Soybean Cell Suspension Research Articles

The effect of cadmium on cell cycle control in suspension culture cells of soybean

Heavy metals inhibit plant growth. This proces may be directly or indirectly connected with mechanisms regulating cell division. We analyzed the effect of Cd2+ on cell cycle progression in partially synchronized soybean (Glycine max) cell suspension culture and followed the expression of cell cycle genes (cyclin B1 and cyclin-dependent kinase A - CDK-A). We have checked the hypothesis that Cd2+-induced impairment of cell division is connected with DNA damage. The [3H]-thymidine incorporation in cell cultures synchronized either with hydroxyurea (HU) or phosphate starvation have shown, that Cd2+ strongly affects the S phase of soybean cell cycle, by causing the earlier entry of cells into S phase and by decreasing the rate of DNA synthesis. RT-PCR analysis indicated that Cd2+ decreases the level of cyclin B1 mRNA and has no effect on CDK-A mRNA. The result of comet assay indicated the damaging effect of Cd2+ on DNA of soybean cells. We suggest that Cd2+ affects plant cell cycle at two major checkpoints: the G1/S — by damaging of DNA, and G2/M - by decreasing the level of cyclin B1 mRNA

Cadmium-induced changes in antioxidant enzymes in suspension culture of soybean cells.

Cadmium (Cd), similarly to other heavy metals, inhibits plant growth. We have recently showed that Cd(2+) either stimulates (1-4 microM) or inhibits (>or= 6 microM) growth of soybean (Glycine max L.) cells in suspension culture (Sobkowiak & Deckert, 2003, Plant Physiol Biochem. 41: 767-72). Here, soybean cell suspension cultures were treated with various concentrations of Cd(2+) (1-10 microM) and the following enzymes were analyzed by native electrophoresis: superoxide dismutase (SOD), catalase (CAT), peroxidase (POX) and ascorbate peroxidase (APOX). We found a significant correlation between the cadmium-induced changes of soybean cell culture growth and the isoenzyme pattern of the antioxidant enzymes. The results suggest that inhibition of growth and modification of antioxidant defense reactions appear in soybean cells when Cd(2+) concentration in culture medium increases only slightly, from 4 to 6 microM.

Cadmium-induced changes in antioxidant enzymes in suspension culture of soybean cells.

Cadmium (Cd), similarly to other heavy metals, inhibits plant growth. We have recently showed that Cd(2+) either stimulates (1-4 microM) or inhibits (>or= 6 microM) growth of soybean (Glycine max L.) cells in suspension culture (Sobkowiak & Deckert, 2003, Plant Physiol Biochem. 41: 767-72). Here, soybean cell suspension cultures were treated with various concentrations of Cd(2+) (1-10 microM) and the following enzymes were analyzed by native electrophoresis: superoxide dismutase (SOD), catalase (CAT), peroxidase (POX) and ascorbate peroxidase (APOX). We found a significant correlation between the cadmium-induced changes of soybean cell culture growth and the isoenzyme pattern of the antioxidant enzymes. The results suggest that inhibition of growth and modification of antioxidant defense reactions appear in soybean cells when Cd(2+) concentration in culture medium increases only slightly, from 4 to 6 microM.

Metabolism of the Nonylphenol Isomer [Ring-U-14c]-4-(3′,5′-Dimethyl-3′-Heptyl)-Phenol by Cell Suspension Cultures of Agrostemma githago and Soybean

