Crystal structure of Arabidopsis thaliana casein kinase 2 α1

Casein kinase 2 (CK2) is a ubiquitous pleiotropic enzyme that is highly conserved across eukaryotic kingdoms. CK2 is singular amongst kinases as it is highly rigid and constitutively active. Arabidopsis thaliana is widely used as a model system in molecular plant research; the biological functions of A. thaliana CK2 are well studied in vivo and many of its substrates have been identified. Here, crystal structures of the alpha subunit of A. thaliana CK2 in three crystal forms and of its complex with the nonhydrolyzable ATP analog AMppNHp are presented. While the C-lobe of the enzyme is highly rigid, structural plasticity is observed for the N-lobe. Small but significant displacements within the active cleft are necessary in order to avoid steric clashes with the AMppNHp molecule. Binding of AMppNHp is influenced by a rigid-body motion of the N-lobe that was not previously recognized in maize CK2

Plant DNA polymerases

Maintenance of genome integrity is a key process in all organisms. DNA polymerases (Pols) are central players in this process as they are in charge of the faithful reproduction of the genetic information, as well as of DNA repair. Interestingly, all eukaryotes possess a large repertoire of polymerases. Three protein complexes, DNA Pol α, δ, and ε, are in charge of nuclear DNA replication. These enzymes have the fidelity and processivity required to replicate long DNA sequences, but DNA lesions can block their progression. Consequently, eukaryotic genomes also encode a variable number of specialized polymerases (between five and 16 depending on the organism) that are involved in the replication of damaged DNA, DNA repair, and organellar DNA replication. This diversity of enzymes likely stems from their ability to bypass specific types of lesions. In the past 10–15 years, our knowledge regarding plant DNA polymerases dramatically increased. In this review, we discuss these recent findings and compare acquired knowledge in plants to data obtained in other eukaryotes. We also discuss the emerging links between genome and epigenome replication

Defects in leaf epidermis of Arabidopsis thaliana plants with CDKA;1 activity reduced in the shoot apical meristem

In Arabidopsis thaliana, like in other dicots, the shoot epidermis originates from protodermis, the outermost cell layer of shoot apical meristem. We examined leaf epidermis in transgenic A. thaliana plants in which CDKA;1.N146, a negative dominant allele of A-type cyclin-dependent kinase, was expressed from the SHOOTMERISTEMLESS promoter, i.e., in the shoot apical meristem. Using cleared whole mount preparations of expanding leaves and sequential in vivo replicas of expanding leaf surface, we show that dominant-negative CDKA;1 expression results in defects in epidermis continuity: loss of individual cells and occurrence of gaps between anticlinal walls of neighboring pavement cells. Another striking feature is ingrowth-like invaginations of anticlinal cell walls of pavement cells. Their formation is related to various processes: expansion of cells surrounding the sites of cell loss, defected cytokinesis, and presumably also, the actual ingrowth of an anticlinal cell wall. The mutant exhibits also increased variation in cell size and locally reduced waviness of anticlinal walls of pavement cells. These unusual features of leaf epidermis phenotype may shed a new light on our knowledge on morphogenesis of jigsaw puzzle-shaped pavement cells and on the CDKA;1 role in regulation of plant development via influence on cytoskeleton and plant cell wall

Functional characterization of the diatom cyclin-dependent kinase A2 as a mitotic regulator reveals plant-like properties in a non-green lineage

Background: Cyclin-dependent kinases (CDKs) are crucial regulators of cell cycle progression in eukaryotes. The diatom CDKA2 was originally assigned to the classical A-type CDKs, but its cell cycle phase-specific transcription at the G2-to-M phase transition is typical for plant-specific B-type CDKs. Results: Here, we report the functional characterization of CDKA2 from the diatom Phaeodactylum tricornutum. Through a yeast two-hybrid library screen, CDKA2 was found to interact with the G2/M-specific CDK scaffolding factor CKS1. Localization of CDKA2 was found to be nuclear in interphase cells, while in cells undergoing cytokinesis, the signal extended to the cell division plane. In addition, overexpression of CDKA2 induced an overall reduction in the cell growth rate. Expression analysis of cell cycle marker genes in the overexpression lines indicates that this growth reduction is primarily due to a prolongation of the mitotic phase. Conclusions: Our study indicates a role for CDKA2 during cell division in diatoms. The functional characterization of a CDK with clear CDKB properties in a non-green organism questions whether the current definition of B-type CDKs being plant-specific might need revision

Root-knot nematode feeding site development is impaired by cyclin-dependent kinase inhibitors

Plant-parasitic nematodes of the genera Meloidogyne trigger the formation of giant cells that undergo recurring acytokinetic mitosis and endocycles. Expression analyses of key cell cycle genes showed their early induction in the nematode feeding site (NFS). Additionally, disturbance in NFS development and juvenile maturation were observed during treatment of infected roots with cell cycle inhibitors. Intense DNA synthesis and enlarged nuclei demonstrated that giant cells undergo additional endocycles. How precisely nematodes manipulate the cell cycle in their favour remains to be understood. A systematic comparison of the temporal and spatial expression pattern of core cell cycle genes between uninfected roots and in galls of Arabidopsis thaliana resulted in the identification of a collection of genes up- or downregulated in NFC. Among them, negative regulators are candidates to control the cell cycle in NFC. Previous work has shown that KRP2, a member of the cyclin-dependent kinase/kip-related proteins (ICK/KRP), regulate mitosis-to-endocycle transition in plant cells, and is expressed in endoreduplicating cells. The KRP2 gene showed to be expressed during gall development. Therefore to study the relevance of the KRP cell cycle inhibitor genes (7 in Arabidopis) for NFS ontogeny, mutant lines over-expressing and knocked-out are being tested to determine their effect on feeding site development. In vivo subcellular localization studies have been carried out to better understand the dynamics of these proteins during giant cell development. Based on these data, three KRP genes are perceived to control giant cell size and consequently nematode reproduction

