Retinoblastoma-related Research Articles

Developmental cues are encoded by the combinatorial phosphorylation of Arabidopsis RETINOBLASTOMA-RELATED protein RBR1.

RETINOBLASTOMA-RELATED (RBR) proteins orchestrate cell division, differentiation, and survival in response to environmental and developmental cues through protein-protein interactions that are governed by multisite phosphorylation. Here we explore, using a large collection of transgenic RBR phosphovariants to complement protein function in Arabidopsis thaliana, whether differences in the number and position of RBR phosphorylation events cause a diversification of the protein's function. While the number of point mutations influence phenotypic strength, phosphosites contribute differentially to distinct phenotypes. RBR pocket domain mutations associate primarily with cell proliferation, while mutations in the C-region are linked to stem cell maintenance. Both phospho-mimetic and a phospho-defective variants promote cell death, suggesting that distinct mechanisms can lead to similar cell fates. We observed combinatorial effects between phosphorylated T406 and phosphosites in different protein domains, suggesting that specific, additive, and combinatorial phosphorylation events fine-tune RBR function. Suppression of dominant phospho-defective RBR phenotypes with a mutation that inhibits RBR interacting with LXCXE motifs, and an exhaustive protein-protein interaction assay, not only revealed the importance of DREAM complex members in phosphorylation-regulated RBR function but also pointed to phosphorylation-independent RBR roles in environmental responses. Thus, combinatorial phosphorylation defined and separated developmental, but not environmental, functions of RBR.

RETINOBLASTOMA-RELATED Has Both Canonical and Noncanonical Regulatory Functions During Thermo-Morphogenic Responses in Arabidopsis Seedlings.

Warm temperatures accelerate plant growth, but the underlying molecular mechanism is not fully understood. Here, we show that increasing the temperature from 22°C to 28°C rapidly activates proliferation in the apical shoot and root meristems of wild-type Arabidopsis seedlings. We found that one of the central regulators of cell proliferation, the cell cycle inhibitor RETINOBLASTOMA-RELATED (RBR), is suppressed by warm temperatures. RBR became hyper-phosphorylated at a conserved CYCLIN-DEPENDENT KINASE (CDK) site in young seedlings growing at 28°C, in parallel with the stimulation of the expressions of the regulatory CYCLIN D/A subunits of CDK(s). Interestingly, while under warm temperatures ectopic RBR slowed down the acceleration of cell proliferation, it triggered elongation growth of post-mitotic cells in the hypocotyl. In agreement, the central regulatory genes of thermomorphogenic response, including PIF4 and PIF7, as well as their downstream auxin biosynthetic YUCCA genes (YUC1-2 and YUC8-9) were all up-regulated in the ectopic RBR expressing line but down-regulated in a mutant line with reduced RBR level. We suggest that RBR has both canonical and non-canonical functions under warm temperatures to control proliferative and elongation growth, respectively.

The MADS-box gene XAANTAL1 participates in Arabidopsis thaliana primary root growth and columella stem cell patterns in response to ROS, via direct regulation of PEROXIDASE 28 and RETINOBLASTOMA RELATED.

The balance between cell growth, proliferation and differentiation emerges from gene regulatory networks coupled to various signal transduction pathways, including reactive oxygen species (ROS) and transcription factors (TFs), enabling developmental responses to environmental cues. The Arabidopsis thaliana's primary root has become a valuable system for unraveling such networks. Recently, the role of TFs that mediate the ROS's inhibition of primary root growth has begun to be characterized. This study demonstrates that the MADS-box transcription factor XAANTAL1 (XAL1) is an essential regulator of hydrogen peroxide (H2O2) in primary root growth and root stem cell niche identity. Interestingly, our findings suggest that XAL1 acts as a positive regulator of H2O2 concentration in the root meristem by directly regulating genes involved in oxidative stress response, such as PEROXIDASE 28 (PER28). Moreover, we found that XAL1 is necessary for the H2O2-induced inhibition of primary root growth through the negative regulation of peroxidase and catalase activities. Furthermore, XAL1, in conjunction with RETINOBLASTOMA-RELATED (RBR), is essential for positively regulating the differentiation of columella stem cells and for participating in primary root growth inhibition in response to oxidative stress induced by H2O2 treatment.

