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T Boller

Publications and source records attributed to T Boller.

At least 73 records · Page 4Linked to original sources
PubMed1993

High affinity binding of a glycopeptide elicitor to tomato cells and microsomal membranes and displacement by specific glycan suppressors.

We have previously isolated glycopeptides derived from yeast invertase that acted as highly potent elicitors in suspension-cultured tomato cells, inducing ethylene biosynthesis and phenylalanine ammonia-lyase activity, and we have found that the high mannose oligosaccharides released from the pure glycopeptide elicitors by endo-beta-N-acetylglucosaminidase H acted as suppressors of elicitor activity (Basse, C. W., Bock, K., and Boller, T. (1992) J. Biol. Chem. 267, 10258-10265). One of the elicitor-active glycopeptides (gp 8c) was labeled with t-butoxycarbonyl-L-[35S]methionine and purified by reversed phase high performance liquid chromatography resulting in a specific radioactivity of the derivative of about 900 Ci/mmol. This radiolabeled glycopeptide showed specific, saturable, and reversible binding to whole tomato cells under conditions in which cells are responsive to elicitors as well as to microsomal membranes derived from these cells. Ligand saturation experiments, performed with microsomal membranes, gave a dissociation constant (Kd) of 3.3 nM as determined by Scatchard analysis. Various glycopeptide elicitors and preparations from yeast invertase were compared with respect to their abilities to compete for binding of 35S-labeled gp 8c to tomato membranes and to induce ethylene biosynthesis in tomato cells. These studies revealed a high degree of correlation between elicitor activities in vivo and displacement activities in vitro. In both tests, a high activity depended on the presence of glycan side chains consisting of more than 8 mannosyl residues. The high mannose oligosaccharides that acted as suppressors of elicitor activity in vivo competed for binding of the labeled elicitor also. The suppressor-active glycan Man11GlcNAc and the elicitor-active gp 8c exhibited very similar displacement activities, and the inhibitory constant (Ki) of the glycan Man11GlcNAc was very similar to the Kd value calculated for 35S-labeled gp 8c, indicating that the glycopeptide elicitors and the glycan suppressors derived from these elicitors competed with similar affinities for the same binding site. The suppressor-inactive glycan Man8GlcNAc had a 200-fold lower capacity to compete for binding of 35S-labeled gp 8c to tomato membranes compared with the suppressor-active glycan Man11GlcNAc. Our results demonstrate the existence of a specific elicitor binding site in tomato cell membranes and suggest that glycopeptides and glycans act as agonists and antagonists for induction of the stress response, respectively, by competing for this binding site.

Amino Acid Sequence↗
PubMed1993

Disruption of TPS2, the gene encoding the 100-kDa subunit of the trehalose-6-phosphate synthase/phosphatase complex in Saccharomyces cerevisiae, causes accumulation of trehalose-6-phosphate and loss of trehalose-6-phosphate phosphatase activity.

Preparations of the trehalose-6-phosphate synthase/phosphatase complex from Saccharomyces cerevisiae contain three polypeptides with molecular masses 56, 100 and 130 kDa, respectively. Recently, we have cloned the gene for the 56-kDa subunit of this complex (TPS1) and found it to be identical with CIF1, a gene essential for growth on glucose and for the activity of trehalose-6-phosphate synthase. Peptide sequencing of the 100-kDa subunit of the trehalose-6-phosphate synthase/phosphatase complex (TPS2) revealed one sequence to be 100% identical with the deduced amino acid sequence of the upstream region of PPH3 on the right arm of chromosome IV. This sequence was used to clone an upstream region of PPH3 containing an open reading frame of 2685 nucleotides, predicted to encode a polypeptide of 102.8 kDa. The N-terminal sequence, as well as three internal amino acid sequences, obtained from peptide sequencing of the 100-kDa subunit, were identical with specific regions of the deduced amino acid sequence. Thus, the sequence cloned represents TPS2, the gene encoding the 100-kDa subunit of the trehalose-6-phosphate synthase/phosphatase complex. Interestingly, a stretch of about 500 amino acids from the first part of TPS2 was 33% identical with the entire TPS1 sequence. Disruption of TPS2 had no effect on trehalose-6-phosphate synthase activity but caused complete loss of trehalose-6-phosphate phosphatase activity, measured in vitro, and accumulation of excessive amounts of trehalose-6-phosphate instead of trehalose upon heat shock or entrance into stationary phase in vivo. These results suggest that TPS2 codes for the structural gene of the trehalose-6-phosphate phosphatase. Heat shock induced an increase in trehalose-6-phosphate phosphatase activity and this was preceded by an accumulation in TPS2 mRNA, suggesting that the trehalose-6-phosphate phosphatase is subjected to transcriptional control under heat-shock conditions.

