Graduate Thesis Or Dissertation
 

Cytokinin biosynthesis in crown gall tumors

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  • Using combined HPLC-RIA, cytokinin levels in a series of tobacco crown gall tumors, having altered morphologies as a result of transposon mutagenesis were examined. Average trans-ribosylzeatin levels in the shoot-producing tumors (tms mutants) and root-producing tumors (tmr mutants) were 1400 pmol/gm and 0.97 pmol/gm, respectively. Cytokinin/auxin ratios were highest in the tms mutants (17-24) and lowest in the tmr mutants (0.004). The evidence suggests that the T-DNA loci which define tumor morphology, also determine phytohormone levels. Cytokinin levels were also examined in several octopine crown gall tumors, with different T[subscript L] and T[subscript R] DNA copy numbers. The trans-ribosylzeatin level in the high T[subscript L] DNA copy number tumor line, 15955/01, was 437 ng/gm compared to 6-30 ng/gm in the other low T[subscript L] DNA copy number tumor lines, suggesting that T-DNA copy number can influence cytokinin levels. At a detection limit of 100 pg/gm, no trans-ribosylzeatin was detected in untransformed tobacco tissue. Some of the properties of the cytokinin biosynthetic enzyme, dimethylallylpyrophosphate:adenosine 5'-monophosphate dimethylallyl transferase (DMA transferase), from Nicotiana tabacum crown gall tumor line 15955/01 were studied. This enzyme demonstrated a substrate specificity for adenosine 5'-monophosphate and catalyzed the synthesis of iso-pentenyladenosine 5'-monophosphate (iPA-P). DMA transferase activity from cell-free extracts of four crown gall tumors and untransformed tobacco tissue was estimated by an assay which included high performance liquid chromatography and immunoaffinity chromatography. No enzyme activity was detected in untransformed tobacco tissue, but was present in all four crown gall tumor lines. Specific activities (1 Unit is defined as the synthesis of 1 fmol iPA-P/min/mg total protein) of the enzyme in the crown gall tumor lines were: A6S/2, 2.0; B₆ 806/E9, 6.6; 15955/1, 2.7 and 15955/01, 5.7. The results strongly indicate that the T-DNA regulates non-tRNA mediated biosynthesis of cytokinins.
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