Retinoblastoma-related Research Articles

TOPOISOMERASE1α Acts through Two Distinct Mechanisms to Regulate Stele and Columella Stem Cell Maintenance.

TOPOISOMERASE1 (TOP1), which releases DNA torsional stress generated during replication through its DNA relaxation activity, plays vital roles in animal and plant development. In Arabidopsis (Arabidopsis thaliana), TOP1 is encoded by two paralogous genes (TOP1α and TOP1β), of which TOP1α displays specific developmental functions that are critical for the maintenance of shoot and floral stem cells. Here, we show that maintenance of two different populations of root stem cells is also dependent on TOP1α-specific developmental functions, which are exerted through two distinct novel mechanisms. In the proximal root meristem, the DNA relaxation activity of TOP1α is critical to ensure genome integrity and survival of stele stem cells (SSCs). Loss of TOP1α function triggers DNA double-strand breaks in S-phase SSCs and results in their death, which can be partially reversed by the replenishment of SSCs mediated by ETHYLENE RESPONSE FACTOR115 In the quiescent center and root cap meristem, TOP1α is epistatic to RETINOBLASTOMA-RELATED (RBR) in the maintenance of undifferentiated state and the number of columella stem cells (CSCs). Loss of TOP1α function in either wild-type or RBR RNAi plants leads to differentiation of CSCs, whereas overexpression of TOP1α mimics and further enhances the effect of RBR reduction that increases the number of CSCs Taken together, these findings provide important mechanistic insights into understanding stem cell maintenance in plants.

A mutation of casein kinase 2 α4 subunit affects multiple developmental processes in Arabidopsis.

Arabidopsis CK2 α4 subunit regulates the primary root and hypocotyl elongation, lateral root formation, cotyledon expansion, rosette leaf initiation and growth, flowering, and anthocyanin biosynthesis. Casein kinase 2 (CK2) is a conserved tetrameric kinase composed of two α and two β subunits. The inhibition of CK2 activity usually results in severe developmental deficiency. Four genes (CKA1-CKA4) encode CK2 α subunit in Arabidopsis. Single mutations of CKA1, CKA2, and CKA3 do not affect the normal growth of Arabidopsis, while the cka1 cka2 cka3 triple mutants are defective in cotyledon and hypocotyl growth, lateral root development, and flowering. The inhibition of CKA4 expression in cka1 cka2 cka3 background further reduces the number of lateral roots and delays the flowering time. Here, we report the characterization of a novel knockout mutant of CKA4, which exhibits various developmental defects including reduced primary root and hypocotyl elongation, increased lateral root density, delayed cotyledon expansion, retarded rosette leaf initiation and growth, and late flowering. The examination of the cellular basis for abnormal root development of this mutant revealed reduced root meristem cells with enhanced RETINOBLASTOMA-RELATED (RBR) expression that promotes cell differentiation in root meristem. Moreover, this cka4-2 mutant accumulates higher anthocyanin in the aerial part and shows an increased expression of anthocyanin biosynthetic genes, suggesting a novel role of CK2 in modulating anthocyanin biosynthesis. In addition, the complementation test using primary root elongation assay as a sample confirms that the changed phenotypes of this cka4-2 mutant are due to the lack of CKA4. Taken together, this study reveals an essential role of CK2 α4 subunit in multiple developmental processes in Arabidopsis.

A noncanonical role for the CKI-RB-E2F cell-cycle signaling pathway in plant effector-triggered immunity.

Effector-triggered immunity (ETI), the major host defense mechanism in plants, is often associated with programmed cell death (PCD). Plants lack close homologs of caspases, the key mediators of PCD in animals. So although the NB-LRR receptors involved in ETI are well studied, how they activate PCD and confer disease resistance remains elusive. We show that the Arabidopsis nuclear envelope protein, CPR5, negatively regulates ETI and the associated PCD through a physical interaction with cyclin-dependent kinase inhibitors (CKIs). Upon ETI induction, CKIs are released from CPR5 to cause overactivation of another core cell-cycle regulator, E2F. In cki and e2f mutants, ETI responses induced by both TIR-NB-LRR and CC-NB-LRR classes of immune receptors are compromised. We further show that E2F is deregulated during ETI, probably through CKI-mediated hyperphosphorylation of retinoblastoma-related 1 (RBR1). This study demonstrates that canonical cell-cycle regulators also play important noncanonical roles in plant immunity.

