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.

A micromanipulation method to measure the mechanical properties of single tomato suspension cells.

A micromanipulation method has been developed to measure the force required to burst single tomato cells (Lycopersicon esculentum vf36) taken from suspension cultures. The method works by compressing a cell between parallel surfaces whilst measuring the force being imposed on the cell, and its deformation. The mean bursting force for two-week-old cells was 3.6 mN (standard error 0.1 mN), at a compression speed of 23 μm s−1. Usually force-deformation curves showed a single bursting event, but sometimes multiple bursts were observed, implying cells could reseal after failure. If cells were deformed without bursting, and then held, they showed a relaxation of the force. This was attributed to water loss, although wall relaxation was also a possibility. The half time of this relaxation was between 1–10 s. Tests on protoplasts gave bursting forces 1000 fold lower than intact cells, and cells treated with Triton to disrupt the membranes and destroy turgor collapsed with no bursting. As expected, both turgor and the presence of a wall were essential to maintaining cell strength.

Production of recombinant rotavirus VP6 from a suspension culture of transgenic tomato (Lycopersicon esculentum Mill.) cells

Recombinant rotavirus VP6 expressed in transgenic tomato cells was found primarily in the intracellular fraction and had a molecular weight of 44 kDa. In a shake flask, transgenic tomato cells produced 0.33 mg recombinant VP6 l−1 after 18 days of incubation. In a high aspect rotating-wall vessel designed by NASA to simulate microgravity, the transgenic cells produced up to 0.15 mg recombinant VP6 l−1.

14-3-3 proteins double the number of outward-rectifying K+ channels available for activation in tomato cells

Outward-rectifying K+ channels are modulated in response to environmental stimuli by a range of intracellular factors, such as cytoplasmic Ca2+, pH, kinases and phosphatases. Here we report that voltage-dependent outward-rectifying K+ channels in tomato cells are also targets for modulation by 14-3-3 proteins. In whole-cell patch-clamp experiments, recombinant 14-3-3 protein (tomato isoform TFT7) was introduced into tomato cell protoplasts via the patch pipette. As a result the steady-state outward K+ current increased twofold and this increase was not dependent upon the presence of cytoplasmic ATP. A phosphorylated peptide that contained a phosphorylated 14-3-3 target-binding motif (RSTS*TP), derived from nitrate reductase, blocked the effect of 14-3-3, thus showing the specific nature of 14-3-3 action. Kinetic parameters of the conductance, like (de)activation kinetics, voltage dependence of gating and activation potential, were not significantly different between control and 14-3-3 infused cells. Analysis of single-channel activity and whole-cell noise indicated that the single-channel conductance was not affected by 14-3-3 infusion. We conclude that 14-3-3 proteins recruit 'sleepy' channels into a voltage-activatable state. The molecular mechanism underlying the 1 : 1 ratio of constitutively active and 14-3-3 recruited channels is discussed in the light of known functions of 14-3-3 dimers.

Salicylic Acid Influences Hsp70/Hsc70 Expression in Lycopersicon esculentum: Dose- and Time-Dependent Induction or Potentiation

Overexpression of heat-shock (HS) proteins (HSP) is often sufficient to protect against lethal environmental stresses. Anti-inflammatory salicylates potentiate the induction of the 70 kDa HSP (Hsp70) in mammals in response to HS and enhance thermotolerance. In plants, salicylic acid (SA) is a natural signalling molecule, mediating resistance in response to avirulent pathogens. The influence of SA on the HS response in plants is, however, unknown. We investigated the effect of SA, alone or with HS, on Hsp70/Hsc70 expression in tomato cells using biometabolic labelling and Western blotting. A dose- and time-dependent influence on Hsp70/Hsc70 accumulation was observed: SA at 1.0 mM (3 h) potentiated heat-induced accumulation, while 1.0 mM (5 h) and 0.5 mM (8 h) induced expression, the latter preceded by increased membrane permeability. These results suggest that in plants, as in mammals, low SA concentrations do not induce Hsp70/Hsc70 expression but potentiate HS induction and confer membrane protection, while cytotoxic levels induce Hsp70/Hsc70.

