Soybean Cell Suspension Cultures Research Articles

Development and validation of a novel and robust cell culture system in soybean (Glycine max (L.) Merr.) for promoter screening.

A novel soybean cell culture was developed, establishing a reliable and rapid promoter assay to enable high-throughput automated screening in soybean protoplasts relevant to shoot tissues in whole plants. Transient reporter gene assays can be valuable to rapidly estimate expression characteristics of heterologous promoters. The challenge for maximizing the value of such screens is to combine relevant cells or tissues with methods that can be scaled for high-throughput screening, especially for crop-rather than model species. We developed a robust and novel soybean cell suspension culture derived from leaf-derived callus for protoplast production: a platform for promoter screening. The protoplasts were transfected with promoter-reporter constructs, of which were chosen and validated against known promoter expression profiles from tissue-derived protoplasts (leaves, stems, and immature cotyledons) and gene expression data from plants. The cell culture reliably produced 2.82 ± 0.94 × 108 protoplasts/g fresh culture mass with a transfection efficiency of 31.06 ± 7.69% at 48h post-incubation. The promoter-reporter gene DNA expression levels of transfected cell culture-derived protoplasts were most similar to that of leaf- and stem-derived protoplasts (correlation coefficient of 0.99 and 0.96, respectively) harboring the same constructs. Cell culture expression was also significantly correlated to endogenous promoter-gene expression in leaf tissues as measured by qRT-PCR (correlation coefficient of 0.80). Using the manual protocols that produced these results, we performed early stage experiments to automate protoplast transformation on a robotic system. After optimizing the protocol, we achieved up to 29% transformation efficiency using our robotic system. We conclude that the soybean cell culture-to-protoplast transformation screen is amenable to automate promoter and gene screens in soybean that could be used to accelerate discoveries relevant for crop improvement. Key features of the system include low-cost, facile protoplast isolation, and transformation for soybean shoot tissue-relevant molecular screening.

The Missing Link in Leguminous Pterocarpan Biosynthesis is a Dirigent Domain-Containing Protein with Isoflavanol Dehydratase Activity.

Pterocarpan forms the basic structure of leguminous phytoalexins, and most of the isoflavonoid pathway genes encoding the enzymes responsible for its biosynthesis have been identified. However, the last step of pterocarpan biosynthesis is a ring closure reaction, and the enzyme that catalyzes this step, 2′-hydroxyisoflavanol 4,2′-dehydratase or pterocarpan synthase (PTS), remains as an unidentified ‘missing link’. This last ring formation is assumed to be the key step in determining the stereochemistry of pterocarpans, which plays a role in their antimicrobial activity. In this study, a cDNA clone encoding PTS from Glycyrrhiza echinata (GePTS1) was identified through functional expression fractionation screening of a cDNA library, which requires no sequence information, and orthologs from soybean (GmPTS1) and Lotus japonicus (LjPTS1) were also identified. These proteins were heterologously expressed in Escherichia coli and biochemically characterized. Surprisingly, the proteins were found to include amino acid motifs characteristic of dirigent proteins, some of which control stereospecific phenoxy radical coupling in lignan biosynthesis. The stereospecificity of substrates and products was examined using four substrate stereoisomers with hydroxy and methoxy derivatives at C-4′. The results showed that the 4R configuration was essential for the PTS reaction, and (−)- and (+)-pterocarpans were produced depending on the stereochemistry at C-3. In suspension-cultured soybean cells, levels of the GmPTS1 transcript increased temporarily prior to the peak in phytoalexin accumulation, strongly supporting the possible involvement of PTS in pterocarpan biosynthesis.

Isoflavone Augmentation in Soybean Cell Cultures Is Optimized Using Response Surface Methodology

Glycine max contains potential therapeutic isoflavones, and its productivity in plants is considerably influenced worldwide by several biotic and abiotic factors. Optimization of soybean cell suspension cultures (Indian variety, JS 335) to maximize the cell growth and isoflavone production in the present study was performed using response surface methodology (RSM) with three independent variables of plant growth regulators, 2,4-dichlorophenoxyacetic acid (2,4-D), 1-naphthalene acetic acid (α-NAA), and kinetin (Kn). The maximum biomass achieved was 70.62 g/L dry weight (dw) using the optimized medium of 2.10 mg/L 2,4-D, 5.52 mg/L α-NAA, and 0.35 mg/L Kn supplemented in the Murashige and Skoog (MS) basal medium. The total isoflavone content of 38.59 mg/g of dw was obtained in the medium with optimized conditions of 1.33 mg/L 2,4-D, 1.76 mg/L α-NAA, and 0.15 mg/L Kn. In comparison to field-grown soybean seeds, the cell suspension cultures profoundly augmented isoflavone concentrations. The optimized conditions for both biomass and total isoflavone content were evaluated by superimposing the contour plots. The results suggested that the optimized medium of cell suspension cultures possibly be used for scale-up studies in bioreactors to offer a high content of bioactive isoflavones.