BIG coordinates auxin and SHORT ROOT to promote asymmetric stem cell divisions in Arabidopsis roots.

BIG regulates ground tissue formative divisions by bridging the auxin gradient with SHR abundance in Arabidopsis roots. The formative divisions of cortex/endodermis initials (CEIs) and CEI daughter cells (CEIDs) in Arabidopsis roots are coordinately controlled by the longitudinal auxin gradient and the radial SHORT ROOT (SHR) abundance. However, the mechanism underlying this coordination remains poorly understood. In this study, we demonstrate that BIG regulates ground tissue formative divisions by bridging the auxin gradient with SHR abundance. Mutations in BIG gene repressed cell cycle progression, delaying the formative divisions within the ground tissues and impairing the establishment of endodermal and cortical identities. In addition, we uncovered auxin's suppressive effect on BIG expression, triggering CYCLIND6;1 (CYCD6;1) activation in an SHR-dependent fashion. Moreover, the degradation of RETINOBLASTOMA-RELATED (RBR) is jointly regulated by BIG and CYCD6;1. The loss of BIG function led to RBR protein accumulation, detrimentally impacting the SHR/SCARECROW (SCR) protein complex and the CEI/CEID formative divisions. Collectively, these findings shed light on a fundamental mechanism wherein BIG intricately coordinates the interplay between SHR/SCR and auxin, steering ground tissue patterning within Arabidopsis root tissue.

RETINOBLASTOMA RELATED 1 switches mitosis to meiosis in rice

The transition from mitosis to meiosis is a critical event in the reproductive development of all sexually reproducing species. However, the mechanisms that regulate this process in plants remain largely unknown. Here, we find that the rice (Oryza sativa L.) protein RETINOBLASTOMA RELATED 1 (RBR1) is essential to the transition from mitosis to meiosis. Loss of RBR1 function results in hyper-proliferative sporogenous-cell-like cells (SCLs) in the anther locules during early stages of reproductive development. These hyper-proliferative SCLs are unable to initiate meiosis, eventually stagnating and degrading at late developmental stages to form pollen-free anthers. These results suggest that RBR1 acts as a gatekeeper of entry into meiosis. Furthermore, cytokinin content is significantly increased in rbr1 mutants, whereas the expression of type-B response factors, particularly LEPTO1, is significantly reduced. Given the known close association of cytokinins with cell proliferation, these findings imply that hyper-proliferative germ cells in the anther locules may be attributed to elevated cytokinin concentrations and disruptions in the cytokinin pathway. Using a genetic strategy, the association between germ cell hyper-proliferation and disturbed cytokinin signaling in rbr1 has been confirmed. In summary, we reveal a unique role of RBR1 in the initiation of meiosis; our results clearly demonstrate that the RBR1 regulatory module is connected to the cytokinin signaling pathway and switches mitosis to meiosis in rice.

A paternal signal induces endosperm proliferation upon fertilization in Arabidopsis.

In multicellular organisms, sexual reproduction relies on the formation of highly differentiated cells, the gametes, which await fertilization in a quiescent state. Upon fertilization, the cell cycle resumes. Successful development requires that male and female gametes are in the same phase of the cell cycle. The molecular mechanisms that reinstate cell division in a fertilization-dependent manner are poorly understood in both animals and plants. Using Arabidopsis, we show that a sperm-derived signal induces the proliferation of a female gamete, the central cell, precisely upon fertilization. The central cell is arrested in S phase by the activity of the RETINOBLASTOMA RELATED1 (RBR1) protein. Upon fertilization, delivery of the core cell cycle component CYCD7;1 causes RBR1 degradation and thus S phase progression, ensuring the formation of functional endosperm and, consequently, viable seeds.

The dual role of the RETINOBLASTOMA-RELATED protein in the DNA damage response is coordinated by the interaction with LXCXE-containing proteins.

