Sara Maes

Functional Analysis of Cyclin-Dependent Kinase Inhibitors of Arabidopsis

Cyclin-dependent kinase inhibitors, such as the mammalian p27Kip1 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 p27Kip1, 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.

Control of proliferation, endoreduplication and differentiation by the Arabidopsis E2Fa-DPa transcription factor.

New plant cells arise at the meristems, where they divide a few times before they leave the cell‐cycle program and start to differentiate. Here we show that the E2Fa–DPa transcription factor of Arabidopsis thaliana is a key regulator determining the proliferative status of plant cells. Ectopic expression of E2Fa induced sustained cell proliferation in normally differentiated cotyledon and hypocotyl cells. The phenotype was enhanced strongly by the co‐expression of E2Fa with its dimerization partner, DPa. In endoreduplicating cells, E2Fa–DPa also caused extra DNA replication that was correlated with transcriptional induction of S phase genes. Because E2Fa–DPa transgenic plants arrested early in development, we argue that controlled exit of the cell cycle is a prerequisite for normal plant development.

The Cyclin-Dependent Kinase Inhibitor KRP2 Controls the Onset of the Endoreduplication Cycle during Arabidopsis Leaf Development through Inhibition of Mitotic CDKA;1 Kinase Complexes

Exit from the mitotic cell cycle and initiation of cell differentiation frequently coincides with the onset of endoreduplication, a modified cell cycle during which DNA continues to be duplicated in the absence of mitosis. Although the mitotic cell cycle and the endoreduplication cycle share much of the same machinery, the regulatory mechanisms controlling the transition between both cycles remain poorly understood. We show that the A-type cyclin-dependent kinase CDKA;1 and its specific inhibitor, the Kip-related protein, KRP2 regulate the mitosis-to-endocycle transition during Arabidopsis thaliana leaf development. Constitutive overexpression of KRP2 slightly above its endogenous level only inhibited the mitotic cell cycle–specific CDKA;1 kinase complexes, whereas the endoreduplication cycle-specific CDKA;1 complexes were unaffected, resulting in an increase in the DNA ploidy level. An identical effect on the endoreduplication cycle could be observed by overexpressing KRP2 exclusively in mitotically dividing cells. In agreement with a role for KRP2 as activator of the mitosis-to-endocycle transition, KRP2 protein levels were more abundant in endoreduplicating than in mitotically dividing tissues. We illustrate that KRP2 protein abundance is regulated posttranscriptionally through CDK phosphorylation and proteasomal degradation. KRP2 phosphorylation by the mitotic cell cycle–specific CDKB1;1 kinase suggests a mechanism in which CDKB1;1 controls the level of CDKA;1 activity through regulating KRP2 protein abundance. In accordance with this model, KRP2 protein levels increased in plants with reduced CDKB1;1 activity. Moreover, the proposed model allowed a dynamical simulation of the in vivo observations, validating the sufficiency of the regulatory interactions between CDKA;1, KRP2, and CDKB1;1 in fine-tuning the mitosis-to-endocycle transition.

The Plant-Specific Cyclin-Dependent Kinase CDKB1;1 and Transcription Factor E2Fa-DPa Control the Balance of Mitotically Dividing and Endoreduplicating Cells in Arabidopsis

Transgenic Arabidopsis thaliana plants overproducing the E2Fa-DPa transcription factor have two distinct cell-specific phenotypes: some cells divide ectopically and others are stimulated to endocycle. The decision of cells to undergo extra mitotic divisions has been postulated to depend on the presence of a mitosis-inducing factor (MIF). Plants possess a unique class of cyclin-dependent kinases (CDKs; B-type) for which no ortholog is found in other kingdoms. The peak of CDKB1;1 activity around the G2-M boundary suggested that it might be part of the MIF. Plants that overexpressed a dominant negative allele of CDKB1;1 underwent enhanced endoreduplication, demonstrating that CDKB1;1 activity was required to inhibit the endocycle. Moreover, when the mutant CDKB1;1 allele was overexpressed in an E2Fa-DPa–overproducing background, it enhanced the endoreduplication phenotype, whereas the extra mitotic cell divisions normally induced by E2Fa-DPa were repressed. Surprisingly, CDKB1;1 transcription was controlled by the E2F pathway, as shown by its upregulation in E2Fa-DPa–overproducing plants and mutational analysis of the E2F binding site in the CDKB1;1 promoter. These findings illustrate a cross talking mechanism between the G1-S and G2-M transition points.

