In planta imaging of pyridine nucleotides using second-generation fluorescent protein biosensors.

Redox changes of pyridine nucleotides in cellular compartments are highly dynamic and their equilibria are under the influence of various reducing and oxidizing reactions. To obtain spatiotemporal data on pyridine nucleotides in living plant cells, typical biochemical approaches require cell destruction. To date, genetically encoded fluorescent biosensors are considered to be the best option to bridge the existing technology gap, as they provide a fast, accurate, and real-time readout. However, the existing pyridine nucleotides genetically encoded fluorescent biosensors are either sensitive to pH change or slow in dissociation rate. Herein, we employed the biosensors which generate readouts that are pH stable for in planta measurement of NADH/NAD+ ratio and NADPH level. We generated transgenic Arabidopsis lines that express these biosensors in plastid stroma and cytosol of whole plants and pollen tubes under the control of CaMV 35S and LAT52 promoters, respectively. These transgenic biosensor lines allow us to monitor real-time dynamic changes in NADH/NAD+ ratio and NADPH level in the plastids and cytosol of various plant tissues, including pollen tubes, root hairs, and mesophyll cells, using a variety of fluorescent instruments. We anticipate that these valuable transgenic lines may allow improvements in plant redox biology studies.

The art of painting chromosome loops.

How to get a metre of DNA into a tiny space while preserving its functional characteristics? This question seems easy to pose, but the answer is far from being trivial. Facing this riddle, salvation came from technical improvements in microscopy and in situ hybridisation techniques applied to cytogenetics. Here, we would like to look into the past at one of these pure cytogenetics articles that makes a breakthrough in addressing this question in plant science. Our choice fell on the work published two decades ago by Fransz et al. (2002). Besides the elegant manner in which DNA probes were organised to bring into light the out-looping arrangement of interphase chromosomes in Arabidopsis thaliana nuclei, this article perfectly illustrates that painting is not reserved to the fine art. As for whether emotional expression prioritised by artists can sometimes hide behind scientific empirical evidence, there is only a small step to make to the general case.

Nuclear envelope dynamics in connection to chromatin remodeling.

The nucleus is a central organelle of eukaryotic cells undergoing dynamic structural changes during cellular fundamental processes such as proliferation and differentiation. These changes rely on the integration of developmental and stress signals at the nuclear envelope (NE), orchestrating responses at the nucleo-cytoplasmic interface for efficient genomic functions such as DNA transcription, replication and repair. While in animals, correlation has already been established between NE dynamics and chromatin remodeling using last-generation tools and cutting-edge technologies, this topic is just emerging in plants, especially in response to mechanical cues. This review summarizes recent data obtained in this field with more emphasis on the mechanical stress response. It also highlights similarities/differences between animal and plant cells at multiples scales, from the structural organization of the nucleo-cytoplasmic continuum to the functional impacts of NE dynamics.

Lysine 27 of histone H3.3 is a fine modulator of developmental gene expression and stands as an epigenetic checkpoint for lignin biosynthesis in Arabidopsis.

Chromatin is a dynamic platform within which gene expression is controlled by epigenetic modifications, notably targeting amino acid residues of histone H3. Among them is lysine 27 of H3 (H3K27), the trimethylation of which by the Polycomb Repressive Complex 2 (PRC2) is instrumental in regulating spatiotemporal patterns of key developmental genes. H3K27 is also subjected to acetylation and is found at sites of active transcription. Most information on the function of histone residues and their associated modifications in plants was obtained from studies of loss-of-function mutants for the complexes that modify them. To decrypt the genuine function of H3K27, we expressed a non-modifiable variant of H3 at residue K27 (H3.3K27A ) in Arabidopsis, and developed a multi-scale approach combining in-depth phenotypical and cytological analyses, with transcriptomics and metabolomics. We uncovered that the H3.3K27A variant causes severe developmental defects, part of them are reminiscent of PRC2 mutants, part of them are new. They include early flowering, increased callus formation and short stems with thicker xylem cell layer. This latest phenotype correlates with mis-regulation of phenylpropanoid biosynthesis. Overall, our results reveal novel roles of H3K27 in plant cell fates and metabolic pathways, and highlight an epigenetic control point for elongation and lignin composition of the stem.

A Microfluidic-Like System (MLS) to Grow, Image, and Quantitatively Characterize Rigidity Sensing by Plant's Roots and Root Hair Cells.

