Suspension-cultured Tomato Cells Research Articles

Induction of an extracellular cyclic nucleotide phosphodiesterase as an accessory ribonucleolytic activity during phosphate starvation of cultured tomato cells.

During growth under conditions of phosphate limitation, suspension-cultured cells of tomato (Lycopersicon esculentum Mill.) secrete phosphodiesterase activity in a similar fashion to phosphate starvation-inducible ribonuclease (RNase LE), a cyclizing endoribonuclease that generates 2':3'-cyclic nucleoside monophosphates (NMP) as its major monomeric products (T. Nürnberger, S. Abel, W. Jost, K. Glund [1990] Plant Physiol 92: 970-976). Tomato extracellular phosphodiesterase was purified to homogeneity from the spent culture medium of phosphate-starved cells and was characterized as a cyclic nucleotide phosphodiesterase. The purified enzyme has a molecular mass of 70 kD, a pH optimum of 6.2, and an isoelectric point of 8.1. The phosphodiesterase preparation is free of any detectable deoxyribonuclease, ribonuclease, and nucleotidase activity. Tomato extracellular phosphodiesterase is insensitive to EDTA and hydrolyzes with no apparent base specificity 2':3'-cyclic NMP to 3'-NMP and the 3':5'-cyclic isomers to a mixture of 3'-NMP and 5'-NMP. Specific activities of the enzyme are 2-fold higher for 2':3'-cyclic NMP than for 3':5'-cyclic isomers. Analysis of monomeric products of sequential RNA hydrolysis with purified RNase LE, purified extracellular phosphodiesterase, and cleared -Pi culture medium as a source of 3'-nucleotidase activity indicates that cyclic nucleotide phosphodiesterase functions as an accessory ribonucleolytic activity that effectively hydrolyzes primary products of RNase LE to substrates for phosphate-starvation-inducible phosphomonoesterases. Biosynthetical labeling of cyclic nucleotide phopshodiesterase upon phosphate starvation suggests de novo synthesis and secretion of a set of nucleolytic enzymes for scavenging phosphate from extracellular RNA substrates.

The enzymatic activity of fungal xylanase is not necessary for its elicitor activity.

Fungal xylanases from Trichoderma spp. are potent elicitors of defense responses in various plants. To determine whether enzymatic activity is necessary for elicitor activity, we used site-directed mutagenesis to reduce the catalytic activity of xylanase II from Trichoderma reesei. For this, the glutamic acid residue at position 210, which is part of the active center in this family of enzymes, was changed to either aspartic acid (E210D) or serine (E210S). Wild-type and mutated forms of xylanase II were expressed in yeast cells and purified to homogeneity. Compared with the wild-type form of xylanase II, E210D had >100-fold and E210S 1,000-fold lower enzymatic activity. In contrast, these mutated forms showed no comparable drop in elicitor activity. They fully stimulated medium alkalinization and ethylene biosynthesis in suspension-cultured tomato (Lycopersicon esculentum) cells, as well as hypersensitive necrosis in leaves of tomato and tobacco (Nicotiana tabacum) plants. These results provide direct evidence that enzyme activity is not necessary for elicitor activity of fungal xylanase.

Monitoring brassinosteroid biosynthetic enzymes by fluorescent tagging and HPLC analysis of their substrates and products

Both the vicinal side chain hydroxyl groups and the 6-oxo function of brassinosteroids were modified by fluorescence tagging. Dansylaminophenylboronic acid was used as a derivatizing agent to form fluorescent esters of brassinosteroids containing a side-chain cis-diol structure. 6-Oxo type brassinosteroids were derivatized by means of dansylhydrazine. The modified brassinosteroids, as far as possible derivatized both at the diol and the oxo group, were separated by HPLC and the optimal emission wavelength was determined. By this approach almost all brassinosteroids, including biosynthetic precursors, were susceptible to highly sensitive analysis in the fmol range. This method has been verified as an analytical tool to determine brassinosteroids in cell culture extracts and to monitor brassinosteroid biosynthetic enzymes. 24-Epibrassinolide has been detected in tomato cell suspension cultures. Several steps of brassinosteroid biosynthesis, including the Baeyer–Villiger oxidation of 24-epicastasterone to give 24-epibrassinolide, were monitored in vitro with protein preparations of the same cell culture line.