The DP-E2F-like Gene DEL1 Controls the Endocycle in Arabidopsis thaliana

Endoreduplication or DNA replication without mitosis is widespread in nature. Well-known examples are fruit fly polytene chromosomes and cereal endosperm. Although endocycles are thought to be driven by the same regulators as those that control the G1-S transition of the mitotic cell cycle, the molecular mechanisms that differentiate mitotically dividing cells from endoreduplicating ones are largely unknown. A novel class of atypical E2F-like proteins has recently been identified and is designated E2F7 in mammals and DP-E2F-like (DEL) in Arabidopsis thaliana . We demonstrate that loss of DEL1 function resulted in increased ploidy levels, whereas ectopic expression of DEL1 reduced endoreduplication. Ploidy changes were correlated with altered expression of a subset of E2F target genes encoding proteins necessary for DNA replication. Because DEL1 proteins were postulated to antagonize the E2F pathway, we generated DEL1-E2Fa-DPa triple transgenics. DEL1 inhibited the endoreduplication phenotype, but not the ectopic cell divisions that resulted from the overexpression of both E2Fa and DPa, illustrating that DEL1 specifically represses the endocycle. Because DEL1 transcripts were detected exclusively in mitotically dividing cells, we conclude that DEL1 is an important novel inhibitor of the endocycle and preserves the mitotic state of proliferating cells by suppressing transcription of genes that are required for cells to enter the DNA endoreduplication cycle.

Atypical E2F activity restrains APC/CCCS52A2 function obligatory for endocycle onset

The endocycle represents an alternative cell cycle that is activated in various developmental processes, including placental formation, Drosophila oogenesis, and leaf development. In endocycling cells, mitotic cell cycle exit is followed by successive doublings of the DNA content, resulting in polyploidy. The timing of endocycle onset is crucial for correct development, because polyploidization is linked with cessation of cell division and initiation of terminal differentiation. The anaphase-promoting complex/cyclosome (APC/C) activator genes CDH1, FZR, and CCS52 are known to promote endocycle onset in human, Drosophila, and Medicago species cells, respectively; however, the genetic pathways governing development-dependent APC/CCDH1/FZR/CCS52 activity remain unknown. We report that the atypical E2F transcription factor E2Fe/DEL1 controls the expression of the CDH1/FZR orthologous CCS52A2 gene from Arabidopsis thaliana. E2Fe/DEL1 misregulation resulted in untimely CCS52A2 transcription, affecting the timing of endocycle onset. Correspondingly, ectopic CCS52A2 expression drove cells into the endocycle prematurely. Dynamic simulation illustrated that E2Fe/DEL1 accounted for the onset of the endocycle by regulating the temporal expression of CCS52A2 during the cell cycle in a development-dependent manner. Analogously, the atypical mammalian E2F7 protein was associated with the promoter of the APC/C-activating CDH1 gene, indicating that the transcriptional control of APC/C activator genes by atypical E2Fs might be evolutionarily conserved.

Moisture Source and Diet affect Development and Reproduction of Orius thripoborus and Orius naivashae, two Predatory Anthocorids from Southern Africa

Abstract The effect of moisture source and diet on the development and reproduction of the pirate bugs, Orius thripoborus (Hesse) and Orius naivashae (Poppius) (Hemiptera: Anthocoridae) was examined in the laboratory. Both species had been collected in and around sugarcane fields in South Africa. Supplementing eggs of the flour moth Ephestia kuehniella (Zeller) (Lepidoptera: Pyralidae) with a green bean pod as a moisture source yielded better nymphal survival and faster development, as compared with free water encapsulated in Parafilm, suggesting that the predators may extract extra nutrients from the bean pod. The impact of two factitious foods and moist honey bee pollen on developmental and reproductive parameters of both predators was also investigated. The overall performance of both Orius species on E. kuehniella eggs and cysts of brine shrimp, Artemia franciscana Kellogg (Crustacea: Artemiidae) was better than on pollen. Nonetheless, a pollen diet alone allowed 66 and 78% of the nymphs of O. thripoborus and O. naivashae, respectively, to reach adulthood. Overall, developmental and reproductive performance of O. thripoborus on the tested diets was superior to that of O. naivashae. The implications of these findings for the mass production of these predators and their potential role in biological control programs in southern Africa are discussed.

