De novo activation of the transposable element Tam2 of Antirrhinum majus

Summary The nivea locus of Antirrhinum majus encodes the enzyme chalcone synthase required for the synthesis of red anthocyanin pigment. The stable allele niv-44 contains an insertion in the nivea gene (Tam2) which has all the structural features of a transposable element. We have shown that this insertion can excise from the nivea locus when niv-44 is combined with another allele (niv-99) in a heterozygote. Activation of Tam2 excision is caused by a factor tightly linked to the niv-99 allele and may be due to complementation between Tam2 and a related element, Tam1. Factors which repress the excision of Tam2 and Tam1 are also described. Repression is not inherited in a simple mendelian way. Many stable mutations may be due to the insertion of transposable elements. Our data suggest that their stability may be due to the absence in the genome of activating factors and to the presence of repressors.     

Interaction between the Tam1 and Tam2 transposable elements of Antirrhinum majus

Summary Two stable derivatives of the highly unstable niv-53::Tam1 allele of Antirrhinum majus were analysed. In both derivatives the Tam1 element is integrated at the same site and in the same orientation as in the parental niv-53::Tam1 allele. In both cases the Tam1 element was found to carry a 5 bp deletion (CACTA) in one of its termini. This explains the excision deficiency of these two alleles of Tam1, niv-53::Tam1-46 and niv-53::Tam1-49. Niv-44::Tam2, another stable nivea mutation, carries the 5 kb element Tam2, which is not a derivative of Tam1 but possesses identical terminal inverted repeats. When the stable lines 46 and 49 were corssed with line 44, suprisingly, a high number of the flowers in the F₁ displayed a variegated phenotype. Sequence analysis of two germinal revertants isolated from the heterozygote niv-53::Tam1-46/niv-44::Tam2 shows excision of the Tam2 element. This indicates that Tam2 is a defective element, which can be complemented by an active Tam1 element. However, the variegated F₁ phenotype observed is not inherited monofactorially. Variegation is seen only at particular times of development of the F₁ plants. These phenomena seem to involve both the Tam1 and Tam2 transposable elements.     

Comparison of genetic behaviour of the transposable element Tam3 at two unlinked pigment loci in Antirrhinum majus

Summary In Antirrhinum majus the transposable element Tam3 has been described at two unlinked loci pallida and nivea, both of which are required for the production of anthocyanin pigment in flowers. In each case the element is inserted in the promoter region and gives a variegated phenotype. We show that the rate of Tam3 excision at both loci is greatly affected by temperature, being approximately 1000-fold higher at 15°C compared with 25°C. Tam3 is also controlled by an unlinked gene Stabiliser, which considerably reduces excision rate. We show that the high degree of sensitivity to temperature and Stabiliser is an intrinsic property of Tam3 which is not shared by an unrelated element, Tam1. The Tam3 insertion at nivea gives rise to a series of alleles which confer reduced pigmentation, novel spatial patterns and changed instability. These are probably a result of imprecise excision and rearrangements of the Tam3 element.     

The homeotic Macho mutant of Antirrhinum majus reverts to wild-type or mutates to the homeotic plena phenotype

Plants of Antirrhinum majus carrying the semidominant Macho alleles of the plena gene display carpelloid sepals and staminoid petals, but the two inner flower whorls of stamens and carpels are normal and produce fertile gametes. In the recessive plena mutant, in contrast, the two outer whorls are normal whereas the stamens are largely or entirely petaloid and the carpels sepaloid, thus producing weakly male-fertile or fully sterile lines. Two new plena and two new Macho alleles have been induced in transposon tagging experiments. Genetic and molecular analysis revealed that the two contrasting mutant phenotypes are caused by mutations in one and the same gene: Several wild-type plants appeared among 27 000 F1 plants of a cross between Macho female plants and wild-type males bearing the active transposons Taml and Tam3. One of these plants segregated plena mutants, three showed reversions to wild-type and another two segregated Macho plants, possibly representing somatic reversions. Additional evidence was provided by an allelism test of Macho × plena. Molecular analysis has independently corroborated the genetical results. Moreover, the double mutant Macho/deficiens shows only carpels and plena/deficiens only sepals, which is in accord with combinatorial models for homeotic flower formation presented recently.     

