The Arabidopsis Cdk1/Cdk2 homolog CDKA;1 controls chromosome axis assembly during plant meiosis.

Meiosis is key to sexual reproduction and genetic diversity. Here, we show that the Arabidopsis cyclin-dependent kinase Cdk1/Cdk2 homolog CDKA;1 is an important regulator of meiosis needed for several aspects of meiosis such as chromosome synapsis. We identify the chromosome axis protein ASYNAPTIC 1 (ASY1), the Arabidopsis homolog of Hop1 (homolog pairing 1), essential for synaptonemal complex formation, as a target of CDKA;1. The phosphorylation of ASY1 is required for its recruitment to the chromosome axis via ASYNAPTIC 3 (ASY3), the Arabidopsis reductional division 1 (Red1) homolog, counteracting the disassembly activity of the AAA+ ATPase PACHYTENE CHECKPOINT 2 (PCH2). Furthermore, we have identified the closure motif in ASY1, typical for HORMA domain proteins, and provide evidence that the phosphorylation of ASY1 regulates the putative self-polymerization of ASY1 along the chromosome axis. Hence, the phosphorylation of ASY1 by CDKA;1 appears to be a two-pronged mechanism to initiate chromosome axis formation in meiosis.

B1-type cyclins control microtubule organization during cell division in Arabidopsis.

Flowering plants contain a large number of cyclin families, each containing multiple members, most of which have not been characterized to date. Here, we analyzed the role of the B1 subclass of mitotic cyclins in cell cycle control during Arabidopsis development. While we reveal CYCB1;5 to be a pseudogene, the remaining four members were found to be expressed in dividing cells. Mutant analyses showed a complex pattern of overlapping, development-specific requirements of B1-type cyclins with CYCB1;2 playing a central role. The double mutant cycb1;1 cycb1;2 is severely compromised in growth, yet viable beyond the seedling stage, hence representing a unique opportunity to study the function of B1-type cyclin activity at the organismic level. Immunolocalization of microtubules in cycb1;1 cycb1;2 and treating mutants with the microtubule drug oryzalin revealed a key role of B1-type cyclins in orchestrating mitotic microtubule networks. Subsequently, we identified the GAMMA-TUBULIN COMPLEX PROTEIN 3-INTERACTING PROTEIN 1 (GIP1/MOZART) as an in vitro substrate of B1-type cyclin complexes and further genetic analyses support a potential role in the regulation of GIP1 by CYCB1s.

The Cdk1/Cdk2 homolog CDKA;1 controls the recombination landscape in Arabidopsis.

Little is known how patterns of cross-over (CO) numbers and distribution during meiosis are established. Here, we reveal that cyclin-dependent kinase A;1 (CDKA;1), the homolog of human Cdk1 and Cdk2, is a major regulator of meiotic recombination in ArabidopsisArabidopsis plants with reduced CDKA;1 activity experienced a decrease of class I COs, especially lowering recombination rates in centromere-proximal regions. Interestingly, this reduction of type I CO did not affect CO assurance, a mechanism by which each chromosome receives at least one CO, resulting in all chromosomes exhibiting similar genetic lengths in weak loss-of-function cdka;1 mutants. Conversely, an increase of CDKA;1 activity resulted in elevated recombination frequencies. Thus, modulation of CDKA;1 kinase activity affects the number and placement of COs along the chromosome axis in a dose-dependent manner.

The retinoblastoma homolog RBR1 mediates localization of the repair protein RAD51 to DNA lesions in Arabidopsis.

The retinoblastoma protein (Rb), which typically functions as a transcriptional repressor of E2F-regulated genes, represents a major control hub of the cell cycle. Here, we show that loss of the Arabidopsis Rb homolog RETINOBLASTOMA-RELATED 1 (RBR1) leads to cell death, especially upon exposure to genotoxic drugs such as the environmental toxin aluminum. While cell death can be suppressed by reduced cell-proliferation rates, rbr1 mutant cells exhibit elevated levels of DNA lesions, indicating a direct role of RBR1 in the DNA-damage response (DDR). Consistent with its role as a transcriptional repressor, we find that RBR1 directly binds to and represses key DDR genes such as RADIATION SENSITIVE 51 (RAD51), leaving it unclear why rbr1 mutants are hypersensitive to DNA damage. However, we find that RBR1 is also required for RAD51 localization to DNA lesions. We further show that RBR1 is itself targeted to DNA break sites in a CDKB1 activity-dependent manner and partially co-localizes with RAD51 at damage sites. Taken together, these results implicate RBR1 in the assembly of DNA-bound repair complexes, in addition to its canonical function as a transcriptional regulator.

