Molecular characterization of Arabidopsis PHO80-like proteins, a novel class of CDKA;1-interacting cyclins.

Cyclins are regulatory proteins that interact with cyclin-dependent kinases (CDKs) to control progression through the cell cycle. In Arabidopsis thaliana, 34 cyclin genes have been described, grouped into five different types (A, B, D, H, and T). A novel class of seven cyclins was isolated and characterized in Arabidopsis, designated P-type cyclins (CYCPs). They all share a conserved central region of 100 amino acids ("cyclin box") displaying homology to the corresponding region of the PHO80 cyclin from Saccharomyces cerevisiae and the related G1 cyclins from Trypanosoma cruzi and T. brucei. The CYCP4;2 gene was able to partially re-establish the phosphate-dependent expression of the PHO5 gene in a pho80 mutant strain of yeast. The CYCPs interact preferentially with CDKA;1 in vivo and in vitro as shown by yeast two-hybrid analysis and co-immunoprecipitation experiments. P-type cyclins were mostly expressed in proliferating cells, albeit also in differentiating and mature tissues. The possible role of CYCPs in linking cell division, cell differentiation, and the nutritional status of the cell is discussed.

Novel complexes of cyclin-dependent kinases and a cyclin-like protein from Arabidopsis thaliana with a function unrelated to cell division.

Although the majority of cyclin-dependent kinases (CDKs) play a key role in cell cycle progression, recent evidence has shown that CDKs are also implicated in transcription regulation. Here, we describe two Arabidopsis CDKs designated Arath;CDKC;1 and Arath; CDKC;2. These CDKs share a PITAIRE signature in the cyclin-binding domain and the structural characteristics of mammalian CDK9. Yeast two-hybrid screens and immunoprecipitation assays identified CDKC-interacting proteins with homology to the animal cyclin T/cyclin K group. We suggest that these Arabidopsis CDKCs may be part of a kinase complex similar to the animal positive transcription elongation factor b, whose activity is essential for transcription control. Expression studies showed that Arath; CDKC transcripts are mainly confined to epidermal tissues and are most abundant in flower tissues. No expression was detected in actively dividing Arabidopsis tissues, suggesting a role for the CDKC proteins in differentiated cells.

Analysis of cell division parameters and cell cycle gene expression during the cultivation of Arabidopsis thaliana cell suspensions.

Arabidopsis thaliana cell suspension cultures were characterized for the first time in detail in terms of biomass accumulation, cell division rate and cell cycle phase durations. Subsequently, this model system was used to follow the transcription profile of key cell cycle genes during a complete cultivation cycle. According to the calculated changes in the relative division rate over time, the cell cycle genes could be classified into four groups based on their transcriptional expression pattern. These differential patterns of gene expression are discussed with respect to the putative roles of the different cell cycle genes in the division cycle. Analysis of protein levels showed that mRNA levels did not correlate with protein levels in all cases. Results obtained in other systems, such as BY-2 cell suspensions or plants, confirm that cell suspension cultures of A. thaliana are suitable for the analysis of cell cycle regulation.

A plant-specific cyclin-dependent kinase is involved in the control of G2/M progression in plants.

Cyclin-dependent kinases (CDKs) control the key transitions in the eukaryotic cell cycle. All the CDKs known to control G(2)/M progression in yeast and animals are distinguished by the characteristic PSTAIRE motif in their cyclin-binding domain and are closely related. Higher plants contain in addition a number of more divergent non-PSTAIRE CDKs with still obscure functions. We show that a plant-specific type of non-PSTAIRE CDKs is involved in the control of the G(2)/M progression. In synchronized tobacco BY-2 cells, the corresponding protein, accumulated in a cell cycle-regulated fashion, peaking at the G(2)/M transition. The associated histone H1 kinase activity reached a maximum in mitosis and required a yet unidentified subunit to be fully active. Down-regulation of the associated kinase activity in transgenic tobacco plants using a dominant-negative mutation delayed G(2)/M transition. These results provide the first evidence that non-PSTAIRE CDKs are involved in the control of the G(2)/M progression in plants.