Detection of point mutations in chloroplast genes of Antirrhinum majus L. I. Identification of a point mutation in the psaB gene of a photosystem I plastome mutant

A point mutation in the plastome-encoded psaB gene of the mutant en:alba-1 of Antirrhinum majus L. was identified by an analysis of chloroplast DNA with a modified PCR-SSCP technique. Application of this technique is indicated when a gene or a group of genes is known in which the point mutation is located. Analysis of primary photosynthetic reactions in the yellowish white plastome mutant indicated a dysfunction of photosystem (PS) 1. The peak wavelength of PS I-dependent chlorophyll (Chl) fluorescence emission at 77 K was shifted by 4 nm to 730 nm, as compared to fluorescence from wild-type. There were no redox transients of the reaction center Chl P₇₀₀ upon illumination of leaves with continuous far-red light or with rate-saturating flashes of white light. The PS I reaction center proteins PsaA and PsaB are not detectable by SDS-PAGE in mutant plastids. Hence, plastome encoded PS I genes were regarded as putative sites of mutation. In order to identify plastome mutations we developed a modified SSCP (single-strand conformation polymorphism) procedure using a large PCR fragment which can be cleaved with various restriction enzymes. When DNA from wild-type and en:alba-1 was submitted to SSCP analysis, a single stranded Hinf I fragment of a PCR product of the psaB gene showed differences in electrophoretic mobility. Sequence analysis revealed that the observed SSCP was caused by a single base substitution at codon 136 (TAT TAG) of the psaB gene. The point mutation produces a new stop codon that leads to a truncated PsaB protein. The results presented indicate that the mutation prevents the assembly of a functional PS I complex. The applicability to other plastome mutants of the new method for detection of point mutations is discussed.     

Molecular cloning of the c2 locus of Zea mays, the gene coding for chalcone synthase

Summary The c2 locus of Zea mays, identified as one of the genes affecting anthocyanin biosynthesis, was cloned using the transposable element En (Spm) as a gene tag. The Spm element present at the c2 locus in the autonomously mutating c2-m1 line was isolated using En1 element specific probes. Sequences flanking the element were identified as c2 locus specific and were used to clone the nonautonomous c2-m2 and wild-type alleles. The cloning and analysis of a cDNA complementary to the c2 locus provided evidence that this gene encodes the enzyme chalcone synthase.     

Robust reporter system based on chalcone synthase rppA gene from Saccharopolyspora erythraea

Industrial overproducing strains present unique hosts for expression of heterologous gene clusters encoding secondary metabolite biosynthesis. For this purpose, efficient gene expression tools and methods are needed. A robust and versatile reporter system based on the rppA gene from Saccharopolyspora erythraea is presented as the method of choice when studying gene expression in actinomycete hosts. The method is easily scalable to accommodate high-throughput procedure, and collected samples can be easily stored and re-tested when needed. The product of RppA is an inert 1,3,6,8-tetrahydroxynaphthalene which spontaneously oxidises to a dark-red quinone flaviolin providing a qualitative visual assessment of gene expression on an agar plate as well as a quantitative spectrophotometric measurement in liquid broth without the need for invasive procedures or external substrate addition. The applicability of the reporter system has been demonstrated by expressing the rppA gene under the control of the heterologous promoters actII-ORF4/P_actI, ermE and its upregulated variant ermE*. The model streptomycete Streptomyces coelicolor, and three industrially important species, Streptomyces tsukubaensis (FK506), Streptomyces cinnamonensis (monensin) and Streptomyces rimosus (oxytetracycline) were used as hosts. The reporter system has shown its utility independently of cultivation conditions or composition of growth medium, from simple laboratory to complex industrial media. The simplicity and robustness of the system, demonstrated even in industrial settings, shows great potential for wider use in different microbial hosts and applications, and may thus represent a new generic and versatile tool useful to a wider scientific community.     

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.     

金鱼草中央细胞中线粒体DNA拷贝数的定量

为探究金鱼草(Antirrhinum majus)中央细胞的线粒体DNA拷贝数,采用竞争型定量PCR技术进行了测定。结果表明,金鱼草中央细胞的体积为(61570±732)μm~3,平均携带(783±25)个拷贝的线粒体DNA,并且中央细胞与卵细胞具有相似的体积/线粒体DNA拷贝数比值。推测金鱼草中央细胞包含如此高丰度线粒体DNA可能是为早期胚乳细胞的发育而储备的。     

A chalcone synthase/stilbene synthase DNA probe for conifers

A probe for chalcone synthase (CHS) was generated by PCR using chalcone synthase conserved sequences. The cloned PCR product has high similarity to both chalcone synthase and stilbene synthase sequences. The probe was used to examine the organization of chalcone synthase and stilbene synthase genes in Abies procera, Pinus lambertiana, P. monticola, Picea glauca, P. sitchensis, Pseudostuga menziesii, Taxus brevifolia, and Thuja plicata. A large number of hybridizing bands were found in all species except T. plicata which did not cross hybridize. The hybridization patterns are highly polymorphic between the species and are also polymorphic within several of them.     

Expression of the chalcone synthase gene from grape and preparation of an anti-CHS antibody

Flavonoids are closely related to a plant's antioxidative ability. Because chalcone synthase (CHS) is the first enzyme to act as part of the flavonoid biosynthesis pathway, its expression and regulation are important. Here we present the expression of a full-length chs cDNA with 1225bp from grape seedlings as well as the preparation of an antibody against the expressed protein. A full-length chs cDNA was introduced into an expressed plasmid pET-30a(+) vector at the EcoRI and SalI restriction sites. pET-chs was found to be highly expressed in Escherichia coli BL21(DE3) pLysS cells with isopropyl-beta-d-thiogalactoside (IPTG) induction. A fusion protein with the His.tag label was purified by Ni-NTA His.Bind Resin and then used as the antigen to immunize a New Zealand rabbit. The resulting antiserum was then further precipitated by 50% saturated ammonium sulfate and DEAE-Sepharose FF column chromatography to obtain the immunoglobulin G (IgG) fraction. The resulting antibody was found capable of immuno-recognizing the CHS of the crude protein extracts from different grape tissues with a molecular mass of 43kDa.     

