MEDIATOR SUBUNIT17 is required for transcriptional optimization of root system architecture in Arabidopsis.

Sucrose and auxin are well-known determinants of root system architecture (RSA). However, the factors that connect the signaling pathways evoked by these two critical factors during root development are poorly understood. In this study, we report the role of MEDIATOR SUBUNIT17 (MED17) in RSA and its involvement in the transcriptional integration of sugar and auxin signaling pathways in Arabidopsis (Arabidopsis thaliana). Sucrose regulates root meristem activation through the TARGET OF RAPAMYCIN-E2 PROMOTER BINDING FACTOR A (TOR-E2FA) pathway, and auxin regulates lateral root (LR) development through AUXIN RESPONSE FACTOR-LATERAL ORGAN BOUNDARIES DOMAIN (ARF-LBDs). Both sucrose and auxin play a vital role during primary and LR development. However, there is no clarity on how sucrose is involved in the ARF-dependent regulation of auxin-responsive genes. This study establishes MED17 as a nodal point to connect sucrose and auxin signaling. Transcription of MED17 was induced by sucrose in an E2FA/B-dependent manner. Moreover, E2FA/B interacted with MED17, which can aid in the recruitment of the Mediator complex on the target promoters. Interestingly, E2FA/B and MED17 also occupied the promoter of ARF7, but not ARF19, leading to ARF7 expression, which then activates auxin signaling and thus initiates LR development. MED17 also activated cell division in the root meristem by occupying the promoters of cell-cycle genes, thus regulating their transcription. Thus, MED17 plays an important role in relaying the transcriptional signal from sucrose to auxin-responsive and cell-cycle genes to regulate primary and lateral root development, highlighting the role of the Mediator as the transcriptional processor for optimal root system architecture in Arabidopsis.

E2FA and E2FB transcription factors coordinate cell proliferation with seed maturation.

The E2F transcription factors and the RETINOBLASTOMA-RELATED repressor protein are principal regulators coordinating cell proliferation with differentiation, but their role during seed development is little understood. We show that in fully developed Arabidopsis thaliana embryos, cell number was not affected either in single or double mutants for the activator-type E2FA and E2FB Accordingly, these E2Fs are only partially required for the expression of cell cycle genes. In contrast, the expression of key seed maturation genes LEAFY COTYLEDON 1/2 (LEC1/2), ABSCISIC ACID INSENSITIVE 3, FUSCA 3 and WRINKLED 1 is upregulated in the e2fab double mutant embryo. In accordance, E2FA directly regulates LEC2, and mutation at the consensus E2F-binding site in the LEC2 promoter de-represses its activity during the proliferative stage of seed development. In addition, the major seed storage reserve proteins, 12S globulin and 2S albumin, became prematurely accumulated at the proliferating phase of seed development in the e2fab double mutant. Our findings reveal a repressor function of the activator E2Fs to restrict the seed maturation programme until the cell proliferation phase is completed.

PP7L is essential for MAIL1-mediated transposable element silencing and primary root growth.

The two paralogous Arabidopsis genes MAINTENANCE OF MERISTEMS (MAIN) and MAINTENANCE OF MERISTEMS LIKE1 (MAIL1) encode a conserved retrotransposon-related plant mobile domain and are known to be required for silencing of transposable elements (TE) and for primary root development. Loss of function of either MAIN or MAIL1 leads to release of heterochromatic TEs, reduced condensation of pericentromeric heterochromatin, cell death of meristem cells and growth arrest of the primary root soon after germination. Here, we show that they act in one protein complex that also contains the inactive isoform of PROTEIN PHOSPHATASE 7 (PP7), which is named PROTEIN PHOSPHATASE 7-LIKE (PP7L). PP7L was previously shown to be important for chloroplast biogenesis and efficient chloroplast protein synthesis. We show that loss of PP7L function leads to the same root growth phenotype as loss of MAIL1 or MAIN. In addition, pp7l mutants show similar silencing defects. Double mutant analyses confirmed that the three proteins act in the same molecular pathway. The primary root growth arrest, which is associated with cell death of stem cells and their daughter cells, is a consequence of genome instability. Our data demonstrate so far unrecognized functions of an inactive phosphatase isoform in a protein complex that is essential for silencing of heterochromatic elements and for maintenance of genome stability in dividing cells.

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.

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.

γ-Tubulin interacts with E2F transcription factors to regulate proliferation and endocycling in Arabidopsis.

