Genetic control of chalcone isomerase activity in anthers of Petunia hybrida

The gene Po in pollen of Petunia hybrida Vilm. controls a discrete step in flavonoid biosynthesis. In recessive genotypes, naringenin-chalcone (4, 2,4,6-tetrahydroxychalcone) is accumulated, whereas, under the influence of the wild-type allele flavonols and anthocyanins are formed. Enzymic investigations on anthers of four genetically defined lines with different pollen colouration revealed a clear correlation between accumulation of naringenin-chalcone and deficiency of chalcone isomerase (EC 5.5.1.6). The results allow the conclusion that chalcone is the first product of the flavanone synthase reaction in anthers of Petunia hybrida and that chalcone isomerase is essential for the formation of flavonols and anthocyanins. These results were similar to those previously obtained with Callistephus chinensis (L.) Nees.Abbreviations EGME ethylen glycol monomethyl ether - MeOH methanol - CI chalcone isomerase - HOAc acetic acid - TLC thinlayer chromatography     

Enzymatic control of anthocyanin expression in the flowers of pea (Pisum sativum) mutants

Using enzymological and immunological methods we have investigated the relationship between chalcone synthase and the A locus, a major gene involved in the control of anthocyanin expression in pea (Pisum sativum L.) flowers. Pea plants containing the dominant allele A usually synthesize anthocyanins in the petal tissue, whereas plants homozygous for the a allele do not produce anthocyanins. We sought to determine whether or not the A locus also controlled the presence or absence of chalcone synthase, the first enzyme of the flavonoid pathway in the flowers of three genetic lines (A, purple-violet flowers; A,am, white flowers with sometimes pink edges; and a, white flowers). Chalcone synthase was found to be present in all three genetic lines by enzyme activity measurement, indirect enzyme-linked immunosorbent assay (ELISA), and Western blotting. Spectroscopic investigations showed that only the genetic lines A and A,am contained anthocyanins and flavonol glycosides, respectively, in the flowers; line a accumulated p-coumaric acid or its derivatives. These data suggest that the A locus in Pisum is not the structural gene for chalcone synthase and it does not appear to regulate the expression of this enzyme.This work was supported by a grant from the Cornell University Biotechnology Program, which is sponsored by the New York State Science and Technology Foundation and a consortium of industries.     

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.     

Genetic control of chalcone-flavanone isomerase activity in Callistephus chinensis

A mutant blocked in anthocyanin synthesis leads to an accumulation of 4,2,4,6-tetrahydroxy-chalcone-2-glucoside (isosalipurposide) in blossoms of Callistephus chinesis (L.) Nees, whereas in geno-types with the wild-type allele, higher oxidized flavonoids and anthocyanins are synthesized. Measurements of chalcone-flavanone isomerase activity of 18 lines of Callistephus chinensis showed a clear correlation between accumulation of chalcone in the recessive genotypes (ch ch) and deficiency of this enzyme activity. Both the chemogenetic and the enzymologic evidence lead to the following conclusions: 1. The first product of the synthesis of the flavonoid skeleton should be tetrahydroxychalcone.-2. The chalcone-flavanone isomerase catalyzes the formation of flavanone from chalcone in a stereospecific way and there-with furnishes the substrate for the further reactions in the flavonoid biosynthesis.Abbreviations EGME ethylene glycol monomethyl ether - HOAc acetic acid - MeOH methanol - PVP polyvinylpyrrolidone - TBA tert. butanol-acetic acid-water, 3:1:1 - TLC thin-layer chromatography     

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.     

