@article{GajdanowiczGarciaMataGonzalezetal.2009, author = {Gajdanowicz, Pawel and Garcia-Mata, Carlos and Gonzalez, Wendy and Morales-Navarro, Samuel El{\"i}as and Sharma, Tripti and Gonzalez-Nilo, Fernando Danilo and Gutowicz, Jan and M{\"u}ller-R{\"o}ber, Bernd and Blatt, Michael R. and Dreyer, Ingo}, title = {Distinct roles of the last transmembrane domain in controlling Arabidopsis K+ channel activity}, issn = {0028-646X}, doi = {10.1111/j.1469-8137.2008.02749.x}, year = {2009}, abstract = {The family of voltage-gated potassium channels in plants presumably evolved from a common ancestor and includes both inward-rectifying (K-in) channels that allow plant cells to accumulate K+ and outward-rectifying (K-out) channels that mediate K+ efflux. Despite their close structural similarities, the activity of Kin channels is largely independent of K+ and depends only on the transmembrane voltage, whereas that of K-out channels responds to the membrane voltage and the prevailing extracellular K+ concentration. Gating of potassium channels is achieved by structural rearrangements within the last transmembrane domain (S6). Here we investigated the functional equivalence of the S6 helices of the Kin channel KAT1 and the K-out channel SKOR by domain-swapping and site-directed mutagenesis. Channel mutants and chimeras were analyzed after expression in Xenopus oocytes. We identified two discrete regions that influence gating differently in both channels, demonstrating a lack of functional complementarity between KAT1 and SKOR. Our findings are supported by molecular models of KAT1 and SKOR in the open and closed states. The role of the S6 segment in gating evolved differently during specialization of the two channel subclasses, posing an obstacle for the transfer of the K+-sensor from K-out to K-in channels.}, language = {en} } @article{VoelkerGomezPorrasBeckeretal.2010, author = {Voelker, Camilla and Gomez-Porras, Judith Lucia and Becker, Dirk and Hamamoto, Shin and Uozumi, Nobuyuki and Gambale, Franco and M{\"u}ller-R{\"o}ber, Bernd and Czempinski, Katrin and Dreyer, Ingo}, title = {Roles of tandem-pore K plus channels in plants : a puzzle still to be solved}, issn = {1435-8603}, doi = {10.1111/j.1438-8677.2010.00353.x}, year = {2010}, abstract = {The group of voltage-independent K+ channels in Arabidopsis thaliana consists of six members, five tandem-pore channels (TPK1-TPK5) and a single K-ir-like channel (KCO3). All TPK/KCO channels are located at the vacuolar membrane except for TPK4, which was shown to be a plasma membrane channel in pollen. The vacuolar channels interact with 14-3-3 proteins (also called General Regulating Factors, GRFs), indicating regulation at the level of protein-protein interactions. Here we review current knowledge about these ion channels and their genes, and highlight open questions that need to be urgently addressed in future studies to fully appreciate the physiological functions of these ion channels.}, language = {en} } @article{GliwickaBalazadehCaldanaetal.2009, author = {Gliwicka, Marta and Balazadeh, Salma and Caldana, Camila and M{\"u}ller-R{\"o}ber, Bernd and Gaj, Malgorzata D.}, title = {The use of multi-qPCR platform and tan1 mutant in identification of TF genes involved in somatic embryogenesis in Arabidopsis}, issn = {0001-5296}, year = {2009}, language = {en} } @article{DreyerPoreeSchneideretal.2004, author = {Dreyer, Ingo and Poree, Fabien and Schneider, A. and Mittelstadt, J. and Bertl, Adam and Sentenac, H. and Thibaud, Jean-Baptiste and M{\"u}ller-R{\"o}ber, Bernd}, title = {Assembly of plant Shaker-like K-out channels requires two distinct sites of the channel alpha-subunit}, issn = {0006-3495}, year = {2004}, abstract = {SKOR and GORK are outward-rectifying plant potassium channels from Arabidopsis thaliana. They belong to the Shaker superfamily of voltage-dependent K+ channels. Channels of this class are composed of four alpha-subunits and subunit assembly is a prerequisite for channel function. In this study the assembly mechanism of SKOR was investigated using the yeast two-hybrid system and functional assays in Xenopus oocytes and in yeast. We demonstrate that SKOR and GORK physically interact and assemble into heteromeric K-out channels. Deletion mutants and chimeric proteins generated from SKOR and the K-in channel alpha-subunit KAT1 revealed that the cytoplasmic C-terminus of SKOR determines channel assembly. Two domains thatchannel a-subunit KAT1 revealed that the cytoplasmic C-terminus of SKOR determines channel assembly. Two domains that are crucial for channel assembly were identified: i), a