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Plant Molecular Biology

Dr. Michael Buettner



Poschet G, Hannich B, Raab S, Jungkunz I, Klemens PA, Krueger S, Neuhaus E, Büttner M. (2011). A Novel Arabidopsis Vacuolar Glucose Exporter is involved in cellular Sugar Homeostasis and affects Composition of Seed Storage Compounds. 1. Plant Physiol. 2011 Oct 7.
Abstract
Subcellular sugar partitioning in plants is strongly regulated in response to developmental cues and changes in external conditions. Besides transitory starch, the vacuolar sugars represent a highly dynamic pool of instantly accessible metabolites which serve as energy source and osmoprotectants. Here, we present the molecular identification and functional characterization of the vacuolar glucose exporter Arabidopsis thaliana ERD6-like 6 (AtERDL6). We demonstrate tonoplast localization of AtERDL6 in plants. In Arabidopsis, AtERDL6 expression is induced in response to factors that activate vacuolar glucose pools like darkness, heat stress and wounding. On the other hand, AtERDL6 transcript levels drop during conditions that trigger glucose accumulation in the vacuole like cold stress and external sugar supply. Accordingly, sugar analyses revealed that Aterdl6 mutants have elevated vacuolar glucose levels and glucose flux across the tonoplast is impaired under stress conditions. Interestingly, overexpressor lines indicated a very similar function for the ERDL6 ortholog BvIMP (beta vulgaris integral membrane protein) from sugar beet. Aterdl6 mutant plants display increased sensitivity against external glucose and mutant seeds exhibit a 10% increase in seed weight due to enhanced levels of seed sugars, proteins and lipids. Our findings underline the importance of vacuolar glucose export during regulation of cellular glucose homeostasis and composition of seed reserves.
Pubmed 
Schulz A, Beyhl D, Marten I, Wormit A, Neuhaus E, Poschet G, Büttner M, Schneider S, Sauer N, Hedrich R. (2011). Proton-driven sucrose symport and antiport are provided by the vacuolar transporters SUC4 and TMT1/2. Plant J.68(1):129-36.
Abstract
The vacuolar membrane is involved in solute uptake into and release from the vacuole, which is the largest plant organelle. In addition to inorganic ions and metabolites, large quantities of protons and sugars are shuttled across this membrane. Current models suggest that the proton gradient across the membrane drives the accumulation and/or release of sugars. Recent studies have associated AtSUC4 with the vacuolar membrane. Some members of the SUC family are plasma membrane proton/sucrose symporters. In addition, the sugar transporters TMT1 and TMT2, which are localized to the vacuolar membrane, have been suggested to function in proton-driven glucose antiport. Here we used the patch-clamp technique to monitor carrier-mediated sucrose transport by AtSUC4 and AtTMTs in intact Arabidopsis thaliana mesophyll vacuoles. In the whole-vacuole configuration with wild-type material, cytosolic sucrose-induced proton currents were associated with a proton/sucrose antiport mechanism. To identify the related transporter on one hand, and to enable the recording of symporter-mediated currents on the other hand, we electrophysiologically characterized vacuolar proteins recognized by Arabidopsis mutants of partially impaired sugar compartmentation. To our surprise, the intrinsic sucrose/proton antiporter activity was greatly reduced when vacuoles were isolated from plants lacking the monosaccharide transporter AtTMT1/TMT2. Transient expression of AtSUC4 in this mutant background resulted in proton/sucrose symport activity. From these studies, we conclude that, in the natural environment within the Arabidopsis cell, AtSUC4 most likely catalyses proton-coupled sucrose export from the vacuole. However, TMT1/2 probably represents a proton-coupled antiporter capable of high-capacity loading of glucose and sucrose into the vacuole.
Pubmed 

