Soybean Suspension Cultures Research Articles

Effect of biotic and abiotic elicitors on isoflavone biosynthesis during seed development and in suspension cultures of soybean (Glycine max L.).

The present investigation aimed to look at the effects of biotic and abiotic elicitors during Soybean seed development and cell suspension culture in isoflavones accumulation. The expression levels of four major genes viz., CHS7, CHS8, IFS2, and IFS1 involved on isoflavones biosynthesis during seed developmental stages from R5L-R7 was seen in both MAUS-2 and JS-335 Soybean varieties. The R7 stage showed 1.24-fold upregulation of IFS1transcript level and considered as the control for Soybean seed development. Both varieties during R6-R8 stages responded differently to the foliar application of 10µM SA, 10µMMJ and 0.1% Aspergillus niger. The IFS2 transcripts were upregulated by SA at the R7 stage with 5.21- and 4.68-fold in JS-335 and MAUS-2, respectively. IFS1 expression was significantly increased by A. niger treatment at R7 stage with 3.98- and 3.21-fold in MAUS-2 and JS-335, respectively. The expression of CHS7 and CHS8 by 10μM SA at R7 level revealed maximum up-regulation of 0.51- and 1.01-fold in MAUS-2; 0.37- and 0.82-fold in JS-335, respectively. In the soybean callus suspension culture, biosynthetic genes were used to validate the effects of elicitor on isoflavones. Both biotic and abiotic treatments contribute to the upregulation of IFS1 and IFS2 expression, that in turn, leads to the accumulation of isoflavone in seed development as well as in suspension cultures. These data further suggested that the IFS2 is the key gene responsible for the isoflavone accumulation during elicitor treatment.

Different glyceollin synthesis-related metabolic content and gene expressions in soybean callus suspension cultures and cotyledon tissues induced by alginate oligosaccharides

Abstract Glyceollins are a group of inducible soybean phytoalexins. The aim of the present study was to investigate the mechanism of glyceollin production in callus suspension cultures (CS) and cotyledon tissues elicited with alginate oligosaccharides (AOS) at 1%, 2%, and 3% concentrations that were applied for three days. HPLC analysis of the extracts from 2% AOS-elicited callus suspension cultures (ECS) revealed that glyceollins content was 85.3-fold and 8.2-fold higher than that in unelicited controls and elicited cotyledon tissues (ECT), respectively. UPLC-Q-Exactive Orbitrap MS analysis showed that the 2% AOS induction resulted in elevated levels of metabolites, the intermediary products of glyceollin biosynthesis, such as daidzein, glycitein, genistein, naringenin, glycinol, glyceollidin I/II, and glyceollin I, II, and III in ECS and ECT. Glyceollin I was prominent among the three glyceollin isomers in ECS, whereas isomer II and III were dominant in the synthetic route for glyceollins in ECT. qRT-PCR analysis of related genes showed that UGT, CYP93A1, G2DT, and G4DT displayed a direct effect on glyceollin synthesis. Our results suggested that 2% AOS-induced CS produced glyceollins in higher content compared to ECT by altering metabolite content and gene expression. Elicitation of CS is thus a promising technology for glyceollin synthesis extensively.

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.

Mutagenesis of Embryogenic Cultures of Soybean and Detecting Polymorphisms Using RAPD Markers

Embryogenic suspension cultures of soybean (Glycine max L. cv. Iroquois) were subjected to mutagenesis using varying concentrations (1, 3, 10, and 30 mM) of ethyl methanesulfonate (EMS). Depending on the concentration of EMS used, the mean survival rate of embryogenic cultures decreased from 74 % (1 mM EMS) to 43 % after 30 mM EMS treatment. Random amplified polymorphic DNA (RAPD) analysis was used to determine whether induction of genetic variability in embryogenic cultures in response to the different EMS treatments may result in identification of polymorphic markers. Two of 35 ‘core’ primers tested revealed polymorphisms. One of the primers, OPO-01/1150, revealed polymorphism in tissue treated with 10 mM EMS, while the other primer, OPO-05/1200, revealed polymorphism in tissue treated with either 1 or 30 mM EMS. These results suggest that RAPD markers are useful in detecting mutations in embryogenic cultures of 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

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 environmental estrogen bisphenol A by plant cell suspension cultures.

