Glycyrrhiza Echinata Research Articles

Chalcone isomerase isozymes with different substrate specificities towards 6'-hydroxy- and 6'-deoxychalcones in cultured cells of Glycyrrhiza echinata, a leguminous plant producing 5-deoxyflavonoids.

Three chalcone isomerase isozymes in Glycyrrhiza echinata (Fabaceae) were separated by chromatofocusing and partially purified to examine their substrate specificities. Two isozymes, one of which was elicitor-inducible, acted on both 6'-hydroxychalcone and 6'-deoxychalcone and presumably are involved in the legume-specific 5-deoxyflavonoid pathway, while another acted on only 6'-hydroxychalcone.

Cloning and characterization of eight cytochrome P450 cDNAs from chickpea ( Cicer arietinum L.) cell suspension cultures

Eight different P450 sequences were isolated from a cDNA library derived from cultured chickpea cells (cultivar ILC3279) elicited with a Phytophthora sojae (formerly megasperma) elicitor ( Pmg-elicitor) by screening with heterologous and homologous probes. Screening with CYP73A1 from Helianthus tuberosus yielded several clones with one identical sequence. A full-length clone could be isolated and this sequence was assigned CYP73A19. Heterologous expression in yeast confirmed that CYP73A19 is the trans-cinnamic acid 4-hydroxylase of chickpea. Screening with a CYP81E2 polymerase chain reaction fragment from chickpea yielded a CYP81E2 full-length sequence and two almost identical CYP81E3 sequences, differing in only 16 out of 498 amino acids; both share more than 85% homology with the isoflavone 2′-hydroxylase from licorice ( Glycyrrhiza echinata L.). Using CYP93A1 as a probe, it was possible to isolate a full-length member of the CYP93 family, CYP93C3, that shares more than 80% homology with isoflavone synthase from soybean. In addition, partial sequences CYP81E3, CYP81E4 and CYP81E5 were also found in this screening. The use of a CYP82A2 probe derived from BAC F10N7 from Arabidopsis thaliana yielded only one sequence, CYP76F1. Rescreening with CYP81E4 and CYP81E5 did not result in the isolation of any new P450 sequences. Northern blot experiments revealed that all but the CYP76F1 are induced rapidly and transiently in cell cultures upon elicitor treatment.

Cloning and functional expression of a cytochrome P450 cDNA encoding 2-hydroxyisoflavanone synthase involved in biosynthesis of the isoflavonoid skeleton in licorice.

Isoflavonoids are distributed predominantly in leguminous plants and play critical roles in plant physiology. A cytochrome P450 (P450), 2-hydroxyisoflavanone synthase, is the key enzyme in their biosynthesis. In cultured licorice (Glycyrrhiza echinata L., Fabaceae) cells, the production of both an isoflavonoid-derived phytoalexin (medicarpin) and a retrochalcone (echinatin) is rapidly induced upon elicitation. In this study, we obtained a full-length P450 cDNA, CYP Ge-8 (CYP93C2), from the cDNA library of elicited G. echinata cells. When the flavanones liquiritigenin and naringenin were incubated with the recombinant yeast microsome expressing CYP93C2, major products emerged and were readily converted to the isoflavones daidzein and genistein by acid treatment. The chemical structures of the products from liquiritigenin (2-hydroxyisoflavanone and isoflavone) were confirmed by mass spectrometry. CYP93C2 was thus shown to encode 2-hydroxyisoflavanone synthase, which catalyzes the hydroxylation associated with 1,2-aryl migration of flavanones. Northern-blot analysis revealed that transcripts of CYP93C2, in addition to those of other P450s involved in phenylpropanoid/flavonoid pathways, transiently accumulate upon elicitation.

Induction of isoflavonoid and retrochalcone branches of the flavonoid pathway in cultured Glycyrrhiza echinata cells treated with yeast extract.

