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Whole tobacco plants containing the root-inducing, left-hand transferred DNA (Ri TL-DNA) display a transformed phenotype, that includes alterations in a number of developmental processes, such as floral induction, flowering and reproduction. We show that the entire Ri TL-DNA is responsible for repression of ornithine and tyrosine decarboxylases while it exerts no effect on transferase and the methyl transferase activities. Evidence is provided that two genes from the Ri TL-DNA, rolA and rolC, alter polyamine metabolism as well as floral induction and flowering. Thus, plants transformed by the rolC gene (under the control of the 35S promoter from cauliflower mosaic virus) were male-sterile (non-viable pollen) and female fertility was reduced by approximatively 80%. A constitutive overexpression of the rolC gene may directly or indirectly cause inhibition of the accumulation of water-insoluble amine conjugates located in the anthers and all the methyl transferases, leading to increases of ornithine decarboxylase, phenylalanine ammonia lyase and putrescine caffeoyl-CoA transferase. The results suggest that male sterility is associated with catabolic processes exerted at the level of water-insoluble amine conjugates and support the view that diamine oxidase may be involved in the regulation of the amine concentration during sexual differentiation, a factor that should be considered when attempting to decipher the mechanisms of control of sexual differentiation. The rolC gene could be useful in determining the role of diamine oxidase in the physiology of flowering. These results suggest that elevated free polyamine and water-soluble polyamine levels (located in the ovaries) contribute to abnormal floral development. The transformed phenotype due to P35S-rolA (the rolA gene fused to the 35S promoter) consisted of inhibited or delayed flowering, and altered floral morphology in the form of flower abortion. The effects of P35S-rolA on flowering and fertility are closely correlated with limitations in the accumulation of the water-soluble and -insoluble amine conjugates and increase in accumulation of free amines, indicating that amine conjugates (via transferases) have important functions in floral induction, floral evocation and reproduction. Spermidine availability as well as tyramine availability (in conjugated forms) could be limiting factor(s) in sexual development in tobacco. [ABSTRACT FROM AUTHOR]

The effects of rolA on root and shoot architecture have been ascribed to a deficiency in gibberellic acid (GA3) and to changes in polyamine metabolism, Using tobacco, we examined interactions among (GA3, 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 (P35S-rolA), caused excised tobacco roots to grow faster with altered root system architeclure. We show that gibberellic acid (GA3) reversed the effects of DEMO 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 P35S-rolA. GA3 treatment alone resulted in increased agrnatine levels, suggesting that the inhibition of the effects of DFMO on architecture was through a stimulation of the arginine decarboxylase (ADC) pathway. GA3 alone also inhibited the accumulation of putrescine and tyramine conjugates in excised roots. In tobacco plants growing in vitro DFMO and P35S-rolA were associated with reduced shoot height, which was partially restored by GA3, treatment; however, GA3 also stimulated shoot height in the controls. GA3 did not lessen the leaf wrinkling associated with P P35S-rolA. P35S-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 P35s-rolA were accompanied by reductions in putrescine titers. GA3 treatment stimulated putrescine accumulation in stems and leaves, and partially reversed the negative effects of DFMO and P35s-rolA on putrescine accumulation in roots, stems and leaves. Again, the restoration of putreseine pools appeared to be through a stimulation of the ADC pathway, since agmatine accumulated in plants exposed to GA3. In general, the effects of DFMO and P35s-rolA on phenotype and polyamine metabolism were coordinated, and in many cases these effects were similarly modulated by GA3, 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 architccture. We were unable, however, to discem consistent evidence for a direct role for GA3 in establishing the RolA phenotype. [ABSTRACT FROM AUTHOR]

Analysis of published sequences for Ri TL-DNA (root-inducing left-hand transferred DNA) of Agrobacterium rhizogenes revealed several unsuspected structural features. First, Ri TL-DNA genes are redundant. Using redundancy as a criterion, three regions (left, middle and right) were discerned. The left one, ORFs (open reading frames) 1-7, contains no detectable redundancy. In the middle region a highly diverged gene family was detected in ORFs 8, 11, 12, 13 and 14. The right region contains an apparently recent duplication (ORF 15 =18+17). We interpret the phenomenon of redundancy, particularly in the central region that encodes the transformed phenotype, to be an adaptation that ensures function in a variety of host species. Comparison of Ri TL-DNA and Ti T-DNAs from Agrobacterium tumefaciens revealed common structures, unpredicted by previous nucleic acid hybridization studies. Ri TL-DNA ORF 8 is a diverged Ti T-DNA tms1. Both Agrobacterium genes consist of a member of the diverged gene family detected in the central part of the Ri TL-DNA, but fused to a sequence similar to iaaM of Pseudomonas savastonoi. Other members of this gene family were found scattered throughout Ti T-DNA. We argue that the central region of Ri and the part of Ti T-DNA including ORFs 5-10 evolved from a common ancestor. We present the hypothesis that the gene family encodes functions that alter developmental plasticity in higher plants. [ABSTRACT FROM AUTHOR]