Plant's roots grow in soils of different rigidities. Understanding how the stiffness of the surrounding environment impacts growth and cell fate of roots and root hair cells is an important and open question. Here, we describe a simple method to setup a microfluidic-like system (MLS) to tackle this question. This system enables to grow plantlets during weeks in microfluidic chips filled with gels of controlled stiffness and to image them under a microscope from a few minutes up to a few days. Furthermore, MLS keeps the numerous benefits of microfluidic chips, such as high-resolution imaging, precise control of the geometry of growth, and standardization of the measurements. In sum, MLS enables one to quantitatively test, even on long time scales, the effect of the rigidity and the geometry of the environment on the growth of roots and root hair cells, including mechanotransduction to the nucleus.

Wheat Type One Protein Phosphatase Participates in the Brassinosteroid Control of Root Growth via Activation of BES1.

Brassinosteroids (BRs) play key roles in diverse plant growth processes through a complex signaling pathway. Components orchestrating the BR signaling pathway include receptors such as kinases, transcription factors, protein kinases and phosphatases. The proper functioning of the receptor kinase BRI1 and the transcription factors BES1/BZR1 depends on their dephosphorylation by type 2A protein phosphatases (PP2A). In this work, we report that an additional phosphatase family, type one protein phosphatases (PP1), contributes to the regulation of the BR signaling pathway. Co-immunoprecipitation and BiFC experiments performed in Arabidopsis plants overexpressing durum wheat TdPP1 showed that TdPP1 interacts with dephosphorylated BES1, but not with the BRI1 receptor. Higher levels of dephosphorylated, active BES1 were observed in these transgenic lines upon BR treatment, indicating that TdPP1 modifies the BR signaling pathway by activating BES1. Moreover, ectopic expression of durum wheat TdPP1 lead to an enhanced growth of primary roots in comparison to wild-type plants in presence of BR. This phenotype corroborates with a down-regulation of the BR-regulated genes CPD and DWF4. These data suggest a role of PP1 in fine-tuning BR-driven responses, most likely via the control of the phosphorylation status of BES1.

Real-time tracking of root hair nucleus morphodynamics using a microfluidic approach.

Root hairs (RHs) are tubular extensions of root epidermal cells that favour nutrient uptake and microbe interactions. RHs show a fast apical growth, constituting a unique single cell model system for analysing cellular morphodynamics. In this context, live cell imaging using microfluidics recently developed to analyze root development is appealing, although high-resolution imaging is still lacking to enable an investigation of the accurate spatiotemporal morphodynamics of organelles. Here, we provide a powerful coverslip based microfluidic device (CMD) that enables us to capture high resolution confocal imaging of Arabidopsis RH development with real-time monitoring of nuclear movement and shape changes. To validate the setup, we confirmed the typical RH growth rates and the mean nuclear positioning previously reported with classical methods. Moreover, to illustrate the possibilities offered by the CMD, we have compared the real-time variations in the circularity, area and aspect ratio of nuclei moving in growing and mature RHs. Interestingly, we observed higher aspect ratios in the nuclei of mature RHs, correlating with higher speeds of nuclear migration. This observation opens the way for further investigations of the effect of mechanical constraints on nuclear shape changes during RH growth and nuclear migration and its role in RH and plant development.

Tissue folding at the organ-meristem boundary results in nuclear compression and chromatin compaction.

Artificial mechanical perturbations affect chromatin in animal cells in culture. Whether this is also relevant to growing tissues in living organisms remains debated. In plants, aerial organ emergence occurs through localized outgrowth at the periphery of the shoot apical meristem, which also contains a stem cell niche. Interestingly, organ outgrowth has been proposed to generate compression in the saddle-shaped organ-meristem boundary domain. Yet whether such growth-induced mechanical stress affects chromatin in plant tissues is unknown. Here, by imaging the nuclear envelope in vivo over time and quantifying nucleus deformation, we demonstrate the presence of active nuclear compression in that domain. We developed a quantitative pipeline amenable to identifying a subset of very deformed nuclei deep in the boundary and in which nuclei become gradually narrower and more elongated as the cell contracts transversely. In this domain, we find that the number of chromocenters is reduced, as shown by chromatin staining and labeling, and that the expression of linker histone H1.3 is induced. As further evidence of the role of forces on chromatin changes, artificial compression with a MicroVice could induce the ectopic expression of H1.3 in the rest of the meristem. Furthermore, while the methylation status of chromatin was correlated with nucleus deformation at the meristem boundary, such correlation was lost in the h1.3 mutant. Altogether, we reveal that organogenesis in plants generates compression that is able to have global effects on chromatin in individual cells.

