The effect of the cyanogenic glucosides linamarin and lotaustralin and their hydrolyzing enzyme linamarase was studied in a B2 generation segregating for the genes Ac and Li. Plants containing the glucosides are protected against grazing by snails both in the seedling stage and as adult plants. In seedlings, however, there is a direct effect on survival, whereas in adult plants the leaf area of plants containing linamarin/lotaustralin is less reduced under intense grazing. Linamarase has no effect on grazing by snails, possibly as a result of the presence of β-glucosidase activity in the gut of these animals. The genes Ac and Li, or genes tightly linked to them, have other effects as well: plants possessing one dominant Ac allele produce fewer flowers than homozygous ac plants. I compared this difference in flower production to the metabolic cost of producing the cyanogenic glucosides. The energy content of the difference in flower head production far exceeded the metabolic cost of cyanoglucoside production in Acac plants. It is possible that the cost of maintaining a certain level of cyanoglucosides is much more important for the plant than the initial cost of biosynthesis. The importance of the effects of Ac and Li in the maintenance of cyanogenic polymorphism in white clover is discussed.

Linamarase (EC 3.2.1.21) is a specialized β-glucosidase that hydrolyses the cyanogenic glucoside linamarin. Two clones of Trifolium repens L. derived from natural populations, of which one clone exhibited linamarase activity, were used in a comparative study to try to establish the localization of linamarase and other β-glucosidases. Two methods were used: the first one was vacuum infiltration of intact leaf cells, followed by centrifugation. A significant amount of linamarase and β-glucosidase activity could be extracted from intact tissue by a 0.25 M NaCl solution, indicating that these activities are localized in the apoplast. The second method, immuno-cytofluorescense of microtome sections, confirmed this. It was found that linamarase and other β-glucosidases are present in the cell walls, especially those of the epidermal cells, and in the cuticle. However their presence in the cell walls of other tissues i.e. walls of the vessels, could not be excluded. No difference in distribution could be detected between linamarase and other β-glucosidases.

Trifolium repens L. and Trifolium nigrescens Viv. are two of the approximately six cyanogenic species known in the genus Trifolium.The two species are closely related: T. nigrescens is considered to be one of the diploid ancestors of the amphidiploid T. repens. We studied morphology, meiosis and the cyanogenic system in T. repens (amphidiploid), T. nigrescens (diploid) and their reciprocal hybrids. A comparison of the enzyme linamarase in the species and hybrids shows that there is a general resemblance between their linamarases. Immunological studies indicate that the linamarases must have a somewhat different three-dimensional structure. These facts are consistent with the view that T. nigrescens (or an ancestral form of this species) has donated the Li gene of T. repens. The other putative parent, T. occidentale has probably not donated an active Li gene. The hypothesis of the origin of the Li gene does not explain its polymorphism in European populations of T. repens, as T. nigrescens is monomorphic for cyanogenesis and amphidiploids do not segregate for genes which are homozygous dominant in one of the parents. Segregation for Li could be caused by a gene mutation or a small exchange between homeologous chromosomes. The latter event is more probable. A nigrescens-repens exchange would give rise to a chromosomal region with reduced homology to both parental chromosomes. The genes in the region of exchange will be tightly linked due to diminished cross-over frequency. It has been known for years that Li has effects on the vegetative and reproductive characters in T. repens and we have recently shown that these effects must be the result of genes linked to Li. As the associated characters influence the fitness of the cyanotypes, not only the origin but also the maintenance of the cyanogenic polymorphism is closely related to the evolutionary history of T. repens.

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Amsterdam SUMMARYThe cyanogenic phenotypes of 19 populations of adult Trifolium repens plants were determined. Substantial differences were found, both in the frequency of Ac-as in Li-phenotypes, The differences found are not attributable to large climatic differences, but to factors acting on a local scale. Ac and Li are not independently assorted: six out of sixteen populations studied showed a positive linkage disequilibrium. As Ac and Li are not genetically linked, the association must be the result of epistatic selection favouring Ac-Li-phenotypes. The relative importance of deterministic forces, i.e. natural selection, and stochastic ones (founder effects, drift) in determining the phenotypic frequencies found in The Netherlands are discussed.