Nathalie Glab

Olomoucine, an inhibitor of the cdc2/cdk2 kinases activity, blocks plant cells at the G1 to S and G2 to M cell cycle transitions

The cdc2/cdk2 protein kinases play key roles in the cell cycle at two control points: the G1/S transition and the entry into mitosis. Olomoucine, a specific inhibitor of these kinases, was tested in two plant cell systems: Petunia mesophyll protoplasts induced to divide and Arabidopsis thaliana cell suspension cultures. The cell cycle status was analysed from DNA histograms or through continuous labelling of cells with 5‐bromodeoxyuridine (BrdUrd) followed by double staining with bis‐benzimide (Hoechst 33258) and propidium iodide (PI). Such analyses resolve cells from several generations according to the extent of their DNA replication. Olomoucine was shown to reversibly arrest differentiated Petunia cells induced to divide at G1 phase and cycling Arabidopsis cells in late G1 and G2. A comparison of the effects of aphidicolin, oryzalin and olomoucine suggests that in the Arabidopsis cell suspension culture, a cdc2/cdk2‐like kinase is activated at a restriction point in late G1.

Comparative Molecular and Functional Analyses of the Tobacco Cyclin-Dependent Kinase Inhibitor NtKIS1a and Its Spliced Variant NtKIS1b

In all eukaryotes, cell cycle progression is controlled by cyclin-dependent kinases (CDKs) whose activity is regulated at several levels including inhibition by CDK inhibitors. Here, we report a comparative molecular and functional analysis of the tobacco (Nicotiana tomentosiformis) CDK inhibitor, NtKIS1a, and its spliced variant, NtKIS1b. The C-terminal end of 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, and tobacco (Nicotiana tabacum) D-type cyclins and an A-type CDK are NtKIS1a, but not NtKIS1b, interacting partners. Although both NtKIS1a and NtKIS1b transcripts are mainly found in flowers and more precisely in stamens, NtKIS1b transcript levels are cell cycle regulated, whereas those of NtKIS1a remain constant during the cell cycle. NtKIS1a and NtKIS1b fused to fluorescent proteins are localized in the nucleus when transiently expressed in onion epidermal cells. Furthermore, there is no competition for their nuclear localization when they are simultaneously overexpressed. In vitro competition toward CDK kinase activity suggests that NtKIS1b is a strong competitor of NtKIS1a. Arabidopsis plants overexpressing NtKIS1a-green fluorescent protein (GFP) or NtKIS1b-GFP fusion proteins were obtained. In these plants, the fusion proteins are still localized in the nucleus. Interestingly, NtKIS1a-GFP-overexpressing plants display strong morphological modifications and a reduced CDK kinase activity, whereas NtKIS1b-GFP-overexpressing plants display a wild-type phenotype including a wild-type CDK kinase activity. Our results strongly suggest that the inhibition of the kinase activity is responsible for the phenotypic modifications.

Phosphorylation of p27KIP1 homologs KRP6 and 7 by SNF1‐related protein kinase–1 links plant energy homeostasis and cell proliferation

SNF1-related protein kinase-1 (SnRK1), the plant kinase homolog of mammalian AMP-activated protein kinase (AMPK), is a sensor that maintains cellular energy homeostasis via control of anabolism/catabolism balance. AMPK-dependent phosphorylation of p27(KIP1) affects cell-cycle progression, autophagy and apoptosis. Here, we show that SnRK1 phosphorylates the Arabidopsis thaliana cyclin-dependent kinase inhibitor p27(KIP1) homologs AtKRP6 and AtKRP7, thus extending the role of this kinase to regulation of cell-cycle progression. AtKRP6 and 7 were phosphorylated in vitro by a recombinant activated catalytic subunit of SnRK1 (AtSnRK1α1). Tandem mass spectrometry and site-specific mutagenesis identified Thr152 and Thr151 as the phosphorylated residues on AtKRP6- and AtKRP7, respectively. AtSnRK1 physically interacts with AtKRP6 in the nucleus of transformed BY-2 tobacco protoplasts, but, in contrast to mammals, the AtKRP6 Thr152 phosphorylation state alone did not modify its nuclear localization. Using a heterologous yeast system, consisting of a cdc28 yeast mutant complemented by A. thaliana CDKA;1, cell proliferation was shown to be abolished by AtKRP6(WT) and by the non-phosphorylatable form AtKRP6(T152A) , but not by the phosphorylation-mimetic form AtKRP6(T152D). Moreover, A. thaliana SnRK1α1/KRP6 double over-expressor plants showed an attenuated AtKRP6-associated phenotype (strongly serrated leaves and inability to undergo callogenesis). Furthermore, this severe phenotype was not observed in AtKRP6(T152D) over-expressor plants. Overall, these results establish that the energy sensor AtSnRK1 plays a cardinal role in the control of cell proliferation in A. thaliana plants through inhibition of AtKRP6 biological function by phosphorylation.

