@article{BalazadehSiddiquiAlluetal.2010, author = {Balazadeh, Salma and Siddiqui, Hamad and Allu, Annapurna Devi and Matallana-Ramirez, Lilian Paola and Caldana, Camila and Mehrnia, Mohammad and Zanor, Maria-In{\´e}s and Koehler, Barbara and M{\"u}ller-R{\"o}ber, Bernd}, title = {A gene regulatory network controlled by the NAC transcription factor ANAC092/AtNAC2/ORE1 during salt-promoted senescence}, issn = {0960-7412}, doi = {10.1111/j.1365-313X.2010.04151.x}, year = {2010}, abstract = {P>The onset and progression of senescence are under genetic and environmental control. The Arabidopsis thaliana NAC transcription factor ANAC092 (also called AtNAC2 and ORE1) has recently been shown to control age-dependent senescence, but its mode of action has not been analysed yet. To explore the regulatory network administered by ANAC092 we performed microarray-based expression profiling using estradiol-inducible ANAC092 overexpression lines. Approximately 46\% of the 170 genes up-regulated upon ANAC092 induction are known senescence-associated genes, suggesting that the NAC factor exerts its role in senescence through a regulatory network that includes many of the genes previously reported to be senescence regulated. We selected 39 candidate genes and confirmed their time-dependent response to enhanced ANAC092 expression by quantitative RT-PCR. We also found that the majority of them (24 genes) are up-regulated by salt stress, a major promoter of plant senescence, in a manner similar to that of ANAC092, which itself is salt responsive. Furthermore, 24 genes like ANAC092 turned out to be stage-dependently expressed during seed growth with low expression at early and elevated expression at late stages of seed development. Disruption of ANAC092 increased the rate of seed germination under saline conditions, whereas the opposite occurred in respective overexpression plants. We also detected a delay of salinity-induced chlorophyll loss in detached anac092-1 mutant leaves. Promoter-reporter (GUS) studies revealed transcriptional control of ANAC092 expression during leaf and flower ageing and in response to salt stress. We conclude that ANAC092 exerts its functions during senescence and seed germination through partly overlapping target gene sets.}, language = {en} } @article{ArvidssonPerezRodriguezMuellerRoeber2011, author = {Arvidsson, Samuel Janne and Perez-Rodriguez, Paulino and M{\"u}ller-R{\"o}ber, Bernd}, title = {A growth phenotyping pipeline for Arabidopsis thaliana integrating image analysis and rosette area modeling for robust quantification of genotype effects}, series = {New phytologist : international journal of plant science}, volume = {191}, journal = {New phytologist : international journal of plant science}, number = {3}, publisher = {Wiley-Blackwell}, address = {Malden}, issn = {0028-646X}, doi = {10.1111/j.1469-8137.2011.03756.x}, pages = {895 -- 907}, year = {2011}, abstract = {To gain a deeper understanding of the mechanisms behind biomass accumulation, it is important to study plant growth behavior. Manually phenotyping large sets of plants requires important human resources and expertise and is typically not feasible for detection of weak growth phenotypes. Here, we established an automated growth phenotyping pipeline for Arabidopsis thaliana to aid researchers in comparing growth behaviors of different genotypes. The analysis pipeline includes automated image analysis of two-dimensional digital plant images and evaluation of manually annotated information of growth stages. It employs linear mixed-effects models to quantify genotype effects on total rosette area and relative leaf growth rate (RLGR) and ANOVAs to quantify effects on developmental times. Using the system, a single researcher can phenotype up to 7000 plants d(-1). Technical variance is very low (typically < 2\%). We show quantitative results for the growth-impaired starch-excessmutant sex4-3 and the growth-enhancedmutant grf9. We show that recordings of environmental and developmental variables reduce noise levels in the phenotyping datasets significantly and that careful examination of predictor variables (such as d after sowing or germination) is crucial to avoid exaggerations of recorded phenotypes and thus biased conclusions.