Publications by authors named "Laszlo Bakó"

27 Publications

  • Page 1 of 1

Conservation analysis of core cell cycle regulators and their transcriptional behavior during limb regeneration in Ambystoma mexicanum.

Mech Dev 2020 12 28;164:103651. Epub 2020 Oct 28.

Molecular and Developmental Complexity Group, Unidad de Genómica Avanzada (U.G.A.-LANGEBIO) CINVESTAV, Irapuato, Mexico. Electronic address:

Ambystoma mexicanum (axolotl) has been one of the major experimental models for the study of regeneration during the past 100 years. Axolotl limb regeneration takes place through a multi-stage and complex developmental process called epimorphosis that involves diverse events of cell reprogramming. Such events start with dedifferentiation of somatic cells and the proliferation of quiescent stem cells to generate a population of proliferative cells called blastema. Once the blastema reaches a mature stage, cells undergo progressive differentiation into the diverse cell lineages that will form the new limb. Such pivotal cell reprogramming phenomena depend on the fine-tuned regulation of the cell cycle in each regeneration stage, where cell populations display specific proliferative capacities and differentiation status. The axolotl genome has been fully sequenced and released recently, and diverse RNA-seq approaches have also been generated, enabling the identification and conservatory analysis of core cell cycle regulators in this species. We report here our results from such analyses and present the transcriptional behavior of key regulatory factors during axolotl limb regeneration. We also found conserved protein interactions between axolotl Cyclin Dependent Kinases 2, 4 and 6 and Cyclins type D and E. Canonical CYC-CDK interactions that play major roles in modulating cell cycle progression in eukaryotes.
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http://dx.doi.org/10.1016/j.mod.2020.103651DOI Listing
December 2020

The chromatin-modifying protein HUB2 is involved in the regulation of lignin composition in xylem vessels.

J Exp Bot 2020 Sep;71(18):5484-5494

Umeå Plant Science Centre, Department of Plant Physiology, Umeå University, Umeå, Sweden.

PIRIN2 (PRN2) was earlier reported to suppress syringyl (S)-type lignin accumulation of xylem vessels of Arabidopsis thaliana. In the present study, we report yeast two-hybrid results supporting the interaction of PRN2 with HISTONE MONOUBIQUITINATION2 (HUB2) in Arabidopsis. HUB2 has been previously implicated in several plant developmental processes, but not in lignification. Interaction between PRN2 and HUB2 was verified by β-galactosidase enzymatic and co-immunoprecipitation assays. HUB2 promoted the deposition of S-type lignin in the secondary cell walls of both stem and hypocotyl tissues, as analysed by pyrolysis-GC/MS. Chemical fingerprinting of individual xylem vessel cell walls by Raman and Fourier transform infrared microspectroscopy supported the function of HUB2 in lignin deposition. These results, together with a genetic analysis of the hub2 prn2 double mutant, support the antagonistic function of PRN2 and HUB2 in deposition of S-type lignin. Transcriptome analyses indicated the opposite regulation of the S-type lignin biosynthetic gene FERULATE-5-HYDROXYLASE1 by PRN2 and HUB2 as the underlying mechanism. PRN2 and HUB2 promoter activities co-localized in cells neighbouring the xylem vessel elements, suggesting that the S-type lignin-promoting function of HUB2 is antagonized by PRN2 for the benefit of the guaiacyl (G)-type lignin enrichment of the neighbouring xylem vessel elements.
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http://dx.doi.org/10.1093/jxb/eraa264DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7501814PMC
September 2020

A Molecular Framework for the Control of Adventitious Rooting by TIR1/AFB2-Aux/IAA-Dependent Auxin Signaling in Arabidopsis.

Mol Plant 2019 11 11;12(11):1499-1514. Epub 2019 Sep 11.

Umeå Plant Science Centre, Department of Plant Physiology, Umeå University, 90736 Umeå, Sweden; Institut Jean-Pierre Bourgin, INRA, AgroParisTech, CNRS, Université Paris-Saclay, 78000 Versailles, France. Electronic address:

In Arabidopsis thaliana, canonical auxin-dependent gene regulation is mediated by 23 transcription factors from the AUXIN RESPONSE FACTOR (ARF) family that interact with auxin/indole acetic acid repressors (Aux/IAAs), which themselves form co-receptor complexes with one of six TRANSPORT INHIBITOR1/AUXIN-SIGNALLING F-BOX (TIR1/AFB) proteins. Different combinations of co-receptors drive specific sensing outputs, allowing auxin to control a myriad of processes. ARF6 and ARF8 are positive regulators of adventitious root initiation upstream of jasmonate, but the exact auxin co-receptor complexes controlling the transcriptional activity of these proteins has remained unknown. Here, using loss-of-function mutants we show that three Aux/IAA genes, IAA6, IAA9, and IAA17, act additively in the control of adventitious root (AR) initiation. These three IAA proteins interact with ARF6 and/or ARF8 and likely repress their activity in AR development. We show that TIR1 and AFB2 are positive regulators of AR formation and TIR1 plays a dual role in the control of jasmonic acid (JA) biosynthesis and conjugation, as several JA biosynthesis genes are up-regulated in the tir1-1 mutant. These results lead us to propose that in the presence of auxin, TIR1 and AFB2 form specific sensing complexes with IAA6, IAA9, and/or IAA17 to modulate JA homeostasis and control AR initiation.
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http://dx.doi.org/10.1016/j.molp.2019.09.001DOI Listing
November 2019

LEAFY activity is post-transcriptionally regulated by BLADE ON PETIOLE2 and CULLIN3 in Arabidopsis.