Nα- and N ε- d-galacturonoyl- l-lysine amides: Properties and possible occurrence in plant cell walls

Three representatives of a novel class of amide (isopeptide) glycoconjugates have been synthesised: Nα- d-galacturonoyl- l-lysine and Nε- d-galacturonoyl- l-lysine and Nε- d-polygalacturonoyl- l-lysine. Galacturonoyl–lysine amide bonds were labile in 2 M trifluoroacetic acid at 120° and in alkali, but relatively stable in cold acid. The amide bonds were resistant to digestion by Driselase, Pronase and trypsin. The polysaccharide backbone of Nε- d-polygalacturonoyl- l-lysine was hydrolysed by Driselase to yield two major ninhydrin-positive compounds which were shown by 1H and 13C NMR spectroscopy to be tri- and tetra- α-(1→4)- d-galacturonoyl- l-lysines. To investigate the possible natural occurrence of N-galacturonoyl isopeptide bonds, we fed cell-suspension cultures of spinach and tomato with d-[6- 14C]glucuronic acid, which radio-labels pectic polysaccharides. The radioactive cell walls were digested with, sequentially, Driselase, mild acid, and proteinases. On electrophoresis at pH 2.0, several of the radioactive digestion-products were cathodic. Some of the cathodic products yielded [ 14C]galacturonic acid upon complete acid hydrolysis. The existence of these products is compatible with the presence of novel N-galacturonoyl isopeptide bonds, which could serve as cross-links in plant cell walls.

Induction of defense-related responses in Cf9 tomato cells by the AVR9 elicitor peptide of cladosporium fulvum is developmentally regulated

The AVR9 elicitor from the fungal pathogen Cladosporium fulvum induces defense-related responses, including cell death, specifically in tomato (Lycopersicon esculentum Mill.) plants that carry the Cf-9 resistance gene. To study biochemical mechanisms of resistance in detail, suspension cultures of tomato cells that carry the Cf-9 resistance gene were initiated. Treatment of cells with various elicitors, except AVR9, induced an oxidative burst, ion fluxes, and expression of defense-related genes. Agrobacterium tumefaciens-mediated transformation of Cf9 tomato leaf discs with Avr9-containing constructs resulted efficiently in transgenic callus formation. Although transgenic callus tissue showed normal regeneration capacity, transgenic plants expressing both the Cf-9 and the Avr9 genes were never obtained. Transgenic F1 seedlings that were generated from crosses between tomato plants expressing the Avr9 gene and wild-type Cf9 plants died within a few weeks. However, callus cultures that were initiated on cotyledons from these seedlings could be maintained for at least 3 months and developed similarly to callus cultures that contained only the Cf-9 or the Avr9 gene. It is concluded, therefore, that induction of defense responses in Cf9 tomato cells by the AVR9 elicitor is developmentally regulated and is absent in callus tissue and cell-suspension cultures, which consists of undifferentiated cells. These results are significant for the use of suspension-cultured cells to investigate signal transduction cascades.

Systemin potentiates the oxidative burst in cultured tomato cells

Plants that have been wounded by insects or other herbivores may be more susceptible to infection by adventitious microbes. Wound-induced signal molecules, which serve to induce responses in the plant that retard further feeding, might also act to prepare a plant for possible pathogen attack. We have examined the effect of a wound-generated systemic messenger (systemin) on a pathogen-stimulated defense-response marker, the oxidative burst. We observed that neither systemin nor its inactive analog (A-17) was able to directly induce H2O2 biosynthesis in suspension-cultured tomato (Lycopersicon esculentum L.) cells, regardless of the duration of exposure of the cells to the two peptides. Similarly, neither systemin nor A-17 was capable of modifying an oligogalacturonide-elicited oxidative burst, as long as elicitor addition occurred within minutes of treatment with systemin or A-17. In contrast, preexposure of the cell cultures to systemin (but not to A-17) led to a time-dependent enhancement of the oligogalacturonide-elicited oxidative burst. By 12 h of exposure, the H2O2 biosynthetic capacity of systemin-treated cells exceeded that of the control cells by a factor of 16 +/- 2. A similar up-regulation by systemin of a mechanically stimulated oxidative burst was also observed. Because the systemin-induced augmentation in oxidant synthesis is quantitatively prevented by coincubation with 2 &mgr;M cycloheximide, and because the oxidative burst of oligogalacturonic acid-elicited control cells (no systemin exposure) is unaffected by preincubation with cycloheximide, we conclude that systemin enhancement of the tomato-cell oxidative burst requires protein synthesis.