}, language = {en} } @article{DortayMuellerRoeber2010, author = {Dortay, Hakan and M{\"u}ller-R{\"o}ber, Bernd}, title = {A highly efficient pipeline for protein expression in Leishmania tarentolae using infrared fluorescence protein as marker}, issn = {1475-2859}, doi = {10.1186/1475-2859-9-29}, year = {2010}, abstract = {Background: Leishmania tarentolae, a unicellular eukaryotic protozoan, has been established as a novel host for recombinant protein production in recent years. Current protocols for protein expression in Leishmania are, however, time consuming and require extensive lab work in order to identify well-expressing cell lines. Here we established an alternative protein expression work-flow that employs recently engineered infrared fluorescence protein (IFP) as a suitable and easy-to-handle reporter protein for recombinant protein expression in Leishmania. As model proteins we tested three proteins from the plant Arabidopsis thaliana, including a NAC and a type-B ARR transcription factor. Results: IFP and IFP fusion proteins were expressed in Leishmania and rapidly detected in cells by deconvolution microscopy and in culture by infrared imaging of 96-well microtiter plates using small cell culture volumes (2 mu L}, 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{OmidbakhshfardWinckArvidssonetal.2014, author = {Omidbakhshfard, Mohammad Amin and Winck, Flavia Vischi and Arvidsson, Samuel Janne and Riano-Pachon, Diego M. and M{\"u}ller-R{\"o}ber, Bernd}, title = {A step-by-step protocol for formaldehyde-assisted isolation of regulatory elements from Arabidopsis thaliana}, series = {Journal of integrative plant biology}, volume = {56}, journal = {Journal of integrative plant biology}, number = {6}, publisher = {Wiley-Blackwell}, address = {Hoboken}, issn = {1672-9072}, doi = {10.1111/jipb.12151}, pages = {527 -- 538}, year = {2014}, abstract = {The control of gene expression by transcriptional regulators and other types of functionally relevant DNA transactions such as chromatin remodeling and replication underlie a vast spectrum of biological processes in all organisms. DNA transactions require the controlled interaction of proteins with DNA sequence motifs which are often located in nucleosome-depleted regions (NDRs) of the chromatin. Formaldehyde-assisted isolation of regulatory elements (FAIRE) has been established as an easy-to-implement method for the isolation of NDRs from a number of eukaryotic organisms, and it has been successfully employed for the discovery of new regulatory segments in genomic DNA from, for example, yeast, Drosophila, and humans. Until today, however, FAIRE has only rarely been employed in plant research and currently no detailed FAIRE protocol for plants has been published. Here, we provide a step-by-step FAIRE protocol for NDR discovery in Arabidopsis thaliana. We demonstrate that NDRs isolated from plant chromatin are readily amenable to quantitative polymerase chain reaction and next-generation sequencing. Only minor modification of the FAIRE protocol will be needed to adapt it to other plants, thus facilitating the global inventory of regulatory regions across species.}, 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{HasnatZupokOlasApeltetal.2021, author = {Hasnat, Muhammad Abrar and Zupok, Arkadiusz and Olas-Apelt, Justyna Jadwiga and M{\"u}ller-R{\"o}ber, Bernd and Leimk{\"u}hler, Silke}, title = {A-type carrier proteins are involved in [4Fe-4S] cluster insertion into the radical S-adenosylmethionine protein MoaA for the synthesis of active molybdoenzymes}, series = {Journal of bacteriology}, volume = {203}, journal = {Journal of bacteriology}, number = {12}, publisher = {American Society for Microbiology}, address = {Washington}, issn = {1098-5530}, doi = {10.1128/JB.00086-21}, pages = {20}, year = {2021}, abstract = {Iron sulfur (Fe-S) clusters are important biological cofactors present in proteins with crucial biological functions, from photosynthesis to DNA repair, gene expression, and bioenergetic processes. For the insertion of Fe-S clusters into proteins, A-type carrier proteins have been identified. So far, three of them have been characterized in detail in Escherichia coli, namely, IscA, SufA, and ErpA, which were shown to partially replace each other in their roles in [4Fe-4S] cluster insertion into specific target proteins. To further expand the knowledge of [4Fe-4S] cluster insertion into proteins, we analyzed the complex Fe-S cluster-dependent network for the synthesis of