Genome-wide identification of RETINOBLASTOMA RELATED 1 binding sites in Arabidopsis reveals novel DNA damage regulators.

Retinoblastoma (pRb) is a multifunctional regulator, which was likely present in the last common ancestor of all eukaryotes. The Arabidopsis pRb homolog RETINOBLASTOMA RELATED 1 (RBR1), similar to its animal counterparts, controls not only cell proliferation but is also implicated in developmental decisions, stress responses and maintenance of genome integrity. Although most functions of pRb-type proteins involve chromatin association, a genome-wide understanding of RBR1 binding sites in Arabidopsis is still missing. Here, we present a plant chromatin immunoprecipitation protocol optimized for genome-wide studies of indirectly DNA-bound proteins like RBR1. Our analysis revealed binding of Arabidopsis RBR1 to approximately 1000 genes and roughly 500 transposable elements, preferentially MITES. The RBR1-decorated genes broadly overlap with previously identified targets of two major transcription factors controlling the cell cycle, i.e. E2F and MYB3R3 and represent a robust inventory of RBR1-targets in dividing cells. Consistently, enriched motifs in the RBR1-marked domains include sequences related to the E2F consensus site and the MSA-core element bound by MYB3R transcription factors. Following up a key role of RBR1 in DNA damage response, we performed a meta-analysis combining the information about the RBR1-binding sites with genome-wide expression studies under DNA stress. As a result, we present the identification and mutant characterization of three novel genes required for growth upon genotoxic stress.

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.

WIND1 Promotes Shoot Regeneration through Transcriptional Activation of ENHANCER OF SHOOT REGENERATION1 in Arabidopsis.

Many plant species display remarkable developmental plasticity and regenerate new organs after injury. Local signals produced by wounding are thought to trigger organ regeneration but molecular mechanisms underlying this control remain largely unknown. We previously identified an AP2/ERF transcription factor WOUND INDUCED DEDIFFERENTIATION1 (WIND1) as a central regulator of wound-induced cellular reprogramming in plants. In this study, we demonstrate that WIND1 promotes callus formation and shoot regeneration by upregulating the expression of the ENHANCER OF SHOOT REGENERATION1 (ESR1) gene, which encodes another AP2/ERF transcription factor in Arabidopsis thaliana The esr1 mutants are defective in callus formation and shoot regeneration; conversely, its overexpression promotes both of these processes, indicating that ESR1 functions as a critical driver of cellular reprogramming. Our data show that WIND1 directly binds the vascular system-specific and wound-responsive cis-element-like motifs within the ESR1 promoter and activates its expression. The expression of ESR1 is strongly reduced in WIND1-SRDX dominant repressors, and ectopic overexpression of ESR1 bypasses defects in callus formation and shoot regeneration in WIND1-SRDX plants, supporting the notion that ESR1 acts downstream of WIND1. Together, our findings uncover a key molecular pathway that links wound signaling to shoot regeneration in plants.

Transcriptional Regulation of Arabidopsis Polycomb Repressive Complex 2 Coordinates Cell-Type Proliferation and Differentiation.

Spatiotemporal regulation of transcription is fine-tuned at multiple levels, including chromatin compaction. Polycomb Repressive Complex 2 (PRC2) catalyzes the trimethylation of Histone 3 at lysine 27 (H3K27me3), which is the hallmark of a repressive chromatin state. Multiple PRC2 complexes have been reported in Arabidopsis thaliana to control the expression of genes involved in developmental transitions and maintenance of organ identity. Here, we show that PRC2 member genes display complex spatiotemporal gene expression patterns and function in root meristem and vascular cell proliferation and specification. Furthermore, PRC2 gene expression patterns correspond with vascular and nonvascular tissue-specific H3K27me3-marked genes. This tissue-specific repression via H3K27me3 regulates the balance between cell proliferation and differentiation. Using enhanced yeast one-hybrid analysis, upstream regulators of the PRC2 member genes are identified, and genetic analysis demonstrates that transcriptional regulation of some PRC2 genes plays an important role in determining PRC2 spatiotemporal activity within a developing organ.