Amino Acid Sequence↗
PubMed1993

Posttranslational processing of a new class of hydroxyproline-containing proteins. Prolyl hydroxylation and C-terminal cleavage of tobacco (Nicotiana tabacum) vacuolar chitinase.

The fungicidal class I chitinases (EC 3.2.1.14) are believed to be important in defending plants against microbial pathogens. The vacuolar isoforms of tobacco (Nicotiana tabacum), chitinases A and B, are the first examples of a new type of hydroxyproline-containing protein with intracellular location, enzymic activity, and a small number of hydroxyprolyl residues restricted to a single, short peptide sequence. We have investigated the posttranslational processing and intracellular transport of transgene-encoded chitinase A in callus cultures of Nicotiana tabacum L. cv Havana 425 and leaves of Nicotiana sylvestris Spegazzini and Comes. Pulse-chase experiments and cell fractionation show that chitinase A is processed in two distinct steps. In the first step, the nascent protein undergoes an increase in apparent M(r) of approximately 1500 detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Experiments with the inhibitor of prolyl hydroxylation, alpha,alpha'-dipyridyl, and pulse-chase labeling of cells expressing recombinant forms of chitinase A indicate that the anomalous increase in M(r) is due to hydroxylation of prolyl residues. This step occurs in the endomembrane system before sorting for secretion and vacuolar transport and does not appear to be required for correct targeting of chitinase A to the vacuole. The second step is a proteolytic cleavage. Sequencing of tryptic peptides of the mature proteins indicates that during processing essentially all molecules of chitinase A and B lose a C-terminal heptapeptide, which has been shown to be a vacuolar targeting signal. This appears to occur primarily in the endomembrane system late in intracellular transport. A model for the posttranslational modification of chitinase A is proposed.

Amino Acid Sequence↗
PubMed1993

Purification and characterization of three soluble invertases from barley (Hordeum vulgare L.) leaves.

Three soluble isoforms of invertase (beta-fructofuranosidase; EC 3.2.1.26) were purified from 7-d-old primary leaves of barley (Hordeum vulgare L.). Invertase I, a monomeric protein of 64 kD, was purified to apparent homogeneity as shown by sodium dodecylsulfate-polyacrylamide gel electrophoresis. Invertases IIA and IIB, multimeric proteins with molecular masses of the 116 and 155 kD, were purified 780- and 1370-fold, respectively, but were not yet homogeneous. Extracts of epidermal strips of leaves contained only invertase IIB. The specific activity of invertase was more than 100-fold higher in the epidermis than in the mesophyll. All three isoforms were acidic invertases, with pH optima of around 5.0 and little activity in the alkaline range. Invertase I had a Km for sucrose of 8.1 mM, and invertases IIA and IIB had much lower values of 1.0 and 1.7 mM, respectively. Invertase I was more than 2-fold more resistant than the other two invertases to the inhibitors HgCl2 and pyridoxal. All three constitutive invertases were found to act also as sucrose-sucrose fructosyltransferases when supplied with high concentrations of sucrose, forming 1-kestose as principal product. However, the fructosyltransferase activity of all three enzymes was inhibited by pyridoxal in the same way as their invertase activity. This characteristic clearly differentiates them from the inducible sucrose-sucrose fructosyltransferase of barley leaves, the activity responsible for the initial steps of fructan biosynthesis, which has previously been shown to be insensitive to pyridoxal.

