A novel protein elicitor (PaNie) from Pythium aphanidermatum induces multiple defense responses in carrot, Arabidopsis, and tobacco.

A novel protein elicitor (PaNie(234)) from Pythium aphanidermatum (Edson) Fitzp. was purified, microsequenced, and the corresponding cDNA was cloned. The deduced amino acid sequence contains a putative eukaryotic secretion signal with a proteinase cleavage site. The heterologously expressed elicitor protein without the secretion signal of 21 amino acids (PaNie(213)) triggered programmed cell death and de novo formation of 4-hydroxybenzoic acid in cultured cells of carrot (Daucus carota). Programmed cell death was determined using the tetrazolium assay and DNA laddering. Infiltration of PaNie(213) into the intercellular space of leaves of Arabidopsis (Columbia-0, wild type) resulted in necroses and deposition of callose on the cell walls of spongy parenchyma cells surrounding the necrotic mesophyll cells. Necroses were also formed in tobacco (Nicotiana tabacum cv Wisconsin W38, wild type) and tomato (Lycopersicon esculentum Mill.) but not in maize (Zea mays), oat (Avena sativa), and Tradescantia zebrina (Bosse), indicating that monocotyledonous plants are unable to perceive the signal. The reactions observed after treatment with the purified PaNie(213) were identical to responses measured after treatment with a crude elicitor preparation from the culture medium of P. aphanidermatum, described previously. The availability of the pure protein offers the possibility to isolate the corresponding receptor and its connection to downstream signaling-inducing defense reactions.

Regulation of anthocyanin biosynthesis in UV-A-irradiated cell cultures of carrot and in organs of intact carrot plants.

Two cell lines of Daucus carota are known to differ with respect to anthocyanin accumulation. cDNA clones encoding enzymes involved in anthocyanidin biosynthesis, namely phenylalanine ammonia-lyase (PAL; EC 4.3.1.5), chalcone synthase (CHS; EC 2.3.1.74), flavanone 3-hydroxylase (F3H; EC 1.14.11.9), dihydroflavonol 4-reductase (DFR; EC 1.1.1.219) and leucoanthocyanidin dioxygenase (LDOX; EC 1.14.11.-), were isolated from libraries derived from cell cultures. Northern blot analysis of anthocyanin-accumulating (DCb) and non-accumulating (DCs) cell cultures of carrot showed that the anthocyanin pathway in these anthocyanin-free DCs cells is blocked. The expression of CHS1, DFR1 and LDOX is not detectable. However, F3H and DFR2 behave differently. In the European wild carrot (Daucus carota ssp. carota) the structural genes coding for the enzymes responsible for anthocyanin biosynthesis are strongly expressed in organs which accumulate anthocyanins. Only the dark-purple coloured petals of the central flowers of the inflorescence and to a certain extent the white flowers and the leaves but not the stems and the roots transcribe these genes. To study the effect of anthocyanins as UV-screens the expression of a protein indispensable for cell proliferation like alpha-tubulin (TUB) was monitored.

Isoforms of chalcone synthase in Daucus carota L. and their differential expression in organs from the European wild carrot and in ultraviolet-A-irradiated cell cultures.

Two isoforms of chalcone synthase (CHS) were isolated from cDNA libraries derived from UV-A-irradiated anthocyanin-accumulating (DCb) and non-accumulating (DCs) cell cultures of carrot (Daucus carota L.). The clones designated as DcCHS1, which were present only in the DCb library, had a deduced primary sequence of 389 amino acids and an expected molecular mass of 42.7 kDa, and seem to be alleles of those cloned by Ozeki et al. (1993). The second isoform (DcCHS2) was present in both libraries. It had the highest degree of similarity (97.7%) to parsley CHS over all 397 amino acids. The expected molecular mass of the corresponding protein was 43.6 kDa. Results obtained from Southern blot analysis indicated the existence of at least two CHS genes in carrot. A transient enhancement of the DcCHS1 mRNA level after continuous irradiation with UV-A light could only be observed in anthocyanin-accumulating cultures, whereas an increase in DcCHS2 mRNA was seen in both cell lines. The maximum accumulation of CHS mRNA occurred 48 h after the onset of UV-A irradiation. In the European wild carrot the accumulation of DcCHS1 mRNA was restricted to the red central flowers, whereas the DcCHS2 mRNA was detectable in all red and white petals, as well as leaves, but was absent in stems and roots. The expression of DcCHS1 was restricted to anthocyanin-accumulating cells or organs. The heterologous expression of both cDNAs in Escherichia coli resulted in immunostainable bands of different sizes on the Western blot and high levels of catalytic CHS activity.