Modification of Expansin Protein Abundance in Tomato Fruit Alters Softening and Cell Wall Polymer Metabolism during Ripening

Modification of Expansin Protein Abundance in Tomato Fruit Alters Softening and Cell Wall Polymer Metabolism during Ripening

Modification of Expansin Protein Abundance in Tomato Fruit Alters Softening and Cell Wall Polymer Metabolism during Ripening

The role of the ripening-specific expansin Exp1 protein in fruit softening and cell wall metabolism was investigated by suppression and overexpression of Exp1 in transgenic tomato plants. Fruit in which Exp1 protein accumulation was suppressed to 3% that of wild-type levels were firmer than controls throughout ripening. Suppression of Exp1 protein also substantially inhibited polyuronide depolymerization late in ripening but did not prevent the breakdown of structurally important hemicelluloses, a major contributor to softening. In contrast, fruit overexpressing high levels of recombinant Exp1 protein were much softer than controls, even in mature green fruit before ripening commenced. This softening was correlated with the precocious and extensive depolymerization of structural hemicelluloses, whereas polyuronide depolymerization was not altered. These data are consistent with there being at least three components to fruit softening and textural changes. One component is a relaxation of the wall directly mediated by Exp1, which indirectly limits part of a second component due to polyuronide depolymerization late in ripening, perhaps by controlling access of a pectinase to its substrate. The third component is caused by depolymerization of hemicelluloses, which occurs independently of or requires only very small amounts of Exp1 protein.

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.

Oligogalacturonic acid and chitosan reduce stomatal aperture by inducing the evolution of reactive oxygen species from guard cells of tomato and Commelina communis.

Stomatal opening provides access to inner leaf tissues for many plant pathogens, so narrowing stomatal apertures may be advantageous for plant defense. We investigated how guard cells respond to elicitors that can be generated from cell walls of plants or pathogens during pathogen infection. The effect of oligogalacturonic acid (OGA), a degradation product of the plant cell wall, and chitosan (beta-1,4-linked glucosamine), a component of the fungal cell wall, on stomatal movements were examined in leaf epidermis of tomato (Lycopersicon esculentum L.) and Commelina communis L. These elicitors reduced the size of the stomatal aperture. OGA not only inhibited light-induced stomatal opening, but also accelerated stomatal closing in both species; chitosan inhibited light-induced stomatal opening in tomato epidermis. The effects of OGA and chitosan were suppressed when EGTA, catalase, or ascorbic acid was present in the medium, suggesting that Ca(2+) and H(2)O(2) mediate the elicitor-induced decrease of stomatal apertures. We show that the H(2)O(2) that is involved in this process is produced by guard cells in response to elicitors. Our results suggest that guard cells infected by pathogens may close their stomata via a pathway involving H(2)O(2) production, thus interfering with the continuous invasion of pathogens through the stomatal pores.

The effect of hydrogen peroxide on the viability of tomato cells and of the fungal pathogen Cladosporium fulvum

An oxidative burst was previously demonstrated to be induced in tomato plants by race specific elicitors of the fungal pathogen Cladosporium fulvum . The in planta levels of H2O2estimated to occur during elicitor treatment, were compared with the levels required to show toxicity to host cells and to the fungal pathogen. Injection of Cf-9 tomato leaves with 100 m m H2O2caused an insignificant degree of necrosis and 1m H2O2was required to cause complete leaf necrosis comparable to that induced by the AVR9 elicitor. Assays with Cf-5 tomato cell suspensions confirmed the low toxicity of H2O2to tomato cells but, as expected, the addition of Fe2+with H2O2(or with intercellular fluids containing AVR5 elicitor) enhanced cell death as determined by the Evans Blue assay. Germination and germ tube growth of conidia of C. fulvum were significantly retarded by 4–5 m m H2O2, and at higher concentrations, death of germ tubes was observed (ED50=22 m m), as determined by the fluorescein diacetate assay. The addition of Fe2+with H2O2had little effect on fungal growth or viability in vitro . These results suggest that the amount of H2O2accumulating during an elicitor-induced response in leaves may be sufficient to affect fungal colonization but not to affect viability of host cells unless the Fe2+status in the apoplast is in some way altered by the elicitor to facilitate OH.production via the Fenton reaction.

Plants have a sensitive perception system for the most conserved domain of bacterial flagellin

The flagellum is an important virulence factor for bacteria pathogenic to animals and plants. Here we demonstrate that plants have a highly sensitive chemoperception system for eubacterial flagellins, specifically targeted to the most highly conserved domain within its N terminus. Synthetic peptides comprising 15-22 amino acids of this domain acted as elicitors of defence responses at sub-nanomolar concentrations in cells of tomato and several other plant species. Peptides comprising only the central 8 to 11 amino acids of the active domain had no elicitor activity but acted as specific, competitive inhibitors in tomato cells. These antagonists suppressed the plant's response to flagellin, crude bacterial extracts and living bacterial cells. Thus, plants have a highly sensitive and selective perception system for the flagellin of motile eubacteria.