Living organisms possess mechanisms to safeguard genome integrity. To avoid spreading mutations, DNA lesions are detected and cell division is temporarily arrested to allow repair mechanisms. Afterward, cells either resume division or respond to unsuccessful repair by undergoing programmed cell death (PCD). How the success rate of DNA repair connects to later cell fate decisions remains incompletely known, particularly in plants. The Arabidopsis thaliana RETINOBLASTOMA-RELATED1 (RBR) protein and its partner E2FA, play both structural and transcriptional functions in the DNA damage response (DDR). Here we provide evidence that distinct RBR protein interactions with LXCXE motif-containing proteins guide these processes. Using the N849F substitution in the RBR B-pocket domain, which specifically disrupts binding to the LXCXE motif, we show that these interactions are dispensable in unchallenging conditions. However, N849F substitution abolishes RBR nuclear foci and promotes PCD and growth arrest upon genotoxic stress. NAC044, which promotes growth arrest and PCD, accumulates after the initial recruitment of RBR to foci and can bind non-focalized RBR through the LXCXE motif in a phosphorylation-independent manner, allowing interaction at different cell cycle phases. Disrupting NAC044-RBR interaction impairs PCD, but their genetic interaction points to opposite independent roles in the regulation of PCD. The LXCXE-binding dependency of the roles of RBR in the DDR suggests a coordinating mechanism to translate DNA repair success to cell survival. We propose that RBR and NAC044 act in two distinct DDR pathways, but interact to integrate input from both DDR pathways to decide upon an irreversible cell fate decision.

The canonical E2Fs together with RETINOBLASTOMA-RELATED are required to establish quiescence during plant development

Maintaining stable and transient quiescence in differentiated and stem cells, respectively, requires repression of the cell cycle. The plant RETINOBLASTOMA-RELATED (RBR) has been implicated in stem cell maintenance, presumably by forming repressor complexes with E2F transcription factors. Surprisingly we find that mutations in all three canonical E2Fs do not hinder the cell cycle, but similarly to RBR silencing, result in hyperplasia. Contrary to the growth arrest that occurs when exit from proliferation to differentiation is inhibited upon RBR silencing, the e2fabc mutant develops enlarged organs with supernumerary stem and differentiated cells as quiescence is compromised. While E2F, RBR and the M-phase regulatory MYB3Rs are part of the DREAM repressor complexes, and recruited to overlapping groups of targets, they regulate distinct sets of genes. Only the loss of E2Fs but not the MYB3Rs interferes with quiescence, which might be due to the ability of E2Fs to control both G1-S and some key G2-M targets. We conclude that collectively the three canonical E2Fs in complex with RBR have central roles in establishing cellular quiescence during organ development, leading to enhanced plant growth.

Predicted landscape of RETINOBLASTOMA-RELATED LxCxE-mediated interactions across the Chloroplastida.

The colonization of land by a single streptophyte algae lineage some 450million years ago has been linked to multiple key innovations such as three-dimensional growth, alternation of generations, the presence of stomata, as well as innovations inherent to the birth of major plant lineages, such as the origins of vascular tissues, roots, seeds and flowers. Multicellularity, which evolved multiple times in the Chloroplastida coupled with precise spatiotemporal control of proliferation and differentiation were instrumental for theevolution of these traits. RETINOBLASTOMA-RELATED (RBR), the plant homolog of the metazoan Retinoblastoma protein (pRB), is a highly conserved and multifunctional core cell cycle regulator that has been implicated in the evolution of multicellularity in the green lineage as well as in plant multicellularity-related processes such as proliferation, differentiation, stem cell regulation and asymmetric cell division. RBR fulfills these roles through context-specific protein-protein interactions with proteins containing the Leu-x-Cys-x-Glu (LxCxE) short-linear motif (SLiM); however, how RBR-LxCxE interactions have changed throughout major innovations in the Viridiplantae kingdom is a question that remains unexplored. Here, we employ an in silico evo-devo approach to predict and analyze potential RBR-LxCxE interactions in different representative species of key Chloroplastida lineages, providing a valuable resource for deciphering RBR-LxCxE multiple functions. Furthermore, our analyses suggest that RBR-LxCxE interactions are an important component of RBR functions and that interactions with chromatin modifiers/remodelers, DNA replication and repair machinery are highly conserved throughout the Viridiplantae, while LxCxE interactions with transcriptional regulators likely diversified throughout the water-to-land transition.

Pickle Recruits Retinoblastoma Related 1 to Control Lateral Root Formation in Arabidopsis.