Regulation of enzymes involved in anthocyanin biosynthesis in carrot cell cultures in response to treatment with ultraviolet light and fungal elicitors.

The accumulation of anthocyanins in cell cultures of Daucus carota L. and the enzymes involved in their biosynthesis were investigated under growth in the dark, continuous irradiation with UV light, incubation with elicitors from Pythium aphanidermatum, and elicitor treatment of UV-irradiated cells. Upon UV irradiation, anthocyanin accumulation was strongly enhanced, and the enzymes of the phenylpropanoid and flavonoid pathways, including the "late" enzymes cyanidin galactosyltransferase, cyanidin galactoside xylosyltransferase, cyanidin triglycoside sinapoyltransferase and sinapic acid glucosyltransferase, all showed transient increases in their activities. The time courses of the enzyme activities exhibited successive maxima with an ordered sequence corresponding to their position in the biosynthetic pathway, suggesting a coordinated induction of the entire set of enzymes. The key enzymes phenylalanine ammonia-lyase and chalcone synthase are regulated on a transcriptional level. Incubation of dark-grown carrot cells with fungal elicitors led to a rapid and transient induction of phenylalanine ammonia-lyase corresponding to the formation of 4-hydroxybenzoic acid, but the amount of anthocyanin did not increase and there was no enhancement of any of the enzyme activities which are part of the anthocyanin pathway, including the enzymes catalyzing glycosylation and acylation reactions. Treatment with UV light and elicitors resulted in a rapid induction of the phenylpropanoid pathway, whereas the inducing effect of UV light on the anthocyanin content, on chalcone synthase and on the enzymes catalyzing the final steps of anthocyanin biosynthesis was suppressed. These results indicate a coordinated regulation of the enzymes involved in anthocyanin biosynthesis, an independent inducibility of the phenylpropanoid pathway, and a hierarchy of the different effectors, as shown by the dominating role of the elicitor-signal over the UV stimulus.

Purification and characterization of glycosyltransferases involved in anthocyanin biosynthesis in cell-suspension cultures of Daucus carota L.

The major anthocyanins accumulated by an Afghan cultivar of Daucus carota L. are cyanidin 3-(xylosylglucosylgalactosides) acylated with sinapic or ferulic acid. The formation of the branched triglycoside present as a common structural element requires an ordered sequence of glycosylation events. Two of these enzymic glycosylation reactions have been detected in protein preparations from carrot cell-suspension cultures. The first step is a galactosyl transfer catalyzed by UDP-galactose: cyanidin galactosyltransferase (CGT) resulting in cyanidin 3-galactoside. The putative second step is the formation of cyanidin 3-(xylosylgalactoside) catalyzed by UDP-xylose: cyanidin 3-galactoside xylosyltransferase (CGXT). Both enzyme activities were characterized from crude protein preparations. The CGT was purified 526-fold from the cytosolic fraction of UV-irradiated cell cultures by ion-exchange chromatography on diethylaminoethyl (DEAE)-Sephacel, affinity chromatography on Blue Sepharose CL-6B, gel permeation chromatography on Sephadex G-75 and elution from the gel matrix after non-dissociating PAGE. Its molecular mass was estimated by SDS-PAGE and by calibrated gel permeation chromatography on Sephadex G-75. In both cases a molecular mass of 52 kDa was determined, indicating that the native protein is a monomer of 52 kDa. The galactosyl transfer and the xylosyl transfer are presumed to be catalyzed by separate enzymes.

