Organization of soybean chalcone synthase gene clusters and characterization of a new member of the family

Chalcone synthase (CHS; EC 2.3.1.74), the first committed enzyme of the multibranched pathway of flavonoid/isoflavonoid biosynthesis is encoded by a multigene family in soybean, (Glycine max L. Merrill). Our results suggest that this gene family comprises at least seven members, some of which are clustered. We have identified four chs clusters in the allo-tetraploid G. max genome and chs5, a newly characterized member of the chs gene family is present in two of them. We describe the complete nucleotide sequence of chs5, the identification of its immediate neighbors and the organization of the four hitherto identified chs clusters in the Gm genome.     

Co-suppression in transgenicPetunia hybrida expressing chalcone synthase A (chsA)

Chalcone synthase A is a key enzyme in the anthocyanin biosynthesis pathway. Expression ofchsA gene in transgenicPetunia hybrida resulted in flower color alterations and co-suppression of transgenes and endogenous genes. We fused the β-glucuronidase (uidA) gene to the C-terminal ofchsA gene, and transferred the fusion gene intoPetunia hybrida viaAgrobacterium tumefaciens. GUS histochemical staining analysis showed that co-suppression occurred specifically during the development of flowers and co-suppression required the mutual interaction of endogenous genes and transgenes. RNAin situ hybridization analysis suggested that co-suppression occurred in the entire plant, and RNA degradation occurred in the cytoplasm.     

Transparent Testa Glabra 1 (TTG1) and TTG1-like genes in Matthiola incana R. Br. and related Brassicaceae and mutation in the WD-40 motif

TTG1 (Transparent Testa Glabra 1), a WD-40 repeat protein, is involved in regulation of flavonoid/anthocyanin biosynthesis, seed coat (mucilage) development/pigmentation and trichome formation in leaves. Here, we characterized the TTG1 gene of Matthiola incana wild type ( e locus), showing 85.3% similarity to TTG1 of A. thaliana on the nucleotide level and 96.2% on the protein level. A white-flowered and glabrous mutant, line 17, of M. incana exhibits one nucleotide change, leading to an amino acid substitution directly in the WD motif (W158R). Correspondingly, the DFR (dihydroflavonol 4-reductase) gene, in which the expression is known to be dependent on TTG1, is not expressed in Matthiola mutant lines 17 (and 19). Comparison of the GC content of the Matthiola TTG1 (54.1%) and Arabidopsis TTG1 (46.1%) genes revealed a strong difference, mostly obtained by neutral substitutions (C to T transitions). To examine whether this is an ecologically influenced trend, a fragment of TTG1 was characterized from another Matthiola species ( M. tricuspidata ) and from Malcolmia flexuosa subsp. naxensis from the eastern Mediterranean, near a beach with sandy and salty soils. Both Matthiola species have a higher GC content in the TTG1 gene than Arabidopsis and the closer-related Malcolmia , indicating that the GC content is rather an evolutionary than an ecological signal. A similar WD-40 repeat protein gene (containing no intron in the 3' untranslated region) with high similarity to the Arabidopsis TTG1 -like ( AtAN11 ) gene was found in Matthiola .     

Precursors and genetic control of anthocyanin synthesis in Matthiola incana R. Br.

After application of dihydroflavonols, naringenin, or suitable substituted chalcones, anthocyanins were synthesized in three genetically defined acyanic lines of Matthiola incana, indicating that the corresponding genetic block concerns the synthesis of the chalcone-flavanone intermediate. Independent of the precursors used, only cyanidin derivatives were produced. This supports the hypothesis that the oxygenation pattern of the B ring in anthocyanin formation is determined at a stage of a C₁₅ intermediate. In addition to the gene responsible for the oxygenation of the 3 position, the genes responsible for the glycosylation in the 3 and 5 positions of the anthocyanin molecule, and those responsible for the acylation with various hydroxycinnamic acids can still exert their influence. Two further genetically defined lines containing flavonol glycosides were not able to synthesize anthocyanins with any of the precursors tested. Their genetic blocks are assumed to be localized after dihydroflavonol synthesis but before anthocyanin formation.     

