The sequence of the S-adenosyl-L-methionine:trans-caffeoyl-CoA O-methyltransferase (CCoAOMT, EC2.... more The sequence of the S-adenosyl-L-methionine:trans-caffeoyl-CoA O-methyltransferase (CCoAOMT, EC2.1.1.104) gene, including the 5'-flanking region of 5 kb, was determined from parsley (Petroselinum crispum) plants. The enzyme appears to be encoded by one or two genes, and the ORF is arranged in five exons spaced by introns from 107 to 263 bp in length. The genomic sequence matches the ORF of the cDNA previously reported from elicited parsley cell cultures, showing only three base changes that do not affect the enzyme polypeptide sequence. S1 nuclease protection assays and primer extension analyses with genomic and cDNA templates revealed the transcription start site 67 bp upstream of the translation start codon, indicating a shorter 5'-UTR than reported previously for the transcript. Promoter regulatory consensus elements such as two 'CAAT' boxes and one 'TATA' box were identified at -196, -127 and -31, respectively, relative to the transcription start site, an...
European journal of biochemistry / FEBS, Jan 15, 1988
Suspension cultures of Ammi majus L. cells produce various linear furanocoumarins in response to ... more Suspension cultures of Ammi majus L. cells produce various linear furanocoumarins in response to treatment with elicitor preparations from either Alternaria carthami Chowdhury or Phytophthora megasperma f.sp. glycinea. Microsomes which were isolated from these cells 14 h after addition of the elicitor efficiently catalyzed the conversion of demethyl [3-14C]suberosin into labelled (+)marmesin in the presence of NADPH and oxygen. In contrast to the chemical cyclization of demethylsuberosin by m-chloroperoxybenzoic acid, the reaction catalyzed by the marmesin synthase proceeded rapidly and no intermediate demethylsuberosin epoxide could be recovered. Significant blue-light-reversible inhibition by carbon monoxide and inhibition by various chemicals known to inhibit reactions dependent on cytochrome P450 suggested that the marmesin synthase is a cytochrome-P450-dependent monooxygenase. Upon prolonged incubation, a subsequent major labelled product originated from (+)marmesin, which was ...
Trans-Caffeoyl-CoA 3-O-methyltransferase is involved in the reinforcement of the plant cell wall ... more Trans-Caffeoyl-CoA 3-O-methyltransferase is involved in the reinforcement of the plant cell wall under conditions that trigger the disease resistance response (Pakusch, A.-E., Kneusel, R.E., and Matern, U. (1989) Arch. Biochem. Biophys. 271, 488-494). Partial amino acid sequences of the enzyme from cultured parsley cells that had been treated with a crude elicitor were identified (Pakusch, A.-E., Matern, U., and Schiltz, E. (1991) Plant Physiol. 95, 137-143), and corresponding degenerated oligonucleotides of 29- and 30-nucleotide length were synthesized. Northern hybridizations with these probes revealed one specific RNA band, and the amount of this RNA appeared to be transiently induced upon elicitation of the cells. De novo enzyme synthesis was confirmed by Western blotting experiments using a specific antiserum. The time course of induction closely followed the pattern observed for phenylalanine ammonia-lyase and suggested the operational coordination of the methyltransferase wit...
Malonylated apigenin 7-O-glucoside was prepared from malonyl-CoA and apigenin 7-O-glucoside using... more Malonylated apigenin 7-O-glucoside was prepared from malonyl-CoA and apigenin 7-O-glucoside using a malonyltransferase from parsley cell cultures. The enzyme product, as well as the flavonoid substrate, was analyzed by high-resolution nuclear magnetic resonance spectroscopy, confirming that malonic acid had been attached to the primary hydroxyl of the glucose moiety of apigenin 7-O-glucoside. During these studies in several solvents, it became apparent that, in particular, the chemical shift of H-6" A and the coupling constants J5",6"A and J5",6"B were dependent on solvent composition and proton concentration. Similar changes of sugar proton resonance frequencies were also observed with methyl 6-O-malonyl-beta-D-glucopyranoside, but not with apigenin 7-O-[6-O-(4-coumaroyl)glucoside]. The change of coupling constants is ascribed to a conformational modification of the sugar portion in the malonylglucosides. Malonylated flavonoid glycosides are exclusively located within the vacuoles of parsley cells. We propose that acylation of flavonoid glycosides which are synthesized in the cytoplasm facilitates transport of the substrates into the vacuole. The conformational modification which is induced by changes in proton concentration may provide a mechanism to trap flavonoids in situ.