The metabolism of the environmental estrogen bisphenol A (BPA) was studied in heterotrophic plant cell suspension cultures of soybean (Glycine max), wheat (Triticum aestivum), foxglove (Digitalis purpurea), and thorn apple (Datura stramonium), which were regarded as metabolic model systems for intact plants. Three main metabolic routes of BPA were observed in the tissues. Most of the radioactivity found in the cell extracts consisted of carbohydrate conjugates of BPA amounting to about 85% (foxglove), 80% (wheat), 7% (soybean) and 15% (thorn apple) of applied 14C. The second main route was formation of non-extractable residues. Portions detected were low in foxglove (3.9% of applied 14C), moderate in wheat (13.5%), high in thorn apple (27.4%) and soybean (49.4%). With thorn apple, BPA derived bound residues were preponderantly resistant towards acid treatment; only traces of BPA were released. The third route was the formation of a highly polar, presumably polymeric material detected in media of soybean and thorn apple (29.3% and 36.0% of applied 14C, respectively). The mechanism of its formation remained unknown. In thorn apple, this highly polar material was formed extremely rapidly, and was considerably stable. Only traces of BPA were liberated by hydrolytic treatment with cellulase or acid. During hydrolysis experiments with glycoside fractions, non-extractable residues and highly polar materials, low amounts of presumably primary metabolites of BPA (up to 6% of applied 14C) were detected besides the parent compound; their chemical structures remained unclear.

Binding site for chitin oligosaccharides in the soybean plasma membrane.

Affinity cross-linking of the plasma membrane fraction to an (125)I-labeled chitin oligosaccharide led to the identification and characterization of an 85-kD, chitin binding protein in plasma membrane-enriched fractions from both suspension-cultured soybean cells and root tissue. Inhibition analysis indicated a binding preference for larger (i.e. degrees of polymerization = 8) N-acetylated chitin molecules with a 50% inhibition of initial activity value of approximately 50 nM. N-Acetyl-glucosamine and chitobiose showed no inhibitory effects at concentrations as high as 250 microM. It is noteworthy that the major lipo-chitin oligosaccharide Nod signal produced by Bradyrhizobium japonicum was also shown to be a competitive inhibitor of ligand binding. However, the binding site appeared to recognize the chitin portion of the Nod signal, and it is unlikely that this binding activity represents a specific Nod signal receptor. Chitooligosaccharide specificity for induction of medium alkalinization and the generation of reactive oxygen in suspension-cultured cells paralleled the binding activity. Taken together, the presence of the chitin binding protein in the plasma membrane fraction and the specificity and induction of a biological response upon ligand binding suggest a role for the protein as an initial response mechanism for chitin perception in soybean (Glycine max).

Phosphatidic acid activates a wound-activated MAPK in Glycine max.

Many plant species demonstrate a systemic increase in phosphatidic acid (PA) levels after being wounded (Lee et al., 1997). To understand the role of PA in wound signal transduction, we investigated if PA can activate protein kinases in soybean (Glycine max L.). We found that a MAPK is activated in soybean seedlings in both wounded and neighboring unwounded leaves. The wound-activated soybean kinase is specifically recognized by an antibody against the alfalfa MAPK, SIMK. When PA production is inhibited with n-butanol, an inhibitor of phospholipase D, the wound-induced activation of the MAPK is suppressed, suggesting that an elevation in PA levels is essential for its activation. Supporting this is the observation that exogenous PA activates the MAPK in suspension-cultured soybean cells. Activation of the 49 kDa MAPK occurs almost exclusively by PA, as other lipids are unable to or can only weakly activate the kinase. PA-induced activation of the MAPK is not a direct effect on the kinase but is mediated by upstream kinases. Our results suggest that PA acts as a second messenger in wound-induced MAPK signaling in plants.

