The B-ring hydroxylation pattern of intermediates of anthocyanin synthesis in pelargonidin-and cyanidin-producing lines of Matthiola incana

In flowers of Matthiola incana, the B-ring hydroxylation pattern of anthocyanins is controlled by the locus b. Recessive genotypes produce pelargonidin and genotypes with wild-type alleles cyanidin as the aglycone. Supplementation experiments on acyanic flowers using extracts of pelargonidin-and cyanidin-producing flowers, respectively, showed not only the presence of compounds with a precursor function for anthocyanin synthesis in the cyanic flowers but also differences in the B-ring hydroxylation pattern of these compounds. Chromatographic investigations proved that flavanones and dihydroflavonols occur in extracts of cyanic flowers. Naringenin, dihydrokaempferol, and their 7-glucosides could be demonstrated in all flower extracts, but in extracts of cyanidin-producing flowers, dihydroquercetin and a further 3, 4-hydroxylated dihydroflavonol, tentatively identified as dihydroquercetin 3-glycoside, were additionally found. In no case, however, could eriodictyol be detected. From these results and from the ready hydroxylation of dihydrokaempferol to dihydroquercetin in a white mutant line of Matthiola incana, it can be concluded that introduction of the 3-hydroxyl group of anthocyanins is not achieved by specific incorporation of caffeic acid during synthesis of the flavonoid skeleton, but by hydroxylation at the dihydroflavonol stage.     

Selection and characterisation of flavanone 3-hydroxylase mutants ofDahlia,Streptocarpus, Verbena andZinnia

Precursor experiments and chromatographic studies indicate that the hydroxylation of flavanones in the 3-position to dihydroflavonols is blocked in special white-flowering mutants ofDahlia, Streptocarpus, Verbena andZinnia. The result of our investigations was confirmed in as much as the activity of the enzyme flavanone 3-hydroxylase, which catalyses the conversion of flavanones to dihydroflavonols, could readily be detected in flower extracts of cyanic strains of the four plant species. It was found to be, however, completely absent in flower extracts of the corresponding acyanic mutants. Thus, the interruption of the anthocyanin pathway in these mutants is clearly caused by a lack of this enzyme activity. Similar to the enzymes from other sources, the 3-hydroxylases ofDahlia, Streptocarpus, Verbena andZinnia are soluble enzymes; they belong to the 2-oxoglutarate-dependent dioxygenases and the reaction is inhibited by ethylenediaminetetraacetic acid, KCN and diethyldithiocarbamate.     

Flavanones and dihydroflavonols as biosynthetic intermediates in Matthiola incana

In anthocyanin-producing flowers of Matthiola incana, the presence of naringenin, naringenin 7-glucoside, dihydrokaempferol and dihydrokaempferol 7-glucoside could be demonstrated. The four isolated compounds initiated anthocyanin synthesis after administration to acyanic flowers of genetically defined lines of Matthiola incana and Antirrhinum majus. Therefore, these compounds cannot be regarded as end-products but rather as intermediates in anthocyanin biosynthesis. Furthermore, naringenin 7-glucoside and dihydrokaempferol 7-glucoside most probably act as a pool for their aglycones, which serve as the actual substrates.     

Enzymatic reduction of (+)-dihydroflavonols to flavan-3,4-cis-diols with flower extracts from Matthiola incana and its role in anthocyanin biosynthesis

A cell-free extract from flowers of Matthiola incana catalyzes a NADPH-dependent stereospecific reduction of (+)-dihydrokaempferol to 3,4-cis-leucopelargonidin (5,7,4-trihydroxyflavan-3,4-cis-diol). The pH-optimum of this reaction is around 6. The rate of reaction with NADH was about 50% of that found with NADPH. (+)-Dihydroquercetin and (+)-dihydromyricetin were also reduced by the enzyme preparation to the corresponding flavan-3,4-cis-diols. Correlation between the genotype of M. incana and the presence of dihydroflavonol 4-reductase is strong evidence that this enzyme is involved in anthocyanin biosynthesis.     