A chalcone isomerase‐like protein enhances flavonoid production and flower pigmentation

Flavonoids are major pigments in plants, and their biosynthetic pathway is one of the best‐studied metabolic pathways. Here we have identified three mutations within a gene that result in pale‐colored flowers in the Japanese morning glory (Ipomoea nil). As the mutations lead to a reduction of the colorless flavonoid compound flavonol as well as of anthocyanins in the flower petal, the identified gene was designated enhancer of flavonoid production (EFP). EFP encodes a chalcone isomerase (CHI)‐related protein classified as a type IV CHI protein. CHI is the second committed enzyme of the flavonoid biosynthetic pathway, but type IV CHI proteins are thought to lack CHI enzymatic activity, and their functions remain unknown. The spatio‐temporal expression of EFP and structural genes encoding enzymes that produce flavonoids is very similar. Expression of both EFP and the structural genes is coordinately promoted by genes encoding R2R3‐MYB and WD40 family proteins. The EFP gene is widely distributed in land plants, and RNAi knockdown mutants of the EFP homologs in petunia (Petunia hybrida) and torenia (Torenia hybrida) had pale‐colored flowers and low amounts of anthocyanins. The flavonol and flavone contents in the knockdown petunia and torenia flowers, respectively, were also significantly decreased, suggesting that the EFP protein contributes in early step(s) of the flavonoid biosynthetic pathway to ensure production of flavonoid compounds. From these results, we conclude that EFP is an enhancer of flavonoid production and flower pigmentation, and its function is conserved among diverse land plant species.     

The effect of petal-size manipulation on pollen removal,seed set,and insect-visitor behavior in Campanula americana

We investigated the effect of petal-size variation in tall bellflower, Campanula americana, on pollen removal rates, seed set, and pollinator behavior. Pollen removal, seed set, and the behavior of two bee visitors (Bombus and Halictus) were assessed in flowers that had 0%, 50%, or 100% of their petal lobes removed. Pollen removal rates did not differ significantly among the three treatment groups, probably due to the presence of an ineffective (ugly) pollinator (Halictus), which quickly removed pollen from completely reduced flowers. Seed set was significantly positively correlated with petal size. Bumblebees discriminated against reduced flowers, thereby lowering pollen movement to reduced flowers. Halictid bees rarely contacted stigmatic surfaces and thus pollen delivery to reduced flowers was limited. The presence of an ugly pollinator probably obscured the effect of petal reduction on pollen receipt, thus masking the impact of attractive structures on male fitness. Given the positive relationship between seed set and petal size, we caution against ascribing expenditure on attractive structures exclusively to male function. In plants with many olules, expenditure on attractive structures may affect seed set.     

The importance of non-pollen plant parts as food sources for the common blossom thrips, Frankliniella schultzei

Like other flower thrips, Frankliniella schultzei Trybom (Thysanoptera: Thripidae) feeds on pollen. However, the influence of a pollen diet on the life history of F. schultzei may not be as significant as reported for other thrips species. Frankliniella schultzei was reared successfully and with low mortalities (20%) on Wax Mallow, (Malvaviscus arboreus Cav.) plant part diets. Development times and fecundity on a petal diet were not significantly different from that on a pollen diet. Fecundity on a diet combining M. arboreus pollen, petal and leaf tissues was significantly higher than those individually containing these tissues. In laboratory choice tests, F. schultzei females encountered petal most often of the three plant parts. Pollen and leaf were encountered with similar frequencies. A significantly higher proportion of petal encounters (0.8) resulted in feeding than did pollen encounters (0.5). Few leaf encounters (0.1) were followed by feeding. Adult and larval F. schultzei were found in M. arboreus flowers but not on leaves. All parts of the flower were inhabited and not just the pollen-bearing petal apices and anthers. We propose that feeding on pollen within M. arboreus flowers is just one of many influences on the life history of F. schultzei and suggest that this may extend to other thrips species/host-plant combinations.     

