Jürgen Ebel

Regulation of enzyme activities related to the biosynthesis of flavone glycosides in cell suspension culture of parsley (Petroselinum hortense)

Abstract 1. 1. In cell suspension cultures of parsley ( Petroselinum hortense ) the degree of increase of activity of phenylalanine ammonia-lyase caused by white light depends on the growth stage of the cultures. Maximum enzyme stimulation is observed at approximately the tenth day of growth. 2. 2. Changes in the activities of eight enzymes directly related to the biosynthesis of apiin ( 7-O-[β- D -apiofuranosyl -(1 → 2)-β- D -glucosyl ]-5,7,4′- trihydroxyflavone and graveobiosid B (3′-methoxyyapiin) were measured over a period of 24 h after illumination of cell suspension cultures of parsley. 3. 3. The enzymes investigated were phenylalanine ammonia-lyase, trans -cinnamic acid 4-hydroxylase, p -coumarete:CoA ligase, chalcone-flavanone isomerase, UDP-glucose:apigenin 7- O -glucosyltransferase, UDP-apiose:apigenin 7- O -glucoside-[1 → 2]-apiosyltransferase, UDP-apiose synthetasem and S -adenosylmethionine:luteolin3′- O -methyltransferase. 4. 4. Two groups of enzymes could be distinguished on the basis of their responses to light. The first group comprises the three enzymes acting on substrates of the phenylpropanoid type (phenylalanine, cinnamic acid and p -coumaric acid); the second group includes all those enzymes involved exclusively in the formation of flavonoid compounds and their glycosides. 5. 5. A hypothesis is discussed which would correlate three other enzymes assumed to take part in the same biosynthetic pathway with one of these groups of enzymes.

Transgenic aequorin monitors cytosolic calcium transients in soybean cells challenged with β-glucan or chitin elicitors

Abstract. Transgenic soybean (Glycine max L.) cells expressing aequorin were used to monitor changes in cytosolic Ca2+ concentrations in response to treatment with fungal elicitors. After an apparent lag phase of about 60 s, both chitin fragments and β-glucan elicitors caused a rapid increase in cytosolic Ca2+ concentration, which peaked within 2–2.5 min of treatment. The Ca2+ concentration then decreased and reached the basal level after about 5 min in the case of the treatment with chitin fragments, while a second rise in the Ca2+ concentration with a maximum occurring after about 7–8 min was observed in the case of β-glucan treatment. Calibration of the signals showed that the elicitors enhanced the cytosolic Ca2+ concentration from resting concentrations as low as 0.1 lM to highest levels of about 2 lM. Dose-response experiments showed that the concentration of elicitors giving a Ca2+ response at the 50% level was 0.4 nM for the chitin fragment and 28 lM and 72 lM, respectively, for a synthetic hepta-β-glucoside and a fungal β-glucan fraction. The β-glucan- or N,N′,N′′,N′′′-tetraacetyl chitotetratose (CH4)-induced Ca2+ signals were inhibited by both the Ca2+ chelator 1,2-bis-(2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid (BAPTA) and by the Ca2+-channel inhibitor La3+. Neomycin, whose target in plant cells has not yet been clearly identified, reduced predominantly the expression of the second peak of the biphasic Ca2+ curve following β-glucan treatment. Bacterial cyclic β-glucans known to suppress β-glucan-induced phytoalexin production were also found to function as a suppressor for the Ca2+ response that was elicited by the fungal β-glucans. The results clearly show that the increase in the cytosolic Ca2+ concentration is an early and rapid event in the elicitor-sensing mechanism of soybean cells, and is probably connected with the subsequent activation of defence responses.

High-affinity binding of a synthetic heptaglucoside and fungal glucan phytoalexin elicitors to soybean membranes

Soybean membranes possess high‐affinity binding sites for fungal β‐glucans that elicit phytoalexin synthesis. The ability of 1,3‐1,6‐β‐glucans, released by acid hydrolysis from mycelial walls of Phytophthora megasperma f.sp. glycinea, to compete for the putative phytoalexin elicitor receptors increases with their average degree of polymerization (DP). The results suggest a function where the probability for glucan fragments of containing a structural determinant that is optimal for binding approaches 1 as the DP tends to infinity. Ligand displacement data obtained against a 125I‐labeled glucan elicitor (average DP= 18) provided a theoretical minimum IC50 (50% inhibitory concentration) for 1,3‐1,6‐β‐glucans of 3 nM. The IC50 value obtained for a synthetic hepta‐β‐glucoside having a known elicitor‐active structure was 8 nM, remarkably close to the predicted value. Displacement of the 125I‐glucan of large DP was uniform and complete showing that the heptaglucoside had access, with similar affinity, to all sites available to the radioligand. Further analysis using a 125I‐labeled aminophenethylamine derivative of the heptaglucoside suggested that the putative glucan‐elicitor receptors bind a basic structural determinant present in all elicitor‐active glucans from the soybean pathogen P. megasperma.

