Annika K. Weimer

Genetic Framework of Cyclin-Dependent Kinase Function in Arabidopsis

Cyclin-dependent kinases (CDKs) are at the heart of eukaryotic cell-cycle control. The yeast Cdc2/CDC28 PSTAIRE kinase and its orthologs such as the mammalian Cdk1 have been found to be indispensable for cell-cycle progression in all eukaryotes investigated so far. CDKA;1 is the only PSTAIRE kinase in the flowering plant Arabidopsis and can rescue Cdc2/CDC28 mutants. Here, we show that cdka;1 null mutants are viable but display specific cell-cycle and developmental defects, e.g., in S phase entry and stem cell maintenance. We unravel that the crucial function of CDKA;1 is the control of the plant Retinoblastoma homolog RBR1 and that codepletion of RBR1 and CDKA;1 rescued most defects of cdka;1 mutants. Our work further revealed a basic cell-cycle control system relying on two plant-specific B1-type CDKs, and the triple cdk mutants displayed an early germline arrest. Taken together, our data indicate divergent functional differentiation of Cdc2-type kinases during eukaryote evolution.

Control of Cell Proliferation, Organ Growth, and DNA Damage Response Operate Independently of Dephosphorylation of the Arabidopsis Cdk1 Homolog CDKA;1

Entry into mitosis is universally controlled by cyclin-dependent kinases (CDKs). A key regulatory event in metazoans and fission yeast is CDK activation by the removal of inhibitory phosphate groups in the ATP binding pocket catalyzed by Cdc25 phosphatases. In contrast with other multicellular organisms, we show here that in the flowering plant Arabidopsis thaliana, cell cycle control does not depend on sudden changes in the phosphorylation pattern of the PSTAIRE-containing Cdk1 homolog CDKA;1. Consistently, we found that neither mutants in a previously identified CDC25 candidate gene nor plants in which it is overexpressed display cell cycle defects. Inhibitory phosphorylation of CDKs is also the key event in metazoans to arrest cell cycle progression upon DNA damage. However, we show here that the DNA damage checkpoint in Arabidopsis can also operate independently of the phosphorylation of CDKA;1. These observations reveal a surprising degree of divergence in the circuitry of highly conserved core cell cycle regulators in multicellular organisms. Based on biomathematical simulations, we propose a plant-specific model of how progression through the cell cycle could be wired in Arabidopsis.

A General G1/S-Phase Cell-Cycle Control Module in the Flowering Plant Arabidopsis thaliana

The decision to replicate its DNA is of crucial importance for every cell and, in many organisms, is decisive for the progression through the entire cell cycle. A comparison of animals versus yeast has shown that, although most of the involved cell-cycle regulators are divergent in both clades, they fulfill a similar role and the overall network topology of G1/S regulation is highly conserved. Using germline development as a model system, we identified a regulatory cascade controlling entry into S phase in the flowering plant Arabidopsis thaliana, which, as a member of the Plantae supergroup, is phylogenetically only distantly related to Opisthokonts such as yeast and animals. This module comprises the Arabidopsis homologs of the animal transcription factor E2F, the plant homolog of the animal transcriptional repressor Retinoblastoma (Rb)-related 1 (RBR1), the plant-specific F-box protein F-BOX-LIKE 17 (FBL17), the plant specific cyclin-dependent kinase (CDK) inhibitors KRPs, as well as CDKA;1, the plant homolog of the yeast and animal Cdc2+/Cdk1 kinases. Our data show that the principle of a double negative wiring of Rb proteins is highly conserved, likely representing a universal mechanism in eukaryotic cell-cycle control. However, this negative feedback of Rb proteins is differently implemented in plants as it is brought about through a quadruple negative regulation centered around the F-box protein FBL17 that mediates the degradation of CDK inhibitors but is itself directly repressed by Rb. Biomathematical simulations and subsequent experimental confirmation of computational predictions revealed that this regulatory circuit can give rise to hysteresis highlighting the here identified dosage sensitivity of CDK inhibitors in this network.