The B″ regulatory subunit of protein phosphatase 2A mediates the dephosphorylation of rice retinoblastoma-related protein-1.

The phosphorylation of plant retinoblastoma-related (RBR) proteins by cyclin-dependent kinases (CDKs) is well documented, but the counteracting phosphatases have not been identified yet. We report here that rice retinoblastoma-related protein-1 (OsRBR1) interacted with the B″ subunit of rice protein phosphatase 2A (OsPP2A B″) and underwent reversible phosphorylation during the cell division cycle. The OsRBR1-OsPP2A B" association required B domain in OsRBR1 and the C-terminal region of OsPP2A B″. We found by immunoprecipitation that OsPP2A B″, OsPP2A catalytic subunit subtype II, PSTAIRE-type CDK and OsRBR1 were in the same protein complex, indicating a physical association between the phosphatase, the kinase and their common substrate. OsPP2A B″ contains three predicted CDK phosphorylation sites: Ser95, Ser102 and Ser119. The in vitro phosphorylation of Ser95 and Ser119 with PSTAIRE-kinases was verified by mass spectrometry. We generated a series of phosphorylation site mutants to mimic the dephosphorylated or phosphorylated states of OsPP2A B″, and confirmed that all of the three predicted sites can be phosphorylated. Yeast two-hybrid experiments suggested that the phosphorylation of OsPP2A B″ promoted the formation of the OsPP2A holoenzyme. A triple phosphorylation mimicking OsPP2A B″ mutant containing holoenzyme showed higher activity in phosphatase assays. Our data collectively show that the phosphatase activity of OsPP2A against OsRBR1 is regulated by the phosphorylation of its B″ regulatory subunit. However, the analysis of the effect of okadaic acid, a phosphatase inhibitor, in rice cell suspension cultures revealed that the dephosphorylation of OsRBR1 was completely inhibited only by high dose (300nM) of the okadaic acid during the cell cycle progression. Therefore the role of the protein phosphatase 1 should be considered as an additional post translational regulatory component of RBR protein function in higher plants.

Irreversible fate commitment in the Arabidopsis stomatal lineage requires a FAMA and RETINOBLASTOMA-RELATED module.

The presumed totipotency of plant cells leads to questions about how specific stem cell lineages and terminal fates could be established. In the Arabidopsis stomatal lineage, a transient self-renewing phase creates precursors that differentiate into one of two epidermal cell types, guard cells or pavement cells. We found that irreversible differentiation of guard cells involves RETINOBLASTOMA-RELATED (RBR) recruitment to regulatory regions of master regulators of stomatal initiation, facilitated through interaction with a terminal stomatal lineage transcription factor, FAMA. Disrupting physical interactions between FAMA and RBR preferentially reveals the role of RBR in enforcing fate commitment over its role in cell-cycle control in this developmental context. Analysis of the phenotypes linked to the modulation of FAMA and RBR sheds new light on the way iterative divisions and terminal differentiation are coordinately regulated in a plant stem-cell lineage.

The inheritance and expression patterns of two classes of retinoblastoma-related genes in the stem of Brassica juncea from both parental genomes

In the present study, two classes of retinoblastoma-related (RBR) genes were isolated from Brassica rapa (AA) and B. nigra (BB). These genes were defined as BraA.RBR.a, BraA.RBR.b in B. rapa and BniB.RBR.a, BniB.RBR.b in B. nigra, and 91 and 90% identity were shared at the amino acid level. Interestingly, alternative splicing was also observed both in BraA.RBR.a and BniB.RBR.a transcripts, which consequently yielded two proteins with different C-termini. In the allotetraploid B. juncea (AABB), the corresponding parental RBR loci were maintained, but they experienced a different evolutionary pattern. BjuB.RBR.b and BjuA.RBR.b were found to contain more base mutations and deletions/insertions than BjuB.RBR.a and BjuA.RBR.a when compared with their counterparts in the presumed parents. A comparison of the transcript levels in various tissues suggests a different expression pattern between RBR1 (BraA.RBR.a, BniB.RBR.a) and RBR2 (BraA.RBR.b, BniB.RBR.b); the former varied in expression among different tissues, whereas the latter was almost constitutively expressed. In B. juncea, we found that four RBR genes from both the A- and B-subgenomes were expressed in the tissues tested, suggesting absence of uniparental silencing of RBR genes. The transcript changes of the RBR family during stem development in tuber mustard (B. juncea (L.) Czern. et Coss. var. tumida Tsen et Lee) were also investigated to find its possible roles involved in this process. The results indicated that expression of BjuB.RBR.a and BjuA.RBR.a peaked in stages III and IV when the stems grow vigorously, which suggested that these genes might perform a specific function in stem swelling, and the constitutive expression of BjuB.RBR.b and BjuA.RBR.b might imply their basic roles in stem development. The lack of parental bias and the altered expression pattern of the two RBR classes from the parents suggest that the RBR members might undergo a subfunctionalisation or neofunctionalisation and that these genes might play important roles in the cell-cycle regulation involved in the stem-swelling process of tuber mustard.

