J. van Brederode

A method for evolutionary studies on RFLP of chloroplast DNA, applicable to a range of plant species

Abstract A fast and low cost method for the isolation of chloroplast DNA (cpDNA), suitable for RFLP analysis, applicable to evolutionary studies in a variety of plant species, is introduced. The method is based on the isolation of chloroplasts in the presence of high salt concentrations and cpDNA isolation using cetyl triammonium bromide (CTAB). The cpDNA preparations obtained can be analysed with restriction endonucleases and are satisfactorily free of nuclear and mitochrondrial DNA contamination. The yield of cpDNA is of the same magnitude as has been described before for other methods

The genetic control of isovitexin-glycosylation in the petals of Melandrium album

SummaryThe glycosylation of flavones in the petals of Melandrium album is shown to be controlled by the genes G, X and A. In the presence of the recessive alleles of these genes, only the aglycone isovitexin (6-C-glucosylapigenin) is found in the petals. The gene G controls the transfer of glucose, the gene X the transfer of xylose to the 7-hydroxyl group of isovitexin. The gene G is epistatic over X. In the presence of the gene A arabinose is coupled to the carbon-carbon bound glucose of isovitexin. In the presence of both G and A, or both X and A the corresponding di-glycosides are formed.The petals of the plants in which all genes for the flavone glycosylation are present in the homozygous recessive form are of a particular phenotype.

Can the spread of agriculture in Europe be followed by tracing the spread of the weed Silene latifolia. A RAPD study

On the basis of gene frequency data of three flavone glycosylating genes, populations of the agricultural weed Silene latifolia (Caryophyllaceae) in Europe can be divided into two chemical races: an eastern and a western race. Morphological data also show a clear east-west division. When the two datasets are combined at least nine different geographical races can be distinguished using cluster analysis. Because these observations are hard to explain by selection, it has been proposed that these different races probably originated as a consequence of migration during the spread of agriculture over Europe in the past. To discriminate between selection and genetic drift many more selectively neutral easy-to-score characters are needed. In order to test whether random amplified polymorphic DNAs (RAPDs) might be suitable for this purpose, we performed a small-scale RAPD analysis on 16 geographical different populations. Using Jaccards coefficient of similarity, we calculated genetic distances by pair-wise comparisons of both unique and shared amplification products, and a dendrogram was subsequently constructed using an unweighted pair-group method with arithmetical averages (UPGMA). On the basis of the dendrogram two clusters were discerned that clearly coincide with the aforementioned east-west division in populations. As there has been little or no artificial selection on this weed, its migration routes may be a good reflection of the different geographical routes agriculture has taken. We propose that a phylogenetic analysis of RAPD data of many more populations may provide additional information on the spread of agriculture over Europe.

Genetic control and biosynthesis of two new flavone-glycosides in the petals of Melandrium album

In a chemicogenetic analysis of the geographical distribution of flavone-glycosides in the petals of Melandrium album, we found two unknown flavone-glycosides in ten Hungarian and four German populations. By means of classical techniques for the identification and structure determination of flavonoids, the structure of these flavones turned out to be 6-C-glucosylglucosylapigenin and 7-O-glucosyl-6-C-glucosylglucosylapigenin, respectively. Genetic analysis showed that the coupling of glucose to the carbon-carbon bound glucose of isovitexin (6-C-glucosylapigenin) was controlled by a single dominant gene, Fg. Fg controls a UDP-glucose: isovitexin 6-C-glucosylglucosyltransferase. By means of ammonium sulfate fractionation and Sephadex chromatography, the enzyme was purified sixfold. The partly purified enzyme had a pH optimum between 8.0 and 8.5. The apparent Km value for UDP-glucose in the presence of 1.0 mm isovitexin was 2.2 mm. The apparent Km value for isovitexin in the presence of 1.8 mm UDP-glucose was 0.08 mm. The glucosyltransferase activity was stimulated by the divalent cations Mn, Mg, Co, and Ca. Neither 7-O-glucosyl nor 7-O-xylosyl isovitexin could serve as an enzyme substrate. Therefore, the biosynthesis of 7-O-glucosyl-6-C-glucosylglucosylapigenin found in the petals of M. album proceeds in a sequential manner: first the formation of 6-C-glucosylglucosylapigenin, followed by 7-O-glucosylation. Isovitexin 6-C-glucosylglucosyltransferase activity controlled by gene Fg could also be demonstrated in leaves of Fg plants. The enzyme probably uses another substrate in these green parts of the plant, because both isovitexin and isovitexin-glycosides are absent.

