Laszlo Bako | Umeå University (original) (raw)

Papers by Laszlo Bako

In Arabidopsis thaliana, canonical auxin-dependent gene regulation is mediated by 23 transcriptio... more In Arabidopsis thaliana, canonical auxin-dependent gene regulation is mediated by 23 transcription factors from the AUXIN RESPONSE FACTOR (ARF) family, most of which interact with 29 auxin/indole acetic acid (Aux/IAA) repressors, themselves forming, in the presence of auxin, coreceptor complexes with one of six TRANSPORT INHIBITOR1/AUXIN-SIGNALLING F-BOX PROTEINS (TIR1/AFB). Different combinations of co-receptors drive specific sensing outputs, allowing auxin to control a myriad of processes. Considerable efforts have been made to discern the temporal and spatial specificity of auxin action. However, owing to a lack of obvious phenotype in single loss-of-function mutants in Aux/IAA genes, most genetic studies have relied on gain-of-function mutants, which are highly pleiotropic. In this article, we describe a molecular framework for the role of several members of the auxin sensing machinery. Using loss-of-function mutants, we demonstrate that TIR1 and AFB2 are positive regulators, w...

The New phytologist, 2018

The Arabidopsis LEAFY (LFY) transcription factor is a key regulator of floral meristem emergence ... more 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 p...

New Phytologist, 2017

Summary The Arabidopsis thaliana gene XYLEM NAC DOMAIN1 (XND1) is upregulated in xylem tracheary ... more Summary 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, E2FTD‐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 ...

eLife, Aug 22, 2017

Both light and temperature have dramatic effects on plant development. Phytochrome photoreceptors... more 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 BLADE-ON-PETIOLE (BOP) 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 BOP2 promotes photo-morphogenesis and modulates thermomorphogenesis by suppressing PIF4 activity, through a reduction in PIF4 protein level. In red-light-grown seedlings PIF4 ubiquitination was reduced in the bop2 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...

Biochemical Journal, 1994

We isolated membrane vesicles from maize (Zea mays L.) coleoptiles and identified in these vesicl... more We isolated membrane vesicles from maize (Zea mays L.) coleoptiles and identified in these vesicles a 58 kDa (pm58) and a 60 kDa (pm60) protein by photoaffinity labelling with 5-azido-[7-3H]indole-3-acetic acid ([3H]N3IAA). Photoaffinity labelling was effectively competed for by auxins as well as by flavonoids. The labelled proteins were solubilized by Triton X-114 from the vesicles and partially purified. Microsequence analysis revealed that pm60 is a beta-glucosidase. This was confirmed by biochemical and immunological analysis. We show that pm60 has a beta-D-glucoside glucohydrolase (EC 3.2.1.21) activity. It uses p-nitro-phenyl beta-D-glucopyranoside (PNPG) as a substrate, with a pH optimum of 5.0. The Km for PNPG is 0.652 mM and the Vmax. 6.24 mumol.min-1.mg-1. The beta-glucosidase activity of pm60 was competitively inhibited by IAA and 1-naphthylacetic acid as well as by gluconolactam and glucose. N-terminal amino-acid-sequence analysis of pm58 revealed similarity to pm60, sug...

Biochemical Society Transactions, 1992

Results and Problems in Cell Differentiation, 1994

The Plant Cell, 2012

Exposure of plants to light intensities that exceed the electron utilization capacity of the chlo... more 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 ciselement 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.

The Plant Cell, 2005

The molecular mechanisms by which the phytohormone auxin coordinates cell division with cell grow... more 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.

The EMBO Journal, 2012

Post-embryonic growth in plants depends on the continuous supply of undifferentiated cells within... more 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 DRB) 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 DRB 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.

PLoS ONE, 2012

Background: A tool for stoichiometric co-expression of effector and target proteins to study intr... more 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.

Plant Physiology, 2011

The circadian clock of the model plant Arabidopsis (Arabidopsis thaliana) is made up of a complex... more 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 o...

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Cell Cycle, 2005

The anaphase-promoting complex (APC), a multisubunit E3 ubiquitin ligase, is an essential regulat... more 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 G 2 until the M-phase exit, ccs52B from G 2 /M to M while ccs52A1 and ccs52A2 were from late M until early G 2 , 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.

Cell, 2012

In plants, where cells cannot migrate, asymmetric cell divisions (ACDs) must be confined to the a... more 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.

Lateral root (LR) formation is an example of plant post-embryonic organogenesis event. LRs are is... more Lateral root (LR) formation is an example of 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 negatively regulates auxin-mediated LR formation through a mechanism that is not yet known. Here we show that PICKLE interacts with RETINOBLASTOMA-RELATED 1 (RBR1) to repress theLATERAL 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.

PLANT PHYSIOLOGY, 1997

Phosphorylation is one of the mechanisms controlling the activity of heat-shock transcription fac... more Phosphorylation is one of the mechanisms controlling the activity of heat-shock transcription factors in yeast and mammalian cells. Here we describe partial purification, identification, and characterization of a protein kinase that phosphorylates the Arabidopsis heat-shock factor AtHSF1 at multiple serine residues. The HSF1 kinase forms a stable complex with AtHSF1, which can be detected by kinase pull-down assays using a histidine-tagged AtHSF1 substrate. The HSF1 kinase interacts with the cell-cycle control protein Suc1p and is immunoprecipitated by an antibody specific for the Arabidopsis cyclin-dependent CDC2a kinase. Phosphorylation by CDC2a in vitro inhibits DNA binding of AtHSF1 to the cognate heat-shock elements, suggesting a possible regulatory interaction between heat-shock response and cell-cycle control in plants.

Genes & Development, 1998