Soybean Suspension Cultures Research Articles

Sonication-assisted Agrobacterium-mediated transformation of soybean [Glycine max (L.) Merrill] embryogenic suspension culture tissue.

Successful transformation of plant tissue using Agrobacterium relies on several factors including bacterial infection, host recognition, and transformation competency of the target tissue. Although soybean [Glycine max (L.) Merrill] embryogenic suspension cultures have been transformed via particle bombardment, Agrobacterium-mediated transformation of this tissue has not been demonstrated. We report here transformation of embryogenic suspension cultures of soybean using "Sonication-Assisted Agrobacterium-mediated Transformation" (SAAT). For SAAT of suspension culture tissue, 10-20 embryogenic clumps (2-4 mm in diameter) were inoculated with 1 ml of diluted (OD600nm 0.1-0.5) log phase Agrobacterium and sonicated for 0-300 s. After 2 days of co-culture in a maintenance medium containing 100 µM acetosyringone, the medium was removed and replaced with fresh maintenance medium containing 400 mg/l Timentin®. Two weeks after SAAT, the tissue was placed in maintenance medium containing 20 mg/l hygromycin and 400 mg/l Timentin®, and the medium was replenished every week thereafter. Transgenic clones were observed and isolated 6-8 weeks following SAAT. When SAAT was not used, hygromycin-resistant clones were not obtained. Southern hybridization analyses of transformed embryogenic tissue confirmed T-DNA integration.

A serine protease from suspension-cultured soybean cells

A serine protease was purified from suspension-cultured soybean cells, by a combination of anion exchange, hydrophobic interaction and affinity chromatography. A 90000 M r subunit, which could be photoaffinity labelled with 3H-diisopropylfluorophosphate (DFP), was identified by SDS-polyacrylamide gel electrophoresis. The enzyme had a broad pH optimum from 5.5 to 8.5, and was strongly inhibited by antipain, leupeptin, aminoethylbenzenesulphonyl fluoride (AEBSF) and DFP, but not by soybean trypsin inhibitor. It cleaved several peptide 4-methylcoumaryl-7-amide derivatives after arginine or lysine residues. Mass spectroscopic analysis of oligopeptide digestion products indicated that the preferred cleavage positions were between paired arginine residues, or C-terminal to single arginine residues, depending on the oligopeptide substrate. Partial amino acid sequences from the purified protein showed sequence identity to bacterial protease II and prolyl peptidase, although the enzyme lacked prolyl endopeptidase activity. We discuss the possible involvement of the protease in plant defense responses.

Molecular cloning of a novel Ca2+-binding protein that is induced by NaCl stress.

Plant responses to high salt stress have been studied for several decades. However, the molecular mechanisms underlying these responses still elude us. In order to understand better the molecular mechanism related to NaCl stress in plants, we initiated the cloning of a large number of NaCl-induced genes in Arabidopsis. Here, we report the cloning of a cDNA encoding a novel Ca2+-binding protein, named AtCP1, which shares sequence similarities with calmodulins. AtCP1 exhibits, in particular, a high degree of amino acid sequence homology to the Ca2+-binding loops of the EF hands of calmodulin. However, unlike calmodulin, AtCP1 appears to have only three Ca2+-binding loops. We examined Ca2+ binding of the protein by a Ca2+-dependent electrophoretic mobility shift assay. A recombinant AtCP1 protein that was expressed in Escherichia coli did show a Ca2+-dependent electrophoretic mobility shift. To gain insight into the expression of the AtCP1 gene, northern blot analysis was carried out. The AtCP1 gene had a tissue-specific expression pattern: high levels of expression in flower and root tissues and nearly undetectable levels in leaves and siliques. Also, the expression of the AtCP1 gene was induced by NaCl treatment but not by ABA treatment. Finally, subcellular localization experiments using an AtCP1:smGFP fusion gene in soybean suspension culture cells and tobacco leaf protoplasts indicate that AtCP1 is most likely a cytosolic protein.

Cytokinin regulates the expression of a soybean beta-expansin gene by a post-transcriptional mechanism.

