant development.

Plant-specific transcriptional regulators called TELOMERE REPEAT BINDING proteins (TRBs) combine two DNA-binding domains, the GH1 domain, which binds to linker DNA and is shared with H1 histones, and the Myb/SANT domain, which specifically recognizes the telobox DNA-binding site motif. TRB1, TRB2, and TRB3 proteins recruit Polycomb group complex 2 (PRC2) to deposit H3K27me3 and JMJ14 to remove H3K4me3 at gene promoters containing telobox motifs to repress transcription. Here, we demonstrate that TRB4 and TRB5, two related paralogs belonging to a separate TRB clade conserved in spermatophytes, regulate the transcription of several hundred genes involved in developmental responses to environmental cues. TRB4 binds to several thousand sites in the genome, mainly at transcription start sites and promoter regions of transcriptionally active and H3K4me3-marked genes, but, unlike TRB1, it is not enriched at H3K27me3-marked gene bodies. However, TRB4 can physically interact with the catalytic components of PRC2, SWINGER, and CURLY LEAF (CLF). Unexpectedly, we show that TRB4 and TRB5 are required for distinctive phenotypic traits observed in clf mutant plants and thus function as transcriptional activators of several hundred CLF-controlled genes, including key flowering genes. We further demonstrate that TRB4 shares multiple target genes with TRB1 and physically and genetically interacts with members of both TRB clades. Collectively, these results reveal that TRB proteins engage in both positive and negative interactions with other members of the family to regulate plant development through both PRC2-dependent and -independent mechanisms.

The plant POLYMERASE-ASSOCIATED FACTOR1 complex links transcription and H2B monoubiquitination genome wide.

The evolutionarily conserved POLYMERASE-ASSOCIATED FACTOR1 complex (Paf1C) participates in transcription, and research in animals and fungi suggests that it facilitates RNA POLYMERASE II (RNAPII) progression through chromatin. We examined the genomic distribution of the EARLY FLOWERING7 (ELF7) and VERNALIZATION INDEPENDENCE3 subunits of Paf1C in Arabidopsis (Arabidopsis thaliana). The occupancy of both subunits was confined to thousands of gene bodies and positively associated 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 a direct functional link between Paf1C activity, monoubiquitination of histone H2B, and the transition of RNAPII to productive elongation. However, for several genes, Paf1C may also act independently of H2Bub deposition or occupy these genes more stable than H2Bub marking, possibly reflecting the dynamic nature of Paf1C association and H2Bub turnover during transcription.

The UBP5 histone H2A deubiquitinase counteracts PRCs-mediated repression to regulate Arabidopsis development.

Polycomb Repressive Complexes (PRCs) control gene expression through the incorporation of H2Aub and H3K27me3. In recent years, there is increasing evidence of the complexity of PRCs' interaction networks and the interplay of these interactors with PRCs in epigenome reshaping, which is fundamental to understand gene regulatory mechanisms. Here, we identified UBIQUITIN SPECIFIC PROTEASE 5 (UBP5) as a chromatin player able to counteract the deposition of the two PRCs' epigenetic hallmarks in Arabidopsis thaliana. We demonstrated that UBP5 is a plant developmental regulator based on functional analyses of ubp5-CRISPR Cas9 mutant plants. UBP5 promotes H2A monoubiquitination erasure, leading to transcriptional de-repression. Furthermore, preferential association of UBP5 at PRC2 recruiting motifs and local H3K27me3 gaining in ubp5 mutant plants suggest the existence of functional interplays between UBP5 and PRC2 in regulating epigenome dynamics. In summary, acting as an antagonist of the pivotal epigenetic repressive marks H2Aub and H3K27me3, UBP5 provides novel insights to disentangle the complex regulation of PRCs' activities.

Histone H1 protects telomeric repeats from H3K27me3 invasion in Arabidopsis.

While the pivotal role of linker histone H1 in shaping nucleosome organization is well established, its functional interplays with chromatin factors along the epigenome are just starting to emerge. Here we show that, in Arabidopsis, as in mammals, H1 occupies Polycomb Repressive Complex 2 (PRC2) target genes where it favors chromatin condensation and H3K27me3 deposition. We further show that, contrasting with its conserved function in PRC2 activation at genes, H1 selectively prevents H3K27me3 accumulation at telomeres and large pericentromeric interstitial telomeric repeat (ITR) domains by restricting DNA accessibility to Telomere Repeat Binding (TRB) proteins, a group of H1-related Myb factors mediating PRC2 cis recruitment. This study provides a mechanistic framework by which H1 avoids the formation of gigantic H3K27me3-rich domains at telomeric sequences and contributes to safeguard nucleus architecture.