Schubert M, Koteyeva NK, Wabnitz PW, Santos P, Büttner M, Sauer N, Demchenko K, Pawlowski K. (2010). Plasmodesmata distribution and sugar partitioning in nitrogen-fixing root nodules of Datisca glomerata. Planta.233(1):139-52.
Abstract
To understand carbon partitioning in roots and nodules of Datisca glomerata, activities of sucrose-degrading enzymes and sugar transporter expression patterns were analyzed in both organs, and plasmodesmal connections between nodule cortical cells were examined by transmission electron microscopy. The results indicate that in nodules, the contribution of symplastic transport processes is increased in comparison to roots, specifically in infected cells which develop many secondary plasmodesmata. Invertase activities are dramatically reduced in nodules as compared to roots, indicating that here the main enzyme responsible for the cleavage of sucrose is sucrose synthase. A high-affinity, low-specificity monosaccharide transporter whose expression is induced in infected cells prior to the onset of bacterial nitrogen fixation, and which has an unusually low pH optimum and may be involved in turgor control or hexose retrieval during infection thread growth.
Pubmed 

Büttner M. (2010). The Arabidopsis sugar transporter (AtSTP) family: an update. 1. Plant Biol (Stuttg). 2010 Sep;12 Suppl 1:35-41.
Abstract
The Arabidopsis sugar transporter (AtSTP) family is one of the best characterised families within the monosaccharide transporter (MST)-like genes. However, several aspects are still poorly investigated or not yet addressed experimentally, such as post-translational modifications and other factors affecting transport activity. This mini-review summarises recent advances in the AtSTP family as well as objectives for future studies.
Pubmed 
Poschet G, Hannich B, Büttner M. (2010). Identification and characterization of AtSTP14, a novel galactose transporter from Arabidopsis. Plant Cell Physiol.51(9):1571-80.
Abstract
AtSTP14, a new Arabidopsis sugar transporter, was identified and characterized on the molecular and physiological level. Reverse transcriptase-PCR analyses and reporter plants demonstrate high AtSTP14 expression levels in the seed endosperm and in cotyledons, as well as in green leaves. Thus, unlike previously characterized monosaccharide transporters, AtSTP14 is expressed in both source and sink tissues and represents the first monosaccharide transporter in the female gametophyte. Heterologous expression in yeast revealed that AtSTP14 does not transport glucose or fructose, but is the first plant transporter specific for galactose. Interestingly, AtSTP14 expression is regulated by factors which also induce cell wall degradation such as extended dark periods or changes in the sugar level, i.e. AtSTP14 is induced 3-fold by 24 h darkness and repressed 3-fold by 2% glucose and 2% sucrose. Two independent Atstp14 mutant lines were identified, but no effect on seed development or other differences during growth under normal conditions could be observed. A putative role for AtSTP14 in the recycling of cell wall-derived galactose during different developmental processes is discussed.
Pubmed 
Büttner M. (2007). The monosaccharide transporter(-like) gene family in Arabidopsis. FEBS Lett.581(12):2318-24.
Abstract
The availability of complete plant genomes has greatly influenced the identification and analysis of phylogenetically related gene clusters. In Arabidopsis, this has revealed the existence of a monosaccharide transporter(-like) gene family with 53 members, which play a role in long-distance sugar partitioning or sub-cellular sugar distribution and catalyze the transport of hexoses, but also polyols and in one case also pentoses and tetroses. An update on the currently available information on these Arabidopsis monosaccharide transporters, on their sub-cellular localization and physiological function will be given.
Pubmed 
Aluri S, Büttner M. (2007). Identification and functional expression of the Arabidopsis thaliana vacuolar glucose transporter 1 and its role in seed germination and flowering. Proc Natl Acad Sci U S A.104(7):2537-42.
Abstract
Sugar compartmentation into vacuoles of higher plants is a very important physiological process, providing extra space for transient and long-term sugar storage and contributing to the osmoregulation of cell turgor and shape. Despite the long-standing knowledge of this subcellular sugar partitioning, the proteins responsible for these transport steps have remained unknown. We have identified a gene family in Arabidopsis consisting of three members homologous to known sugar transporters. One member of this family, Arabidopsis thaliana vacuolar glucose transporter 1 (AtVGT1), was localized to the vacuolar membrane. Moreover, we provide evidence for transport activity of a tonoplast sugar transporter based on its functional expression in bakers' yeast and uptake studies in isolated yeast vacuoles. Analyses of Atvgt1 mutant lines indicate an important function of this vacuolar glucose transporter during developmental processes like seed germination and flowering.
Pubmed 