Molecular evolution and functional specialization of chalcone synthase superfamily from Phalaenopsis Orchid

Plant genomes appear to exploit the process of gene duplication as a primary means of acquiring biochemical and developmental flexibility. The best example is the gene encoding chalcone synthase (CHS, EC2.3.1.74), the first committed step in flavonoid biosynthesis. In this study, we examined the molecular evolution of three CHS family members of Phalaenopsis including a novel chs gene (phchs5), which is slowly evolved. The inferred phylogeny of the chs genes of Phalaenopsis with other two orchid plants, Bromoheadia finlaysoniana and Dendrobium hybrid, suggested that gene duplication and divergence have occurred before divergence of these three genera. Relatively quantitative RT-PCR analysis identified expression patterns of these three chs genes in different floral tissues at different developmental stages. Phchs5 was the most abundantly expressed chs gene in floral organs and it was specifically transcribed in petal and lip at the stages when anthocyanin accumulated (stage1–4). Phchs3 and phchs4 were expressed at much lower levels than phchs5. Phchs3 was expressed in pigmented tissue (including lip, petal and sepal) at middle stages (stages 2–4) and in colorless reproductive tissue at late stage (stage 5). Phchs4 was only expressed in petal at earlier stages (stage 1–3) and in lip at middle stage (stage 4). These results present new data on differentiation of gene expression among duplicate copies of chs genes in Phalaenopsis.     

High intensity and blue light regulated expression of chimeric chalcone synthase genes in transgenic Arabidopsis thaliana plants

Summary To establish a genetic system for dissection of light-mediated signal transduction in plants, we analyzed the light wavelengths and promoter sequences responsible for the light-induced expression of the Arabidopsis thaliana chalcone synthase (CHS) promoter fused to the -glucuronidase (GUS) marker gene. Transgenic A. thaliana lines carrying 1975, 523, 186, and 17 by of the CHS promoter fused to the GUS gene were generated, and the expression of these chimeric genes was monitored in response to high intensity light in mature plants and to different wavelengths of light in seedlings. Fusion constructs containing 1975 and 523 by of CHS promoter sequence behaved identically to the endogenous CHS gene under all conditions. Expression of these constructs was induced specifically in response to high intensity white light and blue light. The response to blue light was seen in the presence of the P_fr form of phytochrome. Fusion constructs containing 186 by of promoter sequence showed reduced basal levels of expression and only weak stimulation by blue light but were induced significantly by high intensity white light. These analyses showed that the expression of the A. thaliana CHS gene is responsive to a specific blue light receptor and that sequences between — 523 and — 186 by are required for optimal basal and blue light-induced expression of this gene. The experiments lay the foundation for a simple genetic screen for light response mutants.     

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     

Molecular characterization and genetic origin of the Brassica napus acetohydroxyacid synthase multigene family

Summary The Brassica napus rapeseed cultivar Topas contains an acetohydroxyacid synthase (AHAS) multigene family consisting of five members (AHAS 1–5). DNA sequence analysis indicate that AHAS1 and AHAS3 share extensive homology. They probably encode the AHAS enzymes essential for plant growth and development. AHAS2 has diverged significantly from AHAS1 and AHAS3 and has unique features in the coding region of the mature polypeptide, transit peptide and upstream non-coding DNA, which raises the possibility that it has a distinct function. AHAS4 and AHAS5 have interrupted coding regions and may be defective. The complexity of the AHAS multigene family in the allotetraploid species B. napus is much greater than reported for Arabidopsis thaliana and Nicotiana tabacum. Analysis of the presumptive progenitor diploid species B. campestris and B. oleracea indicated that AHAS2, AHAS3 and AHAS4 originate from the A genome, whereas AHAS1 and AHAS5 originate from the C genome. Further variation within each of the AHAS genes in these species was found.     

In situ localization of chalcone synthase inLarix needles by indirect immunofluorescence

Summary Chalcone synthase (CHS), the key enzyme of flavonoid biosynthesis, is localized by indirect immunofluorescence in needles ofLarix decidua. In young stages of needle development CHS is present in epidermal cells and individual cells in the region of the vascular bundles which possibly contain tannins. A later developmental stage exhibits immunofluorescence predominantly in the mesophyll of the needle. The epidermis and the cells in the vicinity of the vascular bundles show a considerably weaker and only sporadically detectable fluorescence. CHS is no longer observable at the latest analyzed stages of needle development.     

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