Chemical inhibitors: a tool for plant cell cycle studies

AbstractSynchrony provides a large number of cells at defined points of the cell cycle. Highly synchronised cells are powerful and effective tools for molecular analyses and for studying the biochemical events of the cell cycle in plants. Usually, plant cell suspensions can be synchronised by chemical agents, which arrest the cell cycle by acting on the driving forces of the cell cycle engine such as cyclin-dependent kinase activity, enzymes involved in DNA synthesis or proteolysis of cell cycle regulators or by acting on the cell cycle apparatus (mitotic spindle). The specificity, reversibility and efficiency of each type of cell cycle inhibitor are described and related to their mode of action

Ectopic expression of Kip-related proteins restrains root-knot nematode-feeding site expansion

The development of nematode feeding sites induced by root-knot nematodes involves the synchronized activation of cell cycle processes such as acytokinetic mitoses and DNA amplification. A number of key cell cycle genes are reported to be critical for nematode feeding site development. However, it remains unknown whether plant cyclin-dependent kinase (CDK) inhibitors such as the Arabidopsis interactor/inhibitor of CDK (ICK)/Kip-related protein (KRP) family are involved in nematode feeding site development. This study demonstrates the involvement of Arabidopsis ICK2/KRP2 and ICK1/KRP1 in the control of mitosis to endoreduplication in galls induced by the root-knot nematode Meloidogyne incognita. ! Using ICK/KRP promoter-GUS fusions and mRNA in situ hybridizations, we showed that ICK2/KRP2, ICK3/KRP5 and ICK4/KRP6 are expressed in galls after nematode infection. Loss-of-function mutants have minor effects on gall development and nematode reproduction. Conversely, overexpression of both ICK1/KRP1 and ICK2/KRP2 impaired mitosis in giant cells and blocked neighboring cell proliferation, resulting in a drastic reduction of gall size. ! Studying the dynamics of protein expression demonstrated that protein levels of ICK2/ KRP2 are tightly regulated during giant cell development and reliant on the presence of the nematode. ! This work demonstrates that impeding cell cycle progression by means of ICK1/KRP1 and ICK2/KRP2 overexpression severely restricts gall development, leading to a marked limitation of root-knot nematode development and reduced numbers of offsprin

