B1-type cyclin-dependent kinases are essential for the formation of stomatal complexes in Arabidopsis thaliana

Cyclin-dependent kinases (CDKs) are key regulators of the cell cycle. In yeasts, only one CDK is sufficient to drive cells through the cell cycle, whereas higher eukaryotes developed a family of related CDKs. Curiously, plants contain a unique class of CDKs (B-type CDKs), whose function is still unclear. We show that the CDKB1;1 gene of Arabidopsis (Arabidopsis thaliana) is highly expressed in guard cells and stomatal precursor cells of cotyledons, suggesting a prominent role for B-type CDKs in stomatal development. In accordance, transgenic Arabidopsis plants with reduced B-type CDK activity had a decreased stomatal index because of an early block of meristemoid division and inhibition of satellite meristemoid formation. Many aberrant stomatal cells were observed, all of them blocked in the G2 phase of the cell cycle. Although division of stomatal precursors was inhibited, cells still acquired stomatal identity, illustrating that stomatal cell differentiation is independent of cellular and nuclear division.     

Regulation of cyclin-dependent kinases in Arabidopsis thaliana

In plants, different families of cyclin-dependent kinases (CDKs) and cyclins have been identified, indicating that also in plants the progression through the cell cycle is regulated by CDKs. In all eukaryotes, CDKs exert their activity through well-controlled phosphorylations of specific substrates on serine/threonine residues. Such post-translational modifications are universal mechanisms in signal transduction pathways. They allow the organism to differentiate, regulate growth and/or adapt to environmental changes, the latter being crucial for plants because of their sedentary life-style. This adaptation might explain the occurrence of a special CDK type with plant-specific features. This review focuses on the involvement of plant CDKs in different phases of the cell cycle in Arabidopsis thaliana and outlines their regulation by binding to other proteins, and by phosphorylation and dephosphorylation.     

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.     

Axillary meristem development in Arabidopsis thaliana

Axillary shoot apical meristems initiate post-embryonically in the axils of leaves. Their developmental fate is a main determinant of the final plant body plan. In Arabidopsis, usually a single axillary meristem initiates in the leaf axil even though there is developmental potential for formation of multiple branches. While the wild-type plants rarely form multiple branches in the leaf axil, tfl1-2 plants regularly develop two or more branches in the axils of the rosette leaves. Axillary meristem formation in Arabidopsis occurs in two waves: an acropetal wave forms during plant vegetative development, and a basipetal wave forms during plant reproductive development. We report here the morphological and anatomical changes, and the STM expression pattern associated with the formation of axillary and accessory meristems during Arabidopsis vegetative development.     

Photoperiod regulates flower meristem development in Arabidopsis thaliana

Photoperiod has been known to regulate flowering time in many plant species. In Arabidopsis, genes in the long day (LD) pathway detect photoperiod and promote flowering under LD. It was previously reported that clavata2 (clv2) mutants grown under short day (SD) conditions showed suppression of the flower meristem defects, namely the accumulation of stem cells and the resulting production of extra floral organs. Detailed analysis of this phenomenon presented here demonstrates that the suppression is a true photoperiodic response mediated by the inactivation of the LD pathway under SD. Inactivation of the LD pathway was sufficient to suppress the clv2 defects under LD, and activation of the LD pathway under SD conditions restored clv2 phenotypes. These results reveal a novel role of photoperiod in flower meristem development in Arabidopsis. Flower meristem defects of clv1 and clv3 mutants are also suppressed under SD, and 35S:CO enhanced the defects of clv3, indicating that the LD pathway works independently from the CLV genes. A model is proposed to explain the interactions between photoperiod and the CLV genes.     

Diverse phosphoregulatory mechanisms controlling cyclin-dependent kinase-activating kinases in Arabidopsis

For the full activation of cyclin-dependent kinases (CDKs), not only cyclin binding but also phosphorylation of a threonine (Thr) residue within the T-loop is required. This phosphorylation is catalyzed by CDK-activating kinases (CAKs). In Arabidopsis three D-type CDK genes (CDKD;1-CDKD;3) encode vertebrate-type CAK orthologues, of which CDKD;2 exhibits high phosphorylation activity towards the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II. Here, we show that CDKD;2 forms a stable complex with cyclin H and is downregulated by the phosphorylation of the ATP-binding site by WEE1 kinase. A knockout mutant of CDKD;3, which has a higher CDK kinase activity, displayed no defect in plant development. Instead, another type of CAK - CDKF;1 - exhibited significant activity towards CDKA;1 in Arabidopsis root protoplasts, and the activity was dependent on the T-loop phosphorylation of CDKF;1. We propose that two distinct types of CAK, namely CDKF;1 and CDKD;2, play a major role in CDK and CTD phosphorylation, respectively, in Arabidopsis.     