Amino Acid Sequence↗
PubMed1993

The N-terminal cysteine-rich domain of tobacco class I chitinase is essential for chitin binding but not for catalytic or antifungal activity.

The vacuolar chitinases of class I possess an N-terminal cysteine-rich domain homologous to hevein and chitin-binding lectins such as wheat germ agglutinin and Urtica dioica lectin. To investigate the significance of this domain for the biochemical and functional characteristics of chitinase, chimeric genes encoding the basic chitinase A of tobacco (Nicotiana tabacum) with and without this domain were constructed and constitutively expressed in transgenic Nicotiana sylvestris. The chitinases were subsequently isolated and purified to homogeneity from the transgenic plants. Chromatography on colloidal chitin revealed that only the form with the N-terminal domain, and not the one without it, had chitin-binding properties, demonstrating directly that the domain is a chitin-binding domain (CBD). Under standard assay conditions with radioactive colloidal chitin, both forms of chitinase had approximately the same catalytic activity. However, kinetic analysis demonstrated that the enzyme without CBD had a considerably lower apparent affinity for its substrate. The pH and temperature optima of the two chitinases were similar, but the form with the CBD had an approximately 3-fold higher activation energy and retained a higher activity at low pH values. Both chitinases were capable of inhibiting growth of Trichoderma viride, although the form with the CBD was about three times more effective than the one without it. Thus, the CBD is not necessary for catalytic or antifungal activity of chitinase.

Antifungal Agents↗
PubMed1992

The 70-kilodalton heat-shock proteins of the SSA subfamily negatively modulate heat-shock-induced accumulation of trehalose and promote recovery from heat stress in the yeast, Saccharomyces cerevisiae.

In the yeast, Saccharomyces cerevisiae, the disaccharide trehalose is a stress-related metabolite that accumulates upon exposure of cells to heat shock or a variety of non-heat inducers of the stress response. Here, we describe the influence of mutations in individual heat-shock-protein genes on trehalose metabolism. A strain mutated in three proteins of the SSA subfamily of 70-kDa heat-shock proteins (hsp70) overproduced trehalose during heat shock at 37 degrees C or 40 degrees C and showed abnormally slow degradation of trehalose upon temperature decrease from 40 degrees C to 27 degrees C. The mutant cells were unimpaired in the induction of thermotolerance; however, the decay of thermotolerance during recovery at 27 degrees C was abnormally slow. Since both a high content of trehalose and induced thermotolerance are associated with the heat-stressed state of cells, the abnormally slow decline of trehalose levels and thermotolerance in the mutant cells indicated a defect in recovery from the heat-stressed state. A similar albeit minor defect, as judged from measurements of trehalose degradation during recovery, was detected in a delta hsp104 mutant, but not in a strain deleted in the polyubiquitin gene, UB14. In all our experiments, trehalose levels were closely correlated with thermotolerance, suggesting a thermoprotective function of trehalose. In contrast, heat-shock proteins, in particular hsp70, appear to be involved in recovery from the heat-stressed state rather than in the acquisition of thermotolerance. Cells partially depleted of hsp70 displayed an abnormally low activity of neutral trehalase when shifted to 27 degrees C after heat shock at 40 degrees C. Trehalase activity is known to be under positive control by cAMP-dependent protein kinases, suggesting that hsp70 directly or indirectly stimulate these protein-kinase activities. Alternatively, hsp70 may physically interact with neutral trehalase, thereby protecting the enzyme from thermal denaturation.