proximal interacting region comprising a putative cyclic nucleotide-binding domain together with 33 amino acids just upstream of this domain, and ii), a distal interacting region showing some resemblance to the K-T domain of KAT1. Both regions contributed differently to channel assembly. Whereas the proximal interacting region was found to be active on its own, the distal interacting region required an intact proximal interacting region to be active. K-out alpha-subunits did not assemble with K-in alpha-subunits because of the absence of interaction between their assembly sites}, language = {en} } @article{BeckerGeigerDunkeletal.2004, author = {Becker, Dirk and Geiger, D. and Dunkel, M. and Roller, A. and Bertl, Adam and Latz, A. and Carpaneto, Armando and Dietrich, Peter and Roelfsema, M. R. G. and Voelker, C. and Schmidt, D. and M{\"u}ller-R{\"o}ber, Bernd and Czempinski, Katrin and Hedrich, R.}, title = {AtTPK4, an Arabidopsis tandem-pore K+ channel, poised to control the pollen membrane voltage in a pH- and Ca2+- dependent manner}, issn = {0027-8424}, year = {2004}, abstract = {The Arabidopsis tandem-pore K+ (TPK) channels displaying four transmembrane domains and two pore regions share structural homologies with their animal counterparts of the KCNK family. In contrast to the Shaker-like Arabidopsis channels (six transmembrane domains/one pore region), the functional properties and the biological role of plant TPK channels have not been elucidated yet. Here, we show that AtTPK4 (KCO4) localizes to the plasma membrane and is predominantly expressed in pollen. AtTPK4 (KCO4) resembles the electrical properties of a voltage-independent K+ channel after expression in Xenopus oocytes and yeast. Hyperpolarizing as well as depolarizing membrane voltages elicited instantaneous K+ currents, which were blocked by extracellular calcium and cytoplasmic protons. Functional complementation assays using a K+ transport-deficient yeast confirmed the biophysical and pharmacological properties of the AtTPK4 channel. The features of AtTPK4 point toward a role in potassium homeostasis and membrane voltage control of the growing pollen tube. Thus, AtTPK4 represents a member of plant tandem-pore-K+ channels, resembling the characteristics of its animal counterparts as well as plant-specific features with respect to modulation of channel activity by acidosis and calcium}, language = {en} } @article{KohlerMuellerRoeber2004, author = {Kohler, B. and M{\"u}ller-R{\"o}ber, Bernd}, title = {Remote control - cell and organ communication within plants}, year = {2004}, language = {en} } @article{GomezMerinoBrearleyOrnatowskaetal.2004, author = {Gomez-Merino, Fernando Carlos and Brearley, C. A. and Ornatowska, Magdalena and Abdel-Haliem, Mahmoud E. F. and Zanor, Maria Ines and M{\"u}ller-R{\"o}ber, Bernd}, title = {AtDGK2, a novel diacylglycerol kinase from Arabidopsis thaliana, phosphorylates 1-stearoyl-2-arachidonoyl-sn- glycerol and 1,2-dioleoyl-sn-glycerol and exhibits cold-inducible gene expression}, issn = {0021-9258}, year = {2004}, abstract = {Diacylglycerol kinase (DGK) phosphorylates diacylglycerol (DAG) to generate phosphatidic acid (PA). Both DAG and PA are implicated in signal transduction pathways. DGKs have been widely studied in animals, but their analysis in plants is fragmentary. Here, we report the cloning and biochemical characterization of AtDGK2, encoding DGK from Arabidopsis thaliana. AtDGK2 has a predicted molecular mass of 79.4 kDa and, like AtDGK1 previously reported, harbors two copies of a phorbol ester/DAG-binding domain in its N-terminal region. AtDGK2 belongs to a family of seven DGK genes in A. thaliana. AtDGK3 to AtDGK7 encode similar to55-kDa DGKs that lack a typical phorbol ester/DAG-binding domain. Phylogenetically, plant DGKs fall into three clusters. Members of all three clusters are widely expressed in vascular plants. Recombinant AtDGK2 was expressed in Escherichia coli and biochemically characterized. The enzyme phosphorylated 1,2-dioleoyl-sn-glycerol to yield PA, exhibiting Michaelis-Menten type kinetics. Estimated K-m and V-max values were 125 muM for DAG and 0.25 pmol of PA min(-1) mug(-1), respectively. The enzyme was maximally active at pH 7.2. Its activity was Mg2+-dependent and affected by the presence of detergents, salts, and the DGK inhibitor R59022, but not by Ca2+. AtDGK2 exhibited substrate preference for unsaturated DAG analogues (i.e. 1-stearoyl-2-arachidonoyl-sn-glycerol and 1,2- dioleoyl-sn-glycerol). The AtDGK2 gene is expressed in various tissues of the Arabidopsis plant, including leaves, roots, and flowers, as