Identification of novel cyclin-dependent kinases interacting with the CKS1 protein of Arabidopsis.

The SUC1/CKS1 proteins interact with cyclin-dependent kinases (CDKs) and play an essential, but yet not entirely resolved, role in the regulation of the cell cycle. With the Arabidopsis thaliana CKS1At protein as bait in a two-hybrid screen, two novel Arabidopsis CDKs, Arath;CDKB1;2 and Arath;CDKB2;1, were isolated. A closely related homologue of Arath;CDKB2;1 was discovered in the databases and was nominated Arath;CDKB2;2. Transcript analysis of the five known Arath;CDKA and Arath;CDKB genes revealed that they all had the highest expression in flowers and cell suspensions. Differences in the expression patterns in roots, leaves and stems suggest unique roles for each CDK.

Functional analysis of cyclin-dependent kinase inhibitors of Arabidopsis.

Cyclin-dependent kinase inhibitors, such as the mammalian p27(Kip1) protein, regulate correct cell cycle progression and the integration of developmental signals with the core cell cycle machinery. These inhibitors have been described in plants, but their function remains unresolved. We have isolated seven genes from Arabidopsis that encode proteins with distant sequence homology with p27(Kip1), designated Kip-related proteins (KRPs). The KRPs were characterized by their domain organization and transcript profiles. With the exception of KRP5, all presented the same cyclin-dependent kinase binding specificity. When overproduced, KRP2 dramatically inhibited cell cycle progression in leaf primordia cells without affecting the temporal pattern of cell division and differentiation. Mature transgenic leaves were serrated and consisted of enlarged cells. Although the ploidy levels in young leaves were unaffected, endoreduplication was suppressed in older leaves. We conclude that KRP2 exerts a plant growth inhibitory activity by reducing cell proliferation in leaves, but, in contrast to its mammalian counterparts, it may not control the timing of cell cycle exit and differentiation.

CKS1At overexpression in Arabidopsis thaliana inhibits growth by reducing meristem size and inhibiting cell-cycle progression.

The SUC1/CKS1 proteins associate with cyclin-dependent kinases (CDKs) and play an essential role in the regulation of the cell cycle. Recently, an Arabidopsis thaliana SUC1/CKS1 homologous gene, designated CKS1At, has been cloned. Here, overexpression of CKS1At in Arabidopsis is shown to reduce leaf size and root growth rates. Reduced root growth resulted primarily from an increase of the cell-cycle duration and a shortening of the meristem. Endoreduplication was unaffected. The increased cell-cycle duration was associated with an equal extension of both the G1 and G2 phases. This inhibition was due to the binding of CDK subunits with CDKs. The reduced growth rates in response to altered cell-cycle gene expression demonstrates a direct dependence of plant growth rates on cell-cycle regulation.

Characterization of two distinct DP-related genes from Arabidopsis thaliana.

E2F/DP complexes play a pivotal role in the regulation of the G1/S transition in animals. Recently, plant E2F homologs have been cloned, but DP-related sequences have not been identified so far. Here we report that Arabidopsis thaliana contains at least two different DP-related genes, AtDPa and AtDPb. They exhibit an overall domain organization similar to that of their animal counterparts, although phylogenetic analysis demonstrated that they form a separate subgroup. AtDPs efficiently heterodimerize in vitro with the Arabidopsis E2F-related proteins, AtE2Fa and AtE2Fb through their dimerization domains. AtDPa and AtE2Fa are predominantly produced in actively dividing cells with highest transcript levels in early S phase cells.

Increased leakiness of the tetracycline-inducible Triple-Op promoter in dividing cells renders it unsuitable for high inducible levels of a dominant negative CDC2aAt gene.