Regulators of cyclin-dependent kinases are crucial for maintaining genome integrity in S phase

Maintenance of genome integrity is of critical importance to cells. To identify key regulators of genomic integrity, we screened a human cell line with a kinome small interfering RNA library. WEE1, a major regulator of mitotic entry, and CHK1 were among the genes identified. Both kinases are important negative regulators of CDK1 and -2. Strikingly, WEE1 depletion rapidly induced DNA damage in S phase in newly replicated DNA, which was accompanied by a marked increase in single-stranded DNA. This DNA damage is dependent on CDK1 and -2 as well as the replication proteins MCM2 and CDT1 but not CDC25A. Conversely, DNA damage after CHK1 inhibition is highly dependent on CDC25A. Furthermore, the inferior proliferation of CHK1-depleted cells is improved substantially by codepletion of CDC25A. We conclude that the mitotic kinase WEE1 and CHK1 jointly maintain balanced cellular control of Cdk activity during normal DNA replication, which is crucial to prevent the generation of harmful DNA lesions during replication.     

Time-lapse analysis of stem-cell divisions in the Arabidopsis thaliana root meristem

In the Arabidopsis root, asymmetric stem-cell divisions produce daughters that form the different root cell types. Here we report the establishment of a confocal tracking system that allows the analysis of numbers and orientations of cell divisions in root stem cells. The system provides direct evidence that stem cells have lower division rates than cells in the proximal meristem. It also allows tracking of cell division timing, which we have used to analyse the synchronization of root cap divisions. Finally, it gives new insights into lateral root cap formation: epidermal stem-cell daughters can rotate the orientation of the division plane like the stem cell.     

The phosphoinositide-3-OH-kinase-related kinases of Arabidopsis thaliana

The phosphoinositide-3-OH-kinase-related kinases (PIKKs) are atypical protein kinases exclusive to eukaryotes. They mediate the cellular response to a range of stresses, including genome and RNA surveillance and availability of nutrients for growth. Orthologues of five out of the six PIKK family members are present in plant genomes. Recent studies in plant PIKKs have revealed features unique to, and in common with, other PIKKs. This review summarizes the basic knowledge of these proteins in mammals and yeast in comparison with what is known for Arabidopsis and other plants.     

Differential phosphorylation activities of CDK-activating kinases in Arabidopsis thaliana

Activation of cyclin-dependent kinases (CDKs) requires phosphorylation of a threonine residue within the T-loop by a CDK-activating kinase (CAK). Here we isolated an Arabidopsis cDNA (CAK4At) whose predicted product shows a high similarity to vertebrate CDK7/p40(MO15). Northern blot analysis showed that expressions of the four Arabidopsis CAKs (CAK1At-CAK4At) were not dependent on cell division. CAK2At- and CAK4At-immunoprecipitates of Arabidopsis crude extract phosphorylated CDK and the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II with different preferences. These results suggest the existence of differential mechanisms in Arabidopsis that control CDK and CTD phosphorylation by multiple CAKs.     

Cell fate in the inflorescence meristem and floral buttress of Arabidopsis thaliana

The flowers and leaves of Arabidopsis are arranged in a spiral with successive organs positioned at intervals of approximately 140°. This simple phyllotaxy determines the organization of the flowers of the inflorescence. Here we describe the analysis of X-ray-induced albino sectors in the L2 layer of the Arabidopsis inflorescence. No evidence was found of lineage restrictions within the inflorescence meristem. Comparison of the spatial relationships between albino and green tissue in the sepals of 43 chimeric inflorescences allowed the generation of a three-dimensional fate map. The map relates the initiation of flowers in the plant apex to their final arrangement. The map was found to be a shallow dome with phyllotaxy superimposed on its surface. A similar map was prepared of the floral buttress and this was found to be a ridge with sepal primordia at its edges. Unlike other fate maps of plants these maps relate the relative positions and sizes of organ primordia in terms of the frequency with which they are of the same somatic phenotype, and not the number of cells giving rise to them.     

2-Aminoquinazoline inhibitors of cyclin-dependent kinases

The inhibition of cyclin-dependent kinase 4 (Cdk4) causes cell cycle arrest and restores a checkpoint that is absent in the majority of tumor cells. Compounds that inhibit Cdk4 selectively are targeted for treating cancer. Appropriate substitution of 2-aminoquinazolines is demonstrated to produce high levels of selectivity for Cdk4 versus closely related serine-threonine kinases.     

Axillary meristem development in the branchless Zu-0 ecotype of Arabidopsis thaliana

Axillary meristems form in the leaf axils during post-embryonic development. In order to initiate the genetic dissection of axillary meristem development, we have characterized the late-flowering branchless ecotype of Arabidopsis thaliana (L.) Heynh., Zu-0. The first-formed rosette leaves of Zu-0 plants all initiate axillary meristems, but later-formed leaves of the rosette remain branchless. Alteration in the meristem development is axillary meristem-specific because the shoot apical and floral meristems develop normally. Scanning electron microscopy, histology and RNA in situ analysis with SHOOTMERISTEMLESS ( STM), a marker for meristematic tissues, show that a mound of cells form and STM mRNA accumulates in barren leaf axils, indicating that axillary meristems initiate but arrest in their development prior to organizing a meristem proper. Expression and retention of the STM RNA in barren leaf axils further suggests that STM expression is not sufficient for the establishment of the axillary meristem proper.