Inhibitory Effect of Coronamic Acid Derivatives on Senescence in Cut Carnation Flowers

(+)-(1R,2S)-Allocoronamic acid, (?)-(1S,2R)-allocoronamic acid, and their racemate showed an inhibitory effect on senesence in cut carnation flowers. This antisenescent activity was based on the inhibition of endogenous ethylene formation catalyzed by an ethylene-forming enzyme.     

Purification and characterization of (+)dihydroflavonol (3-hydroxyflavanone) 4-reductase from flowers of Dahlia variabilis

Individual flowers from inflorescences of Dahlia variabilis (cv Scarlet Star) in young developmental stages contained relatively high activity of (+)-dihydroflavonol (DHF) 4-reductase. The DHF reductase was purified from such flowers to apparent homogeneity by a five-step procedure. This included affinity adsorption on Blue Sepharose and elution of the enzyme with NADP+. By gel filtration and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis it was shown that DHF reductase contains only one polypeptide chain with a Mr of about 41,000. The reductase required NADPH as cofactor and catalyzed transfer of the pro-S hydrogen of NADPH to the substrate. Flavanones and dihydroflavonols (3-hydroxyflavanones) were substrates for DHF reductase with pH optima of about 6.0 for flavanones and of about 6.8 for dihydroflavonols. Flavanones were reduced to the corresponding flavan-4-ols and (+)-dihydroflavonols to flavan-3,4-cis-diols. Apparent Michaelis constants determined for (2S)-naringenin, (2S)-eriodicytol, (+)-dihydrokaempferol, (+)-dihydroquercetin, and NADPH were, respectively, 2.3, 2, 10, 15, and 42 microM. V/Km values were higher for dihydroflavonols than for flavanones. Conversion of dihydromyricetin to leucodelphinidin was also catalyzed by the enzyme at a low rate, whereas flavones and flavonols were not accepted as substrates. DHF reductase was not inhibited by metal chelators.     

Genetic and biochemical studies on the conversion of flavanones to dihydroflavonols in flowers of Petunia hybrida

Summary Chemogenetic investigations and precursor experiments on flowers of Petunia hybrida suggest that recessive alleles of the gene An3 block the biosynthetic pathway of flavonols and anthocyanins between the flavanone and dihydroflavonol step. In confirmation of this hypothesis, activity of the enzyme flavanone 3-hydroxylase, which catalyses the conversion of flavanones to dihydroflavonols, was readily demonstrated in enzyme preparations from flowers of lines with the dominant allele An3, whereas no or very low activity could be found in extracts from lines with recessive alleles (an3an3). A second genetic factor is described which clearly reduces the amount of flavonols in the flowers but not the amount of anthocyanins. Crossing experiments revealed that this factor represents a third allele of the An3 gene. It is referred to as an3-1. As expected, a residual flavanone 3-hydroxylase activity of about 10% could be found in enzyme extracts from plants with the an3-1 allele. The decreased level of dihydroflavonol formed under this condition is obviously still sufficient for anthocyanin formation but not for flavonol synthesis.Similar to flavanone 3-hydroxylases from other plants, the enzyme of Petunia is a soluble enzyme and belongs according to its cofactor requirements to the 2-oxoglutarate-dependent dioxygenases. The residual flavanone 3-hydroxylase activity found in plants with the an3-1 allele is identical to the activity extracted from An3-genotypes with regard to cofactors, substrate specificity and most of the inhibitors. The difference observed in the respective pH-optima and the genetic data suggest that the mutation providing the an3-1 phenotype is localized in the structural gene for flavanone 3-hydroxylase.     