Induction of lipid metabolic enzymes during the endoplasmic reticulum stress response in plants.

The endoplasmic reticulum (ER) stress response is a signal transduction pathway activated by the perturbation of normal ER metabolism. We used the maize (Zea mays) floury-2 (fl2) mutant and soybean (Glycine max) suspension cultures treated with tunicamycin (Tm) to investigate the ER stress response as it relates to phospholipid metabolism in plants. Four key phospholipid biosynthetic enzymes, including DG kinase and phosphatidylinositol (PI) 4-phosphate 5-kinase were up-regulated in the fl2 mutant, specifically in protein body fractions where the mutation has its greatest effect. The third up-regulated enzyme, choline-phosphate cytidylyltransferase, was regulated by fl2 gene dosage and developmental signals. Elevated accumulation of the fourth enzyme, PI 4-kinase, was observed in the fl2 endosperm and soybean cells treated with Tm. The activation of these phospholipid biosynthetic enzymes was accompanied by alterations in membrane lipid synthesis and accumulation. The fl2 mutant exhibited increased PI content in protein body membranes at 18 d after pollination and more than 3-fold higher triacylglycerol accumulation in the endosperm by 36 d after pollination. Incorporation of radiolabeled acetate into phospholipids in soybean culture cells increased by about 30% with Tm treatment. The coordinated regulation of ER stress related proteins and multiple components of phospholipid biosynthesis is consistent with signaling through a common pathway. We postulate that the plant ER stress response has an important role in general plant metabolism, and more specifically in integrating the synthesis of protein and lipid reserves to allow proper seed formation.

Photosynthetic properties of two different soybean suspension cultures

The photosynthetic properties of two commonly used suspension cultured lines, embryogenic and photoautotrophic (PA, SB-1 line) cells of soybean [ Glycine max (L.) Merr.] were characterized. We found that compared to the dark green PA cells, the light green embryogenic cells contained fewer and smaller plastids with less-developed thylakoid membranes. The embryogenic cells also contained much lower contents of both chlorophyll and the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) protein, an undetectable level of Rubisco small subunit protein, and a very low rate of photosynthesis. While the DNA contents of the nuclear genomes were similar in these two types of cultured cells, the embryogenic cells possessed a markedly lower content of plastid DNA. The 18-year-old PA suspension culture, SB-1, continues to evolve with higher Rubisco and plastid DNA contents than leaves, and with small decreases in nuclear DNA content that appears to mimic changes in chromosome numbers. These findings may prove useful in the application of plastid transformation, particularly when non-leaf or non-green tissues must be used as targets for transformation and plant regeneration.

Rac-related GTP-binding protein in elicitor-induced reactive oxygen generation by suspension-cultured soybean cells.

Plant cells produce reactive oxygen species (ROS) in response to many stimuli. However, the mechanism of ROS biosynthesis remains unclear. We have explored the hypothesis that the superoxide burst in plants mechanistically resembles the oxidative burst in neutrophils. First we have confirmed that ROS production, which occurs in suspension-cultured soybean (Glycine max) cells in response to hypo-osmotic shock, is inhibited by diphenylene iodonium, an inhibitor of the flavin-dependent oxidase of neutrophils. Because a Rac family G protein is an essential regulator of this NADPH oxidase, and because many plant homologs of Rac have been cloned, we next examined whether Rac-like proteins might be involved in the oxidative burst in the soybean cells. We identified a Rac-like 21-kD soybean protein that cross-reacts with antibodies to human Rac and garden pea Rop and also binds [gamma-(35)S] GTP, a diagnostic trait of small G proteins. This Rac-related protein translocated from the cytosol to microsomes during the oxidative burst. Moreover, soybean cells transiently transformed with either a dominant negative (RacN17) or a dominant positive (RacV12) form of Rac1 showed the anticipated altered responses to three different stimuli: hypo-osmotic shock, oligo-GalUA, and harpin. In response to these stimuli, cells transformed with RacN17 produced less ROS and cells transformed with RacV12 generated more ROS than control cells. These results strongly suggest that a Rac-related protein participates in the regulation of ROS production in soybean cells, possibly via activation of an enzyme complex similar to the NADPH oxidase of phagocytes in animal systems.

