Dynamic changes in mRNA nucleocytoplasmic localization in the nitrate response of Arabidopsis roots.

Nitrate is a nutrient and signal that regulates gene expression. The nitrate response has been extensively characterized at the organism, organ, and cell-type-specific levels, but intracellular mRNA dynamics remain unexplored. To characterize nuclear and cytoplasmic transcriptome dynamics in response to nitrate, we performed a time-course expression analysis after nitrate treatment in isolated nuclei, cytoplasm, and whole roots. We identified 402 differentially localized transcripts (DLTs) in response to nitrate treatment. Induced DLT genes showed rapid and transient recruitment of the RNA polymerase II, together with an increase in the mRNA turnover rates. DLTs code for genes involved in metabolic processes, localization, and response to stimulus indicating DLTs include genes with relevant functions for the nitrate response that have not been previously identified. Using single-molecule RNA FISH, we observed early nuclear accumulation of the NITRATE REDUCTASE 1 (NIA1) transcripts in their transcription sites. We found that transcription of NIA1, a gene showing delayed cytoplasmic accumulation, is rapidly and transiently activated; however, its transcripts become unstable when they reach the cytoplasm. Our study reveals the dynamic localization of mRNAs between the nucleus and cytoplasm as an emerging feature in the temporal control of gene expression in response to nitrate treatment in Arabidopsis roots.

Differences in RAD51 transcriptional response and cell cycle dynamics reveal varying sensitivity to DNA damage among Arabidopsis thaliana root cell types.

Throughout their lifecycle, plants are subjected to DNA damage from various sources, both environmental and endogenous. Investigating the mechanisms of the DNA damage response (DDR) is essential to unravel how plants adapt to the changing environment, which can induce varying amounts of DNA damage. Using a combination of whole-mount single-molecule RNA fluorescence in situ hybridization (WM-smFISH) and plant cell cycle reporter lines, we investigated the transcriptional activation of a key homologous recombination (HR) gene, RAD51, in response to increasing amounts of DNA damage in Arabidopsis thaliana roots. The results uncover consistent variations in RAD51 transcriptional response and cell cycle arrest among distinct cell types and developmental zones. Furthermore, we demonstrate that DNA damage induced by genotoxic stress results in RAD51 transcription throughout the whole cell cycle, dissociating its traditional link with S/G2 phases. This work advances the current comprehension of DNA damage response in plants by demonstrating quantitative differences in DDR activation. In addition, it reveals new associations with the cell cycle and cell types, providing crucial insights for further studies of the broader response mechanisms in plants.

Convergence and molecular evolution of floral fragrance after independent transitions to self-fertilization.

Studying the independent evolution of similar traits provides valuable insights into the ecological and genetic factors driving phenotypic evolution.1 The transition from outcrossing to self-fertilization is common in plant evolution2 and is often associated with a reduction in floral attractive features such as display size, chemical signals, and pollinator rewards.3 These changes are believed to result from the reallocation of the resources used for building attractive flowers, as the need to attract pollinators decreases.2,3 We investigated the similarities in the evolution of flower fragrance following independent transitions to self-fertilization in Capsella.4,5,6,7,8,9 We identified several compounds that exhibited similar changes in different selfer lineages, such that the flower scent composition reflects mating systems rather than evolutionary history within this genus. We further demonstrate that the repeated loss of ß-ocimene emission, one of the compounds most strongly affected by these transitions, was caused by mutations in different genes. In one of the Capsella selfing lineages, the loss of its emission was associated with a mutation altering subcellular localization of the ortholog of TERPENE SYNTHASE 2. This mutation appears to have been fixed early after the transition to selfing through the capture of variants segregating in the ancestral outcrossing population. The large extent of convergence in the independent evolution of flower scent, together with the evolutionary history and molecular consequences of a causal mutation, suggests that the emission of specific volatiles evolved as a response to changes in ecological pressures rather than resource limitation.

Interplay between coding and non-coding regulation drives the Arabidopsis seed-to-seedling transition.

Translation of seed stored mRNAs is essential to trigger germination. However, when RNAPII re-engages RNA synthesis during the seed-to-seedling transition has remained in question. Combining csRNA-seq, ATAC-seq and smFISH in Arabidopsis thaliana we demonstrate that active transcription initiation is detectable during the entire germination process. Features of non-coding regulation such as dynamic changes in chromatin accessible regions, antisense transcription, as well as bidirectional non-coding promoters are widespread throughout the Arabidopsis genome. We show that sensitivity to exogenous ABSCISIC ACID (ABA) during germination depends on proximal promoter accessibility at ABA-responsive genes. Moreover, we provide genetic validation of the existence of divergent transcription in plants. Our results reveal that active enhancer elements are transcribed producing non-coding enhancer RNAs (eRNAs) as widely documented in metazoans. In sum, this study defining the extent and role of coding and non-coding transcription during key stages of germination expands our understanding of transcriptional mechanisms underlying plant developmental transitions.