Purification, characterization and partial peptide microsequencing of progesterone 5 beta-reductase from shoot cultures of Digitalis purpurea.

Progesterone 5 beta-reductase, which catalyzes the reduction of progesterone to 5 beta-pregnane-3,20-dione, was purified 770-fold to homogeneity from the cytosolic fraction of shoot cultures of Digitalis purpurea. This purification involved DEAE-Sephacel, affinity chromatography (Blue-Sepharose CL-6B and adenosine 2',5'-bisphosphate-Sepharose 4B) and elution from a gel matrix after non-dissociating PAGE. The molecular mass determined by SDS/PAGE was 43 kDa and the molecular mass determined by gel-filtration chromatography on calibrated Sephadex G-200 was 280 kDa, thus indicating that the native protein is a polymer consisting of several subunits. The purified enzyme had a Km value of 6 microM for NADPH and 34 microM for progesterone. The enzyme had a strong substrate specificity for progesterone. The relative rates for other steroids such as testosterone, cortisone and cortisol were much lower. The trypsin digestion of the purified progesterone 5 beta-reductase resulted in 100 peptide fragments. The largest fragment after trypsin digestion and sequence analysis consisted of 13 amino acids.

Elicitor-Induced Changes in Ca2+ Influx, K+ Efflux, and 4-Hydroxybenzoic Acid Synthesis in Protoplasts of Daucus carota L.

Suspension-cultured carrot cells (Daucus carota) and their protoplasts respond to a fungal elicitor prepared from the culture medium of Pythium aphanidermatum by accumulating 4-hydroxybenzoic acid (4-HBA). Protoplasts release the compound into the culture medium. Using 45CaCl2 as a tracer, we were able to demonstrate that the secretion of 4-HBA is preceded by a rapid increase in the Ca2+ influx and a concomitant K+ efflux. If the increased Ca2+ influx was prevented by ethyleneglycol-bis([beta]-aminoethylether)-N,N[prime]-tetraacetic acid, 4-HBA synthesis was inhibited by 70%. These results are discussed with regard to signal transduction from the plasma membrane to the nucleus of carrot protoplasts.

Selection of anthocyanin-accumulating potato (Solanum tuberosum L.) cell lines from calli derived from seedlings produced by gamma-irradiated seeds.

Callus cell lines of potato (Solanum tuberosum L. cv. Zarevo) were obtained from seedlings germinated from gamma-irradiated seeds (200 Gy). Some of these cell lines produce red-violet pigments which were identified as acylated anthocyanins. The major anthocyanin was determined to be peonidin 3-O-[6-O-(4-O-E-p-coumaroyl-rhamnosyl)-glucoside]-5-O-glucoside ("peonanin"). Single cell-derived protoclones from non-pigmented protoplasts sometimes also gave rise to pigmented cell clusters thus indicating that the changes in the expression of the anthocyanin pathway can also occur after the stage of initial callus induction.

Anthocyanins from cell suspension cultures of Daucus carota.

Six anthocyanins were isolated from cell suspension cultures of an Afghan cultivar of Daucus carota by PC or HPLC. The structures of these compounds were elucidated by spectroscopic methods as cyanidin 3-O-lathyroside, cyanidin 3-O-(2''-O-beta-D-xylopyranosyl-6''-O-beta-D-glucopyranosyl-beta-D- galactopyranoside), and the latter acylated with 4-coumaric, ferulic, 4-hydroxybenzoic or sinapic acid. Unusual 1H NMR chemical shifts and 1H NOE data indicate an intramolecular copigmentation of the aglycone with these aromatic residues.

Metabolic changes in carrot cells in response to simultaneous treatment with ultraviolet light and a fungal elicitor.