Lateral root (LR) formation is an example of a plant post-embryonic organogenesis event. LRs are issued from non-dividing cells entering consecutive steps of formative divisions, proliferation and elongation. The chromatin remodeling protein PICKLE (PKL) negatively regulates auxin-mediated LR formation through a mechanism that is not yet known. Here we show that PKL interacts with RETINOBLASTOMA-RELATED 1 (RBR1) to repress the LATERAL ORGAN BOUNDARIES-DOMAIN 16 (LBD16) promoter activity. Since LBD16 function is required for the formative division of LR founder cells, repression mediated by the PKL–RBR1 complex negatively regulates formative division and LR formation. Inhibition of LR formation by PKL–RBR1 is counteracted by auxin, indicating that, in addition to auxin-mediated transcriptional responses, the fine-tuned process of LR formation is also controlled at the chromatin level in an auxin-signaling dependent manner.

The F-Box-Like Protein FBL17 Is a Regulator of DNA-Damage Response and Colocalizes with RETINOBLASTOMA RELATED1 at DNA Lesion Sites.

In Arabidopsis (Arabidopsis thaliana), the F-box protein F-BOX-LIKE17 (FBL17) was previously identified as an important cell-cycle regulatory protein. FBL17 is required for cell division during pollen development and for normal cell-cycle progression and endoreplication during the diploid sporophyte phase. FBL17 was reported to control the stability of the CYCLIN-DEPENDENT KINASE inhibitor KIP-RELATED PROTEIN (KRP), which may underlie the drastic reduction in cell division activity in both shoot and root apical meristems observed in fbl17 loss-of-function mutants. However, whether FBL17 has other substrates and functions besides degrading KRPs remains poorly understood. Here we show that mutation of FBL17 leads not only to misregulation of cell cycle genes, but also to a strong upregulation of genes involved in DNA damage and repair processes. This phenotype is associated with a higher frequency of DNA lesions in fbl17 and increased cell death in the root meristem, even in the absence of genotoxic stress. Notably, the constitutive activation of DNA damage response genes is largely SOG1-independent in fbl17 In addition, through analyses of root elongation, accumulation of cell death, and occurrence of γH2AX foci, we found that fbl17 mutants are hypersensitive to DNA double-strand break-induced genotoxic stress. Notably, we observed that the FBL17 protein is recruited at nuclear foci upon double-strand break induction and colocalizes with γH2AX, but only in the presence of RETINOBLASTOMA RELATED1. Altogether, our results highlight a role for FBL17 in DNA damage response, likely by ubiquitylating proteins involved in DNA-damage signaling or repair.

ErbB-3 BINDING PROTEIN 1 Regulates Translation and Counteracts RETINOBLASTOMA RELATED to Maintain the Root Meristem.

The ErbB-3 BINDING PROTEIN 1 (EBP1) drives growth, but the mechanism of how it acts in plants is little understood. Here, we show that EBP1 expression and protein abundance in Arabidopsis (Arabidopsis thaliana) are predominantly confined to meristematic cells and are induced by sucrose and partially dependent on TARGET OF RAPAMYCIN (TOR) kinase activity. Consistent with being downstream of TOR, silencing of EBP1 restrains, while overexpression promotes, root growth, mostly under sucrose-limiting conditions. Inducible overexpression of RETINOBLASTOMA RELATED (RBR), a sugar-dependent transcriptional repressor of cell proliferation, depletes meristematic activity and causes precocious differentiation, which is attenuated by EBP1. To understand the molecular mechanism, we searched for EBP1- and RBR-interacting proteins by affinity purification and mass spectrometry. In line with the double-stranded RNA-binding activity of EBP1 in human (Homo sapiens) cells, the overwhelming majority of EBP1 interactors are part of ribonucleoprotein complexes regulating many aspects of protein synthesis, including ribosome biogenesis and mRNA translation. We confirmed that EBP1 associates with ribosomes and that EBP1 silencing hinders ribosomal RNA processing. We revealed that RBR also interacts with a set of EBP1-associated nucleolar proteins as well as factors that function in protein translation. This suggests EBP1 and RBR act antagonistically on common processes that determine the capacity for translation to tune meristematic activity in relation to available resources.

E2FB Interacts with RETINOBLASTOMA RELATED and Regulates Cell Proliferation during Leaf Development.

