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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.

SummaryChromatin 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), which trimethylation by the Polycomb Repressive Complex 2 (PRC2) is instrumental in regulating spatio-temporal patterns of key developmental genes. H3K27 is also subjected to acetylation, 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.In order 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 reminiscent of PRC2 mutants, part of them 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.

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.

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.

International audience; 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.

International audience; 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.

International audience; Key messageRice 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.AbstractPlants 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/C-cdc20 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.

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.

The spindle assembly checkpoint (SAC) is a refined surveillance mechanism which ensures that chromosomes undergoing mitosis do not segregate until they are properly attached to the spindle microtubules (MT). The SAC has been extensively studied in metazoans and yeast, but little is known about its role in plants. We identified proteins interacting with a MT-associated protein MAP65-3, which plays a critical role in organising mitotic MT arrays, and carried out a functional analysis of previously and newly identified SAC components. We show that Arabidopsis SAC proteins BUB3.1, MAD2, BUBR1/MAD3s and BRK1 interact with each other and with MAP65-3. We found that two BUBR1/MAD3s interacted specifically at centromeres. When stably expressed in Arabidopsis, BRK1 localised to the kinetochores during all stages of the mitotic cell cycle. Early in mitosis, BUB3.1 and BUBR1/MAD3.1 localise to the mitotic spindle, where MAP65-3 organises spindle MTs. A double-knockout mad3.1 mad3.2 mutant presented spindle MT abnormalities, chromosome misalignments on the metaphase plate and the production of lagging chromosomes and micronuclei during mitosis. We conclude that BRK1 and BUBR1/MAD3-related proteins play a key role in ensuring faithful chromosome segregation during mitosis and that their interaction with MAP65-3 may be important for the regulation of MT-chromosome attachment.

The microtubular cytoskeleton plays a major role in cellular organization and proliferation. The first step in construction of a microtubule is microtubule nucleation. Individual microtubules then participate in organization of more complex microtubule arrays. A strong body of evidence suggests that the underlying molecular mechanisms involve protein complexes that are conserved among eukaryotes. However, plant cell specificities, mainly characterized by the presence of a cell wall and the absence of centrosomes, must be taken into account to understand their mitotic processes. The goal of this review is to summarize and discuss current knowledge regarding the mechanisms involved in plant spindle assembly during early mitotic events. The functions of the proteins currently characterized at microtubule nucleation sites and involved in spindle assembly are considered during cell-cycle progression from G2 phase to metaphase.

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.

In plants, different forms of programmed cell death (PCD) have been identified, but they only partially correspond to those described for animals, which is most probably due to structural differences between animal and plant cells. Here, the results show that in tobacco BY-2 cells, bleomycin (BLM), an inducer of double-strand breaks (DSBs), triggers a novel type of non-apoptotic PCD with paraptotic-like features. Analysis of numerous PCD markers revealed an extensive vacuolization, vacuolar rupture, and chromatin condensation, but no apoptotic DNA fragmentation, fragmentation of the nuclei, or sensitivity to caspase inhibitors. BLM-induced PCD was cell cycle regulated, occurring predominantly upon G(2)/M cell cycle checkpoint activation. In addition, this paraptotic-like PCD was at least partially inhibited by caffeine, a known inhibitor of DNA damage sensor kinases ATM and ATR. Interestingly, overexpression of one NtE2F transcriptional factor, whose homologues play a dual role in animal apoptosis and DNA repair, reduced PCD induction and modulated G(2)/M checkpoint activation in BY-2 cells. These observations provide a solid ground for further investigations into the paraptotic-like PCD in plants, which might represent an ancestral non-apoptotic form of PCD conserved among animals, protists, and plants.

Summary • The Arabidopsis genome possesses two confirmed Cytochrome P450 Reductase (CPR) genes, ATR1 and ATR2, together with a third putative homologue, ATR3, which annotation is questionable. • Phylogenetic analysis classified ATR3 as a CPR-like protein sharing homologies with the animal cytosolic dual flavin reductases, NR1 and Fre-1, distinct from the microsomal CPRs, ATR1 and ATR2. Like NR1 and Fre-1, ATR3 lacks the N-terminal endoplasmic reticulum (ER) anchor domain of CPRs and is localized in the cytoplasm. Recombinant ATR3 in plant soluble extracts was able to reduce cytochrome c but failed to reduce the human P450 CYP1A2. • Loss of ATR3 function resulted in early embryo lethality indicating that this reductase activity is essential. A yeast 2-hybrid screen identified a unique interaction of ATR3 with the homologue of the human anti-apoptotic CIAPIN1 and the yeast Dre2 protein. • This interaction suggests two possible roles for ATR3 in the control of cell death and in chromosome segregation at mitosis. Consistent with these results, the promoter of ATR3 is activated during cell cycle progression. Together these results demonstrated that ATR3 belongs to the NR1 subfamily of diflavin reductases whose characterized members are involved in essential cellular functions.