The biotransformation of the nonylphenol isomer [ring-U-14C]-4-(3′,5′-dimethyl-3′-heptyl)-phenol (4-353-NP, consisting of two diastereomers) was studied in soybean and Agrostemma githago cell suspension cultures. With the A. githago cells, a batch two-liquid-phase system (medium/n-hexadecane 200:1, v/v) was used, in order to produce higher concentrations and amounts of 4-353-NP metabolites for their identification; 4-353-NP was applied via the n-hexadecane phase. Initial concentrations of [14C]-4-353-NP were 1 mg L−1 (soybean), and 5 and 10 mg L−1 (A. githago). After 2 (soybean) and 7 days (A. githago) of incubation, the applied 4-353-NP was transformed almost completely by both plant species to four types of products: glycosides of parent 4-353-NP, glycosides of primary 4-353-NP metabolites, nonextractable residues and unknown, possibly polymeric materials detected in the media. The latter two products emerged especially in soybean cultures. Portions of primary metabolites amounted to 19–22% (soybean) and 21–42% of applied 14C (A. githago). After liberation from their glycosides, the primary 4-353-NP metabolites formed by A. githago were isolated by HPLC and examined by GC-EIMS as trimethylsilyl derivatives. In the chromatograms, eight peaks were detected which due to their mass spectra, could be traced back to 4-353-NP. Seven of the compounds were side-chain monohydroxylated 4-353-NP metabolites, while the remaining was a (side-chain) carboxylic acid derivative. Unequivocal identification of the sites of hydroxylation/oxidation of all transformation products was not possible. The main primary metabolites produced by A. githago were supposed to be four diastereomers of 6′-hydroxy-4-353-NP (about 80% of all products identified). It was concluded that plants contribute to the environmental degradation of the xenoestrogen nonylphenol; the toxicological properties of side-chain hydroxylated nonylphenols remain to be examined.

Syringolide elicitor-induced oxidative burst and protein phosphorylation in soybean cells, and tentative identification of two affected phosphoproteins

The bacterial plant pathogen Pseudomonas syringae pv. glycinea carrying an active allele of avirulence gene D (avrD) elicits a hypersensitive defense response (HR) specifically in soybean cultivars carrying the disease resistance gene Rpg4. Expression of avrD leads to production of a low molecular weight elicitor known as syringolide. Infiltration of syringolide into soybean leaves elicits an Rpg4-specific HR and also induces Rpg4-specific Ca2+, H+, and K+ ion fluxes and cell death in soybean cell suspensions. We have demonstrated that syringolide treatment of soybean cell suspensions further induces an Rpg4-specific hydrogen peroxide burst and subsequent shifts in protein phosphorylation. Furthermore, proteins co-migrating with two affected phosphoproteins (45 and 20kDa) were collected from preparative 2D-PAGE gels and internal peptide amino acid sequences were obtained. Partial amino acid sequence from the 45kDa protein showed strong identity to a family of plant EF-hand calcium-binding proteins and sequences from the 20kDa protein showed strong identity to a family of plant glycine-rich RNA-binding proteins (GRPs). The soybean GRP cDNA was cloned and sequenced.

Formation and vacuolar localization of salicylic acid glucose conjugates in soybean cell suspension cultures

The metabolism and intracellular localization of salicylic acid (SA) was investigated in soybean (Glycine max[L.] cv Williams 82) cell suspension cultures. [7–14C]SA was added to the cell cultures, the metabolites were extracted from the cells at various time points and analysed by TLC and HPLC. The [7–14C]SA was taken up rapidly from the culture media and converted primarily to SA 2‐O‐β‐d‐glucose (SAG). Lower levels of glucosylated 2,5‐dihydroxbenzoic acid (gentisic acid) and methyl salicylate 2‐O‐β‐d‐glucose were also formed. Examination of the intracellular localization of the glucose conjugates revealed that all of the conjugates associated with the protoplasts were found in the vacuoles. An SA glucosyltransferase (SAGT) that could catalyse the formation of SAG from SA and UDP‐glucose could be extracted from soybean cells and assayed in vitro. Increasing concentrations of SA added to the culture media induced the SAGT activity. The highest levels of SAGT activity were observed in cells treated with 0.5 mM SA. The SAGT activity in these cells was 88‐fold greater than the SAGT activity in the untreated cells. The intracellular localization of the SAGT activity was also examined and it was determined that the majority of the SAGT activity in the protoplasts was located outside the vacuole. Therefore, it appears as if SAG is formed from SA outside the vacuole, presumably in the cytoplasm, and then subsequently transported into the vacuole where it accumulates.