A tetracycline-inducible promoter system was used to generate transgenic tobacco plants that confer inducible expression of the wild type or a dominant negative allele of the gene coding for the cyclin-dependent kinase (CDK) of Arabidopsis thaliana CDC2aAt. Although the total extractable CDK activity was doubled, the induced expression of the wild-type CDC2aAt did not correlate with any change of the cell cycle kinetics. An increase of CDK activity upon CDC2aAt expression was only seen in dividing cell populations, demonstrating that CDC2aAt expression itself is not sufficient to induce CDK activation. Induced expression of the dominant negative CDC2aAt.N146 correlated with a reduction of CDK activity to 66% of the level found in non-induced cells. This decrease was not sufficient to block cell division. The isolation of plants showing only low inducible levels of CDC2aAt.N146 suggests that a counterselection against strong inducible lines had occurred. Accordingly, Triple-Op promoter activity was found in dividing cells in the absence of tetracycline.

The Arabidopsis thaliana PIN1At gene encodes a single-domain phosphorylation-dependent peptidyl prolyl cis/trans isomerase.

A homologue of the human site-specific prolyl cis/trans isomerase PIN1 was identified in Arabidopsis thaliana. The PIN1At gene encodes a protein of 119 amino acids that is 53% identical with the catalytic domain of the human PIN1 parvulin. Steady-state PIN1At mRNA is found in all plant tissues tested. We show by two-dimensional NMR spectroscopy that the PIN1At is a prolyl cis/trans isomerase with specificity for phosphoserine-proline bonds. PIN1At is the first example of an eukaryotic parvulin without N- or C-terminal extensions. The N-terminal WW domain of 40 amino acids, typical of all the phosphorylation-dependent eukaryotic parvulins, is absent. However, triple-resonance NMR experiments showed that PIN1At contained a hydrophobic helix similar to the alpha1 helix observed in PIN1 that could mediate the protein-protein interactions.

A new D-type cyclin of Arabidopsis thaliana expressed during lateral root primordia formation.

D-type cyclins are believed to regulate the onset of cell division upon mitogenic signaling. Here, the isolation is reported of a new D-type cyclin gene (CYCD4;1) of Arabidopsis thaliana (L.) Heynh. during a two-hybrid screen using the cyclin-dependent kinase CDC2aAt as bait. Transcription of CYCD4;1 can be induced by sucrose. The co-regulated expression of CYCD4;1 and CDC2aAt in starved suspension cultures upon mitogenic stimulation indicates that the formation of a complex between these two partners is important for the resumption of cell division activity. By in-situ hybridizations CYCD4;1 was shown to be expressed during vascular tissue development, embryogenesis, and formation of lateral root primordia. Expression during the latter process suggests that the induced expression of D-type cyclins by mitogenic stimuli might be one of the rate-limiting events for the initiation of lateral roots.

Recombinant production of the p10CKS1At protein from Arabidopsis thaliana and 13C and 15N double-isotopic enrichment for NMR studies.

The CKS1At gene product, p10CKS1At from Arabidopsis thaliana, is a member of the cyclin-dependent kinase subunit (CKS) family of small proteins. These proteins bind the cyclin-dependent kinase (CDK)/cyclin complexes and play an essential, but still not precisely known role in cell cycle progression. To solve the structure of p10CKS1At, a protocol was needed to produce the quantity of protein large enough for nuclear magnetic resonance (NMR) spectroscopy. The first attempt to express CKS1At in Escherichia coli under the control of the T7 promoter was not successful. E. coli BL21(DE3) cotransformed with the CKS1At gene and the E. coli argU gene that encoded the arginine acceptor tRNAUCU produced a sufficient amount of p10CKS1At to start the structural study by NMR. Replacement of four rare codons in the CKS1At gene sequence, including a tandem arginine, by highly used codons in E. coli, restored also a high expression of the recombinant protein. Double-isotopic enrichment by 13C and 15N is reported that will facilitate the NMR study. Isotopically labeled p10CKS1At was purified to yield as much as 55 mg from 1 liter of minimal media by a two-step chromatographic procedure. Preliminary results of NMR spectroscopy demonstrate that a full structural analysis using triple-resonance spectra is feasible for the labeled p10CKS1At protein.

Mutational analysis of two Arabidopsis thaliana cyclin-dependent kinases in fission yeast.