Advanced Image Analysis Methods for Automated Segmentation of Subnuclear Chromatin Domains.

The combination of ever-increasing microscopy resolution with cytogenetical tools allows for detailed analyses of nuclear functional partitioning. However, the need for reliable qualitative and quantitative methodologies to detect and interpret chromatin sub-nuclear organization dynamics is crucial to decipher the underlying molecular processes. Having access to properly automated tools for accurate and fast recognition of complex nuclear structures remains an important issue. Cognitive biases associated with human-based curation or decisions for object segmentation tend to introduce variability and noise into image analysis. Here, we report the development of two complementary segmentation methods, one semi-automated (iCRAQ) and one based on deep learning (Nucl.Eye.D), and their evaluation using a collection of A. thaliana nuclei with contrasted or poorly defined chromatin compartmentalization. Both methods allow for fast, robust and sensitive detection as well as for quantification of subtle nucleus features. Based on these developments, we highlight advantages of semi-automated and deep learning-based analyses applied to plant cytogenetics.

Light, chromatin, action: nuclear events regulating light signaling in Arabidopsis.

The plant nucleus provides a major hub for environmental signal integration at the chromatin level. Multiple light signaling pathways operate and exchange information by regulating a large repertoire of gene targets that shape plant responses to a changing environment. In addition to the established role of transcription factors in triggering photoregulated changes in gene expression, there are eminent reports on the significance of chromatin regulators and nuclear scaffold dynamics in promoting light-induced plant responses. Here, we report and discuss recent advances in chromatin-regulatory mechanisms modulating plant architecture and development in response to light, including the molecular and physiological roles of key modifications such as DNA, RNA and histone methylation, and/or acetylation. The significance of the formation of biomolecular condensates of key light signaling components is discussed and potential applications to agricultural practices overviewed.

Topoisomerase VI participates in an insulator-like function that prevents H3K9me2 spreading.

The organization of the genome into transcriptionally active and inactive chromatin domains requires well-delineated chromatin boundaries and insulator functions in order to maintain the identity of adjacent genomic loci with antagonistic chromatin marks and functionality. In plants that lack known chromatin insulators, the mechanisms that prevent heterochromatin spreading into euchromatin remain to be identified. Here, we show that DNA Topoisomerase VI participates in a chromatin boundary function that safeguards the expression of genes in euchromatin islands within silenced heterochromatin regions. While some transposable elements are reactivated in mutants of the Topoisomerase VI complex, genes insulated in euchromatin islands within heterochromatic regions of the Arabidopsis thaliana genome are specifically down-regulated. H3K9me2 levels consistently increase at euchromatin island loci and decrease at some transposable element loci. We further show that Topoisomerase VI physically interacts with S-adenosylmethionine synthase methionine adenosyl transferase 3 (MAT3), which is required for H3K9me2. A Topoisomerase VI defect affects MAT3 occupancy on heterochromatic elements and its exclusion from euchromatic islands, thereby providing a possible mechanistic explanation to the essential role of Topoisomerase VI in the delimitation of chromatin domains.

Intracellular reactive oxygen species trafficking participates in seed dormancy alleviation in Arabidopsis seeds.

Reactive oxygen species (ROS) release seed dormancy through an unknown mechanism. We used different seed dormancy-breaking treatments to decipher the dynamics and localization of ROS production during seed germination. We studied the involvement of ROS in the breaking of Arabidopsis seed dormancy by cold stratification, gibberellic acid (GA3 ) and light. We characterized the effects of these treatments on abscisic acid and gibberellins biosynthesis and signalling pathways. ROS, mitochondrial redox status and peroxisomes were visualized and/or quantified during seed imbibition. Finally, we performed a cytogenetic characterization of the nuclei from the embryonic axes during seed germination. We show that mitochondria participate in the early ROS production during seed imbibition and that a possible involvement of peroxisomes in later stages should still be analysed. At the time of radicle protrusion, ROS accumulated within the nucleus, which correlated with nuclear expansion and chromatin decompaction. Taken together, our results provide evidence of the role of ROS trafficking between organelles and of the nuclear redox status in the regulation of seed germination by dormancy.