Characterization of a light-dependent glutamate synthase activity in Chlamydomonas reinhardtii

Photosynthetically active vesicles prepared from Chlamydomonas reinhardtii retained a light-dependent glutamate synthase activity which was highly specific for 2-oxoglutarate (K_m=2.1 mM) and L-glutamine (K_m=0.9 mM) as amido group acceptor and donor respectively. This activity was inhibited by azaserine, p-hydroxymercuribenzoate and 3-(p-chlorophenyl)-1,1-dimethyl urea.Light-dependent synthesis of glutamate was also obtained by coupling Chlamydomonas photosynthetic particles to purified ferredoxin-glutamate synthase, using ascorbate and 2,6-dichlorophenol-indophenol as electron donor. This system was also specific for 2-oxoglutarate (K_m=1 mM) and L-glutamine (K_m=0.8 mM) as substrates, and was stimulated by dithioerythritol. Azaserine and p-hydroxymercuribenzoate, but not 3-(p-chlorophenyl)-1,1-dimethyl urea, inhibited the reconstituted activity; high concentrations of 2-oxoglutarate were inhibitory.Abbreviations A Absorbance - CCP p-Trichlorometoxi-carbonylcyanide-phenylhydrazone - Chl Chlorophyll - CMU 3-(p-Chlorophenyl)-1,1-dimethyl urea - DPIP 2,6-Dichlorophenol-indophenol - DTE Dithioerythritol - MSX L-Methionine, D-L, sulfoximine - MV Methyl viologen     

Population genetics of Matthiola bolleana (Brassicaceae) in the Canary Islands

Genetic variation at 11 isozyme loci was used to explore the levels and apportionment of genetic variation in the eight known populations of Matthiola bolleana, an endemic Brassicaceae to the Eastern Canarian islands of Fuerteventura and Lanzarote. Within islands, ecosystem antiquity and uniformity, lack of evidence for selection or genetic bottlenecks, the high genetic identities between the populations, and the low values of F _ST converge to suggest that the high levels of variation detected have been maintained in a context of prolonged environmental stability through an overall predominance of outbreeding and unrestricted gene flow. Despite the geographical closeness between Fuerteventura and Lanzarote and the long range dispersal features of the propagules of M. bolleana, we detected a striking qualitative genetic differentiation between the two islands. We suggest that wind direction has made seed and pollen flow between Fuerteventura and Lanzarote much less likely than expected, and that the lack of inter-island dispersal has far overriden the influence of stochastic forces and of the reproductive attributes of M. bolleana in shaping the patterns of inter-island genetic differentiation. However, genetic similarity in M. bolleana is within the ranges defined by Canarian taxa for which the extent of genetic differentiation has also been assessed using the values of isozyme genetic identity.     

Immunofluorescence localization of chalcone synthase in roots ofPisum sativum L. andPhaseolus vulgaris L. and comparable immunochemical analysis of chalcone synthase from pea leaves

Summary Chalcone synthase in roots ofPisum sativum andPhaseolus vulgaris was demonstrated enzymatically and immunochemically. In situ localization by indirect immunofluorescence revealed that chalcone synthase is chiefly present in the lateral regions of the calyptra, in the rhizodermis, and the cortex. In the central cylinder the enzyme protein is no longer detectable a short distance behind the meristem. Chalcone synthase was not found in root tips ofZea mays. Two isoforms of chalcone synthase were separated by chromatofocusing of protein extracts from pea leaves. The two forms differed in their subunit molecular masses. The smaller isoform was not detected in roots.Abbreviations CHS chalcone synthase - FITC fluorescein isothiocyanate - IgG immunoglobulin G - DTE dithioerythritol     

Isolation and sequence analysis of a chalcone synthase cDNA ofMatthiola incana R. Br. (Brassicaceae)

A cDNA clone (pcM12) of the chalcone synthase (CHS) ofMatthiola incana R. Br. (Brassicacease) was isolated from a cDNA library, sequenced and analysed. It comprises the complete coding sequence for the CHS and 5 and 3 untranslated regions. The deduced amino acid sequence shows that theMatthiola incana CHS consists of 394 amino acid residues. Comparison with CHS amino acid sequences of other plants indicates more than 82% homology.     

Molecular and biochemical characterization of benzalacetone synthase and chalcone synthase genes and their proteins from raspberry (Rubus idaeus L.)