Messenger RNA-binding properties of nonpolysomal ribonucleoproteins from heat-stressed tomato cells

Most cells experiencing heat stress reprogram their translational machinery to favor the synthesis of heat-stress proteins. Translation of other transcripts is almost completely repressed, but most untranslated messengers are not degraded. In contrast to yeast, Drosophila melanogaster, and HeLa cells, plant cells store repressed messengers in cytoplasmic nonpolysomal ribonucleoproteins (RNPs). To follow the fate of untranslated transcripts, we studied protein composition, mRNA content, and RNA-binding properties of nonpolysomal RNPs from heat-stressed tomato (Lycopersicon peruvianum) cells. Contrary to the selective interaction in vivo, RNPs isolated from tomato cells bound both stress-induced and repressed messengers, suggesting that the selection mechanism resides elsewhere. This binding was independent of a cap or a poly(A) tail. The possible role of proteasomes and heat-stress granules (HSGs) in mRNA storage is a topic of debate. We found in vitro messenger-RNA-binding activity in messenger RNP fractions free of C2-subunit-containing proteasomes and HSGs. In addition, mRNAs introduced into tobacco (Nicotiana plumbaginifolia) protoplasts were found in the cytoplasm but were not associated with HSGs.

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.

Characterization of a tomato karyopherin that interacts with the Tomato Yellow Leaf Curl Virus (TYLCV) capsid protein

cDNA from poly(A)+ RNA which has been extracted from A karyopherin a (LeKAPa1) cDNA was isolated from tomato Lycopersicon esculentum cv. MoneyMaker leaves and used as plants. The deduced LeKAPa1 protein sequence of 527 amino template for reverse transcription. The cDNA preparation was acids showed similarity to other plant karyopherin a proteins. then subcloned using Eco RI adapters into the Eco RI site of When LeKAPa1 was expressed in a yeast two-hybrid system the lgt10 phage vector (Stratagene). The recombinant l phage together with the gene coding for the capsid protein (CP) of the DNA was packaged in vitro using the Stratagene lambda packtomato yellow curl leaf virus (TYLCV), it interacted directly with aging extract Gigapack II according to the manufacturer’s CP. Thus, LeKAPa1 may be involved in the nuclear import of instructions, and used to infect the Escherichia coli C600 hfl’ TYLCV CP and, potentially, the TYLCV genomes during viral strain. The resulting cDNA library (2.0◊106 plaques) was screinfection of the host tomato cells. ened with the AtKAPa probe as described (Ausubel et al., 1994). Briefly, a 32P-labelled 2 kb fragment of AtKAPa cDNA containing the complete open reading frame was hybridized for 16 h

Metal Stress Response and Tolerance of Cultured Cells from Silene vulgaris and Lycopersicon peruvianum: Role of Heat Stress Proteins

Summary The influence of the heavy metal ions Hg ++ , Cu ++ , Cd ++ and Zn ++ and of arsenite on growth, amino acid uptake, protein- and heat shock protein synthesis was investigated in cell cultures of a heavy metal tolerant Silene vulgaris and the sensitive Lycopersicon peruvianum . A distinct tolerance of Silene cell growth in comparison to tomato cells against Cu, Cd and Zn was observed. Synthesis of the small heat stress proteins was induced in both species, however, with quantitative differences depending on species and metal ion. While in tomato cells all metals induce HSP synthesis, in Silene the induction by Cu and Cd was found to be significantly lower in comparison to Hg and arsenite, and Zn did not induce HSP. Therefore, generally a lower tolerance of the cells against a metal is connected with a higher HSP synthesis. From comparison of cell growth and HSP accumulation in the presence of metal ions it was further concluded that HSP synthesis is a part of HM stress response of tolerant and non tolerant cells as under heat shock, but HSPs are not responsible for the heritable metal tolerance of Silene . In contrast to heat shock, metal stress does not inhibit the cell protein synthesis directly. In cultured tomato and Silene cells the inhibition of protein synthesis under metal stress was found to be a consequence of the inhibition of amino acid uptake. Zn has no effect on amino acid uptake of Silene cells . It is concluded that only Zn tolerance of Silene seems to be related with membrane stability.

Phytochelatins and heavy metal tolerance

The induction and heavy metal binding properties of phytochelatins in heavy metal tolerant ( Silene vulgaris ) and sensitive (tomato) cell cultures, in water cultures of these plants and in Silene vulgaris grown on a medieval copper mining dump were investigated. Application of heavy metals to cell suspension cultures and whole plants of Silene vulgaris and tomato induces the formation of heavy metal–phytochelatin-complexes with Cu and Cd and the binding of Zn and Pb to lower molecular weight substances. The binding of heavy metal ions to phytochelatins seems to play only a transient role in the heavy metal detoxification, because the Cd- and Cu-complexes disappear in the roots of water cultures of Silene vulgaris between 7 and 14 days after heavy metal exposition. Free heavy metal ions were not detectable in the extracts of all investigated plants and cell cultures. Silene vulgaris plants grown under natural conditions on a mining dump synthesize low molecular weight heavy metal binding compounds only and show no complexation of heavy metal ions to phytochelatins. The induction of phytochelatins is a general answer of higher plants to heavy metal exposition, but only some of the heavy metal ions are able to form stable complexes with phytochelatins. The investigation of tolerant plants from the copper mining dump shows that phytochelatins are not responsible for the development of the heavy metal tolerant phenotypes.