TDM1 Regulation Determines the Number of Meiotic Divisions.

Cell cycle control must be modified at meiosis to allow two divisions to follow a single round of DNA replication, resulting in ploidy reduction. The mechanisms that ensure meiosis termination at the end of the second and not at the end of first division are poorly understood. We show here that Arabidopsis thaliana TDM1, which has been previously shown to be essential for meiotic termination, interacts directly with the Anaphase-Promoting Complex. Further, mutations in TDM1 in a conserved putative Cyclin-Dependant Kinase (CDK) phosphorylation site (T16-P17) dominantly provoked premature meiosis termination after the first division, and the production of diploid spores and gametes. The CDKA;1-CYCA1.2/TAM complex, which is required to prevent premature meiotic exit, phosphorylated TDM1 at T16 in vitro. Finally, while CYCA1;2/TAM was previously shown to be expressed only at meiosis I, TDM1 is present throughout meiosis. These data, together with epistasis analysis, lead us to propose that TDM1 is an APC/C component whose function is to ensure meiosis termination at the end of meiosis II, and whose activity is inhibited at meiosis I by CDKA;1-TAM-mediated phosphorylation to prevent premature meiotic exit. This provides a molecular mechanism for the differential decision of performing an additional round of division, or not, at the end of meiosis I and II, respectively.

Functional Conservation in the SIAMESE-RELATED Family of Cyclin-Dependent Kinase Inhibitors in Land Plants.

The best-characterized members of the plant-specific SIAMESE-RELATED (SMR) family of cyclin-dependent kinase inhibitors regulate the transition from the mitotic cell cycle to endoreplication, also known as endoreduplication, an altered version of the cell cycle in which DNA is replicated without cell division. Some other family members are implicated in cell cycle responses to biotic and abiotic stresses. However, the functions of most SMRs remain unknown, and the specific cyclin-dependent kinase complexes inhibited by SMRs are unclear. Here, we demonstrate that a diverse group of SMRs, including an SMR from the bryophyte Physcomitrella patens, can complement an Arabidopsis thaliana siamese (sim) mutant and that both Arabidopsis SIM and P. patens SMR can inhibit CDK activity in vitro. Furthermore, we show that Arabidopsis SIM can bind to and inhibit both CDKA;1 and CDKB1;1. Finally, we show that SMR2 acts to restrict cell proliferation during leaf growth in Arabidopsis and that SIM, SMR1/LGO, and SMR2 play overlapping roles in controlling the transition from cell division to endoreplication during leaf development. These results indicate that differences in SMR function in plant growth and development are primarily due to differences in transcriptional and posttranscriptional regulation, rather than to differences in fundamental biochemical function.

Modulation of plant growth in vivo and identification of kinase substrates using an analog-sensitive variant of CYCLIN-DEPENDENT KINASE A;1.

BACKGROUND: Modulation of protein activity by phosphorylation through kinases and subsequent de-phosphorylation by phosphatases is one of the most prominent cellular control mechanisms. Thus, identification of kinase substrates is pivotal for the understanding of many - if not all - molecular biological processes. Equally, the possibility to deliberately tune kinase activity is of great value to analyze the biological process controlled by a particular kinase. RESULTS: Here we have applied a chemical genetic approach and generated an analog-sensitive version of CDKA;1, the central cell-cycle regulator in Arabidopsis and homolog of the yeast Cdc2/CDC28 kinases. This variant could largely rescue a cdka;1 mutant and is biochemically active, albeit less than the wild type. Applying bulky kinase inhibitors allowed the reduction of kinase activity in an organismic context in vivo and the modulation of plant growth. To isolate CDK substrates, we have adopted a two-dimensional differential gel electrophoresis strategy, and searched for proteins that showed mobility changes in fluorescently labeled extracts from plants expressing the analog-sensitive version of CDKA;1 with and without adding a bulky ATP variant. A pilot set of five proteins involved in a range of different processes could be confirmed in independent kinase assays to be phosphorylated by CDKA;1 approving the applicability of the here-developed method to identify substrates. CONCLUSION: The here presented generation of an analog-sensitive CDKA;1 version is functional and represent a novel tool to modulate kinase activity in vivo and identify kinase substrates. Our here performed pilot screen led to the identification of CDK targets that link cell proliferation control to sugar metabolism, proline proteolysis, and glucosinolate production providing a hint how cell proliferation and growth are integrated with plant development and physiology.