Desensitization of the perception system for chitin fragments in tomato cells

Suspension-cultured tomato (Lycopersicon esculentum) cells react to stimulation by chitin fragments with a rapid, transient alkalinization of the growth medium, but behave refractory to a second treatment with the same stimulus (G. Felix, M. Regenass, T. Boller [1993] Plant J 4: 307-316). We analyzed this phenomenon and found that chitin fragments caused desensitization in a time- and concentration-dependent manner. Partially desensitized cells exhibited a clear shift toward lower sensitivity of the perception system. The ability of chitin oligomers to induce desensitization depended on the degree of polymerization (DP), with DP5 approximately DP4 >> DP3 >> DP2 > DP1. This correlates with the ability of these oligomers to induce the alkalinization response and to compete for the high-affinity binding site on tomato cells and microsomal membranes, indicating that the alkalinization response and the desensitization process are mediated by the same receptor. The dose required for half-maximal desensitization was about 20 times lower than the dose required for half-maximal alkalinization; desensitization could therefore be used as a highly sensitive bioassay for chitin fragments and chitin-related stimuli such as lipochitooligosaccharides (nodulation factors) from Rhizobium leguminosarum. Desensitization was not associated with increased inactivation of the stimulus or with a disappearance of high-affinity binding sites from the cell surface, and thus appears to be caused by an intermediate step in signal transduction.

Elicitor-induced extensin insolubilization in suspension-cultured tomato cells

Insolubilization of cell wall hydroxyproline-rich glycoproteins is a common plant defence response when plants are challenged with either pathogens or elicitors derived from pathogens. A yeast elicitor that insolubilized cell wall extensin of suspension-cultured tomato cells was purified by 80% ethanol precipitation of yeast extract followed by anion-exchange chromatography, gel-filtration chromatography and reverse phase HPLC. MALDI/TOF-mass spectrum analysis of the purified elicitor preparation generated one major peak at 410 ± Da. Elicitor activity was inactivated at 100° for 10 min or after incubation with pronase. No loss of elicitor activity was observed after periodate treatment. Polyclonal antiserum raised against native tomato extensin was used to demonstrate extensin insolubilization in vivo. Salt-elutability of extensin from tomato cells was dependent upon concentration and time of incubation of the cells with elicitor. SDS-PAGE/western blotting of salt-eluted protein from elicited cells also demonstrated insolubilization of HRGPs in the cell wall. The elicitor was found to stimulate cell wall peroxidase activity and extensin insolubilization in isolated tomato cell walls and in intact cells. The elicitor also induced a transient oxidative burst which began after 5 min and was maximal after 20 min incubation with the cells. Other typical plant defence responses known to be triggered by elicitors, such as changes in H +, K + or Ca 2+ fluxes, enhanced activities of lipoxygenase, phenylalanine ammonia lyase and superoxide dismutase, were not observed when tomato cells were challenged with this elicitor. The Ca 2+ channel blocker verapamil did not prevent elicitor-induced extensin insolubilization in whole cells. This study suggests that biochemical events, such as transient oxidative burst and enhanced peroxidase activity, which accompany extensin insolubilization involve the cell wall without any apparent plasma membrane participation. The above cell wall events may be sufficient to form the early plant defence responses to microbial challenge. © 1997 Elsevier Science Ltd. All rights reserved

Formation of Di-Isodityrosine and Loss of Isodityrosine in the Cell Walls of Tomato Cell-Suspension Cultures Treated with Fungal Elicitors or H2O2.