Caractérisation d'inhibiteurs de complexes CDK cycline chez Arabidopsis thaliana

Comme pour tous les organismes pluricellulaires, la croissance et le développement des plantes nécessitent une coordination de la production de cellules via la mitose et la différenciation cellulaire. La progression du cycle cellulaire est contrôlée par les complexes CDK-cycline. Les inhibiteurs de ces complexes, les CKIs, représentent d excellents candidats pour réguler cet équilibre entre les processus de prolifération et différentiation cellulaires qui ont lieu au cours du développement. Afin de mettre en évidence le rôle d intégrateurs potentiel des CKIs, le développement floral a été utilisé en tant que modèle.Grâce à l utilisation de la qRT-PCR, nous avons montré que durant le développement floral d Arabidopsis thaliana, un groupe restreint de CKIs était exprimé. Nous avons choisi de travailler sur les deux CKIs les plus exprimés, KRP6 et KRP7. Une caractérisation fine de leur profil d expression durant le développement a été réalisée en utilisant des approches complémentaires telles que l analyse de l activité de leur promoteur, de la dynamique de leur transcrit, de leur expression protéique et de leur régulation post-traductionnelle.Jusqu à présent, seules des approches gain de fonction ont été utilisées pour étudier le rôle des CKIs chez les plantes. C est pour cela que nous avons choisi des approches perte de fonction pour analyser le rôle de KRP6 et de KRP7 au cours du développement floral. Ainsi, nous avons généré des doubles mutants d insertion krp6-krp7, krp3-krp6, krp3-krp7, des triples mutants d insertion krp3-krp6-krp7 et diverses lignées ARN interférence avec des promoteurs spécifiques. Malgré l étude de ces nombreuses lignées, nous n avons pas réussi à mettre en évidence des effets phénotypiques associés à l absence de la fonction CKI au cours du développement floral. Ces résultats mettent en évidence la redondance fonctionnelle qui semble exister entre les KRPs, ainsi un quadruple mutant pourrait être nécessaire pour entrainer des modifications développementales. Afin de mieux comprendre cette fonction d intégrateurs des KRPs au cours du développement floral, les partenaires de KRP6 et de KRP7 ont été recherchés. Des criblages double-hybride ont été réalisés afin d identifier des ADNc, spécifiques du développement floral, codant des protéines capables d interagir avec KRP6 et KRP7. De façon intéressante, mis à part les cyclines de type D, un nouveau type d interaction a pu être mis en évidence. Un sous-groupe de la famille des rémorines est capable d interagir avec KRP6 ou KRP7 en système double-hybride. Les rémorines sont des protéines spécifiques du règne végétal, associées à la membrane plasmique mais dont la fonction reste à clarifier. Une approche BiFC en protoplastes BY-2 a permis de confirmer l existence de ce type d interaction. De plus, l influence des rémorines sur la localisation intracellulaire des KRPs a été étudiée. En présence de ces nouveaux partenaires, KRP7 est capable d adopter une localisation nucléo-cytoplasmique.Enfin, des résultats récents ont montré que l AMPK était capable de phosphoryler p27KIP1, l homologue fonctionnel des KRPs chez les mammifères. Ces évènements de phosphorylation entrainent des modifications de sa localisation intracellulaire et de son activité inhibitrice vis-à-vis des complexes CDK-cycline. Après la réalisation d analyses in silico ayant permis de prédire des sites putatifs de phosphorylation par SnRK1, l homologue de l AMPK chez A. thaliana, pour certains KRPs, la protéine KRP6 sous forme recombinante a été utilisée pour réaliser des essais kinase in vitro. Une phosphorylation de KRP6 est détectée en présence de la sous unité catalytique activée de SnRK1. Contrairement aux mammifères, cet évènement de phosphorylation entraine une altération de l activité inhibitrice de KRP6 sans modification de sa localisation intracellulaire. Cette abolition de l activité de KRP6 a été confirmée in planta. En effet, les phénotypes associés à la surexpression de KRP6 peuvent être atténués par la surexpression simultanée de la sous-unité catalytique de SnRK1. L existence de ce lien entre KRP6 et SnRK1 met en évidence une relation directe entre l homéostasie énergétique et la prolifération cellulaire.As in all multicellular organisms, growth and development in plants require the coordination of cell production by division and cell differentiation. Progression through cell cycle is controlled by the kinase activity of CDK/cyclin complexes. Inhibitors of these complexes, CKIs, represent excellent candidates to regulate the balance between proliferation and differentiation processes during development. To get insight in the potential integrator role of CKIs, floral development was chosen as a developmental model. Using a real time quantitative PCR approach, we bring to light that during floral development of Arabidopsis thaliana, a restricted subset of CKIs was preferentially expressed. It was decided to focus our work on the two major expressed CKIs, KRP6 and KRP7. A better characterization of their expression patterns of during development was undertaken using complementary approaches such as promoter activity analysis, mRNA dynamics, protein expression and post-translational regulation analysis. Because until now gain of function approaches have been largely applied to unravel the role of plant CKIs, our challenge was to detect a floral phenotype for KRP6 and KRP7 loss of function mutants, either using knock-out mutants or RNAi lines. We generated krp6-krp7, krp3-krp6, krp3-krp7 double mutants and krp3-krp6-krp7 triple mutant and also several RNAi lines with specifics promoters. Despite the study of these numerous lines, we were not able to highlight phenotypic effects associated with the absence of CKI function during floral development. All these results emphasis functional redundancy which appears to exist between all KRPs, thus quadruple mutant might be needed to provoke some developmental modification.In order to better understand the integrative function of KRPs during floral development, partners of KRP6 and KRP7 were assessed. Two-hybrid screens were performed to identify cDNAs from a floral-buds-development library encoding proteins that are able to interact with KRP6 and KRP7. Interestingly, apart from D-type cyclins, we brought to light a new type of interaction. Indeed, a sub-class of the remorin protein family was able to interact with KRP6 or KRP7 in yeast two-hybrid. Remorins are plant specific plasma membrane associated proteins with unknown function. A BiFC approach in BY-2 protoplasts allowed us to confirm remorins/KRP6-7 interactions. Furthermore, the influence of the presence of remorin proteins on KRP6/7 localisation was assessed. KRP7 is able to adopt a nucleo-cytoplasmic localisation in presence of its new partners.Finally, recent results have shown that AMPK is phosphorylating p27KIP1, KRPs functional counterpart in mammals. These phosphorylation events lead to changes in its cellular localisation and its inhibitory activity toward CDK-cyclin complexes. After in silico analysis aiming to predict potential AMPK Arabidopsis homologue SnRK1 phosphorylation sites within some KRPs protein sequences, recombinant KRP6 was used in order to perform in vitro kinase assays. Phosphorylation occurs efficiently on KRP6 when activated SnRK1 catalytic subunit is present. Furthermore, unlike in mammals, this phosphorylation event leads to an alteration of KRP6 inhibitory activity without modification of its cellular localisation. This abolition of KRP6 activity was confirmed by in planta analysis. Indeed, KRP6 overexpression phenotype can be attenuated by simultaneous SnRK1 catalytic subunit overexpression. The existence of this link between KRP6 and SnRK1 underscores a direct relationship between energy homeostasis and cell proliferation.PARIS11-SCD-Bib. électronique (914719901) / SudocSudocFranceF