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.

DET1-mediated COP1 regulation avoids HY5 activity over second-site gene targets to tune plant photomorphogenesis.

DE-ETIOLATED 1 (DET1) and CONSTITUTIVE PHOTOMORPHOGENESIS 1 (COP1) are two essential repressors of Arabidopsis photomorphogenesis. These proteins can associate with CULLIN4 to form independent CRL4-based E3 ubiquitin ligases that mediate the degradation of several photomorphogenic transcription factors, including ELONGATED HYPOCOTYL 5 (HY5), thereby controlling multiple gene-regulatory networks. Despite extensive biochemical and genetic analyses of their multi-subunit complexes, the functional links between DET1 and COP1 have long remained elusive. Here, we report that DET1 associates with COP1 in vivo, enhances COP1-HY5 interaction, and promotes COP1 destabilization in a process that dampens HY5 protein abundance. By regulating its accumulation, DET1 avoids HY5 association with hundreds of second-site genomic loci, which are also frequently targeted by the skotomorphogenic transcription factor PHYTOCHROME-INTERACTING FACTOR 3. Accordingly, ectopic HY5 chromatin enrichment favors local gene repression and can trigger fusca-like phenotypes. This study therefore shows that DET1-mediated regulation of COP1 stability tunes down the HY5 cistrome, avoiding hyper-photomorphogenic responses that might compromise plant viability.

Multifaceted activities of the plant SAGA complex.

From yeast to human, the Spt-Ada-GCN5-acetyltransferase (SAGA) gigantic complex modifies chromatin during RNA polymerase II initiation and elongation steps to facilitate transcription. Its enzymatic activity involves a histone acetyltransferase module (HATm) that acetylates multiple lysine residues on the N-terminal tails of histones H2B and H3 and a deubiquitination module (DUBm) that triggers co-transcriptional deubiquitination of histone H2B. With a few notable exceptions described in this review, most SAGA subunits identified in yeast and metazoa are present in plants. Studies from the last 20 years have unveiled that different SAGA subunits are involved in gene expression regulation during the plant life cycle and in response to various types of stress or environmental cues. Their functional analysis in the Arabidopsis thaliana model species is increasingly shedding light on their intrinsic properties and how they can themselves be regulated during plant adaptive responses. Recent biochemical studies have also uncovered multiple associations between plant SAGA and chromatin machineries linked to RNA Pol II transcription. Still, considerably less is known about the molecular links between SAGA or SAGA-like complexes and chromatin dynamics during transcription in Arabidopsis and other plant species. We summarize the emerging knowledge on plant SAGA complex composition and activity, with a particular focus on the best-characterized subunits from its HAT (such as GCN5) and DUB (such as UBP22) modules, and implication of these ensembles in plant development and adaptive responses.

The Salt Sensitivity Induced by Disruption of Cell Wall-Associated Kinase 1 (SlWAK1) Tomato Gene Is Linked to Altered Osmotic and Metabolic Homeostasis.

Tomato cell wall-associated kinase 1 (SlWAK1) has only been studied in biotic stress response and hence its function in abiotic stress remains unknown. In a screening under salinity of an insertional mutant collection of tomato (Solanum lycopersicum L.), a mutant exhibiting lower degree of leaf chlorosis than wild type (WT) together with reduced leaf Na+ accumulation was selected. Genetic analysis of the mutation revealed that a single T-DNA insertion in the SlWAK1 gene was responsible of the mutant phenotype. Slwak1 null mutant reduced its shoot growth compared with WT, despite its improved Na+ homeostasis. SlWAK1 disruption affected osmotic homeostasis, as leaf water content was lower in mutant than in WT under salt stress. In addition, Slwak1 altered the source-sink balance under salinity, by increasing sucrose content in roots. Finally, a significant fruit yield reduction was found in Slwak1 vs. WT under long-term salt stress, mainly due to lower fruit weight. Our results show that disruption of SlWAK1 induces a higher sucrose transport from source leaf to sink root, negatively affecting fruit, the main sink at adult stage.

Linker histones are fine-scale chromatin architects modulating developmental decisions in Arabidopsis.

