PRODUCTION OF THE ESCHERICHIA-COLI BETAINE-ALDEHYDE DEHYDROGENASE, ANENZYME REQUIRED FOR THE SYNTHESIS OF THE OSMOPROTECTANT GLYCINE BETAINE, IN TRANSGENIC PLANTS

In several organisms osmotic stress tolerance is mediated by the accum ulation of the osmoprotective compound glycine betaine. With the ambit ion to transfer the betaine biosynthetic pathway into plants not capab le of synthesizing this osmoprotectant, the Escherichia coli gene betB encoding the second enzyme in the pathway, betaine-aldehyde dehydroge nase was introduced into Nicotiana tabacum. The betB structural gent w as fused to the promoter of ats1a, a gene coding for the small subunit of Rubisco in Arabidopsis thaliana. Two types of constructs were made , either encoding the N-terminal transit peptide for chloroplast targe ting or without the targeting signal for cytoplasmic localization of t he BetB polypeptide. Analysis of transgenic N. tabacum plants harborin g these constructs showed that in both cases the transgenes were expre ssed. Northern analysis of the plants demonstrated the accumulation of betB-related mRNA of the correct size. The production and processing of the corresponding polypeptides could be demonstrated by immunoblott ing using polyclonal antisera raised against the BetB polypeptide. The transit peptide encoded by ats1a was able to direct BetB to the chlor oplast, as suggested by the presence of the correctly processed sets p olypeptide in the chloroplast fraction. High betaine-aldehyde dehydrog enase activity was detected in transgenic plants, both in those where the chimeric gene product was targeted to the chloroplast and those wh ere it remained in the cytoplasm. The transgenic tobacco acquired resi stance to the toxic intermediate, betaine aldehyde, in the betaine bio synthetic pathway indicating that the bacterial enzyme is biologically active in its new host. Furthermore, these transgenic plants were abl e to convert exogenously supplied betaine aldehyde efficiently to glyc ine betaine.