About 84% of the hydroxyproline residues in a cell culture of tomato (Lycopersicon esculentum x Lycopersicon peruvianum) were present in phenol-inextractable (i.e. covalently wall-bound) material. Treatment of the cells with any of three fungal elicitors (wall fragments from Phytophthora megasperma and Pythium aphanidermatum and xylanase from Aureobasidium pullulans) or with 1 mM H2O2 had little effect on the quantity of phenolinextractable hydroxyproline per milligram of freeze-dried cells. However, each treatment induced a decrease in the content of phenol-inextractable isodityrosine (Idt) residues. Each treatment, except with the P. megasperma fragments, also induced an increase in phenol-inextractable di- (Di-Idt). The increase in Di-Idt partly accounted for the loss of Idt. We conclude that the elicitors and H2O2 acted to reinforce the existing cross-linking of cell wall (glyco)proteins by evoking oxidative coupling reactions to convert Idt to Di-Idt plus unidentified products. The promotion of cross-linking by elicitor treatment is proposed to be a defensive response that restricts the penetration of pathogens.

Differential induction of seven 1-aminocyclopropane-1-carboxylate synthase genes by elicitor in suspension cultures of tomato (Lycopersicon esculentum).

The key enzyme of ethylene biosynthesis, ACC synthase, is encoded by a multigene family. We describe three new DNA sequences encoding members of the ACC synthase family of the tomato. One of these sequences encodes a novel ACC synthase, LE-ACS6, which is phylogenetically related to the ACC synthases LE-ACS1A and LE-ACS1B. Gene-specific probes for seven tomato ACC synthase genes were prepared. They were used for RNase protection assays to study the accumulation of ACC synthase transcripts in suspension-cultured tomato cells after the addition of an elicitor. The ACC synthase genes LE-ACS2, LE-ACS5 and LE-ACS6 were strongly induced by the elicitor. In contrast, the genes LE-ACS1B, LE-ACS3 and LE-ACS4 were constitutively expressed and LE-ACS1B was present at all times at a particularly high level. Thus, there are two groups of ACC synthase transcripts expressed in these cells, either elicitor-induced or constitutive. A transcript of LE-ACS1A was not detected. Despite the presence of LE-ACS1B, LE-ACS2, LE-ACS3, LE-ACS4 and LE-ACS5, there was only little ethylene produced in the absence of the elicitor. Increased ethylene production is usually correlated with the accumulation of ACC synthase transcripts, indicating that ethylene production is controlled via the transcriptional activation of ACC synthase genes. However, the abundance of several ACC synthase mRNAs studied was not strictly correlated with the rate of elicitor-induced ethylene production. Our data provide evidence that the activity of these ACC synthases may not solely be controlled by the transcriptional activation of ACC synthase genes.

Solubilization, Partial Purification, and Characterization of a Binding Site for a Glycopeptide Elicitor from Microsomal Membranes of Tomato Cells.

Purified glycopeptides derived from yeast invertase act as highly potent elicitors in suspension-cultured tomato (Lycopersicon esculentum [L.] Mill) cells, inducing ethylene biosynthesis and phenylalanine ammonia lyase half-maximally at concentrations of 1 to 5 nM. We previously demonstrated the presence of a high-affinity binding site that specifically recognized these glycopeptides in cells and microsomal membranes of tomato (C.W. Basse, A. Fath, T. Boller [1993] J Biol Chem 268: 14724-14731). This elicitor-binding site was solubilized in an active form from the microsomal membranes using the neutral detergents n-dodecylmaltoside and n-dodecanoylsucrose and purified 67-fold in a single step by anion-exchange chromatography. Ligand saturation studies and competition experiments with unlabeled glycopeptides and glycans demonstrated that the detergent-solubilized elicitor-binding site retained the high affinity (Kd approximately 1-4 nM) and selectivity of the membrane-bound form. The binding site was found to have a high affinity for N-linked glycans with nine mannosyl residues from fungal glycoproteins, whereas it did not recognize the typical mammalian glycans with nine mannosyl residues, demonstrating further its high selectivity.

The Pto kinase mediates a signaling pathway leading to the oxidative burst in tomato.

The Pto gene encodes a serine/threonine kinase that confers resistance in tomato to Pseudomonas syringae pv. tomato strains that express the avirulence gene avrPto. Partial characterization of the Pto signal transduction pathway and the availability of transgenic tomato lines (+/- Pto) make this an ideal system for exploring the molecular basis of disease resistance. In this paper, we test two transgenic tomato cell suspension cultures (+/-Pto) for production of H2O2 following independent challenge with two strains of P. syringae pv. tomato (+/-avrPto). Only when Pto and avrPto are present in the corresponding organisms are two distinct phases of the oxidative burst seen, a rapid first burst followed by a slower and more prolonged second burst. In the remaining three plant-pathogen interactions, we observe either no burst or only a first burst, indicating that the second burst is correlated with disease resistance. Further support for this observation comes from the finding that both resistant and susceptible tomato lines produce the critical second oxidative burst when challenged with P. syringae pv. tabaci, a nonhost pathogen that elicits a hypersensitive response on both tomato lines. The Pto kinase is not required, however, for the oxidative burst initiated by non-specific elicitors such as oligogalacturonides or osmotic stress. A model describing a possible role for the Pto kinase in the overall scheme of oxidative burst signaling is proposed.