Deep functional redundancy between FAMA and FOUR LIPS in stomatal development

Functional redundancy arises between gene paralogs as well as non-homologous genes that play a common role at a shared node. The bHLH transcription factor FAMA, along with the paralogous MYB genes, FOUR LIPS (FLP) and MYB88 all ensure that Arabidopsis stomata contain just two guard cells (GCs) by enforcing a single symmetric precursor cell division before stomatal maturity. Consistent with this function, FLP and FAMA exhibit the same expression pattern in which both translational GFP fusions emit fluorescence just before and after symmetric division; however, FAMA but not FLP is required to confer GC fate. Strikingly, swapping the genes and promoters of the FLP and FAMA genes results in the reciprocal complementation of respective loss-of-function mutants. Thus, an FLP transgene can restore GC fate to a fama mutant background. FAMA, FLP and the FLP paralog MYB88 were previously shown to influence higher order functions in stomatal development, including maintaining and stabilizing stomatal fate. Here we show that these overlapping functions are likely to also involve interactions between FLP and FAMA with the RETINOBLASTOMA-RELATED (RBR) protein.

Arabidopsis Retinoblastoma-related and Polycomb group proteins: cooperation during plant cell differentiation and development

RETINOBLASTOMA (RB) is a tumour suppressor gene originally discovered in patients that develop eye tumours. The pRb protein is now well established as a key cell-cycle regulator which suppresses G1-S transition via interaction with E2F-DP complexes. pRb function is also required for a wide range of biological processes, including the regulation of stem-cell maintenance, cell differentiation, permanent cell-cycle exit, DNA repair, and genome stability. Such multifunctionality of pRb is thought to be facilitated through interactions with various binding partners in a context-dependent manner. Although the molecular network in which RB controls various biological processes is not fully understood, it has been found that pRb interacts with transcription factors and chromatin modifiers to either suppress or promote the expression of key genes during the switch from cell proliferation to differentiation. RETINOBLASTOMA-RELATED (RBR) is the plant orthologue of RB and is also known to negatively control the G1-S transition. Similar to its animal counterpart, plant RBR has various roles throughout plant development; however, much of its molecular functions outside of the G1-S transition are still unknown. One of the better-characterized molecular mechanisms is the cooperation of RBR with the Polycomb repressive complex 2 (PRC2) during plant-specific developmental events. This review summarizes the current understanding of this cooperation and focuses on the processes in Arabidopsis in which the RBR-PRC2 cooperation facilitates cell differentiation and developmental transitions.

Novel roles of plant RETINOBLASTOMA-RELATED (RBR) protein in cell proliferation and asymmetric cell division

The retinoblastoma (Rb) protein was identified as a human tumour suppressor protein that controls various stages of cell proliferation through the interaction with members of the E2F family of transcription factors. It was originally thought to be specific to animals but plants contain homologues of Rb, called RETINOBLASTOMA-RELATED (RBR). In fact, the Rb-E2F module seems to be a very early acquisition of eukaryotes. The activity of RBR depends on phosphorylation of certain amino acid residues, which in most cases are well conserved between plant and animal proteins. In addition to its role in cell-cycle progression, RBR has been shown to participate in various cellular processes such as endoreplication, transcriptional regulation, chromatin remodelling, cell growth, stem cell biology, and differentiation. Here, we discuss the most recent advances to define the role of RBR in cell proliferation and asymmetric cell division. These and other reports clearly support the idea that RBR is used as a landing platform of a plethora of cellular proteins and complexes to control various aspects of cell physiology and plant development.