Phytoalexin synthesis in soybean cells: Elicitor induction of phenylalanine ammonia-lyase and chalcone synthase mRNAs and correlation with phytoalexin accumulation

A glucan elicitor from cell walls of the fungus Phytophthora megasperma f. sp. glycinea, a pathogen of soybean (Glycine max), induced large and rapid increases in the activities of enzymes of general phenylpropanoid metabolism, phenylalanine ammonia-lyase, and of the flavonoid pathway, acetyl-CoA carboxylase and chalcone synthase, in suspension-cultured soybean cells. The changes in phenylalanine ammonia-lyase and chalcone synthase activities were correlated with corresponding changes in the mRNA activities encoding these enzymes, as determined by enzyme synthesis in vitro in a mRNA-dependent reticulocyte lysate. The time courses of the elicitor-induced changes in mRNA activities for both enzymes were very similar with respect to each other. Following the onset of induction, the two mRNA activities increased significantly at 3 h, reached highest levels at 5 to 7 h, and subsequently returned to low values at 10 h. Similar degrees of induction of mRNA activities and of the catalytic activities of phenylalanine ammonia-lyase and chalcone synthase were observed in response to three diverse microbial compounds, the glucan elicitor from P. megasperma, xanthan, an extracellular polysaccharide from Xanthomonas campestris, and endopolygalacturonase from Aspergillus niger. However, whereas the glucan elicitor induced the accumulation of large amounts of the phytoalexin, glyceollin, in soybean cells, endopolygalacturonase induced only low, albeit significant, amounts; xanthan did not enhance glyceollin accumulation under the conditions of this study. This result might imply that enzymes other than phenylalanine ammonia-lyase or chalcone synthase exert an important regulatory function in phytoalexin synthesis in soybean cells.

Molecular cloning and expression of 4-coumarate:coenzyme A ligase, an enzyme involved in the resistance response of soybean (Glycine max L.) against pathogen attack.

We have isolated three classes of cDNAs that probably encode three 4-coumarate:coenzyme A ligase (4CL) isoenzymes in soybean (Glycine max L.). The deduced amino acid sequences reveal several regions of extended sequence identity among 4CLs of all plants analyzed to date. The sequences of two of these regions are consistent with a domain structure proposed for a group of enzymes catalyzing the ATP-dependent covalent binding of AMP to their substrates during the reaction sequence. By using two cDNA fragments that do not cross-hybridize under the conditions used, we demonstrate that 4CL in soybean is very likely encoded by a small gene family. Members of this family are differentially expressed in soybean cell cultures treated with [beta]-glucan elicitors of Phytophthora megasperma f. sp. glycinea or in soybean roots infected with either an incompatible or compatible race of the fungus. These results are in agreement with our previous observation that elicitor treatment of soybean cells caused a preferential enhancement in the activity level of one of the 4CL isoenzymes. In soybean, 4CL isoenzymes possessing different substrate affinities for substituted cinnamic acids, and showing differential regulation to environmental stress, may play a pivotal role in distributing substituted cinnamate intermediates at a branch point of general phenylpropanoid metabolism into subsequent specific pathways.

Coordinated changes in enzyme activities of phenylpropanoid metabolism during the growth of soybean cell suspension cultures

Abstract Variations in teh activities of several enzymes of phenylpropanoid metabolism were studied in fermenter-grown cell suspension cultures of soyben ( Glycine max ). Concomitant large increases and subsequent decreases in the activities of phenylalanine ammonina-lyase (EC 4.3.1.5), cinnamic acid 4-hydroxylase, and two isoenzymes of p -coumarate:CoA ligase occurred prior to the stationary phase of the cell cultures. These findings represent a further example of an interdependent regulation of these enzymes of the general phenylpropanoid metabolism. The increases in all of these enzyme activities could be further enhanced by illunination of the cells. No comparable light effects and no significant changes were observed for the specific activity of an S -adenosylmethionine: o -dihydric phenol m - O -mehyltransferase and for the overall rate of the two-step reduction of feruloyl-CoA to coniferyl alcohol. These enzymatic reactions therefore appear to be regulated independently of the enzymes of the general phenylpropanoid metabolism.