Vignault C, Vachaud M, Cakir B, Glissant D, Dédaldéchamp F, Büttner M, Atanassova R, Fleurat-Lessard P, Lemoine R, Delrot S. (2005). VvHT1 encodes a monosaccharide transporter expressed in the conducting complex of the grape berry phloem. J Exp Bot.56(415):1409-18.
Abstract
The accumulation of sugars in grape berries requires the co-ordinate expression of sucrose transporters, invertases, and monosaccharide transporters. A monosaccharide transporter homologue (VvHT1, Vitis vinifera hexose transporter 1) has previously been isolated from grape berries at the veraison stage, and its expression was shown to be regulated by sugars and abscisic acid. The present work investigates the function and localization of VvHT1. Heterologous expression in yeast indicates that VvHT1 encodes a monosaccharide transporter with maximal activity at acidic pH (pH 4.5) and high affinity for glucose (K(m)=70 muM). Fructose, mannose, sorbitol, and mannitol are not transported by VvHT1. In situ hybridization shows that VvHT1 transcripts are primarily found in the phloem region of the conducting bundles. Immunofluorescence and immunogold labelling experiments localized VvHT1 in the plasma membrane of the sieve element/companion cell interface and of the flesh cells. The expression and functional properties of VvHT1 suggests that it retrieves the monosaccharides needed to provide the energy necessary for cell division and cell growth at an early stage of berry development.
Pubmed 

von Schweinichen C, Büttner M. (2005) Root-specific expression of a plant cell wall invertase leads to early flowering and an increase in whole plant biomass in Arabidopsis. Plant Biology 5(7):469-475Pubmed 
Schneidereit A, Scholz-Starke J, Sauer N, Büttner M. (2004). AtSTP11, a pollen tube-specific monosaccharide transporter in Arabidopsis. Planta.221(1):48-55.
Abstract
Pollen development, as well as pollen germination and pollen tube growth, requires a highly regulated supply of sugars. In this paper we describe the molecular, kinetic, and physiological characterization of AtSTP11, a new member of the H+/monosaccharide transporter family in Arabidopsis thaliana (L.) Heynh. Heterologous expression in yeast (Saccharomyces cerevisiae) showed that AtSTP11 is a high-affinity (Km = 25 microM), broad-spectrum, and uncoupler-sensitive monosaccharide transporter of the plasma membrane. In reverse transcription-polymerase chain reaction analyses we found that AtSTP11 expression is restricted to flowers. Furthermore, AtSTP11-promoter::GFP plants revealed that AtSTP11 expression is only found in pollen tubes. Using a specific antibody we could also detect the AtSTP11 protein exclusively in pollen tubes but not in other flower tissues or in pollen grains of any developmental stage. These results suggest that the newly identified AtSTP11 transporter plays a role in the supply of monosaccharides to growing pollen tubes.
Pubmed 
Stadler R, Büttner M, Ache P, Hedrich R, Ivashikina N, Melzer M, Shearson SM, Smith SM, Sauer N. (2003). Diurnal and light-regulated expression of AtSTP1 in guard cells of Arabidopsis. Plant Physiol.133(2):528-37.
Abstract
Guard cell chloroplasts are unable to perform significant photosynthetic CO2 fixation via Rubisco. Therefore, guard cells depend on carbon supply from adjacent cells even during the light period. Due to their reversible turgor changes, this import cannot be mediated by plasmodesmata. Nevertheless, guard cells of several plants were shown to use extracellular sugars or to accumulate sucrose as an osmoticum that drives water influx to increase stomatal aperture. This paper describes the first localization of a guard cell-specific Arabidopsis sugar transporter involved in carbon acquisition of these symplastically isolated cells. Expression of the AtSTP1 H+-monosacharide symporter gene in guard cells was demonstrated by in situ hybridization and by immunolocalization with an AtSTP1-specific antiserum. Additional RNase protection analyses revealed a strong increase of AtSTP1 expression in the dark and a transient, diurnally regulated increase during the photoperiod around midday. This transient increase in AtSTP1 expression correlates in time with the described guard cell-specific accumulation of sucrose. Our data suggest a function of AtSTP1 in monosaccharide import into guard cells during the night and a possible role in osmoregulation during the day.
Pubmed 
Schneidereit A, Scholz-Starke J, Büttner M. (2003). Functional characterization and expression analyses of the glucose-specific AtSTP9 monosaccharide transporter in pollen of Arabidopsis. Plant Physiol.133(1):182-90.
Abstract
A genomic clone and the corresponding cDNA of a new Arabidopsis monosaccharide transporter AtSTP9 were isolated. Transport analysis of the expressed protein in yeast showed that AtSTP9 is an energy-dependent, uncoupler-sensitive, high-affinity monosaccharide transporter with a K(m) for glucose in the micromolar range. In contrast to all previously characterized monosaccharide transporters, AtSTP9 shows an unusual specificity for glucose. Reverse transcriptase-polymerase chain reaction analyses revealed that AtSTP9 is exclusively expressed in flowers, and a more detailed approach using AtSTP9 promoter/reporter plants clearly showed that AtSTP9 expression is restricted to the male gametophyte. AtSTP9 expression is not found in other floral organs or vegetative tissues. Further localization on the cellular level using a specific antibody revealed that in contrast to the early accumulation of AtSTP9 transcripts in young pollen, the AtSTP9 protein is only found weakly in mature pollen but is most prominent in germinating pollen tubes. This preloading of pollen with mRNAs has been described for genes that are essential for pollen germination and/or pollen tube growth. The pollen-specific expression found for AtSTP9 is also observed for other sugar transporters and indicates that pollen development and germination require a highly regulated supply of sugars.
Pubmed 
Scholz-Starke J, Büttner M, Sauer N. (2003). AtSTP6, a new pollen-specific H+-monosaccharide symporter from Arabidopsis. Plant Physiol.131(1):70-7.
Abstract
This paper describes the molecular, kinetic, and physiological characterization of AtSTP6, a new member of the Arabidopsis H(+)/monosaccharide transporter family. The AtSTP6 gene (At3g05960) is interrupted by two introns and encodes a protein of 507 amino acids containing 12 putative transmembrane helices. Expression in yeast (Saccharomyces cerevisiae) shows that AtSTP6 is a high-affinity (K(m) = 20 microM), broad-spectrum, and uncoupler-sensitive monosaccharide transporter that is targeted to the plasma membrane and that can complement a growth deficiency resulting from the disruption of most yeast hexose transporter genes. Analyses of AtSTP6-promoter::GUS plants and in situ hybridization experiments detected AtSTP6 expression only during the late stages of pollen development. A transposon-tagged Arabidopsis mutant was isolated and homozygous plants were analyzed for potential effects of the Atstp6 mutation on pollen viability, pollen germination, fertilization, and seed production. However, differences between wild-type and mutant plants could not be observed.
Pubmed 