γ-Tubulin is associated with microtubule nucleation, but evidence is accumulating in eukaryotes that it also functions in nuclear processes and in cell division control independently of its canonical role. We found that in Arabidopsis thaliana, γ-tubulin interacts specifically with E2FA, E2FB, and E2FC transcription factors both in vitro and in vivo. The interaction of γ-tubulin with the E2Fs is not reduced in the presence of their dimerization partners (DPs) and, in agreement, we found that γ-tubulin interaction with E2Fs does not require the dimerization domain. γ-Tubulin associates with the promoters of E2F-regulated cell cycle genes in an E2F-dependent manner, probably in complex with the E2F-DP heterodimer. The up-regulation of E2F target genes PCNA, ORC2, CDKB1;1, and CCS52A under γ-tubulin silencing suggests a repressive function for γ-tubulin at G1/S and G2/M transitions, and the endocycle, which is consistent with an excess of cell division in some cells and enhanced endoreduplication in others in the shoot and young leaves of γ-tubulin RNAi plants. Altogether, our data show ternary interaction of γ-tubulin with the E2F-DP heterodimer and suggest a repressive role for γ-tubulin with E2Fs in controlling mitotic activity and endoreduplication during plant development.

Transcriptional repression by MYB3R proteins regulates plant organ growth.

In multicellular organisms, temporal and spatial regulation of cell proliferation is central for generating organs with defined sizes and morphologies. For establishing and maintaining the post-mitotic quiescent state during cell differentiation, it is important to repress genes with mitotic functions. We found that three of the Arabidopsis MYB3R transcription factors synergistically maintain G2/M-specific genes repressed in post-mitotic cells and restrict the time window of mitotic gene expression in proliferating cells. The combined mutants of the three repressor-type MYB3R genes displayed long roots, enlarged leaves, embryos, and seeds. Genome-wide chromatin immunoprecipitation revealed that MYB3R3 binds to the promoters of G2/M-specific genes and to E2F target genes. MYB3R3 associates with the repressor-type E2F, E2FC, and the RETINOBLASTOMA RELATED proteins. In contrast, the activator MYB3R4 was in complex with E2FB in proliferating cells. With mass spectrometry and pairwise interaction assays, we identified some of the other conserved components of the multiprotein complexes, known as DREAM/dREAM in human and flies. In plants, these repressor complexes are important for periodic expression during cell cycle and to establish a post-mitotic quiescent state determining organ size.

Cell cycle control by the target of rapamycin signalling pathway in plants.

Cells need to ensure a sufficient nutrient and energy supply before committing to proliferate. In response to positive mitogenic signals, such as light, sugar availability, and hormones, the target of rapamycin (TOR) signalling pathway promotes cell growth that connects to the entry and passage through the cell division cycle via multiple signalling mechanisms. Here, we summarize current understanding of cell cycle regulation by the RBR-E2F regulatory hub and the DREAM-like complexes, and highlight possible functional relationships between these regulators and TOR signalling. A genetic screen recently uncovered a downstream signalling component to TOR that regulates cell proliferation, YAK1, a member of the dual specificity tyrosine phosphorylation-regulated kinase (DYRK) family. YAK1 activates the plant-specific SIAMESE-RELATED (SMR) cyclin-dependent kinase inhibitors and therefore could be important to regulate both the CDKA-RBR-E2F pathway to control the G1/S transition and the mitotic CDKB1;1 to control the G2/M transition. TOR, as a master regulator of both protein synthesis-driven cell growth and cell proliferation is also central for cell size homeostasis. We conclude the review by briefly highlighting the potential applications of combining TOR and cell cycle knowledge in the context of ensuring future food security.

[Prevalence, potential risk factors and sequelae of diastasis recti abdominis]. / A rectus diastasis prevalenciája, lehetséges rizikófaktorai és szövodményei.

INTRODUCTION: There is scant knowledge on diastasis recti which occurs mostly in 3rd trimester of pregnancy. AIM: Our aim was to assign the prevalence of diastasis recti and the possible risk factors and to investigate its association with some chronical diseases, like low back pain and urinary incontinence. METHOD: 200 women's interrectus distance was measured who filled out a self-made diastasis recti questionnaire, the SF-36, Oswestry Disability Index and the International Consultation on Incontinence Modular Questionnaire - Urinary Incontinence Short Form questionnaires. RESULTS: Prevalence of the condition was 46.5%. In case of risk factors, relationship between number of deliveries and interrectus distance was significant. We found a significant difference in quality of life, in presence of low back pain and urinary incontinence between the normal and the abnormal group. CONCLUSIONS: In line with the literature we found, that diastasis recti can predispose on serious sequelae, hence on decreased quality of life. Orv. Hetil., 2017, 158(12), 454-460.