Reactive oxygen intermediates modulate nitric oxide signaling in the plant hypersensitive disease-resistance response

The mechanisms involved in plant defense show several similar characteristics with the innate immune systems of vertebrates and invertebrates. In animals, nitric oxide (NO) cooperates with reactive oxygen intermediates (ROI) to kill tumor cells and is also required for macrophage killing of bacteria. Such cytotoxic events occur because unregulated levels of NO determine its diffusion-limited reaction with O 2 – generating peroxynitrite (ONOO –), a mediator of cellular injury in many biological systems. In soybean suspension cells, unregulated NO production during the onset of a pathogen-induced hypersensitive response (HR) is not sufficient to activate the hypersensitive cell death, which is triggered only by fine tuning the NO/ROI ratio. Furthermore, that hypersensitive cell death is activated following interaction of NO with H 2O 2, rather than O 2 –. Increasing O 2 – levels reduces NO-derived toxicity, and the addition of ONOO – to soybean suspensions does not affect cell viability. Consistently with the fact that ONOO – is not an essential mediator of NO/RO-induced cell death, during the HR superoxide dismutase (SOD) accelerates O 2 – dismutation to H 2O 2 and therefore minimizes the loss of NO by reaction with O 2 – and triggers hypersensitive cell death through the NO/H 2O 2 synergism. Consequently, the rates of production and dismutation of O 2 – generated during the oxidative burst play a crucial role in modulating NO signaling through the cell death pathway, which proceeds through mechanisms different from those commonly observed in animals.

Induction of H2O2 synthesis by β-glucan elicitors in soybean is independent of cytosolic calcium transients

Soybean cell suspension cultures have been used to investigate the role of the elevation of the cytosolic Ca2+ concentration in β-glucan elicitors-induced defence responses, such as H2O2 and phytoalexin production. The intracellular Ca2+ concentration was monitored in transgenic cells expressing the Ca2+-sensing aequorin. Two lines of evidence showed that a transient increase of the cytosolic Ca2+ concentration is not necessarily involved in the induction of H2O2 generation: (i) a Bradyrhizobium japonicum cyclic β-glucan induced the H2O2 burst without increasing the cytosolic Ca2+ concentration; (ii) two ion channel blockers (anthracene-9-carboxylate, A9C; 5-nitro-2-(3-phenylpropylamino)-benzoate, NPPB) could not prevent a Phytophthora soja β-glucan elicitor-induced H2O2 synthesis but did prevent a cytosolic Ca2+ concentration increase. Moreover, A9C and NPPB inhibited P. sojae β-glucan-elicited defence-related gene inductions as well as the inducible accumulation of phytoalexins, suggesting that the P. sojae β-glucan-induced transient cytosolic Ca2+ increase is not necessary for the elicitation of H2O2 production but is very likely required for phytoalexin synthesis

The role of lipid peroxidation in aluminium toxicity in soybean cell suspension cultures.

The primary reactions leading to Al toxicity in plant cells have not yet been elucidated. We used soybean (Glycine max [L.] Merr.) cell suspension cultures to address the question whether lipid peroxidation plays an important role in Al toxicity. Upon transfer to an Al-containing culture medium with a calculated Al3+ activity of 15 microM soybean cells showed a distinct and longtime increase in lipid peroxidation within 4 h. At the same time a drastic loss of cell viability was observed. Butylated hydroxyanisole (BHA) and N,N'-diphenyl-p-phenylenediamine (DPPD), two lipophilic antioxidants, were able to almost completely suppress lipid peroxidation in Al-treated cells at a concentration of 20 microM. This effect was dose-dependent for DPPD and was observed at minimum concentrations of 1-2 microM. When lipid peroxidation was suppressed by DPPD or BHA cell viability remained high even in the presence of toxic Al concentrations. These results suggest that Al-induced enhancement of lipid peroxidation is a decisive factor for Al toxicity in suspension cultured soybean cells.