shown by Northern blot analysis and promoter-reporter gene fusions. We found that AtDGK2 is induced by exposure to low temperature (4degreesC), pointing to a role in cold signal transduction}, language = {en} } @article{WittZanorMuellerRoeber2004, author = {Witt, Isabell and Zanor, Maria Ines and M{\"u}ller-R{\"o}ber, Bernd}, title = {Transcription factor function search : how do individual factors regulate agronomical important processes in plants? (Subproject A)}, isbn = {3-00-011587-0}, year = {2004}, language = {en} } @article{XuBrearleyLinetal.2005, author = {Xu, J. and Brearley, C. A. and Lin, W. H. and Wang, Y. and Ye, R. and M{\"u}ller-R{\"o}ber, Bernd and Xu, Z. H. and Xue, H. W.}, title = {A role of Arabidopsis inositol polyphosphate kinase, AtIPK2 alpha, in pollen germination and root growth}, issn = {0032-0889}, year = {2005}, abstract = {Inositol polyphosphates, such as inositol trisphosphate, are pivotal intracellular signaling molecules in eukaryotic cells. In higher plants the mechanism for the regulation of the type and the level of these signaling molecules is poorly understood. In this study we investigate the physiological function of an Arabidopsis (Arabidopsis thaliana) gene encoding inositol polyphosphate kinase (AtIPK2alpha), which phosphorylates inositol 1,4,5-trisphosphate successively at the D-6 and D-3 positions, and inositol 1,3,4,5-tetrakisphosphate at D-6, resulting in the generation of inositol 1,3,4,5,6-pentakisphosphate. Semiquantitative reverse transcription-PCR and promoter-beta-glucuronidase reporter gene analyses showed that AtIPK2alpha is expressed in various tissues, including roots and root hairs, stem, leaf, pollen grains, pollen tubes, the flower stigma, and siliques. Transgenic Arabidopsis plants expressing the AtIPK2alpha antisense gene under its own promoter were generated. Analysis of several independent transformants exhibiting strong reduction in AtIPK2alpha transcript levels showed that both pollen germination and pollen tube growth were enhanced in the antisense lines compared to wild-type plants, especially in the presence of nonoptimal low Ca2+ concentrations in the culture medium. Furthermore, root growth and root hair development were also stimulated in the antisense lines, in the presence of elevated external Ca2+ concentration or upon the addition of EGTA. In addition, seed germination and early seedling growth was stimulated in the antisense lines. These observations suggest a general and important role of AtIPK2alpha, and hence inositol polyphosphate metabolism, in the regulation of plant growth most likely through the regulation of calcium signaling, consistent with the well-known function of inositol trisphosphate in the mobilization of intracellular calcium stores}, language = {en} } @article{RianoPachonDreyerMuellerRoeber2005, author = {Riano-Pachon, Diego Mauricio and Dreyer, Ingo and M{\"u}ller-R{\"o}ber, Bernd}, title = {Orphan transcripts in Arabidopsis thaliana : identification of several hundred previously unrecognized genes}, issn = {0960-7412}, year = {2005}, abstract = {Expressed sequence tags (ESTs) represent a huge resource for the discovery of previously unknown genetic information and functional genome assignment. In this study we screened a collection of 178 292 ESTs from Arabidopsis thaliana by testing them against previously annotated genes of the Arabidopsis genome. We identified several hundreds of new transcripts that match the Arabidopsis genome at so far unassigned loci. The transcriptional activity of these loci was independently confirmed by comparison with the Salk Whole Genome Array Data. To a large extent, the newly identified transcriptionally active genomic regions do not encode 'classic' proteins, but instead generate non-coding RNAs and/or small peptide-coding RNAs of presently unknown biological function. More than 560 transcripts identified in this study are not represented by the Affymetrix GeneChip arrays currently widely used for expression profiling in A. thaliana. Our data strongly support the hypothesis that numerous previously unknown genes exist in the Arabidopsis genome}, language = {en} } @article{LinWangMuellerRoeberetal.2005, author = {Lin, W. H. and Wang, Y. and M{\"u}ller-R{\"o}ber, Bernd and Brearley, C. A. and Xu, Z. H. and Xue, H. W.