Parsley cell cultures produce linear furanocoumarins and the linear benzodipyrandione, graveolone... more Parsley cell cultures produce linear furanocoumarins and the linear benzodipyrandione, graveolone, in response to treatment with an elicitor from either Plzj~phthora mc~gaspernia or Alfernaria curthami. Activities of enzymes involved in general phenylpropanoid metabolism, phenylalanine ammonia-lyase and 4-coumarate : CoA ligase, as well as of an enzyme involved specifically in furanocoumarin biosynthesis, dimethylallyl diphosphate : umbelliferone dimethylallyltransferase, were monitored over several days after treatment with A . carthami elicitor. In addition, the activities of chalcone synthase, an enzyme involved in flavonoid formation, and of glucose-6-phosphate: NADP 1 -oxidoreductase were also monitored. The lyase and the ligase activities increased steadily for 48 h and the dimethylallyltransferase activity for 54 h, while the synthase activity was not altered and the oxidoreductase activity decreased gradually. In some experiments, phenylalanine ammonia-lyase activity reached a maximum value of 250 pkat/kg, twice the inaximal activity observed previously in parsley cells after treatment with either ultraviolet light or an elicitor preparation from P. mrgaspwma. In crude extracts, phenylalanine ammonia-lyase activity was shown to be inhibited by unidentified small-molecular-weight compounds which were formed in proportion to the elicitor treatment.
The concept of systemic acquired resistance (SAR) enables a novel approach to crop protection, an... more The concept of systemic acquired resistance (SAR) enables a novel approach to crop protection, and particular pathogenesis-related proteins, i.e. an acidic chitinase, have been classified as markers of the SAR response. Basic class I (VCHIT1b) and a class III (VCH3) chitinase cDNAs were cloned from cultured Vitis vinifera L. cv Pinot Noir cells and used to probe the induction response of grapevine cells to salicylic acid or yeast elicitor. Furthermore, the cells were treated with the commercial SAR activators 2,6-dichloroiso-nicotinic acid or benzo(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester. Elicitor or salicylic acid induced both VCHIT1b and VCH3 transcript abundances, whereas 2,6-dichloroiso-nicotinic acid or benzo(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester enhanced exclusively the expression of VCH3. To assess the systemic sensation of chitinase expression, single leaves of Vitis vinifera L. cv Pinot Noir or Vitis rupestris plants were inoculated with Plasmopara viticola spore suspensions, and the VCH3 and VCHIT1b mRNA amounts in the infected versus the adjacent, healthy leaf were monitored. Two VCH3 mRNA maxima were observed 2 and 6 d postinoculation in the infected, susceptible V. vinifera tissue, whereas in the healthy leaf the transcript increased from low levels d 2 postinoculation to prominent levels d 6 to 8 postinoculation. The level of VCH3 mRNA increased also over 4 d in the inoculated, resistant V. rupestris tissue. However, necrotic spots rapidly limited the infection, and the VCH3 transcript was undetectable in the upper-stage, healthy leaf. The expression of VCHIT1b remained negligible under either experimental condition. Overall, the results suggest that the selective expression of VCH3 might be a reliable indicator of the SAR response in V. vinifera L.
The activity of caffeoyl-coenzyme A (CoA) 3-0-methyltransferase, an enzyme widely distributed in ... more The activity of caffeoyl-coenzyme A (CoA) 3-0-methyltransferase, an enzyme widely distributed in plants and involved in cell wall reinforcement in a disease resistance response, appears to be subject to a complex type of regulation in vivo. In cultured parsley (Petroselinum crispum) cells treated with an elicitor from Phytophthora megasperma f.sp. glycinea, the enzyme activity is rapidly induced by a transient increase in the rate of de novo transcription. Parsley caffeoyl-CoA-specific methyltransferase differs in several aspects from other plant 0-methyltransferases but shows limited homology to bacterial adenine-specific DNA methyltransferases. Kinetic analysis revealed an Ordered Bi Bi mechanism for catalysis, with caffeoyl-CoA bound prior to Sadenosyl-L-methionine and feruloyl-CoA released last from the enzyme. The small inhibitory constant determined in vitro for feruloyl-CoA suggests that, in vivo, the enzyme activity is also under tight control by the steady-state product concentration in addition to the rate of transcription that becomes affected upon elicitor challenge.