Büttner M, Truernit E, Baier K Scholz-Starke J, Sontheim M, Lauterbach C, Huß VAR, Sauer N. (2000). AtSTP3, a green leaf-specific, low affinity monosaccharide-H+ symporter of Arabidopsis thaliana. Plant, Cell & Environment 23.75-184

Büttner M, Sauer N. (2000). Monosaccharide transporters in plants: structure, function and physiology. Biochim Biophys Acta.1465(1-2):263-74.
Abstract
Monosaccharide transport across the plant plasma membrane plays an important role both in lower and higher plants. Algae can switch between phototrophic and heterotrophic growth and utilize organic compounds, such as monosaccharides as additional or sole carbon sources. Higher plants represent complex mosaics of phototrophic and heterotrophic cells and tissues and depend on the activity of numerous transporters for the correct partitioning of assimilated carbon between their different organs. The cloning of monosaccharide transporter genes and cDNAs identified closely related integral membrane proteins with 12 transmembrane helices exhibiting significant homology to monosaccharide transporters from yeast, bacteria and mammals. Structural analyses performed with several members of this transporter superfamily identified protein domains or even specific amino acid residues putatively involved in substrate binding and specificity. Expression of plant monosaccharide transporter cDNAs in yeast cells and frog oocytes allowed the characterization of substrate specificities and kinetic parameters. Immunohistochemical studies, in situ hybridization analyses and studies performed with transgenic plants expressing reporter genes under the control of promoters from specific monosaccharide transporter genes allowed the localization of the transport proteins or revealed the sites of gene expression. Higher plants possess large families of monosaccharide transporter genes and each of the encoded proteins seems to have a specific function often confined to a limited number of cells and regulated both developmentally and by environmental stimuli.
Pubmed 