}, title = {At5PTase13 modulates cotyledon vein development through regulating auxin homeostasis}, issn = {0032-0889}, year = {2005}, abstract = {Phosphatidylinositol signaling pathway and the relevant metabolites are known to be critical to the modulation of different aspects of plant growth, development, and stress responses. Inositol polyphosphate 5-phosphatase is a key enzyme involved in phosphatidylinositol metabolism and is encoded by an At5PTase gene family in Arabidopsis thaliana. A previous study shows that At5PTase11 mediates cotyledon vascular development probably through the regulation of intracellular calcium levels. In this study, we provide evidence that At5PTase13 modulates the development of cotyledon veins through its regulation of auxin homeostasis. A T-DNA insertional knockout mutant, At5pt13-1, showed a defect in development of the cotyledon vein, which was rescued completely by exogenous auxin and in part by brassinolide, a steroid hormone. Furthermore, the mutant had reduced auxin content and altered auxin accumulation in seedlings revealed by the DR5:beta-glucuronidase fusion construct in seedlings. In addition, microarray analysis shows that the transcription of key genes responsible for auxin biosynthesis and transport was altered in At5pt13-1. The At5pt13-1 mutant was also less sensitive to auxin inhibition of root elongation. These results suggest that At5PTase13 regulates the homeostasis of auxin, a key hormone controlling vascular development in plants}, language = {en} } @article{GomezMerinoAranaCeballosTrejoTellezetal.2005, author = {Gomez-Merino, Fernando Carlos and Arana-Ceballos, Fernando Alberto and Trejo-Tellez, L. I. and Skirycz, Aleksandra and Brearley, C. A. and Dormann, P. and M{\"u}ller-R{\"o}ber, Bernd}, title = {Arabidopsis AtDGK7, the smallest member of plant diacylglycerol kinases (DGKs), displays unique biochemical features and saturates at low substrate concentration : the DGK inhibitor R59022 differentially affects AtDGK2 and AtDGK7 activity in vitro and alters plant growth and development}, issn = {0021-9258}, year = {2005}, abstract = {Diacylglycerol kinase (DGK) regulates the level of the second messenger diacylglycerol and produces phosphatidic acid (PA), another signaling molecule. The Arabidopsis thaliana genome encodes seven putative diacylglycerol kinase isozymes (named AtDGK1 to -7), structurally falling into three major clusters. So far, enzymatic activity has not been reported for any plant Cluster II DGK. Here, we demonstrate that a representative of this cluster, AtDGK7, is biochemically active when expressed as a recombinant protein in Escherichia coli. AtDGK7, encoded by gene locus At4g30340, contains 374 amino acids with an apparent molecular mass of 41.2 kDa. AtDGK7 harbors an N-terminal catalytic domain, but in contrast to various characterized DGKs (including AtDGK2), it lacks a cysteine-rich domain at its N terminus, and, importantly, its C-terminal DGK accessory domain is incomplete. Recombinant AtDGK7 expressed in E. coli exhibits Michaelis-Menten type kinetics with 1,2-dioleoyl-sn-glycerol as substrate. AtDGK7 activity was affected by pH, detergents, and the DGK inhibitor R59022. We demonstrate that both AtDGK2 and AtDGK7 phosphorylate diacylglycerol molecular species that are typically found in plants, indicating that both enzymes convert physiologically relevant substrates. AtDGK7 is expressed throughout the Arabidopsis plant, but expression is strongest in flowers and young seedlings. Expression of AtDGK2 is transiently induced by wounding. R59022 at similar to 80 mu M inhibits root elongation and lateral root formation and reduces plant growth, indicating that DGKs play an important role in plant development}, language = {en} } @article{MichardLacombePoreeetal.2005, author = {Michard, Erwan and Lacombe, Beno{\^i}t and Poree, Fabien and M{\"u}ller-R{\"o}ber, Bernd and Sentenac, Herv{\´e} and Thibaud, Jean-Baptiste and Dreyer, Ingo}, title = {A unique voltage sensor sensitizes the potassium channel AKT2 to phosphoregulation}, year = {2005}, abstract = {Among all voltage-gated K+ channels from the model plant Arabidopsis thaliana, the weakly rectifying K+ channel (K-weak channel) AKT2 displays unique gating properties. AKT2 is exceptionally regulated by phosphorylation: when nonphosphorylated AKT2 behaves as an inward-rectifying potassium channel; phosphorylation of AKT2 abolishes inward rectification by shifting its activation threshold far positive (>200 mV) so that it closes only at voltages positive of + 100 mV. In its phosphorylated form, AKT2 is thus locked in the open state in the entire physiological voltage range. To understand the molecular grounds of this unique gating behavior, we generated chimeras between AKT2 and the conventional inward-rectifying channel KAT1. The transfer of the pore from KAT1 to AKT2 altered the permeation properties of the