Yeast extract-treated suspension cultures of a new cell line, AK-1, of Glycyrrhiza echinata were induced to produce an isoflavonoid phytoalexin (medicarpin) and metabolites of retrochalcone/flavone pathway (echinatin, licodione, and 7,4'-dihydroxyflavone). From these cells, putative full-length cDNAs encoding cytochrome P450s, (2S)-flavanone 2-hydroxylase and isoflavone 2'-hydroxylase, were cloned.

Identification of a cytochrome P450 cDNA encoding (2 S)-flavanone 2-hydroxylase of licorice ( Glycyrrhiza echinata L.; Fabaceae) which represents licodione synthase and flavone synthase II

The microsome of insect cells expressing CYP Ge-5 (CYP93B1), a cytochrome P450 cDNA of licorice ( Glycyrrhiza echinata L.), catalyzed the formation of [ 14C]licodione and [ 14C]-2-hydroxynaringenin from (2 S)-[ 14C]liquiritigenin and (2 S)-[ 14C]naringenin, respectively. On acid treatment, the products were converted to 14C-labeled 7,4′-dihydroxyflavone and apigenin. Eriodictyol was also converted to luteolin by the reaction with the microsome of yeast expressing CYP93B1 and subsequent acid treatment. CYP93B1 was thus shown to encode (2 S)-flavanone 2-hydroxylase, which has previously been designated to licodione synthase and flavone synthase II depending on the substrates employed.

Anthocyanin-producing dandelion callus as a chalcone synthase source in recombinant polyketide reductase assay

Purple-coloured dandelion ( Taraxacum officinale) callus cultures producing anthocyanin pigments were established on a cytokinin-rich medium under the light. When the cells were placed in the dark, only grey cells proliferated. Anthocyanin productivity of these cells was partially restored in the light. The major pigment was identified as cyanidin 3-(6″-malonylglucoside). The lower stem of the original plant contained the same pigment. Chalcone synthase (CHS) activity was detected in the extracts of these purple cells, whereas no activity was observed in grey cells propagated in the dark. When the CHS-active cell-free extract was combined with the extract of Escherichia coli over expressing polyketide reductase (PKR) cDNA of licorice ( Glycyrrhiza echinata), isoliquiritigenin (a 6′-deoxychalcone), in addition to naringenin (a 5-hydroxyflavanone), was detected as the reaction product from 4-coumaroyl-CoA, malonyl-CoA and NADPH. This result confirms the catalytic function of the PKR gene product.

Cloning of cytochrome P450 cDNAs from cultured Glycyrrhiza echinata L. cells and their transcriptional activation by elicitor-treatment

From a cDNA library of cultured Glycyrrhiza echinata L. (Fabaceae) cells treated with yeast extract (YE) elicitor, eight cytochrome P450 (P450) fragments (Ge-1 to 8) were isolated using the PCR method based on conserved sequences of P450s. Comparison of amino acid sequences revealed that two of the fragments (Ge-1 and 2) belong to the CYP73 family encoding trans-cinnamic acid 4-hydroxylase. Ge-4 had 63% identity with the sequence included in CYP93, which has recently been reported to be induced by methyl jasmonate in soybean cells (Suzuki et al., FEBS Lett., 383 (1996) 83–86), and Ge-6 and 7 were highly homologous to the partial sequence of CYP84 encoding ferulic acid 5-hydroxylase of Arabidopsis thaliana (Meyer et al., Proc. Natl. Acad. Sci. USA, 93 (1996) 6869–6874). Others (Ge-3, 5 and 8) displayed homology less than 43% with known plant P450s. A full-length cDNA (CYP73A14) containing Ge-1 sequence was isolated from the cDNA library, and its amino acid sequence showed 91–93% identity with CYP73 of other leguminous plant species. Northern blot analysis of mRNAs of G. echinata cells indicated that CYP73 (Ge-1 and 2) genes were transcribed at the early stages post-elicitation (<10 h). The transcription level of the Ge-1 gene was much higher than that of Ge-2. Transcription of Ge-3, 5, 6 and 7 was also activated by elicitation, but no clear signal from Ge-4 and 8 was observed. When total RNAs of cultured cells of Medicago sativa were analyzed by Northern blotting, transcripts hybridized with G. echinata cDNA clones containing Ge-1, 3, 5 and 6 sequences could be detected. Possible involvement of cloned P450s in elicitor-induced phenylpropanoid/flavonoid biosynthesis is discussed.