The cytokinin-inducible soybean mRNA Cim1 accumulates 20-60-fold upon cytokinin addition to cytokinin-starved soybean suspension cultures. In this report, we demonstrate that cytokinin-induced stability of the Cim1 mRNA plays an important role in the accumulation of the message. We also present evidence that cytokinin-induced Cim1 stability is blocked by the addition of the protein phosphatase inhibitor okadaic acid. Thus, we suggest that protein phosphatase activity is required for the cytokinin-induced stability and subsequent accumulation of Cim1 in soybean cells. The deduced amino acid sequence of the Cim1 protein product is similar to the group I pollen allergens from various plants, which constitute a subfamily of expansin proteins. The relatedness between Cim1 and the expansins supports our hypothesis that the protein product of Cim1 is localized to the cell wall and suggests a role for Cim1 in cytokinin-regulated cell wall expansion. Thus, post-transcriptional regulation of Cim1 by cytokinin may represent a molecular link between cytokinin and changes in cell shape and size.

Metabolic Studies of the Growth Regulator, Maleic Hydrazide

Abstract The metabolism of the plant growth regulator maleic hydrazide (MH) has been studied in sterile cell suspension cultures of tobacco, soybean, maize and wheat under standardized conditions. Maleic hydrazide was converted to its O-b -D-glucoside in yields between 9.0 % (tobacco) 15.0 % (soybean), 5.1 % (maize) and 2.2 % (wheat) respectively. This glucoside was completely cleaved under simulated conditions (pH 1, 37°C, 24 h) of a ruminant stomach. From these results it is concluded that MH-O-b-D-glucoside (MHG) belongs to a small group of acid-labile pesticide conjugates (11). The participation of a glucosyltransferase (GT) (EC 2.4.1-) in this conjugation reaction of MH was demonstrated in vitro for the first time. In addition, up to 18 % of the applied maleic hydrazide became associated with nonextractable residues (NER) in soybean, whereas in tobacco only 0.2 % could be detected in this fraction. The residue from soybean cells was solubilized only to a low degree (about 3 %) under simulated stomach conditions, but up to 20 % by the white rot fungus Phanerochaetechrysosporium.

Polyamine Sparing May Be Involved in the Prolongation of Cell Division Due to Inhibition of Phenylpropanoid Synthesis in Cytokinin-Starved Soybean Cells

Effects on growth, mostly of an inhibitory nature, have been attributed to phenolic compounds in vivo and in vitro. This suggests that l-α-aminooxy-β-phenylpropionic acid (l-AOPP), a competitive inhibitor of phenylalanine ammonia-lyase (PAL), the enzyme controlling the first step in phenylpropanoid synthesis, might stimulate growth in soybean suspension cultures (Glycine max, cv. Acme). The promotive effect of l-AOPP, measured as an increase in cell number, was more clearly detected in the growth-limiting condition of cytokinin starvation. At least one more cell division cycle was completed in the presence of l-AOPP before growth by division ceased and growth continued by expansion only. Phenolic acids are known to conjugate with polyamines, modulating the free levels of these plant growth substances. Thus, the effect of l-AOPP on the titers of free and conjugated polyamines (putrescine, spermidine, and spermine) was investigated by high performance liquid chromatography in the course of cytokinin starvation. An increased level of free putrescine was detected in the presence of l-AOPP relative to controls, especially in the initial period before growth became restricted to cell expansion. The decrease in free putrescine associated with the cessation of cell division was temporarily delayed, suggesting that an interaction between phenolic acids and polyamines is involved in the mechanism of growth promotion by l-AOPP.