Integrating analog and digital modes of gene expression at Arabidopsis FLC.

Quantitative gene regulation at the cell population level can be achieved by two fundamentally different modes of regulation at individual gene copies. A 'digital' mode involves binary ON/OFF expression states, with population-level variation arising from the proportion of gene copies in each state, while an 'analog' mode involves graded expression levels at each gene copy. At the Arabidopsis floral repressor FLOWERING LOCUS C (FLC), 'digital' Polycomb silencing is known to facilitate quantitative epigenetic memory in response to cold. However, whether FLC regulation before cold involves analog or digital modes is unknown. Using quantitative fluorescent imaging of FLC mRNA and protein, together with mathematical modeling, we find that FLC expression before cold is regulated by both analog and digital modes. We observe a temporal separation between the two modes, with analog preceding digital. The analog mode can maintain intermediate expression levels at individual FLC gene copies, before subsequent digital silencing, consistent with the copies switching OFF stochastically and heritably without cold. This switch leads to a slow reduction in FLC expression at the cell population level. These data present a new paradigm for gradual repression, elucidating how analog transcriptional and digital epigenetic memory pathways can be integrated.

Whole-mount smFISH allows combining RNA and protein quantification at cellular and subcellular resolution.

Multicellular organisms result from complex developmental processes largely orchestrated through the quantitative spatiotemporal regulation of gene expression. Yet, obtaining absolute counts of messenger RNAs at a three-dimensional resolution remains challenging, especially in plants, owing to high levels of tissue autofluorescence that prevent the detection of diffraction-limited fluorescent spots. In situ hybridization methods based on amplification cycles have recently emerged, but they are laborious and often lead to quantification biases. In this article, we present a simple method based on single-molecule RNA fluorescence in situ hybridization to visualize and count the number of mRNA molecules in several intact plant tissues. In addition, with the use of fluorescent protein reporters, our method also enables simultaneous detection of mRNA and protein quantity, as well as subcellular distribution, in single cells. With this method, research in plants can now fully explore the benefits of the quantitative analysis of transcription and protein levels at cellular and subcellular resolution in plant tissues.

Plant chromatin on the move: an overview of chromatin mobility during transcription and DNA repair.

It has become increasingly clear in recent years that chromosomes are highly dynamic entities. Chromatin mobility and re-arrangement are involved in many biological processes, including gene regulation and the maintenance of genome stability. Despite extensive studies on chromatin mobility in yeast and animal systems, up until recently, not much had been investigated at this level in plants. For plants to achieve proper growth and development, they need to respond rapidly and appropriately to environmental stimuli. Therefore, understanding how chromatin mobility can support plant responses may offer profound insights into the functioning of plant genomes. In this review, we discuss the state of the art related to chromatin mobility in plants, including the available technologies for their role in various cellular processes.

The plant-specific DDR factor SOG1 increases chromatin mobility in response to DNA damage.

Homologous recombination (HR) is a conservative DNA repair pathway in which intact homologous sequences are used as a template for repair. How the homology search happens in the crowded space of the cell nucleus is, however, still poorly understood. Here, we measure chromosome and double-strand break (DSB) site mobility in Arabidopsis thaliana, using lacO/LacI lines and two GFP-tagged HR reporters. We observe an increase in chromatin mobility upon the induction of DNA damage, specifically at the S/G2 phases of the cell cycle. This increase in mobility is lost in the sog1-1 mutant, a central transcription factor of the DNA damage response in plants. Also, DSB sites show particularly high mobility levels and their enhanced mobility requires the HR factor RAD54. Our data suggest that repair mechanisms promote chromatin mobility upon DNA damage, implying a role of this process in the early steps of the DNA damage response.

ANCHOR: A Technical Approach to Monitor Single-Copy Locus Localization in Planta.

Together with local chromatin structure, gene accessibility, and the presence of transcription factors, gene positioning is implicated in gene expression regulation. Although the basic mechanisms are expected to be conserved in eukaryotes, less is known about the role of gene positioning in plant cells, mainly due to the lack of a highly resolutive approach. In this study, we adapted the use of the ANCHOR system to perform real-time single locus detection in planta. ANCHOR is a DNA-labeling tool derived from the chromosome partitioning system found in many bacterial species. We demonstrated its suitability to monitor a single locus in planta and used this approach to track chromatin mobility during cell differentiation in Arabidopsis thaliana root epidermal cells. Finally, we discussed the potential of this approach to investigate the role of gene positioning during transcription and DNA repair in plants.