Retinoblastoma related1 regulates asymmetric cell divisions in Arabidopsis.

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.

OSD1 promotes meiotic progression via APC/C inhibition and forms a regulatory network with TDM and CYCA1;2/TAM.

Cell cycle control is modified at meiosis compared to mitosis, because two divisions follow a single DNA replication event. Cyclin-dependent kinases (CDKs) promote progression through both meiosis and mitosis, and a central regulator of their activity is the APC/C (Anaphase Promoting Complex/Cyclosome) that is especially required for exit from mitosis. We have shown previously that OSD1 is involved in entry into both meiosis I and meiosis II in Arabidopsis thaliana; however, the molecular mechanism by which OSD1 controls these transitions has remained unclear. Here we show that OSD1 promotes meiotic progression through APC/C inhibition. Next, we explored the functional relationships between OSD1 and the genes known to control meiotic cell cycle transitions in Arabidopsis. Like osd1, cyca1;2/tam mutation leads to a premature exit from meiosis after the first division, while tdm mutants perform an aberrant third meiotic division after normal meiosis I and II. Remarkably, while tdm is epistatic to tam, osd1 is epistatic to tdm. We further show that the expression of a non-destructible CYCA1;2/TAM provokes, like tdm, the entry into a third meiotic division. Finally, we show that CYCA1;2/TAM forms an active complex with CDKA;1 that can phosphorylate OSD1 in vitro. We thus propose that a functional network composed of OSD1, CYCA1;2/TAM, and TDM controls three key steps of meiotic progression, in which OSD1 is a meiotic APC/C inhibitor.

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.

Identification of kinase substrates by bimolecular complementation assays.

As a consequence of the transient nature of kinase-substrate interactions, the detection of kinase targets, although central for understanding many biological processes, has remained challenging. Here we present a straightforward procedure that relies on the comparison of wild type with activation-loop mutants in the kinase of interest by bimolecular complementation assays. As a proof of functionality, we present the identification and in vivo confirmation of substrates of the major cell-cycle kinase in Arabidopsis, revealing a direct link between cell proliferation and the control of the redox state.

A PSTAIRE-type cyclin-dependent kinase controls light responses in land plants.

Light is a critical signal perceived by plants to adapt their growth rate and direction. Although many signaling components have been studied, how plants respond to constantly fluctuating light remains underexplored. Here, we showed that in the moss Physcomitrium (Physcomitrella) patens, the PSTAIRE-type cyclin-dependent kinase PpCDKA is dispensable for growth. Instead, PpCDKA and its homolog in Arabidopsis thaliana control light-induced tropisms and chloroplast movements by probably influencing the cytoskeleton organization independently of the cell cycle. In addition, lower PpCDKA kinase activity was required to elicit light responses relative to cell cycle regulation. Thus, our study suggests that plant CDKAs may have been co-opted to control multiple light responses, and owing to the bistable switch properties of PSTAIRE-type CDKs, the noncanonical functions are widely conserved for eukaryotic environmental adaptation.

Auxin is required for the assembly of A-type cyclin-dependent kinase complexes in tobacco cell suspension culture.