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.

In eukaryotes, both microtubules (MTs) and microfilaments (MFs) are cytoskeletal polymers involved in meristematic cell proliferation. While animal cells build their MT arrays from structured organelles, such as centrosomes, and while they depolymerise their MFs and become round during mitosis, vascular plant cells lack centrosomes, maintain a filamentous actin cage around the spindle and are surrounded by a cell wall, preventing cellular mobility. During the cell cycle, plants activate specific dispersed MT nucleating sites, revealing successive plant-specific cytoskeletal arrays. The MF meshwork surrounding the spindle eventually drives the centrifugal growth of MTs, which leads Golgi-derived vesicles to fuse and separate each daughter cell during cytokinesis. The main orientation of actin fibres is parallel to that of spindle MTs, while a perpendicular constriction ring ensures animal daughter cell separation. However, despite the differences in their cytoskeleton behaviour and dynamics, both cell types succeed in controlled chromosome segregation. Key Concepts Mitosis is a short cell cycle period during which the cytoskeleton ensures balanced segregation of duplicated sister chromatids into two daughter cells. Plant cells have to deal with their pecto-cellulosic cell wall, constraining the orientation of the division axis in order to control morphogenesis. In plants, primary stem cells are located in shoot and root apical meristems (SAM and RAM) and secondary growth is ensured by cambium activation. Besides the conservation of microtubules (MTs) and actin filaments (MFs), required for successful karyokinesis and cytokinesis, respectively, in all eukaryotes, plant mitotic cells present an original cytoskeleton organisation. All the somatic cells of vascular plants lack centrosomes, and microtubule organising centres are spread all over the nuclear envelope, at the cell cortex and along pre-existing microtubules. Preceding the mitotic step per se, maintenance of centromere/kinetochore cohesion and integrity is required for the building of a bipolar spindle and accurate chromosome segregation. During mitosis, the activity of MT nucleating complexes (TuRCs) and MT-associated proteins (MAPs) leads to a succession of MT arrays that reorganise in the cytoplasm: cortical MTs/perinuclear MTs/pre-prophase band MTs/spindle MTs/phragmoplast MTs. The spindle apparatus, consisting of kinetochore fibres and interpolar MTs, is barrel-shaped and lacks polar asters. Some of the MAPs also connect MFs. Gamma-tubulin containing complexes and augmin complexes participate in the nucleation of new highly dynamic MTs that ensure spindle robustness. Actin filaments intermingle with MTs and do not depolymerise during mitosis, except in the PPB, which becomes an actin-deplete zone (ADZ). The cell plate expands centrifugally to the cell edges thanks to actin filaments, preceding MTs on which Golgi-derived vesicles move towards the equator and fuse together. Keywords: plants; cell division; mitosis; cytoskeleton; microtubules; actin microfilaments; preprophase band; spindle; phragmoplast; centromere/kinetochore; centromere cohesion

The promoters of several E2F-regulated genes identified in plants contain a variety of E2F motifs, notably a composite element consisting of a “CDE-like element” C/GGCGG on one strand, described as repressor in animals, associated with an E2F element on the complementary strand. This detailed study throughout plant development using ribonucleotide reductase promoters, allows us to propose a model, where E2F and composite elements play a dual role. Such regulation is mainly conditioned by the availability of E2F factors in tissues and during the cell cycle in tobacco.

*† Summary Accurate DNA replication is one of the most important events in the life of a cell. To perform this task, the cell utilizes several DNA polymerase complexes. We investigated the role of DNA polymerase e during gametophyte and seed development using forward and reverse genetic approaches. In Arabidopsis, the catalytic subunit of this complex is encoded by two genes, AtPOL2a and AtPOL2b, whereas the second largest regulatory subunit AtDPB2 is present as a unique complete copy. Disruption of AtPOL2a or AtDPB2 resulted in a sporophytic embryo-defective phenotype, whilst mutations in AtPOL2b produced no visible effects. Loss of AtDPB2 function resulted in a severe reduction in nuclear divisions, both in the embryo and in the endosperm. Mutations in AtPOL2a allowed several rounds of mitosis to proceed, often with aberrant planes of division. Moreover, AtDPB2 was not expressed during development of the female gametophyte, which requires three post-meiotic nuclear divisions. Since a consensus binding site for E2F transcription factors was identified in the promoter region of both genes, the promoter–reporter fusion technique was used to show that luciferase activity was increased at specific phases of the cell cycle in synchronized tobacco BY-2 cells. Our results support the idea that fertilization may utilize the mechanisms of cell cycle transcriptional regulation of genes to reactivate the divisions of the oosphere and central cell.

