László Bakó

• Umeå University

Lateral root (LR) formation is an example of a plant post-embryonic organogenesis event. LRs are issued from non-dividing cells entering consecutive steps of formative divisions, proliferation and elongation. The chromatin remodeling protein PICKLE (PKL) negatively regulates auxin-mediated LR formation through a mechanism that is not yet known. Her...

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...

PIRIN2 (PRN2) was earlie rreported to suppress syringyl (S)-type lignin accumulation of xylem vessels of Arabidopsis (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 developme...

In Arabidopsis thaliana, canonical auxin-dependent gene regulation is mediated by 23 transcription factors from the AUXIN RESPONSE FACTOR (ARF) family interacting with 29 auxin/indole acetic acid repressors (Aux/IAA), themselves forming coreceptor complexes with one of six TRANSPORT INHIBITOR1/AUXIN-SIGNALLING F-BOX (TIR1/AFB) PROTEINS. Different c...

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 sho...

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...

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 beco...

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 high...

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 which developed into tadpoles. More recently, induced expression of transcription factors, oct4, sox2, klf4 and c-myc has evidenced the plast...

In order to gain insights into the regulatory pathways controlling phloem development, we characterized three genes encoding membrane proteins from the G subfamily of ABC transporters (ABCG9, ABCG11 and ABCG14), whose expressions in the phloem were confirmed. Mutations in the genes encoding these dimerizing "half transporters" are semidominant and...

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...

Sorting of 2A polyprotein transfected protoplasts using FACS. (A) Scheme of fluorescent-activated cell sorting (FACS) of protoplasts transfected with 2A polyprotein in order to obtain a homogenous mutant population. Protoplasts were transfected with GS-CFP-2A-RABD2a vector (v), resulting in a heterogeneous mixture of transfected (v, about 10% of to...

Accumulation of GS-CFP-2A in vacuole like structures when co-expressed with ARF1 (T31N). Bright field (A) and confocal (B) images of Arabidopsis protoplast transiently expressing GS-CFP-2A-ARF1 (T31N). In addition to the phenotype shown in Figure 5G–H, some protoplasts showed CFP fluorescence in vacuole like structures as shown here. Bars = 5 µm. (...

Supporting Material and Methods. (DOCX)

Endo H is a valid marker for protein trafficking. (A) Immunoblot analysis of ultracentrifuged protein extracts from Nicotiana benthamiana transiently expressing HACAH1-2A-RABD2a (wt) using anti-HA, anti-Rubisco and anti-BiP antibodies. Soluble stroma-containing and pelleted ER-containing microsome fractions are indicated. Samples were subjected (+)...

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 RET...

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...

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 w...

Phylogenetic analysis of the ANT family of AP2 transcription factors in Arabidopsis and Populus. Arabidopsis AINTEGUMENTA (ANT) groups with four Populus genes, which were named AINTEGUMENTALIKE 1-4 (AIL1-AIL4). AIL1 and AIL3 analysed in detail are marked in bold letters. (TIF)

Expression of AIL1 and AIL3 in transgenic hybrid aspen. Expression in the apex of AIL1 in the wild type hybrid aspen (T89) and two transgenic lines (AIL1oe line 2B and 3B) expressing AIL1 cDNA under the control of 35S promoter. Y-axis shows the transcript levels of AIL1 relative to that of the reference gene (18S rRNA). Data from 2 independent expe...

Analysis of bud set in amiRNA expressing lines. Analysis of bud set phenotype in wild type hybrid aspen (T89), and amiRNA lines 255-6 and 256-23. The transition from active growth to completion of bud set was divided into 4 stages where 0 is a completely developed bud and 3 correspond to an actively growing apex. Number of plants used for each geno...

Primers for amiRNA construction. (DOCX)

Expression of AIL1 in amiRNA expressing lines. A). Expression of AIL1 in apices of tissue culture grown wild type (T89) and amiRNA expressing hybrid aspen (255-6). A). Expression of AIL1 in apices of tissue culture grown wild type (T89) and amiRNA expressing hybrid aspen (256-23). Expression values are median of three technical replicates. (TIF)

Primers for CYCD3:2 promoter fragments. (DOCX)

Expression of AIL genes is downregulated after short day treatment during growth cessation. Expression of AINTEGUMENTALIKE genes (AIL1-AIL4) was analysed in the apex of wild type hybrid aspen after 6 weeks of SD treatment (8h day). Y-axis indicates the expression levels after 6 weeks of short day treatment normalized to the level prior to the start...

Real-time PCR primer sequences. *Please note that cross-reactivity could occur between primer pairs used for the detection of AIL1 and AIL2 gene expression. (DOCX)

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 in...