Identification of Alternaria alternata mycotoxins by LC-SPE-NMR and their cytotoxic effects to soybean (Glycine max) cell suspension culture.

This present work describes the application of liquid chromatograpy-solid phase extraction-nuclear magnetic resonance spectroscopy to analyse Alternaria alternata crude extracts. Altenusin (1), alternariol (2), 3'-hydroxyalternariol monomethyl ether (3), and alternariol monomethyl ether (4), were separated and identified. High-resolution mass spectrometry confirmed the proposed structures. The cytotoxic effects of these compounds towards plants were determined using soybean (Glycine max) cell cultures as a model. EC50 values which range from 0.11 (±0.02) to 4.69 (±0.47) μM showed the high cytotoxicity of these compounds.

Chalcone suppresses lignin biosynthesis in illuminated soybean cells

Lignin and its related metabolites play critical roles in plant growth and development. Thus, lignin biosynthesis has attracted interest as a novel target site of plant growth inhibitors. Chalcone has been shown to not only inhibit lignin biosynthesis in plants, but also to suppress the growth of many annual plant species. In order to know the direct effect of chalcone on plant metabolism, the effects of chalcone on the activities of key enzymes in lignin biosynthesis and on the related metabolites were clarified with a time‐course study by using light‐induced suspension cultures of soybean cells. The fresh weight and packed cell volume of the soybean cells were inhibited after 8 h of chalcone treatment. The activities of phenylalanine ammonia lyase (EC 4.3.1.24) and 4‐coumarate: coenzyme A ligase (4CL; EC 6.2.1.12) were largely inhibited 4 h after the treatment with 0.15 mmol L−1 chalcone. Unlike these two enzymes, the activity of cinnamyl alcohol dehydrogenase (EC 1.1.1.195) was not inhibited until 16 h after the chalcone treatment. The content of the 4CL substrates and lignin in the soybean cells became relatively lower than the control under the light condition within 4 h and 8 h after the chalcone treatment, respectively. These results suggest that the growth suppression of soybean cells is positively associated with the inhibition of lignin biosynthesis by exogenous chalcone.

Structure–activity studies of GmSubPep, a soybean peptide defense signal derived from an extracellular protease

GmSubPep, a 12-amino acid peptide isolated from soybean leaves, induces the expression of genes in soybean suspension-cultured cells that encode proteins involved in defense against pathogens. The peptide is derived from an extracellular subtilisin-like protease (subtilase) and binds a putative cell-surface receptor that initiates a defense signaling cascade. Interaction of the peptide with its receptor results in alkalinization of soybean suspension cell media which can be utilized to analyze the kinetics of receptor binding. Substitutions of alanine at each of the 12 amino acid positions revealed that the amino acids at positions 10 (arginine) and 12 (histidine) were essential for activity. Both analogs were able to reduce the physiological effects of GmSubPep associated with receptor binding. Deletion of the C-terminal histidine [GmSubPep(1–11)] abolished the alkalinizing activity and this peptide was also a strong competitor for receptor binding. Deletion of N-terminal amino acids from GmSubPep caused a sequential loss of activity with no alkalinizing activity for GmSubPep(4–12). However, the N-terminal deleted peptides did not compete with GmSubPep for receptor binding. Further modifications at the arginine-10 position indicated that an ionizable proton was not essential for activity as an attenuated response was found for a citrulline substitution. Substitution of the histidine-12 with methylated histidine at position N-1 of the imidazole group abolished activity, whereas substitution at N-3 was completely active, indicating that the N-3 analog retains important receptor binding properties. This study indicates that the extreme C-terminal of GmSubPep has important signal transduction properties while the C-terminal is essential for receptor interaction.