A novel compound, serinol phosphate, was identified in sugarcane (Saccharum offlcinarum) done 51N... more A novel compound, serinol phosphate, was identified in sugarcane (Saccharum offlcinarum) done 51NG97. It was produced by an enzyme-mediated transamination of dibydroxyacetone phosphate with either alanine, glubtmate, aspartate, or glutamine serving equally well as an amino donor. Some detectable phosphatase activity was present in crude leaf enzyme preparation that hydrolyzed serinol phosphate. A proposal for a pathway of the biosynthesis of serinol in sugarcane was formulated.
Flavonols and conditionally also anthocyanins, aside from flavonols, are the predominant polyphen... more Flavonols and conditionally also anthocyanins, aside from flavonols, are the predominant polyphenols accumulated in various tissues of the model plant Arabidopsis thaliana L. In vitro experiments suggested that the dioxygenases involved in their biosynthesis, flavonol synthase and anthocyanidin synthase, are ''multifunctional" enzymes showing distinct side activities. The in vivo relevance of the additional activities attributed to these enzymes, however, has remained obscure. In this review we summarize the most recent results and present final proof of the complementing activities of these synthases for flavonol and anthocyanidin formation in the model plant A. thaliana. The impact of their modification on the biosynthetic pathway and the pattern of flavonoids in different plant tissues are discussed.
Two dimethylallyl diphosphate:umbelliferone dimethylallyltransferase (prenyltransferase) activiti... more Two dimethylallyl diphosphate:umbelliferone dimethylallyltransferase (prenyltransferase) activities, catalysing the 6-prenylation and the 7-O-prenylation, respectively, of umbelliferone in the course of phytoalexin synthesis, increased in Ammi majus cell suspension cultures in response to elicitor treatment. Both enzyme activities were dependent on Mg2+ or Mn2+ with significant preference for Mg2+ in the 6-prenylation reaction. Whereas dark-grown cells did not contain these activities, both prenyltransferase activities were induced rapidly by the addition of elicitor reaching a first maximum after 10-14 hr and a second maximum beyond 30 hr. Other coumarin specific, elicitor-induced enzyme activities of A. majus cells, in contrast, showed only one maximum of activity within the 50 hr experimental period, while the pattern of induction of phenylalanine ammonia-lyase activity resembled that of the prenyltransferases with maxima at ca 8 hr and 20-30 hr. Preliminary data suggest that the apparent biphasic induction of these enzyme activities is due to post-translational enzyme modifications.
Proceedings of The National Academy of Sciences, 1978
Alternaria solani, the causal agent of early blight disease in potato, produces two host-specific... more Alternaria solani, the causal agent of early blight disease in potato, produces two host-specific, lipidlike toxins in culture. Both compounds are required in the leaf bioassay for the elicitation of typical early blight symptoms, but the compounds are individually inactive. The procedures for the preparation of both compounds are outlined. These compounds can be used effectively to select for toxin-insensitive
Acridone synthase II cDNA was cloned from irradiated cell suspension cultures of Ruta graveolens ... more Acridone synthase II cDNA was cloned from irradiated cell suspension cultures of Ruta graveolens L. and expressed in Escherichia coli. The translated polypeptide of M r 42 681 revealed a high degree of similarity to heterologous chalcone and stilbene synthases (70^75%), and the sequence was 94% identical to that of acridone synthase I cloned previously from elicited Ruta cells. Highly active recombinant acridone synthases I and II were purified to apparent homogeneity by a four-step purification protocol, and the affinities to N-methylanthraniloyl-CoA and malonyl-CoA were determined. The molecular mass of acridone synthase II was estimated from size exclusion chromatography on a Fractogel EMD BioSEC (S) column at about 45 kDa, as compared to a mass of 44 þ 3 kDa found for the acridone synthase I on Superdex 75. Nevertheless, the sedimentation analysis by ultracentrifugation revealed molecular masses of 81 þ 4 kDa for both acridone synthases. It is proposed, therefore, that the acridone synthases of Ruta graveolens are typical homodimeric plant polyketide synthases.
