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Abstract
The activity of the tms 2 locus, ultimately responsible for IAA biosynthesis in Agrobacterium tumefaciens- transformed cells, was investigated. Two different approaches were used to examine tms 2 activity. In the first approach, suppressed copies of this gene in the pith tissues of regenerated plants (containing $\geq$40 to 60 copies per cell) were examined to determine why they were suppressed and the requirements for reactivating them. First, suppression was not due to instability or gross structural re-arrangements of the T-DNA since it was faithfully and stably inherited. Secondly, the plants tissues minimally required mechanical wounding, fresh nutrients and a transient exposure to IAA to reinitiate auxin autonomy. Thirdly, in some of the plants, auxin autonomy could only be induced in the presence of 5-azacytidine, in addition to the other requirements. While the mechanism responsible for T-DNA suppression remains unclear, in at least some plants DNA hypermethylation may play a role. These experiments clearly demonstrated that the host plant can strongly influence T-DNA gene expression.
The second approach utilized a straightforward assay for indoleacetamide hydrolase (AH) (encoded at tms 2) to determine whether any consistent correlation existed between levels of AH and IAA levels in the transformed cell's growth cycle. After devising a suitable micro-assay for AH, its enzyme activity was studied in a number of cloned lines. The findings were: first, AH activity is modulated in a consistent, reproducible pattern throughout the growth cycle of the transformed cells. Second, this level of activity correlated with the IAA levels in the transformed cells. Third, both wounding and fresh nutrients, two of the requirements for auxin autonomy reinitiation, contributed to the increase in AH activity. These results were not due to an overall change in cellular metabolism since other non-T-DNA enzymes, e.g. G6PdH and PAL behaved as expected. The results reported here demonstrate that not only can the host affect T-DNA gene activity, but that such interactions may be essential for the survival of a transformed cell.