New Phytol 2018 10 11;220(2):579-592. Epub 2018 Jul 11.

Univ. Grenoble Alpes, CNRS, CEA, INRA, BIG-LPCV, 38054, Grenoble Cedex 9, France.

The Arabidopsis LEAFY (LFY) transcription factor is a key regulator of floral meristem emergence and identity. LFY interacts genetically and physically with UNUSUAL FLORAL ORGANS, a substrate adaptor of CULLIN1-RING ubiquitin ligase complexes (CRL1). The functionally redundant genes BLADE ON PETIOLE1 (BOP1) and -2 (BOP2) are potential candidates to regulate LFY activity and have recently been shown to be substrate adaptors of CULLIN3 (CUL3)-RING ubiquitin ligases (CRL3). We tested the hypothesis that LFY activity is controlled by BOPs and CUL3s in plants and that LFY is a substrate for ubiquitination by BOP-containing CRL3 complexes. When constitutively expressed, LFY activity is fully dependent on BOP2 as well as on CUL3A and B to regulate target genes such as APETALA1 and to induce ectopic flower formation. We also show that LFY and BOP2 proteins interact physically and that LFY-dependent ubiquitinated species are produced in vitro in a reconstituted cell-free CRL3 system in the presence of LFY, BOP2 and CUL3. This new post-translational regulation of LFY activity by CRL3 complexes makes it a unique transcription factor subjected to a positive dual regulation by both CRL1 and CRL3 complexes and suggests a novel mechanism for promoting flower development.
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http://dx.doi.org/10.1111/nph.15329DOI Listing
October 2018

Transcriptional landscapes of Axolotl (Ambystoma mexicanum).

Dev Biol 2018 01 25;433(2):227-239. Epub 2017 Dec 25.

Molecular and Developmental Complexity Group, Unidad de Genómica Avanzada-Laboratorio Nacional de Genómica para la Biodiversidad (LANGEBIO), CINVESTAV, Irapuato, Guanajuato, Mexico. Electronic address:

The axolotl (Ambystoma mexicanum) is the vertebrate model system with the highest regeneration capacity. Experimental tools established over the past 100 years have been fundamental to start unraveling the cellular and molecular basis of tissue and limb regeneration. In the absence of a reference genome for the Axolotl, transcriptomic analysis become fundamental to understand the genetic basis of regeneration. Here we present one of the most diverse transcriptomic data sets for Axolotl by profiling coding and non-coding RNAs from diverse tissues. We reconstructed a population of 115,906 putative protein coding mRNAs as full ORFs (including isoforms). We also identified 352 conserved miRNAs and 297 novel putative mature miRNAs. Systematic enrichment analysis of gene expression allowed us to identify tissue-specific protein-coding transcripts. We also found putative novel and conserved microRNAs which potentially target mRNAs which are reported as important disease candidates in heart and liver.
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http://dx.doi.org/10.1016/j.ydbio.2017.08.022DOI Listing
January 2018

BLADE-ON-PETIOLE proteins act in an E3 ubiquitin ligase complex to regulate PHYTOCHROME INTERACTING FACTOR 4 abundance.

Elife 2017 08 22;6. Epub 2017 Aug 22.

Umeå Plant Science Centre, Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, Umeå, Sweden.

Both light and temperature have dramatic effects on plant development. Phytochrome photoreceptors regulate plant responses to the environment in large part by controlling the abundance of PHYTOCHROME INTERACTING FACTOR (PIF) transcription factors. However, the molecular determinants of this essential signaling mechanism still remain largely unknown. Here, we present evidence that the () genes, which have previously been shown to control leaf and flower development in Arabidopsis, are involved in controlling the abundance of PIF4. Genetic analysis shows that promotes photo-morphogenesis and modulates thermomorphogenesis by suppressing activity, through a reduction in PIF4 protein level. In red-light-grown seedlings PIF4 ubiquitination was reduced in the mutant. Moreover, we found that BOP proteins physically interact with both PIF4 and CULLIN3A and that a CULLIN3-BOP2 complex ubiquitinates PIF4 in vitro. This shows that BOP proteins act as substrate adaptors in a CUL3 E3 ubiquitin ligase complex, targeting PIF4 proteins for ubiquitination and subsequent degradation.
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http://dx.doi.org/10.7554/eLife.26759DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5582868PMC
August 2017

XYLEM NAC DOMAIN1, an angiosperm NAC transcription factor, inhibits xylem differentiation through conserved motifs that interact with RETINOBLASTOMA-RELATED.

New Phytol 2017 Oct 25;216(1):76-89. Epub 2017 Jul 25.