Microsomes prepared from cultured Ammi mujus cells that had been challenged for 14 h with an elic... more Microsomes prepared from cultured Ammi mujus cells that had been challenged for 14 h with an elicitor derived from the cell walls of Phytophthora megasperma f.sp. glycineu (Pmg) converted psoralen to bergaptol(5-hydroxypsoralen) in the presence of NADPH and oxygen. The enzymatic activity was characterized as an inducible cytochrome-P-450-dependent monooxygenase associated with the endoplasmic reticulum. All of the steps involved in bergapten (5methoxypsoralen) biosynthesis in Ammi majus have now been demonstrated in vitro. The results suggest that bergaptol and not hydroxymarmesin in the precursor of bergapten.
Cell suspension cultures of carnation (Ditr~?~hus ccrr~~~p/~~//~.s L.) accumulate, upon challenge... more Cell suspension cultures of carnation (Ditr~?~hus ccrr~~~p/~~//~.s L.) accumulate, upon challenge with crude fungal elicitor, various dianthramide phytoalexins, all of which derive from N-benzoylanthranilate. In vitro, microsomes from the elicited carnation cells hydroxylated N-benzoylanthranilate in the 4-and/or 2'-positions to yield the hydroxyanthranilate and/or salicyloyl derivatives. 2'-Hydroxylation was shown to precede 4-hydroxylation in the formation of N-salicyloyl-4-hydroxyanthranilate, and both these activities depended strictly on NADPH and molecular oxygen. 4-Hydroxylation was shown to be catalyzed by cytochrome P-450-dependent monooxygenase(s), whereas the 2'-hydroxylating activity appeared to be due to a novel class of enzymes. also responding synergistically to NADH in combination with NADPH and showing apparent inhibition by cytochromc (' but not by carbon monoxide. The difference in type of 4-and 2'-hydroxylases was corroborated by the exclusive inhibition of either activity in imidazolc vs. MOPS buffers as well as their differential heat sensitivities. In the course of these studies, low concentrations of N-salicyloylanthranilatc turned out to inhibit the cytochrome P-4SO-dependent 4-hydroxylation more strongly than any of the commercial inhibitor chemicals tested, while neither the substrate, N-benzoylanthrdnilate, nor the final product, Ksalicyloyl-4_hydroxyanthranilate, exhibited such signkant inhibition. In addition, 2'-hydroxylation activity was affected much less by N-benzoyianthranilate, N-salicyloylanthranilate or by inhibitor chemicals. The results demonstrate the requirement of two different classes of hydroxylase activities that appear to introduce the antimycotic quality to the dianthramides for phytoalexin defense.
Two malonyltransferases, malonyl-CoA:flavone/flavonol i'-0-glucoside malonyltransferase and malon... more Two malonyltransferases, malonyl-CoA:flavone/flavonol i'-0-glucoside malonyltransferase and malonyl-CoA:flavonol 3-0-glucoside malonyltransferase, were purified to apparent homogeneity from uv-irradiated parsley cell cultures. Both purified enzymes appear to be specific for flavonoid glycosides. Additional malonyltransferases, active toward several phenol glucosides other than flavonoids, were present in partially purified 7-0-glucoside malonyltransferase preparations. Antibodies raised against the purified 3-0-glucoside malonyltransferase did not inhibit the activity of the 7-0-glucoside malonyltransferase over a wide antibody concentration range. Determination of the rate of synthesis in viva of the 3-0-glucoside malonyltransferase after ultraviolet lightpulse induction of parsley cells revealed two maxima at 6 and 30 h, respectively. These results indicate that the induced changes in 3-0-glucoside malonyltransferase activity were the consequence of either a repeated change in the rate of synthesis of one enzyme species or changes in the synthesis rates of more than one enzyme species.
