Wheat transformation with glutenin gene.

The rheological properties of wheat grains are associated with the composition of the starchy endosperm in high molecular weight (HMW) glutenin proteins. The HMW glutenin 1xDy12 subunit gene was co introduced with the screenable bar and the reporter gus marker genes in the commercial spring wheat Minaret cultivar (cv). The gene of interest and the marker genes were carried by two separated plasmids, the pBS10BH1 and the pAhC25 respectively. Seven days old calli initiated from immature embryos were bombarded by use of the PDS-1000/He device. A number of different bombardment and culture conditions were tested. These parameters were evaluated on the basis of GUS transient expression. Among those a 4 degrees C cold pre-treatment of the spikes before immature embryos were excised, the culture medium incorporating activated charcoal, silver nitrate and glucose yielded higher GUS transient expression rate. The selective agent bialaphos was maintained at various stages during culture from induction of somatic embryogenesis to rooting of regenerated plantlets. 137 bialaphos resistant plants were obtained among which 109 were carried to maturity. Transgenic plants were characterized by PCR, Southern and SDS-PAGE analysis of the glutenin content.

In vivo evaluation of the context sequence of the translation initiation codon in plants.

Statistical analysis of the AUG initiation codon context in several plant organisms identified a nucleotide preference in some positions around the AUG. Sixteen AUG contexts were studied using transient expression in tobacco, maize and Norway spruce. Besides the importance of A or G at position -3, we revealed the role of positions -2, -1 for which AA or CC were found to be the best for tobacco and maize, respectively. GC (positions +4, +5) were also found to be important in both tobacco and maize. Finally, we identified a variation in context efficiency according to cell type, since A was better than G at position -3 in tobacco leaf protoplasts, while both nucleotides were equally efficient in tobacco suspension cells.

Signs of translational regulation within the transcript leader of a plant plasma membrane H(+)-ATPase gene.

Transcripts of most plant plasma membrane H(+)-ATPase genes possess a leader (5' untranslated region) that is unusually long and that contains a short upstream open reading frame (uORF), two features which suggest post-transcriptional regulation. To investigate the putative role of the transcript leader, we have placed the leader of pma3, one of the Nicotiana plumbaginifolia H(+)-ATPase genes, between the CaMV 35S promoter and the sequence coding for the beta-glucuronidase (GUS) reporter gene. Transient expression of this chimeric gene and of derived mutants was analysed in electroporated tobacco protoplasts. The whole leader had a positive effect on translation, since deletion of most of its sequence reduced GUS activity. Suppression of the uORF by point mutation of its initiating AUG increased GUS activity by about 55%. Analysis of various deletions and mutations suggested that the uORF is translated by at least two-thirds of scanning ribosomes, half of which subsequently reinitiate downstream translation under our experimental conditions. Reinitiation did not depend on the nucleotide sequence of the uORF, nor on that separating the uORF and the main open reading frame. We conclude that the pma3 transcript possesses features of translational regulation, whose mode of functioning has yet to be discovered.