EPILOBIUM AND THE PLASMONE HYPOTHESIS
The willow-herb Epilobium was chosen by P. Michaelis for his intensive and extensive investigations of non-Mendelian inheritance carried out over the last 40-odd years, beginning with his studies on the cytology and embryonic development of Epilobium published in 1925 (19). In an extensive review summarizing his observations and conclusions in 1954, Michaelis stated, "We cannot distinguish manifestations of single plasmagene units, but are limited to recognition of the effect of all those hereditary components of the cell that do not show Mendelian segregation;" (21). He uses the term plasmone, originated by von Wettstein, to include all extranuclear hereditary elements of the cell (21). More recently, Michaelis has summarized his principal fundings and conclusions in a series of short papers (22-25).
In choosing an example to illustrate plasmone inheritance Michaelis has often described the following set of experiments illustrated with one of his diagrams in Fig. 6.6. The figure shows typical F1 plants resulting from reciprocal crosses between two species of Epilobium, E. hirsutum I and E. luteum. The smaller plant on the right is pollen sterile, female X E. hirsutum male has fertile pollen. In Fig. 6.7 we see a similar cross between two geographic races of E. hirsutum. Michaelis carried out a series of backcrosses starting with the healthy F1 hybrid from the original E. luteum X E. hirsutum Jena cross. The normal appearance of the F1 hybrid indicated that the E. hirsutum genes which came from the pollen were functioning adequately in the cytoplasm of the E. luteum female parent.
|Fig. 6.6. Typical F1 plants resulting from reciprocal crosses between two species of Epilobium, E. luteum and E. hirsutum. The plant on the right, with stunted growth and sterile pollen, received its cytoplasm principally from the E. hirsutum parent, while the healthy plant on the left received its cytoplasm from the E. luteum parent. From (22).|
|Fig. 6.7. Typical F1 plants from reciprocal crosses between two geographical races of E. hirsutum, (E. hirsutum Jena and E. hirsutum Munchen). As in Fig. 6.6, the plants resulting from reciprocal crosses are very different. From (22).|
What would happen if all the E. luteum genes were replaced by E. hirsutum genes by means of repeated backcrosses with the E. hirsutum parent as male? Michaelis performed this celebrated series of backcrosses for twenty-five generations, testing the hybrids for their response in crosses with E. luteum. The question was whether totally replacing the E. luteum genome would produce a plant like the original homozygous E. hirsutum which, used as the female parent, gave poor F1 progeny in crosses with E. luteum males. The answer was very clear. Even after twenty-five generations, crosses between the hybrid (E. hirsutum genes in a E. luteum cytoplasm) and E. luteum males gave normal progeny.
As Michaelis concluded, this experiment demonstrated the constancy and relative autonomy of the E. luteum cytoplasm in cells containing E. hirsutum genes.
Subsequently Michaelis undertook reciprocal crosses of a similar sort with a large assortment of different species and races in the genus Epilobium. He concluded, "This series of crosses leads to the result that reciprocal differences are not produced by the cytoplasm alone, but by the interaction of the cytoplasm with the nuclei. All the different plants possess the same cytoplasm as Epilobium hirsutum Jena but different nuclei. It can be said that the reciprocal differences are produced by difficulties in the interactions between nuclei and cytoplasm. In reciprocally equal hybrids the cytoplasm and the nuclei work together in a normal way."
It should be emphasized that the differences in reciprocal crosses which Michaelis observed included not only effects upon chloroplast development, but also more general morphogenetic effects such as stunted growth of plants, deformed flowers, and pollen sterility. In no case is the biochemical basis of the phenotypic change known. Poor growth may be attributed to poorly functioning chloroplasts, and by implication to mutations in chloroplast DNA. However, mutations affecting only flower formation and pollen development in plants that are otherwise normal can hardly be viewed as chloroplast mutations. Perhaps they are indications, as Michaelis has proposed, of mutations in some other extranuclear genetic system.
In other studies Michaelis attempted to analyze the pattern of variegation in leaf development. In particular, he attempted to evaluate the number of genetic copies per cell on the basis of a simple mathematical model of segregation. To develop a workable model it was necessary to make certain simplifying assumptions, in particular to assume that plastids, whether normal or mutant, multiply at the same rate and that plastids are distributed to daughter cells at cell division in a random and equal manner like green and white marbles in a black box. As might have been anticipated, his calculations indicated that one needed a system with a low number of units to account for the variegation pattern observed, a lower number than his plasmone hypothesis had envisaged.
Indeed, Michaelis tried to distinguish between genes present in many copies (plasmone genes) and those present in a few copies (plastome genes located in plastids) by analyzing segregation patterns in variegated seedlings. Applying this method to green-white variegation in striped seedlings, Michaelis found a result not too different from that in Pelargoniun—that very few copies need be invoked to explain the data. Uncertainties in cell lineage and cell division rates make the quantitation imprecise. Nonetheless, it is a striking observation that far fewer DNA copies are necessary than would be expected on the basis of estimates of the number of plastids. This result is puzzling and reminiscent of a similar problem with mitochondrial DNA in yeast (cf. p. 120).