Ultraviolet light induces anthocyanin biosynthesis in cell cultures of an Afghan cultivar of Daucus carota (Daucus carota L. ssp. sativus). Simultaneous treatment with a fungal elicitor from Pythium aphanidermatum results in an inhibition of the catalytic activity of chalcone synthase (CHS), which in turn correlates with an inhibition of anthocyanin biosynthesis. On immunoblots, one isoenzyme (40 kDa) of CHS disappears upon elicitor treatment. On an mRNA level, only the mRNA for the 40-kDa-CHS is active after treatment with ultraviolet light. After inhibition of anthocyanin biosynthesis by the elicitor the enzyme protein disappears and the CHS mRNA is strongly diminished. This inhibition depends on the concentration of the elicitor. In addition, elicitor treatment leads to an induction of the general phenylpropanoid pathway as well as to the accumulation of 4-hydroxybenzoic acid which is covalently bound to wall polysaccharides of the carrot cells. The possible function of phenylalanine ammonia-lyase in providing precursors for 4-hydroxybenzoic acid is discussed.

A stereospecific enzyme of the putative biosynthetic pathway of cardenolides. Characterization of a progesterone 5 beta-reductase from leaves of Digitalis purpurea L.

Leaves of Digitalis purpurea contain an enzyme activity which catalyzes the conversion of progesterone to 5 beta-pregnane-3,20-dione. Since cardenolides without exception possess a 5 beta-configuration, 5 beta-pregnane-3,20-dione can serve as a precursor for this class of secondary metabolites. It is assumed that the enzyme is part of the putative biosynthetic pathway of cardenolides. This enzyme activity was spotted in the soluble fraction of a crude homogenate. Product formation was detected by gas chromatography and by gas chromatography/mass spectroscopy (g.c./m.s.). The enzyme had a pH optimum at 8.0 and an apparent Km value of 6 microM for progesterone. It required NADPH as a co-substrate with an apparent Km value of 22 microM. The optimum temperature in vitro was 30 degrees C. The activity was not dependent on monovalent and bivalent cations.

3 beta-hydroxysteroid oxidoreductase in suspension cultures of Digitalis lanata EHRH.

A 3 beta-hydroxysteroid oxidoreductase was isolated and characterized in the microsomes of Digitalis lanata cell cultures. The enzyme catalyzes the conversion of 5 alpha-pregnane-3,20-dione to 5 alpha-pregnan-3 beta-ol-20-one and requires NAD(P)H2. The enzyme was found to have a pH optimum of 8.0. The reaction had an optimum incubation temperature of 25 degrees C with linear reduction for the first 4 h, reaching maximum enzyme activity after 7 h. Substrate kinetics for 5 alpha-pregnane-3,20-dione and NADPH2 resulted in apparent Km-values of 18.5-20 microM for 5 alpha-pregnane-3,20-dione and 50-120 microM for the co-substrate NADPH2. In order to localize 3 beta-hydroxysteroid oxidoreductase differential centrifugation as well as linear sucrose density gradient centrifugation were performed. The results obtained lead to the conclusion that 3 beta-hydroxysteroid oxidoreductase is not associated with a single cell compartment, but consists of a major soluble part and a markedly smaller part of endoplasmic reticulum-associated activity.

Characterization and localization of progesterone 5 alpha-reductase from cell cultures of foxglove (Digitalis lanata EHRH).

Progesterone 5 alpha-reductase, which catalyses the reduction of progesterone to 5 alpha-pregnane-3,20-dione, was isolated and characterized from cell cultures of Digitalis lanata (foxglove). Optimum enzyme activity was observed at pH 7.0, and the enzyme had an apparent Km value of 30 microM for its substrate progesterone. The enzyme needs NADPH as reductant, which could not be replaced by NADH. For NADPH, the apparent Km value is 130 microM. The optimum temperature was 40 degrees C; at temperatures below 45 degrees C, the product 5 alpha-pregnane-3,20-dione was reduced by a second reaction to 5 alpha-pregnan-3 beta-ol-20-one. Progesterone 5 alpha-reductase activity was not dependent on bivalent cations. In the presence of EDTA, 0.1 mM-Mn2+ had no influence on enzyme activity, whereas 0.1 mM-Ca2+, -Co2+ and -Zn2+ decreased progesterone 5 alpha-reductase activity. Only 0.1 mM-Mg2+ was slightly stimulatory. EDTA and thiol reagents such as dithiothreitol stimulate progesterone 5 alpha-reductase activity. By means of linear sucrose gradient fractionation of the cellular membranes, progesterone 5 alpha-reductase was found to be located in the endoplasmic reticulum.

