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.     

Chalcone synthase in rice (Oryza sativa L.): Detection of the CHS protein in seedlings and molecular mapping of the chs locus

The chalcone synthase is a key enzyme that catalyses the first dedicated reaction of the flavonoid pathway in higher plants. The chs gene and its protein product in rice has been investigated. The presence of a chalcone synthase (CHS) protein in rice seedlings and its developmental stage-specific expression has been demonstrated by western analysis. The chalcone synthase of rice was found to be immunologically similar to that of maize. A rice cDNA clone, Os-chs cDNA, encoding chalcone synthase, isolated from a leaf cDNA library of an indica rice variety Purpleputtu has been mapped to the centromeric region of chromosome 11 of rice. It was mapped between RFLP markers RG2 and RG103. RG2 is the nearest RFLP marker located at a genetic distance of 3.3 cM. Some segments of chromosome 11 of rice including chs locus are conserved on chromosome 4 of maize. The markers, including chs locus on chromosome 11 of rice are located, though not in the same order, on chromosome 4 of maize. Genetic analysis of purple pigmentation in two rice lines, Abhaya and Shyamala, used in the present mapping studies, indicated the involvement of three genes, one of which has been identified as a dominant inhibitor of leaf pigmentation. The Os-chs cDNA shows extensive sequence homology, both for DNA and protein (deduced), to that of maize, barley and also to different monocots and dicots.     

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     

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.     

Root restriction affected anthocyanin composition and up-regulated the transcription of their biosynthetic genes during berry development in ‘Summer Black’ grape

Root restriction was applied to ‘Summer black’ grape (Vitis vinifera L. × Vitis labrusca L.) to investigate its effect on anthocyanin biosynthesis in grape berry during development. Anthocyanin composition and expression patterns of 16 genes in anthocyanin pathway were thus analyzed. The results showed that the anthocyanin levels in berry skin were significantly increased and the anthocyanin profile was enriched. Gene expression pattern revealed that the increased anthocyanins coincide with the up-regulated expression of all 16 genes investigated, including phenylalanine ammonia-lyase, 4-coumarate CoA ligase, chalcone synthase 1, chalcone synthase 2, chalcone synthase 3, chalcone isomerase, flavanone 3-hydroxylase 1, flavanone 3-hydroxylase 2, flavonoid 3′-hydroxylase (F3′H), flavonoid 3′,5′-hydroxylase (F3′5′H), di-hydroflavonol 4-reductase, leucoanthocyanidin dioxygenase, O-methyltransferases (OMT), UDP-glucose:flavonoid 3-O-glucosyl-transferase (3GT), UDP-glucose:flavonoid 5-O-glucosyl-transferase (5GT) and glutathione S-transferase (GST). The increased total anthocyanins predominantly resulted from the increase of tri-hydroxylated, methoxylated and mono-glycosylated rather than di-hydroxylated, non-methoxylated, and di-glycosylated forms, which might be due to the differential regulation of F3′5′H/F3′H, OMT and 3GT, respectively.     

Transformation of antisense constructs of the chalcone synthase gene superfamily into Gerbera hybrida: differential effect on the expression of family members

Suppression of gene expression using antisense technology has been successful in various applications. In this paper we report differential inhibition of gene expression of the chalcone synthase (chs) gene superfamily members in transgenic Gerbera hybrida (Asteraceae) plants. We have transformed two different cDNAs of the chs gene family, gchs 1 [4] and gchs2, in antisense orientation under control of the CaMV 35S promoter into gerbera. Gchs1 codes for an enzyme with chalcone synthase activity while gchs2 is a more diverged member of the gene family having distinct structure and expression pattern. Furthermore, gchs2 is evidently not involved in anthocyanin synthesis and encodes an enzyme with novel catalytic properties. In both cases effective blocking of the resident sense gene expression was detected. In addition, the transformation affected differentially the expression of other members of the chs gene family. The degree of inhibition appeared to depend on the sequence homology between the antisense and the target genes. In the unevenly coloured inflorescences detected among anti-gchs1 transformants during their growth, relaxation of the antisense effect was here shown to start from the most distant member of the gene family, further demonstrating the influence of sequence homology in the stability of antisense inhibition.     