Phosphate Induces Rapid H2O2 Generation in Soybean Suspension Cells

Abstract: Involvement of reactive oxygen species has been implicated in plant defence against pathogens. We report here a novel pathway of H2O2 generation induced by the addition of phosphate in soybean (Glycine max L.) cell suspension cultures. This H2O2 generation was initiated shortly after the addition of phosphate, and lasted only approximately one hour, as opposed to several hours observed during an attack by an avirulent strain of the bacterial pathogen Pseudomonas syringae pv. glycinea (Psg). In addition, when cell cultures were treated with both phosphate and the avirulent pathogen, two distinct oxidative burst events were observed. In contrast to DPI-sensitive Psg-induced H2O2 generation, phosphate-induced H2O2 generation was insensitive to this NADPH oxidase inhibitor. This suggests that an NADPH oxidase-independent pathway may be involved in the phosphate-induced H2O2 accumulation, which could be involved in sensing of phosphate availability in the environment.

Growth Inhibition in Suspension-cultured Soybean Cells in the Presence of Ferulic Acid is Mediated through Spermine Accumulation

Summary The effects of ferulic acid on the growth and polyamine levels of suspension-cultured soybean { Glycine max [L.] Merrill cv. Baek Tae) cells were investigated. When soybean suspension cells were cultured in the presence of ferulic acid, cell growth was significantly inhibited. Ferulic acid treatment resulted in a higher spermine level and lower levels of putrescine and spermidine in soybean suspension cells. The growth of soybean cells cultured in the presence of ferulic acid did not recover as a result of putrescine and spermidine addition. 5'-methylthioadenosine, an inhibitor of spermine biosynthesis, caused a reduced level of spermine in soybean suspension cells cultured in the presence of ferulic acid. The growth of soybean cells cultured in the presence of ferulic acid was completely recovered after addition of MTA. Our cumulative results suggest that spermine accumulation is a factor causing growth inhibition of suspension-cultured soybean cells in the presence of ferulic acid.

Differential Gene Expression in Plants Stressed by the Peroxidizing Herbicide Oxyfluorfen§

The response of plants to the peroxidizing herbicide oxyfluorfen was investigated. The action of this p-nitrodiphenyl ether is based on inhibition of plastidic protoporphyrinogen oxidase, which leads to accumulation of protoporphyrin IX in the cytosol yielding reactive oxygen species by light activation. The induction of activities of antioxidative enzymes was followed in Nicotiana tabacum plants, var. BelW3. Glutathione reductase activity was elevated by 75% compared to control, monodehydroascorbate reductase by 65% and glutathione 5-transferase by 110% . The mRNA of ascorbate peroxidase and catalase isoform 2 was induced, the catalase isoform 1 was reduced. These findings were confirmed and supported by measuring enzymatic activity changes in photoheterotrophically grown soybean (Glycine max) suspension cultures. To find a possible involvement of compounds regulating oxidative stress response, we investigated the influence of salicylic acid and BTH (benzo(1,2,3)thiadiazole- 7-carbothioic acid 5-methylester), both inducers of pathogen defense, on soybean cell suspension cultures. The specific activities of glutathione reductase, monodehydroascorbate reductase and glutathione 5-transferase increased strongly, comparable to oxyfluorfen treatment. Both compounds protected the cells against oxyfluorfen-induced lipid peroxidation and alleviated the accumulation of protoporphyrin IX

Metabolism of the Herbicide Isoproturon in Wheat and Soybean Cell Suspension Cultures