Department of Horticulture, Virginia Tech, Blacksburg, VA, 24061, USA.

The Arabidopsis thaliana gene XYLEM NAC DOMAIN1 (XND1) is upregulated in xylem tracheary elements. Yet overexpression of XND1 blocks differentiation of tracheary elements. The molecular mechanism of XND1 action was investigated. Phylogenetic and motif analyses indicated that XND1 and its homologs are present only in angiosperms and possess a highly conserved C-terminal region containing linear motifs (CKII-acidic, LXCXE, E2F -like and LXCXE-mimic) predicted to interact with the cell cycle and differentiation regulator RETINOBLASTOMA-RELATED (RBR). Protein-protein interaction and functional analyses of XND1 deletion mutants were used to test the importance of RBR-interaction motifs. Deletion of either the LXCXE or the LXCXE-mimic motif reduced both the XND1-RBR interaction and XND1 efficacy as a repressor of differentiation, with loss of the LXCXE motif having the strongest negative impacts. The function of the XND1 C-terminal domain could be partially replaced by RBR fused to the N-terminal domain of XND1. XND1 also transactivated gene expression in yeast and plants. The properties of XND1, a transactivator that depends on multiple linear RBR-interaction motifs to inhibit differentiation, have not previously been described for a plant protein. XND1 harbors an apparently angiosperm-specific combination of interaction motifs potentially linking the general differentiation regulator RBR with a xylem-specific pathway for inhibition of differentiation.
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http://dx.doi.org/10.1111/nph.14704DOI Listing
October 2017

The Role of microRNAs in Animal Cell Reprogramming.

Stem Cells Dev 2016 07 27;25(14):1035-49. Epub 2016 Jun 27.

1 Molecular and Developmental Complexity Group, Unidad de Genómica Avanzada (U.G.A.-LANGEBIO) CINVESTAV , Irapuato, México.

Our concept of cell reprogramming and cell plasticity has evolved since John Gurdon transferred the nucleus of a completely differentiated cell into an enucleated Xenopus laevis egg, thereby generating embryos that developed into tadpoles. More recently, induced expression of transcription factors, oct4, sox2, klf4, and c-myc has evidenced the plasticity of the genome to change the expression program and cell phenotype by driving differentiated cells to the pluripotent state. Beyond these milestone achievements, research in artificial cell reprogramming has been focused on other molecules that are different than transcription factors. Among the candidate molecules, microRNAs (miRNAs) stand out due to their potential to control the levels of proteins that are involved in cellular processes such as self-renewal, proliferation, and differentiation. Here, we review the role of miRNAs in the maintenance and differentiation of mesenchymal stem cells, epimorphic regeneration, and somatic cell reprogramming to induced pluripotent stem cells.
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http://dx.doi.org/10.1089/scd.2015.0359DOI Listing
July 2016

ABCG9, ABCG11 and ABCG14 ABC transporters are required for vascular development in Arabidopsis.

Plant J 2013 Dec;76(5):811-24

Umeå Plant Science Centre, Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, S-90187, Umeå, Sweden; Umeå Plant Science Centre, Department of Plant Physiology, Umeå University, S-90183, Umeå, Sweden; UMR 1318, AgroParisTech, Institut Jean-Pierre Bourgin, Institut National de la Recherche Agronomique Centre de Versailles, RD10, 78026, Versailles Cedex, France.

In order to obtain insights into the regulatory pathways controlling phloem development, we characterized three genes encoding membrane proteins from the G sub-family of ABC transporters (ABCG9, ABCG11 and ABCG14), whose expression in the phloem has been confirmed. Mutations in the genes encoding these dimerizing 'half transporters' are semi-dominant and result in vascular patterning defects in cotyledons and the floral stem. Co-immunoprecipitation and bimolecular fluorescence complementation experiments demonstrated that these proteins dimerize, either by flexible pairing (ABCG11 and ABCG9) or by forming strict heterodimers (ABCG14). In addition, metabolome analyses and measurement of sterol ester contents in the mutants suggested that ABCG9, ABCG11 and ABCG14 are involved in lipid/sterol homeostasis regulation. Our results show that these three ABCG genes are required for proper vascular development in Arabidopsis thaliana.
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http://dx.doi.org/10.1111/tpj.12334DOI Listing
December 2013

Use of the foot-and-mouth disease virus 2A peptide co-expression system to study intracellular protein trafficking in Arabidopsis.

PLoS One 2012 14;7(12):e51973. Epub 2012 Dec 14.

Umeå Plant Science Centre, Department of Plant Physiology, Umeå University, Umeå, Sweden.

Background: A tool for stoichiometric co-expression of effector and target proteins to study intracellular protein trafficking processes has been provided by the so called 2A peptide technology. In this system, the 16-20 amino acid 2A peptide from RNA viruses allows synthesis of multiple gene products from single transcripts. However, so far the use of the 2A technology in plant systems has been limited.