Nod factors and chitooligomers elicit an increase in cytosolic calcium in aequorin-expressing soybean cells.

Rhizobial Nod factors (NFs) function as nodulation signals that trigger symbiotic responses of leguminous host plants. NFs consist of a chitin oligomer backbone carrying a fatty acid at the non-reducing end. Depending on the rhizobial strain, NFs carry additional substituents, which may determine host specificity. Transgenic suspension-cultured soybean (Glycine max [L.] Merr.) cells expressing aequorin have been used to record cytosolic [Ca(2+)] changes upon treatment with purified NFs and chitin fragments. Both compounds elicited an increase of cytosolic [Ca(2+)] at nanomolar concentrations. The shape and amplitude of cytosolic [Ca(2+)] changes was similar to the response elicited by un-derivatized chitin oligomers. Cells challenged first with NFs did not respond to a subsequent treatment with chitin oligomers and vice versa. Dose-response experiments showed that un-derivatized chitin oligomers were more active compared with NFs. The capacity of NFs to elicit the calcium response depended on their structure. The presence of reducing end substituents in methylfucosylated NFs from Rhizobium sp. NGR234 and the O-acetyl group at the non-reducing end in NFs from Sinorhizobium meliloti attenuated the activity to cause the calcium changes. The sulfate group in NFs from Rhizobium tropici did not affect the elicitor activity. Pentameric S. meliloti NFs were more active than tetrameric molecules, whereas trimeric or dimeric degradation products were inactive. Substituents in NFs may have the function to avoid stimulation of defense reactions mediated by the perception system for chitin oligomers.

Cloning and Characterization of Glyoxalase I from Soybean

Glyoxalase I and glutathione transferase (GST) are two glutathione-dependent enzymes which are enhanced in plants during cell division and in response to diverse stress treatments. In soybean, a further connection between these two enzymes has been suggested by a clone (Accession No. X68819) resembling a GST being described as a glyoxalase I. To characterize glyoxalase I in soybean, GmGlyox I resembling the dimeric enzyme from animals has been cloned from a cDNA library prepared from soybean suspension cultures. When expressed in Escherichia coli, GmGlyox I was found to be a 38-kDa dimer composed of 21-kDa subunits and unlike the enzyme from mammals showed activity in the absence of metal ions. GmGlyox I was active toward the hemithioacetal adducts formed by reacting methylglyoxal, or phenylglyoxal, with glutathione, homoglutathione, or γ-glutamylcysteine, showing no preference for homoglutathione adducts over glutathione adducts, even though homoglutathione is the dominant thiol in soybean. When the clone X68819 was expressed in E. coli, the respective recombinant enzyme was active as a GST rather than a glyoxalase and was termed GmGST 3. GmGST 3 was active as a homodimer (45 kDa) composed of 26-kDa subunits and showed a preference for glutathione over homoglutathione when conjugating 1-chloro-2,4-dinitrobenzene. Both enzymes are associated with cell division in soybean cultures, but GmGST 3 (0.4% total protein) was 40 times more abundant than GmGlyox I (0.01%).

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.

パラコート耐性ダイズ懸濁培養細胞の選抜とその耐性機構

パラコート耐性ダイズ懸濁培養細胞の選抜とその耐性機構

Transformation of soybean [Glycine max (L.) Merrill] using proliferative embryogenic tissue maintained on semi-solid medium