Geographic trends in flavone-glycosylation genes and seed morphology in EuropeanSilene pratensis (Caryophyllaceae)

Three of the loci controlling isovitexin glycosylation inSilene pratensis are polymorphic and show geographic trends which are compared with geographic trends in seed morphology (and other phenotypic characters) as demonstrated by multivariate analysis. Various lines of evidence support the hypothesis thatS. pratensis spread into Europe from at least two genetically differentiated sources.S. dioica, by contrast, shows little interpretable geographic variation in morphology or flavonoid content.

Identification, properties and genetic control of UDP-glucose: Isovitexin 7-O-glucosyltransferase isolated from petals of Melandrium album

SummaryIn extracts of petals of M. album, an enzyme has been demonstrated which catalyzes the transfer of the glucosyl moiety of UDP-glucose to the 7-hydroxylgroup of isovitexin.This enzyme is controlled by a dominant gene G; in plants with the recessive genotype no glucosyltransferase activity could be detected.The enzyme was purified 16-fold by (NH4)2SO4 fractionation and Sephadex-chromatography.The glucosyltransferase had a pH optimum of pH 7.5, was not stimulated by divalent metal ions, and had a “true Km” value of 1.2x10-4 M for UDP-glucose and a “true Km” value of 4.6x10-4 M for isovitexin.Several flavones with an apigenin hydroxylation pattern could serve as glucosyl acceptors. The highest activity was found, however, with isovitexin.The enzyme was unable to catalyze the transfer of xylose from UDP-xylose to the 7-hydroxylgroup of isovitexin, although isovitexin 7-O-xyloside has been found in petals of M. album plants.ADP-glucose could not serve as a glucosyl donor.The transferase activity was also present in leaves and calyces of GG plants. In these organs the transferase uses another flavone as a substrate. Neither isovitexin 7-O-glucoside nor isovitexin could be detected in these organs.

The possible evolution of Silene pratensis as deduced from present day variation patterns

Abstract Variation within and between European populations of Silene pratensis has been determined at different levels: morphological, biochemical and genetic. The various data sets were analysed separately and comparison of the patterns led to a number of conclusions concerning the evolution of the species. Coinciding patterns were found for flavone glycosylating genes, seed, pollen and capsule morphology. Together with observations on habitat and with some historical evidence, these patterns elucidated the evolutionary history of S. pratensis in Europe since the last Ice Age. The isozyme data and the flower morphology, on the other hand, presents us with knowledge about the ontogenesis and the influence of the environment on S. pratensis. Finally we can begin to determine the evolutionary relationships within section Elisanthe by comparing the variation of S. pratensis with the variation known for other species.

Morphological effects of the flavone isovitexin in a non-glycosylating genotype ofSilene pratensis (Caryophyllaceae)

Genetic studies have shown that the unglycosylated flavone isovitexin causes an aberrant petal morphology inSilene pratensis. Scanning electron micrographs show that the individuals with free isovitexin have abnormal upper epidermal cells.

The geographic distribution of flavone-glycosylating genes in Silene pratensis (Rafn.) Godron and Gren. (Caryophyllaceae)

In Silene pratensis three loci (g, gl and fg) control the glycosylation of isovitexin. Three alleles are known for both the g-locus (g, gGand gX) and the gl-locus (gl, glAand glR); for the fg-locus there are only two alleles (fg and Fg). The distribution of these alleles over 285 European populations of S. pratensis has been investigated. It was concluded that there are three different chemical races within S. pratensis in Europe. The first race contains the populations in western and southern Europe and displays high frequencies of gG, gl and fg. The frequencies of gGand glRare very high in the second chemical race, which can be found in the USSR, Scandinavia and eastern Poland. The third chemical race occurs in central Europe and in this race the frequencies of both g and glRare high, Fg has low to moderate frequencies in the second and third groups. The alleles glAand gXare seldom found in S. pratensis, but are present in the closely related S. dioica. They do occur with low frequencies in some populations of S. pratensis, possibly as a result of hybridization with S. dioica.

Evolutionary Aspects of Crassulacean Acid Metabolism in the Crassulaceae

The CAM pathway is taxonomically widespread and is believed to be used by about 16 000 species of vascular plants (see p. 9), a figure that includes more than 6% of all angiosperms. In addition, CAM has been reported in certain advanced groups of lower vascular plants (Isoetes; see Chap. 18) and in the specialized gymnosperm Welxvitschia mirabilis (Schulze et al. 1976; Winter 1985).