Influence of Pathogenic and Non‐pathogenic Bacteria on Soybean Suspension Cells

AbstractCo‐cultivations of plant suspension cultures of soybean (Glycine max) with compatible phytopathogenic (Pseudomonas syringae pv. glycinea), incompatible phytopathogenic (Pseudomonas syringae pv. tomato), and different non‐pathogenic (Erwinia herbicola, Escherichia coli) bacteria were carried out. Growth and viability (triphenyltetrazolium chloride activity) of plant cells and bacteria as well as enzyme activities of peroxidase (PO), polyphenoloxidase (PPO), and phenylalanine ammonialyase (PAL) within the plant cells were investigated over an incubation period of 7 days. The compatible pathogen inhibited growth and viability of the plant cells after 1 day and led to the death of the majority of the plant cells by the seventh day. In contrast, the incompatible pathogen directly reduced growth and viability of the soybean cells and caused a strong induction of enzyme activities of PO and PAL to more than 4 times of the untreated control by the seventh day. The epiphytic bacterium Erwinia herbicola caused a slight inhibition of growth and viability of the plant cells after the second day of co‐cultivation. The PO activity increased in the same manner as in the incompatible interaction. The saprophytic Escherichia coli strain had a negligible influence on soybean suspension cells. All the bacteria tested except for Escherichia coli multiplied rapidly during co‐cultivation and reached populations of 108‐109 colonyfoming units/ml in the stationary phase. The results from this study demonstrate that the soybean suspension cells react differently to compatible, incompatible and saprophytic bacteria.

Release and biological activity of diffusible signal compounds from elicited plant cells

Summary Treatment of cell suspension cultures of soybean, alfalfa, or tobacco with macromolecular elicitors resulted in the release of low molecular mass (500–2000 u (Da)) endogenous elicitors that could pass through a dialysis membrane into the surrounding culture medium. The active compounds induced phenylalanine ammonia-lyase (PAL) activity in cells of all three species, isoflavonoid phytoalexin accumulation in soybean and alfalfa cells, and an oxidative burst in soybean cells. Release of factors from soybean cells was inhibited by the RNA synthesis inhibitor actinomycin D, but not by diphenylene iodonium, an inhibitor of the oxidative burst. The activity of the factors released from tobacco cells in response to the proteinaceous elicitor cryptogein, or from alfalfa cells in response to yeast elicitor, was unaffected by pretreatment with sodium periodate or proteinase K. In contrast, treatment of the factor(s) from soybean cells with proteinase K or trypsin destroyed its ability to induce the phytoalexin glyceollin. Ion-exchange chromatography revealed that the active material from cell cultures of each species consisted of at least two components. One component released from soybean cells appears to be a small peptide.

Biotransformation of Pyrene by Cell Cultures of Soybean (Glycine max L.), Wheat (Triticum aestivum L.), Jimsonweed (Datura stramonium L.), and Purple Foxglove (Digitalis purpurea L.)

The metabolism of the four-ringed polycyclic aromatic hydrocarbon (PAH) pyrene was investigated using cell suspension cultures of soybean, wheat, purple foxglove, and jimsonweed and callus cultures of soybean and foxglove. In all species, nonextractable residues were found (soybean, jimsonweed, and foxglove suspensions, <10% of applied 14 C; soybean and foxglove callus cultures, 20-25%; wheat, 30-40%); soluble metabolites were detected in only foxglove and wheat. About 90% of applied pyrene was transformed in wheat. Corresponding data from soybean and foxglove callus cultures were about 30% and those from soybean, jimsonweed, and foxglove suspensions about 7%. In foxglove, 1-hydroxypyrene methyl ether was identified as the main metabolite, whereas a complex mixture of carbohydrate conjugates of 1-hydroxypyrene was found in wheat. Due to the present results, crop and wild plants may be metabolic sinks for PAHs in the environment. Concentrations and toxicological implications of 1-hydroxypyrene, its derivatives, and analogous metabolites of other PAHs should be investigated.

Transformation of Soybean Embryogenic Cultures by Microprojectile Bombardment.