Glucose↗
PubMed1992

Characterization of the 56-kDa subunit of yeast trehalose-6-phosphate synthase and cloning of its gene reveal its identity with the product of CIF1, a regulator of carbon catabolite inactivation.

Trehalose-6-phosphate synthase is the key enzyme for biosynthesis of trehalose, the major soluble carbohydrate in resting cells of yeast. This enzyme was purified from a strain of Saccharomyces cerevisiae lacking vacuolar proteases. It was found to be a multimeric protein of 630 kDa. Monoclonal antibodies were raised against its smallest subunit (56 kDa) and used for screening a yeast cDNA library. This yielded an immunopositive cDNA clone of 1.7 kb, containing an open reading frame of 1485 base pairs. Its sequence, called TPS1 (for trehalose-6-phosphate synthase), was represented by a single gene in the yeast genome and was found to be almost identical with the recently sequenced CIF1, a gene important for carbon catabolite inactivation, believed to be allelic with FDP1. A mutant obtained by disruption of TPS1 had a very low activity of trehalose-6-phosphate synthase, indicating that TPS1 is an important component of the enzyme. The mutant also showed a growth defect when transferred from glycerol to glucose, a phenotype similar to that of the cif1 and fdp1 mutants deficient in carbon catabolite inactivation. Thus, the smallest subunit of the biosynthetic enzyme trehalose-6-phosphate synthase appears to have, in addition, a central regulatory role in the carbohydrate metabolism of yeast.

Amino Acid Sequence↗
PubMed1992

Elicitors and suppressors of the defense response in tomato cells. Purification and characterization of glycopeptide elicitors and glycan suppressors generated by enzymatic cleavage of yeast invertase.

Cleavage of yeast invertase by alpha-chymotrypsin produced a number of small glycopeptides that were highly active as elicitors of ethylene biosynthesis and phenylalanine ammonia-lyase in suspension-cultured tomato cells. Five of these elicitors were purified and their amino acid sequence determined. They all had sequences corresponding to known sequences of yeast invertase, and all contained an asparagine known to carry a N-linked small high mannose glycan. The most active glycopeptide elicitor induced ethylene biosynthesis and phenylalanine ammonia-lyase half-maximally at a concentration of 5-10 nM. Structure-activity relationships of the peptide part were analyzed by further cleavage of a defined glycopeptide elicitor with various proteolytic enzymes. Removal of the C-terminal phenylalanine enhanced the elicitor activity, whereas removal of N-terminal arginine impaired it. A glycopeptide with the peptide part trimmed to the dipeptide arginine-asparagine was still fully active as elicitor. Glycopeptides with identical amino acid sequences were further separated into fractions differing in the oligosaccharide side chain. A given peptide had high elicitor activity when carrying a glycan with 10-12 mannosyl residues (Man10-12GlcNAc2), a 3-fold lower activity when carrying Man9GlcNAc2 and a 100-fold lower activity when carrying Man8GlcNAc2. The oligosaccharides, released by endo-beta-N-acetylglucosaminidase H from the pure glycopeptide elicitors, acted as suppressors of elicitor-induced ethylene biosynthesis and phenylalanine ammonia-lyase activity. A series of such oligosaccharides in the size range of Man8-13GlcNAc was purified. The structure and composition of the purified oligosaccharides corresponded to the known small high mannose glycans of yeast invertase as verified by 1H NMR spectroscopy at 600 MHz. The highest suppressor activities were obtained with the oligosaccharides containing 10-12 mannosyl residues (Man10-12GlcNAc). The oligosaccharide Man8 GlcNAc was ineffective as a suppressor. Thus, the structural requirements for the free oligosaccharides to act as efficient suppressors were the same as for the oligosaccharide side chains of the glycopeptides for high elicitor activity. We propose that the glycan suppressors bind to the same recognition site as the glycopeptide elicitors without inducing a response.

Amino Acid Sequence↗
PubMed1992

Synthesis and degradation of polyphosphate in the fission yeast Schizosaccharomyces pombe: mutations in phosphatase genes do not affect polyphosphate metabolism.