The sequence of the S-adenosyl-L-methionine:trans-caffeoyl-CoA O-methyltransferase (CCoAOMT, EC2.... more The sequence of the S-adenosyl-L-methionine:trans-caffeoyl-CoA O-methyltransferase (CCoAOMT, EC2.1.1.104) gene, including the 5'-flanking region of 5 kb, was determined from parsley (Petroselinum crispum) plants. The enzyme appears to be encoded by one or two genes, and the ORF is arranged in five exons spaced by introns from 107 to 263 bp in length. The genomic sequence matches the ORF of the cDNA previously reported from elicited parsley cell cultures, showing only three base changes that do not affect the enzyme polypeptide sequence. S1 nuclease protection assays and primer extension analyses with genomic and cDNA templates revealed the transcription start site 67 bp upstream of the translation start codon, indicating a shorter 5'-UTR than reported previously for the transcript. Promoter regulatory consensus elements such as two 'CAAT' boxes and one 'TATA' box were identified at -196, -127 and -31, respectively, relative to the transcription start site, an...
European journal of biochemistry / FEBS, Jan 15, 1988
Suspension cultures of Ammi majus L. cells produce various linear furanocoumarins in response to ... more Suspension cultures of Ammi majus L. cells produce various linear furanocoumarins in response to treatment with elicitor preparations from either Alternaria carthami Chowdhury or Phytophthora megasperma f.sp. glycinea. Microsomes which were isolated from these cells 14 h after addition of the elicitor efficiently catalyzed the conversion of demethyl [3-14C]suberosin into labelled (+)marmesin in the presence of NADPH and oxygen. In contrast to the chemical cyclization of demethylsuberosin by m-chloroperoxybenzoic acid, the reaction catalyzed by the marmesin synthase proceeded rapidly and no intermediate demethylsuberosin epoxide could be recovered. Significant blue-light-reversible inhibition by carbon monoxide and inhibition by various chemicals known to inhibit reactions dependent on cytochrome P450 suggested that the marmesin synthase is a cytochrome-P450-dependent monooxygenase. Upon prolonged incubation, a subsequent major labelled product originated from (+)marmesin, which was ...
Trans-Caffeoyl-CoA 3-O-methyltransferase is involved in the reinforcement of the plant cell wall ... more Trans-Caffeoyl-CoA 3-O-methyltransferase is involved in the reinforcement of the plant cell wall under conditions that trigger the disease resistance response (Pakusch, A.-E., Kneusel, R.E., and Matern, U. (1989) Arch. Biochem. Biophys. 271, 488-494). Partial amino acid sequences of the enzyme from cultured parsley cells that had been treated with a crude elicitor were identified (Pakusch, A.-E., Matern, U., and Schiltz, E. (1991) Plant Physiol. 95, 137-143), and corresponding degenerated oligonucleotides of 29- and 30-nucleotide length were synthesized. Northern hybridizations with these probes revealed one specific RNA band, and the amount of this RNA appeared to be transiently induced upon elicitation of the cells. De novo enzyme synthesis was confirmed by Western blotting experiments using a specific antiserum. The time course of induction closely followed the pattern observed for phenylalanine ammonia-lyase and suggested the operational coordination of the methyltransferase wit...
Malonylated apigenin 7-O-glucoside was prepared from malonyl-CoA and apigenin 7-O-glucoside using... more Malonylated apigenin 7-O-glucoside was prepared from malonyl-CoA and apigenin 7-O-glucoside using a malonyltransferase from parsley cell cultures. The enzyme product, as well as the flavonoid substrate, was analyzed by high-resolution nuclear magnetic resonance spectroscopy, confirming that malonic acid had been attached to the primary hydroxyl of the glucose moiety of apigenin 7-O-glucoside. During these studies in several solvents, it became apparent that, in particular, the chemical shift of H-6" A and the coupling constants J5",6"A and J5",6"B were dependent on solvent composition and proton concentration. Similar changes of sugar proton resonance frequencies were also observed with methyl 6-O-malonyl-beta-D-glucopyranoside, but not with apigenin 7-O-[6-O-(4-coumaroyl)glucoside]. The change of coupling constants is ascribed to a conformational modification of the sugar portion in the malonylglucosides. Malonylated flavonoid glycosides are exclusively located within the vacuoles of parsley cells. We propose that acylation of flavonoid glycosides which are synthesized in the cytoplasm facilitates transport of the substrates into the vacuole. The conformational modification which is induced by changes in proton concentration may provide a mechanism to trap flavonoids in situ.
