The timing and the frequency of Ds excisions differ between maize kernels which carry the Ac elements present in the wx-m7 and wx-m9 alleles, respectively, although the two elements are identical in sequence and are located within the same gene in identical orientation (Heinlein and Starlinger, Maydica 36:309-316, 1991). Immunohistochemical staining of 30 DAP (days after pollination) endosperm cells for the TPase revealed that during this late stage of endosperm development the wx-m7 endosperm contains much more of the TPase aggregates than wx-m9 endosperm. The two Ac elements therefore might be differentially expressed. This is further corroborated by the observation that the Ac alleles can not be replaced by each other in kernels carrying the bz-m2(DI) allele without altering the pattern of revertant Bz sectors in the aleurone. Whereas wx-m7/wx-m7/wx-m7 endosperms are characterized by very large revertant sectors due to excision events having occurred during early developmental stages, the phenotype of the wx-m7/wx-m7/wx-m9 endosperms exhibits very rare and unicellular sectors due to late events. However, the history of the wx-m7 and wx-m9 maize lines used in the crosses are not known in detail and these lines therefore cannot be considered isogenic. Consequently, we performed genetic experiments aimed to reveal whether other gene products (encoded by modifier genes) are involved in the control and regulation of transposition.

We started our experiments with heterozygotes between wx-m7 and wx-m9 and crossed this line to an appropriate tester strain, e.g. bz-m2(DI). If the differences between the wx-m7 and wx-m9 lines were due to the presence of modifier genes, we expected either a new variegation pattern in the progeny of this cross if several unlinked or loosely linked modifier genes were involved, or the reappearance of the two previously known patterns segregating independently of the Wx alleles. The outcome of this experiment was the reappearance of the two previous patterns, which segregated in the expected ratios. The particular patterns seen in the aleurone were concordant with the respective wx-m7 and wx-m9 variegation patterns in the inner endosperm. However, this was not taken to be diagnostic for the presence of the specific Wx alleles since the variegation patterns of these alleles to some degree might underlie the action of the putative modifier genes also. Hence, we extracted the DNA from 120 kernels showing either of the two variegation patterns and probed this DNA by PCR for the presence of the respective waxy-alleles. In all but two cases (which might be misselections) we found that the kernels which exhibit an Ac-specific variegation phenotype also carry the respective Ac element. The outcome of this experiment therefore excluded the presence of a modifier gene linked to Ac, unless this gene maps very close (within 2 map units) to Ac.

As a next step we crossed plants grown from selected kernels that did not receive an Ac element due to a meiotic excision event to plants that carried the other Ac element combined with the bz-m2(DI) allele. The chromosome that previously carried Ac should still carry the putative tightly linked modifier gene. For the case that this modifier gene gives rise to the "Ac specific" variegation pattern rather than Ac itself, we expected the appearance of the weakly variegated phenotype characteristic for Ac7/Ac9 heterozygotes (see above) on 25% of the progeny kernels. However, this phenotype was not found on the ears. Instead, we solely observed variegation patterns characteristic for the Ac element brought in by the tester plant.

Taken together, these results strongly suggest that the Ac allele specific variegation patterns are not due to modifier genes. Accordingly, it seems reasonable to assume that the differences between Ac-specific variegation patterns are due to differential expression of the Ac elements. 

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