Methodology/principal Findings: The aim of this work was to assess the suitability of the 2A peptide technology to study the effects exerted by dominant mutant forms of three small GTPase proteins, RABD2a, SAR1, and ARF1 on intracellular protein trafficking in plant cells. Special emphasis was given to CAH1 protein from Arabidopsis, which is trafficking to the chloroplast via a poorly characterized endoplasmic reticulum-to-Golgi pathway. Dominant negative mutants for these GTPases were co-expressed with fluorescent marker proteins as polyproteins separated by a 20 residue self-cleaving 2A peptide. Cleavage efficiency analysis of the generated polyproteins showed that functionality of the 2A peptide was influenced by several factors. This enabled us to design constructs with greatly increased cleavage efficiency compared to previous studies. The dominant negative GTPase variants resulting from cleavage of these 2A peptide constructs were found to be stable and active, and were successfully used to study the inhibitory effect on trafficking of the N-glycosylated CAH1 protein through the endomembrane system.

Conclusions/significance: We demonstrate that the 2A peptide is a suitable tool when studying plant intracellular protein trafficking and that transient protoplast and in planta expression of mutant forms of SAR1 and RABD2a disrupts CAH1 trafficking. Similarly, expression of dominant ARF1 mutants also caused inhibition of CAH1 trafficking to a different extent. These results indicate that early trafficking of the plastid glycoprotein CAH1 depends on canonical vesicular transport mechanisms operating between the endoplasmic reticulum and Golgi apparatus.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0051973PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3522588PMC
September 2013

A bistable circuit involving SCARECROW-RETINOBLASTOMA integrates cues to inform asymmetric stem cell division.

Cell 2012 Aug 23;150(5):1002-15. Epub 2012 Aug 23.

Molecular Genetics, Department of Biology, University of Utrecht, 3584 CH Utrecht, The Netherlands.

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.
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http://dx.doi.org/10.1016/j.cell.2012.07.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3500399PMC
August 2012

The CRYPTOCHROME1-dependent response to excess light is mediated through the transcriptional activators ZINC FINGER PROTEIN EXPRESSED IN INFLORESCENCE MERISTEM LIKE1 and ZML2 in Arabidopsis.

Plant Cell 2012 Jul 10;24(7):3009-25. Epub 2012 Jul 10.

Umeå Plant Science Centre, Department of Plant Physiology, Umeå University, S-901 87 Umea, Sweden.

Exposure of plants to light intensities that exceed the electron utilization capacity of the chloroplast has a dramatic impact on nuclear gene expression. The photoreceptor Cryptochrome 1 (cry1) is essential to the induction of genes encoding photoprotective components in Arabidopsis thaliana. Bioinformatic analysis of the cry1 regulon revealed the putative cis-element CryR1 (GnTCKAG), and here we demonstrate an interaction between CryR1 and the zinc finger GATA-type transcription factors ZINC FINGER PROTEIN EXPRESSED IN INFLORESCENCE MERISTEM LIKE1 (ZML1) and ZML2. The ZML proteins specifically bind to the CryR1 cis-element as demonstrated in vitro and in vivo, and TCTAG was shown to constitute the core sequence required for ZML2 binding. In addition, ZML2 activated transcription of the yellow fluorescent protein reporter gene driven by the CryR1 cis-element in Arabidopsis leaf protoplasts. T-DNA insertion lines for ZML2 and its homolog ZML1 demonstrated misregulation of several cry1-dependent genes in response to excess light. Furthermore, the zml1 and zml2 T-DNA insertion lines displayed a high irradiance-sensitive phenotype with significant photoinactivation of photosystem II (PSII), indicated by reduced maximum quantum efficiency of PSII, and severe photobleaching. Thus, we identified the ZML2 and ZML1 GATA transcription factors as two essential components of the cry1-mediated photoprotective response.
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http://dx.doi.org/10.1105/tpc.112.100099DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3426129PMC
July 2012

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

EMBO J 2012 Mar 3;31(6):1480-93. Epub 2012 Feb 3.

Royal Holloway, University of London, School of Biological Sciences, Centre for Systems and Synthetic Biology, Egham, UK.

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.
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http://dx.doi.org/10.1038/emboj.2012.13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3321179PMC
March 2012

Short day-mediated cessation of growth requires the downregulation of AINTEGUMENTALIKE1 transcription factor in hybrid aspen.

PLoS Genet 2011 Nov 3;7(11):e1002361. Epub 2011 Nov 3.

Umeå Plant Science Centre, Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, Umeå, Sweden.