Production of yellow colour in flowers: redirection of flavonoid biosynthesis in Petunia

Chalcones are intermediates in the biosynthesis of all flavonoids. In addition, in some species they constitute the major yellow flower pigments. There are two types of chalcones, distinguished by the presence (6′-hydroxychalcones) or absence (6′-deoxychalcones) of a hydroxyl group at the 6′ position of the A-ring. The 6′-deoxychalcones are formed when the enzyme chalcone reductase (CHR) is active in conjunction with chalcone synthase (CHS). In Petunia, only 6′-hydroxychalcones are synthesized, and except in the pollen of some genotypes, they are ephemeral intermediates in flavonoid metabolism. By introducing a CHR cDNA from Medicago sativa under the control of the 35S CaMV promoter into acyanic- or cyanic-flowered lines of Petunia, flower colour was changed from either white to pale yellow or deep purple to pale purple, respectively. Lines were generated that accumulated up to 60% of their petal flavonoids as 6′-deoxychalcones. Several different 6′-deoxychalcones accumulated in the petals of the CHR transgenics. The structures of three of these were determined: one, butein 4-O-glucoside, is a novel plant chalcone. Another chalcone compound was identified in the pollen of the transgenics. The results show that the Petunia chalcone isomerase is unable to use 6′-deoxychalcones as substrates so that 6′-deoxychalcones are stable in Petunia petals, leaves and pollen, but some Petunia flavonoid enzymes can use 6′-deoxychalcones as substrates to modify their structures. The introduction of CHR provides a method to redirect the flavonoid pathway into chalcone production, in order to modify flower colour or to reduce the biosynthesis of other flavonoid types.     

Enzymatic Reactions by Five Chalcone Synthase Homologs from Hop (Humulus lupulus L.)

The enzyme activities encoded in five cDNAs for chalcone synthase (CHS) homologs from hop were investigated. Only valerophenone synthase (VPS) and CHS_H1 showed both naringenin-chalcone and phlorisovalerophenone forming activity. Narigenin-chalcone production by VPS was much lower than by CHS_H1. Therefore, it is highly possible that flavonoid depends mainly on CHS_H1, while bitter acid biosynthesis depends mainly on VPS and CHS_H1.     

Tissue-distribution of secondary phenolic biosynthesis in developing primary leaves of Avena sativa L.

Primary leaves of oats (Avena sativa L.) have been used to study the integration of secondary phenolic metabolism into organ differentiation and development. In particular, the tissue-specific distribution of products and enzymes involved in their biosynthesis has been investigated. C-Glucosylflavones along with minor amounts of hydroxycinnamic-acid esters constitute the soluble phenolic compounds in these leaves. In addition, considerable amounts of insoluble products such as lignin and wall-bound ferulic-acid esters are formed. The tissue-specific activities of seven enzymes were determined in different stages of leaf growth. The rate-limiting enzyme of flavonoid biosynthesis in this system, chalcone synthase, together with chalcone isomerase (EC 5.5.1.6) and the terminal enzymes of the vitexin and isovitexin branches of the pathway (a flavonoid O-methyltransferase and an isovitexin arabinosyltransferase) are located in the leaf mesophyll. Since the flavonoids accumulate predominantly (up to 70%) in both epidermal layers, an intercellular transport of products is postulated. In contrast to the flavonoid enzymes, L-phenylalanine ammonia-lyase (EC 4.3.1.5), 4-coumarate: CoA ligase (EC 6.2.1.12), and S-adenosyl-L-methionine: caffeate 3-O-methyltransferase (EC 2.1.1.-), all involved in general phenylpropanoid metabolism, showed highest activities in the basal leaf region as well as in the epidermis and the vascular bundles. We suggest that these latter enzymes participate mainly in the biosynthesis of non-flavonoid phenolic products, such as lignin in the xylem tissue and wall-bound hydroxycinnamic acid-esters in epidermal, phloem, and sclerenchyma tissues.Abbreviations CHI chalcone isomerase - CHS chalcone synthase - 4CL 4-coumarate: CoA ligase - CMT S-adenosyl-L-methionine:caffeate 3-O-methyltransferase - FMT S-adenosyl-L-methionine:vitexin 2-O-rhamnoside 7-O-methyltransferase - HPLC high-performance liquid chromatography - IAT uridine 5-diphosphate L-arabinose:isovitexin 2-O-arabinosyltransferase - PAL L-phenylalanine ammonia-lyase     