Genetically controlled anthocyanin synthesis in cell cultures of Matthiola incana

Callus cultures were derived from different parts of 8 anthocyanin producing and 2 white flowering lines of the crucifer Matthiola incana. The tissue cultures of the cyanic lines were shown to produce genotype specific anthocyanin patterns, whereas in the calli of the acyanic lines no anthocyanin synthesis occured.Abbreviations IAA indoleacetic acid - 2,4-D dichlorophenoxyacetic acid - MeOH methanol - Et₂O ether - ETOAc ethylacetate     

Cytokinins in cut carnation flowers. II. Relationship between endogenous ethylene and cytokinin levels in the petals

Tentative identification using HPLC and RIA techniques indicated the presence of zeatin-O-glucoside, zeatin, ribosylzeatin, dihydrozeatin, iso-pentenyladenine and iso-pentenyladenosine in the petals of carnation flowers. Dihydrozeatin is apparently responsible for most of the biological activity. Within the petals most activity was detected in the basal parts which also senesced much slower than the upper parts of the petals. Treatment with AOA extended petal longevity and reduced ethylene production. This was associated with higher cytokinin-like activity in the basal parts of the petals.These higher levels of cytokinins were not observed in the petals of ACC treated flowers or in the detached control flowers. It is suggested that cytokinin transport and/or metabolism may play an important role in regulating ethylene production in cut carnations.     

Anthocyanin synthesis in tissue cultures of Callistephus chinensis (China aster)

Callus cultures were derived from stems and leaves of 3 anthocyanin producing and 3 acyanic lines of Callistephus chinensis (Compositae). The tissue cultures of the cyanic lines were shown to produce cyanidin whereas in the calli of the acyanic lines no anthocyanin synthesis occurred Culture conditions were improved in order to enhance both anthocyanin production and growth of the tissue cultures.Abbreviations IAA indoleacetic acid - NAA naphtaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - MS-medium Murashige and Skoog medium     

Inhibition of wilting and autocatalytic ethylene production in cut carnation flowers by cis-propenylphosphonic acid

The effect of cis-propenylphosphonic acid (PPOH), a structural analoge of ethylene, on flower wilting and ethylene production was investigated using cut carnation flowers which are very sensitive to ethylene. Wilting (petal in-rolling) of the flowers was delayed by continuously immersing the stems in a 5–20 mM PPOH solution. In addition, the continuous treatment with PPOH markedly reduced autocatalytic ethylene production of the petals accompanying senescence. This reduction of autocatalytic ethylene production was considered responsible for the inhibitory effect of PPOH on flower wilting. The inhibitory activity of trans-propenylphosphonic acid (trans-PPOH), on both flower wilting and the autocatalytic ethylene production accompanying senescence was markedly lower than that of PPOH, suggesting that PPOH action is stereoselective. PPOH may be of interest as a new, water-soluble inhibitor of wilting and autocatalytic ethylene production in cut carnation flowers.     

Inhibition by 1-Aminocyclobutane-l-Carboxylate of the Activity of 1-Aminocyclopropane-l-Carboxylate Oxidase Obtained from Senescing Petals of Carnation {Dianthus caryophyllus L.) Flowers

We partially purified 1-aminocyclopropane-l-carboxy-late (ACC)oxidase from senescing petals of carnation {Dianthus caryophyllusL. cv. Nora) flowers and investigated its general characteristics,and, in particular, the inhibition of its activity by ACC analogs.The enzyme had an optimum pH at 7-7.5 and required Fe²⁺, ascorbateand NaHCO₃ for its maximal activity. The K_m for ACC was calculatedas 111-125 µM in the presence of NaHCO₃. Its M_r was estimatedto be 35 and 36 kDa by gel-filtration chromatography on HPLCand SDS-PAGE, respectively, indicating that the enzyme existsin a monomeric form. These properties were in agreement withthose reported previously with ACC oxidases from different planttissues including senescing carnation petals. Among six ACCanalogs tested, l-aminocyclobutane-l-carboxylate (ACBC) inhibitedmost severely the activity of ACC oxidase from carnation petals.ACBC acted as a competitive inhibitor with the K_i of 20-31 µM.The comparison between the K_m for ACC and the K_i for ACBC indicatedthat ACBC had an affinity which was ca. 5-fold higher than thatof ACC. Whereas ACC inactivated carnation ACC oxidase in a time-dependentmanner during incubation, ACBC did not cause the inactiva-tionof the enzyme. Preliminary experiments showed that ACBC andits N-substituted derivatives delayed the onset of senescencein cut carnation flowers. (Received August 19, 1996; Accepted November 26, 1996)     