Mercuric chloride induced membrane damage in tomato cultured cells

Suspension cultures of tomato cells were used to study the membrane injury by the toxic concentration of mercuric chloride. Assessment of electrolyte leakage, UV-absorbance of the tissue leachates, relative leakage ratio, injury index, membrane lipid peroxidation, lipoxygenase activity, α-amino nitrogen and total soluble carbohydrate contents showed the extent of membrane damage as a function of the increasing concentration of mercuric chloride. It is suggested that the selected parameters can be used as qualitative tests for determination of stress-induced membrane damage

Identification of Diacylglycerol Pyrophosphate as a Novel Metabolic Product of Phosphatidic Acid during G-protein Activation in Plants

We provide evidence that phosphatidic acid (PtdOH) formed during signaling in plants is metabolized by a novel pathway. In much of this study, 32Pi-labeled Chlamydomonas cells were used, and signaling was activated by adding the G-protein activator mastoparan. Within seconds of activation, large amounts of [32P]PtdOH were formed, with peak production at about 4 min, when the level was 5-25-fold higher than the control. As the level of [32P]PtdOH subsequently decreased, an unknown phospholipid (PLX) increased in radiolabeling; before activation it was barely detectable. The chromatographic properties of PLX resembled those of lyso-PtdOH and CMP.PtdOH but on close inspection were found to be different. PLX was shown to be diacylglycerol pyrophosphate (DGPP), the product of a newly discovered enzyme, phosphatidate kinase, whose in vitro activity was described recently (Wissing, J. B., and Behrbohm, H. (1993) Plant Physiol. 102, 1243-1249). The identity of DGPP was established by co-chromatrography with a standard and by degradation analysis as follows: [32P]DGPP was deacylated, and the product (glycerolpyrophosphate, GroPP) was hydrolyzed by mild acid treatment or pyrophosphatase to produce GroP and Pi as the only radioactive products. Since DGPP is the pyrophosphate derivative of PtdOH and is formed as the concentration of PtdOH decreases, we assumed that PtdOH was converted in vivo to DGPP. This was confirmed by showing that during a short labeling protocol while the specific radioactivity of DGPP was increasing, the specific radioactivity of the 32Pi derived from DGPP as above was higher than that of [32P]GroP. DGPP was also formed in suspension cultures of tomato and potato cells, and its synthesis was activated by mastoparan. Moreover, it was also found in intact tissues of a number of higher plants, for example, carnation flower petals, vetch roots, leaves of fig-leaved goosefoot, and common persicaria and microspores of rape seed. Our results suggest that DGPP is a common but minor plant lipid that increases in concentration when signaling is activated. Possible functions of DGPP in phospholpase C and D signaling cascades are discussed.

Transformation of the mycotoxin ochratoxin A in plants. 2. Time course and rates of degradation and metabolite production in cell-suspension cultures of different crop plants.

Ochratoxin A, one of the most toxic mycotoxins, can be metabolized nearly completely by suspension cultures of various plant cells. The transformation products identified in this study were almost the same in the cell-suspension cultures of maize, carrot, tomato, potato, soybean, wheat and barley, but the quantitative distribution differed strongly depending on incubation time and species of plant-cell culture. The compounds were extracted with ethyl acetate and detected by reversed-phase HPLC with gradient elution. From the result it is supposed that besides ochratoxin A also ochratoxin derivatives may occur in food and feedstuff of plant origin.