Chromatin Manipulation and Editing: Challenges, New Technologies and Their Use in Plants.

An ongoing challenge in functional epigenomics is to develop tools for precise manipulation of epigenetic marks. These tools would allow moving from correlation-based to causal-based findings, a necessary step to reach conclusions on mechanistic principles. In this review, we describe and discuss the advantages and limits of tools and technologies developed to impact epigenetic marks, and which could be employed to study their direct effect on nuclear and chromatin structure, on transcription, and their further genuine role in plant cell fate and development. On one hand, epigenome-wide approaches include drug inhibitors for chromatin modifiers or readers, nanobodies against histone marks or lines expressing modified histones or mutant chromatin effectors. On the other hand, locus-specific approaches consist in targeting precise regions on the chromatin, with engineered proteins able to modify epigenetic marks. Early systems use effectors in fusion with protein domains that recognize a specific DNA sequence (Zinc Finger or TALEs), while the more recent dCas9 approach operates through RNA-DNA interaction, thereby providing more flexibility and modularity for tool designs. Current developments of "second generation", chimeric dCas9 systems, aiming at better targeting efficiency and modifier capacity have recently been tested in plants and provided promising results. Finally, recent proof-of-concept studies forecast even finer tools, such as inducible/switchable systems, that will allow temporal analyses of the molecular events that follow a change in a specific chromatin mark.

GIP1 and GIP2 Contribute to the Maintenance of Genome Stability at the Nuclear Periphery.

The maintenance of genetic information is important in eukaryotes notably through mechanisms occurring at the nuclear periphery where inner nuclear membrane proteins and nuclear pore-associated components are key factors regulating the DNA damage response (DDR). However, this aspect of DDR regulation is still poorly documented in plants. We addressed here how genomic stability is impaired in the gamma-tubulin complex component 3-interacting protein (gip1gip2) double mutants showing defective nuclear shaping. Using neutral comet assays for DNA double-strand breaks (DSBs) detection, we showed that GIP1 and GIP2 act redundantly to maintain genome stability. At the cellular level, γ-H2AX foci in gip1gip2 were more abundant and heterogeneous in their size compared to wild-type (WT) in root meristematic nuclei, indicative of constitutive DNA damage. This was linked to a constitutive activation of the DDR in the gip1gip2 mutant, with more emphasis on the homologous recombination (HR) repair pathway. In addition, we noticed the presence of numerous RAD51 foci which did not colocalize with γ-H2AX foci. The expression of GIP1-GFP in the double mutant rescued the cellular response to DNA damage, leading to the systematic colocalization of RAD51 and γ-H2AX foci. Interestingly, a significant proportion of RAD51 foci colocalized with GIP1-GFP at the nuclear periphery. Altogether, our data suggest that GIPs may partly contribute to the spatio-temporal recruitment of RAD51 at the nuclear periphery.

Is the plant nucleus a mechanical rheostat?

Beyond its biochemical nature, the nucleus is also a physical object. There is accumulating evidence that its mechanics plays a key role in gene expression, cytoskeleton organization, and more generally in cell and developmental biology. Building on data mainly obtained from the animal literature, we show how nuclear mechanics may orchestrate development and gene expression. In other words, the nucleus may play the additional role of a mechanical rheostat. Although data from plant systems are still scarce, we pinpoint recent advances and highlight some differences with animal systems. Building on this survey, we propose a list of prospects for future research in plant nuclear mechanotransduction and development.

Corrigendum: The GIP Gamma-Tubulin Complex-Associated Proteins are Involved in Nuclear Architecture in Arabidopsis Thaliana.

[This corrects the article DOI: 10.3389/fpls.2013.00480/full.].

The F-Box-Like Protein FBL17 Is a Regulator of DNA-Damage Response and Colocalizes with RETINOBLASTOMA RELATED1 at DNA Lesion Sites.