Day length is a key environmental cue regulating the timing of major developmental transitions in plants. For example, in perennial plants such as the long-lived trees of the boreal forest, exposure to short days (SD) leads to the termination of meristem activity and bud set (referred to as growth cessation). The mechanism underlying SD-mediated induction of growth cessation is poorly understood. Here we show that the AIL1-AIL4 (AINTEGUMENTALIKE) transcription factors of the AP2 family are the downstream targets of the SD signal in the regulation of growth cessation response in hybrid aspen trees. AIL1 is expressed in the shoot apical meristem and leaf primordia, and exposure to SD signal downregulates AIL1 expression. Downregulation of AIL gene expression by SDs is altered in transgenic hybrid aspen plants that are defective in SD perception and/or response, e.g. PHYA or FT overexpressors. Importantly, SD-mediated regulation of growth cessation response is also affected by overexpression or downregulation of AIL gene expression. AIL1 protein can interact with the promoter of the key cell cycle genes, e.g. CYCD3.2, and downregulation of the expression of D-type cyclins after SD treatment is prevented by AIL1 overexpression. These data reveal that execution of SD-mediated growth cessation response requires the downregulation of AIL gene expression. Thus, while early acting components like PHYA and the CO/FT regulon are conserved in day-length regulation of flowering time and growth cessation between annual and perennial plants, signaling pathways downstream of SD perception diverge, with AIL transcription factors being novel targets of the CO/FT regulon connecting the perception of SD signal to the regulation of meristem activity.
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http://dx.doi.org/10.1371/journal.pgen.1002361DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3207903PMC
November 2011

Importance of post-translational modifications for functionality of a chloroplast-localized carbonic anhydrase (CAH1) in Arabidopsis thaliana.

PLoS One 2011 10;6(6):e21021. Epub 2011 Jun 10.

Department of Plant Physiology, Umeå Plant Science Centre, Umeå University, Umeå, Sweden.

Background: The Arabidopsis CAH1 alpha-type carbonic anhydrase is one of the few plant proteins known to be targeted to the chloroplast through the secretory pathway. CAH1 is post-translationally modified at several residues by the attachment of N-glycans, resulting in a mature protein harbouring complex-type glycans. The reason of why trafficking through this non-canonical pathway is beneficial for certain chloroplast resident proteins is not yet known. Therefore, to elucidate the significance of glycosylation in trafficking and the effect of glycosylation on the stability and function of the protein, epitope-labelled wild type and mutated versions of CAH1 were expressed in plant cells.

Methodology/principal Findings: Transient expression of mutant CAH1 with disrupted glycosylation sites showed that the protein harbours four, or in certain cases five, N-glycans. While the wild type protein trafficked through the secretory pathway to the chloroplast, the non-glycosylated protein formed aggregates and associated with the ER chaperone BiP, indicating that glycosylation of CAH1 facilitates folding and ER-export. Using cysteine mutants we also assessed the role of disulphide bridge formation in the folding and stability of CAH1. We found that a disulphide bridge between cysteines at positions 27 and 191 in the mature protein was required for correct folding of the protein. Using a mass spectrometric approach we were able to measure the enzymatic activity of CAH1 protein. Under circumstances where protein N-glycosylation is blocked in vivo, the activity of CAH1 is completely inhibited.

Conclusions/significance: We show for the first time the importance of post-translational modifications such as N-glycosylation and intramolecular disulphide bridge formation in folding and trafficking of a protein from the secretory pathway to the chloroplast in higher plants. Requirements for these post-translational modifications for a fully functional native protein explain the need for an alternative route to the chloroplast.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0021021PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3112209PMC
October 2011

Partners in time: EARLY BIRD associates with ZEITLUPE and regulates the speed of the Arabidopsis clock.

Plant Physiol 2011 Apr 7;155(4):2108-22. Epub 2011 Feb 7.

Department of Plant Physiology, Umea Plant Science Centre, Umea University, SE-901 87 Umea, Sweden.

The circadian clock of the model plant Arabidopsis (Arabidopsis thaliana) is made up of a complex series of interacting feedback loops whereby proteins regulate their own expression across day and night. early bird (ebi) is a circadian mutation that causes the clock to speed up: ebi plants have short circadian periods, early phase of clock gene expression, and are early flowering. We show that EBI associates with ZEITLUPE (ZTL), known to act in the plant clock as a posttranslational mediator of protein degradation. However, EBI is not degraded by its interaction with ZTL. Instead, ZTL counteracts the effect of EBI during the day and increases it at night, modulating the expression of key circadian components. The partnership of EBI with ZTL reveals a novel mechanism involved in controlling the complex transcription-translation feedback loops of the clock. This work highlights the importance of cross talk between the ubiquitination pathway and transcriptional control for regulation of the plant clock.
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http://dx.doi.org/10.1104/pp.110.167155DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3091123PMC
April 2011

Activity-dormancy transition in the cambial meristem involves stage-specific modulation of auxin response in hybrid aspen.

Proc Natl Acad Sci U S A 2011 Feb 2;108(8):3418-23. Epub 2011 Feb 2.

Department of Forest Genetics and Plant Physiology, Umea Plant Science Centre, Swedish University of Agricultural Sciences, SE-90183 Umea, Sweden.

