Biomedical subjects

L Willmitzer

Publications and source records attributed to L Willmitzer.

At least 19 recordsLinked to original sources

PubMed1999

Electric signaling and pin2 gene expression on different abiotic stimuli depend on a distinct threshold level of endogenous abscisic acid in several abscisic acid-deficient tomato mutants

Experiments were performed on three abscisic acid (ABA)-deficient tomato (Lycopersicon esculentum Mill.) mutants, notabilis, flacca, and sitiens, to investigate the role of ABA and jasmonic acid (JA) in the generation of electrical signals and Pin2 (proteinase inhibitor II) gene expression. We selected these mutants because they contain different levels of endogenous ABA. ABA levels in the mutant sitiens were reduced to 8% of the wild type, in notabilis they were reduced to 47%, and in flacca they were reduced to 21%. In wild-type and notabilis tomato plants the induction of Pin2 gene expression could be elicited by heat treatment, current application, or mechanical wounding. In flacca and sitiens only heat stimulation induced Pin2 gene expression. JA levels in flacca and sitiens plants also accumulated strongly upon heat stimulation but not upon mechanical wounding or current application. Characteristic electrical signals evolved in the wild type and in the notabilis and flacca mutants consisting of a fast action potential and a slow variation potential. However, in sitiens only heat evoked electrical signals; mechanical wounding and current application did not change the membrane potential. In addition, exogenous application of ABA to wild-type tomato plants induced transient changes in membrane potentials, indicating the involvement of ABA in the generation of electrical signals. Our data strongly suggest the presence of a minimum threshold value of ABA within the plant that is essential for the early events in electrical signaling and mediation of Pin2 gene expression upon wounding. In contrast, heat-induced Pin2 gene expression and membrane potential changes were not dependent on the ABA level but, rather, on the accumulation of JA.

Journal Article↗

PubMed1998

Differences in chain length distribution of inulin from Cynara scolymus and Helianthus tuberosus are reflected in a transient plant expression system using the respective 1-FFT cDNAs.

A newly isolated cDNA clone, Cy3, encoding the fructan fructan 1-fructosyltransferase (1-FFT) from artichoke was expressed using tobacco protoplasts as expression system. Analysis of the inulin molecules synthesized upon incubation of protoplast extracts with a mixture of oligofructans (DP3-5) shows the production of inulins with a degree of polymerization (DP) of up to 23, whereas parallel experiments performed using a 1-FFT cDNA from Jerusalem artichoke led to the production of fructans with a DP of up to only 12. The results of in vitro fructan synthesis catalyzed by transiently expressed enzymes therefore reflect the difference of in vivo fructan composition of Jerusalem artichoke (M(DP) = 8-10) and artichoke (M(DP) = 65). These data suggest that the fructan pattern in a given species is mainly defined by the enzymatic characteristics of 1-FFT.

Amino Acid Sequence↗

PubMed1998

Inhibition of a starch-granule-bound protein leads to modified starch and repression of cold sweetening.

We have cloned a gene involved in starch metabolism that was identified by the ability of its product to bind to potato starch granules. Reduction in the protein level of transgenic potatoes leads to a reduction in the phosphate content of the starch. The complementary result is obtained when the protein is expressed in Escherichia coli, as this leads to an increased phosphate content of the glycogen. It is possible that this protein is responsible for the incorporation of phosphate into starch-like glucans, a process that is not understood at the biochemical level. The reduced phosphate content in potato starch has some secondary effects on its degradability, as the respective plants show a starch excess phenotype in leaves and a reduction in cold-sweetening in tubers.

Amino Acid Sequence↗

PubMed1998

Transgenic plants changed in carbon allocation pattern display a shift in diurnal growth pattern.

Photosynthesis, partitioning of carbohydrates and growth have to be highly orchestrated to enable an efficient performance of plants. To study the diurnal relationships between carbon distribution and growth, we analysed transgenic potato plants with altered carbon allocation patterns. To modify carbohydrate supply of growing sinks, we used plants that accumulated starch as a consequence of inhibition in triose-phosphate export from chloroplasts and plants that were genetically inhibited in starch production. Carbon assimilation was analysed by gas exchange and single cell analysis of source leaves. Export was determined by microanalysis of phloem exudates and internodal growth rates were measured by displacement transducers. Gas exchange measurements showed similar assimilation rates in the wild-type and transgenic plants during the light period. Sugar analysis of phloem exudates and epidermal cells revealed a severe shift of sucrose concentrations in the individual plant lines. Moreover, epidermal cells turned out to be a potential storage site for carbohydrates in potato. Finally, we could demonstrate that changing the diurnal rhythm of carbon allocation results in a change in the diurnal growth pattern.

