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 posttranscriptional 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.