Influence of a specific xyloglucan-nonasaccharide derived from cell walls of suspension-cultured cells of Daucus carota L. on regenerating carrot protoplasts.

A xyloglucan oligosaccharide was isolated from cell walls of Daucus carota L. suspension-cultured cells. From analytical data (gel-permeation chromatography, thin-layer chromatography, monosaccharide analysis, methylation analysis) it can be concluded that this oligosaccharide preparation consists mainly of a nonasaccharide known as XG9 (Glc4Xyl3GalFuc). This nonasaccharide showed excellent "anti-auxin" properties in the pea-stem bioassay, with 80% inhibition of 2,4-dichlorophenoxyacetic acid (2,4-D)-induced longitudinal growth of etiolated pea stem segments at concentrations of 1-0.1 nM. Applied in nanomolar concentrations to protoplasts regenerating in a medium containing 4.52 µM 2,4-D, the nonasaccharide influenced the viability of the protoplasts and the activities of glycan synthases in vitro. The effects were similar to those achieved by the omission of 2,4-D from the regeneration medium. The composition of the regenerated cell wall was not changed significantly by the use of 2,4-D-depleted medium or the addition of XG9 to 2,4-D-containing medium.

Induction of chalcone synthase in cell suspension cultures of carrot (Daucus carota L. spp. sativus) by ultraviolet light: evidence for two different forms of chalcone synthase.

Two cell lines of carrot (Daucus carota L. spp. sativus), grown as cell-suspension cultures in the dark, were irradiated with ultraviolet light (315-420 nm) 10 d after the onset of cultivation. Chalcone synthase (CHS) enzyme activity was induced in both cell lines. Anthocyanin synthesis was only stimulated in the anthocyanin-containing cell line DCb. Parallel to the increase in CHS activity there was an increase with time in the amount of one CHS form with an isoelectric point of 6.5 and a molecular weight of 40 kilodaltons (kDa) per subunit. Whereas the anthocyanin-free cell line DCs failed to accumulate anthocyanin, it did stimulate another CHS form with an isoelectric point at pH 5.5 and a molecular weight of 43 kDa per subunit. Both enzyme activities could be separated by isoelectric focusing and stabilized using sodium hydrosulfite as an oxidation protectant. In carrot plants, CHS was restricted to the dark purple petals of the inflorescence (40 kDa) and to the leaves (43 kDa).

Reconstitution of cytochrome P-450-dependent digitoxin 12 beta-hydroxylase from cell cultures of foxglove (Digitalis lanata EHRH.).

Cytochrome P-450-dependent digitoxin 12 beta-hydroxylase from cell cultures of foxglove (Digitalis lanata) was solubilized from microsomal membranes with CHAPS (3-[(3-cholamidopropyl)dimethylammonio]propane-1-sulphonic acid). Cytochrome P-450 was separated from NADPH: cytochrome c (P-450) reductase by ion-exchange chromatography on DEAE-Sephacel. NADPH:cytochrome c (P-450) reductase was further purified by affinity chromatography on 2',5'-ADP-Sepharose 4B. This procedure resulted in a 248-fold purification of the enzyme; on SDS/polyacrylamide-gel electrophoresis after silver staining, only one band, corresponding to a molecular mass of 80 kDa, was present. The digitoxin 12 beta-hydroxylase activity could be reconstituted by incubating partially purified cytochrome P-450 and NADPH:cytochrome c (P-450) reductase together with naturally occurring microsomal lipids and flavin nucleotides. This procedure yielded about 10% of the original amount of digitoxin 12 beta-hydroxylase.