The plant metabolism of the phenylurea herbicide isoproturon (IPU) was studied in aseptic cell suspension cultures of soybean and wheat. After incubation with the14C-labeled herbicide (1 ppm) over 48 h, total metabolic rates were 45–54%. The predominant metabolites in soybean were identified as monodesmethyl-IPU, 2-hydroxy-IPU, and 2-hydroxy-monodesmethyl-IPU. The major metabolic pathway identified for wheat was the conversion of IPU to 2-hydroxy-IPU as the primary metabolite and then to 2-hydroxy-monodesmethyl-IPU. 2-Hydroxy-IPU and an olefinic metabolite (isopropenyl-IPU) are described here for the first time. Metabolite identification was based on high-performance liquid chromatography retention times as well as time-of-flight/secondary-ion mass spectrometry and1H nuclear magnetic resonance spectroscopy. Wheat microsomes catalyzed the NADPH-dependent conversion of isoproturon to monodesmethyl and 2-hydroxy-monodesmethyl metabolites. 2-Hydroxy-IPU was only a minor enzymatic product so there was a remarkable difference between the primary cellular and enzymatic metabolites.14C Incorporation into the wheat-insoluble residue fraction increased from 3% (48 h) to 14% (7 days). A sequential solubilization procedure indicated that binding occurred mainly to the operationally defined hemicellulose (65%) and lignin (10%) fractions. Simulated stomach conditions (pH 1, 37° C, 24 h) released only about 5% of total bound radioactivity. The similarity of IPU metabolism in microbial, plant, and animal systems is discussed, with 2-hydroxy-monodesmethyl-IPU being a common metabolite.

Metabolism and cell wall incorporation of phenoxyacetic acid in soybean cell suspension culture

Metabolism and cell wall incorporation of phenoxyacetic acid in soybean cell suspension culture

Metabolism and cell wall incorporation of phenoxyacetic acid in soybean cell suspension culture

The metabolism of [ 14 C]phenoxyacetic acid (POA) was studied in cell suspension culture of soybean (Glycine max). POA was metabolized to 4-HO-POA, 4-HO-POA glucoside and 4-HO-POA glycosidic ester. A large part of the 4-HO-POA glucoside and small amounts of the glycosidic ester were recovered in the medium. POA was also converted to non-extractable residues bound to cell walls. Sequential extraction of cell-wall polymers showed that non-extractable residues, partly identified with 4-HO-POA and POA, were mainly associated with hemicelluloses and lignin. Comparison of the metabolism of [carboxy- 14 C]- and [phenyl- 14 C]POA revealed some degradation of the POA side-chain, followed in all probability by the incorporation of the aromatic moiety into cell walls. However, the sturdiness of the resulting bonds prevented precise identification of these bound aromatic structures. In summary, the degradation of POA in soybean cell culture provided a good model to study the formation of non-extractable residues of pesticides.

Metabolism and cell wall incorporation of phenoxyacetic acid in soybean cell suspension culture

The metabolism of [14C]phenoxyacetic acid (POA) was studied in cell suspension culture of soybean (Glycine max). POA was metabolized to 4-HO-POA, 4-HO-POA glucoside and 4-HO-POA glycosidic ester. A large part of the 4-HO-POA glucoside and small amounts of the glycosidic ester were recovered in the medium. POA was also converted to non-extractable residues bound to cell walls. Sequential extraction of cell-wall polymers showed that non-extractable residues, partly identified with 4-HO-POA and POA, were mainly associated with hemicelluloses and lignin. Comparison of the metabolism of [carboxy-14C]- and [phenyl-14C]POA revealed some degradation of the POA side-chain, followed in all probability by the incorporation of the aromatic moiety into cell walls. However, the sturdiness of the resulting bonds prevented precise identification of these bound aromatic structures. In summary, the degradation of POA in soybean cell culture provided a good model to study the formation of non-extractable residues of pesticides. © 1999 Society of Chemical Industry

Metabolism and cell wall incorporation of phenoxyacetic acid in soybean cell suspension culture

Metabolism and cell wall incorporation of phenoxyacetic acid in soybean cell suspension culture

Potentiation of the oxidative burst and isoflavonoid phytoalexin accumulation by serine protease inhibitors