Animals↗

PubMed1998

Inactivation of a glycyl-tRNA synthetase leads to an arrest in plant embryo development.

Embryo formation is the first patterning process during vegetative plant growth. Using transposons as insertional mutagens in Arabidopsis, we identified the mutant edd1 that shows embryo-defective development. The insertion mutation is lethal, arresting embryo growth between the globular and heart stages of embryonic development. The mutant phenotype cosegregates with a transposed Dissociation element. Sequences flanking the transposed element were isolated and used to isolate a full-length cDNA clone representing the wild-type EDD1 gene. Complementation of the mutant through Agrobacterium-mediated gene transfer of an EDD1 wild-type copy as well as loss of the transposon concomitant with phenotypic reversion demonstrated that the transposon had caused the mutation. Based on homology to Escherichia coli, the EDD1 gene is predicted to encode a novel glycyl-tRNA synthetase (GlyRS) that has not been identified previously in higher plants. An N-terminal portion of the plant protein is able to direct a marker protein into pea chloroplasts. Thus, the gene identified by the embryo-defective insertion mutation encodes a GlyRS homolog, probably acting within the plastidic compartment.

Amino Acid Sequence↗

PubMed1998

Production of 1-kestose in transgenic yeast expressing a fructosyltransferase from Aspergillus foetidus.

Sucrose-inducible secretory sucrose:sucrose 1-fructosyltransferase (1-SST) from Aspergillus foetidus has been purified and subjected to N-terminal amino acid sequence determination. The enzyme is extensively glycosylated, and the active form is probably represented by a dimer of identical subunits with an apparent molecular mass of 180 kDa as judged from mobility in seminative acrylamide gels. The enzyme catalyzes fructosyl transfer from sucrose to sucrose producing glucose and 1-kestose. Oligosaccharides with a higher degree of polymerization are not obtained with sucrose as the substrate. The cDNA encoding the A. foetidus 1-SST has been cloned and sequenced. Sequence homology was found to be highest to levanases, but no hydrolytic activity was observed when levan was incubated with the enzyme. Expression of the cloned gene in an invertase-deficient mutant of Saccharomyces cerevisiae resulted in 1-kestose production, with 6-kestose and neokestose being side products of the reaction. Products were well distinguishable from those formed by yeast transformants expressing a cytosolic invertase.

Amino Acid Sequence↗

PubMed1997

Solute accumulation and decreased photosynthesis in leaves of potato plants expressing yeast-derived invertase either in the apoplast, vacuole or cytosol.

Potato (Solanum tuberosum cv. Désirée) plants expressing yeast invertase directed either to the apoplast, vacuole or cytosol were biochemically and physiologically characterised. All lines of transgenic plants showed similarities to plants growing under water stress. Transformants were retarded in growth, and accumulated hexoses and amino acids, especially proline, to levels up to 40-fold higher than those of the wild types. In all transformants rates of CO2 assimilation and leaf conductance were reduced. From the unchanged intercellular partial pressure of CO2 and apoplastic cis-abscisic acid (ABA) content of transformed leaves it was concluded that the reduced rate of CO2 assimilation was not caused by a limitation in the availability of CO2 for the ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco). In the transformants the amount of Rubisco protein was not reduced, but both activation state and carboxylation efficiency of photosynthesis were lowered. In vacuolar and cytosolic transformants this inhibition of Rubisco might be caused by a changed ratio of organic bound and inorganic phosphate, as indicated by a doubling of phosphorylated intermediates. But in apoplastic transformants the pattern of phosphorylated intermediates resembled that of leaves of water-stressed potato plants, although the cause of inhibition of photosynthesis was not identical. Whereas in water-stressed plants increased contents of the phytohormone ABA are supposed to mediate the adaptation to water stress, no contribution of ABA to reduction of photosynthesis could be detected in invertase transformants.

Abscisic Acid↗

PubMed1997

Two genes encoding extension-like proteins are predominantly expressed in tomato root hair cells.

A differential screen of a tomato root hair cDNA library resulted in the cloning of two cDNAs, Dif10 and Dif54, whose corresponding genes are preferentially expressed in root hair cells as determined by analysis of mRNA levels in various tomato organs. Transcript levels showed no increase in leaves subjected to hormonal and environmental stress treatments. Sequence analysis of the cDNAs revealed high similarity to members of the extension family. Extensions are hydroxyproline-rich glycoproteins (HRGPs) located in the cell wall. In order to study the functional significance of HRGPs in root hairs, tomato seedling roots were treated with micromolar concentrations of 3,4-dehydro-L-proline (Dhp), a selective inhibitor of prolyl hydroxylase. Dhp treatment resulted in changes in root growth and the development of root hairs with reduced hair length, suggesting an important role of HRGPs in hair morphogenesis.