Gerbera hybrida (Asteraceae) imposes regulation at several anatomical levels during inflorescence development on the gene for dihydroflavonol-4-reductase

In the ornamental cut flower plant Gerbera hybrida the spatial distribution of regulatory molecules characteristic of differentiation of the composite inflorescence is visualized as the various patterns of anthocyanin pigmentation of different varieties. In order to identify genes that the plant can regulate according to these anatomical patterns, we have analysed gene expression affecting two enzymatic steps, chalcone synthase (CHS) and dihydroflavonol-4-reductase (DFR), in five gerbera varieties with spatially restricted anthocyanin pigmentation patterns. The dfr expression profiles vary at the levels of floral organ, flower type and region within corolla during inflorescence development according to the anthocyanin pigmentation of the cultivars. In contrast, chs expression, although regulated in a tissue-specific manner during inflorescence development, varies only occasionally. The variation in the dfr expression profiles between the varieties reveals spatially specific gene regulation that senses the differentiation events characteristic of the composite inflorescence.     

Chalcone synthase-like genes active during corolla development are differentially expressed and encode enzymes with different catalytic properties in Gerbera hybrida (Asteraceae)

Recent studies on chalcone synthase (CHS) and the related stilbene synthase (STS) suggest that the structure of chs-like genes in plants has evolved into different forms, whose members have both different regulation and capacity to code for different but related enzymatic activities. We have studied the diversity of chs-like genes by analysing the structure, expression patterns and catalytic properties of the corresponding enzymes of three genes that are active during corolla development in Gerbera hybrida. The expression patterns demonstrate that chs-like genes are representatives of three distinct genetic programmes that are active during organ differentiation in gerbera. Gchs1 and gchs3 code for typical CHS enzymes, and their gene expression pattern temporally correlates with flavonol (gchs1, gchs3) and anthocyanin (gchs1) synthesis during corolla development. Gchs2 is different. The expression pattern does not correlate with the pigmentation pattern, the amino acid sequence deviates considerably from the consensus of typical CHSs, and the catalytic properties are different. The data indicate that it represents a new member in the large superfamily of chs and chs-related genes.     

查尔酮合成酶基因

查尔酮合成酶基因是苯丙氨酸代谢途径中的关键基因,在类黄酮类物质合成中扮演着重要的角色,调控着色素合成、防御反应、植物育性等生理生化过程,对植物的生长发育起着至关重要的作用。现对查尔酮合成酶在苯丙氨酸代谢途径中的地位、基因表达特性、基因功能以及基因进化等方面的进展做一介绍。     

Genetic transformation of green bean callus via Agrobacterium mediated DNA transfer

Summary Kanamycin resistant callus was produced from leaf disc or hypocotyl expiants of green bean (Phaseolus vulgaris L.) when cultured on a defined medium containing 50 mg/l kanamycin after 4 days of co-cultivation with Agrobacterium tumefaciens strain EHA101 containing the binary vector pKYLX71GUS. The presence of neomycin phosphotransferase II (NPT-II) in crude cellular extracts from the kanamycin resistant callus was confirmed by ELISA. The expression of the ß-glucuronidase (GUS) reporter gene was confirmed by histochemical and fluorimetric analyses. Southern blot border analysis confirmed the integration of the foreign DNA. In addition to the evidence obtained from Southern analysis, the absence of Agrobacterium in the transformed callus cultures was confirmed by microscopic observation and through test cultures. Using the above protocol, bean callus cultures were also transformed with a bean chalcone synthase promoter-GUS fusion. These cultures, when treated with the elicitor glutathione, showed higher levels of GUS expression than the unelicited callus clumps.     

