Marie-Edith Chabouté

Cell Cycle Regulation of the Tobacco Ribonucleotide Reductase Small Subunit Gene Is Mediated by E2F-like Elements

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.

Genomic organization and nucleotide sequences of two histone H3 and two histone H4 genes of Arabidopsis thaliana

SummaryTwo 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 GCP3-Interacting Proteins GIP1 and GIP2 Are Required for γ-Tubulin Complex Protein Localization, Spindle Integrity, and Chromosomal Stability

The stabilization of a robust mitotic spindle is required for correct chromosome segregation. GIP proteins interact with microtubule nucleation complexes and localize on mitotic microtubule arrays. The analysis of knockdown mutants suggests that GIP proteins act in both the recruitment of these complexes at nucleation sites and the maintenance of spindle efficiency. 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.

Nucleotide sequences of two corn histone H3 genes. Genomic organization of the corn histone H3 and H4 genes.

SummaryTwo 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 GIP gamma-tubulin complex-associated proteins are involved in nuclear architecture in Arabidopsis thaliana.

During interphase, the microtubular cytoskeleton of cycling plant cells is organized in both cortical and perinuclear arrays. Perinuclear microtubules (MTs) are nucleated from γ-Tubulin Complexes (γ-TuCs) located at the surface of the nucleus. The molecular mechanisms of γ-TuC association to the nuclear envelope (NE) are currently unknown. The γ-TuC Protein 3 (GCP3)-Interacting Protein 1 (GIP1) is the smallest γ-TuC component identified so far. AtGIP1 and its homologous protein AtGIP2 participate in the localization of active γ-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 γ-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 γ-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.

Molecular characterization of tobacco ribonucleotide reductase RNR1 and RNR2 cDNAs and cell cycle-regulated expression in synchronized plant cells

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.

Ribonucleotide Reductase Regulation in Response to Genotoxic Stress in Arabidopsis

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-like catalytic subunit genes participate in unique aspects of the cellular response to DNA damage in Arabidopsis.

Plant γH2AX foci are required for proper DNA DSB repair responses and colocalize with E2F factors

Cellular responses to DNA double-strand breaks (DSBs) are linked in mammals and yeasts to the phosphorylated histones H2AX (γH2AX) 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 γH2AX 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 DSB-induced foci containing tobacco E2F transcription factors, in both A. thaliana roots and BY-2 tobacco cells. These E2F foci partially colocalize with γH2AX 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.

Microtubule nucleation and establishment of the mitotic spindle in vascular plant cells

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.

ATR3 encodes a diflavin reductase essential for Arabidopsis embryo development

*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.