Influence of minerals and elicitation on Datura stramonium L. tropane alkaloid production: Modelization of the in vitro biochemical response

Secondary metabolites are mostly involved in the relationship of the organism with its environment, e.g. in plant defence mechanisms or as signal compound; their synthesis is often induced by different kinds of biotic and/or abiotic stresses. In general the major strategies to improve their productivity are: the optimization of growth and production media or induction of secondary metabolite pathways by elicitors and metabolic engineering. From this point of view, elicitation is largely employed as a strategy to mimic stress effects, which then lead to a physiological response involving secondary metabolite synthesis and accumulation into the hairy root (HR). Thus, studies on the response to elicitation for HR in accordance with nutritional status are necessary. In the present study, which focuses on tropane alkaloid production in Datura stramonium L. hairy roots, the effects of minerals in the B5 culture medium [O.L. Gamborg, R.A. Miller, K. Ojima, Nutrient requirements of suspension cultures of soybean root cells, Exp. Cell Res. 50 (1968) 151–158] and elicitor (jasmonic acid) are studied using a mathematical model approach (screening analysis) and factorial design 2 3 was carried out to study the response to elicitation in accordance with nutritional status of HR for [NO 3 −], [H 2PO 4 −] and [Ca 2+]. The modelization of different minerals effects showed a response to elicitation only after the 8th day of cultivation. The study of models coefficients revealed that nitrate [NO 3 −], phosphorus [H 2PO 4 −] and calcium [Ca 2+] levels and combinations [NO 3 −/Ca 2+] and [NO 3 −/H 2PO 4 −/Ca 2+] were positive with elicitation; however, interaction effects [NO 3 −/H 2PO 4 −] and [H 2PO 4 −/Ca 2+] were negative. The coefficients position on the 28th day of cultivation, with the Pareto chart allowed to adjust the mathematical model by eliminating the no significant effects [NO 3 −/H 2PO 4 −], [H 2PO 4 −/Ca 2+] and [NO 3 −/H 2PO 4 −/Ca 2+]. As a result, we show that simulation with the adjusted model allows predicting the response to elicitation in accordance with the nutritional state of HR in nitrate and calcium. The measured values on the 28th day were statistically identical with calculated values obtained by mathematical models. These results revealed that HR, with limited nitrate, showed lower response to elicitation by jasmonic acid. Moreover, the adjusted model, of first degree, was not predictably beyond the experimental domain. As a general conclusion, NO 3 − (62.5 mM) must be added to the B5 medium at the beginning of the culture. The lack of these compounds reduced alkaloid metabolism, even in combination with elicitor treatments like jasmonic acid. The same trend was observed with Ca 2+ (11.1 mM), H 2PO 4 − (6.06 mM) and nitrate/calcium ratio (62.5 mM/11.1 mM), which also increased alkaloid production when combined with jasmonic acid 10 mM. These positive effects appeared at the 2nd week after initiation of the culture.

Abstracts of Nippon Dojo-Hiryogaku Zasshi

Abstracts of Nippon Dojo-Hiryogaku Zasshi

Isolation and aharacterization of genes induced by a short time of treatment with Nod factors in soybean suspension-cultured cells using a PCR-based cDNA subtraction method

Isolation and aharacterization of genes induced by a short time of treatment with Nod factors in soybean suspension-cultured cells using a PCR-based cDNA subtraction method

Analysis of the limitations of hepatitis B surface antigen expression in soybean cell suspension cultures

Soybean cell suspension cultures were transformed using Agrobacterium tumefaciens harboring pHBS/pHER constructs to express hepatitis B surface antigen (HBsAg). The transformed colonies were selected and analyzed for the expression of HBsAg by PCR, reverse transcription (RT) PCR, Western blot and ELISA analysis. The maximum expression of 700 ng/g F.W. was noted in pHER transformed cells. The highest expressing colonies were used to initiate the cell suspension cultures and the expression of HBsAg was estimated periodically. The expression levels were reduced drastically in cell suspension cultures compared to the colonies maintained on semi-solid medium. Various parameters were studied to maximize the cell growth and to retain the expression levels. The supplementation of culture medium with a protease inhibitor, leupeptin hemisulfate could restore up to 50% of HBsAg expression in cell suspension cultures. This is the first report to investigate the possible cause and solution to the loss of recombinant protein expression levels in plant cell suspension cultures.