Localization of HA-tagged CAH1 by immunogold labelling and electron microscopy. (A and B) Transmission Electron Microscopy (TEM) images of wt Arabidopsis suspension culture cells. (A) Detailed image of a chloroplast. (B) General view of the cell with ER, Golgi (G), cell wall (CW), vacuole (V), and chloroplast (C). The immunogold (IG) labelling over...

CAH1 contains an intramolecular disulphide bridge. (A) ClustalW2 [47] protein sequence analysis of Arabidopsis α-CA homologues. Cysteine residues at positions 27 and 191 (red) in CAH1 sequence are conserved (*) in seven of the eight homologues described by Fabre et al. [4]. CA8 is substantially larger than CA1-7, possibly indicating that this gene...

TargetP analysis of Arabidopsis α-type CAs. TargetP [8] analysis of Arabidopsis α-type CAs protein sequences described by Fabre et al. [4]. Seven of the eight homologues have an N-terminal signal sequence (SP) for the ER. (TIF)

Cycloheximide treatment results in a higher proportion of Endo H resistant HC. (A) Protoplasts from Arabidopsis cell suspension culture were transiently transfected with HA-tagged wt CAH1 (HC). Prior to protein extraction, protoplasts were either incubated in presence or absence of the protein biosynthesis inhibitor cycloheximide (CHX) for 8 h. Ext...

HA-tagged CAH1 is glycosylated in plant cells, harbouring four or five N-glycans. Migration patterns of HA-tagged CAH1 forms in protoplasts of Arabidopsis suspension culture cells (A) and Arabidopsis mesophyll cells (B) expressing wt (HC) or mutant CAH1. Antibody specificity was verified using water-transfected protoplasts (−). (A) Protoplasts from...

Enrichment of ER marker in microsome fraction. Western blot using antibodies against the ER localized protein BiP to verify that microsome fractions in Figure 4a and b were intact. S and I, soluble supernatant and insoluble microsome pellet, respectively. (TIF)

Nomenclature of constructs used to transfect plant cells and protoplasts. (DOC)

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...

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 s...

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 exp...

Plant retinoblastoma-related (RBR) proteins are primarily considered as key regulators of G1/S phase transition, with functional roles in a variety of cellular events during plant growth and organ development. Polyclonal antibody against the C-terminal region of the Arabidopsis RBR1 protein also specifically recognizes the alfalfa 115 kDa MsRBR pro...

Perennial plants such as the long-lived trees of boreal forest cycle between periods of active growth and dormancy. Transition from active growth to dormancy is induced by short day (SD) signal. Once dormancy is established, prolonged exposure to low temperature is required for breaking dormancy before warm temperatures can induce growth. We have s...

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 biogenesi...

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 transc...

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...

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 proliferat...

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 proliferat...

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, Cd...

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) protei...

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 regulatio...

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 k...

Hormones as auxins and cytokinins trigger the division cycle in differentiated plant cells and are required for the maintenance of proliferation in cultured cells in vitro. Northern analysis showed that the expression pattern of alfalfa cdc2 genes is significantly different in cells of primary explants exposed to hormone treatment and of rapidly cy...

Synchronous cell systems are among the most suitable models to study the plant cell cycle. However, to date, the tobacco BY-2 cell line is the plant cell line, which can be synchronized to high levels. The aim of the present work is to produce a fast growing rice cell suspensions and to elaborate the synchronization method of the rice suspension cu...

Reversible phosphorylation of proteins by kinases and phosphatases plays a key regulatory role in several eukaryotic cellular functions including the control of the division cycle. Increasing numbers of sequence and biochemical data show the involvement of cyclin-dependent kinases (CDKs) and cyclins in regulation of the cell cycle progression in hi...

A beta-glucosidase that cleaves the biologically inactive hormone conjugates cytokinin-O- and kinetin-N3-glucosides is encoded by the maize Zm-p60.1 gene. The expression of the Zm-p60.1 gene was analyzed by Northern blot analysis and in-situ hybridization. It was found that the expression levels of the Zm-p60.1-specific mRNA changed after pollinati...

Mutation of the PRL1 gene, encoding a regulatory WD protein, results in glucose hypersensitivity and derepression of glucose-regulated genes in Arabidopsis. The yeast SNF1 protein kinase, a key regulator of glucose signaling, and Arabidopsis SNF1 homologs AKIN10 and AKIN11, which can complement the Δsnf1 mutation, were found to interact with an N-t...

The prl1 mutation localized by T-DNA tagging on Arabidopsis chromosome 4-44 confers hypersensitivity to glucose and sucrose. The prl1 mutation results in transcriptional derepression of glucose responsive genes defining a novel suppressor function in glucose signaling. The prl1 mutation also augments the sensitivity of plants to growth hormones inc...

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 c...