Microarray analysis of E2Fa-DPa-overexpressing plants uncovers a cross-talking genetic network between DNA replication and nitrogen assimilation

Previously we have shown that overexpression of the heterodimeric E2Fa-DPa transcription factor in Arabidopsis thaliana results in ectopic cell division, increased endoreduplication, and an early arrest in development. To gain a better insight into the phenotypic behavior of E2Fa-DPa transgenic plants and to identify E2Fa-DPa target genes, a transcriptomic microarray analysis was performed. Out of 4,390 unique genes, a total of 188 had a twofold or more up- (84) or down-regulated (104) expression level in E2Fa-DPa transgenic plants compared to wild-type lines. Detailed promoter analysis allowed the identification of novel E2Fa-DPa target genes, mainly involved in DNA replication. Secondarily induced genes encoded proteins involved in cell wall biosynthesis, transcription and signal transduction or had an unknown function. A large number of metabolic genes were modified as well, among which, surprisingly, many genes were involved in nitrate assimilation. Our data suggest that the growth arrest observed upon E2Fa-DPa overexpression results at least partly from a nitrogen drain to the nucleotide synthesis pathway, causing decreased synthesis of other nitrogen compounds, such as amino acids and storage proteins.

Dispersal potential of native and exotic predatory ladybirds as measured by a computer-monitored flight mill

The performance of three species of predatory ladybirds was compared in a flight mill and the effect of diet on their flight parameters was tested. The invasive ladybird Harmonia axyridis Pallas (Coleoptera: Coccinellidae) outperformed Cryptolaemus montrouzieri Mulsant (Coleoptera: Coccinellidae) and Adalia bipunctata (L.) (Coleoptera: Coccinellidae) in terms of flight distance, duration and velocity. Harmonia axyridis flew at least two times further, needed three times less breaks and flew two times faster than C. montrouzieri and A. bipunctata fed the same diet. Ladybirds reared on eggs of Ephestia kuehniella (Zeller) (Lepidoptera: Pyralidae) performed better than their counterparts reared on natural prey (aphids for H. axyridis and A. bipunctata, mealybugs for C. montrouzieri). The findings of this study indicate that comparative flight studies can be useful to identify candidate biocontrol agents with pronounced dispersal abilities and thus can yield significant evidence to be used in an environmental risk assessment. However, it also demonstrates that variability related to mass rearing conditions should not be ignored when standardizing a risk assessment procedure for candidate biocontrol agents.

The influence of acclimation, endosymbionts and diet on the supercooling capacity of the predatory bug Macrolophus pygmaeus

The generalist predator Macrolophus pygmaeus Rambur (Hemiptera: Miridae) is a key biological control agent in European greenhouses. The influence of acclimation, infection with endosymbiotic bacteria and diet on the cold tolerance of the Mediterranean biocontrol population was assessed by determining the supercooling point, i.e. the temperature at which the insect’s body fluids freeze. This parameter provides a first indication of an insect’s establishment potential in a new region and of its possible geographical range. Allowing the predatory bugs to adapt to lower temperatures resulted in an increase in supercooling ability. Macrolophus pygmaeus bugs exposed to antibiotics in their artificial diet and hence cured from their infection with the endosymbiotic bacteria Wolbachia pipientis, Rickettsia bellii and R. limoniae were more tolerant to freezing than infected bugs. The diet of the predators also affected the freezing temperature of the body fluids. Predators fed an artificial diet based on egg yolk were less resistant to freezing than those fed Ephestia kuehniella Zeller (Lepidoptera: Pyralidae) eggs. These findings illustrate that several factors may affect the cold hardiness of a biocontrol agent and may thus complicate the evaluation of its establishment potential in the framework of an environmental risk assessment.