Arabidopsis E2FA stimulates proliferation and endocycle separately through RBR-bound and RBR-free complexes.

Post-embryonic growth in plants depends on the continuous supply of undifferentiated cells within meristems. Proliferating cells maintain their competence for division by active repression of differentiation and the associated endocycle entry. We show by upregulation and downregulation of E2FA that it is required for maintaining proliferation, as well as for endocycle entry. While E2FB-RBR1 (retinoblastoma-related protein 1) complexes are reduced after sucrose addition or at elevated CYCD3;1 levels, E2FA maintains a stable complex with RBR1 in proliferating cells. Chromatin immunoprecipitation shows that RBR1 binds in the proximity of E2F promoter elements in CCS52A1 and CSS52A2 genes, central regulators for the switch from proliferation to endocycles. Overexpression of a truncated E2FA mutant (E2FA(ΔRB)) lacking the RBR1-binding domain interferes with RBR1 recruitment to promoters through E2FA, leading to decreased meristem size in roots, premature cell expansion and hyperactivated endocycle in leaves. E2F target genes, including CCS52A1 and CCS52A2, are upregulated in E2FA(ΔRB) and e2fa knockout lines. These data suggest that E2FA in complex with RBR1 forms a repressor complex in proliferating cells to inhibit premature differentiation and endocycle entry. Thus, E2FA regulates organ growth via two distinct, sequentially operating pathways.

Antioxidant effect of the active metabolites of tibolone.

Certain steroidal compounds have an antioxidant effect in humans. Our aim was to test whether the synthetic steroid tibolone and its metabolites are also able to display such a property. For this, granulocytes from healthy men and women were incubated for two hours with different concentrations (10(-7), 10(-8), 10(-9 )M) of either estradiol, tibolone, 3α-hydroxytibolone, 3ß-hydroxytibolone, Δ(4)-tibolone, 3α-sulfated-tibolone, 3α-17ß-disulfated-tibolone, 3ß-sulfated-tibolone or 3ß-17ß-disulfated-tibolone. Superoxide anion generation of neutrophils was measured by photometry. Results of different steroids were given as percentages of their controls. A more simple superoxide generating system, the xanthine-xanthine oxidase reaction was also tested. We found that granulocyte superoxide production did not differ from the control using 10(-9 )M of steroids. Using 10(-8 )M concentration: estradiol (80.9 ± 2.5%); 3ß-sulfated-tibolone (83.3 ± 4.7%); 3ß-17ß-disulfated-tibolone (81.0 ± 4.2%) caused a significant decrease in superoxide production, compared to the control. In addition at 10(-7 )M, 3ß-hydroxytibolone and 3α-sulfated-tibolone also showed antioxidant effects. In the xanthine-xanthine oxidase system estradiol (67.4 ± 1.0%), 3α-sulfated-tibolone (85.8 ± 5.3%), 3α-17ß-disulfated-tibolone (71.9 ± 2.5%), 3ß-sulfated-tibolone (73.9 ± 5.0%), and 3ß-17ß-disulfated-tibolone (65.8 ± 3.4%) caused a significant decrease in superoxide production. Conclusively, although tibolone itself did not show significant antioxidant capacity, most of its active metabolites have antioxidant effects.

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.

Balancing act: matching growth with environment by the TOR signalling pathway.

One of the most fundamental aspects of growth in plants is its plasticity in relation to fluctuating environmental conditions. Growth of meristematic cells relies predominantly on protein synthesis, one of the most energy-consuming activities in cells, and thus is tightly regulated in accordance with the available nutrient and energy supplies. The Target of Rapamycin (TOR) signalling pathway takes a central position in this regulation. The core of the TOR signalling pathway is conserved throughout evolution, and can be traced back to the last eukaryotic common ancestor. In plants, a single complex constitutes the TOR signalling pathway. Manipulating the components of the TOR complex in Arabidopsis highlighted its common role as a major regulator of protein synthesis and metabolism, that is also involved in other biological functions such as cell-wall integrity, regulation of cell proliferation, and cell size. TOR, as an integral part of the auxin signalling pathway, connects hormonal and nutrient pathways. Downstream of TOR, S6 kinase and the ribosomal S6 protein have been shown to mediate several of these responses, although there is evidence of other complex non-linear TOR signalling pathway structures.

Auxin-dependent cell cycle reactivation through transcriptional regulation of Arabidopsis E2Fa by lateral organ boundary proteins.

