Are small GTPases signal hubs in sugar-mediated induction of fructan biosynthesis?
(2009)
Journal - PloS one (United States )
Abstract :
External sugar initiates biosynthesis of the reserve carbohydrate fructan, but the molecular processes mediating this response remain obscure. Previously it was shown that a phosphatase and a general kinase inhibitor hamper fructan accumulation. We use various phosphorylation inhibitors both in barley and in Arabidopsis and show that the expression of fructan biosynthetic genes is dependent on PP2A and different kinases such as Tyr-kinases and PI3-kinases. To further characterize the phosphorylation events involved, comprehensive analysis of kinase activities in the cell was performed using a PepChip, an array of >1000 kinase consensus substrate peptide substrates spotted on a chip. Comparison of kinase activities in sugar-stimulated and mock(sorbitol)-treated Arabidopsis demonstrates the altered phosphorylation of many consensus substrates and documents the differences in plant kinase activity upon sucrose feeding. The different phosphorylation profiles obtained are consistent with sugar-mediated alterations in Tyr phosphorylation, cell cycling, and phosphoinositide signaling, and indicate cytoskeletal rearrangements. The results lead us to infer a central role for small GTPases in sugar signaling.
Kinome profiling of Arabidopsis using arrays of kinase consensus substrates.
(2007)
Journal - Plant methods (England )
Abstract :
BACKGROUND: Kinome profiling aims at the parallel analysis of kinase activities in a cell. Novel developed arrays containing consensus substrates for kinases are used to assess those kinase activities. The arrays described in this paper were already used to determine kinase activities in mammalian systems, but since substrates from many organisms are present we decided to test these arrays for the determination of kinase activities in the model plant species Arabidopsis thaliana. RESULTS: Kinome profiling using Arabidopsis cell extracts resulted in the labelling of many consensus peptides by kinases from the plant, indicating the usefulness of this kinome profiling tool for plants. Method development showed that fresh and frozen plant material could be used to make cell lysates containing active kinases. Dilution of the plant extract increased the signal to noise ratio and non-radioactive ATP enhances full development of spot intensities.Upon infection of Arabidopsis with an avirulent strain of the bacterial pathogen Pseudomonas syringae pv. tomato, we could detect differential kinase activities by measuring phosphorylation of consensus peptides. CONCLUSION: We show that kinome profiling on arrays with consensus substrates can be used to monitor kinase activities in plants. In a case study we show that upon infection with avirulent P. syringae differential kinase activities can be found. The PepChip can for example be used to purify (unknown) kinases that play a role in P. syringae infection.This paper shows that kinome profiling using arrays of consensus peptides is a valuable new tool to study signal-transduction in plants. It complements the available methods for genomics and proteomics research.
Developing fructan-synthesizing capability in a plant invertase via mutations in the sucrose-binding box.
(2006)
Journal - The Plant journal : for cell and molecular biology (England )
Abstract :
Fructans are fructose polymers that are synthesized from sucrose by fructosyltransferases. Fructosyltransferases are present in unrelated plant families suggesting a polyphyletic origin for their transglycosylation activity. Based on sequence comparisons and enzymatic properties, fructosyltransferases are proposed to have evolved from vacuolar invertases. Between 1% and 5% of the total activity of vacuolar invertase is transglycosylating activity. We investigated the nature of the changes that can convert a hydrolysing invertase into a transglycosylating enzyme. Remarkably, replacing 33 amino acids (amino acids 143-175) corresponding to the N-terminus of the mature onion vacuolar invertase with the corresponding region of onion fructan:fructan 6G-fructosyltransferase (6G-FFT) led to a shift in activity from hydrolysis of sucrose towards transglycosylation between two sucrose molecules. The substituted N-terminal region contains the sucrose-binding box that harbours the nucleophile involved in sucrose hydrolysis (Asp164). Subsequent research into the individual amino acids responsible for the enhanced transglycosylation activity revealed that mutations in amino acids Trp161 and Asn166, can give rise to a shift towards polymerase activity. Changing the amino acid at either of these positions in the sucrose-binding box increases the transglycosylation capacity of invertases two- to threefold compared to wild type. Combining the two mutations had an additive effect on transglycosylation ability, resulting in an approximately fourfold enhancement. The mutations generated correspond with natural variation present in the sucrose-binding boxes of vacuolar invertases and fructosyltransferases. These relatively small changes that increase the transglycosylation capacity of invertases might explain the polyphyletic origin of the fructan accumulation trait.