Enzymic O-methylation of isoliquiritigenin and licodione in alfalfa and licorice cultures

S- Adenosyl- l-methionine (SAM): isoliquiritigenin (2′,4,4′-trihydroxychalcone) 2′- O-methyltransferase (CHMT) of alfalfa ( Medicago sativa) catalyses the formation of 4,4′-dihydroxy-2′-methoxychalcone, which is the most potent inducer of nodulation-genes of Rhizobium meliloti, the symbiont of alfalfa which forms nitrogen-fixing nodules. SAM: licodione 2′- O-methyltransferase (LMT) is involved in the biosynthesis of a retrochalcone in cultured licorice ( Glycyrrhiza echinata) cells and has been shown to be induced as a defence response of the cells. Because licodione exists in an equilibrium mixture of tautomeric 2′,4,4′,β-tetrahydroxychalcone (major) and 1-(2,4-dihydroxyphenyl)-3-(4-hydroxyphenyl)-1,3-propanedione (minor), the apparent mode of action of both enzymes is very similar. In this study, cultured alfalfa cells were shown to exhibit rapid and transient increases in the extractable activities of both CHMT and LMT after treatment with yeast extract (YE). Treatment of solution-cultured alfalfa seedlings with YE also resulted in a similar induction of both CHMT and LMT activities in the roots, but no activity was detected in the shoots. These activities were attributed to a single gene product, the CHMT protein, as extracts of Escherichia coli transformed with the CHMT cDNA exhibited both CHMT and LMT activities. In contrast, in G. echinata cells, LMT was induced after YE treatment, but no CHMT activity was observed. It is concluded that alfalfa CHMT and licorice LMT are distinct enzymes, the former displaying the wider substrate specificity.

Licodione Synthase, a Cytochrome P450 Monooxygenase Catalyzing 2-Hydroxylation of 5-Deoxyflavanone, in Cultured Glycyrrhiza echinata L. Cells.

Cultured Glycyrrhiza echinata L. (Leguminosae) cells produce a retrochalcone echinatin (4,4[prime]-dihydroxy-2-methoxychalcone) and its biosynthetic intermediate licodione [1-(2,4-dihydroxyphenyl)-3-(4-hydroxyphenyl)-1,3-propanedione, a dibenzoylmethane (keto form) or its enol tautomer ([beta]-hydroxychalcone)], when treated with elicitor-active substances, e.g. yeast extract. A microsomal fraction (160,000g pellet) prepared from yeast extract-induced suspension cultures of G. echinata catalyzed the formation of licodione from (2S)-liquiritigenin (7,4[prime]-dihydroxyflavanone) in the presence of NADPH and air. This licodione synthase activity was shown to be dependent on cytochrome P450 by its microsomal localization, requirement of NAD(P)H and O2 for activity, and inhibition by typical cytochrome P450 inhibitors. Licodione synthase activity transiently increased in the cells after treatment with yeast extract. When (2S)-naringenin (5,7,4[prime]-trihydroxyflavanone) and NADPH were incubated with the same microsomal preparation, a polar compound, which further converted into apigenin (5,7,4[prime]-trihydroxyflavone) when treated with acid, was produced. The reaction mechanism of licodione synthase is likely to be 2-hydroxylation of the flavanone molecule and subsequent hemiacetal opening and is possibly the same as the previously suggested mechanism of flavone synthase II from soybean and, furthermore, closely related to isoflavone synthase from Pueraria lobata.

