Abstract
In a screen for light regulated transcription factors the ATB2 gene was identified. The ATB2 gene belongs to the S-basic domain/leucine zipper (bZIP) class. The spatial expression of ATB2 correlates with transport of metabolites from tissues that are metabolic active. Research has shown that high concentrations of sucrose repress GUS
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activity of promoter-GUS lines. This effect is sucrose specific, even glucose or fructose cannot mimic this effect. Regulation by sucrose is controlled post-transcriptionally because high concentrations of sucrose upregulate transcript levels while expression is repressed. To date ATB2 is the only gene discovered which is regulated at the post-transcriptional level by sucrose. Ectopic expression of a 5’ UTR construct shows that the repression system is functional in all tissues of the shoot.
To identify mutants in the sucrose regulatory pathway, a non destructive screen, utilizing a dual reporter system, was devised. Both GUS and LUC reporter genes were used and are driven individually by ATB2 regulatory sequences. Repression of both GUS and LUC expression was tested when seedlings, containing the dual reporter system, were grown on solid medium. Lines which displayed sufficient expression and repression were identified and used for a EMS mutagenesis. Many putative mutants were identified but closer analysis revealed that mutants which displayed an altered regulatory phenotype for both of the reporter genes of the dual reporter system, were not among them.
Analysis of the ATB2 sequences (genomic and cDNA) reveals an unusually long (383 bp) leader sequence. Literature studies have shown that long 5’ sequences often contain regulatory elements. This is also the case for the ATB2 gene as deletion of the 5’ UTR relieves the sucrose regulation. Closer examination of the 5’UTR did not reveal any secondary structures but 4 uORFs were identified. The major uORF is 129 nt long and encodes a 42 aa protein. Arabidopsis contains two ATB2 homologs and both have the long leader sequence.
A series of leader-deletion constructs have been made to locate the position of the sucrose regulating element. When the leader of the ATB2 gene is sequentially deleted the results show that the sucrose regulation is relieved when the major uORF is deleted (sucrose control uORF, SC-uORF). It seems that translation of the major uORF is important for the sucrose specific regulation. Only deletion of the startcodon of the major uORF relieves the regulation by sucrose. Deleting the startcodons from the other uORFs does not abolish the sucrose regulation. Closer analysis of the SC-uORF revealed a high amino acid conservation to other plant uORFs. These uORFs are located upstream of bZIP transcription factors. Remarkably, these transcription factors all belong to the S-type bZIP transcription factor group.
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