Caractérisation d'un inhibiteur de kinases cycline-dépendantes de N. tomentosiformis (analyse de son rôle au cours du développement de la plante)

Le développement d'une plante nécessite un contrôle précis entre prolifération cellulaire et différenciation. Le cycle cellulaire est contrôlé par des kinases dépendantes des cyclines (CDKs) dont l'activité est régulée à plusieurs niveaux, en particulier par des inhibiteurs (CKIs, cyclin dépendent kinase inhibitors). Le criblage d'une banque double hybride de la suspension cellulaire de tabac BY-2 avec une CDKA comme appât à permis l'isolement de deux ADNc, nommés NtKIS1a et NtKIS1b. Les deux ARNm proviennent d'un même gène de N. tomentosiformis par épissage alternatif. La séquence protéique déduite de NtKIS1a présente de fortes similarités de séquence avec les CKIs de mammifères de la famille CIP/KIP, alors que ce n'est pas le cas de NtKIS1b. En accord avec cette observation, NtKIS1a mais pas NtKIS1b inhibe in vitro l'activité kinase de complexes CDK/cycline. Pour élucider le rôle de NtKIS1a et NtKIS1b au cours du développement, leur surexpression dans différentes espèces végétales a été réalisée. Les plantes d'Arabidopsis thaliana surexprimant NtKIS1b ont un phénotype sauvage, tandis que celles surexprimant NtKIS1a présentent d'importantes modifications morphologiques. L'ensemble de nos résultats suggèrent que les modifications phénotypiques proviennent d'une inhibition de la division et montrent donc que NtKIS1a est un inhibiteur de la division in planta. Des plantes surexprimant simultanément NtKIS1a et AtCycD3;1 ont été obtenues. Leur analyse montre que la surexpression du CKI NtKIS1a restaure un développement normal des plantes surexprimant AtCycD3;l, fournissant la première évidence d'une coopération CKI-cycline in planta. Dans le but d'appréhender les liens qui existent entre le cycle cellulaire et le développement, l'expression de deux gènes a été modifiée simultanément in planta : KNAT1 (knottedl-like from Arabidopsis thaliana), impliqué dans le développement et la fonction du méristème apical caulinaire, et NtKIS1a, impliqué dans l'inhibition du cycle cellulaire. L'analyse des plantes F1 montre que la co-expression de NtKIS1a et KNAT1 renforce le phénotype des plantes 35S::KNAT1, suggérant que les produits des deux gènes coopèrent au cours du développement.Plant development requires stringent controls between cell proliferation and cell differentiation. Proliferation is positively regulated by cyclin dependent kinases (CDKs), whose activity is regulated at several levels including inhibition by CDK inhibitors (CKIs). The screen of a two-hybrid BY-2 cell suspension library with a CDKA as a bait, allows the isolation of two cDNA, named NtKIS1a and NtKIS1b. NtKIS1a and NtKIS1b mRNAs arise from the same N. tomentosiformis gene by alternative splicing. The deduced polypeptide from NtKIS1a shares strong sequence similarity with mammalian CIP/KIP inhibitors, which is not the case for NtKIS1b. Consistent with this, NtKIS1a but not NtKIS1b inhibits in vitro the kinase activity of CDK/cyclin complexes. To gain insight into the role of NtKIS1a and NtKIS1b during plant development, their overexpression in different species was achieved. Arabidopsis thaliana plants overexpressing NtKIS1b display a wild type phenotype, whereas plants overexpressing NtKIS1a display strong morphological modifications. Our results suggest that the inhibition of cell division is responsible for the phenotypic modifications and thus that NtKIS1a is a cell division inhibitor in planta. Plants overexpressing simultaneously NtKIS1a and AtCycD3;1 were achieved. Their analyze demonstrates that overexpression of the CKI NtKIS1a restores essentially normal development in AtCycD3;1 overexpressing plants, providing for the first time, evidence of Cyclin D-CKI co-operation within the context of a living plant. At the aim of highlighting the links between cell cycle and development, the expression of two genes was modify simultaneously in planta: KNAT1 (knotted1-like from Arabidopsis thaliana) involved in shoot apical meristem development and function, and NtKIS1a involved in cell cycle regulation. The analysis of the F1 plants shows that co-expression of NtKIS1a and KNAT1 enhance the KNAT1 phenotype, suggesting that the two gene products co-operate with each other during plant development.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