We have analyzed five mutant alleles of two cyclin-dependent kinases from Arabidopsis thaliana, CDC2aAt and CDC2bAt, in Schizosaccharomyces pombe. Two of the five mutant alleles produced similar phenotypes for both cyclin-dependent kinases. The other three mutants caused phenotypes dependent on the particular cyclin-dependent kinase. Of all the mutant alleles, only two were found to possess a detectable kinase activity. Our mutational analysis lends further support for CDC2aAt being the true orthologue of the yeast cdc2. CDC2bAt, even though quite divergent from S. pombe cdc2, still retains the ability to interact with at least some essential cell cycle regulators, suggesting some functional homology with the yeast protein. Additionally, we demonstrated that the three amino acid deletion in the DL50 mutants results in the loss of the ability to interact with the suc1/CKS1 proteins.

Expression of CKS1At in Arabidopsis thaliana indicates a role for the protein in both the mitotic and the endoreduplication cycle.

Although endoreduplication is common in plants, little is known about the mechanisms regulating this process. Here, we report the patterns of endoreduplication at the cellular level in the shoot apex of Arabidopsis thaliana L. Heynh. plants grown under short-day conditions. We show that polyploidy is developmentally established in the pith, maturing leaves, and stipules. To investigate the role of the cell cycle genes CDC2aAt, CDC2bAt, CYCB1;1, and CKS1At in the process of endoreduplication, in-situ hybridizations were performed on the vegetative shoot apices. Expression of CDC2aAt, CDC2bAt, and CYCB1;1 was restricted to mitotically dividing cells. In contrast, CKS1At expression was present in both mitotic and endoreduplicating tissues. Our data indicate that CDC2aAt, CDC2bAt, and CYCB1;1 only operate during mitotic divisions, whereas CKS1At may play a role in both the mitotic and endoreduplication cycle.

The Arabidopsis Cks1At protein binds the cyclin-dependent kinases Cdc2aAt and Cdc2bAt.

In Arabidopsis, two cyclin-dependent kinases (CDK), Cdc2aAt and Cdc2bAt, have been described. Here, we have used the yeast two-hybrid system to identify Arabidopsis proteins interacting with Cdc2aAt. Three different clones were isolated, one of which encodes a Suc1/Cks1 homologue. The functionality of the Arabidopsis Suc1/Cks1 homologue, designed Cks1At, was demonstrated by its ability to rescue the temperature-sensitive cdc2-L7 strain of fission yeast at low and intermediate expression levels. In contrast, high cks1At expression levels inhibited cell division in both mutant and wild-type yeast strains. Cks1At binds both Cdc2aAt and Cdc2bAt in vivo and in vitro. Furthermore, we demonstrate that the fission yeast Suc1 binds Cdc2aAt but only weakly Cdc2bAt, whereas the human CksHs1 associated exclusively with Cdc2aAt.

Herbicide safener-inducible gene expression in Arabidopsis thaliana.

The potential use of a new chemical-inducible gene expression system in Arabidopsis thaliana has been examined. The system is based on the maize In2-2 promoter which is activated by benzenesulfonamide herbicide safeners. Plants transformed with the beta-glucuronidase (gus) reporter gene under the control of the In2-2 promoter were grown in the presence of different safeners and the induced GUS activity pattern was studied histochemically. In the absence of safeners, the In2-2 promoter was not active. Application of different safeners induced distinct gus expression patterns, including expression in the root, hydathodes, and the shoot apical meristem. Plants maintained continuously on inducing concentrations of the safeners were retarded in growth. The growth inhibition effects of the Sa5 safener could be overcome in a sulfonylurea-resistant background. In2-2 promoter activity could also be induced by the sulfonylurea herbicide chlorsulfuron. In the sulfonylurea-resistant background, which derives from herbicide-resistant acetolactate synthase activity, induction of the In2-2 promoter by chlorsulfuron was lower. Furthermore, branched-chain amino acids, known to inhibit acetolactate synthase activity, also induced In2-2 promoter activity. Our data suggest a strong correlation between In2-2 expression and inhibition of the acetolactate synthase activity.