Although activation of A-type cyclin-dependent kinase (CDKA) is required for plant cell division, little is known about how CDKA is activated before commitment to cell division. Here, we show that auxin is required for the formation of active CDKA-associated complexes, promoting assembly of the complex in tobacco suspension culture Bright Yellow-2 (BY-2) cells. Protein gel blot analysis revealed that CDKA levels increased greatly after stationary-phase BY-2 cells were subcultured into fresh medium to re-enter the cell cycle. However, these increasing levels subsided when cells were subcultured into auxin-deprived medium, and a subtle increase was observed after subculturing into sucrose-deprived medium. Additionally, p13(suc1)-associated kinase activity did not increase significantly after subculturing into either auxin- or sucrose-deprived medium, but increased strongly after subculturing into medium containing both auxin and sucrose. Using gel filtration, we found that p13(suc1)-associated kinase activity against tobacco retinoblastoma-related protein was maximal in fractions corresponding to the molecular mass of the cyclin/CDKA complex. Interestingly, this peak distribution of high molecular-mass fractions of CDKA disappeared after cells were subcultured into auxin-deprived medium. These findings suggest an important role for auxin in the assembly of active CDKA-associated complexes.

RETINOBLASTOMA RELATED1 mediates germline entry in Arabidopsis.

To produce seeds, flowering plants need to specify somatic cells to undergo meiosis. Here, we reveal a regulatory cascade that controls the entry into meiosis starting with a group of redundantly acting cyclin-dependent kinase (CDK) inhibitors of the KIP-RELATED PROTEIN (KRP) class. KRPs function by restricting CDKA;1-dependent inactivation of the Arabidopsis Retinoblastoma homolog RBR1. In rbr1 and krp triple mutants, designated meiocytes undergo several mitotic divisions, resulting in the formation of supernumerary meiocytes that give rise to multiple reproductive units per future seed. One function of RBR1 is the direct repression of the stem cell factor WUSCHEL (WUS), which ectopically accumulates in meiocytes of triple krp and rbr1 mutants. Depleting WUS in rbr1 mutants restored the formation of only a single meiocyte.

Integration of developmental and environmental signals into cell proliferation and differentiation through RETINOBLASTOMA-RELATED 1.

Plants continuously form new organs during post-embryonic development, thus progression of the proliferative cell cycle and subsequent transition into differentiation must be tightly controlled by developmental and environmental cues. Recent studies have begun to uncover how cell proliferation and cell differentiation are coordinated at the molecular level through tight transcriptional regulation of cell cycle and/or developmental regulators. Accumulating evidence suggests that RETINOBLASTOMA-RELATED 1 (RBR1), the Arabidopsis homolog of the human tumor suppressor Retinoblastoma (Rb), functions as a molecular hub linking cell proliferation, differentiation, and environmental response. In this review we will discuss recent findings on cell cycle regulation, highlighting the emerging roles of RBR1 as a key integrator of internal differentiation cues and external stimuli into the cell cycle machinery.

PRC2 represses dedifferentiation of mature somatic cells in Arabidopsis.

Plant somatic cells are generally acknowledged to retain totipotency, the potential to develop into any cell type within an organism. This astonishing plasticity may contribute to a high regenerative capacity on severe damage, but how plants control this potential during normal post-embryonic development remains largely unknown(1,2). Here we show that POLYCOMB REPRESSIVE COMPLEX 2 (PRC2), a chromatin regulator that maintains gene repression through histone modification, prevents dedifferentiation of mature somatic cells in Arabidopsis thaliana roots. Loss-of-function mutants in PRC2 subunits initially develop unicellular root hairs indistinguishable from those in wild type but fail to retain the differentiated state, ultimately resulting in the generation of an unorganized cell mass and somatic embryos from a single root hair. Strikingly, mutant root hairs complete the normal endoreduplication programme, increasing their nuclear ploidy, but subsequently reinitiate mitotic division coupled with successive DNA replication. Our data show that the WOUND INDUCED DEDIFFERENTIATION3 (WIND3) and LEAFY COTYLEDON2 (LEC2) genes are among the PRC2 targets involved in this reprogramming, as their ectopic overexpression partly phenocopies the dedifferentiation phenotype of PRC2 mutants. These findings unveil the pivotal role of PRC2-mediated gene repression in preventing unscheduled reprogramming of fully differentiated plant cells.