Two new members of the polyketide synthase (PKS) gene family (RiPKS4 and RiPKS5) were cloned from raspberry fruits (Rubus idaeus L., cv Royalty) and expressed in Escherichia coli. Characterization of the recombinant enzyme products indicated that RiPKS4 is a bifunctional polyketide synthase producing both 4-hydroxybenzalacetone and naringenin chalcone. The recombinant RiPKS4 protein, like the native protein from raspberry fruits [W. Borejsza-Wysocki, G. Hrazdina, Plant Physiol. 1996;110: 791-799] accepted p-coumaryl-CoA and ferulyl-CoA as starter substrates and catalyzed the formation of both naringenin chalcone, 4-hydroxy-benzalacetone and 3-methoxy-4-hydroxy-benzalacetone. Although activity of RiPKS4 was higher with ferulyl-CoA than with p-coumaryl-CoA, the corresponding product, 3-methoxy-4-hydroxy phenylbutanone could not be detected in raspberries to date. Sequence analysis of the genes and proteins suggested that this feature of RiPKS4 was created by variation in the C-terminus due to DNA recombination at the 3′ region of its coding sequence. RiPKS5 is a typical chalcone synthase (CHS) that uses p-coumaryl-CoA only as starter substrate and produces naringenin chalcone exclusively as the reaction product.     

Cross-reaction of chalcone synthase and stilbene synthase overexpressed in Escherichia coli

Chalcone synthase (CHS) and stilbene synthase (STS) are related plant polyketide synthases belonging to the CHS superfamily. CHS and STS catalyze common condensation reactions of p-coumaroyl-CoA and three C₂-units from malonyl-CoA but different cyclization reactions to produce naringenin chalcone and resveratrol, respectively. Using purified Pueraria lobata CHS and Arachis hypogaea STS overexpressed in Escherichia coli, bisnoryangonin (BNY, the derailed lactone after two condensations) and p-coumaroyltriacetic acid lactone (the derailed lactone after three condensations) were detected from the reaction products. More importantly, we found a cross-reaction between CHS and STS, i.e. resveratrol production by CHS (2.7–4.2% of naringenin) and naringenin production by STS (1.4–2.3% of resveratrol), possibly due to the conformational flexibility of their active sites.     

Characterization of a thermostable dihydrodipicolinate synthase from Thermoanaerobacter tengcongensis

Dihydrodipicolinate synthase (DHDPS) catalyses the first reaction of the (S)-lysine biosynthesis pathway in bacteria and plants. The hypothetical gene for dihydrodipicolinate synthase (dapA) of Thermoanaerobacter tengcongensis was found in a cluster containing several genes of the diaminopimelate lysine–synthesis pathway. The dapA gene was cloned in Escherichia coli, DHDPS was subsequently produced and purified to homogeneity. The T. tengcongensis DHDPS was found to be thermostable (T _0.5 = 3 h at 90°C). The specific condensation of pyruvate and (S)-aspartate-β -semialdehyde was catalyzed optimally at 80°C at pH 8.0. Enzyme kinetics were determined at 60°C, as close as possible to in vivo conditions. The established kinetic parameters were in the same range as for example E. coli dihydrodipicolinate synthase. The specific activity of the T. tengcongensis DHDPS was relatively high even at 30°C. Like most dihydrodipicolinate synthases known at present, the DHDPS of T. tengcongensis seems to be a tetramer. A structural model reveals that the active site is well conserved. The binding site of the allosteric inhibitor lysine appears not to be conserved, which agrees with the fact that the DHDPS of T. tengcongensis is not inhibited by lysine under physiological conditions.     

Immunochemical localization of phenylalanine ammonia-lyase and chalcone synthase in anthers

Phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS) from anthers of the garden tulip Apeldoorn have been purified to apparent homogeneity as revealed by sodium dodecyl sulfate disc-gel electrophoresis. Phenylalanine ammonia-lyase was either purified by successive chromatography on Sephacryl S 300 Superfine, HA Ultrogel and on diethylaminoethyl Sephacel or by immunoaffinity chromatography in a single step. Purification of CHS was achieved by chromatography on Sephadex G 200 and on HA Ultrogel followed by chromatofocusing. The purified enzymes were used for the immunization of rabbits. The specificity of the antisera against both PAL and CHS was tested by diverse methods. Antisera against PAL and CHS were employed to detect the localization of the enzymes in cross sections of tulip anthers using an indirect immunofluorometric method. The results show that PAL and CHS are located predominantly in the tapetum cells. These observations strengthen the view that the tapetum plays an important role in the regulation of phenylpropanoid metabolism within the loculus of anthers.Abbreviations CHS chalcone synthase - PAL phenylalanine ammonia-lyase - SDS sodium dodecyl sulfate Some of the results were presented at the meeting of German Botanical Society in Freiburg, FRG, September 1982, and at the meeting of the Groupe Polyphenols in Toulouse, France, September/October 1982     