Enzymatic synthesis of 6?-deoxychalcone in cultured Glycyrrhiza echinata cells

5-Deoxy-(iso)flavonoids are biosynthesized from 6'-deoxychalcone (isoliquiritigenin). The coaction of a reductase with chalcone synthase (CHS) has been established in soybean cells to be responsible for the synthesis of 6'-deoxychalcone. Western blot analysis of crude extracts from cultured cells of Glycyrrhiza echinata, another member of the Leguminosae, revealed proteins which cross-react with an antiserum raised against the soybean reductase. DEAE-Cellulose chromatography of the extract yielded fractions which showed CHS activity but not deoxychalcone synthase activity, and these fractions were also negative in Western blot analysis. In contrast, fractions displaying positive signals with the antiserum were also able to synthesize 6'-deoxychalcone/5-deoxyflavanone. These results indicate that in G. echinata, too, synthesis of 6'-deoxychalcone is likely to be performed by the coaction of the reductase and CHS. Induction of the reductase and CHS by yeast extract treatment of the cells was demonstrated.

Notizen: A Novel Triterpenoid Isolated from the Roots of Glycyrrhiza echinata L.

3β,21 α-dihydroxy-oleane-11,13-dien-28-oic acid was isolated from the roots of Glycyrrhiza echinata L. and its structure was elucidated by spectral analysis.

カンゾウ培養細胞のフラボノイド生合成調節

カンゾウ培養細胞のフラボノイド生合成調節

Biotransformation of phenylcarboxylic acids by plant cell cultures

A suspension culture of Glycyrrhiza echinata converted benzoic acid into its glucosyl ester. Suspension cultures of Aconitum japonicum, Coffea arabica, Dioscoreophyllum cumminsii and Nicotiana tabacum, transformed benzoic acid into its gentiobiosyl ester in addition to the glucosyl ester. The suspension cultures of A.japonicum and G. echinata converted phenylacetic acid into the esters attached to the C-6 position of glucose, that is, 6- O-phenylacetyl- d-glucose and ethyl 6- O-phenylacetyl-β- d-glucopyranoside. That of D. cumminsii converted phenylacetic acid into the glucose ester and also into phenethyl β- d-glucopyranoside showing glucosylation after the reduction of the carboxylic group. These suspension cultures converted cinnamic acid into p-coumaric acid and its glucosyl ester and p-coumaric acid into its glucosyl ester. However, the conversion of caffeic acid was not observed. The suspension cultures of A.japonicum and C. arabica converted 3-phenylpropionic acid into its gentiobiosyl ester. On the other hand, the culture of D. cumminsii did not produce the glycosyl ester but instead 3-(4-hydroxyphenyl)propionic acid was formed, thus showing hydroxylation capability.

NAD(P)H-dependent 6′-deoxychalcone synthase activity in Glycyrrhiza echinata cells induced by yeast extract

The crude extract prepared from Glycyrrhiza echinata cells treated with yeast extract catalyzed the formation of liquiritigenin (5-deoxyflavanone) and isoliquiritigenin (6′-deoxychalcone) in addition to naringenin (5-hydroxyflavanone) when incubated with 4-coumaroyl-CoA and malonyl-CoA in the presence of high concentrations (0.1 m m or higher) of NADPH. Incubation without NADPH, or with low concentrations (0.01 m m or lower), gave only naringenin as a reaction product. With NADH (1 m m), the major product was naringenin accompanied by a small quantity of liquiritigenin. The initial product of the assay with 1 m m NADPH was isoliquiritigenin, indicating a reaction catalyzed by 6′-deoxychalcone synthase (DOCS). Subsequent formation of liquiritigenin was attributed to the presence of chalcone isomerase in the crude extract. The results constitute the first demonstration in vitro of DOCS activity which, in G. echinata cells and other leguminous plants, is involved in the biosynthesis of retrochalcone and 5-deoxyisoflavonoid-derived phytoalexins.