Transgenic soybean can be efficiently produced by particle bombardment of embryogenic suspension culture material. Unfortunately, the time required to obtain a transformation-competent soybean suspension culture line is often lengthy and can result in reduced fertility of regenerated plants. In addition, establishment and maintenance of embryogenic suspension cultures can be very difficult. The objective of this work was to minimize the time required to obtain transformation-competent embryogenic tissue and optimize DNA delivery into that tissue. Somatic embryos were induced from immature cotyledons of soybean [Glycine max (L.) Merrill cv ‘Jack’] by placement of cotyledons, adaxial side up, on a MS-based induction medium containing 40 mg (181 µM) 2,4-dichlorophenoxyacetic acid (2,4-D) per 1 and 6% sucrose. Embryogenic tissues, which formed from the surface of the cotyledons within 2–4 wk, were transferred to an embryo proliferation medium containing 20 mg (90 µM) 2,4-D per 1 and 3% sucrose. After 4 wk, proliferative embryogenic tissue could be used for transformation via particle bombardment. Desiccation of target tissue, period of subculture prior to bombardment, and the number of bombardments per target tissue were evaluated for enhancement of transient β-glucuronidase (GUS) expression. The highest number of blue foci was observed when the target tissue was desiccated for 10 min in an uncovered Petri plate containing proliferation medium, subcultured on the same day of bombardment, and bombarded three times on a single day. For stable transformation, selection was started 20 d after bombardment using 9 mg hygromycin per 1 for 4 wk, and 18 mg per 1 thereafter. Stably transformed clones were obtained from tissue bombarded once and twice on a single day. GUS assays and Southern hybridization analysis of DNA from putative clones confirmed stable integration of the introduced genes. Fertile transgenic plants were obtained in 11–12 mo following culture initiation.

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.

Research Articles

The metabolism of phenanthrene was studied both in cell suspension cultures of wheat (Triticum aestivum) and soybean (Glycine max), and in intact plants of the water mossFontinalis antipyretica. Metabolism in cell suspension cultures strongly differed between the monocotyle and the dicotyle plant. Only small amounts oftrans-phenanthrene-9,10-dihydrodiole and phenanthrene-9,10-dione were detectable in the wheat culture. Soybean cultures, in contrast demonstrated a strong turnover resulting in a 75% reduction of the initial phenanthrene concentration. Metabolites were phenanthrene-9,10-dione, not further characterized polar metabolites and bound residues. Intact plants ofFontinalis antipyretica metabolized only small amounts of phenanthrene. Data obtained from cell cultures did not provide information for the metabolic potential in intact plants. Therefore standardized tests with model systems like suspension cultures lead to inadequate assessment of the ecological risk of certain xenobiotics.

Kinetic and calcium-binding properties of three calcium-dependent protein kinase isoenzymes from soybean.

Calmodulin-like domain protein kinases (CDPKs) are a family of calcium- but not calmodulin-dependent protein kinases found in a wide variety of plants and in protists. CDPKs are encoded by large multigene families, and to assess whether family members play distinct or redundant roles in vivo, we characterized soybean CDPK isoforms alpha, beta, and gamma, which share 60-80% identity in amino acid sequence. RNA blot analysis showed that the three CDPKs were expressed in most plant tissues examined and in suspension-cultured soybean cells. Recombinant CDPKalpha, -beta, and -gamma phosphorylated peptide substrates containing the four-residue motif R/K-X-X-S/T, but CDPKalpha was the most selective for residues outside of the motif. The CDPKs were inhibited by the general protein kinase inhibitors K252a and staurosporine and by calphostin C, which is an inhibitor of protein kinase C. The calcium-binding properties of each CDPK were distinct. The Kd's for Ca2+ determined by flow dialysis in the absence of substrates were 51, 1.4, and 1.6 micro M for CDPKalpha, -beta, and -gamma, respectively. In the presence of the peptide substrate syntide-2 the Kd of CDPKalpha decreased to 0.6 microM. Also, the sensitivity of this isoenzyme's activity to calcium varied with protein substrate. The concentrations of Ca2+ required for half-maximal activity (K0.5) for each CDPK with syntide-2 as substrate were 0.06, 0.4, and 1 micro M, respectively. These results show that members of the CDPK family differ in biochemical properties and support the hypothesis that each isoform may have a distinct role in calcium signal transduction.