We succeeded in visualizing aggregates of the Ac transposase (TPase) by immunofluorescence in transgenic tobacco protoplasts. Protoplasts were prepared from tobacco plants carrying the full length Ac TPase gene and a truncated TPase gene lacking 102 amino acids from the amino terminus, respectively, under control of the cauliflower mosaic virus 35S promoter. Protoplasts were centrifuged onto polylysine-precoated microscopic slides. After fixation and alcohol-extraction, they were stained by the indirect immunofluorescence procedure described by Heinlein et al. (Plant J. 5:705-714, 1994).

In protoplasts containing the full length TPase gene, the protein is detected in the nuclei in the form of many, rod-like aggregates, similar to those that were observed with a very low frequency in maize. In protoplasts expressing the truncated TPase derivative, in addition to nuclear complexes large bodies of coalesced TPase are detected in the cytoplasm. We conclude from these results that the full length TPase and the truncated TPase, which is lacking one of its three nuclear localization signals (Boehm et al., Plant J., in press, 1995), both exhibit very similar properties as in transiently transfected Petunia hybrida protoplasts. Individual batches of protoplasts always display immunofluorescence signals of very similar intensity and uniformly shaped aggregates. In several independent transformants the immunofluorescence signal (supposedly reflecting the amount of TPase) is always higher in plants homozygous for the transgene than in heterozygotes. The age of the plant material (between 6 and 19 weeks) used for protoplast preparation apparently has no influence on amount or shape of the aggregates.

Scofield et al. (Cell 75:507-517, 1993) have recently shown that high levels of Ac TPase inhibit transposition in transgenic tobacco plants. Our results indicate that the TPase forms large aggregates, and in Petunia similar aggregates are supposed to be transpositionally inactive (Heinlein et al., Plant J. 5:705-714, 1994). Though there is only indirect evidence for the inactivity of the TPase aggregates and we cannot exclude a specific function of them during transposition, it is conceivable that the TPase aggregates are responsible for the effects described by Scofield et al.

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