E2F factors are implicated in various cellular processes including specific gene induction at the G1/S transition of the cell cycle. We present in this study a novel regulatory aspect for the tobacco large subunit of ribonucleotide reductase (R1a) and its encoding gene (RNR1a) in the UV-C response. By structural analyses, two E2F sites were identified on the promoter of this gene. Functional analysis showed that, in addition to their role in the specific G1/S induction of the RNR1a gene, both E2F sites were important for regulating specific RNR1a gene expression in response to UV-C irradiation in non-synchronized and synchronized cells. Concomitantly, western blot and cellular analyses showed an increase of a 60 kDa E2F factor and a transient translocation of a GFP-R1a protein fusion from cytoplasm to nucleus in response to UV irradiation.

Centromeres are chromatin structures that are required for proper separation of chromosomes during mitosis and meiosis. The centromere is composed of centromeric DNA, often enriched in satellite repeats, and kinetochore complex proteins. To date, over 100 kinetochore components have been identified in various eukaryotes. Kinetochore assembly begins with incorporation of centromeric histone H3 variant CENH3 into centromeric nucleosomes. Protein components of the kinetochore are either present at centromeres throughout the cell cycle or localize to centromeres transiently, prior to attachment of microtubules to each kinetochore in prometaphase of mitotic cells. This is the case for the spindle assembly checkpoint (SAC) proteins in animal cells. The SAC complex ensures equal separation of chromosomes between daughter nuclei by preventing anaphase onset before metaphase is complete, i.e. the sister kinetochores of all chromosomes are attached to spindle fibers from opposite poles. In this review, we focus on the organization of centromeric DNA and the kinetochore assembly in plants. We summarize recent advances regarding loading of CENH3 into the centromere, and the subcellular localization and protein-protein interactions of Arabidopsis thaliana proteins involved in kinetochore assembly and function. We describe the transcriptional activity of corresponding genes based on in silico analysis of their promoters and cell cycle-dependent expression. Additionally, barley homologs of all selected A. thaliana proteins have been identified in silico, and their sequences and domain structures are presented.

Centromeres play a pivotal role in maintaining genome integrity by facilitating the recruitment of kinetochore and sister-chromatid cohesion proteins, both required for correct chromosome segregation. Centromeres are epigenetically specified by the presence of the histone H3 variant (CENH3). In this study, we investigate the role of the highly conserved gamma-tubulin complex protein 3-interacting proteins (GIPs) in Arabidopsis centromere regulation. We show that GIPs form a complex with CENH3 in cycling cells. GIP depletion in the gip1gip2 knockdown mutant leads to a decreased CENH3 level at centromeres, despite a higher level of Mis18BP1/KNL2 present at both centromeric and ectopic sites. We thus postulate that GIPs are required to ensure CENH3 deposition and/or maintenance at centromeres. In addition, the recruitment at the centromere of other proteins such as the CENP-C kinetochore component and the cohesin subunit SMC3 is impaired in gip1gip2. These defects in centromere architecture result in aneuploidy due to severely altered centromeric cohesion. Altogether, we ascribe a central function to GIPs for the proper recruitment and/or stabilization of centromeric proteins essential in the specification of the centromere identity, as well as for centromeric cohesion in somatic cells.

The RB/E2F pathway is involved in the control of the G(1)/S transition of the eukaryotic cell cycle where various S phase genes are activated by specific E2F factors. Ribonucleotide reductase (RNR) plays an essential role in the DNA synthesis pathway. Earlier studies showed that there are at least two RNR1 genes (RNR1a and RNR1b) and one RNR2 gene in tobacco. In synchronized tobacco BY2 cells, RNR1b gene expression is at its highest level in S phase. To investigate transcriptional regulation of the RNR1b gene, its promoter region was cloned and sequenced. Unlike its animal counterparts, the tobacco RNR1b promoter contains a consensus E2F-binding site. Surprisingly, this site is found in the leader sequence of the gene. We show here by gel shift analysis and antibody competition that one nuclear complex specifically binds this motif, and an E2F factor is part of this complex. Using reporter gene analysis, tobacco RNR1b promoter activity was detected during S phase in synchronized cells and in plant meristematic tissues. Mutation of the E2F element substantially reduced both activities. For the first time in plants, a single E2F motif found in the leader sequence plays an important role in the meristem and S phase-specific expression of the tobacco RNR1b gene.