Amino Acid Sequence↗

PubMed1997

Increased potato tuber size resulting from apoplastic expression of a yeast invertase.

The role of sucrose cleavage in determining sink strength in potato was investigated by generating transgenic potato plants that expressed a yeast invertase in either the cytosol or apoplast of tubers. Cytosolic localization gave rise to a reduction in tuber size and an increase in tuber number per plant whereas apoplastic targeting led to an increase in tuber size and a decrease in tuber number per plant. Sink organ size can be manipulated through modification of sucrose metabolism.

Chimera↗

PubMed1997

Identification of mutants in metabolically regulated gene expression.

Sucrose is the main transported form of assimilates, but, significantly, it also regulates a variety of processes such as photosynthesis and carbon or nitrogen storage. The effects of high sucrose levels are mediated directly by modulation of gene expression. The regulation of storage protein accumulation, here patatin from potato tubers, was used as a model system to study sucrose mediated signal transduction. The transcriptional regulation of patatin genes in conserved in transgenic Arabidopsis, as shown by the analysis of expression of two classes of patatin promoters fused to uidA. Two distinctly different patterns of gene expression were observed. In roots, class I promoter expression is strongly dependent on the exogenous supply of sugars. 3-O-methylglucose induction indicates that the sensor is located upstream of hexokinase. In contrast, the class II promoter is constitutively active in root tips and hydatodes. The progeny of a homozygous class I line was mutagenized with ethyl methane sulphonate and screened for signal transduction mutants using a non-destructive screening system for GUS activity. Four mutants showing reduced sucrose responses (rsr) and two mutants with modified expression patterns (mep) regarding the root tip were identified. In backcross analyses, it was shown that rsr1-1 carries a recessive trans mutation whereas rsr4-1 seems to be a semi-dominant trans mutation in sugar-mediated gene regulation.

Arabidopsis↗

PubMed1997

Antisense inhibition of cytosolic phosphorylase in potato plants (Solanum tuberosum L.) affects tuber sprouting and flower formation with only little impact on carbohydrate metabolism.

To determine the function of cytosolic phosphorylase (Pho2; EC 2.4.1.1), transgenic potato plants were created in which the expression of the enzyme was inhibited by introducing a chimeric gene containing part of the coding region for cytosolic phosphorylase linked in antisense orientation to the 35S CaMV promotor. As revealed by Northern blot analysis and native polyacrylamide gel electrophoresis, the expression of cytosolic phosphorylase was strongly inhibited in both leaves and tubers of the transgenic plants. The transgenic plants propagated from stem cuttings were morphologically indiscernible from the wild-type. However, sprouting of the transgenic potato tubers was significantly altered: compared with the wild-type, transgenic tubers produced 2.4 to 8.1 times more sprouts. When cultivated in the greenhouse, transgenic seed tubers produced two to three times more shoots than the wild-type. Inflorescences appeared earlier in the resulting plants. Many of the transgenic plants flowered two or three times successively. Transgenic plants derived from seed tubers formed 1.6 to 2.4 times as many tubers per plant as untransformed controls. The size and dry matter content of the individual tubers was not noticeably altered. Tuber yield was significantly higher in the transgenic plants. As revealed by carbohydrate determination of freshly harvested and stored tubers, starch and sucrose pools were not noticeably affected by the antisense inhibition of cytosolic phosphorylase; however, glucose and fructose levels were markedly reduced after prolonged storage. These results favour the view that cytosolic phosphorylase does not participate in starch degradation. The possible links between the reduced levels of cytosolic phosphorylase and the observed changes with respect to sprouting and flowering are discussed.

Agrobacterium tumefaciens↗

PubMed1997

Transgenic potato tubers accumulate high levels of 1-kestose and nystose: functional identification of a sucrose sucrose 1-fructosyltransferase of artichoke (Cynara scolymus) blossom discs.