The fission yeast Schizosaccharomyces pombe was found to accumulate large amounts of polyphosphate, particularly when grown on arginine as the nitrogen source. Upon transfer to a medium without phosphate, polyphosphate was degraded and served as an endogenous phosphate reserve. When phosphate was added again after a prolonged period of phosphate starvation, fission yeast cells synthesized more polyphosphate than they had contained before starvation, a phenomenon known as over-compensation. Strains carrying mutated structural genes for three different phosphatases, pho1, pho2 or pho3, degraded polyphosphate at the same rate as the wild-type strain during phosphate starvation and showed the same type of over-compensation when phosphate was added again.

Genes, Fungal↗
PubMed1992

Cloning and disruption of a gene required for growth on acetate but not on ethanol: the acetyl-coenzyme A synthetase gene of Saccharomyces cerevisiae.

A DNA fragment of Saccharomyces cerevisiae with high homology to the acetyl-coenzyme A (acetyl-CoA) synthetase genes of Aspergillus nidulans and Neurospora crassa has been cloned, sequenced and mapped to chromosome I. It contains an open reading frame of 2139 nucleotides, encoding a predicted gene product of 79.2 kDa. In contrast to its ascomycete homologs, there are no introns in the coding sequence. The first ATG codon of the open reading frame is in an unusual context for a translational start site, while the next ATG, 24 codons downstream, is in a more conventional context. Possible implications of two alternative translational start sites for the cellular localization of the enzyme are discussed. A stable mutant of this gene, obtained by the gene disruption technique, had the same low basal activity of acetyl-CoA synthetase as wild-type cells when grown on glucose but completely lacked the strong increase in activity upon entering the stationary phase, providing direct proof that the gene encodes an inducible acetyl-CoA synthetase (ACS1) of yeast. As expected, the mutant was unable to grow on acetate as sole carbon source. Nevertheless, it showed normal induction of isocitrate lyase on acetate media, indicating that activity of acetyl-CoA synthetase is dispensable for induction of the glyoxylate cycle in S. cerevisiae. Surprisingly, disruption of the ACS1 gene did not affect growth on media containing ethanol as the sole carbon source, demonstrating that there are alternative pathways leading to acetyl-CoA under these conditions.

Acetate-CoA Ligase↗
PubMed1992

Glycopeptide elicitors of stress responses in tomato cells: N-linked glycans are essential for activity but act as suppressors of the same activity when released from the glycopeptides.

Induction of ethylene, an early symptom of the stress response in tomato (Lycopersicon esculentum [L.] Mill) cells, was used as a bioassay to purify elicitor activity from yeast extract. The purified elicitor preparation consisted of small glycopeptides (mean relative molecular weight of approximately 2500) and induced ethylene biosynthesis and phenylalanine ammonia-lyase activity half-maximally at 15 nanograms per milliliter. Elicitor activity was partially abolished by pronase and almost completely by endo-beta-N-acetylglucosaminidase H, alpha-mannosidase, or periodate. The oligosaccharides released upon treatment with endo-beta-N-acetylglucosaminidase H competitively inhibited the elicitor activity of the glycopeptides. This suppressor activity was abolished by periodate oxidation and alpha-mannosidase treatment. The suppressors were chromatographically separated into four active fractions with sizes corresponding to 7 to 10 monosaccharides. They consisted predominantly of mannose and contained also N-acetylglucosamine and glucose. The suppressors had no effect on the response of the tomato cells to a different elicitor, derived from cell walls of Phytophthora megasperma f. sp. glycinea. This strongly suggests that different recognition sites exist for different elicitors in tomato cells, and that the oligosaccharide suppressors act specifically on the perception of just one elicitor. The hypothesis is put forward that the suppressors bind to one of the elicitor recognition sites nonproductively, i.e. without producing a signal, thereby preventing induction of the stress responses by the corresponding elicitor.