Induction of chalcone synthase in cell suspension cultures of carrot (Daucus carota L. spp. sativus) by ultraviolet light: evidence for two different forms of chalcone synthase

Two cell lines of carrot (Daucus carota L. spp. sativus), grown as cell-suspension cultures in the dark, were irradiated with ultraviolet light (315–420 nm) 10 d after the onset of cultivation. Chalcone synthase (CHS) enzyme activity was induced in both cell lines. Anthocyanin synthesis was only stimulated in the anthocyanin-containing cell line DCb. Parallel to the increase in CHS activity there was an increase with time in the amount of one CHS form with an isoelectric point of 6.5 and a molecular weight of 40 kilodaltons (kDa) per subunit. Whereas the anthocyanin-free cell line DCs failed to accumulate anthocyanin, it did stimulate another CHS form with an isoelectric point at pH 5.5 and a molecular weight of 43 kDa per subunit. Both enzyme activities could be separated by isoelectric focusing and stabilized using sodium hydrosulfite as an oxidation protectant. In carrot plants, CHS was restricted to the dark purple petals of the inflorescence (40 kDa) and to the leaves (43 kDa).Abbreviations BSA bovine serum albumin - CHS chalcone synthase - IEF isoelectric focusing - kDa kilodaltons - KP_i potassium phosphate buffer - PAL phenylalanine ammonialyase - pI isoelectric point - UV ultraviolet     

Genetic differentiation of three species of Matthiola (Brassicaceae) in the Sicilian insular system

We examined the genetic variation of 12 isozyme loci in 14 populations of Matthiola (Brassicaceae) representing the geographic distribution of the species M. incana, M. fruticulosa ssp. fruticulosa and M. tricuspidata in the Sicilian insular system and the adjacent mainland areas to estimate the levels and apportionment of genetic variation in the insular populations and to understand their population dynamics. The disparity in the distribution of polymorphism in populations of M. incana ssp. incana (low within populations but with high values of F_ST and G_ST) contrasts with the homogeneity in the inter-population distribution of the high genetic variation detected in M. tricuspidata and M. fruticulosa ssp. fruticulosa. While the low polymorphism found in M. incana ssp. incana is consistent with its origin through cultivation and the associated lack of gene flow, the Sicilian populations of the other two taxa probably derived from multiple founder events from nearby continental areas and, according to our estimates, have maintained high interpopulational gene flow. Unlike M. incana, the Sicilian populations of M. tricuspidata and M. fruticulosa ssp. fruticulosa could have survived the glaciations in refugia. This higher antiquity, together with the maintenance of abundant gene flow, largely explains their high values of genetic variation. In contrast, M. incana ssp. pulchella and M. incana ssp. rupestris have low indices of polymorphism and they are probably neo-endemics, as their distribution areas were severely affected by the Plio-Pleistocene glaciations.     

Analysis ofcis-regulatory elements involved in the activation of a member of chalcone synthase gene family (PsChs1) in pea

Cis-regulatory elements involved in the activation of the plant defense-related gene encoding chalcone synthase 1 (PsChs1) in pea (Pisum sativum L.) were examined by transient transfection, gel mobility shift assay andin vitro DNase I-footprinting analysis. Transient transfection assay revealed that a 61 bp DNA fragment spanning from –242 to –182 ofPsChs1 was required for the maximal promoter activity and possibly involved in the enhancement of elicitor-mediated activation. Nuclear isolate from elicitor-treated pea epicotyl tissues contained some factor(s) that specifically bound to this DNA fragment to form a complex with low mobility (LMC, low mobility complex) in gel mobility shift assay. DNase I-footprinting analysis of LMC revealed that among three protected regions detected in a 61 bp DNA fragment, two regions contained identical AT-rich sequence, TAAAATACT. Site directed mutation in either or both identical sequences, TAAAATACT to TGGAATACT, resulted in the reduction or loss in the ability to form LMC. Detailed analysis of 61 bp DNA fragment demonstrated that the region from –242 to –226 containing promoter-distal TAAAATACT motif was imperative for the maximal elicitor-mediated activation ofPsChs1.