Although the basic mechanisms which control the progression through the cell cycle appear to be conserved in all higher eukaryotes, the unique features of the plant developmental programme must be somehow reflected in a plant-specific regulation of the factors which control cell division. In the last few years, considerable progress has been achieved in identifying the major components of the cell cycle in plants. The question of how these components direct expression of specific genes at specific stages of the cell cycle, and how they are themselves regulated, constitutes a challenge for the present and the next years. This review summarizes our current knowledge at molecular and biochemical levels of cell cycle-regulated expression in the model system, the synchronized tobacco BY2 cell suspension, and discusses the results in comparison to those obtained by different methods and in other plant systems.

Eukaryotic ribonucleotide reductase (RNR), the enzyme involved in the synthesis of the deoxyribonucleotides, consists of two R1 and R2 subunits whose activities and gene expression are differentially regulated during the cell cycle and are preferentially induced at the G1/S transition. We have isolated three cDNA clones from a tobacco S phase library, two encoding the large R1 subunit, the first cloned in plants, and one encoding the small R2 subunit. From Southern blot hybridization we deduce that RNR2 is encoded by a single-copy gene whereas RNR1 is encoded by a small multigene family. The level of RNR mRNA is cell-cycle regulated showing a maximum in S phase. In mid-S phase, RNR2 transcripts show a higher maximum level than RNR1 transcripts. Analysis of the effects of various cell cycle inhibitors added to freshly subcultured stationary phase cells leads to the conclusion that RNR gene induction at the entry of the cells into the cell cycle takes place in late G1-early S phase. Addition of DNA synthesis-blocking agents to cycling cells synchronized in mid-S phase resulted in an enhancement of RNR transcript level, thus suggesting that RNR gene expression may be linked to the DNA synthesis rate by a feedback-like regulatory mechanism.

The spindle assembly checkpoint (SAC) is a refined surveillance mechanism which ensures that chromosomes undergoing mitosis do not segregate until they are properly attached to the spindle microtubules (MT). The SAC has been extensively studied in metazoans and yeast, but little is known about its role in plants. We identified proteins interacting with a MT-associated protein MAP65-3, which plays a critical role in organising mitotic MT arrays, and carried out a functional analysis of previously and newly identified SAC components. We show that Arabidopsis SAC proteins BUB3.1, MAD2, BUBR1/MAD3s and BRK1 interact with each other and with MAP65-3. We found that two BUBR1/MAD3s interacted specifically at centromeres. When stably expressed in Arabidopsis, BRK1 localised to the kinetochores during all stages of the mitotic cell cycle. Early in mitosis, BUB3.1 and BUBR1/MAD3.1 localise to the mitotic spindle, where MAP65-3 organises spindle MTs. A double-knockout mad3.1 mad3.2 mutant presented spindle MT abnormalities, chromosome misalignments on the metaphase plate and the production of lagging chromosomes and micronuclei during mitosis. We conclude that BRK1 and BUBR1/MAD3-related proteins play a key role in ensuring faithful chromosome segregation during mitosis and that their interaction with MAP65-3 may be important for the regulation of MT-chromosome attachment.

The functional organization of the nuclear envelope (NE) is only just emerging in plants with the recent characterization of NE protein complexes and their molecular links to the actin cytoskeleton. The NE also plays a role in microtubule (MT) nucleation by recruiting γ-Tubulin Complexes (γ-TuCs) which contribute to the establishment of a robust mitotic spindle. γ-tubulin Complex Protein 3 (GCP3)-interacting proteins (GIPs) have been identified recently as integral components of γ-TuCs. GIPs have been conserved throughout evolution and are also named MZT1 (mitotic-spindle organizing protein 1). This review focuses on recent data investigating the role of GIP/MZT1 at the NE, including insights from the study of GIP partners. It also uncovers new functions for GIP/MZT1 during interphase and highlights a current view of NE-associated components which are critical for nuclear shaping during both cell division and differentiation.

During interphase, the microtubular cytoskeleton of cycling plant cells is organized in both cortical and perinuclear arrays. Perinuclear microtubules (MTs) are nucleated from gamma-Tubulin Complexes (gamma-TuCs) located at the surface of the nucleus. The molecular mechanisms of gamma-TuC association to the nuclear envelope (NE) are currently unknown. The gamma-TuC Protein 3 (GCP3)-Interacting Protein 1 (GIP1) is the smallest gamma-TuC component identified so far. AtGIP1 and its homologous protein AtGIP2 participate in the localization of active gamma-TuCs at interphasic and mitotic MT nucleation sites. Arabidopsis gip1gip2 mutants are impaired in establishing a fully functional mitotic spindle and exhibit severe developmental defects. In this study, gip1gip2 knock down mutants were further characterized at the cellular level. In addition to defects in both the localization of gamma-TuC core proteins and MT fiber robustness, gip1gip2 mutants exhibited a severe alteration of the nuclear shape associated with an abnormal distribution of the nuclear pore complexes. Simultaneously, they showed a misorganization of the inner nuclear membrane protein AtSUN1. Furthermore, AtGIP1 was identified as an interacting partner of AtTSA1 which was detected, like the AtGIP proteins, at the NE. These results provide the first evidence for the involvement of a gamma-TuC component in both nuclear shaping and NE organization. Functional hypotheses are discussed in order to propose a model for a GIP-dependent nucleo-cytoplasmic continuum.