RETINOBLASTOMA-RELATED Protein Stimulates Cell Differentiation in theArabidopsisRoot Meristem by Interacting with Cytokinin Signaling

Maintenance of mitotic cell clusters such as meristematic cells depends on their capacity to maintain the balance between cell division and cell differentiation necessary to control organ growth. In the Arabidopsis thaliana root meristem, the antagonistic interaction of two hormones, auxin and cytokinin, regulates this balance by positioning the transition zone, where mitotically active cells lose their capacity to divide and initiate their differentiation programs. In animals, a major regulator of both cell division and cell differentiation is the tumor suppressor protein RETINOBLASTOMA. Here, we show that similarly to its homolog in animal systems, the plant RETINOBLASTOMA-RELATED (RBR) protein regulates the differentiation of meristematic cells at the transition zone by allowing mRNA accumulation of AUXIN RESPONSE FACTOR19 (ARF19), a transcription factor involved in cell differentiation. We show that both RBR and the cytokinin-dependent transcription factor ARABIDOPSIS RESPONSE REGULATOR12 are required to activate the transcription of ARF19, which is involved in promoting cell differentiation and thus root growth.

A SCARECROW-RETINOBLASTOMA Protein Network Controls Protective Quiescence in the Arabidopsis Root Stem Cell Organizer

Author SummaryIn the plant Arabidposis thaliana, root meristems (in the growing tip of the root) contain slowly dividing cells that act as an organizing center for the root stem cells that surround them. This centre is called the quiescent centre (QC). In this study, we show that the slow rate of division in the QC is regulated by the interaction between two proteins: Retinoblastoma homolog (RBR) and SCARECROW (SCR), a transcription factor that controls stem cell maintenance. RBR and SCR regulate quiescence in the QC by repressing an asymmetric cell division that generates short-term stem cells. Here we genetically manipulate the cells in the QC to alter their quiescence by regulating the RBR/SCR interaction to demonstrate that quiescence is not needed for the organizing capacity of the QC but instead provides cells with a higher resistance to genotoxic stress, allowing stem cells in the QC to survive even if more rapidly cycling stem cells are damaged. A role for mitotic quiescence has been reported in animal stem cells, in which Rb has been implicated. These findings indicate that it might serve a similar role in plant stem cells.

Transcriptional Reprogramming by TOR

Photosynthesis or glucose stimulates TOR signaling to activate the root meristem.

A Bistable Circuit Involving SCARECROW-RETINOBLASTOMA Integrates Cues to Inform Asymmetric Stem Cell Division

In plants, where cells cannot migrate, asymmetric cell divisions (ACDs) must be confined to the appropriate spatial context. We investigate tissue-generating asymmetric divisions in a stem cell daughter within the Arabidopsis root. Spatial restriction of these divisions requires physical binding of the stem cell regulator SCARECROW (SCR) by the RETINOBLASTOMA-RELATED (RBR) protein. In the stem cell niche, SCR activity is counteracted by phosphorylation of RBR through a cyclinD6;1-CDK complex. This cyclin is itself under transcriptional control of SCR and its partner SHORT ROOT (SHR), creating a robust bistable circuit with either high or low SHR-SCR complex activity. Auxin biases this circuit by promoting CYCD6;1 transcription. Mathematical modeling shows that ACDs are only switched on after integration of radial and longitudinal information, determined by SHR and auxin distribution, respectively. Coupling of cell-cycle progression to protein degradation resets the circuit, resulting in a "flip flop" that constrains asymmetric cell division to the stem cell region.

Characterization and Expression Analysis of a Retinoblastoma-Related Gene from Chinese Wild Vitis pseudoreticulata

Retinoblastoma-related (RBR) genes, a conserved gene family in higher eukaryotes, play important roles in cell differentiation, development, and mammalian cell death; however, little is known of their function in plants. In this study, a RBR gene was isolated from the Chinese wild grape, Vitis pseudoreticulata W. T. Wang clone “Baihe-35-1”, and designated as VpRBR. The cDNA sequence of VpRBR was 3,030 bp and contained an open reading frame of 3,024 bp. Conceptual translation of this gene indicated a composition of 1,007 amino acids with a predicted molecular mass of 117.3 kDa. The predicted protein showed a retinoblastoma-associated protein domain A from amino acid residues 416 to 579, and domain B from residues 726 to 855. The result of expression analysis indicated that VpRBR was expressed in tissues, leaves, stem, tendrils, flower, and grape skin at different expression levels. Further quantitative reverse transcription-PCR (qRT-PCR) data indicated that VpRBR levels were higher in Erysiphe necator-treated “Baihe-35-1” and “Baihe-13-1”, two resistant clones of Chinese wild V. pseudoreticulata, than in E. necator-treated “Hunan-1”, a susceptible clone of V. pseudoreticulata. Furthermore, the expression of VpRBR in response to salicylic acid (SA), methyl jasmonate (MeJA), and ethylene (Eth) in grape leaves was also investigated. Taken together, these data indicate that VpRBR may contribute to some aspect of powdery mildew resistance in grape.