Hauska G, Büttner M. (1997). The cytochrome b6f/bc1-complexes, in Bioelectrochemistry: Principles and Practice, Vol.4 "Bioenergetics" (P. Gräber and G. Milazzo, eds), Birkhäuser Verlag, Basel, pp. 389-417

Büttner M, Singh KB. (1997). Arabidopsis thaliana ethylene-responsive element binding protein (AtEBP), an ethylene-inducible, GCC box DNA-binding protein interacts with an ocs element binding protein. Proc Natl Acad Sci U S A.94(11):5961-6.
Abstract
Ocs elements are a group of promoter sequences required for the expression of both pathogen genes in infected plants and plant defense genes. Genes for ocs element binding factors (OBFs), belonging to a specific class of basic-region leucine zipper (bZIP) transcription factors, have been isolated in a number of plants. Using protein-protein interaction screening with OBF4 we have isolated AtEBP, an Arabidopsis protein that contains a novel DNA-binding domain, the AP2/EREBP domain. One class of proteins that contain this domain are the tobacco ethylene-responsive element binding proteins (EREBPs). The EREBPs bind the GCC box that confers ethylene responsiveness to a number of pathogenesis related (PR) gene promoters. AtEBP expression is inducible by exogenous ethylene in wild-type plants and AtEBP transcripts are increased in the ctr1-1 mutant, where ethylene-regulated pathways are constitutively active. Electrophoretic mobility-shift assay and DNase I footprint analysis revealed that AtEBP can specifically bind to the GCC box. Interestingly, the highest level of AtEBP expression was detected in callus tissue, where ocs elements are very active. Synergistic effects of the GCC box with ocs elements or the related G-box sequence have been previously observed, for example, in the ethylene-induced expression of a PR gene promoter. Our results suggest that cross-coupling between EREBP and bZIP transcription factors occurs and may therefore be important in regulating gene expression during the plant defense response.
Pubmed 
Zhang B, Chen W, Foley RC, Büttner M, Singh KB. (1995). Interactions between distinct types of DNA binding proteins enhance binding to ocs element promoter sequences. Plant Cell.7(12):2241-52.
Abstract
Octopine synthase (ocs) elements are a group of promoter elements that have been exploited by plant pathogens to express genes in plants. ocs elements are components of the promoters of certain plant glutathione S-transferase genes and may function as oxidative stress response elements. Genes for ocs element binding factors (OBFs), which belong to a specific class of highly conserved, plant basic domain-leucine zipper transcription factors, have been isolated and include the Arabidopsis OBF4 and OBF5 genes. To characterize proteins that modulate the activity of the OBF proteins, we screened an Arabidopsis cDNA library with the labeled OBF4 protein and isolated OBP1 (for OBF binding protein). OBP1 contains a 51-amino acid domain that is highly conserved with two plant DNA binding proteins, which we refer to as the MOA domain. OBP1 is also a DNA binding protein and binds to the cauliflower mosaic virus 35S promoter at a site distinct from the ocs element in the 35S promoter. OBP1 specifically increased the binding of the OBF proteins to ocs element sequences, raising the possibility that interactions between these proteins are important for the activity of the 35S promoter.
Pubmed 
Hager-Braun C, Xie DL, Jarosch U, Herold E, Büttner M, Zimmermann R, Deutzmann R, Hauska G, Nelson N. (1995). Stable photobleaching of P840 in Chlorobium reaction center preparations: presence of the 42-kDa bacteriochlorophyll a protein and a 17-kDa polypeptide. Biochemistry.34(29):9617-24.
Abstract
Simple procedures for the anaerobic preparation of photoactive and stable P840 reaction centers from Chlorobium tepidum and Chlorobium limicola in good yield are presented and quantitated. The subunit composition was tested by cosedimentation in sucrose density gradients. For C. limicola, it minimally comprises four subunits: the P840 reaction center protein PscA, the BChla antenna protein FMO, the FeS protein PscB with centers A and B, and a positively charged 17-kDa protein denoted PscD. The preparation from Chlorobium tepidum additionally contained PscC, a cytochrome c-551. The BChla absorption peak of the purified complexes was at 810 nm, with a shoulder at 835 nm. The ratio of the shoulder to the peak was 0.25, which corresponds to 1 reaction center per 70 BChla molecules if a uniform extinction coefficient of BChla is assumed. However, bleaching at 610 nm in continuous light corresponded up to 1 photoactive reaction center per 50 BChla molecules. Therefore, either the extinction coefficient of BChla in the reaction center is overestimated or the one for photobleaching is underestimated. In any case, the major portion of the reaction center was photoactive in the preparations. A P840 reaction center subcomplex, lacking PscD and deficient in FMO and PscB, but retaining the cytochrome c subunit, was obtained as a side product. It was photoinactive and had an absorption peak at 814 nm and a 835/814 absorbance ratio of 0.42. FMO and PscB show the tendency to form a complementary subcomplex. FMO and PscD are apparently required to stabilize the photoactive reaction center, while the cytochrome c subunit is not.
Pubmed 