International audience; Cellular responses to DNA double-strand breaks (DSBs) are linked in mammals and yeasts to the phosphorylated histones H2AX (cH2AX) repair foci which are multiproteic nuclear complexes responsible for DSB sensing and signalling. However, neither the components of these foci nor their role are yet known in plants. In this paper, we describe the effects of cH2AX deficiency in Arabidopsis thaliana plants challenged with DSBs in terms of genotoxic sensitivity and E2F-mediated transcriptional responses.We further establish the existence, restrictive to the G1 ⁄ S transition, of specific DSBinduced foci containing tobacco E2F transcription factors, in both A. thaliana roots and BY-2 tobacco cells. These E2F foci partially colocalize with cH2AX foci while their formation is ataxia telangiectasia mutated (ATM)-dependent, requires the E2F transactivation domain with its retinoblastoma-binding site and is optimal in the presence of functional H2AXs. Overall, our results unveil a new interplay between plant H2AX and E2F transcriptional activators during the DSB response.

Ribonucleotide reductase (RNR) is an essential enzyme that provides dNTPs for DNA replication and repair. Arabidopsis (Arabidopsis thaliana) encodes three AtRNR2-like catalytic subunit genes (AtTSO2, AtRNR2A, and AtRNR2B). However, it is currently unclear what role, if any, each gene contributes to the DNA damage response, and in particular how each gene is transcriptionally regulated in response to replication blocks and DNA damage. To address this, we investigated transcriptional changes of 17-d-old Arabidopsis plants (which are enriched in S-phase cells over younger seedlings) in response to the replication-blocking agent hydroxyurea (HU) and to the DNA double-strand break inducer bleomycin (BLM). Here we show that AtRNR2A and AtRNR2B are specifically induced by HU but not by BLM. Early AtRNR2A induction is decreased in an atr mutant, and this induction is likely required for the replicative stress checkpoint since rnr2a mutants are hypersensitive to HU, whereas AtRNR2B induction is abolished in the rad9-rad17 double mutant. In contrast, AtTSO2 transcription is only activated in response to double-strand breaks (BLM), and this activation is dependent upon AtE2Fa. Both TSO2 and E2Fa are likely required for the DNA damage response since tso2 and e2fa mutants are hypersensitive to BLM. Interestingly, TSO2 gene expression is increased in atr versus wild type, possibly due to higher ATM expression in atr. On the other hand, a transient ATR-dependent H4 up-regulation was observed in wild type in response to HU and BLM, perhaps linked to a transient S-phase arrest. Our results therefore suggest that individual RNR2-likecatalytic subunit genes participate in unique aspects of the cellular response to DNA damage in Arabidopsis.

Heavy metal contamination in soils is easily transmitted to human through plants via the food chain. A major concern is to understand the plant response to heavy metal soil contamination to develop phytoremediation. Two plant models have been investigated in our study, the tomato, which is of agronomical importance, Arabidopsis, which is used as a model for molecular genetics. Heavy metal toxicity is described to induce oxidative stress linked to oxidation of proteins and membrane lipids but also to alterations of DNA damage response. We have investigated the metabolic response of cadmium and copper in parallel in both plant models and analyzed the transcriptional response of Arabidopsis RNR genes coding for isoforms of ribonucleotide reductase, an essential enzyme involved in DNA synthesis. Both Cd and Cu had a dose-dependent effect on plant growth. We also observed a rapid increase of catalase activity upon Cd or Cu treatments in tomato and Arabidopsis. At the transcriptional level, treatment with Cd resulted in a biphasic induction of two RNR genes in Arabidopsis; the first induction peak could be paralleled to the increase of the catalase activity.

*† ‡ Summary Re-activation of cell division after fertilization involves the specific regulation of a set of genes. To identify genes involved in the gametophytic to sporophytic transition, we screened Arabidopsis T-DNA insertion lines for early seed abortion at the zygote (zeus) or one-cell embryo stages (cyclops), and characterized a sporophytic zygote-lethal mutation, zeus1. ZEUS1 encodes a thymidylate kinase (AtTMPK) that synthesizes dTDP and is involved in the regulation of DNA replication. Unlike in yeast and animals, the single AtTMPK gene is capable of producing two proteins by alternative splicing; the longer isoform is targeted to the mitochondria, the shorter to the cytosol. Transcription of AtTMPK is activated during the G 1/S-phase transition of the cell cycle, similarly to yeast and mammalian orthologues. In AtTMPK:GUS plants, the reporter gene was preferentially expressed in cells undergoing division, but was not detected during the male and female gametophytic mitoses. GUS expression was observed in mature embryo sacs prior to fertilization, and this expression may indicate the time of synchronization of the gamete cell-cycle phases. Identification of ZEU1 emphasizes the importance of control of the metabolism of DNA in the regulation of the G 1/S-phase transition at fertilization.