Petunia p34cdc2 protein kinase activity in G2/M cells obtained with a reversible cell cycle inhibitor, mimosine

Protoplasts isolated from petunia leaf mesophyll are non‐cycling cells mostly with 2C content. Cells regenerating from protoplast culture enter mitosis after 48 h. This experimental model is used to relate p34cd2 kinase activity to cell cycle phase. Our results show that the histone Hl phosphorylation, and hence p34cdc2 kinase activity, peaks with G2+ early M cell cycle phase. However, a trace kinase activity was already present when most cells were entering S phase. To obtain a maximum of cells in G1+S phases, the protoplast culture was treated with the rare amino acid, mimosine. Mimosine blocked plant cells derived from protoplast culture both at g1 and in early and mid S phase. Despite the increased G1+S level, p34cdc2 kinase activity did not increase. This suggests that the trace activity appearing when the majority of cells are entering S does not correspond to any putative P34cdc2 activation at G1/S transition but to the activation of the minor 4C population initially present in the leaf: the hypothesis remains that p34cdc2 kinase activity is solely related to G2+M phase in petunia.

The Cyclin-Dependent Kinase Inhibitor KRP6 Induces Mitosis and Impairs Cytokinesis in Giant Cells Induced by Plant-Parasitic Nematodes in Arabidopsis

This work points to an unexpected role for KRP6 during mitosis, suggesting that not all KRPs regulate the cell cycle in the same manner. The findings support the idea that plant-parasitic nematodes have evolved the ability to exploit plant cell cycle genes to the benefit of gall establishment. In Arabidopsis thaliana, seven cyclin-dependent kinase (CDK) inhibitors have been identified, designated interactors of CDKs or Kip-related proteins (KRPs). Here, the function of KRP6 was investigated during cell cycle progression in roots infected by plant-parasitic root-knot nematodes. Contrary to expectations, analysis of Meloidogyne incognita–induced galls of KRP6-overexpressing lines revealed a role for this particular KRP as an activator of the mitotic cell cycle. In accordance, KRP6-overexpressing suspension cultures displayed accelerated entry into mitosis, but delayed mitotic progression. Likewise, phenotypic analysis of cultured cells and nematode-induced giant cells revealed a failure in mitotic exit, with the appearance of multinucleated cells as a consequence. Strong KRP6 expression upon nematode infection and the phenotypic resemblance between KRP6 overexpression cell cultures and root-knot morphology point toward the involvement of KRP6 in the multinucleate and acytokinetic state of giant cells. Along these lines, the parasite might have evolved to manipulate plant KRP6 transcription to the benefit of gall establishment.

Flow and image cytometry of the uncoupling activity of heterologous proteins expressed in yeast

1 Biosystfimes Membranaires, CNRS, Gif-sur-Yvette, France. 2 Centre de G~n~tique Mol~culaire, CNRS, Gif-sur-Yvette, France. 3 CEREMOD, CNRS, Meudon, France. 4 Service de Cytom~trie, ISV, CNRS, Gif-sur-Yvette, France. 5 Laboratoire de Cytom~trie Analytique et Preparative, Institut Pasteur, Paris, France. 6 Service dImagerie Cellulaire, CNRS/Universit~, Orsay, France 7 Centro de Investigationes Biologicas, CSIC, Madrid, Spain.