Regenerable embryogenic suspension cultures of soybean (Glycine max [L.] Merrill, cv. Jack) were transformed by microprojectile bombardment with a β-glucuronidase (gus) gene and a hygromycin phosphotransferase (hpt) gene under control of the cauliflower mosaic virus (CaMV) 35S promoter on different plasmids mixed together. Many independent transgenic clones were obtained by selection for hygromycin resistance. GUS activity was detected in 45% of the transgenic clones showing that cotransformation occurred at high frequency. Stable integration of both the gus reporter gene and the hpt selectable marker were further confirmed by polymerase chain reaction (PCR) amplification of both genes using double primer sets together in the same reaction. Southern blot hybridization analysis also showed the presence of the foreign genes in genomic DNA. The frequency of embryo development was increased and the time span required for embryo development was reduced by the use of a liquid medium to induce embryo development. An average of 3645 GUS-positive spots and ten transgenic clones were produced per bombardment by this procedure giving a 0.27% ratio of stable to transient expression. The procedure described here can be used for modification of agronomic traits and for study of gene regulation in soybean.

Synthesis and Turnover of Cell-Wall Polysaccharides and Starch in Photosynthetic Soybean Suspension Cultures.

Soybean (Glycine max [L.] Merr.) suspension cultures grown under photoautotrophic and photomixotrophic (1% sucrose) culture conditions were used in 14CO2 pulse-chase experiments to follow cell-wall polysaccharide and starch biosynthesis and turnover. Following a 30-min pulse with 14CO2, about one-fourth of the 14C of the photoautotrophic cells was incorporated into the cell wall; this increased to about 80% during a 96-h chase in unlabeled CO2. Cells early in the cell culture cycle (3 d) incorporated more 14C per sample and also exhibited greater turnover of the pectin and hemicellulose fractions as shown by loss of 14C during the 96-h chase than did 10- and 16-d cells. When the chase occurred in the dark, less 14C was incorporated into the cell wall because of the cessation of growth and higher respiratory loss. The dark effect was much less pronounced with the photomixotrophic cells. Even though the cell starch levels were much lower than in leaves, high 14C incorporation was found during the pulse, especially in older cells. The label was largely lost during the chase, indicating that starch is involved in the short-term storage of photosynthate. Thus, these easily labeled and manipulated photosynthetic cells demonstrated extensive turnover of the cell-wall pectin and hemicellulose fractions and starch during the normal growth process.

The rare occurrence of plant tissue culture contamination by Methylobacterium mesophilicum

A pink-pigmented, facultative methylotrophic (PPFM) bacterium, Methylobacterium mesophilicum, which is found on the leaf surface of most plants, has been reported to be a covert contaminant of tissue cultures initiated from Glycine max (soybean) leaves and seeds by Holland and Polacco (1992). The bacteria can be detected as pink colonies when leaves are pressed or tissue culture homogenates are plated on a medium with methanol as the sole carbon source. Since the presence of contaminating bacteria can confound any biochemical results obtained with such cultures (Holland and Polacco 1992), we wanted to determine the extent of the contamination of our tissue cultures of soybean and other species. No PPFMs were detected in any soybean culture we have, and previous results describing the biochemical characteristics of ureide utilization by one of our soybean suspension cultures (27C) also indicates that PPFM bacteria were not present. Analysis of about 200 other strains of 11 different species maintained in this lab showed that only three of about 160 callus cultures, recently initiated from Datura innoxia leaves, contained PPFMs. The D. innoxia leaves did have PPFMs on their surface but in most cases they did not survive the surface disinfestation and culture regimes. Thus PPFM bacterial contamination should not be a serious problem in most plant tissue cultures.

Immunocytochemical study of cell cycle control by cytokinin in cultured soybean cells

An immunocytochemical method was used to determine the proportion of cells in the DNA synthesis (S phase) of the mitotic cycle in suspension cultures of soybean (Glycine max (L.) Merr. cv. Acme) callus of cotyledonary origin, the stably cytokinin-dependent tissue used in the cytokinin bioassay devised by Carlos O. Miller. A standard cell synchronization protocol involving hydroxyurea was used to demonstrate the applicability of the immunocytochemical method to this cell culture. Cells were brought to mitotic arrest by cytokinin withdrawal, and the cell division cycle was restarted by the addition of cytokinin. We have followed the pattern of resumption of S phase after the readdition of cytokinin. This pattern reveals the existence of three subpopulations of cells in cytokinin-starved cultures, consistent with the occurrence of three cytokinin-requiring events in the cell cycle: one in mitosis, one in S phase, and one in the G1 phase.