Accurate DNA replication is one of the most important events in the life of a cell. To perform this task, the cell utilizes several DNA polymerase complexes. We investigated the role of DNA polymerase epsilon during gametophyte and seed development using forward and reverse genetic approaches. In Arabidopsis, the catalytic subunit of this complex is encoded by two genes, AtPOL2a and AtPOL2b, whereas the second largest regulatory subunit AtDPB2 is present as a unique complete copy. Disruption of AtPOL2a or AtDPB2 resulted in a sporophytic embryo-defective phenotype, whilst mutations in AtPOL2b produced no visible effects. Loss of AtDPB2 function resulted in a severe reduction in nuclear divisions, both in the embryo and in the endosperm. Mutations in AtPOL2a allowed several rounds of mitosis to proceed, often with aberrant planes of division. Moreover, AtDPB2 was not expressed during development of the female gametophyte, which requires three post-meiotic nuclear divisions. Since a consensus binding site for E2F transcription factors was identified in the promoter region of both genes, the promoter-reporter fusion technique was used to show that luciferase activity was increased at specific phases of the cell cycle in synchronized tobacco BY-2 cells. Our results support the idea that fertilization may utilize the mechanisms of cell cycle transcriptional regulation of genes to reactivate the divisions of the oosphere and central cell.

A new Arabidopsis thaliana (ecotype Columbia) genomic library has been constructed in Yeast Artificial Chromosomes: the CIC library (for CEPH, INRA and CNRS). Optimization of plant culture conditions and protoplast preparation allowed the recovery of large amounts of viable protoplasts. Mechanical shearing of DNA was minimized by isolation of DNA from protoplasts embedded in agarose. Cloning of large inserts was favored by including two successive size fractionation steps (after partial EcoRI digestion and after ligation with the vector arms), which selected DNA fragments larger than 350 kb. The library consists of 1152 clones with an average insert size of 420 kb. Clones carrying chloroplast DNA and various nuclear repeated sequences have been identified. Twenty-one per cent of the clones are found to contain chloroplast DNA. Therefore, the library represents around four nuclear genome equivalents. The clones containing 5S rDNA genes, 18S-25S rDNA sequences and the 180 bp paracentromeric repeated element account for 3.6%, 8.9% and 5.8%, respectively. Only one clone was found to carry the 160 bp paracentromeric repeated element. Given the smaller size of clones carrying Arabidopsis repeated DNA, the average size of remaining clones is around 480 kb. The library was screened by PCR amplification using pairs of primers corresponding to sequences dispersed in the genome. Seventy out of 76 pairs of primers identified from one to seven YAC clones. Thus at least 92% of the genome is represented in the CIC library. The survey of the library for clones containing unlinked DNA sequences indicates that the proportion of chimeric clones is lower than 10%.

Ribonucleotide reductase (RNR) is a key enzyme involved in the DNA synthesis pathway. The RNR-encoded genes are cell cycle regulated and specifically expressed in S phase. The promoter of the RNR2 gene encoding for the small subunit was isolated from tobacco. Both in vivo and in vitro studies of the DNA–protein interactions in synchronized BY2 tobacco cells showed that two E2F-like motifs were involved in multiple specific complexes, some of which displayed cell cycle–regulated binding activities. Moreover, these two elements could specifically interact with a purified tobacco E2F protein. Involvement of the E2F elements in regulating the RNR2 promoter was checked by functional analyses in synchronized transgenic BY2 cells transformed with various RNR2 promoter constructs fused to the luciferase reporter gene. The two E2F elements were involved in upregulation of the promoter at the G1/S transition and mutation of both elements prevented any significant induction of the RNR promoter. In addition, one of the E2F elements sharing homology with the animal E2F/cell cycle–dependent element motif behaved like a repressor when outside of the S phase. These data provide evidence that E2F elements play a crucial role in cell cycle regulation of gene transcription in plants.