Induction of phytoalexin synthesis in soybean. Elicitor-induced increase in enzyme activities of flavonoid biosynthesis and incorporation of mevalonate into glyceollin.

Soybean (Glycine max) cotyledons removed from 6-to 8-day-old seedlings were treated with mycelial walls of Phytophthora megasperma var. sojae (elicitor) and the activity changes of the enzymes phenylalanine ammonia-lyase and flavanone synthase were determined. Large increases and subsequent decreases in the activities of both enzymes were found in the elicitor-induced cotyledons. No changes in enzyme activities, occurred in control cut cotyledons with elicitor omitted. Concomitant with the elicitor-stimulated changes in enzyme activities there was accumulation of the soybean phytoalexin, glyceollin. [2-14C] Mevalonic acid was incorporated into glyceollin in elicitor-treated but not in untreated cotyledons. The results prove that stimulation of the enzymes of flavonoid biosynthesis and probably glyceollin biosynthesis are caused by the effect of the P. megasperma elicitor and not merely by wounding the plant material. The induced enzyme levels could control glyceollin biosynthesis.

Purification and properties of an o-dihydricphenol meta-O-methyltransferase from cell suspension cultures of parsley and its relation to flavonoid biosynthesis

Abstract 1. 1. An O -methyltransferase directly related to flavone glycoside biosynthesis has been isolated from cell suspension cultures of parsley. The enzyme has been purified 82-fold by MnCl 2 and (NH 4 ) 2 SO 4 precipitation and chromatography on DEAE-cellulose, Sephadex G-100 and hydroxylapatite. The enzyme catalyses the transfer of the methyl group of S -adenosyl- l -methionine to the meta position of o -dihydric phenols. 2. 2. Highest enzyme activities were obtained after illumination of the cells for 24 h with high intensities of white light from fluorescent lamps. 3. 3. The enzyme has a pH optimum around pH 9.7. It requires Mg 2+ and is not inhibited by p -chloromercuribenzoate or iodoacetamide. A molecular weight of about 48 000 was estimated from the elution volume after chromatography on a Sephadex G-100 column. 4. 4. Only o -dihydric phenols can function as substrates for the enzyme and only the meta -hydroxyl group is methylated. Luteolin (5,7,3′,4′-tetrahydroxyflavone) and its 7- O -glucoside with apparent K m values of 4.6·10 −5 and 3.1·10 −5 M, respectively, are the best substrates tested, whereas eriodictyol (5,7,3′,4′-tetrahydroxyflavanone) ( K m = 1.2·10 −3 M) and caffeic acid ( K m = 1.6·10 −3 M) had much lower affinity for the enzyme. With luteolin as substrate the apparent K m value for S -adenosyl- l -methionine is 1.5·10 −4 M. 5. 5. Cell cultures also contain enzymatic activity for the synthesis of S -adenosyl- l -methionine from l -methionine and ATP. In contrast to the O -methyltransferase the activity of this enzyme is not influenced by illumination of the cultures.

Determination of Specific Growth Stages of Plant Cell Suspension Cultures by Monitoring Conductivity Changes in the Medium

SummaryConductivity changes in the medium of cultured soybean (Glycine max L.) cells were shown to be strictly correlated with nitrate uptake and growth of the cultures. A continuous record of the conductivity was used as a simple and reliable method of determining specific growth stages and concomitant peaks in the activities of nitrate reductase and phenylalanine ammonia-lyase.

Phytoalexin synthesis in soybean (Glycine max): Similar time courses of mRNA induction in hypocotyls infected with a fungal pathogen and in cell cultures treated with fungal elicitor

Rapid, transient increases in mRNA activities related to phytoalexin synthesis were observed after inoculation of soybean seedlings (Glycine max) with the fungus, Phytophthora megasperma f. sp. glycinea, or treatment of cultured soybean cells with an elicitor preparation from the same fungus. The time courses of changes in activity were similar for mRNAs coding for enzymes of general phenylpropanoid metabolism and for chalcone synthase mRNA in both systems. Cloned cDNA was used to demonstrate that the induced changes in chalcone synthase mRNA activity coincided with changes in amount of mRNA. The results suggest that phytoalexin synthesis, one mechanism of induced resistance of plants to pathogens, is regulated by temporary gene activation.