Visualizing and Measuring Single Locus Dynamics in Arabidopsis thaliana.

In eukaryotes, DNA is packed into an incredibly complex structure called chromatin. Although chromatin was often considered as a static entity, it is now clear that chromatin proteins and the chromatin fiber itself are in fact very dynamic. For instance, the packaging of the DNA into the nucleus requires an extraordinary degree of compaction but this should be achieved without compromising the accessibility to the transcription machinery and other nuclear processes. Approaches such as gene tagging have been established for living cells in order to detect, track, and analyze the mobility of single loci. In this chapter, we provide an experimental protocol for performing locus tracking in Arabidopsis thaliana roots and for characterizing locus mobility behavior via a Mean Square Displacement analysis.

Tidying-up the plant nuclear space: domains, functions, and dynamics.

Understanding how the packaging of chromatin in the nucleus is regulated and organized to guide complex cellular and developmental programmes, as well as responses to environmental cues is a major question in biology. Technological advances have allowed remarkable progress within this field over the last years. However, we still know very little about how the 3D genome organization within the cell nucleus contributes to the regulation of gene expression. The nuclear space is compartmentalized in several domains such as the nucleolus, chromocentres, telomeres, protein bodies, and the nuclear periphery without the presence of a membrane around these domains. The role of these domains and their possible impact on nuclear activities is currently under intense investigation. In this review, we discuss new data from research in plants that clarify functional links between the organization of different nuclear domains and plant genome function with an emphasis on the potential of this organization for gene regulation.

Noncoding SNPs influence a distinct phase of Polycomb silencing to destabilize long-term epigenetic memory at Arabidopsis FLC.

In Arabidopsis thaliana, the cold-induced epigenetic regulation of FLOWERING LOCUS C (FLC) involves distinct phases of Polycomb repressive complex 2 (PRC2) silencing. During cold, a PHD-PRC2 complex metastably and digitally nucleates H3K27me3 within FLC On return to warm, PHD-PRC2 spreads across the locus delivering H3K27me3 to maintain long-term silencing. Here, we studied natural variation in this process in Arabidopsis accessions, exploring Lov-1, which shows FLC reactivation on return to warm, a feature characteristic of FLC in perennial Brassicaceae This analysis identifies an additional phase in this Polycomb silencing mechanism downstream from H3K27me3 spreading. In this long-term silencing (perpetuated) phase, the PHD proteins are lost from the nucleation region and silencing is likely maintained by the read-write feedbacks associated with H3K27me3. A combination of noncoding SNPs in the nucleation region mediates instability in this long-term silencing phase with the result that Lov-1 FLC frequently digitally reactivates in individual cells, with a probability that diminishes with increasing cold duration. We propose that this decrease in reactivation probability is due to reduced DNA replication after flowering. Overall, this work defines an additional phase in the Polycomb mechanism instrumental in natural variation of silencing, and provides avenues to dissect broader evolutionary changes at FLC.

Temperature Sensing Is Distributed throughout the Regulatory Network that Controls FLC Epigenetic Silencing in Vernalization.

Many organisms need to respond to complex, noisy environmental signals for developmental decision making. Here, we dissect how Arabidopsis plants integrate widely fluctuating field temperatures over month-long timescales to progressively upregulate VERNALIZATION INSENSITIVE3 (VIN3) and silence FLOWERING LOCUS C (FLC), aligning flowering with spring. We develop a mathematical model for vernalization that operates on multiple timescales-long term (month), short term (day), and current (hour)-and is constrained by experimental data. Our analysis demonstrates that temperature sensing is not localized to specific nodes within the FLC network. Instead, temperature sensing is broadly distributed, with each thermosensory process responding to specific features of the plants' history of exposure to warm and cold. The model accurately predicts FLC silencing in new field data, allowing us to forecast FLC expression in changing climates. We suggest that distributed thermosensing may be a general property of thermoresponsive regulatory networks in complex natural environments.

Mechanisms Underlying the Environmentally Induced Plasticity of Leaf Morphology.