Journal Article↗
PubMed1991

A short C-terminal sequence is necessary and sufficient for the targeting of chitinases to the plant vacuole.

Tobacco contains different isoforms of chitinase (EC 3.2.1.14), a hydrolase thought to be involved in the defense against pathogens. Deduced amino acid sequences for putatively vacuolar, basic chitinases differ from the homologous extracellular, acidic isoforms by the presence of a C-terminal extension. To examine the role of this C-terminal extension in protein sorting, Nicotiana silvestris plants were stably transformed with chimeric genes coding for tobacco basic chitinase A with and without the seven C-terminal amino acids. In plants expressing unmodified chitinase A, the enzyme activity was low in the intercellular wash fluid but high in protoplasts and isolated vacuoles. In contrast, in plants expressing mutant chitinase lacking the C terminus, the activity was high in the intercellular wash fluid but low in protoplasts. N. silvestris plants were also transformed with similar constructions coding for a structurally unrelated, extracellular cucumber chitinase. In plants expressing unmodified cucumber chitinase, its activity was present in the intercellular wash fluid and absent from protoplasts. In plants expressing cucumber chitinase with the C-terminal extension from tobacco chitinase A, activity was low in intercellular wash fluids but high in protoplasts and vacuoles. These results demonstrate that the C-terminal extension of tobacco chitinase A is necessary and sufficient for the vacuolar localization of chitinases and, therefore, that it comprises a targeting signal for plant vacuoles.

Amino Acid Sequence↗
PubMed1991

A constitutive, heat shock-activated neutral trehalase occurs in Schizosaccharomyces pombe in addition to the sporulation-specific acid trehalase.

Trehalase was studied in Schizosaccharomyces pombe cells growing vegetatively on minimal medium and in sporulating cultures. Acid trehalase activity, measured at pH 4.2, was absent in vegetative cells and occurred only in asci, indicating that this activity represented the sporulation-specific trehalase reported previously. In contrast, neutral trehalase, measured at pH 6.0, was constitutively present in vegetative cells during the exponential and stationary growth phase as well as in asci. In vegetative cells, neutral trehalase did not sediment with cell walls, suggesting a cytoplasmic localization. Its activity increased ten-fold when growing cells were subjected to heat treatment of 2 h. Neutral trehalase from heat-treated cells had a pH optimum of 6.0 and was almost completely inhibited by 3 mM ZnCl2. Acid trehalase activity could be measured in intact asci, indicating that it is localized in the ascus cell walls, while neutral trehalase was not detectable in intact asci and appeared to be present primarily in the walls of ascospores and in the ascus epiplasm.

Cell Wall↗
PubMed1991

A method to study the rapid phosphorylation-related modulation of neutral trehalase activity by temperature shifts in yeast.

Heat shock enhanced the synthesis of neutral trehalase in growing cells of Saccharomyces cerevisiae, as detected by immunological methods. The activity of the enzyme was measured in extracts obtained by two methods: cells were either harvested by filtration and subsequent disruption with glass beads at 0-4 degrees C or immediately frozen with liquid nitrogen in the presence of Triton X-100, followed by thawing at 30 degrees C. The first procedure yielded artificially high activities of neutral trehalase in heat-shocked cells due to rapid (less than 1 min) activation during handling at 4 degrees C before homogenization. Activity of the enzyme in these homogenates decreased 75-90% upon a treatment with alkaline phosphatase, indicating that activation was due to phosphorylation. The second procedure yielded low trehalase activities for heat-shock treated cells, much higher activities for cells shifted back for some seconds to 27 degrees C, and very low activities again for cells shifted from 27 to 40 degrees C for a second time. Thus, permeabilization of cells following rapid freezing in Triton X-100 is a method of choice to study post-translational modulation of the neutral trehalase of S. cerevisiae by phosphorylation and dephosphorylation.