| ISSN : | 0960-7412 |
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| Mesh Heading : | Amino Acid Sequence Binding Sites Codon, Nonsense Evolution, Molecular Fructans Glycosylation Hexosyltransferases Hydrolysis Models, Molecular Molecular Sequence Data Onions Phylogeny Plant Proteins Recombinant Fusion Proteins Sequence Alignment Sucrose beta-Fructofuranosidase chemistry genetics metabolism metabolism genetics metabolism genetics |
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| Mesh Heading Relevant : | biosynthesis chemistry metabolism metabolism chemistry metabolism |
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Using natural variation to investigate the function of individual amino acids in the sucrose-binding box of fructan:fructan 6G-fructosyltransferase (6G-FFT) in product formation.
(2005)
Journal - Plant molecular biology (Netherlands )
Abstract :
Enzymes of the glycosyl hydrolase family 32 are highly similar with respect to primary sequence but catalyze divergent reactions. Previously, the importance of the conserved sucrose-binding box in determining product specificity of onion fructan:fructan 6G-fructosyltransferase (6G-FFT) was established [Ritsema et al., 2004, Plant Mol. Biol. 54: 853-863]. Onion 6G-FFT synthesizes the complex fructan neo-series inulin by transferring fructose residues to either a terminal fructose or a terminal glucose residue. In the present study we have elucidated the molecular determinants of product specificity by substitution of individual amino acids of the sucrose binding box with amino acids that are present on homologous positions in other fructosyltransferases or vacuolar invertases. Substituting the presumed nucleophile Asp85 of the beta-fructosidase motif resulted in an inactive enzyme. 6G-FFT mutants S87N and S87D did not change substrate or product specificities, whereas mutants N84Y and N84G resulted in an inactive enzyme. Most interestingly, mutants N84S, N84A, and N84Q added fructose residues preferably to a terminal fructose and hardly to the terminal glucose. This resulted in the preferential production of inulin-type fructans. Combining mutations showed that amino acid 84 determines product specificity of 6G-FFT irrespective of the amino acid at position 87.
| ISSN : | 0167-4412 |
|---|
| Mesh Heading : | Amino Acid Sequence Amino Acids Amino Acids, Aromatic Asparagine Binding Sites Cell Line Consensus Sequence Fructose Genetic Variation Hexosyltransferases Kinetics Mutation, Missense Protein Binding Sequence Homology, Amino Acid Substrate Specificity Sucrose Time Factors Vacuoles beta-Fructofuranosidase metabolism genetics metabolism genetics metabolism genetics genetics metabolism metabolism enzymology genetics metabolism |
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| Mesh Heading Relevant : | genetics genetics metabolism |
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Fructosyltransferase mutants specify a function for the beta-fructosidase motif of the sucrose-binding box in specifying the fructan type synthesized.
(2004)
Journal - Plant molecular biology (Netherlands )
Abstract :
The onion fructosyltransferase fructan:fructan 6G-fructosyltransferase (6G-FFT) synthesizes fructans of the inulin neo-series using 1-kestose as a substrate. 6G-FFT couples a fructosyl residue to either the terminal glucose via a beta (2-6) linkage or a terminal fructose via a beta (2-1) linkage. The sucrose-binding box is present at the N-terminus of invertases and fructosyltransferases. We tested its function by producing swaps of the first 36 amino acids of 6G-FFT with that of onion sucrose:sucrose 1-fructosyltransferase (1-SST) (SST-GFT) and vacuolar invertase (INV-GFT). In contrast to 6G-FFT, invertase and 1-SST are able to utilize sucrose as their only substrate. The chimerical enzymes were unable to use sucrose, but were active when incubated with 1-kestose. INV-GFT synthesized a similar array of fructans as 6G-FFT, in contrast, SST-GFT showed a dramatic shift in activity towards synthesis of beta (2-1) linkages. Thus the region containing the sucrose-binding box is directing the fructan type synthesized. In invertases, the beta -fructosidase motif, which is part of the sucrose-binding box, consists of NDPNG/A. This motif is variable in fructosyltransferases and consists of NDPSG in 6G-FFT and ADPNA in 1-SST of onion. We studied the importance of the 6G-FFT beta -fructosidase motif using mutants S87N (NDPNG) and N84A;S87N (ADPNG). S87N has 6G-FFT activity, whereas N84A;S87N has a activity that was shifted towards synthesis of beta (2-1) linkages. This is in agreement with the observed activities of the chimerical proteins and indicates that the beta -fructosidase motif of the sucrose-binding box is specifying the fructan type synthesized.