The primary function of leaves is to provide an interface between plants and their environment for gas exchange, light exposure and thermoregulation. Leaves have, therefore a central contribution to plant fitness by allowing an efficient absorption of sunlight energy through photosynthesis to ensure an optimal growth. Their final geometry will result from a balance between the need to maximize energy uptake while minimizing the damage caused by environmental stresses. This intimate relationship between leaf and its surroundings has led to an enormous diversification in leaf forms. Leaf shape varies between species, populations, individuals or even within identical genotypes when those are subjected to different environmental conditions. For instance, the extent of leaf margin dissection has, for long, been found to inversely correlate with the mean annual temperature, such that Paleobotanists have used models based on leaf shape to predict the paleoclimate from fossil flora. Leaf growth is not only dependent on temperature but is also regulated by many other environmental factors such as light quality and intensity or ambient humidity. This raises the question of how the different signals can be integrated at the molecular level and converted into clear developmental decisions. Several recent studies have started to shed the light on the molecular mechanisms that connect the environmental sensing with organ-growth and patterning. In this review, we discuss the current knowledge on the influence of different environmental signals on leaf size and shape, their integration as well as their importance for plant adaptation.

Presynaptic Biogenesis Requires Axonal Transport of Lysosome-Related Vesicles.

Nervous system function relies on the polarized architecture of neurons, established by directional transport of pre- and postsynaptic cargoes. While delivery of postsynaptic components depends on the secretory pathway, the identity of the membrane compartment(s) supplying presynaptic active zone (AZ) and synaptic vesicle (SV) proteins is unclear. Live imaging in Drosophila larvae and mouse hippocampal neurons provides evidence that presynaptic biogenesis depends on axonal co-transport of SV and AZ proteins in presynaptic lysosome-related vesicles (PLVs). Loss of the lysosomal kinesin adaptor Arl8 results in the accumulation of SV- and AZ-protein-containing vesicles in neuronal cell bodies and a corresponding depletion of SV and AZ components from presynaptic sites, leading to impaired neurotransmission. Conversely, presynaptic function is facilitated upon overexpression of Arl8. Our data reveal an unexpected function for a lysosome-related organelle as an important building block for presynaptic biogenesis.

Measuring Dynamics of Histone Proteins by Photobleaching in Arabidopsis Roots.

Histone proteins play an important role in determining chromatin structure and gene expression. Studies in several systems have established that histones are in continuous turnover within the chromatin. It is therefore important to quantitatively measure the binding dynamics of these proteins in vivo. Photobleaching-based approaches such as fluorescence recovery after photobleaching (FRAP) are advantageous in that they are applied to living cells at a single cell level. In this chapter, I provide a detailed experimental protocol on how to perform histone FRAP experiments in Arabidopsis thaliana roots and how to analyze the most important parameters.

Gaining insight into plant gene transcription using smFISH.

Single molecule RNA fluorescent in situ hybridization (smFISH) enables gene transcription to be assessed at the cellular level. In this point of view article, we describe our recent smFISH research in the model plant Arabidopsis thaliana and discuss how this technique could further knowledge of plant gene transcription in the future.

Disruption of an RNA-binding hinge region abolishes LHP1-mediated epigenetic repression.

Epigenetic maintenance of gene repression is essential for development. Polycomb complexes are central to this memory, but many aspects of the underlying mechanism remain unclear. LIKE HETEROCHROMATIN PROTEIN 1 (LHP1) binds Polycomb-deposited H3K27me3 and is required for repression of many Polycomb target genes in Arabidopsis Here we show that LHP1 binds RNA in vitro through the intrinsically disordered hinge region. By independently perturbing the RNA-binding hinge region and H3K27me3 (trimethylation of histone H3 at Lys27) recognition, we found that both facilitate LHP1 localization and H3K27me3 maintenance. Disruption of the RNA-binding hinge region also prevented formation of subnuclear foci, structures potentially important for epigenetic repression.

Cell-Size-Dependent Transcription of FLC and Its Antisense Long Non-coding RNA COOLAIR Explain Cell-to-Cell Expression Variation.

Single-cell quantification of transcription kinetics and variability promotes a mechanistic understanding of gene regulation. Here, using single-molecule RNA fluorescence in situ hybridization and mathematical modeling, we dissect cellular RNA dynamics for Arabidopsis FLOWERING LOCUS C (FLC). FLC expression quantitatively determines flowering time and is regulated by antisense (COOLAIR) transcription. In cells without observable COOLAIR expression, we quantify FLC transcription initiation, elongation, intron processing, and lariat degradation, as well as mRNA release from the locus and degradation. In these heterogeneously sized cells, FLC mRNA number increases linearly with cell size, resulting in a large cell-to-cell variability in transcript level. This variation is accounted for by cell-size-dependent, Poissonian FLC mRNA production, but not by large transcriptional bursts. In COOLAIR-expressing cells, however, antisense transcription increases with cell size and contributes to FLC transcription decreasing with cell size. Our analysis therefore reveals an unexpected role for antisense transcription in modulating the scaling of transcription with cell size.