The Arabidopsis thaliana Checkpoint Kinase WEE1 Protects against Premature Vascular Differentiation during Replication Stress

Because of their sessile lifestyle, plants need to react promptly to factors that affect meristem integrity. This work shows that the WEE1 checkpoint kinase maintains the root meristem activity under replication stress by controlling S-phase progression, thereby preventing premature onset of vascular cell differentiation. A sessile lifestyle forces plants to respond promptly to factors that affect their genomic integrity. Therefore, plants have developed checkpoint mechanisms to arrest cell cycle progression upon the occurrence of DNA stress, allowing the DNA to be repaired before onset of division. Previously, the WEE1 kinase had been demonstrated to be essential for delaying progression through the cell cycle in the presence of replication-inhibitory drugs, such as hydroxyurea. To understand the severe growth arrest of WEE1-deficient plants treated with hydroxyurea, a transcriptomics analysis was performed, indicating prolonged S-phase duration. A role for WEE1 during S phase was substantiated by its specific accumulation in replicating nuclei that suffered from DNA stress. Besides an extended replication phase, WEE1 knockout plants accumulated dead cells that were associated with premature vascular differentiation. Correspondingly, plants without functional WEE1 ectopically expressed the vascular differentiation marker VND7, and their vascular development was aberrant. We conclude that the growth arrest of WEE1-deficient plants is due to an extended cell cycle duration in combination with a premature onset of vascular cell differentiation. The latter implies that the plant WEE1 kinase acquired an indirect developmental function that is important for meristem maintenance upon replication stress.

RETINOBLASTOMA RELATED1 Regulates Asymmetric Cell Divisions in Arabidopsis

Formative cell divisions produce daughter cells with different identities and are of key importance for the development of multicellular organisms. Here, formative divisions in the root and shoot of Arabidopsis are shown to be modulated by a common mechanism that relies on the activity level of a core cell cycle regulator that integrates cell proliferation with cell differentiation. Formative, also called asymmetric, cell divisions produce daughter cells with different identities. Like other divisions, formative divisions rely first of all on the cell cycle machinery with centrally acting cyclin-dependent kinases (CDKs) and their cyclin partners to control progression through the cell cycle. However, it is still largely obscure how developmental cues are translated at the cellular level to promote asymmetric divisions. Here, we show that formative divisions in the shoot and root of the flowering plant Arabidopsis thaliana are controlled by a common mechanism that relies on the activity level of the Cdk1 homolog CDKA;1, with medium levels being sufficient for symmetric divisions but high levels being required for formative divisions. We reveal that the function of CDKA;1 in asymmetric cell divisions operates through a transcriptional regulation system that is mediated by the Arabidopsis Retinoblastoma homolog RBR1. RBR1 regulates not only cell cycle genes, but also, independent of the cell cycle transcription factor E2F, genes required for formative divisions and cell fate acquisition, thus directly linking cell proliferation with differentiation. This mechanism allows the implementation of spatial information, in the form of high kinase activity, with intracellular gating of developmental decisions.

The regulatory network of cell cycle progression is fundamentally different in plants versus yeast or metazoans

Plant growth and proliferation control is coming into a global focus due to recent ecological and economical developments. Plants represent not only the largest food supply for mankind but also may serve as a global source of renewable energies. However, plant breeding has to accomplish a tremendous boost in yield to match the growing demand of a still rapidly increasing human population. Moreover, breeding has to adjust to changing environmental conditions, in particular increased drought. Regulation of cell-cycle control is a major determinant of plant growth and therefore an obvious target for plant breeding. Furthermore, cell-cycle control is also crucial for the DNA damage response, for instance upon irradiation. Thus, an in-depth understanding of plant cell-cycle regulation is of importance beyond a scientific point of view. The mere presence of many conserved core cell-cycle regulators, e.g. CDKs, cyclins, or CDK inhibitors, has formed the idea that the cell cycle in plants is exactly or at least very similarly controlled as in yeast or human cells. Here together with a recent publication we demonstrate that this dogma is not true and show that the control of entry into mitosis is fundamentally different in plants versus yeast or metazoans. Our findings build an important base for the understanding and ultimate modulation of plant growth not only during unperturbed but also under harsh environmental conditions.