By screening a cDNA library of artichoke (Cynara scolymus) blossom discs for fructosyltransferases, we isolated a clone designated Cy21. The deduced amino acid sequence shows homology to acid beta-fructosyl hydrolases and to the sucrose-fructan 6-fructosyltransferase (6-SFT) of barley. Transiently expressed in Nicotiana tabacum protoplasts, the Cy21 gene-product synthesized 1-kestose, indicating that Cy21 codes for a sucrose sucrose 1-fructosyltransferase (1-SST). The enzyme worked at physiologically relevant sucrose concentrations (25 mM sucrose). In the protoplast system, 1-kestose seemed to be the only fructan product of the 1-SST. The enzyme activity was not affected by pyridoxal-HCl, an inhibitor of both the beta-fructosyl hydrolase and the fructosyltransferase activity of invertases. The fructosyltransferase activity of the Cy21 gene-product, however, could be inhibited by Zn2+, Ag+ and Cu2+ ions. In artichoke plants the Cy21 transcript was highly abundant in primary roots and blossom discs. Transgenic potato tubers expressing Cy21 contain high levels of 1-kestose along with nystose and traces of fructosyl-nystose, supporting the conclusion that the Cy21 clone encodes a sucrose sucrose 1-fructosyltransferase.

Amino Acid Sequence↗

PubMed1997

Two cDNAs from potato are able to complement a phosphate uptake-deficient yeast mutant: identification of phosphate transporters from higher plants.

Acquisition as well as translocation of phosphate are essential processes for plant growth. In many plants, phosphate uptake by roots and distribution within the plant are presumed to occur via a phosphate/proton cotransport mechanism. Here, we describe the isolation of two cDNAs, StPT1 and StPT2, from potato (Solanum tuberosum) that show homology to the phosphate/proton cotransporter PHO84 from the yeast Saccharomyces cerevisiae. The predicted products of both cDNAs share 35% identity with the PHO84 sequence. The deduced structure of the encoded proteins revealed 12 membrane-spanning domains with a central hydrophilic region. The molecular mass was calculated to be 59 kD for the StPT1 protein and 58 kD for the StPT2 protein. When expressed in a PHO84-deficient yeast strain, MB192, both cDNAs complemented the mutant. Uptake of radioactive orthophosphate by the yeast mutant expressing either StPT1 or StPT2 was dependent on pH and reduced in the presence of uncouplers of oxidative phosphorylation, such as 2,4-dinitrophenol or carbonyl cyanide m-chlorophenylhydrazone. The K(m) for Pi uptake of the StPT1 and StPT2 proteins was determined to be 280 and 130 microM, respectively. StPT1 is expressed in roots, tubers, and source leaves as well as in floral organs. Deprivation of nitrogen, phosphorus, potassium, and sulfur changed spatial expression as well as the expression level of StPT1. StPT2 expression was detected mainly in root organs when plants were deprived of Pi and to a lesser extent under sulfur deprivation conditions. No expression was found under optimized nutrition conditions or when other macronutrients were lacking.

Amino Acid Sequence↗

PubMed1997

Cloning and characterization of the gene for amylosucrase from Neisseria polysaccharea: production of a linear alpha-1,4-glucan.

The gene for the amylosucrase from Neisseria polysaccharea (ATCC 43768) was cloned by use of a functional expression system in Escherichia coli XL1-Blue. The deduced amino acid sequence of the protein has homology to the sequences of the alpha-amylase class of enzymes, with the highest similarities being found to the sequences of the trehalose synthase from Pimelobacter sp. strain R48 (17) and amylomaltase from Thermotoga maritima (11). However, the regions of highest homology within the alpha-amylase class of enzymes, which are essential for the catalytic activity, are only scarcely found in the sequence of amylosucrase. By using the enzyme isolated from culture supernatants of transformed E. coli cells, it is possible to synthesize linear alpha-1,4-glucans from sucrose, indicating that the enzyme is not capable of producing alpha-1,6-glycosidic linkages on its own.

Amino Acid Sequence↗

PubMed1996

Expression analysis of a sucrose synthase gene from sugar beet (Beta vulgaris L.).

To investigate the expression pattern of sucrose synthase, a cDNA from tap roots of sugar beet (Beta vulgaris L.) was isolated using a heterologous sucrose synthase cDNA from potato. The 2762 bp long cDNA clone designated SBSS 1 encodes for a 822 amino acid polypeptide of a predicted molecular mass of 93.7 kDa. The deduced amino acid sequence of sugar beet sucrose synthase has homologies of 65-70% when compared to predicted amino acid sequences of sucrose synthases from other species. RNA blot analysis shows that SBSS1 is expressed most predominant in tap root under normal growth conditions. Cold treatment and anaerobiosis lead to an increase in the steady-state levels of SBSS 1 mRNA in leaf and root tissue. In tap root slices, sugars in various concentrations had no influence on the SBSS 1 transcript level. On the other hand, wounding resulted in a decreased transcript level.