Linalool and linalool oxide production in transgenic carnation flowers expressing the Clarkia breweri linalool synthase gene

Most modern cut-flower cultivars, including those of carnation(Dianthus caryophyllus), lack distinct fragrance.Carnationcv. Eilat flowers produce and emit various fragrance compounds, includingbenzoic acid derivatives and sesquiterpenes, but not monoterpenes. Based onGC-MS analysis, benzoic acid, benzyl benzoate, phenylethyl benzoate, methylbenzoate, cis-3-hexenyl benzoate and -caryophylleneare the major fragrance compounds, representing ca. 60% of the total volatilesgenerated by these flowers. The level of these compounds increases dramaticallyduring petal development. To evaluate the possibility of producing monoterpenesin carnation cv. Eilat, we generated transgenic plants expressing the linaloolsynthase gene from Clarkia breweri under the regulation ofthe CaMV 35S constitutive promoter. The product of this gene catalyzes theproduction of the monoterpene linalool from geranyl diphosphate. HeadspaceGC-MSanalysis revealed that leaves and flowers of transgenic, but not controlplants,emit linalool and its derivatives, cis- andtrans-linalool oxide. GC-MS analysis of petal extractrevealed the accumulation of trans-linalool oxide but notlinalool. The emission of linalool by the transgenic flowers did not lead todetectable changes in flower scent for human olfaction.     

Excision of transposable elements from the chalcone isomerase and dihydroflavonol 4-reductase genes may contribute to the variegation of the yellow-flowered carnation (Dianthus caryophyllus)

In the "Rhapsody" cultivar of the carnation, which bears white flowers variegated with red flecks and sectors, a transposable element, dTdic1, belonging to the Ac/Ds superfamily, was found within the dihydroflavonol 4-reductase (DFR) gene. The red flecks and sectors of "Rhapsody" may be attributable to a reversion to DFR activity after the excision of dTdic1. The yellow color of the carnation petals is attributed to the synthesis and accumulation of chalcone 2'-glucoside. In several of the carnation cultivars that bear yellow flowers variegated with white flecks and sectors, both the chalcone isomerase (CHI) and DFR genes are disrupted by dTdic1.     

Changes in 1-aminocyclopropane-1-carboxylic-acid content of cut carnation flowers in relation to their senescence

The rise in ethylene production accompanying the respiration climacteric and senescence of cut carnation flowers (Dianthus caryophyllus L. cv. White Sim) was associated with a 30-fold increase in the concentration of 1-aminocyclopropane-1-carboxylic acid (ACC) in the petals (initial content 0.3 nmol/g fresh weight). Pretreatment of the flowers with silver thiosulfate (STS) retarded flower senescence and prevented the increase in ACC concentration in the petals. An increase in ACC in the remaining flower parts, which appeared to precede the increase in the petals, was only partially prevented by the STS pretreatment. Addition of aminoxyacetic acid (2 mM) to the solution in which the flowers were kept completely inhibited accumulation of ACC in all flower parts.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AOA -aminoxyacetic acid - STS silver thiosulfate complex     

Chalcone synthesis and hydroxylation of flavonoids in 3′-position with enzyme preparations from flowers of Dianthus caryophyllus L. (carnation)