Cis-acting elements of the CHS1 gene from white mustard controlling promoter activity and spatial patterns of expression

Chalcone synthase (CHS) catalyses the first regulatory step in the branch pathway of phenylpropanoid biosynthesis specific for synthesis of ubiquitous flavonoid pigments and UV protectants. External stimuli such as stress, light and wounding induce CHS expression that is both tissue-specific and under developmental control. In order to identify cis-acting elements involved in organ and tissue specifity, we fused varying parts of the CHS1 promoter of white mustard (Sinapis alba L.) to the GUS-coding region and analysed the expression of these constructs in stably transformed Arabidopsis plants. Two different stages of development were examined, seedlings as an early stage and flowers as the final stage of development. In seedlings, the full-length promoter showed expression in all organs except the hypocotyl; in flowers expression could be observed in all whorls. Unit 1 of the mustard CHS1 promoter, an element conserved in several CHS genes, which has been recently identified as a light responsive element, is able to mediate a tissue-specific expression pattern similar to that obtained with the full-length promoter in seedlings as well as in flowers. Other elements enhance or repress expression in combination with Unit 1, or mediate defined spatial expression independently of Unit 1. One such element, located between-907 and -655, directs expression similar to that of the full-length promoter in flowers but not in seedlings and differs therefore in function to Unit 1. Our data suggest a dominant regulation of CHS1 expression by Unit 1. Other elements within this promoter might interact with Unit 1 or confer a subset of spatial expression patterns when Unit 1 is deleted.Abbreviations ADH alcohol dehydrogenase - CaMV cauliflower mosaic virus - CHS chalcone synthase - GUS -glucuronidase     

Serologically-Related Anionic Peroxidases from Petunia and Cucumber can Substitute Flavonoid Antioxidants

In this study we have investigated whether naturally occurring flavonoid-deficient mutant Red Star of Petunia hybrida is capable of metabolizing H₂O₂ by invoking other antioxidant enzyme system. We demonstrated that reduced flower pigmentation due to a reduction in the chalcone synthase mRNA expression results in strong H₂O₂ accumulation accompanied by the induction of a specific set of anionic peroxidase (PRX), serologically-related to main cucumber srPRX. We found correlation between rate of H₂O₂ accumulation and qualitative, as well as quantitative changes in the srPRX expression which seems to be determined by flower phenotype. In detached flower buds cultured in vitro both abscisic acid and anther extirpation prevented anthocyanin pigmentation, and thus flavonoid biosynthesis, resulting in a marked accumulation of immunoprecipitable srPRX. In contrast, pigmented flowers cultivated under the same conditions did not accumulate corresponding srPRX. The results suggest that a specific set of anionic PRX can substitute for the absence of flavonoid antioxidants.     

Temporal and spatial regulation of anthocyanin biosynthesis provide diverse flower colour intensities and patterning in <Emphasis Type="Italic">Cymbidium</Emphasis> orchid