Parsley cell cultures produce linear furanocoumarins and the linear benzodipyrandione, graveolone... more Parsley cell cultures produce linear furanocoumarins and the linear benzodipyrandione, graveolone, in response to treatment with an elicitor from either Plzj~phthora mc~gaspernia or Alfernaria curthami. Activities of enzymes involved in general phenylpropanoid metabolism, phenylalanine ammonia-lyase and 4-coumarate : CoA ligase, as well as of an enzyme involved specifically in furanocoumarin biosynthesis, dimethylallyl diphosphate : umbelliferone dimethylallyltransferase, were monitored over several days after treatment with A . carthami elicitor. In addition, the activities of chalcone synthase, an enzyme involved in flavonoid formation, and of glucose-6-phosphate: NADP 1 -oxidoreductase were also monitored. The lyase and the ligase activities increased steadily for 48 h and the dimethylallyltransferase activity for 54 h, while the synthase activity was not altered and the oxidoreductase activity decreased gradually. In some experiments, phenylalanine ammonia-lyase activity reached a maximum value of 250 pkat/kg, twice the inaximal activity observed previously in parsley cells after treatment with either ultraviolet light or an elicitor preparation from P. mrgaspwma. In crude extracts, phenylalanine ammonia-lyase activity was shown to be inhibited by unidentified small-molecular-weight compounds which were formed in proportion to the elicitor treatment.
The concept of systemic acquired resistance (SAR) enables a novel approach to crop protection, an... more The concept of systemic acquired resistance (SAR) enables a novel approach to crop protection, and particular pathogenesis-related proteins, i.e. an acidic chitinase, have been classified as markers of the SAR response. Basic class I (VCHIT1b) and a class III (VCH3) chitinase cDNAs were cloned from cultured Vitis vinifera L. cv Pinot Noir cells and used to probe the induction response of grapevine cells to salicylic acid or yeast elicitor. Furthermore, the cells were treated with the commercial SAR activators 2,6-dichloroiso-nicotinic acid or benzo(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester. Elicitor or salicylic acid induced both VCHIT1b and VCH3 transcript abundances, whereas 2,6-dichloroiso-nicotinic acid or benzo(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester enhanced exclusively the expression of VCH3. To assess the systemic sensation of chitinase expression, single leaves of Vitis vinifera L. cv Pinot Noir or Vitis rupestris plants were inoculated with Plasmopara viticola spore suspensions, and the VCH3 and VCHIT1b mRNA amounts in the infected versus the adjacent, healthy leaf were monitored. Two VCH3 mRNA maxima were observed 2 and 6 d postinoculation in the infected, susceptible V. vinifera tissue, whereas in the healthy leaf the transcript increased from low levels d 2 postinoculation to prominent levels d 6 to 8 postinoculation. The level of VCH3 mRNA increased also over 4 d in the inoculated, resistant V. rupestris tissue. However, necrotic spots rapidly limited the infection, and the VCH3 transcript was undetectable in the upper-stage, healthy leaf. The expression of VCHIT1b remained negligible under either experimental condition. Overall, the results suggest that the selective expression of VCH3 might be a reliable indicator of the SAR response in V. vinifera L.
The activity of caffeoyl-coenzyme A (CoA) 3-0-methyltransferase, an enzyme widely distributed in ... more The activity of caffeoyl-coenzyme A (CoA) 3-0-methyltransferase, an enzyme widely distributed in plants and involved in cell wall reinforcement in a disease resistance response, appears to be subject to a complex type of regulation in vivo. In cultured parsley (Petroselinum crispum) cells treated with an elicitor from Phytophthora megasperma f.sp. glycinea, the enzyme activity is rapidly induced by a transient increase in the rate of de novo transcription. Parsley caffeoyl-CoA-specific methyltransferase differs in several aspects from other plant 0-methyltransferases but shows limited homology to bacterial adenine-specific DNA methyltransferases. Kinetic analysis revealed an Ordered Bi Bi mechanism for catalysis, with caffeoyl-CoA bound prior to Sadenosyl-L-methionine and feruloyl-CoA released last from the enzyme. The small inhibitory constant determined in vitro for feruloyl-CoA suggests that, in vivo, the enzyme activity is also under tight control by the steady-state product concentration in addition to the rate of transcription that becomes affected upon elicitor challenge.
