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

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

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

Emerging role of the plant ERF transcription factors in coordinating wound defense responses and repair

Plants react to wounding through the activation of both defense and repair pathways, but how these two responses are coordinated is unclear. Here, we put forward the hypothesis that diverse members of the subfamily X of the plant-specific ethylene response factor (ERF) transcription factors coordinate stress signaling with the activation of wound repair mechanisms. Moreover, we highlight the observation that tissue repair is strongly boosted through the formation of a heterodimeric protein complex that comprises ERF and transcription factors of the GRAS domain type. This interaction turns ERFs into highly potent and stress-responsive activators of cell proliferation. The potency to induce stem cell identity suggests that these heterodimeric transcription factor complexes could become valuable tools to increase crop regeneration and transformation efficiency

Evidence for a role of Arabidopsis CDT1 proteins in gametophyte development and maintenance of genome integrity

Meristems retain the ability to divide throughout the life cycle of plants, which can last for over 1000 years in some species. Furthermore, the germline is not laid down early during embryogenesis but originates from the meristematic cells relatively late during development. Thus, accurate cell cycle regulation is of utmost importance to avoid the accumulation of mutations during vegetative growth and reproduction. The Arabidopsis thaliana genome encodes two homologs of the replication licensing factor CDC10 Target1 (CDT1), and overexpression of CDT1a stimulates DNA replication. Here, we have investigated the respective functions of Arabidopsis CDT1a and CDT1b. We show that CDT1 proteins have partially redundant functions during gametophyte development and are required for the maintenance of genome integrity. Furthermore, CDT1-RNAi plants show endogenous DNA stress, are more tolerant than the wild type to DNA-damaging agents, and show constitutive induction of genes involved in DNA repair. This DNA stress response may be a direct consequence of reduced CDT1 accumulation on DNA repair or may relate to the ability of CDT1 proteins to form complexes with DNA polymerase e, which functions in DNA replication and in DNA stress checkpoint activation. Taken together, our results provide evidence for a crucial role of Arabidopsis CDT1 proteins in genome stability

The long non-coding RNA LINDA restrains cellular collapse following DNA damage in Arabidopsis thaliana

The genomic integrity of every organism is endangered by various intrinsic and extrinsic stresses. To maintain genomic integrity, a sophisticated DNA damage response (DDR) network is activated rapidly after DNA damage. Notably, the fundamental DDR mechanisms are conserved in eukaryotes. However, knowledge about many regulatory aspects of the plant DDR is still limited. Important, yet little understood, regulatory factors of the DDR are the long non-coding RNAs (lncRNAs). In humans, 13 lncRNAs functioning in DDR have been characterized to date, whereas no such lncRNAs have been characterized in plants yet. By meta-analysis, we identified the putative long intergenic non-coding RNA induced by DNA damage (LINDA) that responds strongly to various DNA double-strand break-inducing treatments, but not to replication stress induced by mitomycin C. After DNA damage, LINDA is rapidly induced in an ATM- and SOG1-dependent manner. Intriguingly, the transcriptional response of LINDA to DNA damage is similar to that of its flanking hypothetical protein-encoding gene. Phylogenetic analysis of putative Brassicales and Malvales LINDA homologs indicates that LINDA lncRNAs originate from duplication of a flanking small protein-encoding gene followed by pseudogenization. We demonstrate that LINDA is not only needed for the regulation of this flanking gene but also fine-tuning of the DDR after the occurrence of DNA double-strand breaks. Moreover, Δlinda mutant root stem cells are unable to recover from DNA damage, most likely due to hyper-induced cell death