Green fluorescent protein and its derivatives as versatile markers for gene expression in living Drosophila melanogaster, plant and mammalian cells

We have investigated the utility of the green fluorescent protein (GFP) as a marker for gene expression in living adult Drosophila melanogaster (Dm) and cultured plant and mammalian cells. Using Dm, we generated transgenic flies bearing a glass-responsive gfp fusion gene to test the utility of GFP as a spatial reporter. In the adult living fly, GFP is clearly visible in the ocelli and the eye. We have optimized the use of filters for distinguishing the GFP signal from abundant autofluorescence in living Dm. In addition, we have used GFP to identify photoreceptor cells in pupal eye cultures that have been fixed and stained according to standard histological procedures. GFP was also detected in individual living plant cells following transient transfection of soybean suspension cultures, demonstrating that GFP is an effective transformation marker in plant cells. Similarly, transient transfection of mammalian cells with a modified form of GFP, S65T, allowed detection of single living cells expressing the reporter. This modified form of GFP gave a robust signal that was resistant to photobleaching. We then used a CellScan system exhaustive photon reassignment (EPR) deconvolution algorithm to generate high-resolution three-dimensional images of GFP fluorescence in the living cell.

Phytochrome signal-transduction: characterization of pathways and isolation of mutants.

The study of phytochrome signalling has yielded a wealth of data describing both the perception of light by the receptor, and the terminal steps in phytochrome-regulated gene expression by a number of transcription factors. We are now focusing on establishing the intervening steps linking phytochrome photoactivation to gene expression, and the regulation and interactions of these signalling pathways. Recent work has utilized both a pharmacological approach in phototrophic soybean suspension cultures and microinjection techniques in tomato to establish three distinct phytochrome signal-transduction pathways: (i) a calcium-dependent pathway that regulates the expression of genes encoding the chlorophyll a/b binding protein (CAB) and other components of photosystem II; (ii) a cGMP-dependent pathway that regulates the expression of the gene encoding chalcone synthase (CHS) and the production of anthocyanin pigments; and (iii) a pathway dependent upon both calcium and cGMP that regulates the expression of genes encoding components of photosystem I and is necessary for the production of mature chloroplasts. To study the components and the regulation of phytochrome signal-transduction pathways, mutants with altered photomorphogenic responses have been isolated by a number of laboratories. However, with several possible exceptions, little real progress has been made towards the isolation of mutants in positive regulatory elements of the phytochrome signal-transduction pathway. We have characterized a novel phytochrome A (PhyA)-mediated far-red light (FR) response in Arabidopsis seedlings which we are currently using to screen for specific phyA signal-transduction mutants.

Plant metabolic studies of the growth regulator maleic hydrazide

The metabolism of maleic hydrazide has been studied in cell suspension cultures of soybean, wheat, and maize under standardized conditions (40 mL flasks, 1 ppm, 48 h). Maleic hydrazide was converted to its beta-D-glucoside as the predominant soluble metabolite in yields of between 2 and 15%. The latter was completely cleaved under simulated stomach conditions (pH 1, 37 degrees C, 24 h). In addition, up to 18% of the applied maleic hydrazide became associated with the nonextractable residue. The residue from soybean cells was solubilized only to a low degree (approximately 3%) under simulated stomach conditions. The lignin and hemicellulose components appeared to contain most of the radioactivity in the nonextractable residue from soybean cells. It is concluded that metabolism in cultured plant cells resembled that in whole plants and that the beta-D-glucoside of maleic hydrazide belongs to the small group of acid-labile pesticidal conjugates.