A novel compound, serinol phosphate, was identified in sugarcane (Saccharum offlcinarum) done 51N... more A novel compound, serinol phosphate, was identified in sugarcane (Saccharum offlcinarum) done 51NG97. It was produced by an enzyme-mediated transamination of dibydroxyacetone phosphate with either alanine, glubtmate, aspartate, or glutamine serving equally well as an amino donor. Some detectable phosphatase activity was present in crude leaf enzyme preparation that hydrolyzed serinol phosphate. A proposal for a pathway of the biosynthesis of serinol in sugarcane was formulated.
Flavonols and conditionally also anthocyanins, aside from flavonols, are the predominant polyphen... more Flavonols and conditionally also anthocyanins, aside from flavonols, are the predominant polyphenols accumulated in various tissues of the model plant Arabidopsis thaliana L. In vitro experiments suggested that the dioxygenases involved in their biosynthesis, flavonol synthase and anthocyanidin synthase, are ''multifunctional" enzymes showing distinct side activities. The in vivo relevance of the additional activities attributed to these enzymes, however, has remained obscure. In this review we summarize the most recent results and present final proof of the complementing activities of these synthases for flavonol and anthocyanidin formation in the model plant A. thaliana. The impact of their modification on the biosynthetic pathway and the pattern of flavonoids in different plant tissues are discussed.
Two dimethylallyl diphosphate:umbelliferone dimethylallyltransferase (prenyltransferase) activiti... more Two dimethylallyl diphosphate:umbelliferone dimethylallyltransferase (prenyltransferase) activities, catalysing the 6-prenylation and the 7-O-prenylation, respectively, of umbelliferone in the course of phytoalexin synthesis, increased in Ammi majus cell suspension cultures in response to elicitor treatment. Both enzyme activities were dependent on Mg2+ or Mn2+ with significant preference for Mg2+ in the 6-prenylation reaction. Whereas dark-grown cells did not contain these activities, both prenyltransferase activities were induced rapidly by the addition of elicitor reaching a first maximum after 10-14 hr and a second maximum beyond 30 hr. Other coumarin specific, elicitor-induced enzyme activities of A. majus cells, in contrast, showed only one maximum of activity within the 50 hr experimental period, while the pattern of induction of phenylalanine ammonia-lyase activity resembled that of the prenyltransferases with maxima at ca 8 hr and 20-30 hr. Preliminary data suggest that the apparent biphasic induction of these enzyme activities is due to post-translational enzyme modifications.
Proceedings of The National Academy of Sciences, 1978
Alternaria solani, the causal agent of early blight disease in potato, produces two host-specific... more Alternaria solani, the causal agent of early blight disease in potato, produces two host-specific, lipidlike toxins in culture. Both compounds are required in the leaf bioassay for the elicitation of typical early blight symptoms, but the compounds are individually inactive. The procedures for the preparation of both compounds are outlined. These compounds can be used effectively to select for toxin-insensitive
Acridone synthase II cDNA was cloned from irradiated cell suspension cultures of Ruta graveolens ... more Acridone synthase II cDNA was cloned from irradiated cell suspension cultures of Ruta graveolens L. and expressed in Escherichia coli. The translated polypeptide of M r 42 681 revealed a high degree of similarity to heterologous chalcone and stilbene synthases (70^75%), and the sequence was 94% identical to that of acridone synthase I cloned previously from elicited Ruta cells. Highly active recombinant acridone synthases I and II were purified to apparent homogeneity by a four-step purification protocol, and the affinities to N-methylanthraniloyl-CoA and malonyl-CoA were determined. The molecular mass of acridone synthase II was estimated from size exclusion chromatography on a Fractogel EMD BioSEC (S) column at about 45 kDa, as compared to a mass of 44 þ 3 kDa found for the acridone synthase I on Superdex 75. Nevertheless, the sedimentation analysis by ultracentrifugation revealed molecular masses of 81 þ 4 kDa for both acridone synthases. It is proposed, therefore, that the acridone synthases of Ruta graveolens are typical homodimeric plant polyketide synthases.