Selection of tomato tissue cultures able to grow on ribose as the sole carbon source

When tomato (Lycopersicon esculentum Mill.) callus or cell cultures were placed on media containing ribose as the sole carbon source, the tissues turned dark brown and ceased growth. However, after approximately 60 days bright green tissue able to grow on ribose emerged from 3 % of the brown necrotic callus tissue pieces plated. The selected tissue was highly organized, consisting of leafy primordia and associated meristematic tissues, sustained growth on ribose, and demonstrated the capacity to regenerate whole plants for at least 3 years. Cultures able to grow on ribose could not be selected from liquid suspension cultured tomato cells or from callus which had been mechanically macerated into cell aggregates containing less than approximately 100 cells. Plants regenerated from ribose adapted cultures were abnormal, having shortened internodes and thicker greener leaves. Regenerated plants were both male and female sterile.

Evidence for rishitin biosynthesis in tomato cultures

Rishitin accumulation as well as its biosynthesis from added [2- 14C]MVA was observed in tomato cell suspension cultures elicited by yeast extract.

Perception of Fungal Sterols in Plants (Subnanomolar Concentrations of Ergosterol Elicit Extracellular Alkalinization in Tomato Cells).

Suspension-cultured cells of tomato (Lycopersicon esculentum Mill.) reacted to spores and spore exudates of the pathogen Cladosporium fulvum with a rapid, transient alkalinization of their growth medium that resembled the previously described alkalinization response elicited by chitin fragments (G. Felix, M. Regenass, T. Boller [1993] Plant J 4: 307-316) and was likewise inhibited by the protein kinase inhibitor K-252a. However, the spore factor recognized by the cells differed from chitin fragments in that it was butanol soluble and active in cells refractory to stimulation by chitin fragments. The spore factor was purified and identified as ergosterol, the main sterol of most higher fungi. With pure ergosterol, half-maximal induction was reached at about 10 pm. After treatment with ergosterol, tomato cells became refractory to a subsequent stimulation by C. fulvum and vice versa, indicating that ergosterol was the principal component of the spores recognized by the plant cells. Most other sterols were inactive, including cholesterol, a range of animal steroid hormones, and all natural plant sterols tested, except for stigmasterol, which was about 106 times less active than ergosterol. Our data demonstrate that tomato cells perceive ergosterol with a selectivity and sensitivity that resembles the perception of steroid hormones in animals.

Effects of Clomazone on IPP Isomerase and Prenyl Transferase Activities in Cell Suspension Cultures and Cotyledons of Solanaceous Species

Laboratory assays were conducted to determine the sensitivity of tomato and tobacco cell suspension cultures and tomato and pepper cotyledons to clomazone. A comparison of fresh weight and carotenoid content indicated up to a three-fold difference between the clomazone-tolerant tobacco and clomazone-susceptible tomato cell suspension cultures. In contrast, an approximate 60-fold difference between the tolerant pepper and susceptible tomato cotyledons was observed when total chlorophyll and carotenoid contents were measured. The effect of clomazone and its possible metabolites on in vivo and in vitro extractable IPP isomerase (EC 5.3.3.2) and prenyltransferase (EC 2.5.1.29) activity was investigated. There was no clear inhibitory effect of clomazone or possible clomazone metabolites upon enzyme activity in tomato or tobacco cell suspension cultures or on light or dark grown tomato or pepper cotyledons. No specific enzymatic target site of clomazone was identified in correlation with the reduction in total chlorophyll or carotenoid content.

Subcellular localization of 1-aminocyclopropane-1-carboxylate oxidase in tomato cells

The localization of 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase was examined in suspension-cultured cells of tomato (Lycopersicon esculentum Mill.), using cell-fractionation techniques, followed by immunoblot analysis with monospecific antibodies raised against a tomato ACC oxidase expressed in Escherichia coli. When assayed in vivo, ACC oxidase had a low activity in untreated tomato cells but was strongly induced when the cells were supplied with its substrate, ACC. Immunoblots showed that this induction was accompanied by the accumulation of a single protein corresponding to ACC oxidase, with an apparent molecular mass (Mr) of 36 kDa. The level of this protein in induced cells, estimated by immunoblotting, was compared with that in protoplasts and vacuoles, and with that in various particulate and soluble fractions obtained by differential centrifugation of cell homogenates. It was found that the ACC oxidase antigen was absent from the vacuole, and that most of it was localized in the cytoplasm of the protoplasts without being associated with membranes. Measurements of ACC oxidase activity in preparations of protoplasts and vacuoles supported these results.