the molybdenum cofactor (Moco) and the expression of genes encoding nitrate reductase in E. coli. Our studies include the identification of the A-type carrier proteins ErpA and IscA, involved in [4Fe-4S] cluster insertion into the radical Sadenosyl-methionine (SAM) enzyme MoaA. We show that ErpA and IscA can partially replace each other in their role to provide [4Fe-4S] clusters for MoaA. Since most genes expressing molybdoenzymes are regulated by the transcriptional regulator for fumarate and nitrate reduction (FNR) under anaerobic conditions, we also identified the proteins that are crucial to obtain an active FNR under conditions of nitrate respiration. We show that ErpA is essential for the FNR-dependent expression of the narGHJI operon, a role that cannot be compensated by IscA under the growth conditions tested. SufA does not appear to have a role in Fe-S cluster insertion into MoaA or FNR under anaerobic growth employing nitrate respiration, based on the low level of gene expression.
IMPORTANCE Understanding the assembly of iron-sulfur (Fe-S) proteins is relevant to many fields, including nitrogen fixation, photosynthesis, bioenergetics, and gene regulation. Remaining critical gaps in our knowledge include how Fe-S clusters are transferred to their target proteins and how the specificity in this process is achieved, since different forms of Fe-S clusters need to be delivered to structurally highly diverse target proteins. Numerous Fe-S carrier proteins have been identified in prokaryotes like Escherichia coli, including ErpA, IscA, SufA, and NfuA. In addition, the diverse Fe-S cluster delivery proteins and their target proteins underlie a complex regulatory network of expression, to ensure that both proteins are synthesized under particular growth conditions.}, language = {en} } @article{RibeiroAraujoFernieetal.2012, author = {Ribeiro, Dimas M. and Araujo, Wagner L. and Fernie, Alisdair R. and Schippers, Jos H. M. and M{\"u}ller-R{\"o}ber, Bernd}, title = {Action of Gibberellins on growth and metabolism of arabidopsis plants Associated with high concentration of carbon dioxide}, series = {Plant physiology : an international journal devoted to physiology, biochemistry, cellular and molecular biology, biophysics and environmental biology of plants}, volume = {160}, journal = {Plant physiology : an international journal devoted to physiology, biochemistry, cellular and molecular biology, biophysics and environmental biology of plants}, number = {4}, publisher = {American Society of Plant Physiologists}, address = {Rockville}, issn = {0032-0889}, doi = {10.1104/pp.112.204842}, pages = {1781 -- 1794}, year = {2012}, abstract = {Although the positive effect of elevated CO2 concentration [CO2] on plant growth is well known, it remains unclear whether global climate change will positively or negatively affect crop yields. In particular, relatively little is known about the role of hormone pathways in controlling the growth responses to elevated [CO2]. Here, we studied the impact of elevated [CO2] on plant biomass and metabolism in Arabidopsis (Arabidopsis thaliana) in relation to the availability of gibberellins (GAs). Inhibition of growth by the GA biosynthesis inhibitor paclobutrazol (PAC) at ambient [CO2] (350 mu mol CO2 mol(-1)) was reverted by elevated [CO2] (750 mu mol CO2 mol(-1)). Thus, we investigated the metabolic adjustment and modulation of gene expression in response to changes in growth of plants imposed by varying the GA regime in ambient and elevated [CO2]. In the presence of PAC (low-GA regime), the activities of enzymes involved in photosynthesis and inorganic nitrogen assimilation were markedly increased at elevated [CO2], whereas the activities of enzymes of organic acid metabolism were decreased. Under ambient [CO2], nitrate, amino acids, and protein accumulated upon PAC treatment; however, this was not the case when plants were grown at elevated [CO2]. These results suggest that only under ambient [CO2] is GA required for the integration of carbohydrate and nitrogen metabolism underlying optimal biomass determination. Our results have implications concerning the action of the Green Revolution genes in future environmental conditions.