| ISSN : | 0167-4412 |
|---|
| Mesh Heading : | Amino Acid Motifs Amino Acid Sequence Binding Sites Cell Line Fructans Hexosyltransferases Molecular Sequence Data Mutagenesis, Site-Directed Sequence Homology, Amino Acid Substrate Specificity Sucrose Trisaccharides beta-Fructofuranosidase genetics genetics genetics metabolism genetics |
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| Mesh Heading Relevant : | Point Mutation biosynthesis metabolism metabolism metabolism |
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Fructans: beneficial for plants and humans.
(2003)
Journal - Current opinion in plant biology (United States )
Abstract :
The recent cloning of genes encoding fructosyltransferases and fructan exohydrolases has been a major breakthrough in fructan research. Now, fructan metabolism and fructosyltransferase enzymes can be studied at the molecular level. In addition, fructan synthesis and breakdown can be adapted in such a way that tailor-made fructans are produced in plants for use as healthy food ingredients.
| ISSN : | 1369-5266 |
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| Mesh Heading : | Bifidobacterium Evolution, Molecular Fructans Humans Inulin Lactobacillus Phylogeny Plants drug effects chemistry metabolism pharmacology drug effects genetics |
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| Mesh Heading Relevant : | biosynthesis metabolism |
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Engineering fructan metabolism in plants.
(2003)
Journal - Journal of plant physiology (Germany )
Abstract :
Fructans, or polyfructosylsucroses, are storage carbohydrates present in many higher plants. They are also considered healthy food ingredients. Engineering crops into high level production of specific fructan molecules is one of the mayor strategic research goals. Understanding the properties of fructosyltransferases is important, in order to direct the synthesis of fructans. In plants at least two fructosyltransferases are needed to synthesise fructans. One enzyme synthesises the fructan trisaccharide 1-kestose, the next enzyme uses 1-kestose for elongation and/or modification, producing longer fructans. The specificity of fructosyltransferases determines the type of glycosidic bond formed and the donor and acceptor substrates used. This enables the synthesis of many structurally diverse fructans. The production of these molecules in crops such as sugar beet and potato makes the commercial use of fructans feasible.
| ISSN : | 0176-1617 |
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| Mesh Heading : | Biotechnology Crops, Agricultural Fructans Hexosyltransferases Plants, Genetically Modified enzymology genetics metabolism chemistry metabolism genetics metabolism enzymology genetics |
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| Mesh Heading Relevant : | Genetic Engineering methods biosynthesis metabolism |
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Kinome profiling of Arabidopsis using arrays of kinase consensus substrates
(2007)
Journal - Plant Methods
Abstract :
BackgroundKinome profiling aims at the parallel analysis of kinase activities in a cell. Novel developed arrays containing consensus substrates for kinases are used to assess those kinase activities. The arrays described in this paper were already used to determine kinase activities in mammalian systems, but since substrates from many organisms are present we decided to test these arrays for the determination of kinase activities in the model plant species Arabidopsis thaliana.ResultsKinome profiling using Arabidopsis cell extracts resulted in the labelling of many consensus peptides by kinases from the plant, indicating the usefulness of this kinome profiling tool for plants. Method development showed that fresh and frozen plant material could be used to make cell lysates containing active kinases. Dilution of the plant extract increased the signal to noise ratio and non-radioactive ATP enhances full development of spot intensities.Upon infection of Arabidopsis with an avirulent strain of the bacterial pathogen Pseudomonas syringae pv. tomato, we could detect differential kinase activities by measuring phosphorylation of consensus peptides.ConclusionWe show that kinome profiling on arrays with consensus substrates can be used to monitor kinase activities in plants. In a case study we show that upon infection with avirulent P. syringae differential kinase activities can be found. The PepChip can for example be used to purify (unknown) kinases that play a role in P. syringae infection.This paper shows that kinome profiling using arrays of consensus peptides is a valuable new tool to study signal-transduction in plants. It complements the available methods for genomics and proteomics research.