BACKGROUND: Chromatin provides a tunable platform for gene expression control. Besides the well-studied core nucleosome, H1 linker histones are abundant chromatin components with intrinsic potential to influence chromatin function. Well studied in animals, little is known about the evolution of H1 function in other eukaryotic lineages for instance plants. Notably, in the model plant Arabidopsis, while H1 is known to influence heterochromatin and DNA methylation, its contribution to transcription, molecular, and cytological chromatin organization remains elusive. RESULTS: We provide a multi-scale functional study of Arabidopsis linker histones. We show that H1-deficient plants are viable yet show phenotypes in seed dormancy, flowering time, lateral root, and stomata formation-complemented by either or both of the major variants. H1 depletion also impairs pluripotent callus formation. Fine-scale chromatin analyses combined with transcriptome and nucleosome profiling reveal distinct roles of H1 on hetero- and euchromatin: H1 is necessary to form heterochromatic domains yet dispensable for silencing of most transposable elements; H1 depletion affects nucleosome density distribution and mobility in euchromatin, spatial arrangement of nanodomains, histone acetylation, and methylation. These drastic changes affect moderately the transcription but reveal a subset of H1-sensitive genes. CONCLUSIONS: H1 variants have a profound impact on the molecular and spatial (nuclear) chromatin organization in Arabidopsis with distinct roles in euchromatin and heterochromatin and a dual causality on gene expression. Phenotypical analyses further suggest the novel possibility that H1-mediated chromatin organization may contribute to the epigenetic control of developmental and cellular transitions.

Arabidopsis S2Lb links AtCOMPASS-like and SDG2 activity in H3K4me3 independently from histone H2B monoubiquitination.

BACKGROUND: The functional determinants of H3K4me3, their potential dependency on histone H2B monoubiquitination, and their contribution to defining transcriptional regimes are poorly defined in plant systems. Unlike in Saccharomyces cerevisiae, where a single SET1 protein catalyzes H3K4me3 as part of COMPlex of proteins ASsociated with Set1 (COMPASS), in Arabidopsis thaliana, this activity involves multiple histone methyltransferases. Among these, the plant-specific SET DOMAIN GROUP 2 (SDG2) has a prominent role. RESULTS: We report that SDG2 co-regulates hundreds of genes with SWD2-like b (S2Lb), a plant ortholog of the Swd2 axillary subunit of yeast COMPASS. We show that S2Lb co-purifies with the AtCOMPASS core subunit WDR5, and both S2Lb and SDG2 directly influence H3K4me3 enrichment over highly transcribed genes. S2Lb knockout triggers pleiotropic developmental phenotypes at the vegetative and reproductive stages, including reduced fertility and seed dormancy. However, s2lb seedlings display little transcriptomic defects as compared to the large repertoire of genes targeted by S2Lb, SDG2, or H3K4me3, suggesting that H3K4me3 enrichment is important for optimal gene induction during cellular transitions rather than for determining on/off transcriptional status. Moreover, unlike in budding yeast, most of the S2Lb and H3K4me3 genomic distribution does not rely on a trans-histone crosstalk with histone H2B monoubiquitination. CONCLUSIONS: Collectively, this study unveils that the evolutionarily conserved COMPASS-like complex has been co-opted by the plant-specific SDG2 histone methyltransferase and mediates H3K4me3 deposition through an H2B monoubiquitination-independent pathway in Arabidopsis.

Plant Chromatin Catches the Sun.

Plants use solar radiation as energy source for photosynthesis. They also take advantage of the information provided by the varying properties of sunlight, such as wavelength, orientation, and periodicity, to trigger physiological and developmental adaptations to a changing environment. After more than a century of research efforts in plant photobiology, multiple light signaling pathways converging onto chromatin-based mechanisms have now been identified, which in some instances play critical roles in plant phenotypic plasticity. In addition to locus-specific changes linked to transcription regulation, light signals impact higher-order chromatin organization. Here, we summarize current knowledge on how light can affect the global composition and the spatial distribution of chromatin domains. We introduce emerging questions on the functional links between light signaling and the epigenome, and further discuss how different chromatin regulatory layers may interconnect during plant adaptive responses to light.

DET1-mediated degradation of a SAGA-like deubiquitination module controls H2Bub homeostasis.