Two histone H3 and two histone H4 genes have been cloned from a λgtWESλ·B Arabidopsis thaliana gene library. From their nucleotide sequences and from studies on their genomic organization, the following conclusions can be drawn: : 1) The nucleotide sequences of the two H3 coding regions show only 85% homology, but encode the same proteins. The Arabidopsis H3 has the same amino acid sequence as its counterpart in corn, but differs from that of pea and wheat by replacement in position 90 of a serine by an alanine. The two H4 coding regions have 97% sequence homology and encode the same protein, identical to the sequence of their counterpart in pea, corn and one H4 variant in wheat. 2) The 5'-flanking regions of the 4 genes contain the classical histone-gene-specific consensus sequences, except H3A725 which lacks the GATCC-like pentamer. The conserved octanucleotide 5'-CGCGGATC-3' which was previously found in the 5'-flanking sequences of corn and wheat H3 and H4 genes is also present in all four genes described here approximately 200 to 250 nucleotides upstream from the initiation ATG. The 5'-flanking regions of the H4 genes display extensive sequence homology, whereas those of the H3 genes do not. 3) The 3'-flanking regions do not possess the classical histone-gene-specific T hyphenated dyad symmetry motif. 4) Each H3 and H4 gene exists as 5 to 7 copies per haploid genome.

The general structure of the plant histone genes has been deduced from the comparison of the nucleotide sequences of ten H3 and H4 genes of maize (3 H3 and 3 H4) and Arabidopsis thaliana (2 H3 and 2 H4). The five H3 and five H4 genes encode the same proteins, respectively. The 5′-flanking regions contain the classical histone gene-specific consensus sequences. In addition, a conserved octanucleotide CGCGGATC was found in all plant histone genes at 200–250 nucleotides before the initiation codon. All six maize H3 and H4 genes are transcribed during early germination as shown by nuclease S1 mapping and reverse transcriptase primer extension experiments. The mRNA 5′-ends are located within the consensus sequence CCAA/CT/C. The 3′-ends lack the classical T-hyphenated dGC-rich palindromic structure and possess long nontranslated sequences. In both plants the multiple copies of the H3 and H4 genes are organized into multigenic families. The genes of each family show a similar proximal environment, suggesting that they originate from the duplication of a common ancestor.

Two histone H3 genes have been cloned from a λgtWESλ.B corn genomic library. The nucleotide sequences show 96% homology and both encode the same protein, which differs from its counterpart in wheat and pea by one amino acid substitution. The 5'-flanking regions of the two corn H3 genes contain the classical histone-gene-specific consensus sequences and possess several regions of extensive nucleotide homology. A conserved octanucleotide 5'-CGCGGATC-3' occurs at approximately 200 nucleotides upstream from the initiation ATG codon. This octanucleotide was found to exist in all of the 7 plant histone genes sequenced so far. Codon usage is characterized by a very high frequency of C (67%) and G (28%) at the third position of the codons, those ending by A (1%) and T (4%) being practically excluded.Comparison of Southern blots of EcoRI, EcoRV and BamHI digested genomic DNA suggests that the corn H3 and H4 genes are not closely associated. The H3 genes exist as 60 to 80 copies and the H4 genes as 100 to 120 copies per diploid genome. re]19851002 rv]19851212 ac]19851216.

The histone H3 and H4 genes are shown to be expressed in both Arabidopsis plantlets and transitory multicellular suspension. The 5′- and 3′-ends of the H4 mRNAs have been localized on two H4 genes previously sequenced, H4A748 and H4A777. S1-nuclease mapping and reverse-transcriptase-primer-elongation experiments revealed the existence of two start points for transcription, located 31 and 37 nucleotides downstream from the TATA-box. The 3′-end of the mRNA corresponding to H4A748 was localized at 177 nt after the stop codon. The other gene, H4A777, most probably is not expressed. In addition to a long 3′-untranslated region, the H4 mRNA was shown to be polyadenylated in both plantlets and cell-suspension. This observation was extended to the H3 mRNAs of Arabidopsis and of two other dicots, tobacco and sunflower. Previous results on maize H3 and H4 mRNAs suggest that polyadenylation is a common feature for histone mRNAs in higher plants.

Northern blot analysis revealed that the histone H3 and H4 mRNAs are of unusual large size in germinating maize embryos. S1-mapping experiments show that the 3'-untranslated regions of the mRNAs transcribed from 3 H3 and 2 H4 maize genes previously described are much longer than in the non-polyadenylated histone mRNAs which represent a major class in animals. Moreover, oligo d(T) cellulose fractionation of RNAs isolated at different developmental stages indicates that more than 99% of the maize H3 and H4 mRNAs are polyadenylated. A putative polyadenylation signal is present in all five genes 17 to 27 nucleotides before the 3'-ends of the mRNAs.