Acclimation of photosystem II to high temperature in a suspension culture of soybean (Glycine max) cells requires proteins that are associated with the thylakoid membrane

In a study of the responses of photosystem II (PSII) to high temperature in suspension-cultured cells of soybean (Glycine max L. Merr.), we found that high temperatures inactivated PSII via two distinct pathways. Inactivation of PSII by moderately high temperatures, such as 41 degrees C, was reversed upon transfer of cells to 25 degrees C. The recovery of PSII required light, but not the synthesis of proteins de novo. By contrast, temperatures higher than 45 degrees C inactivated PSII irreversibly. An increase in the growth temperature from 25 to 35 degrees C resulted in an upward shift of 3 degrees C in the profile of the heat-induced inactivation of PSII, which indicated that the thermal stability of PSII had been enhanced. This acclimative response was reflected by the properties of isolated thylakoid membranes: PSII in thylakoid membranes from cells that had been grown at 35 degrees C exhibited greater thermal stability than that from cells grown at 25 degrees C. Disruption of the vesicular structure of thylakoid membranes with 0.05% Triton X-100 decreased the thermal stability of PSII to a similar level in both types of thylakoid membrane. Proteins released by Triton X-100 from thylakoid membranes from cells grown at 35 degrees C were able to increase the thermal stability of Triton-treated thylakoid membranes. These observations suggest that proteins that are associated with thylakoid membranes might be involved in the enhancement of the thermal stability of PSII.

Calcium-mediated perception and defense responses activated in plant cells by metabolite mixtures secreted by the biocontrol fungus Trichoderma atroviride.

BackgroundCalcium is commonly involved as intracellular messenger in the transduction by plants of a wide range of biotic stimuli, including signals from pathogenic and symbiotic fungi. Trichoderma spp. are largely used in the biological control of plant diseases caused by fungal phytopathogens and are able to colonize plant roots. Early molecular events underlying their association with plants are relatively unknown.ResultsHere, we investigated the effects on plant cells of metabolite complexes secreted by Trichoderma atroviride wild type P1 and a deletion mutant of this strain on the level of cytosolic free Ca2+ and activation of defense responses. Trichoderma culture filtrates were obtained by growing the fungus alone or in direct antagonism with its fungal host, the necrotrophic pathogen Botrytis cinerea, and then separated in two fractions (>3 and <3 kDa). When applied to aequorin-expressing soybean (Glycine max L.) cell suspension cultures, Trichoderma and Botrytis metabolite mixtures were distinctively perceived and activated transient intracellular Ca2+ elevations with different kinetics, specific patterns of intracellular accumulation of reactive oxygen species and induction of cell death. Both Ca2+ signature and cellular effects were modified by the culture medium from the knock-out mutant of Trichoderma, defective for the production of the secreted 42 kDa endochitinase.ConclusionNew insights are provided into the mechanism of interaction between Trichoderma and plants, indicating that secreted fungal molecules are sensed by plant cells through intracellular Ca2+ changes. Plant cells are able to discriminate signals originating in the single or two-fungal partner interaction and modulate defense responses.

Isolation of a novel ABC-transporter gene from soybean induced by salicylic acid

This paper reports on the identification and characterization of a new ATP-binding cassette (ABC) transporter which was identified as a salicylic acid-induced gene from soybean (Glycine max cv. Williams 82) in a subtractive suppression hybridization approach. A fragment of an ABC-transporter gene was used to isolate a full-length cDNA clone for this gene with a length of 4750 bp. The encoded protein has a length of 1447 amino acids and is composed of two similar repeat units typical of full-size ABC transporters. The sequence displays a close relationship to plant pleiotropic drug resistance (PDR)-type transporters and, on a homology basis, clusters together with the Arabidopsis thaliana PDR12 gene, suggesting GmPDR12 as a name for the gene isolated from soybean. GmPDR12 is rapidly responsive to salicylic acid and methyl jasmonate. The mRNA starts to accumulate 30 min after the addition of the signalling compounds. Salicylic acid is required for the execution of the hypersensitive reaction in soybean cell suspension cultures inoculated with Pseudomonas syringae pv. glycinea. It has been demonstrated previously that salicylic acid can be substituted by a variety of functional analogues of salicylic acid. All of these compounds lead to a strong and rapid transcriptional activation of GmPDR12, suggesting a shared signalling pathway.

Proteins induced by cadmium in soybean cells

The cadmium (Cd)-induced changes in protein pattern and identification of metal-stimulated polypeptides were analyzed in soybean cell suspension culture. The cell cultures were treated with various concentrations of Cd(2+) (3-10microM) for 24, 48 and 72h. The synthesis of [(35)S]-labeled proteins and their accumulation were analyzed by SDS-PAGE, whereas the identification of selected protein bands was performed by mass spectrometry. It is shown that Cd induced the appearance of the following proteins in soybean cells: superoxide dismutase, histone H2B, chalcone synthase and glutathione transferase.

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 (&gt;or= 6 microM) growth of soybean (Glycine max L.) cells in suspension culture (Sobkowiak &amp; 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.

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

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

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.