Mutagenesis and selection for oligomycin resistance in soybean (Glycine max L. Merr) suspension culture cells

Soybean suspension culture cells were subjected to mutagenesis with ethyl methane sulfonate and cells resistant to the ATP synthase inhibitor oligomycin were recovered. Suspension cultures showed high levels of resistance over a period of 17 months after mutagenesis. The level of resistance to oligomycin decreased during prolonged growth in the absence of the selection agent. Although mitochondrial inheritance of the resistance phenotype was not tested, this observation is consistent with segregation of resistant and sensitive mitochondria in progeny cells. Cross resistance to venturicidin was also observed which suggested the possible involvement of the mitochondrial ATP synthase subunit 9 gene, however, sequences of ATP synthase subunit 9 cDNA clones from resistant cells were identical to the wild type sequence. In addition, Southern blot analyses of DNA from wild type and resistant cultures did not identify rearrangements. These results indicated that resistance to oligomycin could not be directly attributed to a mutation in the primary gene sequence, an alteration in the RNA editing pattern of transcripts, or gross rearrangements in the region containing and directly adjacent to the ATP synthase subunit 9 gene in the mitochondrial DNA from resistant cultures.

Metabolism of the Insecticide Phoxim in Plants and Cell Suspension Cultures of Soybean

The metabolism of phoxim (Z-alpha-[[(diethuxyphosphinothioyl) oxy]imino]phenylacetonitrile), Volaton, was investigated using heterotrophic cell suspension cultures of soybean (Glycine max L.) and isolated organs (roots, stems, cotyledons, and leaves) of aseptically grown soybean plants. In both systems phoxim was first hydrolyzed to the corresponding oxime, which was then reduced to a primary nitrile amine. The primary amino group was N-malonylated as the terminal step of the catabolic sequence. The structure of this terminal metabolite was elucidated by spectroscopic (UV, IR, NMR, and MS) methods and chemical synthesis. In the plant no organ-specific differences in phoxim metabolism were observed. In the cell culture system phoxim was quantitatively converted to the N-malonate within 18-20 h, whereas in plant organs such extensive conversion could not be observed even within incubation times of about 5 days. The N-malonate was found to be excreted from the cultured cells into the medium; this effect could not be shown for the plant tissues.

Demonstration of a β-type adaptin at the plant plasma membrane

Highly purified plasma membrane was prepared from etiolated zucchini hypocotyls, developing pea cotyledons and suspension-cultured soybean cells by phase partitioning and sucrose density gradient centrifugation. The brain β-adaptin monoclonal antibody B1/M6, which recognizes a similar adaptin in zucchini clathrin coated vesicles, cross-reacted with an electrophoretically identical polypeptide in the plant plasma membrane fractions. Immunogold labelling of thin sections of soybean cells confirmed the presence of the β-adaptin at the plasma membrane. The presence of this adaptin at the Golgi apparatus could not be demonstrated unequivocally.

Xenobiotic glucosyltransferase activity from suspension-culturedGlycine maxcells

Proteins extracted from suspension-cultured soybean (Glycine max (L.) Merr. Corsoy 79) cells contained O- and N-glucosyltransferases (GTs; EC 2.4.1) that catalyzed glucosylation of several xenobiotic compounds including (a) the hydroxylated herbicide metabolites 6-hydroxybentazone (B-6-OH), 8-hydroxybentazone and 5-hydroxydiclofop (b) the herbicide chloramben and (c) the environmental contaminants 2,4-dichlorophenol and 3,4-dichloroaniline. The O-GT that catalyzes B-6-OH glucosylation, UDP-glucose: B-6-OH glucosyltransferase (B6GT), was chosen for further study. A rapid and sensitive B6GT assay was developed that uses ethyl acetate extraction to separate the product 6-O-[14C]glucosylbentazone from the glucose donor uridine[5′]diphospho-[1]-α-D-[U-14C]glucose. B6GT, recovered in the soluble protein fraction, was extracted in consistently high amounts from Corsoy 79 cells cultured for one to seven days. 2-Mercaptoethanol in the extraction buffer increased B6GT activity as did sodium, potassium, calcium, magnesium or manganese(II) chlorides in the assay buffer. B6GT activity in an ammonium sulfate fraction (30–50% saturation pellet) displayed two pH optima, one at pH 5.5 and another at pH 10 to 11. Apparent Km values for UDP-glucose and B-6-OH in the ammonium sulfate fraction were 90 and 4.5μM, respectively. Thus, we have partially characterized glucosylation of B-6-OH, one example of the several xenobiotic GT activities present in Corsoy 79 soybean.