Purification of an autocatalytic protein-glycosylating enzyme from cell suspensions of Daucus carota L.

A glycosyltransferase was identified in the 174 000 · g membrane pellet and the supernatant from extracts of cell suspensions of Daucus carota L. The enzyme from the supernatant was enriched 475-fold, and sodium dodecyl sulfate-gel electrophoresis and fluorography of this purified sample showed that the only enriched protein band (40 000 Da) was simultaneously an enzyme and a glucose-acceptor. Gel filtration and electrophoresis under non-denaturing conditions proved that in vivo this protein provides the subunits for a very large molecule. Radio-gas-liquid chromatography demonstrated that only one glucosyl moiety was transferred from UDP-glucose to the protein.

The uptake of acylated anthocyanin into isolated vacuoles from a cell suspension culture of Daucus carota.

Anthocyanin-containing vacuoles were isolated from protoplasts of a cell suspension culture of Daucus carota. The vacuoles were stable for at least 2 h as demonstrated by the fact that they showed no efflux of anthocyanin. The uptake of radioactively labelled anthocyanin was time-dependent with a pH optimum at 7.5, and could be inhibited by the protonophore carbonylcyanide m-chlorophenylhydrazone. Furthermore, the transport was specific, since vacuoles from other plant species showed no uptake of labelled anthocyanin, and strongly depended on acylation with sinapic acid, as deacylated glycosides were not taken up by isolated vacuoles. Hence, it is suggested that the acylation of anthocyanin, which is also required for the stabilization of colour in vacuoles, is important for transport, and that acylated anthocyanin is transported by a selective carrier and might be trapped by a pH-dependent conformational change of the molecule inside the acid vacuolar sap.

Immobilization of digitoxin 12ß-hydroxylase, a cytochrome P-450-dependent enzyme from cell cultures of Digitalis lanata EHRH.

Attempts were made to immobilize digitoxin 12ß-hydroxylase, a membrane-bound, cytochrome P-450-dependent monooxygenase from cell cultures of Digitalis lanata. The optimum procedure was the entrapment of microsomes in 2% alginate by crosslinking the polysaccharide chains with CaCl2. After the immobilization of the enzyme about 70% of its activity was retained. The kinetic data such as the pH optimum and the optimum substrate concentrations were identical for the immobilized enzyme and freely suspended microsomes. Using ß-methyldigitoxin as a substrate enzyme activity could be observed for more than 20 h. A continuous flow system for immobilized digitoxin 12ß-hydroxylase is described.

In vitro inhibition of carrot chalcone synthase by 3'-nucleotidase: the role of the 3'-phosphate group of malonyl-coenzyme A in flavonoid biosynthesis.

In mixing experiments with extracts derived from two cell lines of Daucus carota tissue cultures with and without chalcone synthase activity, strong inhibition of chalcone synthase (CHS) became obvious. This inhibition was due to the presence of a heatlabile protein in extracts from cells devoid of CHS activity. This protein was partially purified and identified as 3'-nucleotidase (EC 3.1.3.6). Inhibition was also observed in the presence of purified 3'-nucleotidase from Lolium multiflorum. The phosphate group in the 3'-position of adenosine, a part of the CoA thioester substrates of CHS, was hydrolyzed by this enzyme. The dephosphorylated form of malonyl-CoA was no longer a substrate, whereas 4-coumaryl-3'-dephospho-CoA as well as 4-coumaryl-CoA was still able to act as a primer for the CHS reaction. Further studies showed that malonyl-3'-dephospho-CoA was an efficient CHS inhibitor. On the other hand, CoA-SH lost its inhibitory activity after dephosphorylation in the 3'-position. These results are discussed with respect to the mechanism of chalcone synthesis.