Stimulation of chalcone synthase activity by yeast extract in cultured Glycyrrhiza echinata cells and 5-deoxyflavanone formation by isolated protoplasts

Chalcone synthase activity catalyzing the formation of naringenin (5-hydroxyflavanone) was detected in cell suspension cultures of Glycyrrhiza echinata. This activity rapidly increased by treatment of the cells with yeast extract, while non-treated cells showed a constant low activity. Isolated G. echinata protoplasts accumulated retrochalcone (echinatin) and its biosynthetic intermediate (licodione) during 24 h of culture. When the protoplasts were incubated with [(14)C(U)]phenylalanine, liquiritigenin (5-deoxyflavanone) was transiently labeled, indicating the induction of 6'-deoxychalcone synthase. The formation of liquiritigenin, in addition to naringenin, was observed when the crude extracts from the protoplasts were assaved for CHS activity.

Regulation of retrochalcone biosynthesis: Activity changes of O-methyltransferases in the yeast extract-induced Glycyrrhiza echinata cells

Three O-methyltransferases which catalyze S-adenosyl-L-methionine (SAM)-dependent O-methylation of licodione (LMT), flavone/flavonol (FMT), and caffeic acid (CMT) were separated from the callus culture of Glycyrrhiza echinata, and characteristic differences between their pH optima and Mg(2+) requirement for activity were demonstrated. The activity of LMT, which is involved in retrochalcone (echinatin) biosynthesis, but not of FMT or CMT, was found to be stimulated when suspension-cultured G. echinata cells were treated with yeast extract (YE), which causes rapid production of echinatin in the cells. Cycloheximide suppressed both the YE-induced echinatin formation and LMT enhancement. The results indicate a selective induction of retrochalcone pathway in Glycyrrhiza cells in response to stress.

Induction of stress metabolites in immobilized Glycyrrhiza echinata cultured cells

Transfer into a fresh medium or immobilization by entrapment in calcium alginate gels of cultured Glycyrrhiza echinata cells caused a rapid and transient accumulation of a retrochalcone, echinatin, in both the cells and the medium. The higher level and longer duration of echinatin production was observed in the immobilized cells than in freely suspended cells. Transfer of the cells into the medium containing either sodium alginate or calcium chloride, and the addition of sodium alginate into the suspension culture, caused the same effect as observed in cell immobilization. A novel metabolite was also detected in the induced cells. Activities of the enzymes involved in echinatin biosynthesis were shown to rapidly increase by immobilization of the cells.

Stress-induced formation of echinatin and a metabolite, 5′-prenyl-licodione, in cultured Glycyrrhiza echinata cells

The production of a retrochalcone, echinatin, by isoflavone-rich Glycyrrhiza echinata (M-2) cultured cells was stimulated by the addition of yeast extract or calcium alginate beads to the culture medium. Combined addition of yeast extract and cycloheximide suppressed the formation of retrochalcone, suggesting de novo synthesis. A new metabolite was isolated from the induced cells and its structure was determined to be 1-[2,4-dihydroxy-5-(3-methyl-2-butenyl)phenyl]-3-(4-hydroxyphenyl)-1,3-propanedione (5′-prenyl-licodione).

Isoechinatic acid from the roots of Glycyrrhiza echinata

Isoechinatic acid from the roots of Glycyrrhiza echinata

甘草の栽培研究 (第1報)

In order to examine the possibility of cultivating licorice in Hokkaido, Glycyrrhiza echinata, G. uralensis, and G. glabra were cultivated since 1955, and the state of growth and seeding were observed. The plants harvested in 1958 were examined for the content of extract and glycyrrhizin. It was found from the state of growth and content of glycyrrhizin that it is possible to cultivate licorice in cold area.