Amino Acid Sequence↗

PubMed1996

Synthesis of fructans in tubers of transgenic starch-deficient potato plants does not result in an increased allocation of carbohydrates.

Inhibition of starch biosynthesis in transgenic potato (Solanum tuberosum L. cv. Désirée) plants (by virtue of antisense inhibition of ADP-glucose pyrophosphorylase) has recently been reported to influence tuber formation and drastically reduce dry matter content of tubers, indicating a reduction in sink strength (Müller-Röber et al. 1992, EMBO J 11: 1229-1238). Transgenic tubers produced low levels of starch, but instead accumulated high levels of soluble sugars. We wanted to know whether these changes in tuber development/sink strength could be reversed by the production of a new high-molecular-weight polymer, i.e. fructan, that incorporates sucrose and thereby should reduce the level of osmotically active compounds. To this end the enzyme levan sucrase from the gram-negative bacterium Erwinia amylovora was expressed in tubers of transgenic potato plants inhibited for starch biosynthesis. Levan sucrase was targeted to different subcellular compartments (apoplasm, vacuole and cytosol). Only in the case of apoplastic and vacuolar targeting was significant accumulation of fructan observed, leading to fructan representing between 12% and 19% of the tuber dry weight. Gel filtration and 13C-nuclear magnetic resonance spectroscopy showed that the molecular weight and structure of the fructan produced in transgenic plants is identical to levan isolated from E. amylovora. Whereas apoplastic expression of levansucrase had deleterious effects on tuber development, tubers containing the levansucrase in the vacuole did not differ in phenotype from tubers of the starch-deficient plants used as starting material for transformation with the levansucrase. When tuber yield was analysed, no increase but rather a further decrease relative to ADP-glucose pyro-phosphorylase antisense plants was observed.

Carbohydrate Metabolism↗

PubMed1996

Cloning and functional analysis of a cDNA encoding a novel 139 kDa starch synthase from potato (Solanum tuberosum L.).

Three isoforms of starch synthase were shown to be present in soluble potato tuber extracts by activity staining after native gel electrophoresis. An antibody directed against a domain conserved in starch synthases was used to clone a cDNA for one of these isoforms by screening a tuber-specific expression library. A partial cDNA of 2.6 kbp was obtained and used to isolate a full-length cDNA of 4167 bp. The deduced amino acid sequence identifies the protein as a novel type of starch synthase from potato with a molecular mass of 139.2 kDa for the immature enzyme including its transit peptide. This novel isoform was designated SS III. An analysis of the expression pattern of the gene indicates that SS III is equally expressed in tubers of different developmental stages as well as in sink and source leaves. In several independent transgenic potato lines, where the expression of SS III was repressed using the antisense approach, the activity of a specific starch synthase isoform was reduced to non-detectable levels as determined through activity staining after native gel electrophoresis. The reduction of this isoform of starch synthase leads to the synthesis of a structurally modified starch in the transgenic plants: there is a drastic change in granule morphology and an increased level of covalently linked phosphate.

Amino Acid Sequence↗

PubMed1996

Structure and expression of mitochondrial citrate synthases from higher plants.

Mitochondrial citrate synthase (EC 4.1.3.7) represents the first enzyme of the tricarboxylic acid cycle, catalyzing the condensation of acetyl-CoA and oxaloacetate, finally yielding citrate and CoA. We report here the isolation of cDNA clones encoding citrate synthase from Nicotiana tabacum, Beta vulgaris and Populus. Nucleotide and deduced amino acid sequences were compared with previously published sequences of mitochondrial citrate synthases from Arabidopsis thaliana and potato, as well as with the sequence of glyoxysomal citrate synthase from pumpkin. Homologies between the various plant mitochondrial enzymes were in the range from 77.2% (potato vs. Arabidopsis) to 94.2% (potato vs. tobacco) on the nucleotide level (coding regions only), and in the range from 70.1% to 90.4% (potato vs. Arabidopsis, and potato vs. tobacco, respectively) on the amino acid level. Identities of the mitochondrial isozymes to the pumpkin glyoxysomal enzyme were below 30% on the nucleotide and amino acid level. In Northern blot experiments citrate synthase mRNA was detected in all tissues analyzed. However, levels of expression showed tissue dependency despite the fact that citrate synthase is usually considered a house-keeping enzyme. Whether these different levels of expression reflect tissue-specific variations with respect to basic metabolism awaits further analysis.

Amino Acid Sequence↗