In Arabidopsis (Arabidopsis thaliana), the F-box protein F-BOX-LIKE17 (FBL17) was previously identified as an important cell-cycle regulatory protein. FBL17 is required for cell division during pollen development and for normal cell-cycle progression and endoreplication during the diploid sporophyte phase. FBL17 was reported to control the stability of the CYCLIN-DEPENDENT KINASE inhibitor KIP-RELATED PROTEIN (KRP), which may underlie the drastic reduction in cell division activity in both shoot and root apical meristems observed in fbl17 loss-of-function mutants. However, whether FBL17 has other substrates and functions besides degrading KRPs remains poorly understood. Here we show that mutation of FBL17 leads not only to misregulation of cell cycle genes, but also to a strong upregulation of genes involved in DNA damage and repair processes. This phenotype is associated with a higher frequency of DNA lesions in fbl17 and increased cell death in the root meristem, even in the absence of genotoxic stress. Notably, the constitutive activation of DNA damage response genes is largely SOG1-independent in fbl17 In addition, through analyses of root elongation, accumulation of cell death, and occurrence of γH2AX foci, we found that fbl17 mutants are hypersensitive to DNA double-strand break-induced genotoxic stress. Notably, we observed that the FBL17 protein is recruited at nuclear foci upon double-strand break induction and colocalizes with γH2AX, but only in the presence of RETINOBLASTOMA RELATED1. Altogether, our results highlight a role for FBL17 in DNA damage response, likely by ubiquitylating proteins involved in DNA-damage signaling or repair.

Mechanical Shielding in Plant Nuclei.

In animal single cells in culture, nuclear geometry and stiffness can be affected by mechanical cues, with important consequences for chromatin status and gene expression. This calls for additional investigation into the corresponding physiological relevance in a multicellular context and in different mechanical environments. Using the Arabidopsis root as a model system, and combining morphometry and micro-rheometry, we found that hyperosmotic stress decreases nuclear circularity and size and increases nuclear stiffness in meristematic cells. These changes were accompanied by enhanced expression of touch response genes. The nuclear response to hyperosmotic stress was rescued upon return to iso-osmotic conditions and could even lead to opposite trends upon hypo-osmotic stress. Interestingly, nuclei in a mutant impaired in the functions of the gamma-tubulin complex protein 3 (GCP3) interacting protein (GIP)/MZT1 proteins at the nuclear envelope were almost insensitive to such osmotic changes. The gip1gip2 mutant exhibited constitutive hyperosmotic stress response with stiffer and deformed nuclei, as well as touch response gene induction. The mutant was also resistant to lethal hyperosmotic conditions. Altogether, we unravel a stereotypical geometric, mechanical, and genetic nuclear response to hyperosmotic stress in plants. Our data also suggest that chromatin acts as a gel that stiffens in hyperosmotic conditions and that the nuclear-envelope-associated protein GIPs act as negative regulators of this response.

The wheat TdRL1 is the functional homolog of the rice RSS1 and promotes plant salt stress tolerance.

KEY MESSAGE: Rice rss1 complementation assays show that wheat TdRL1 and RSS1 are true functional homologs. TdRL1 over-expression in Arabidopsis conferred salt stress tolerance and alleviated ROS accumulation. Plants have developed highly flexible adaptive responses to their ever-changing environment, which are often mediated by intrinsically disordered proteins (IDP). RICE SALT SENSITIVE 1 and Triticum durum RSS1-Like 1 protein (TdRL1) are both IDPs involved in abiotic stress responses, and possess conserved D and DEN-Boxes known to be required for post-translational degradation by the APC/Ccdc20 cyclosome. To further understand their function, we performed a computational analysis to compare RSS1 and TdRL1 co-expression networks revealing common gene ontologies, among which those related to cell cycle progression and regulation of microtubule (MT) networks were over-represented. When over-expressed in Arabidopsis, TdRL1::GFP was present in dividing cells and more visible in cortical and endodermal cells of the Root Apical Meristem (RAM). Incubation with the proteasome inhibitor MG132 stabilized TdRL1::GFP expression in RAM cells showing a post-translational regulation. Moreover, immuno-cytochemical analyses of transgenic roots showed that TdRL1 was present in the cytoplasm and within the microtubular spindle of mitotic cells, while, in interphasic cells, it was rather restricted to the cytoplasm with a spotty pattern at the nuclear periphery. Interestingly in cells subjected to stress, TdRL1 was partly relocated into the nucleus. Moreover, TdRL1 transgenic lines showed increased germination rates under salt stress conditions as compared to wild type. This enhanced salt stress tolerance was associated to an alleviation of oxidative damage. Finally, when expressed in the rice rss1 mutant, TdRL1 suppressed its dwarf phenotype upon salt stress, confirming that both proteins are true functional homologs required for salt stress tolerance in cereals.

Genome wide identification of wheat and Brachypodium type one protein phosphatases and functional characterization of durum wheat TdPP1a.