Chalcone synthase activity was demonstrated in enzyme preparations from flowers of defined genotypes of Dianthus caryophyllus L. (carnation). In the absence of chalcone isomerase activity, which could be completely excluded by genetic methods, the first product formed from malonyl-CoA and 4-coumaroyl-CoA proved to be naringenin chalcone, followed by formation of naringenin as a result of chemical cyclization. In the presence of chalcone isomerase activity, however, naringenin was the only product of the synthase reaction. In vitro, both 4-coumaryl-CoA and caffeoyl-CoA were found to be used as substrates for the condensation reaction with respective pH optima of 8.0 and 7.0. The results of chemogenetic and enzymatic studies, however, showed that in vivo only 4-coumaroyl-CoA serves as substrate for the formation of the flavonoid skeleton. In confirmation of these results, an NADPH-dependent microsomal 3-hydroxylase activity could be demonstrated, catalyzing hydroxylation of naringenin and dihydrokaempferol in 3-position. Furthermore, a strict correlation was found between 3-hydroxylase activity and the gene r which is known to control the formation of 3, 4-hydroxylated flavonoid compounds.     

The development of Botrytis cinerea on cut flowers of carnation

Enclosing carnation blooms in polythene increased rotting associated with naturally occurring Botrytis infections and accelerated the rate of conidia development when flowers were artificially inoculated before being stored at 1·7 °C. Petal infections were observed 16 and 48 h after inoculation and incubation at 18·3 and 1·7 °C respectively. The products of exosmosis from petals kept at 18·3 °C for 7 days stimulated germination of B. cinerea conidia. Slight pectic enzyme activity was detected in a liquid medium, derived from carnation petals, after incubating B. cinerea cultures at 18·3 °C for 6 days. Although cultures of Botrytis evolved virtually no ethylene, the surge of ethylene produced by uncontaminated ageing flowers occurred sooner after inoculation. A spray containing 1·2% a.i. of 2-aminobutane controlled rotting but damaged flowers.     

Ethylene production and β-cyanoalanine synthase activity in carnation flowers

The relationship between ethylene production and the CN^--assimilating enzyme -cyanoalanine synthase (CAS; EC 4.4.1.9) was examined in the carnation (Dianthus caryophyllus L.) flower. In petals from cut flowers aged naturally or treated with ethylene to accelerate senescence the several hundred-fold increase in ethylene production which occurred during irreversible wilting was accompanied by a one- to twofold increase in CAS activity. The basal parts of the petal, which produced the most ethylene, had the highest CAS activity. Studies of flower parts (styles, ovaries, receptacles, petals) showed that the styles had a high level of CAS together with the ethylene-forming enzyme (EFE) system for converting 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene. The close association between CAS and EFE found in styles could also be observed in detached petals after induction by ACC or ethylene. Treatment of the cut flowers with cycloheximide reduced synthesis of CAS and EFE. The data indicate that CAS and ethylene production are associated, and are discussed in relation to the hypothesis that CN^- is formed during the conversion of ACC to ethylene.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglyoine - CAS -cyanoalanine synthase - CHI cycloheximide - EFE ethylene-forming enzyme     

Changes in ethylene production and 1-aminocyclopropane-1-carboxylic acid content of pollinated carnation flowers

Pollination of flowers of standard carnation (Dianthus caryophyllus L. cv. White Sim) with pollen from flowers of miniature carnations (D. caryophyllus L. cv. Exquisite) caused them to wilt irreversibly within 1 to 2 days. Pollination stimulated a sequential increase in ethylene production by stigmas, ovaries, receptacles, and petals of the flowers. The ACC content of the stigmas increased rapidly in the first few hours after pollination. The possibility that subsequent production of ethylene by other parts of the flower is stimulated by translocated ACC is discussed. Ethylene production and ACC content of other parts of the flower reached their maximum 24 h after pollination. The petal tissues contributed the bulk of the ethylene productionper flower thereafter. There appears to be a qualitative difference between the enzyme in the stigmas converting ACC to ethylene and that in other parts of the flower.