Main conclusion

This study confirmed pigment profiles in different colour groups, isolated key anthocyanin biosynthetic genes and established a basis to examine the regulation of colour patterning in flowers of Cymbidium orchid. Cymbidium orchid (Cymbidium hybrida) has a range of flower colours, often classified into four colour groups; pink, white, yellow and green. In this study, the biochemical and molecular basis for the different colour types was investigated, and genes involved in flavonoid/anthocyanin synthesis were identified and characterised. Pigment analysis across selected cultivars confirmed cyanidin 3-O-rutinoside and peonidin 3-O-rutinoside as the major anthocyanins detected; the flavonols quercetin and kaempferol rutinoside and robinoside were also present in petal tissue. β-carotene was the major carotenoid in the yellow cultivars, whilst pheophytins were the major chlorophyll pigments in the green cultivars. Anthocyanin pigments were important across all eight cultivars because anthocyanin accumulated in the flower labellum, even if not in the other petals/sepals. Genes encoding the flavonoid biosynthetic pathway enzymes chalcone synthase, flavonol synthase, flavonoid 3′ hydroxylase (F3′H), dihydroflavonol 4-reductase (DFR) and anthocyanidin synthase (ANS) were isolated from petal tissue of a Cymbidium cultivar. Expression of these flavonoid genes was monitored across flower bud development in each cultivar, confirming that DFR and ANS were only expressed in tissues where anthocyanin accumulated. Phylogenetic analysis suggested a cytochrome P450 sequence as that of the Cymbidium F3′H, consistent with the accumulation of di-hydroxylated anthocyanins and flavonols in flower tissue. A separate polyketide synthase, identified as a bibenzyl synthase, was isolated from petal tissue but was not associated with pigment accumulation. Our analyses show the diversity in flower colour of Cymbidium orchid derives not from different individual pigments but from subtle variations in concentration and pattern of pigment accumulation.

     

Flower color modifications of Torenia hybrida by cosuppression of anthocyanin biosynthesis genes

White and blue/white varieties of Torenia hybrida were successfully obtained from the blue variety cv. Summerwave (SWB) by cosuppressing expression of two of the genes involved in anthocyanin biosynthesis; chalcone synthase (CHS) and dihydroflavonol 4-reductase (DFR). Such molecular breeding is the only precise and efficient way to create flower color variation in SWB due to its male and female sterility. Flower color and the degree of suppression varied between transgenic lines, and anthocyanin biosynthesis was more consistently suppressed in the dorsal petal lobes, ventral petal lobes and corolla tube than lateral petal lobes. A pink variety was obtained by cosuppressing the flavonoid 3,5-hydroxylase (F35H) gene. Yellow torenia was obtained from T-33, an in-house cultivar that contained both carotenoids and anthocyanins, by cosuppression of CHS or DFR genes.     

Inhibition of flavonoid biosynthesis by gibberellic acid in cell suspension cultures of Daucus carota L.

In carrot cells (Daucus carota L.), cultured in the presence of gibberellic acid, anthocyanin synthesis is blocked at the level of chalcone synthase. By feeding suitable precursors for anthocyanins (naringenin, eriodictyol, dihydroquercetin) biosynthesis of cyanidin glycosides can be restored. After addition of these substrates to the culture medium in the presence of gibberellic acid, the activity of chalcone synthase remained as low as in the control without precursors. The highest increase in anthocyanin content was achieved using dihydroquercetin as the added precursor. The time course of this supplementation showed a rapid response; within 4 h a substantial increase in anthocyanin could be observed. In contranst, the flavonol quercetin is not a precursor for cyanidin. The fact that naringenin was also accepted for cyanidin synthesis leads to the conclusion that hydroxylation in 3-position of ring B in Daucus carota takes place at the flavonoid stage.Abbreviations CHI Chalcone isomerase - CHS chalcone synthase - DMSO dimethylsulfoxide - GA₃ gibberellic acid - PAL phenylalanine ammonia-lyase     

Germinal reversion of an unstable mutation for anthocyanin pigmentation in soybean

Summary Plants of the w4-mutable line of soybean [Glycine max (L.) Merr.] are chimeral for anthocyanin pigmentation. Mutable plants produce both near-white and purple flowers, as well as flowers of mutable phenotype with purple sectors on near-white petals. It is established here that the mutable trait is conditioned by an unstable recessive allele of the w4 locus that conditions anthocyanin biosynthesis. The gene symbol w4-m is assigned to the mutable allele. Allele w4-m was derived from a stable, wild-type W4 progenitor allele and reverts at high frequency to a stable, wild-type W4 allele. Reversion occurs both early and late during the development of the germ line. Several experiments give estimates of germinal reversion frequency, indicating that approximately 6% of mutable alleles revert to wild-type from one generation to the next. Allele w4-m exhibits many features typical of an allele controlled by a transposable element.