The molecular basis of short-day-induced growth cessation and dormancy in the meristems of perennial plants (e.g., forest trees growing in temperate and high-latitude regions) is poorly understood. Using global transcript profiling, we show distinct stage-specific alterations in auxin responsiveness of the transcriptome in the stem tissues during short-day-induced growth cessation and both the transition to and establishment of dormancy in the cambial meristem of hybrid aspen trees. This stage-specific modulation of auxin signaling appears to be controlled via distinct mechanisms. Whereas the induction of growth cessation in the cambium could involve induction of repressor auxin response factors (ARFs) and down-regulation of activator ARFs, dormancy is associated with perturbation of the activity of the SKP-Cullin-F-box(TIR) (SCF(TIR)) complex, leading to potential stabilization of repressor auxin (AUX)/indole-3-acetic acid (IAA) proteins. Although the role of hormones, such as abscisic acid (ABA) and gibberellic acid (GA), in growth cessation and dormancy is well established, our data now implicate auxin in this process. Importantly, in contrast to most developmental processes in which regulation by auxin involves changes in cellular auxin contents, day-length-regulated induction of cambial growth cessation and dormancy involves changes in auxin responses rather than auxin content.
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http://dx.doi.org/10.1073/pnas.1011506108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3044397PMC
February 2011

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

J Exp Bot 2011 Mar 31;62(6):2155-68. Epub 2010 Dec 31.

Institute of Plant Biology, Biological Research Center, Hungarian Academy of Sciences, Temesvári krt. 62, H-6726 Szeged, Hungary.

Plant retinoblastoma-related (RBR) proteins are primarily considered as key regulators of G(1)/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 G(2)/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 G(1) cells. Its amount increased upon entry into the S phase and remained high during the G(2)/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 G(2)/M phase regulation in plant cells is discussed.
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http://dx.doi.org/10.1093/jxb/erq413DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3060694PMC
March 2011

Real-time classification of datasets with hardware embedded neuromorphic neural networks.

Authors:
Laszlo Bako

Brief Bioinform 2010 May 6;11(3):348-63. Epub 2010 Jan 6.

Petru MaiorUniversity, Tirgu-Mures, Romania.

Neuromorphic artificial neural networks attempt to understand the essential computations that take place in the dense networks of interconnected neurons making up the central nervous systems in living creatures. This article demonstrates that artificial spiking neural networks--built to resemble the biological model--encoding information in the timing of single spikes, are capable of computing and learning clusters from realistic data. It shows how a spiking neural network based on spike-time coding can successfully perform unsupervised and supervised clustering on real-world data. A temporal encoding procedure of continuously valued data is developed, together with a hardware implementation oriented new learning rule set. Solutions that make use of embedded soft-core microcontrollers are investigated, to implement some of the most resource-consuming components of the artificial neural network. Details of the implementations are given, with benchmark application evaluation and test bench description. Measurement results are presented, showing real-time and adaptive data processing capabilities, comparing these to related findings in the specific literature.
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http://dx.doi.org/10.1093/bib/bbp066DOI Listing
May 2010

EBP1 regulates organ size through cell growth and proliferation in plants.

EMBO J 2006 Oct 5;25(20):4909-20. Epub 2006 Oct 5.

Laboratory of Plant Breeding, Department of Plant Sciences, Graduate School of Experimental Plant Sciences, Wageningen University and Research Centre, Wageningen, The Netherlands.

Plant organ size shows remarkable uniformity within species indicating strong endogenous control. We have identified a plant growth regulatory gene, functionally and structurally homologous to human EBP1. Plant EBP1 levels are tightly regulated; gene expression is highest in developing organs and correlates with genes involved in ribosome biogenesis and function. EBP1 protein is stabilised by auxin. Elevating or decreasing EBP1 levels in transgenic plants results in a dose-dependent increase or reduction in organ growth, respectively. During early stages of organ development, EBP1 promotes cell proliferation, influences cell-size threshold for division and shortens the period of meristematic activity. In postmitotic cells, it enhances cell expansion. EBP1 is required for expression of cell cycle genes; CyclinD3;1, ribonucleotide reductase 2 and the cyclin-dependent kinase B1;1. The regulation of these genes by EBP1 is dose and auxin dependent and might rely on the effect of EBP1 to reduce RBR1 protein level. We argue that EBP1 is a conserved, dose-dependent regulator of cell growth that is connected to meristematic competence and cell proliferation via regulation of RBR1 level.
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http://dx.doi.org/10.1038/sj.emboj.7601362DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1618091PMC
October 2006

Activation of an alfalfa cyclin-dependent kinase inhibitor by calmodulin-like domain protein kinase.

Plant J 2006 Apr;46(1):111-23

Institute of Plant Biology, Biological Research Centre, Hungarian Academy of Sciences, Szeged, H-6726, Temesvári krt. 62, Hungary.