Cell cycle entry and quiescence are regulated by the E2F transcription factors in association with RETINOBLASTOMA-RELATED (RBR). E2FB is considered to be a transcriptional activator of cell cycle genes, but its function during development remains poorly understood. Here, by studying E2FB-RBR interaction, E2F target gene expression, and epidermal cell number and shape in e2fb mutant and overexpression lines during leaf development in Arabidopsis (Arabidopsis thaliana), we show that E2FB in association with RBR plays a role in the inhibition of cell proliferation to establish quiescence. In young leaves, both RBR and E2FB are abundant and form a repressor complex that is reinforced by an autoregulatory loop. Increased E2FB levels, either by expression driven by its own promoter or ectopically together with DIMERIZATION PARTNER A, further elevate the amount of this repressor complex, leading to reduced leaf cell number. Cell overproliferation in e2fb mutants and in plants overexpressing a truncated form of E2FB lacking the RBR binding domain strongly suggested that RBR repression specifically acts through E2FB. The increased number of small cells below the guard cells and of fully developed stomata indicated that meristemoids preferentially hyperproliferate. As leaf development progresses and cells differentiate, the amount of RBR and E2FB gradually declined. At this stage, elevation of E2FB level can overcome RBR repression, leading to reactivation of cell division in pavement cells. In summary, E2FB in association with RBR is central to regulating cell proliferation during organ development to determine final leaf cell number.

RETINOBLASTOMA-RELATED Genes Specifically Control Inner Floral Organ Morphogenesis and Pollen Development in Rice.

RETINOBLASTOMA-RELATED (RBR) is an essential gene in plants, but its molecular function outside of its role in cell cycle entry remains poorly understood. We characterized the functions of OsRBR1 and OsRBR2 in plant growth and development in rice using both forward- and reverse-genetics methods. The two genes were coexpressed and performed redundant roles in vegetative organs but exhibited separate functions in flowers. OsRBR1 was highly expressed in the floral meristem and regulated the expression of floral homeotic genes to ensure floral organ formation. Mutation of OsRBR1 caused loss of floral meristem identity, resulting in the replacement of lodicules, stamens, and the pistil with either a panicle-like structure or whorls of lemma-like organs. OsRBR2 was preferentially expressed in stamens and promoted pollen formation. Mutation of OsRBR2 led to deformed anthers without pollen. Similar to the protein interaction between AtRBR and AtMSI1 that is essential for floral development in Arabidopsis, OsMSI1 was identified as an interaction partner of OsRBR1 and OsRBR2. OsMSI1 was ubiquitously expressed and appears to be essential for development in rice (Oryza sativa), as the mutation of OsMSI1 was lethal. These results suggest that OsRBR1 and OsRBR2 function with OsMSI1 in reproductive development in rice. This work characterizes further functions of RBRs and improves current understanding of specific regulatory pathways of floral specification and pollen formation in rice.

Chromatin Remodeling and Epigenetic Regulation in Plant DNA Damage Repair.

DNA damage response (DDR) in eukaryotic cells is initiated in the chromatin context. DNA damage and repair depend on or have influence on the chromatin dynamics associated with genome stability. Epigenetic modifiers, such as chromatin remodelers, histone modifiers, DNA (de-)methylation enzymes, and noncoding RNAs regulate DDR signaling and DNA repair by affecting chromatin dynamics. In recent years, significant progress has been made in the understanding of plant DDR and DNA repair. SUPPRESSOR OF GAMMA RESPONSE1, RETINOBLASTOMA RELATED1 (RBR1)/E2FA, and NAC103 have been proven to be key players in the mediation of DDR signaling in plants, while plant-specific chromatin remodelers, such as DECREASED DNA METHYLATION1, contribute to chromatin dynamics for DNA repair. There is accumulating evidence that plant epigenetic modifiers are involved in DDR and DNA repair. In this review, I examine how DDR and DNA repair machineries are concertedly regulated in Arabidopsis thaliana by a variety of epigenetic modifiers directing chromatin remodeling and epigenetic modification. This review will aid in updating our knowledge on DDR and DNA repair in plants.

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 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.

