Antisense transgenesis of tobacco with a flax pectin methylesterase affects pollen ornamentation.

Antisense transgenesis of tobacco (Nicotiana tabacum) with a partial flax (Linum usitatissimum L.) pectin methylesterase (Lupme3) cDNA sequence yielded plants with altered pollen content. Moreover, the characteristically sculptured cell wall surrounding the pollen grains was modified in transgenic tobacco plants: the wavy ornamentation was dramatically reduced, suggesting the involvement of the demethylation of pectin in the pollen cell wall-specific structure. Germination of pollen was decreased and the pollen tube surface aspect was also different in transgenic plants.

Isolation of a flax pectin methylesterase promoter and its expression in transgenic tobacco.

Pectin methylesterases (PME) catalyze the de-esterification of methoxylated pectins in plant cell walls. We have isolated a 1.9 kb regulatory region upstream from the Lupme3 coding sequence of Linum usitatissimum L. (flax) using a 'Polymerase Chain Reaction (PCR) walking' strategy. Two 5' truncated deletion fragments (1.5 and 0.44 kb) of this potential promoter sequence were inserted upstream of the gus reporter gene in order to study their expression in transgenic plants. These constructs were transferred into Nicotiana tabacum, a heterologous system using Agrobacterium tumefaciens. Expression of the reporter gene was analyzed in regenerated transgenic plants and calli to study the promoter activities of these sequences. This expression was observed in calli with both constructs. In contrast, expression in organs was only detected in tobacco plants transformed with the largest (1.5 kb) construct. This long fragment triggered expression in roots and immature or vitrified leaves. Expression in both organs was localized in the vasculature, but also detected in the root meristem. These results are the first evidence, to our knowledge, of the spatial and temporal regulation of a specific pme promoter of flax. Localization of Lupme3 promoter activity in vascular tissues of immature organs provides an insight into the role of this PME isoform in cell elongation and differentiation.

Tropane alkaloid production by hairy roots of Atropa belladonna obtained after transformation with Agrobacterium rhizogenes 15834 and Agrobacterium tumefaciens containing rol A, B, C genes only.

Atropa belladonna leaf disks were infected by a wild strain Agrobacterium rhizogenes 15834 harboring the Ri-TL-DNA and by a disarmed Agrobacterium tumefaciens strain harboring a construction with only rol ABC and npt II genes. Thirteen root lines were established and examined for their growth rate and alkaloid productivity to evaluate the possible role of rol genes in morphological differentiation and in tropane alkaloid formation. A great diversity has been observed in the growth rate of these 13 root lines. The root biomass increased up to 75 times. The total alkaloid contents were similar in the root lines obtained by infection with A. rhizogenes 15834 and A. tumefaciens rol ABC. The last ones accumulated between 4 (1.1 mg g(-1) DW) and 27 (8 mg g(-1) DW) times more alkaloids than the intact roots (0.3 mg g(-1) DW). This work has shown that the rol ABC genes were sufficient to increase tropane alkaloid production in A. belladonna hairy root cultures.