Kip-related proteins (KRPs) play a central role in the regulation of the cell cycle and differentiation through modulation of cyclin-dependent kinase (CDK) functions. We have identified a CDK inhibitor gene from Medicago truncatula (Mt) by a yeast two-hybrid screen. The KRPMt gene was expressed in all plant organs and cultured cells, and its transcripts accumulated after abscisic acid and NaCl treatment. The KRPMt protein exhibits seven conserved sequence domains and a PEST motif that is also detected in various Arabidopsis KRPs. In the yeast two-hybrid test, the KRPMt protein interacted with CDK (Medsa;CDKA;1) and D-type cyclins. However, in the pull-down assays, B-type CDK complexes were also detectable. Recombinant KRPMt differentially inhibited various alfalfa CDK complexes in phosphorylation assays. The immunoprecipitated Medsa;CDKA;1/A;2 complex was strongly inhibited, whereas the mitotic Medsa;CDKB2;1 complex was the most sensitive to inhibition. Function of Medsa;CDKB1;1 complex was not inhibited by the KRPMt protein. The mitotic Medsa;CYCB2 and Medsa;CYCA2;1 complexes responded weakly to this inhibitor protein. Kinase complexes from G2/M cells showed increased sensitivity towards the inhibitor compared with those isolated from G1/S-phase cells. In vitro phosphorylation of Medicago retinoblastoma-related protein was also reduced in the presence of KRPMt. Phosphorylation of this inhibitor protein by the recombinant calmodulin-like domain protein kinase (MsCPK3) resulted in enhanced inhibition of CDK function. The data presented emphasize the selective sensitivity of various cyclin-dependent kinase complexes to this inhibitor protein, and suggest a role for CDK inhibitors and CPKs in cross-talk between Ca2+ signalling and regulation of cell-cycle progression in plants.
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http://dx.doi.org/10.1111/j.1365-313X.2006.02677.xDOI Listing
April 2006

Evidence for a protein transported through the secretory pathway en route to the higher plant chloroplast.

Nat Cell Biol 2005 Dec 13;7(12):1224-31. Epub 2005 Nov 13.

Umeå Plant Science Centre, Department of Plant Physiology, Umeå University, S-90187 Umeå, Sweden.

In contrast to animal and fungal cells, green plant cells contain one or multiple chloroplasts, the organelle(s) in which photosynthetic reactions take place. Chloroplasts are believed to have originated from an endosymbiotic event and contain DNA that codes for some of their proteins. Most chloroplast proteins are encoded by the nuclear genome and imported with the help of sorting signals that are intrinsic parts of the polypeptides. Here, we show that a chloroplast-located protein in higher plants takes an alternative route through the secretory pathway, and becomes N-glycosylated before entering the chloroplast.
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http://dx.doi.org/10.1038/ncb1330DOI Listing
December 2005

The role of the Arabidopsis E2FB transcription factor in regulating auxin-dependent cell division.

Plant Cell 2005 Sep 29;17(9):2527-41. Epub 2005 Jul 29.

Royal Holloway University of London, School of Biological Sciences, Egham TW20 0EX, United Kingdom.

The molecular mechanisms by which the phytohormone auxin coordinates cell division with cell growth and differentiation are largely unknown. Here, we show that in Arabidopsis thaliana E2FB, accumulation and stability are positively regulated by auxin. Coexpression of E2FB, but not of E2FA, with its dimerization partner A, stimulated cell proliferation in the absence of auxin in tobacco (Nicotiana tabacum) Bright Yellow-2 cells. E2FB regulated the entry into both S- and M-phases, the latter corresponding to the activation of a plant-specific mitotic regulator, CDKB1;1. Increased E2FB levels led to shortened cell cycle duration, elevated cell numbers, and extremely small cell sizes. In the absence of auxin, cells elongated with concomitant increase in their ploidy level, but both were strongly inhibited by E2FB. We conclude that E2FB is one of the key targets for auxin to determine whether cells proliferate or whether they exit the cell cycle, enlarge, and endoreduplicate their DNA.
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http://dx.doi.org/10.1105/tpc.105.033761DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1197432PMC
September 2005

Arabidopsis anaphase-promoting complexes: multiple activators and wide range of substrates might keep APC perpetually busy.

Cell Cycle 2005 Aug 18;4(8):1084-92. Epub 2005 Aug 18.

Institut des Sciences du Végétal, Gif sur Yvette, France.

The anaphase-promoting complex (APC), a multisubunit E3 ubiquitin ligase, is an essential regulator of the cell cycle from metaphase until S phase in yeast and metazoans. APC mediates degradation of numerous cell cycle-related proteins, including mitotic cyclins and its activation and substrate-specificity are determined by two adaptor proteins, Cdc20 and Cdh1. Plants have multiple APC activators and the Cdh1-type proteins, in addition, are represented by two subclasses, known as Ccs52A and Ccs52B. The Arabidopsis genome contains five cdc20 genes as well as ccs52A1, ccs52A2 and ccs52B. In Schizosaccharomyces pombe, expression of the three Atccs52 genes elicited distinct phenotypes supporting nonredundant function of the AtCcs52 proteins. Consistent with these activities, the AtCcs52 proteins were able to bind both to the yeast and the Arabidopsis APCs. In synchronized Arabidopsis cell cultures the cdc20 transcripts were present from early G2 until the M-phase exit, ccs52B from G2/M to M while ccs52A1 and ccs52A2 were from late M until early G2, suggesting consecutive action of these APC activators in the plant cell cycle. The AtCcs52 proteins interacted with different subsets of mitotic cyclins, in accordance with their expression profiles, either in free- or CDK-bound forms. Expression of most APC subunits was constitutive, whereas cdc27a and cdc27b, corresponding to two forms of apc3, and ubc19 and ubc20 encoding E2-C type ubiquitin-conjugating enzymes displayed differences in their cell cycle regulation. These data indicate the existence of numerous APC(Cdc20/Ccs52/Cdc27) forms in Arabidopsis, which in conjunction with different E2 enzymes might have distinct or complementary functions at distinct stages of the cell cycle.
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August 2005

The Medicago CDKC;1-CYCLINT;1 kinase complex phosphorylates the carboxy-terminal domain of RNA polymerase II and promotes transcription.