}, language = {en} } @article{SharmaDangSinghetal.2018, author = {Sharma, Niharika and Dang, Trang Minh and Singh, Namrata and Ruzicic, Slobodan and M{\"u}ller-R{\"o}ber, Bernd and Baumann, Ute and Heuer, Sigrid}, title = {Allelic variants of OsSUB1A cause differential expression of transcription factor genes in response to submergence in rice}, series = {Rice}, volume = {11}, journal = {Rice}, number = {2}, publisher = {Springer Open}, address = {London}, issn = {1939-8425}, doi = {10.1186/s12284-017-0192-z}, pages = {19}, year = {2018}, abstract = {Background: Flooding during seasonal monsoons affects millions of hectares of rice-cultivated areas across Asia. Submerged rice plants die within a week due to lack of oxygen, light and excessive elongation growth to escape the water. Submergence tolerance was first reported in an aus-type rice landrace, FR13A, and the ethylene-responsive transcription factor (TF) gene SUB1A-1 was identified as the major tolerance gene. Intolerant rice varieties generally lack the SUB1A gene but some intermediate tolerant varieties, such as IR64, carry the allelic variant SUB1A-2. Differential effects of the two alleles have so far not been addressed. As a first step, we have therefore quantified and compared the expression of nearly 2500 rice TF genes between IR64 and its derived tolerant near isogenic line IR64-Sub1, which carries the SUB1A-1 allele. Gene expression was studied in internodes, where the main difference in expression between the two alleles was previously shown. Results: Nineteen and twenty-six TF genes were identified that responded to submergence in IR64 and IR64-Sub1, respectively. Only one gene was found to be submergence-responsive in both, suggesting different regulatory pathways under submergence in the two genotypes. These differentially expressed genes (DEGs) mainly included MYB, NAC, TIFY and Zn-finger TFs, and most genes were downregulated upon submergence. In IR64, but not in IR64-Sub1, SUB1B and SUB1C, which are also present in the Sub1 locus, were identified as submergence responsive. Four TFs were not submergence responsive but exhibited constitutive, genotype-specific differential expression. Most of the identified submergence responsive DEGs are associated with regulatory hormonal pathways, i.e. gibberellins (GA), abscisic acid (ABA), and jasmonic acid (JA), apart from ethylene. An in-silico promoter analysis of the two genotypes revealed the presence of allele-specific single nucleotide polymorphisms, giving rise to ABRE, DRE/CRT, CARE and Site II cis-elements, which can partly explain the observed differential TF gene expression. Conclusion: This study identified new gene targets with the potential to further enhance submergence tolerance in rice and provides insights into novel aspects of SUB1A-mediated tolerance.}, language = {en} } @article{WinckKwasniewskiWienkoopetal.2011, author = {Winck, Flavia Vischi and Kwasniewski, Miroslaw and Wienkoop, Stefanie and M{\"u}ller-R{\"o}ber, Bernd}, title = {An optimized method for the isolation of nuclei from chlamydomas Reinhardtii (Chlorophyceae)}, series = {Journal of phycology}, volume = {47}, journal = {Journal of phycology}, number = {2}, publisher = {Wiley-Blackwell}, address = {Malden}, issn = {0022-3646}, doi = {10.1111/j.1529-8817.2011.00967.x}, pages = {333 -- 340}, year = {2011}, abstract = {The cell nucleus harbors a large number of proteins involved in transcription, RNA processing, chromatin remodeling, nuclear signaling, and ribosome assembly. The nuclear genome of the model alga Chlamydomonas reinhardtii P. A. Dang. was recently sequenced, and many genes encoding nuclear proteins, including transcription factors and transcription regulators, have been identified through computational discovery tools. However, elucidating the specific biological roles of nuclear proteins will require support from biochemical and proteomics data. Cellular preparations with enriched nuclei are important to assist in such analyses. Here, we describe a simple protocol for the isolation of nuclei from Chlamydomonas, based on a commercially available kit. The modifications done in the original protocol mainly include alterations of the differential centrifugation parameters and detergent-based cell lysis. The nuclei-enriched fractions obtained with the optimized protocol show low contamination with mitochondrial and plastid proteins. The protocol can be concluded within only 3 h, and the proteins extracted can be used for gel-based and non-gel-based proteomic approaches.}, language = {en} }