Arabidopsis RETINOBLASTOMA RELATED directly regulates DNA damage responses through functions beyond cell cycle control

The rapidly proliferating cells in plant meristems must be protected from genome damage. Here, we show that the regulatory role of the Arabidopsis RETINOBLASTOMA RELATED (RBR) in cell proliferation can be separated from a novel function in safeguarding genome integrity. Upon DNA damage, RBR and its binding partner E2FA are recruited to heterochromatic γH2AX‐labelled DNA damage foci in an ATM‐ and ATR‐dependent manner. These γH2AX‐labelled DNA lesions are more dispersedly occupied by the conserved repair protein, AtBRCA1, which can also co‐localise with RBR foci. RBR and AtBRCA1 physically interact in vitro and in planta. Genetic interaction between the RBR‐silenced amiRBR and Atbrca1 mutants suggests that RBR and AtBRCA1 may function together in maintaining genome integrity. Together with E2FA, RBR is directly involved in the transcriptional DNA damage response as well as in the cell death pathway that is independent of SOG1, the plant functional analogue of p53. Thus, plant homologs and analogues of major mammalian tumour suppressor proteins form a regulatory network that coordinates cell proliferation with cell and genome integrity.

Two maize Kip-related proteins differentially interact with, inhibit and are phosphorylated by cyclin D–cyclin-dependent kinase complexes

The family of maize Kip-related proteins (KRPs) has been studied and a nomenclature based on the relationship to rice KRP genes is proposed. Expression studies of KRP genes indicate that all are expressed at 24 h of seed germination but expression is differential in the different tissues of maize plantlets. Recombinant KRP1;1 and KRP4;2 proteins, members of different KRP classes, were used to study association to and inhibitory activity on different maize cyclin D (CycD)-cyclin-dependent kinase (CDK) complexes. Kinase activity in CycD2;2-CDK, CycD4;2-CDK, and CycD5;3-CDK complexes was inhibited by both KRPs; however, only KRP1;1 inhibited activity in the CycD6;1-CDK complex, not KRP4;2. Whereas KRP1;1 associated with either CycD2;2 or CycD6;1, and to cyclin-dependent kinase A (CDKA) recombinant proteins, forming ternary complexes, KRP4;2 bound CDKA and CycD2;2 but did not bind CycD6;1, establishing a differential association capacity. All CycD-CDK complexes included here phosphorylated both the retinoblastoma-related (RBR) protein and the two KRPs; interestingly, while KRP4;2 phosphorylated by the CycD2;2-CDK complex increased its inhibitory capacity, when phosphorylated by the CycD6;1-CDK complex the inhibitory capacity was reduced or eliminated. Evidence suggests that the phosphorylated residues in KRP4;2 may be different for every kinase, and this would influence its performance as a cyclin-CDK inhibitor.

Functional characterization of the ribosome biogenesis factors PES, BOP1, and WDR12 (PeBoW), and mechanisms of defective cell growth and proliferation caused by PeBoW deficiency in Arabidopsis

The nucleolar protein pescadillo (PES) controls biogenesis of the 60S ribosomal subunit through functional interactions with Block of Proliferation 1 (BOP1) and WD Repeat Domain 12 (WDR12) in plants. In this study, we determined protein characteristics and in planta functions of BOP1 and WDR12, and characterized defects in plant cell growth and proliferation caused by a deficiency of PeBoW (PES-BOP1-WDR12) proteins. Dexamethasone-inducible RNAi of BOP1 and WDR12 caused developmental arrest and premature senescence in Arabidopsis, similar to the phenotype of PES RNAi. Both the N-terminal domain and WD40 repeats of BOP1 and WDR12 were critical for specific associations with 60S/80S ribosomes. In response to nucleolar stress or DNA damage, PeBoW proteins moved from the nucleolus to the nucleoplasm. Kinematic analyses of leaf growth revealed that depletion of PeBoW proteins led to dramatically suppressed cell proliferation, cell expansion, and epidermal pavement cell differentiation. A deficiency in PeBoW proteins resulted in reduced cyclin-dependent kinase Type A activity, causing reduced phosphorylation of histone H1 and retinoblastoma-related (RBR) protein. PeBoW silencing caused rapid transcriptional modulation of cell-cycle genes, including reduction of E2Fa and Cyclin D family genes, and induction of several KRP genes, accompanied by down-regulation of auxin-related genes and up-regulation of jasmonic acid-related genes. Taken together, these results suggest that the PeBoW proteins involved in ribosome biogenesis play a critical role in plant cell growth and survival, and their depletion leads to inhibition of cell-cycle progression, possibly modulated by phytohormone signaling.