Endoreplication as a potential driver of cell wall modifications

Endoreplication represents a variant of the mitotic cell cycle during which cells replicate their DNA without mitosis and/or cytokinesis, resulting in an increase in the cells’ ploidy level. This process is especially prominent in higher plants, where it has been correlated with cell differentiation, metabolic output and rapid cell growth. However, different reports argue against a ploidy-dependent contribution to cell growth. Here, we review accumulating data suggesting that endocycle onset might exert an effect on cell growth through transcriptional control of cell wall-modifying genes to drive cell wall changes required to accommodate turgor-driven rapid cell expansion, consistent with the idea that vacuolar expansion rather than a ploidy-driven increase in cellular volume represents the major force driving cell growth

Associated bacteria affect sexual reproduction by altering gene expression and metabolic processes in a biofilm inhabiting diatom

Diatoms are unicellular algae with a fundamental role in global biogeochemical cycles as major primary producers at the base of aquatic food webs. In recent years, chemical communication between diatoms and associated bacteria has emerged as a key factor in diatom ecology, spurred by conceptual and technological advancements to study the mechanisms underlying these interactions. Here, we use a combination of physiological, transcriptomic, and metabolomic approaches to study the influence of naturally coexisting bacteria, Maribacter sp. and Roseovarius sp., on the sexual reproduction of the biofilm inhabiting marine pennate diatom Seminavis robusta. While Maribacter sp. severely reduces the reproductive success of S. robusta cultures, Roseovarius sp. slightly enhances it. Contrary to our expectation, we demonstrate that the effect of the bacterial exudates is not caused by altered cell-cycle regulation prior to the switch to meiosis. Instead, Maribacter sp. exudates cause a reduced production of diproline, the sexual attraction pheromone of S. robusta. Transcriptomic analyses show that this is likely an indirect consequence of altered intracellular metabolic fluxes in the diatom, especially those related to amino acid biosynthesis, oxidative stress response, and biosynthesis of defense molecules. This study provides the first insights into the influence of bacteria on diatom sexual reproduction and adds a new dimension to the complexity of a still understudied phenomenon in natural diatom populations

Tissue-specific control of the endocycle by the anaphase promoting complex/cyclosome inhibitors UVI4 and DEL1

The endocycle represents a modified mitotic cell cycle that in plants is often coupled to cell enlargement and differentiation. Endocycle onset is controlled by activity of the Anaphase Promoting Complex/Cyclosome (APC/C), a multisubunit E3 ubiquitin ligase targeting cell-cycle factors for destruction. CELL CYCLE SWITCH52 (CCS52) proteins represent rate-limiting activator subunits of the APC/C. In Arabidopsis (Arabidopsis thaliana), mutations in either CCS52A1 or CCS52A2 activators result in a delayed endocycle onset, whereas their overexpression triggers increased DNA ploidy levels. Here, the relative contribution of the APC/C-CCS52A1 and APC/C-CCS52A2 complexes to different developmental processes was studied through analysis of their negative regulators, being the ULTRAVIOLET-B-INSENSITIVE4 protein and the DP-E2F-Like1 transcriptional repressor, respectively. Our data illustrate cooperative activity of the APC/C-CCS52A1 and APC/C-CCS52A2 complexes during root and trichome development, but functional interdependency during leaf development. Furthermore, we found APC/C-CCS52A1 activity to control CCS52A2 expression. We conclude that interdependency of CCS52A-controlled APC/C activity is controlled in a tissue-specific manner

Suppressor of gamma response 1 modulates the DNA damage response and oxidative stress response in leaves of cadmium-exposed Arabidopsis thaliana

Cadmium (Cd) exposure causes an oxidative challenge and inhibits cell cycle progression, ultimately impacting plant growth. Stress-induced effects on the cell cycle are often a consequence of activation of the DNA damage response (DDR). The main aim of this study was to investigate the role of the transcription factor SUPPRESSOR OF GAMMA RESPONSE 1 (SOG1) and three downstream cyclin-dependent kinase inhibitors of the SIAMESE-RELATED (SMR) family in the Cd-induced DDR and oxidative challenge in leaves of Arabidopsis thaliana. Effects of Cd on plant growth, cell cycle regulation and the expression of DDR genes were highly similar between the wildtype and smr4/5/7 mutant. In contrast, sog1-7 mutant leaves displayed a much lower Cd sensitivity within the experimental time-frame and significantly less pronounced upregulations of DDR-related genes, indicating the involvement of SOG1 in the Cd-induced DDR. Cadmium-induced responses related to the oxidative challenge were disturbed in the sog1-7 mutant, as indicated by delayed Cd-induced increases of hydrogen peroxide and glutathione concentrations and lower upregulations of oxidative stress-related genes. In conclusion, our results attribute a novel role to SOG1 in regulating the oxidative stress response and connect oxidative stress to the DDR in Cd-exposed plants