The plant‐specific CDKB1‐CYCB1 complex mediates homologous recombination repair in Arabidopsis

Upon DNA damage, cyclin‐dependent kinases (CDKs) are typically inhibited to block cell division. In many organisms, however, it has been found that CDK activity is required for DNA repair, especially for homology‐dependent repair (HR), resulting in the conundrum how mitotic arrest and repair can be reconciled. Here, we show that Arabidopsis thaliana solves this dilemma by a division of labor strategy. We identify the plant‐specific B1‐type CDKs (CDKB1s) and the class of B1‐type cyclins (CYCB1s) as major regulators of HR in plants. We find that RADIATION SENSITIVE 51 (RAD51), a core mediator of HR, is a substrate of CDKB1‐CYCB1 complexes. Conversely, mutants in CDKB1 and CYCB1 fail to recruit RAD51 to damaged DNA. CYCB1;1 is specifically activated after DNA damage and we show that this activation is directly controlled by SUPPRESSOR OF GAMMA RESPONSE 1 (SOG1), a transcription factor that acts similarly to p53 in animals. Thus, while the major mitotic cell‐cycle activity is blocked after DNA damage, CDKB1‐CYCB1 complexes are specifically activated to mediate HR.

Aurora Kinases Throughout Plant Development

Aurora kinases are evolutionarily conserved key mitotic determinants in all eukaryotes. Yeasts contain a single Aurora kinase, whereas multicellular eukaryotes have at least two functionally diverged members. The involvement of Aurora kinases in human cancers has provided an in-depth mechanistic understanding of their roles throughout cell division in animal and yeast models. By contrast, understanding Aurora kinase function in plants is only starting to emerge. Nevertheless, genetic, cell biological, and biochemical approaches have revealed functional diversification between the plant Aurora kinases and suggest a role in formative (asymmetric) divisions, chromatin modification, and genome stability. This review provides an overview of the accumulated knowledge on the function of plant Aurora kinases as well as some major challenges for the future.

Phosphorylation of MAP65-1 by Arabidopsis Aurora Kinases Is Required for Efficient Cell Cycle Progression.

Arabidopsis Aurora kinases phosphorylate MAP65-1 at its unfolded tail domain and dynamic switching of its phosphorylation status throughout mitosis is required for proper cell cycle progression Aurora kinases are key effectors of mitosis. Plant Auroras are functionally divided into two clades. The alpha Auroras (Aurora1 and Aurora2) associate with the spindle and the cell plate and are implicated in controlling formative divisions throughout plant development. The beta Aurora (Aurora3) localizes to centromeres and likely functions in chromosome separation. In contrast to the wealth of data available on the role of Aurora in other kingdoms, knowledge on their function in plants is merely emerging. This is exemplified by the fact that only histone H3 and the plant homolog of TPX2 have been identified as Aurora substrates in plants. Here we provide biochemical, genetic, and cell biological evidence that the microtubule-bundling protein MAP65-1—a member of the MAP65/Ase1/PRC1 protein family, implicated in central spindle formation and cytokinesis in animals, yeasts, and plants—is a genuine substrate of alpha Aurora kinases. MAP65-1 interacts with Aurora1 in vivo and is phosphorylated on two residues at its unfolded tail domain. Its overexpression and down-regulation antagonistically affect the alpha Aurora double mutant phenotypes. Phospho-mutant analysis shows that Aurora contributes to the microtubule bundling capacity of MAP65-1 in concert with other mitotic kinases.