Microsomes prepared from cultured Ammi mujus cells that had been challenged for 14 h with an elic... more Microsomes prepared from cultured Ammi mujus cells that had been challenged for 14 h with an elicitor derived from the cell walls of Phytophthora megasperma f.sp. glycineu (Pmg) converted psoralen to bergaptol(5-hydroxypsoralen) in the presence of NADPH and oxygen. The enzymatic activity was characterized as an inducible cytochrome-P-450-dependent monooxygenase associated with the endoplasmic reticulum. All of the steps involved in bergapten (5methoxypsoralen) biosynthesis in Ammi majus have now been demonstrated in vitro. The results suggest that bergaptol and not hydroxymarmesin in the precursor of bergapten.
Cell suspension cultures of carnation (Ditr~?~hus ccrr~~~p/~~//~.s L.) accumulate, upon challenge... more Cell suspension cultures of carnation (Ditr~?~hus ccrr~~~p/~~//~.s L.) accumulate, upon challenge with crude fungal elicitor, various dianthramide phytoalexins, all of which derive from N-benzoylanthranilate. In vitro, microsomes from the elicited carnation cells hydroxylated N-benzoylanthranilate in the 4-and/or 2'-positions to yield the hydroxyanthranilate and/or salicyloyl derivatives. 2'-Hydroxylation was shown to precede 4-hydroxylation in the formation of N-salicyloyl-4-hydroxyanthranilate, and both these activities depended strictly on NADPH and molecular oxygen. 4-Hydroxylation was shown to be catalyzed by cytochrome P-450-dependent monooxygenase(s), whereas the 2'-hydroxylating activity appeared to be due to a novel class of enzymes. also responding synergistically to NADH in combination with NADPH and showing apparent inhibition by cytochromc (' but not by carbon monoxide. The difference in type of 4-and 2'-hydroxylases was corroborated by the exclusive inhibition of either activity in imidazolc vs. MOPS buffers as well as their differential heat sensitivities. In the course of these studies, low concentrations of N-salicyloylanthranilatc turned out to inhibit the cytochrome P-4SO-dependent 4-hydroxylation more strongly than any of the commercial inhibitor chemicals tested, while neither the substrate, N-benzoylanthrdnilate, nor the final product, Ksalicyloyl-4_hydroxyanthranilate, exhibited such signkant inhibition. In addition, 2'-hydroxylation activity was affected much less by N-benzoyianthranilate, N-salicyloylanthranilate or by inhibitor chemicals. The results demonstrate the requirement of two different classes of hydroxylase activities that appear to introduce the antimycotic quality to the dianthramides for phytoalexin defense.
Two malonyltransferases, malonyl-CoA:flavone/flavonol i'-0-glucoside malonyltransferase and malon... more Two malonyltransferases, malonyl-CoA:flavone/flavonol i'-0-glucoside malonyltransferase and malonyl-CoA:flavonol 3-0-glucoside malonyltransferase, were purified to apparent homogeneity from uv-irradiated parsley cell cultures. Both purified enzymes appear to be specific for flavonoid glycosides. Additional malonyltransferases, active toward several phenol glucosides other than flavonoids, were present in partially purified 7-0-glucoside malonyltransferase preparations. Antibodies raised against the purified 3-0-glucoside malonyltransferase did not inhibit the activity of the 7-0-glucoside malonyltransferase over a wide antibody concentration range. Determination of the rate of synthesis in viva of the 3-0-glucoside malonyltransferase after ultraviolet lightpulse induction of parsley cells revealed two maxima at 6 and 30 h, respectively. These results indicate that the induced changes in 3-0-glucoside malonyltransferase activity were the consequence of either a repeated change in the rate of synthesis of one enzyme species or changes in the synthesis rates of more than one enzyme species.
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