Chalcone synthase and flavonoid products in primary-leaf tissues of rye and maize

During cell and tissue differentiation of developing rye (Secale cereale L.) and maize (Zea mays L.) primary leaves, various flavonoids are synthesized and accumulate in both epidermal and mesophyll tissues. In order to prove either the biosynthetic autonomy of each tissue type and- or intercellular transport of flavonoids, the tissue distributions of chalcone synthase (CHS; EC 2.3.1.74), the key enzyme of the pathway, and of flavonoids have been comparatively investigated. Monoclonal antibodies raised against CHS from rye were used to relate enzyme activity in a particular tissue extract to the corresponding amount of CHS protein. A close correlation was found between CHS activities and amounts of CHS protein during leaf development and in the various tissues. The simultaneous occurrence of CHS in both epidermal layers as well as in the mesophyll correlated with the accumulation of flavonoid products in these tissues, indicating tissue autonomy of flavonoid biosynthesis. These data are in contrast to previous reports (Knogge and Weissenböck, 1986, Planta 167, 196–205) on primary leaves of oat (Avena sativa) where CHS and several subsequent enzymes were located mainly in the mesophyll whereas major flavonoid products accumulated predominantly in both epidermal cell layers, indicating that intertissue transport of flavonoids might occur.     

Regulation of phenylalanine ammonia-lyase genes in carrot suspension cultured cells

Induction of anthocyanin synthesis occurs during metabolic differentiation in carrot suspension cultured cells grown in medium lacking 2,4-dichlorophenoxyacetic acid (2,4-D), and is closely correlated with embryogenesis. Anthocyanin synthesis may also be induced by light-irradiation under different culture conditions. The phenylalanine ammonia-lyase (PAL) gene (TRN-PAL), which was transiently induced by the transfer effect, was also rapidly induced after light-irradiation. However, TRN-PAL was not involved in anthocyanin synthesis. A second PAL gene, ANT-PAL, was involved in anthocyanin synthesis. ANT-PAL was induced during metabolic differentiation in medium lacking 2,4-D parallel with the induction of chalcone synthase (CHS). PAL genes in the carrot genome are expressed differentially depending on the nature of the environmental stimulus, e.g. transfer effect and light, and other parameters which also affect anthocyanin synthesis.Abbreviations CHS chalcone synthase - 2,4-D 2,4-dichlorophenoxyacetic acid - GUS -glucuronidase - Luc firefly luciferase - PAL phenylalanine ammonia-lyase - UV ultraviolet     

Genetic control of chalcone isomerase activity in flowers of Dianthus caryophyllus

In flowers of Dianthus caryophyllus (carnation), the gene I is concerned with a discrete step in flavonoid biosynthesis, Genotypes with recessive (ii) alleles produce yellow flowers, which contain the chalcone isosalipurposide (naringenin-chalcone-2-glucoside) as the major petal pigment, but in genotypes with wild-type alleles flavonols and anthocyanins can be formed and the flowers are white or red. Enzymatic measurements on petal extracts of four strains with different flower coloration revealed a clear correlation between accumulation of chalcone in recessive genotypes and deficiency of chalcone isomerase (E.C. 5.5.1.6) activity. From the chemogenetic and enzymological evidence it can be concluded that naringenin-chalcone is the first product of the synthesis of the flavonoid skeleton and that only the conversion of naringenin-chalcone to naringenin furnishes the substrate for the further reactions to flavonol and anthocyanin.These investigations were supported by a grant from Deutsche Forschungsgemeinschaft.     

Chalcone synthases from spinach (Spinacia oleracea L.)

The two chalcone-synthase forms from leaves ofSpinacia oleracea L. were purified to apparent homogeneity. Antibodies were raised against both proteins in rabbits. The specificity of the antibodies was tested using immunotitration, immunoblotting, and immunoelectrophoresis techniques. The antibodies exhibited exclusive specificity for chalcone synthase and did not discriminate between the two antigens. The homodimeric chalcone synthases had the same subunit molecular weight but differed in their apparent native molecular weights. The peptide maps indicated extensive homology between the proteins. Chalcone-synthase activity was not detected in isolated spinach chloroplasts. Both enzyme forms were present in spinach cell-suspension cultures in which they were induced by light.Abbreviations DEAE diethylaminoethyl - DTE 1,4-dithioerythritol - EDTA ethylenediaminetetraacetic acid - HPLC high-performance liquid chromatography - IgG immunoglobulin G - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis Parts of the results were presented at the 14^th International Botanical Congress at Berlin in July 1987