The extensin multigene family responds differentially to superoxide or hydrogen peroxide in tomato cell cultures

Changes in extensin gene expression were examined in cultured tomato cells following treatments leading to the production of activated oxygen species. Digitonin, a steroid glycoalkaloid compound, has been shown to trigger a rapid and transient production of superoxide anion, O −⋅ 2. 6 h after application of 50 or 100 μM of digitonin, the accumulation of four extensin transcripts (1.5, 2.6, 4.0 and 6.1 kb) was observed. Superoxide dismutase strongly inhibited the digitonin-mediated response, suggesting a key role of O −⋅ 2 in the signalling cascade. Furthermore, cells treated with enzymatically produced O −⋅ 2 generated by xanthine oxidase (0.015 U/ml) gave a similar extensin response and again, SOD exerted a strong inhibitory effect on the response. On the other hand, H 2O 2 (2 mM) or the enzymatic H 2O 2 generator, glucose oxidase (0.34 U/ml), elicited the accumulation of only three of the four transcripts (1.5, 2.6 and 4.0 kb), indicating that the corresponding genes could be regulated either by H 2O 2 or O −⋅ 2 but that the gene encoding the 6.1 kb transcript was exclusively expressed in response to O −⋅ 2. Finally, it was shown that lipid peroxidation, which was only induced when cells were exposed to H 2O 2, did not participate in the AOS-mediated gene expression for extensin. It can be concluded from these results that tomato cells are able to discriminate H 2O 2 from O −⋅ 2 and they probably sense the latter by the specific oxidation of an extracellular component.

Expression of the tomato peroxidase gene TPX1 in NaCl-adapted and unadapted suspension cells

The expression pattern of a peroxidase – (EC 1.11.1.7) encoding gene, TPX1, was studied in NaCl-adapted and unadapted tomato (Lycopersicon esculentum) cells growing in suspension culture. We followed the transcript level during the cell growth cycle and under different salt treatments. There was a cell-cycle-dependent TPX1 expression, the maximum level of transcripts coinciding with the period of cell enlargement in both salt-adapted and unadapted tomato cells. However, enzyme activity of the encoded isoperoxidase was only detected in the culture medium of the salt-adapted cells. NaCl treatment decreased TPX1 transcripts in both cell lines, but the threshold concentration for this down-regulation was higher in adapted cells. In conclusion, salt-adapted tomato cells show significant changes in comparison with unadapted cells in the expression of a gene encoding a cell-wall-targeted peroxidase.

Negative selection driven by cytosine deaminase gene in Lycopersicon esculentum hairy roots

The bacterial cytosine deaminase ( cd) gene was employed as a negative selection marker in transgenic tomato ( Lycopersicon esculentum var. cerasiforme) hairy roots. The cd gene was introduced in tomato cells using a modified binary vector in the Agrobacterium rhizogenes wild type strain 8196. The transconjugant strain was obtained through triparental mating and then inoculated on tomato stem fragments. The roots that appeared at the inoculation site were isolated and subcultured until axenic root clones were established. These roots were tested for their ability to grow in medium containing 5-fluorocytosine (5-FC), which becomes toxic in the presence of cytosine deaminase. The data, together with molecular analysis of the CD-T-DNA transferred, clearly show that expression of cd in root cells provides a strong negative selection, completely inhibiting root growth. This negative selection was obtained in roots where only one copy of the cd gene was integrated in the cell genome, providing a potential tool for mutagenesis and transposon mobilization studies.

Purification and characterization of an endo-polygalacturonase from Verticillium albo-atrum

A polygalacturonase was isolated from the culture filtrate of the fungal plant pathogen Verticillium albo-atrum and purified 22-fold to homogeneity as judged by SDS-electrophoresis. The enzyme was a basic protein with a molecular weight of 37 kDa, an isoelectric point ≥8·6 and containing 1·7% carbohydrate. The enzyme was an endo-polygalacturonase and hydrolysed a wide range of pectic substrates including polygalacturonic acid, 93% methylated pectin and pectins in tomato cell walls. The best substrate was 31% methylated pectin. Relative reaction rates on pectins with different degrees of methylation could be explained by considering both the number of susceptible bonds and non-specific enzyme-substrate interactions. The principal products of long-term hydrolysis were di- and mono-galacturonate. Maximum activity was observed at pH 4·6–5·0 and 46 °C. However, the enzyme lost activity above 30 °C in the absence of substrate. Enzyme activity was very sensitive to changes in ionic strength at low salt levels. It was stable in the pH range 3–11 at 30 °C.