Cell Membrane Permeability↗
PubMed1991

Rapid changes of protein phosphorylation are involved in transduction of the elicitor signal in plant cells.

Plant cells have an acute sense for pathogen-derived chemical stimuli, so-called elicitors, which induce the plant's defense response. To investigate the molecular basis of chemosensory transduction, elicitor-treated tomato cells were labeled with 1-min pulses of [32P] phosphate. This technique revealed drastic changes in protein phosphorylation in vivo within minutes of stimulation. The protein kinase inhibitors K-252a and staurosporine completely prevented these elicitor-induced changes in protein phosphorylation. They also blocked two early biochemical responses to elicitors, extracellular alkalinization and biosynthesis of ethylene. The ability of K-252a, staurosporine, and benzoylated staurosporine derivatives to inhibit elicitor responses in vivo correlated with their ability to inhibit tomato microsomal protein kinase in vitro. When K-252a was given to elicited cells 1 min after the[32] phosphate, the radioactivity in certain newly labeled phosphoprotein bands disappeared again within minutes. This correlated with an arrest of alkalinization within minutes when K-252a was applied in midcourse of elicitation. These data show that phosphorylation of protein substrates by K-252a-sensitive protein kinases is essential for transduction of elicitor signals in plant cells and that continuous phosphorylation of these proteins is required to maintain the elicited state.

Journal Article↗
PubMed1991

Acquisition of thermotolerance in Saccharomyces cerevisiae without heat shock protein hsp 104 and in the absence of protein synthesis.

Acquisition of thermotolerance in response to a preconditioning heat treatment at 40 degrees C was studied in mutants of the yeast Saccharomyces cerevisiae lacking a specific heat shock protein or the ability to synthesize proteins at 40 degrees C. A mutant carrying a deletion of heat shock protein hsp 104 and the corresponding wildtype strain were both highly sensitive to heat stress at 50.4 degrees C without preconditioning but both acquired almost the same level of thermotolerance after 60 min of preconditioning. Both strains showed equal induction of trehalose-6-phosphate synthase and accumulated equal levels of trehalose during the treatment. The conditional mutant ts--187 synthesized no proteins during the preconditioning heat treatment but nevertheless acquired thermotolerance, albeit to a lesser degree than the corresponding wildtype strain. Induction of trehalose-6-phosphate synthase and accumulation of trehalose were reduced to a similar extent. These results show that acquisition of thermotolerance and accumulation of trehalose are closely correlated during heat preconditioning and are modulated by protein synthesis but do not require it.

Adaptation, Physiological↗
PubMed1991

Analysis and cloning of the ethylene-forming enzyme from tomato by functional expression of its mRNA in Xenopus laevis oocytes.

The last step in biosynthesis of the plant hormone ethylene, oxidation of 1-aminocyclopropane-1-carboxylic acid (ACC), is catalysed by the elusive ethylene-forming enzyme (EFE). EFE is induced by fungal elicitors in suspension-cultured tomato cells. We demonstrate that Xenopus laevis oocytes injected with RNA from elicitor-treated tomato cells gain the ability to convert ACC to ethylene. The enzyme expressed in the oocytes under the direction of plant RNA is indistinguishable from genuine plant EFE with regard to its saturation kinetics, its iron dependency and its stereospecificity to the diastereomeric ethyl derivatives of ACC, allocoronamic acid and coronamic acid. In tomato cells stimulated for different times with elicitor, the level of EFE correlates with the level of RNA directing EFE expression in oocytes. Hybridization and co-injection experiments demonstrate that the tomato RNA species directing EFE expression in oocytes are homologous to clone pTOM13 which has been shown to inhibit ethylene production in plants when expressed in antisense. Using a cDNA library from elicitor-stimulated tomato cells, we have isolated several homologues of pTOM13 and identified one of them, pHTOM5, as a clone of EFE on the basis of its functional expression in the Xenopus oocytes.

Amino Acid Sequence↗