Previous studies (Genetics 9:969-978, 1980; Sci. 213:1515-1517, 1981) have indicated that Mu acts in the germ line tissue very late in development, meiotically or post-meiotically. In the past few growing seasons, we have learned much more about when Mu acts to produce new mutations. Since mutational events are presumably the result of the transposition of Mu1-like elements, these studies provide information as to when transposition is occurring.
New evidence on the timing of Mu activity has been obtained from studies of 1) mutants that occur in unexpected frequencies, 2) discordant endosperm mutants, and 3) gamma and ultraviolet ray treatment. Mutants occurring in unexpected frequencies: Frequently, when new Mu-induced mutants are found segregating on selfed ears in Mu outcross families, they are observed in ratios much lower than the 3:1 ratio expected if the Mu-induced mutant had been carried by the gamete of the Mu parent. Such "low ratio mutants" are possible if the mutational event did not occur in the Mu parent but occurred very early in the development of the embryo of the seed producing the plant that was selfed. If such a mutation occurred before the cell lineages giving rise to the tassel and ear separated, both reproductive structures could have some germ line cells heterozygous for the new mutant. Depending upon the extent of the male and female germ tissue carrying this new mutant allele, various frequencies could be generated, varying from 25 percent mutant individuals to less than one percent. Frequencies as low as one mutant per ear have been observed. To test this explanation for "low ratio" ears, normal seeds were planted from ears segregating endosperm mutants in a low frequency. If the above explanation for these ears is correct, then one-half of the normal seeds from the ear will be heterozygous for the mutant allele, if for example, all tissue of the ear received the new mutant allele while only a very small portion of the tassel did. If only a small portion of the tassel and ear carries the mutant allele, very few of the plants from the normal seeds will be heterozygous for the allele. However, when a segregating selfed ear is found it should segregate in a 3:1 ratio. Results from the tests of two "low ratio" mutants support the suggestions that Mu can induce mutants early in development (Table 1). Another prediction can be made if Mu is inducing mutants at this time in development. In our standard Mu tests, the procedure is to self pollinate the Mu parent and the second ear of the outcross parent and screen the self progenies for new mutants. The selfing of the Mu and the outcross parents is essential because, if either parent is segregating for a new mutant, it would confuse our test results. However, occasionally, in spite of finding no mutants in the selfs, about half of the plants of the outcross progeny will segregate for a new mutant. Such a situation can be explained on the basis of a bookkeeping error or an environmentally sensitive mutant (e.g., a mutant that requires high temperature for expression, but the self progenies were tested at low temperature and the selfs of the outcross tested at high temperature). However, these exceptions could also be the result of a mutation in the cell lineage giving rise to the tassel, and not in the cell lineage giving rise to the ear of the Mu plant or vice versa. If this plant is selfed and outcrossed as a male, the self progeny will not segregate for the mutant, but the selfs of the outcrosses will. If the above explanation is correct, half of the plants from the seeds of the selfed Mu parent should be heterozygous for the mutant found segregating in the selfs of the outcross plants. These tests are yet to be made.
Discordant mutant seeds: For the past few years we have been studying the induction by Mu of mutants at the y1 locus. We have induced mutants by using Mu plants as females and also as males. These mutants were induced by using Y1 Y1 Mu plants crossed onto or by y1 y1 wx wx gl8 gl8 (See Mol. Gen. Genet. 200:9-13, 1985). When the mutant seeds (white or pale yellow endosperm) from these tests were planted and the resulting plants selfed, it was found that in some instances the selfed ears segregated for yellow seeds. In these discordant seeds the endosperm had the putative mutant allele, while the embryo was heterozygous for the nonmutant Y1 allele. These discordant seeds are found in greater frequency in crosses in which the Mu Y1 Y1 parent is used as a male than when the Mu parent is used as a female (Table 2). Such results are expected if Mu is inducing mutations in the gametophytes, because of the differences in the development of the male and female gametophyte. If mutations that result in the observed discordant seeds (i.e., white endosperm, Y1 y1 embryo) are occurring in the gametophytes, the two sperm of the male gametophyte would be expected to differ with respect to mutant and nonmutant alleles much more frequently than would the polar fusion nucleus and the egg nucleus of the female gametophyte. In the male gametophyte only one mutational event is necessary to generate such a seed, while in the female gametophyte two mutational events must occur, one in each of the two cell lineages giving rise to the two nuclei of the polar fusion nucleus.
Pollen irradiation: Irradiation of Mu pollen with ultraviolet light and gamma rays has demonstrated that they have an effect on the mutation inducing ability of Mu. Ultraviolet light seems to enhance the effect of Mu (MGNL 56:2-4, 1982; 58:19-20, 1984), while gamma rays seem to diminish its effect (MGNL 58:15-16, 1985). Because DNA replication does not occur in mature pollen, it is unlikely that Mu transposition leading to mutations takes place at the time of irradiation. These effects on Mu's mutational activity are most likely restricted to zygote or early developmental time periods, suggesting Mu might normally be active at these stages.
Figure 1 summarizes the evidence to date bearing on the time of Mu mutator activity.
Donald S. Robertson
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