Schütz M, Zirngibl S, le Coutre J, Büttner M, Xie DL, Nelson N, Deutzmann R, Hauska G. (1994). A transcription unit for the Rieske FeS-protein and cytochrome b in Chlorobium limicola. Photosynthesis Research 39.163-174
Büttner M, Xie DL, Nelson H, Pinther W, Hauska G, Nelson N. (1992). Photosynthetic reaction center genes in green sulfur bacteria and in photosystem 1 are related. Proc Natl Acad Sci U S A.89(17):8135-9.
Abstract
Oxygenic photosynthesis of chloroplasts and cyanobacteria involves two photosystems, which originate from different prokaryotic ancestors. The reaction center of photo-system 2 (PS2) is related to the well-characterized reaction center of purple bacteria, while the reaction center of photosystem 1 (PS1) is related to the green sulfur bacteria, as is convincingly documented here. An operon encoding the P840 reaction center of Chlorobium limicola f.sp. thiosulfatophilum has been cloned and sequenced. It contains two structural genes, coding for proteins of 730 and 232 amino acids. The first protein resembles the large subunits of the PS1 reaction center. Putative binding elements for the primary donor, P840 in Chlorobium and P700 in PS1, and for the acceptors A0, A1, and FeS center X are conserved. The second protein is related to the PS1 subunit carrying the FeS centers A and B. An adjacent third gene, not belonging to the reaction center, encodes a protein related to dolichyl-phosphate-D-mannose synthase from yeast. The different origins of PS1 and PS2 are discussed.
Pubmed 
Büttner M, Xie DL, Nelson H, Pinther W, Hauska G, Nelson N. (1992). The photosystem I-like P840-reaction center of green S-bacteria is a homodimer. Biochim Biophys Acta.1101(2):154-6.
Abstract
An operon encoding the P840 reaction center of Chlorobium limicola f.sp.thiosulfatophilum has been cloned and sequenced. It contains two structural genes coding for proteins of 730 and 232 amino acids. The first protein resembles the large subunits of the Photosystem I (PS I) reaction center. Putative binding elements for the primary donor, P840 in Chlorobium and P700 in PS I and for the acceptors A(o), A(1) and FeS-center X are conserved. The second protein is related to the PS I subunit carrying the FeS-centers A and B. Since all our efforts to find a gene for a second, large subunit failed, the P840 reaction center probably is homodimeric.
Pubmed 

Xie DL, Büttner M, Nelson H, Chitnis P, Pinther W, Hauska G, Nelson N. (1992) A transcription unit for the PS I-like P840-reaction center of the green S-Bacterium Chlorobium limicola. in Research in Photosynthesis (Murata, N. ed.) pp. I.4.513-520, Kluwer Academic Publishers

Zirngibl S, Xie DL, Riedl A, Nitschke W, Nelson H, Nelson N, Liebl U, le Coutre J, Hauska G, Kellner E, Grodzitzki D, Büttner M. (1992). Low potential Rieske FeS-centers of menaquinol oxidizing cytochrome bc complexes. in Research in Photosynthesis (Murata, N. ed.) pp. II.7.471-478, Kluwer Academic Publishers


/var/www/cos/ / https://www.cos.uni-heidelberg.de/ Dr. Michael Buettner _e