MRGI-1, a dominant allele that confers methomyl resistance in yeast expressing the cytoplasmic male sterility T-urfI3 gene from maize

International audienceWe have previously described a eukaryotic het-erologous expression system, with the urf13TW gene in yeast, which mimics the disease susceptibility associated with the Texas cytoplasmic male sterility in maize. This yeast model was used to isolate yeast nuclear mutants conferring methomyl resistance. The genetic strategy we have developed focused on screening for nuclear dominant yeast mutations which restore methomyl resistance. MRGI-1, a yeast nuclear dominant allele, was identified as a methomyl-resistance restorer. We have shown that methomyl resistance co-segregated with a pleiotropic phenotype in the heterozygous MRGI-1/MRG1 diploids, detectable even in the absence of the maize-derived mito-chondrial protein and/or methomyl. We observed an increase in oxygen uptake, a significant decrease of the levels of cytochrome aa 3, and a decrease in the growth yield. This phenotype is influenced by the carbon source and the results suggest a defect in the adaptation to the respiratory pathway in MRGI-1 yeast cells

M phase-specific activation of the Nicotiana sylvestris Cyclin B1 promoter involves multiple regulatory elements

B-type cyclins are cell cycle regulatory proteins specifically expressed in G2-M phases. To understand the mechanisms that regulate their expression, it is important to identify the required promoter element(s). By using synchronized BY-2 cell cultures stably transformed with chimeric genes composed of sequential Nicsy; CycB1; 1 promoter deletions fused to the beta-glucuronidase reporter gene (gus), we show that at least five distinct promoter regions are required for maximal M-phase expression. Furthermore, two distinct promoter regions contain sufficient element(s) to induce M-phase-specific expression. In one of these regions, a 23 bp promoter element is able to activate reporter gene expression in cells induced to divide in an orientation-independent manner but without an M-phase specific expression. Therefore, this 23 bp element, which contains a 5 bp element identical to the MYB binding core, is a good upstream activating sequence (UAS) candidate. Moreover, electrophoretic mobility shift assays show that this putative UAS specifically binds protein complexes that appear to differ whether cells are cycling or not. The constitutive transcriptional activation mediated by this UAS would suggest that the Nicsy; CycB1; 1 gene promoter is regulated through an activation/repression mechanism

Identification of proteins interacting with the Arabidopsis Cdc2aAt protein

Cyclin-dependent kinases (CDKs) control the progression through the cell cycle, Using a two-hybrid approach, two clones encoding proteins interacting with the Arabidopsis thaliana CDK Cdc2aAt were identified, One clone encoded a novel putative substrate of Cdc2aAt, whereas the second clone was identified as a D-type cyclin (cycD1;1)

Decreased glycolate oxidase activity leads to altered carbon allocation and leaf senescence after a transfer from high CO₂ to ambient air in Arabidopsis thaliana