Reversible phosphorylation is an essential mechanism regulating signal transduction during development and environmental stress responses. An important number of dephosphorylation events in the cell are catalyzed by type one protein phosphatases (PP1), which catalytic activity is driven by the binding of regulatory proteins that control their substrate specificity or subcellular localization. Plants harbor several PP1 isoforms accounting for large functional redundancies. While animal PP1s were reported to play relevant roles in controlling multiple cellular processes, plant orthologs remain poorly studied. To decipher the role of plant PP1s, we compared PP1 genes from three monocot species, Brachypodium, common wheat and rice at the genomic and transcriptomic levels. To gain more insight into the wheat PP1 proteins, we identified and characterized TdPP1a, the first wheat type one protein phosphatase from a Tunisian durum wheat variety Oum Rabiaa3. TdPP1a is highly conserved in sequence and structure when compared to mammalian, yeast and other plant PP1s. We demonstrate that TdPP1a is an active, metallo-dependent phosphatase in vitro and is able to interact with AtI2, a typical regulator of PP1 functions. Also, TdPP1a is capable to complement the heat stress sensitivity of the yeast mutant indicating that TdPP1a is functional also in vivo. Moreover, transient expression of TdPP1a::GFP in tobacco leaves revealed that it is ubiquitously distributed within the cell, with a strong accumulation in the nucleus. Finally, transcriptional analyses showed similar expression levels in roots and leaves of durum wheat seedlings. Interestingly, the expression in leaves is significantly induced following salinity stress, suggesting a potential role of TdPP1a in wheat salt stress response.

Nuclear envelope: a new frontier in plant mechanosensing?

In animals, it is now well established that forces applied at the cell surface are propagated through the cytoskeleton to the nucleus, leading to deformations of the nuclear structure and, potentially, to modification of gene expression. Consistently, altered nuclear mechanics has been related to many genetic disorders, such as muscular dystrophy, cardiomyopathy and progeria. In plants, the integration of mechanical signals in cell and developmental biology has also made great progress. Yet, while the link between cell wall stresses and cytoskeleton is consolidated, such cortical mechanical cues have not been integrated with the nucleoskeleton. Here, we propose to take inspiration from studies on animal nuclei to identify relevant methods amenable to probing nucleus mechanics and deformation in plant cells, with a focus on microrheology. To identify potential molecular targets, we also compare the players at the nuclear envelope, namely lamina and LINC complex, in both plant and animal nuclei. Understanding how mechanical signals are transduced to the nucleus across kingdoms will likely have essential implications in development (e.g. how mechanical cues add robustness to gene expression patterns), in the nucleoskeleton-cytoskeleton nexus (e.g. how stress is propagated in turgid/walled cells), as well as in transcriptional control, chromatin biology and epigenetics.

MGO3 and GIP1 act synergistically for the maintenance of centromeric cohesion.

The control of genomic maintenance during S phase is crucial in eukaryotes. It involves the establishment of sister chromatid cohesion, ensuring faithful chromosome segregation, as well as proper DNA replication and repair to preserve genetic information. In animals, nuclear periphery proteins - including inner nuclear membrane proteins and nuclear pore-associated components - are key factors which regulate DNA integrity. Corresponding functional homologues are not so well known in plants which may have developed specific mechanisms due to their sessile life. We have already characterized the Gamma-tubulin Complex Protein 3-interacting proteins (GIPs) as essential regulators of centromeric cohesion at the nuclear periphery. GIPs were also shown to interact with TSA1, first described as a partner of the epigenetic regulator MGOUN3 (MGO3)/BRUSHY1 (BRU1)/TONSOKU (TSK) involved in genomic maintenance. Here, using genetic analyses, we show that the mgo3gip1 mutants display an impaired and pleiotropic development including fasciation. We also provide evidence for the contribution of both MGO3 and GIP1 to the regulation of centromeric cohesion in Arabidopsis.

GIP Contributions to the Regulation of Centromere at the Interface Between the Nuclear Envelope and the Nucleoplasm.

Centromeres are known as specific chromatin domains without which eukaryotic cells cannot divide properly during mitosis. Despite the considerable efforts to understand the centromere/kinetochore assembly during mitosis, until recently, comparatively few studies have dealt with the regulation of centromere during interphase. Here, we briefly review and discuss past and recent advances about the architecture of centromeres and their regulation during the cell cycle. Furthermore, we highlight and discuss new findings and hypotheses regarding the specific regulation of centromeres in both plant and animal nuclei, especially with GIP proteins at the interface between the nuclear envelope and the nucleoplasm.