Treatment of soybean (Glycine max L. cv Williams 82) cell-suspension cultures with Pseudomonas syringae pv glycinea (Psg) harboring an avirulence gene (avrA) or with yeast elicitor resulted in an oxidative burst characterized by the accumulation of H2O2. This burst, and the resultant induction of glutathione S-transferase transcripts, occurred more rapidly and was more prolonged if cells were simultaneously treated with serine protease inhibitors such as phenylmethylsulfonyl fluoride (PMSF) or diisopropylfluorophosphate. PMSF and diisopropylfluorophosphate potentiate a large oxidative burst in cells exposed to Psg harboring the avrC avirulence gene, which is not recognized by the soybean cultivar used in this study. The potentiated burst was inhibited by diphenylene iodonium, an inhibitor of NADPH oxidase, and by the protein kinase inhibitor K252a. PMSF treatment of elicited cells or cells exposed to Psg:avrA caused a large increase in the accumulation of the isoflavonoid phytoalexin glyceollin; however, this was not associated with increased levels of transcripts encoding key phytoalexin biosynthetic enzymes. Glyceollin accumulation was inhibited by diphenylene iodonium; however, the oxidative burst in cells treated with Psg:avrC and PMSF was not followed by phytoalexin accumulation. We conclude that active oxygen species from the oxidative burst are necessary but not sufficient for inducing isoflavonoid phytoalexin accumulation in soybean cells.

H2O2 Generation and the influence of antioxidants during the 2,3,5-triiodobenzoic acid-mediated induction of glutathione s-transferase in soybean

The induction of glutathione S-transferase (GST, EC 2.5.1.18) with 2,3,5-triiodobenzoic acid (TIBA) in soybean ( Glycine max) hypocotyls was diminished by the natural antioxidants ascorbate and glutathione but not with 1,4-dithio-threitol (DTT). DTT, 1,2-benzenediol and 1,4-benzenediol induced GST activity in soybean hypocotyls. Furthermore, in the presence of luminol TIBA-treated soybean suspension cells exhibited strong biphasic chemiluminescence. The induced chemiluminescence was inhibited by added catalase (EC 1.11.1.6) and superoxide dismutase (EC 1.15.1.1) but not by the peroxidase inhibitor azide. These findings indicate that as a response to TIBA-treatment, soybean cells produce H 2O 2 and O 2− without involvement of peroxidase (EC 1.11.1.7). The time course of active oxygen species production was nearly identical to that described for cells treated with an incompatible pathogen.

Involvement of an NAD(P)H oxidase in the elicitor-inducible oxidative burst of soybean

The production of H 2O 2 in soybean ( Glycine max L. cv Harosoy 63) suspension cells upon treatment with a 1,3-1,6-β- glucan elicitor fraction from the cell walls of Phytophthora sojae was investigated. The β-glucan elicitor used in these studies was specifically recognized by the soybean β-glucan-binding proteins and induced phytoalexin synthesis in the cell culture. Production of H 2O 2 was measured by the in situ oxidation of o-dianisidine and was both a time- and a concentration-dependent process, with a maximal response occurring after 20–25 min. Inhibitor studies showed that catalase, peroxidase inhibitors and ascorbate abolished the H 2O 2-mediated o-dianisidine oxidation. Diphenyleneiodonium, but not p-hydroxymercury benzoic acid, both known inhibitors of the mammalian NADPH oxidase involved in the oxidative burst of phagocytes, was a strong inhibitor of the inducible H 2O 2 production with a concentration for 50% inhibition of 4 μM. The inhibitor studies also indicated that the oxidase might be at least in part responsible for H 2O 2 production by the cells. Furthermore, diphenyleneiodonium was the only inhibitor of O 2 − synthesis in cell-free analyses with a concentration for 50% inhibition of 2.2 μM. Neither ascorbate nor diphenyleneiodonium were able to inhibit elicitor-induced phytoalexin accumulation, indicating that in soybean H 2O 2 and O 2 − may not be directly responsible for phytoalexin production, but may be an independent defence response. © 1997 Elsevier Science Ltd. All rights reserved