Multicellular organisms depend on cell production, cell fate specification, and correct patterning to shape their adult body. In plants, auxin plays a prominent role in the timely coordination of these different cellular processes. A well-studied example is lateral root initiation, in which auxin triggers founder cell specification and cell cycle activation of xylem pole-positioned pericycle cells. Here, we report that the E2Fa transcription factor of Arabidopsis thaliana is an essential component that regulates the asymmetric cell division marking lateral root initiation. Moreover, we demonstrate that E2Fa expression is regulated by the LATERAL ORGAN BOUNDARY DOMAIN18/LATERAL ORGAN BOUNDARY DOMAIN33 (LBD18/LBD33) dimer that is, in turn, regulated by the auxin signaling pathway. LBD18/LBD33 mediates lateral root organogenesis through E2Fa transcriptional activation, whereas E2Fa expression under control of the LBD18 promoter eliminates the need for LBD18. Besides lateral root initiation, vascular patterning is disrupted in E2Fa knockout plants, similarly as it is affected in auxin signaling and lbd mutants, indicating that the transcriptional induction of E2Fa through LBDs represents a general mechanism for auxin-dependent cell cycle activation. Our data illustrate how a conserved mechanism driving cell cycle entry has been adapted evolutionarily to connect auxin signaling with control of processes determining plant architecture.

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.

Light-dependent regulation of DEL1 is determined by the antagonistic action of E2Fb and E2Fc.

Endoreduplication represents a variation on the cell cycle in which multiple rounds of DNA replication occur without subsequent chromosome separation and cytokinesis, thereby increasing the cellular DNA content. It is known that the DNA ploidy level of cells is controlled by external stimuli such as light; however, limited knowledge is available on how environmental signals regulate the endoreduplication cycle at the molecular level. Previously, we had demonstrated that the conversion from a mitotic cell cycle into an endoreduplication cycle is controlled by the atypical E2F transcription factor, DP-E2F-LIKE1 (DEL1), that represses the endocycle onset. Here, the Arabidopsis (Arabidopsis thaliana) DEL1 gene was identified as a transcriptional target of the classical E2Fb and E2Fc transcription factors that antagonistically control its transcript levels through competition for a single E2F cis-acting binding site. In accordance with the reported opposite effects of light on the protein levels of E2Fb and E2Fc, DEL1 transcription depended on the light regime. Strikingly, modified DEL1 expression levels uncoupled the link between light and endoreduplication in hypocotyls, implying that DEL1 acts as a regulatory connection between endocycle control and the photomorphogenic response.

The effect of certain steroid hormones on the expression of genes involved in the metabolism of free radicals.

Steroid hormones influence the antioxidant processes of cells. However, the molecular mechanism of this effect is not fully clear. Our aim was to examine how steroid hormones affect the expression of certain genes that play a role in antioxidant processes. Blood was taken from ten healthy volunteers. Neutrophil granulocytes were separated and treated either with 17-ß-estradiol, progesterone, testosterone, or cortisol. Whole RNA was isolated and reverse transcription was carried out in treated and control groups. Relative quantification was performed with SYBR Green assay and gene-specific oligonucleotides. We found that the expression of Mn-superoxide dismutase was significantly increased by 17-ß-estradiol and testosterone, myeloperoxidase expression was significantly elevated by cortisol and progesterone, and the expression of NADPH oxidase was significantly decreased by progesterone. We conclude that the antioxidant effect of steroid hormones is in part carried out through transcriptional regulation of certain enzymes. Subsequent studies are required in order to examine the non-genomic, membrane receptor mediated effect of steroids on antioxidant processes.

Proteome-wide landscape of solubility limits in a bacterial cell.

Proteins are prone to aggregate when expressed above their solubility limits. Aggregation may occur rapidly, potentially as early as proteins emerge from the ribosome, or slowly, following synthesis. However, in vivo data on aggregation rates are scarce. Here, we classified the Escherichia coli proteome into rapidly and slowly aggregating proteins using an in vivo image-based screen coupled with machine learning. We find that the majority (70%) of cytosolic proteins that become insoluble upon overexpression have relatively low rates of aggregation and are unlikely to aggregate co-translationally. Remarkably, such proteins exhibit higher folding rates compared to rapidly aggregating proteins, potentially implying that they aggregate after reaching their folded states. Furthermore, we find that a substantial fraction (~ 35%) of the proteome remain soluble at concentrations much higher than those found naturally, indicating a large margin of safety to tolerate gene expression changes. We show that high disorder content and low surface stickiness are major determinants of high solubility and are favored in abundant bacterial proteins. Overall, our study provides a global view of aggregation rates and hence solubility limits of proteins in a bacterial cell.