Metabolic and physiological analyses of Arabidopsis thaliana glycolate oxidase (GOX) mutant leaves were performed to understand the development of the photorespiratory phenotype after transfer from high CO₂ to air. We show that two Arabidopsis genes, GOX1 and GOX2, share a redundant photorespiratory role. Air-grown single gox1 and gox2 mutants grew normally and no significant differences in leaf metabolic levels and photosynthetic activities were found when compared with wild-type plants. To study the impact of a highly reduced GOX activity on plant metabolism, both GOX1 and GOX2 expression was knocked-down using an artificial miRNA strategy. Air-grown amiRgox1/2 plants with a residual 5% GOX activity exhibited a severe growth phenotype. When high-CO₂-grown adult plants were transferred to air, the photosynthetic activity of amiRgox1/2 was rapidly reduced to 50% of control levels, and a high non-photochemical chlorophyll fluorescence quenching was maintained. (13)C-labeling revealed that daily assimilated carbon accumulated in glycolate, leading to reduced carbon allocation to sugars, organic acids, and amino acids. Such changes were not always mirrored in leaf total metabolite levels, since many soluble amino acids increased after transfer, while total soluble protein, RuBisCO (ribulose-1,5-bisphosphate carboxylase/oxygenase), and chlorophyll amounts decreased in amiRgox1/2 plants. The senescence marker, SAG12, was induced only in amiRgox1/2 rosettes after transfer to air. The expression of maize photorespiratory GOX in amiRgox1/2 abolished all observed phenotypes. The results indicate that the inhibition of the photorespiratory cycle negatively impacts photosynthesis, alters carbon allocation, and leads to early senescence in old rosette leaves.This work was supported by a public grant overseen by the French National Research Agency (ANR) as part of the ‘Investissement d’Avenir’ program, through the ‘Lidex-3P’ project and a French State grant (ANR-10-LABX- 0040-SPS) funded by the IDEX Paris-Saclay, ANR-11-IDEX-0003-02. We wish to thank the Ile-de-France region for financial support via a Sesame project. YD was supported by a PhD grant from the French Ministry of Higher Education and Research

Characterization of cis-acting element involved in cell cycle phase-independent activation of Arath;CycB1;1 transcription and identification of putative regulatory proteins

Progression through the cell cycle is driven by cyclin-dependent kinases (CDKs) whose activity is controlled by regulatory subunits called cyclins. The expression of cyclins is subject to numerous controls at multiple levels, not least at the level of transcription. As a first step to unravel the mechanisms that regulate expression of B-cyclins in plants, we undertook the identification of the required promoter elements of the Arath;CycB1;1 gene. A detailed analysis of different promoter fragments consisted in analysing their ability to mediate cell cycle-dependent transcriptional oscillations of the gus reporter gene in transformed BY-2 cell lines. We showed that different promoter regions took part in transcriptional activation. Furthermore, 202 bp upstream of the ATG were sufficient to induce M-phase-specific expression. This region contains an 18 bp sequence including a Myb-binding core (AACGG) which is able to activate reporter gene without leading to M-phase-specific expression. Electrophoretic mobility shift assays showed that this 18 bp sequence specifically binds protein complexes from Arabidopsis cell suspension enriched either in G1 or G2 phase. Furthermore, the Myb core, AACGG, was characterized as necessary for the binding of proteins. DNA affinity purification of the complexes bound to the 18 bp sequence allowed the isolation of three different complexes and two proteins from these complexes were identified by mass spectrometry analyses. A new putative Myb transcription factor and a hypothetical protein, HYP containing with a leucine zipper and Myc-type dimerization domains were identified. When over-expressed in plants, HYP factor is able to trans-activate the expression of gus reporter gene downstream from the -202 promoter fragment as well as the endogenous CycB1;1 gene

Cell cycle regulation by plant growth regulators: involvement of auxin and cytokinin in the re-entry of Petunia protoplasts into the cell cycle

In order to understand the mode of action of auxins and cytokinins in the induction of cell division, the effects of the two plant growth regulators 2,4-dichlorophenoxyacetic acid (2,4-D) and N-6-benzyladenine (BA) were investigated using mesophyll protoplasts of Petunia hybrida, cultivated in either complete medium or in medium deficient in cytokinin, auxin or both. Firstly we studied DNA synthesis, using 5-bromodeoxyuridine/bisbenzimide Hoechst/propidium iodide flow cytometry analyses and by the monitoring of histone H4 transcript levels. Roscovitine, a cyclin-dependent kinase (CDK) inhibitor, was found to block the cell cycle prior to entry into the S and M phases in the cultured P. hybrida protoplasts. This suggests that in Petunia cells there is a requirement for CDK activity in order to complete the G1 and G2 phases. Further experiments using roscovitine showed that neither 2,4-D nor BA were individually able to induce cell cycle progression beyond the roscovitine G1 arrest. We also monitored the phytohormonal induction of S phase by studying variations in transcript levels of the gene for mitogenactivated protein kinase (PMEK1) and transcript levels of the cell division cycle gene cdc2Pet. Only 2,4-D, and not BA, was able to stimulate PMEK1 gene transcription; thus, the more rapid S-phase induction in 2,4-D-treated protoplasts may be attributable to the activation of this transduction pathway. In contrast, both plant growth regulators were required to induce the appearance of cdc2Pet mRNA transcripts prior to S-phase engagement