The Arabidopsis Transcription Factor AtTCP15 Regulates Endoreduplication by Modulating Expression of Key Cell-cycle Genes

Plant cells frequently undergo endoreduplication, a modified cell cycle in which genome is repeatedly replicated without cytokinesis. As the key step to achieve final size and function for cells, endoreduplication is prevalent during plant development. However, mechanisms to control the balance between endoreduplication and mitotic cell division are still poorly understood. Here, we show that the Arabidopsis TCP (CINCINNATA-like TEOSINTE BRANCHED1-CYCLOIDEA-PCF)-family transcription factor gene AtTCP15 is expressed in trichomes, as well as in rapidly dividing and vascular tissues. Expression of AtTCP15SRDX, AtTCP15 fused with a SRDX repressor domain, induces extra endoreduplication in trichomes and cotyledon cells in transgenic Arabidopsis. On the contrary, overexpression of AtTCP15 suppresses endoreduplication in trichomes and other examined cells. Misregulation of AtTCP15 affects the expression of several important genes involved in cell-cycle regulation. AtTCP15 protein binds directly to the promoter regions of CYCA2;3 and RETINOBLASTOMA-RELATED (RBR) genes, which play key roles in endoreduplication. Taken together, AtTCP15 plays an important role in regulating endoreduplication during Arabidopsis development.

The E2F transcription factor family regulates CENH3 expression in Arabidopsis thaliana

To elucidate the epigenetic maintenance mechanism for functional plant centromeres, we studied transcriptional regulation of the centromere-specific histone H3 variant CENH3 in Arabidopsis thaliana. We focused on the structure and activity of the CENH3 promoter (CENH3pro) and its regulation by E2F transcription factors. Use of CENH3pro::GUS reporter gene constructs showed that CENH3pro is active in dividing tissues, and that full expression in root meristems depends on intragenic regulatory elements within the second intron. Chromatin immunoprecipitation identified CENH3 as an E2F target gene. Transient co-expression of a CENH3pro::GUS reporter gene construct with various E2F transcription factors in A.thaliana protoplasts showed that E2Fa and E2Fb (preferentially with dimerization protein DPb) activate CENH3pro. Stable over-expression of E2Fa and E2Fb increased the CENH3 transcript level in planta, whereas over-expression of E2Fc decreased the CENH3 transcript level. Surprisingly, mutation of the two E2F binding sites of CENH3pro, in particular the more upstream one (E2F2), caused an increase in CENH3pro activity, indicating E2F-dependent transcriptional repression. CENH3pro repression may be triggered by the interplay of typical and atypical E2Fs in a cell cycle-dependent manner, and/or by interaction of typical E2Fs with retinoblastoma-related (RBR) protein. We speculate that E2Fs are involved in differential transcriptional regulation of CENH3 versus H3, as H3 promoters lack E2F binding motifs. E2F binding motifs are also present in human and Drosophila CENH3pro regions, thus cell cycle-dependent transcriptional regulation of CENH3 may be highly conserved.

Distinct Cell-Autonomous Functions of RETINOBLASTOMA-RELATED in Arabidopsis Stem Cells Revealed by the Brother of Brainbow Clonal Analysis System

Mutations that cause lethality in the gametophyte phase pose a major challenge for studying postfertilization gene function. When both male and female haploid cells require a functional gene copy, null alleles cause developmental arrest before the formation of the zygote, precluding further investigation. The Arabidopsis thaliana Rb homolog RETINOBLASTOMA-RELATED (RBR) has an important function in the stem cell niche, but its requirement in both male and female gametophytes has prevented full loss-of-function studies. To circumvent this obstacle, we designed a clonal deletion system named BOB (Brother of Brainbow) in which null mutant sectors marked by double fluorescence are generated in a fully complemented wild-type background. In this system, both copies of a complementing RBR transgene are eliminated by tissue-specific and inducible CRE expression, and homozygous mutant clones can be distinguished visually. Since mutant sectors can be produced in a homozygous, rather than a heterozygous, background, this system facilitates clonal deletion analysis not only for gametophytic lethal alleles but also for any type of mutation. Using the BOB system, we show that RBR has unique cell-autonomous functions in different cell types within the root stem cell niche.