DE-ETIOLATED 1 (DET1) is an evolutionarily conserved component of the ubiquitination machinery that mediates the destabilization of key regulators of cell differentiation and proliferation in multicellular organisms. In this study, we provide evidence from Arabidopsis that DET1 is essential for the regulation of histone H2B monoubiquitination (H2Bub) over most genes by controlling the stability of a deubiquitination module (DUBm). In contrast with yeast and metazoan DUB modules that are associated with the large SAGA complex, the Arabidopsis DUBm only comprises three proteins (hereafter named SGF11, ENY2 and UBP22) and appears to act independently as a major H2Bub deubiquitinase activity. Our study further unveils that DET1-DDB1-Associated-1 (DDA1) protein interacts with SGF11 in vivo, linking the DET1 complex to light-dependent ubiquitin-mediated proteolytic degradation of the DUBm. Collectively, these findings uncover a signaling path controlling DUBm availability, potentially adjusting H2Bub turnover capacity to the cell transcriptional status.

The SlCBL10 Calcineurin B-Like Protein Ensures Plant Growth under Salt Stress by Regulating Na+ and Ca2+ Homeostasis.

Characterization of a new tomato (Solanum lycopersicum) T-DNA mutant allowed for the isolation of the CALCINEURIN B-LIKE PROTEIN 10 (SlCBL10) gene whose lack of function was responsible for the severe alterations observed in the shoot apex and reproductive organs under salinity conditions. Physiological studies proved that SlCBL10 gene is required to maintain a proper low Na+/Ca2+ ratio in growing tissues allowing tomato growth under salt stress. Expression analysis of the main responsible genes for Na+ compartmentalization (i.e. Na+/H+ EXCHANGERs, SALT OVERLY SENSITIVE, HIGH-AFFINITY K+ TRANSPORTER 1;2, H+-pyrophosphatase AVP1 [SlAVP1] and V-ATPase [SlVHA-A1]) supported a reduced capacity to accumulate Na+ in Slcbl10 mutant leaves, which resulted in a lower uploading of Na+ from xylem, allowing the toxic ion to reach apex and flowers. Likewise, the tomato CATION EXCHANGER 1 and TWO-PORE CHANNEL 1 (SlTPC1), key genes for Ca2+ fluxes to the vacuole, showed abnormal expression in Slcbl10 plants indicating an impaired Ca2+ release from vacuole. Additionally, complementation assay revealed that SlCBL10 is a true ortholog of the Arabidopsis (Arabidopsis thaliana) CBL10 gene, supporting that the essential function of CBL10 is conserved in Arabidopsis and tomato. Together, the findings obtained in this study provide new insights into the function of SlCBL10 in salt stress tolerance. Thus, it is proposed that SlCBL10 mediates salt tolerance by regulating Na+ and Ca2+ fluxes in the vacuole, cooperating with the vacuolar cation channel SlTPC1 and the two vacuolar H+-pumps, SlAVP1 and SlVHA-A1, which in turn are revealed as potential targets of SlCBL10.

Profiling Developmentally and Environmentally Controlled Chromatin Reprogramming.

Dynamic reshuffling of the chromatin landscape is a recurrent theme orchestrated in many, if not all, plant developmental transitions and adaptive responses. Spatiotemporal variations of the chromatin properties on regulatory genes and on structural genomic elements trigger the establishment of distinct transcriptional contexts, which in some instances can epigenetically be inherited. Studies on plant cell plasticity during the differentiation of stem cells, including gametogenesis, or the specialization of vegetative cells in various organs, as well as the investigation of allele-specific gene regulation have long been impaired by technical challenges in generating specific chromatin profiles in complex or hardly accessible cell populations. Recent advances in increasing the sensitivity of genome-enabled technologies and in the isolation of specific cell types have allowed for overcoming such limitations. These developments hint at multilevel regulatory events ranging from nucleosome accessibility and composition to higher order chromatin organization and genome topology. Uncovering the large extent to which chromatin dynamics and epigenetic processes influence gene expression is therefore not surprisingly revolutionizing current views on plant molecular genetics and (epi)genomics as well as their perspectives in eco-evolutionary biology. Here, we introduce current methodologies to probe genome-wide chromatin variations for which protocols are detailed in this book chapter, with an emphasis on the plant model species Arabidopsis.