Mirella Pomponi

Overexpression of Arabidopsis phytochelatin synthase in tobacco plants enhances Cd2+ tolerance and accumulation but not translocation to the shoot

Phytochelatins (PCs) are metal binding peptides involved in heavy metal detoxification. To assess whether enhanced phytochelatin synthesis would increase heavy metal tolerance and accumulation in plants, we overexpressed the Arabidopsis phytochelatin synthase gene (AtPCS1) in the non-accumulator plant Nicotiana tabacum. Wild-type plants and plants harbouring the Agrobacterium rhizogenes rolB oncogene were transformed with a 35SAtPCS1 construct. Root cultures from rolB plants could be easily established and we demonstrated here that they represent a reliable system to study heavy metal tolerance. Cd2+ tolerance in cultured rolB roots was increased as a result of overexpression of AtPCS1, and further enhanced when reduced glutathione (GSH, the substrate of PCS1) was added to the culture medium. Accordingly, HPLC analysis showed that total PC production in PCS1-overexpressing rolB roots was higher than in rolB roots in the presence of GSH. Overexpression of AtPCS1 in whole seedlings led to a twofold increase in Cd2+ accumulation in the roots and shoots of both rolB and wild-type seedlings. Similarly, a significant increase in Cd2+ accumulation linked to a higher production of PCs in both roots and shoots was observed in adult plants. However, the percentage of Cd2+ translocated to the shoots of seedlings and adult overexpressing plants was unaffected. We conclude that the increase in Cd2+ tolerance and accumulation of PCS1 overexpressing plants is directly related to the availability of GSH, while overexpression of phytochelatin synthase does not enhance long distance root-to-shoot Cd2+ transport.

Different promoter regions control level and tissue specificity of expression of Agrobacterium rhizogenes rolB gene in plants.

Expression of the rolB gene of A. rhizogenes T-DNA triggers root differentiation in transformed plant cells. In order to study the regulation of this morphogenetic gene, the GUS reporter gene was placed under the control of several deleted fragments of the rolB 5′ non-coding region: carrot disc transformations and the analysis of transgenic tobacco plants containing these constructions identified the presence of distinct regulatory domains in the rolB promoter. Two regions (located from positions −623 to −471 and from −471 to −341, from the translation start codon) control the level but not the tissue specificity of rolB expression: progressive deletions of the rolB promoter starting from position −1185 to −341, although at different levels, maintained the same pattern of GUS expression — maximal in root meristems and less pronounced in the vascular tissue of aerial organs. Further deletion of 35 bp, from −341 to −306, drastically affected tissue specificity: GUS activity was still clearly detectable in the vascular tissue of the aerial organs while expression in the root meristem was totally suppressed. Analysis of transgenic embryos and seedlings confirmed that distinct promoter domains are responsible for meristematic (root) and differentiated (vascular) expression of rolB. Finally, we present data concerning the effects of plant hormones on the expression of rolB-GUS constructions.

Transcription in sea urchin. I. Initiation sites on DNA and chromatin

Abstract The interaction of Escherichia coli RNA polymerase with sea urchin (SU) DNA and chromatin was studied under conditions that permit direct measurement of sites able to support chain initiation. The results show that the number of initiation sites (IS) is much smaller on chromatin than on DNA; we obtain a value of one IS per 2.5 × 106 base pairs (bp) on chromatin, and 2 × 104 bp for native DNA. The availability of IS was tested over a range of temperatures between 12 and 37°C. It was found that on SU DNA template there is a five-fold increase of available sites with increase of temperature; no such increase was observed with chromatin template.

Morphogenetic Genes in the T-DNA of Ri Plasmids

Agrobacterium rhizogenes is responsible for the hairy root syndrome of dicotyledonous plants which consists in an abundant proliferation of roots at the wounded site of bacterial infection (Elliot, 1951). Hairy roots contain a portion (T-DNA) of a large bacterial plasmid (Ri plasmid), which directs growth and differentiation of the transformed plant cells (Chilton et al., 1982; Spano et al-, 1982b; White et al., 1982; Willmitzer et al.,1982). Hairy roots grow very actively in vitro in the absence of hormones with a characteristic highly branched and plagiotropic pattern (David et al., 1984); they synthesize specific opines of which three major types have been so far identified, agropine, mannopine and cucumopine (Petit and Tempe., 1985), corresponding to different families of Ri plasmids (Costantino et al., 1981). Whole, fertile plants of different species have been regenerated from hairy root tissues in several laboratories (Chilton et al., 1982; Spano et al., 1982a; Tepfer, 1984; Spano et al., 1987).