An attempt to discriminate respiratory dysfunction or mitochondrial uncoupling in yeast mutants by confocal microscopy, image and flow cytometry

Phytoplankton community structure of the mediterranean Than lagoon has been studied over a year (1991-1992). Flow eytometry has allowed to describe the population structure by using properties of light scatter and fluorescence induced by cell pigment contents. The algal community is mainly composed o[ centric diatoms, dinoflagcllatcs, eryptophytcs, unspecified phototrophic micro-organisms and unknown picophytoplankton. The size of most of organisms estimated from size fractionation by filtration and from cytometric analysis ranges between 0.4 to 30 p.m. The relative abundance of the different algal sub-populations determines a spatio-temporal phytoplankton structure relatively stable in the lagoon. However in the ostreiculture zone one should note a significant decrease in the cell abundances specially of large size (> 2p.m) under the influence of filterfeeding animals. Flow cytometry has allowed to detect picoalgae (0.4 to 0.Sp.m) which form the major population of the algal assemblage. Cell concentrations vary from 2.103 cells ml -I (in winter) to 1.8.105 cells ml l (in summer). Picoplankton accounted for 20 to 95% of total algal cell abundance and for 10 to 50% of phototrophic biomass (0.3 to 7 mg m 3 as chlorophyll a) according to seasons and stations. These picoalgae emit only red fluorescence and arc characterized by high chlorophyll b on chlorophyll 11 ratio. This lagunar picoplanktonic population is quite different from marine picoplankton observed in areas under influence of Mediterranean Sea. It may be considered as typical of the particular physical and chemical features of lagunar ecosystem and specially of the Thau lagoon. The quantitative significance of this picophytoplankton unknown until now raises questions about the part and the status of this population in the phytoplankton community of the Thau lagoon. As well it raises questions about its contribution to the functioning of the aquatic system (relationships with the different compartments of II~c ecosystem).

Mitochondrial dysfunction in yeast expressing the cytoplasmic male sterility T-urf13 gene from maize: An image and flow cytometric analysis

Phytoplankton community structure of the mediterranean Than lagoon has been studied over a year (1991-1992). Flow eytometry has allowed to describe the population structure by using properties of light scatter and fluorescence induced by cell pigment contents. The algal community is mainly composed o[ centric diatoms, dinoflagcllatcs, eryptophytcs, unspecified phototrophic micro-organisms and unknown picophytoplankton. The size of most of organisms estimated from size fractionation by filtration and from cytometric analysis ranges between 0.4 to 30 p.m. The relative abundance of the different algal sub-populations determines a spatio-temporal phytoplankton structure relatively stable in the lagoon. However in the ostreiculture zone one should note a significant decrease in the cell abundances specially of large size (> 2p.m) under the influence of filterfeeding animals. Flow cytometry has allowed to detect picoalgae (0.4 to 0.Sp.m) which form the major population of the algal assemblage. Cell concentrations vary from 2.103 cells ml -I (in winter) to 1.8.105 cells ml l (in summer). Picoplankton accounted for 20 to 95% of total algal cell abundance and for 10 to 50% of phototrophic biomass (0.3 to 7 mg m 3 as chlorophyll a) according to seasons and stations. These picoalgae emit only red fluorescence and arc characterized by high chlorophyll b on chlorophyll 11 ratio. This lagunar picoplanktonic population is quite different from marine picoplankton observed in areas under influence of Mediterranean Sea. It may be considered as typical of the particular physical and chemical features of lagunar ecosystem and specially of the Thau lagoon. The quantitative significance of this picophytoplankton unknown until now raises questions about the part and the status of this population in the phytoplankton community of the Thau lagoon. As well it raises questions about its contribution to the functioning of the aquatic system (relationships with the different compartments of II~c ecosystem).