channel. However, the gating properties were unaffected, suggesting that the pore region of AKT2 is not responsible for the unique K-weak gating. Instead, a lysine residue in S4, highly conserved among all K-weak channels but absent from other plant K+ channels, was pinpointed in a site-directed mutagenesis approach. Substitution of the lysine by serine or aspartate abolished the "open-lock" characteristic and converted AKT2 into an inward- rectifying channel. Interestingly, phosphoregulation of the mutant AKT2-K197S appeared to be similar to that of the K-in channel KAT1: as suggested by mimicking the phosphorylated and dephosphorylated states, phosphorylation induced a shift of the activation threshold of AKT2-K197S by about +50 mV. We conclude that the lysine residue K197 sensitizes AKT2 to phosphoregulation. The phosphorylation-induced reduction of the activation energy in AKT2 is similar to 6 kT larger than in the K197S mutant. It is discussed that this hypersensitive response of AKT2 to phosphorylation equips a cell with the versatility to establish a potassium gradient and to make efficient use of it}, language = {en} } @article{JohanssonWulfetangePoreeetal.2006, author = {Johansson, Ingela and Wulfetange, Klaas and Poree, Fabien and Michard, Erwan and Gajdanowicz, Pawel and Lacombe, Benoit and Sentenac, Herve and Thibaud, Jean-Baptiste and M{\"u}ller-R{\"o}ber, Bernd and Blatt, Michael R. and Dreyer, Ingo}, title = {External K+ modulates the activity of the Arabidopsis potassium channel SKOR via an unusual mechanism}, issn = {0960-7412}, doi = {10.1111/j.1365-313X.2006.02690.X}, year = {2006}, abstract = {Plant outward-rectifying K+ channels mediate K+ efflux from guard cells during stomatal closure and from root cells into the xylem for root-shoot allocation of potassium (K). Intriguingly, the gating of these channels depends on the extracellular K+ concentration, although the ions carrying the current are derived from inside the cell. This K+ dependence confers a sensitivity to the extracellular K+ concentration ([K+]) that ensures that the channels mediate K+ efflux only, regardless of the [K+] prevailing outside. We investigated the mechanism of K+-dependent gating of the K+ channel SKOR of Arabidopsis by site-directed mutagenesis. Mutations affecting the intrinsic K+ dependence of gating were found to cluster in the pore and within the sixth transmembrane helix (S6), identifying an 'S6 gating domain' deep within the membrane. Mapping the SKOR sequence to the crystal structure of the voltage-dependent K+ channel KvAP from Aeropyrum pernix suggested interaction between the S6 gating domain and the base of the pore helix, a prediction supported by mutations at this site. These results offer a unique insight into the molecular basis for a physiologically important K+-sensory process in plants}, language = {en} } @article{FengNiElgeetal.2006, author = {Feng, Xiao-Li and Ni, Wei-Min and Elge, Stephan and M{\"u}ller-R{\"o}ber, Bernd and Xu, Zhi-Hong and Xue, Hong-Wei}, title = {Auxin flow in anther filaments is critical for pollen grain development through regulating pollen mitosis}, issn = {0167-4412}, doi = {10.1007/s11103-006-0005-z}, year = {2006}, abstract = {It was well known that auxin is critical for anther/pollen grain development, however, the clear distribution and detailed effects of auxin during floral development are still unclear. We have shown here that, through analyzing GUS activities of Arabidopsis lines harboring auxin response elements DR5-GUS, auxin was mainly accumulated in the anther during flower stages 10-12. Further studies employing the indoleacetic acid-lysine synthetase (iaaL) coding gene from Pseudomonas syringae subsp. savastanoi under control of the promoter region of Arabidopsis phosphatidylinositol monophosphate 5-kinase 1 gene, which conducts the anther filament-specific expression, showed that block of auxin flow of filaments resulted in shortened filaments and significantly defective pollen grains. Similar phenotype was observed in tobacco plants transformed with the same construct, confirming the effects of auxin flow in filaments on anther development. Detailed studies further revealed that the meiosis process of pollen grain was normal while the mitosis at later stage was significantly defected, indicating the effects of auxin flow in filaments on pollen grain mitosis process. Analysis employing [C-14]IAA, as well as the observation on the expression of AtPIN1, coding for auxin efflux carrier, demonstrated the presence of polar auxin transport in anther filaments and pollen grains}, language = {en} }