The effects of
rolA on root and shoot architecture have been ascribed to a deficiency in gibberellic acid (GA
3) and to changes in polyamine metabolism. Using tobacco, we examined interactions among GA
3, a polyamine accumulation inhibitor (α-DL-difluoromethylornithine or DFMO) and the
rolA gene controlled by the 35S CaMV promoter. We measured the effects of these three agents on architecture and polyamine accumulation in excised roots and whole plants grown in vitro. Previous work showed that DFMO or genetic transformation with the
rolA gene from
Agrobacterium rhizogenes, controlled by the 35S promoter (P
35S-
rolA), caused excised tobacco roots to grow faster with altered root system architecture. We show that gibberellic acid (GA
3) reversed the effects of DFMO on the architecture of excised root systems, but neither reversed the effects of DFMO on growth, nor the changes in growth and architecture associated with P
35S-
rolA. GA
3 treatment alone resulted in increased agmatine levels, suggesting that the inhibition of the effects of DFMO on architecture was through a stimulation of the arginine decarboxylase (ADC) pathway, GA
3 alone also inhibited the accumulation of putrescine and tyramine conjugates in excised roots. In tobacco plants growing in vitro DFMO and P
35S-
rolA were associated with reduced shoot height, which was partially restored by GA
3 treatment; however, GA
3 also stimulated shoot height in the controls. GA
3 did not lessen the leaf wrinkling associated with P
35S-
rolA. P
35S-
rolA increased root number in young seedlings in vitro, and increased root system length in seedlings grown in soil. As in excised roots, the developmental changes linked to DFMO and P
35S-
rolA were accompanied by reductions in putrescine titers. GA
3 treatment stimulated putrescine accumulation in stems and leaves, and partially reversed the negative effects of DFMO and P
35S-
rolA on putrescine accumulation in roots, stems and leaves. Again, the restoration of putrescine pools appeared to be through a stimulation of the ADC pathway, since agmatine accumulated in plants exposed to GA
3. In general, the effects of DFMO and P
35S-
rolA on phenotype and polyamine metabolism were coordinated, and in many cases these effects were similarly modulated by GA
3, reinforcing the previous conclusion that the phenotypic effects of
rolA in roots and shoots occur through interference with polyamine metabolism and that the putrescine conjugates are particularly important in regulating root system growth and architecture. We were unable, however, to discem consistent evidence for a direct role for GA
3 in establishing the RolA phenotype.
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