Retinoblastoma protein is essential for early meiotic events inArabidopsis

We have analysed the role of RBR (retinoblastoma related), the Arabidopsis homologue of the tumour suppressor Retinoblastoma protein (pRb), during meiosis. We characterise the rbr-2 mutation, which causes a loss of RBR in male meiocytes. The rbr-2 plants exhibit strongly reduced fertility, while vegetative growth is generally unaffected. The reduced fertility is due to a meiotic defect that results in reduced chiasma formation and subsequent errors in chromosome disjunction. Immunolocalisation studies in wild-type meiocytes reveal that RBR is recruited as foci to the chromosomes during early prophase I in a DNA double-strand-break-dependent manner. In the absence of RBR, expression of several meiotic genes is reduced. The localisation of the recombinases AtRAD51 and AtDMC1 is normal. However, localisation of the MutS homologue AtMSH4 is compromised. Additionally, polymerisation of the synaptonemal complex protein AtZYP1 is abnormal. Together, these data indicate that loss of RBR during meiosis results in a reduction of crossover formation and an associated failure in chromosome synapsis. Our results indicate that RBR has an important role in meiosis affecting different aspects of this complex process.

Immunodetection of retinoblastoma-related protein and its phosphorylated form in interphase and mitotic alfalfa cells

Plant retinoblastoma-related (RBR) proteins are primarily considered as key regulators of G1/S phase transition, with functional roles in a variety of cellular events during plant growth and organ development. Polyclonal antibody against the C-terminal region of the Arabidopsis RBR1 protein also specifically recognizes the alfalfa 115 kDa MsRBR protein, as shown by the antigen competition assay. The MsRBR protein was detected in all cell cycle phases, with a moderate increase in samples representing G2/M cells. Antibody against the human phospho-pRb peptide (Ser807/811) cross-reacted with the same 115 kDa MsRBR protein and with the in vitro phosphorylated MsRBR protein C-terminal fragment. Phospho-MsRBR protein was low in G1 cells. Its amount increased upon entry into the S phase and remained high during the G2/M phases. Roscovitine treatment abolished the activity of alfalfa MsCDKA1;1 and MsCDKB2;1, and the phospho-MsRBR protein level was significantly decreased in the treated cells. Colchicine block increased the detected levels of both forms of MsRBR protein. Reduced levels of the MsRBR protein in cells at stationary phase or grown in hormone-free medium can be a sign of the division-dependent presence of plant RBR proteins. Immunolocalization of the phospho-MsRBR protein indicated spots of variable number and size in the labelled interphase nuclei and high signal intensity of nuclear granules in prophase. Structures similar to phospho-MsRBR proteins cannot be recognized in later mitotic phases. Based on the presented western blot and immunolocalization data, the possible involvement of RBR proteins in G2/M phase regulation in plant cells is discussed.

Arabidopsis S6 kinase mutants display chromosome instability and altered RBR1–E2F pathway activity

The 40S ribosomal protein S6 kinase (S6K) is a conserved component of signalling pathways controlling growth in eukaryotes. To study S6K function in plants, we isolated single- and double-knockout mutations and RNA-interference (RNAi)-silencing lines in the linked Arabidopsis S6K1 and S6K2 genes. Hemizygous s6k1s6k2/++ mutant and S6K1 RNAi lines show high phenotypic instability with variation in size, increased trichome branching, produce non-viable pollen and high levels of aborted seeds. Analysis of their DNA content by flow cytometry, as well as chromosome counting using DAPI staining and fluorescence in situ hybridization, revealed an increase in ploidy and aneuploidy. In agreement with this data, we found that S6K1 associates with the Retinoblastoma-related 1 (RBR1)-E2FB complex and this is partly mediated by its N-terminal LVxCxE motif. Moreover, the S6K1-RBR1 association regulates RBR1 nuclear localization, as well as E2F-dependent expression of cell cycle genes. Arabidopsis cells grown under nutrient-limiting conditions require S6K for repression of cell proliferation. The data suggest a new function for plant S6K as a repressor of cell proliferation and required for maintenance of chromosome stability and ploidy levels.