The eukaryotic cell division cycle is coordinated by cyclin-dependent kinases (CDKs), represented by a single major serine/threonine kinase in yeasts (Cdc2/CDC28) and a family of kinases (CDK1 to CDK8) in human cells. Previously, two cdc2 homologs, cdc2MsA and cdc2MsB, have been identified in alfalfa (Medicago sativa). By isolating cDNAs using a cd...

The eukaryotic cell division cycle is coordinated by cyclin-dependent kinases (CDKs), represented by a single major serine/threonine kinase in yeasts (Cdc2/CDC28) and a family of kinases (CDK1 to CDK8) in human cells. Previously, two cdc2 homologs, cdc2MsA and cdc2MsB, have been identified in alfalfa (Medicago sativa). By isolating cDNAs using a cd...

Similar to other higher eukaryotic organisms, in flowering plants, embryo development is the consequence of fertilization events. Union of gametes as the male sperm nucleus and the female egg results in the zygote which later develops into an embryo within the ovule. During the sexual reproductive cycle, the egg cell is prepared for initiation of t...

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 solubilize...

Transcription in plants, as in other eukaryotes, is catalyzed by three RNA polymerases (RNAPs). Catalytically active forms of RNAPs were first isolated by Roeder and Rutter (1969) and designated as RNAPI(A), II(B) and III(C). RNAPI transcribes rRNA genes, RNAPII synthesizes the precursors of mRNAs and RNAPIII is involved in the transcription of 5S...

A beta-glucoside encoded by a cloned Zea mays complementary DNA (Zm-p60.1) cleaved the biologically inactive hormone conjugates cytokinin-O-glucosides and kinetin-N3-glucoside, releasing active cytokinin. Tobacco protoplasts that transiently expressed Zm-p60.1 could use the inactive cytokinin glucosides to initiate cell division. The ability of pro...

This review article provides a comprehensive summary of the basic molecular and cellular events underlying the induction of the cell division cycle in auxin-treated somatic plant cells. Various pathways of signal transduction chains are discussed as mediators between auxin receptors and alteration of the gene expression pattern. The central role of...

Recent studies have demonstrated a connection between xanthine oxidase-generated reactive oxygen intermediates and histamine release during ischemia-reperfusion. In the present work, the effect of modulation of the endogenous histamine level on the xanthine oxidase activity was examined during the reperfusion of a canine ileal segment following a 2...

A biologically active and photolabile auxin analog, 5-azido-[7-3H]indole-3-acetic acid ([3H]N3IAA), was used to search for auxin-binding proteins in cytosolic extracts from maize coleoptiles (Zea mays L.) and identified a protein with a molecular mass of 60 kDa (p60). Binding of [3H]N3IAA is highly specific as demonstrated by competition analysis w...

Northern analysis has revealed substantial differences in mRNA accumulation of the two histone H3 gene variants represented by pH3c-1 and pH3c-11 cDNA clones. Both in partially synchronized cell suspension cultures and in protoplast-derived cells from alfalfa, Medicago varia, the maximal level of the histone H3-1 gene transcript coincided with the...

The cdc2 protein kinase plays a central role in control of the eukaryotic cell cycle of animals and yeasts. We have isolated a cDNA clone (cdc2Ms) from alfalfa (Medicago sativa L.) that is homologous to the yeast cdc2/CDC28 genes. The encoded protein is 64% identical to the yeast and mammalian counterparts and shows all the prominent structural fea...

Conflicting data have been reported on the relationship between reactive oxygen intermediates and the formation of oxygenase-derived eicosanoids. Plasma levels of prostacyclin (PGI2, measured as the stable metabolite 6-keto-PGF1 alpha) and thromboxane A2 (TxA2, measured as TxB2) in the effluent blood of a canine ileal segment were determined follow...

Protein phosphorylation plays an essential part in the regulation of cell cycle of eukaryotes (Norbury and Nurse, 1989; Nurse, 1990). Several components of this regulatory mechanism are well characterized in fission and budding yeasts (Moreno et al., 1989; Wittenberg and Reed, 1988), Xenopus oocytes (Cyert and Kirschner, 1988) and mammalian cells (...

Az Arabidopsis SnRK heterotrimer kináz komplex epitópjelölt alegységeinek szuszpenziós sejtkultúrában és növényekben való expressziójával a komplexek alegységszerkezetét és kölcsönhatásait vizsgáltuk. Megállapítottuk, ha a katalitikus AKIN10/11 kináz alegység a PRL1 inhibítor fehérjével való asszociációnak köszönhetoen inaktív, nem foszforilált for...

A támogatási időszak során a brasszinoszteroidok (BR-ok, növényi szteroid hormonok) bioszintézisében résztvevő gének funkcióit és szabályozását vizsgáltuk Arabidopsis modellnövényben. Molekuláris genetikai módszerek segítségével meghatároztuk a CYP90C1 és CYP90D1 gének által kódolt ezimek szerepét a hormon szintézisében, és eredményeink alapján új...