Specialization of CDK regulation under DNA damage

A common response to DNA damage, such as DNA double strand breaks (DSBs), is the inhibition of cell division to provide time for repair and prevent propagation of mutations. DSBs are sensed by the kinase Ataxia telangiectasia-mutated (Atm) that initiates a regulatory cascade resulting in the inactivation of cyclin-dependent kinases (CDKs; Fig. 1). However, CDK activity has been found to be required for homology-dependent repair (HR), also called homologous recombination repair, giving rise to the question how this prominent DNA repair pathway can be triggered at times of mitotic arrest. In the recent issue of The EMBO Journal, we present data that address this question and describe how this apparent conflict of CDK regulation in the DNA damage response is tackled in Arabidopsis thaliana. Here, we briefly summarize these findings and raise some questions for future work. Following up earlier observations that CYCLIN B1;1 (CYCB1;1), a mitotic cyclin in the model plant Arabidopsis thaliana, is often upregulated under DNA damage, e.g. after ionizing radiation, we tested the growth of mutants of CYCB1;1 and its 3 sister genes in the B1-cyclin class, i.e. CYCB1;2, CYCB1;3 and CYCB1;4, on different DNAdamage inducing media. While mutants in all 4 cyclin genes grew indistinguishably from the wildtype on the Sphase stress-triggering substance hydroxy urea and the DSB-inducing drug Bleomycin, all mutants were hypersensitive to cisplatin, which induces DNA crosslinks that require homology dependent repair for dissolution. This growth reduction was associated with high levels of DNA damage as revealed by a large number of yH2AX foci and an increased tail in comet assays. Subsequently, we found that homologous recombination is severely reduced in all cycb1 mutants. In search for the kinase partner of the B1-type cyclins, we tested growth of mutants in CDKA;1, the combined Cdk1 and Cdk2 homolog of Arabidopsis, on cisplatin. CDKA;1 is constitutively expressed during the cell cycle and represents the major cell-cycle promoting kinase in Arabidopsis. However, plants with reduced CDKA;1 activity levels were not hypersensitive to cisplatin or other DNA damage-inducing drugs. Plants contain a specific class of B-type CDKs whose transcription accumulates from S-phase till mitosis when a sister chromatid is available for repair. Double mutants of both B1-type CDKs (referred to here as cdkb1) in Arabidopsis have been previously analyzed and only showed mild developmental defects under unperturbed conditions. In contrast, when grown on cisplatin and bleomycin, cdkb1 mutants were much more affected than the wildtype. Quantification of the DNA damage in cdkb1 mutants revealed that they accumulated as much damage as atm mutants, placing them in the group of major DNA damage response regulators in plants. Since the triple mutant cycb1;1 cdkb1;1 cdkb1;2 showed no additional sensitivity on cisplatin, we concluded that indeed CDKB1, but not other Cdks, are the major partners of CYCB1s during DNA damage response. In search of targets of CDKB1-CYCB1 action, we found that this complex could efficiently phosphorylate RAD51 in vitro, the central HR protein. Conversely, we found that the number of RAD51 foci was strongly reduced in cycb1 and cdkb1 mutants, consistent with their low levels of recombination activity and strong hypersensitivity to cisplatin. However, it remains to be seen whether phosphorylation is directly required for proper RAD51 accumulation at DNA damage sites. In addition, it is likely that the CDKB1-CYCB1 complex has other, yet to be identified targets during HR. As a last question, we addressed how CYCB1 accumulates upon DNA damage. To this end, we first confirmed that CYCB1, as well as CDKB1;1, are transcriptionally upregulated in plants treated with cisplatin. While a p53 homolog is not present in Arabidopsis and other plants analyzed so far, plants do contain a transcriptional regulator, called SUPRESSOR OF GAMMA RESPONSE 1 (SOG1) that