International audience; Microtubules (MTs) are crucial for both the establishment of cellular polarity and the progression of all mitotic phases leading to karyokinesis and cytokinesis. MT organization and spindle formation rely on the activity of γ-tubulin and associated proteins throughout the cell cycle. To date, the molecular mechanisms modulating γ-tubulin complex location remain largely unknown. In this work, two Arabidopsis thaliana proteins interacting with gamma-tubulin complex protein3 (GCP3), GCP3-interacting protein1 (GIP1) and GIP2, have been characterized. Both GIP genes are ubiquitously expressed in all tissues analyzed. Immunolocalization studies combined with the expression of GIP-green fluorescent protein fusions have shown that GIPs colocalize with γ-tubulin, GCP3, and/or GCP4 and reorganize from the nucleus to the prospindle and the preprophase band in late G2. After nuclear envelope breakdown, they localize on spindle and phragmoplast MTs and on the reforming nuclear envelope of daughter cells. The gip1 gip2 double mutants exhibit severe growth defects and sterility. At the cellular level, they are characterized by MT misorganization and abnormal spindle polarity, resulting in ploidy defects. Altogether, our data show that during mitosis GIPs play a role in γ-tubulin complex localization, spindle stability and chromosomal segregation.

Salt stress is one of the main environmental factors limiting plant growth and yield in cereal crops. It is therefore imperative to develop varieties more tolerant to salt stress in order to increase yield and ensure food security. The signaling pathway linking salt stress perception to cellular response was addressed here by studying RSS1-like proteins in plants. RSS1 (Rice Salt Sensitive 1) protein plays an important role in salt stress tolerance. It acts at the interface of stress perception and developmental control and division in meristems. During this work, the RSS1 counterpart named TdRL1 (Triticum durum RSS-Like 1) was isolated from the durum wheat Tunisian variety "Oum Rabiaa". We have demonstrated that TdRL1 carries the conserved D and DEN-Box motifs involved in the post-translational regulation of the protein. In addition, we show that TdRL1 is the functional homologue of RSS1 since it was able to complement the loss-of-function mutant rss1, hypersensitive to salt stress. In addition, heterologous expression of TdRL1 enhances salt stress tolerance in yeast and in Arabidopsis by increasing germination and reducing the accumulation of reactive oxygen species. Our cytological studies have shown that the TdRL1 protein is cytoplasmic in interphase and is localized at the spindle during mitosis. Remarkably, TdRL1 changes its subcellular localization under salt stress treatment and shows a partial accumulation in the nucleus, highlighting the multifunctional nature of this protein during salt stress response. Our data suggest that under salt stress, TdRL1 plays a role in the regulation of the cell cycle in relation with the microtubule network. Pursuing the study of RSS1-like multifunctional proteins will open up new research areas for the creation of wheat varieties that are more resilient to environmental stresses.; Le stress salin est l'un des principaux facteurs environnementaux limitant la croissance des plantes et entraînant des pertes de rendement des cultures céréalières. Il est ainsi impératif de développer des variétés plus tolérantes à la salinité afin d’augmenter leurs rendements et assurer la sécurité alimentaire. La voie signalétique reliant la perception du stress salin à la réponse cellulaire, encore peu connue, a été abordée ici par l’étude des protéines RSS1-like conservées chez les plantes. La protéine RSS1 (Rice Salt Sensitive 1) du riz joue un rôle primordial dans la tolérance au stress salin en agissant à l’interface entre la perception des stress et le contrôle du développement et de la division dans les méristèmes. Lors de ce travail, l'homologue de RSS1 nommé TdRL1 (Triticum durum RSS-Like 1) a été isolé à partir de la variété tunisienne de blé dur “Oum Rabiaa“. Nous avons démontré que TdRL1 porte les motifs D et DEN-Box conservés impliqués dans la régulation post-traductionnelle de la protéine. En outre nous avons apporté la preuve que TdRL1 est l’homologue fonctionnel de RSS1 puisqu'il est capable de complémenter le mutant de perte de fonction rss1, hypersensible au stress salin. En outre, l’expression hétérologue de TdRL1 améliore la tolérance au stress salin chez la levure ainsi que chez Arabidopsis et ce par l’augmentation du pouvoir germinatif et la réduction de l’accumulation des espèces oxygénées réactives. Nos études cytologiques ont montré que la protéine TdRL1 est cytoplasmique en interphase et se localise au niveau des microtubules kinétochoriens pendant la mitose. Remarquablement, TdRL1 change de localisation cellulaire sous stress salin et montre une accumulation partielle dans le noyau, soulignant le caractère multifonctionnel de cette protéine dans la réponse au stress salin. L’ensemble des données suggère que sous contrainte saline, TdRL1 joue un rôle dans la régulation du cycle cellulaire en relation avec le réseau microtubulaire. L‘étude de la famille RSS1-like multifonctionnelle permettra ainsi d’aborder de nouvelles voies de recherche pour la création variétale de blé plus résilientes aux stress de l'environnement.