Plant J 2005 Jun;42(6):810-20

Institute of Plant Biology, Biological Research Center of the Hungarian Academy of Sciences, H-6701 Szeged, Hungary.

The Ms;CDKC;1 kinase is structurally similar to those cyclin-dependent kinases (CDKs) that are not involved directly in cell cycle regulation. The presence of a PITAIRE motif in Ms;CDKC;1 suggests that it interacts with cyclins different from known PSTAIRE/PPTALRE kinase regulatory subunits. Here we demonstrate that a Medicago CYCLINT (CYCT) protein is a specific interactor of Ms;CDKC;1 and the interaction between these two proteins gives rise to an active kinase complex that localizes to the nucleus and phosphorylates the carboxy-terminal YSPTSPS heptapeptide repeat domain (CTD) of the largest subunit of RNA polymerase II in vitro. Mutation of Ser to Ala at position 5 within the heptapeptide repeat abolishes substrate phosphorylation by the Ms;CDKC;1 kinase complex. Furthermore, our data show that addition of the Medicago CDKC;1-CYCT;1 heterodimer completely restored the transcriptional activity of a HeLa nuclear extract depleted of endogeneous CDK9 kinase complexes. Together, these results indicate that the Medicago CDKC;1-CYCT;1 complex is a positive regulator of transcription in plants and has a role similar to the CDK9/cyclin T complex of human positive transcription elongation factor P-TEFb.
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http://dx.doi.org/10.1111/j.1365-313X.2005.02421.xDOI Listing
June 2005

Differential stage-specific regulation of cyclin-dependent kinases during cambial dormancy in hybrid aspen.

Plant J 2004 May;38(4):603-15

Department of Forest Genetics and Plant Physiology, Umea Plant Science Centre, Swedish University of Agricultural Sciences, 90183 Umea, Sweden.

The cambium of woody plants cycles between active and dormant states. Dormancy can be subdivided into eco- and endodormant stages. Ecodormant trees resume growth upon exposure to growth-promotive signals, while the establishment of endodormant state results in loss of the ability to respond to these signals. In this paper, we analysed the regulation of cyclin-dependent kinases (CDKs) to understand the differential response of cell division machinery to growth-promotive signals during the distinct stages of dormancy in hybrid aspen. We show that 4 weeks of short-day (SD) treatment causes termination of the cambial cell division and establishment of the ecodormant state. This coincides with a steady decline in the histone H1 kinase activity of the PSTAIRE-type poplar CDKA (PttCDKA) and the PPTTLRE-type PttCDKB kinase complexes. However, neither the transcript nor the polypeptide levels of PttCDKA and PttCDKB are reduced during ecodormancy. In contrast, 6 weeks of SD treatment establishes endodormancy, which is marked by the reduction and disappearance of the PttCDKA and PttCDKB protein levels and the PttCDKB transcript levels. The transition to endodormancy is preceded by an elevated E2F (adenosine E2 promoter binding factor) phosphorylation activity of the PttCDKA kinase that reduces the DNA-binding activity of E2F in vitro. The transition to endodormancy is followed by a reduction of retinoblastoma (Rb) phosphorylation activity of PttCDKA protein complexes. Both phosphorylation events could contribute to block the G1 to S phase transition upon the establishment of endodormancy. Our results indicate that eco- and endodormant stages of cambial dormancy involve a stage-specific regulation of the cell cycle effectors at multiple levels.
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http://dx.doi.org/10.1111/j.1365-313X.2004.02070.xDOI Listing
May 2004

The VirD2 pilot protein of Agrobacterium-transferred DNA interacts with the TATA box-binding protein and a nuclear protein kinase in plants.

Proc Natl Acad Sci U S A 2003 Aug 4;100(17):10108-13. Epub 2003 Aug 4.

Max Planck Institute for Plant Breeding Research, Carl-von-Linne-Weg 10, D-50829 Cologne (Köln), Germany.

The bacterial virulence protein VirD2 plays an important role in nuclear import and chromosomal integration of Agrobacterium-transferred DNA in fungal, plant, animal, and human cells. Here we show that in nuclei of alfalfa cells, VirD2 interacts with and is phosphorylated by CAK2Ms, a conserved plant ortholog of cyclin-dependent kinase-activating kinases. CAK2Ms binds to and phosphorylates the C-terminal regulatory domain of RNA polymerase II largest subunit, which can recruit the TATA box-binding protein. VirD2 is found in tight association with the TATA box-binding protein in vivo. These results indicate that recognition of VirD2 is mediated by widely conserved nuclear factors in eukaryotes.
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http://dx.doi.org/10.1073/pnas.1733208100DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC187781PMC
August 2003