G. van Nigtevecht

Genetic studies in dioecious melandrium. I: Sex-linked and sex-influenced inheritance inMelandrium album and Melandrium dioicum

Sex-linked and sex-influenced inheritance are of interest because of their relation to the still intriguing problem of sex detrmination. Genes involved in the formation of the sex organs are regarded to be sex-determining genes. These genes may be present in all chromosomes including the sex-chromosomes. Other genes present in the sex-chromosomes, but not involved in sex determination, are the sex-linked genes. A mutation for narrow leaves we came across in ourM. ablum material is regarded as a case of sex linkage. Also the certation effect observed inM. album andM. dioicum must have been caused by genes on the sex-chromosomes. In both cases, however, it is not altogether unikekely, that the genes, regarded as sex-linked ones, actually take in the process of sex-determination.Sex-determining genes might influence the effect of other genes, that are therefore called sex-influenced genes. We observed a number of such sex-influenced characters inMelandrium.InM. album, female plants are, on the whole, larger than male plants, having larger stems and leaves. The petals, however, are larger in male plants, except in families with very broad petals. The leaves and petals are narrower in female plants than in male ones, except in families with very broad leaves and families with broad petals, where the difference in shape was no longer present. Usually, slightly more anthocyanin is formed in male plants than in females both in petals and the green parts. More glandular hairs were observed on male plants than on female ones.Insofar the observations were made inM. dioicum the same results were obtained.We regard these phenomena to be an expression of the different physiological conditions in female and in male plants, these conditions being provoked by the sex-determinging genes and more favourable for vegetative growth in female than in male plants.

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.

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.

Genetic studies in dioeciousmelandrium. II

The process of sex determination leads to the formation of the sex organs. Formerly two different groups of sex-determining genes were distinguished: the sex-promoting genes causing the actual development of the sex organs, and sex-deciding genes, which decide whether female-promoting genes, or male-promoting genes, or both come into action, thus giving rise to female flowers, male and hermaphrodite ones respectively.Obsrvations on our own material and data from the literature lead us to the conclusion that no such sharp distinction could be made. In this respect especially the hypothesis ofHeslop-Harrison (1957) on the role of auxin in sex determinations is of interest. It is argued that the sex-determing genes together provoke in flower primordia a certain net auxin activity that determines the development of the sex organs. A genothype that realizes a relatively high net auxin activity will favour the develoment of pistils and will work against the formation of stamina. A genotype that causes a relatively low auxin activity has the opposite effect.Changes of distinctly different sex-determining genes might have a similar effect on sex-expression. For example changes of genes in the Y-chromosome of an XY plant may cause the development of a pistil in the flowers of an XY plant and simultaneously work against the formation of stamina, as is demonstrated by some mutations in ourMelandrium material. In one mutation cytologically no aberration of the Y-chromosome was observed. The Y-chromosome of the other mutations appered to lack a part of their non-homologous arms. We observed the same phenotypical change after autosomal selection started in threeM. dioicum populations.A new feature of the distal part of the non-homologous arm of the Y-chromosome showed up in our hermaphrodite gerontogones. This part appeared to be indispensable for the functioning of both female and male gametes.In the discussion the formation of unisexual flowers in monoecious species and in dioecious ones was regarded to be fully comparable. In both instances auxin was assumed to play a dominant part in the differentiation of the flowers. We discussed the possible role of auxin in the regulating mechanism of protein synthesis.The origin of dioecism was explaned in terms of building up an uneven distribution of + genes (increasing the net auxin activity) and-genes (decreasing the net auxin activity) in a non-homologous pair of chromosomes.It is suggested that apomictic plants are like female plants characterized by a relatively high auxin activity in the flower primordia. We assumed therefore apomixis and dioecism to be by their origin related phenomena.With regard to the discrepancy in occurrence of dioecism and polyploidy in the plant kingdom and in the animal kingdom, it is argued that in plant species polyploidisation interferes with the building up of the balanced dioecious system, a situation not met with in the animal kingdom, because of the unfavourable aspects of polyploidy in animals.

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 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.

Genetics of anthocyanin formation in petals of the Red Campion (Silene dioica (L) Clairv.)

Apart from the genes I1 and Ia which control the intensity of flower colour, the genes C, A, P, M, N and AC are involved in anthocyanin biosynthesis in Silene dioica. In c/c plants no anthocyanins are present in any part of the plant. Gene A controls the formation of anthocyanin in the petals. Gene M governs the glucosylation of the 5-hydroxyl group of anthocyanins, whereas gene N controls the attachment of rharnnose to the 3-O-bound glucose. Gene AC governs the binding of a cinnamic acid derivative to the sugar at the 3-position; gene P determines whether this acyl moiety is p-coumaric acid (p/p plants) or caffeic acid (in P/P plants). This gene P is pleiotropic in its action; in P/P plants cyanidin-glycosides are present, whereas in p/p plants pelargonidin-glycosides are found. It is suggested that gene P acts by hydroxylating p-coumaroyl-CoA to caffeoyl-CoA, which is then both used as precursor for the synthesis of the anthocyanidin skeleton and in the acylation step.

Dominance relations between isovitexin: 7-0-glycosyltransferase alleles in Melandrium

SummaryIn a hybrid swarm of Melandrium dioicum and Melandrium album, a plant was found in which the genes gG and gX, controlling an isovitexin 7-O-glucosyltransferase, respectively an isovitexin 7-O-xylosyltransferase, were co-dominant. In earlier experiments, gene gG was always dominant over its allele gX. Genetical and biochemical analysis revealed that this co-dominance was caused by an allele of gX, further designated gX′. Comparison of the enzyme-kinetic properties of the gX′ controlled xylosyltransferase with the gX controlled xylosyltransferase revealed no difference in substrate affinity for UDP-xylose nor for isovitexin. However, the maximal activity of the gX′ controlled transferase was 8+9 times higher than the gX controlled activity. These results support the competition model previously proposed to explain the dominance relationship between gG and gX. This model was based on the assumptions that 1) both the xylosyl- and the glucosyltransferase work at saturating isovitexin concentration and that 2) the lower Vmax of the xylosyltransferase, makes this enzyme a poorer competitor for isovitexin than the glucosyltransferase.

Dominance relationships between allelic glycosyltransferase genes in Melandrium: An enzyme-kinetic approach

SummaryIn the petals of Melandrium the glycosylation of the 7-hydroxylgroup of isovitexin is governed by a series of 4 multiple alleles: gG, g, gX, and