PHYSIOLOGICAL ANALYSIS OF TRANSGENIC ARABIDOPSIS-THALIANA PLANTS EXPRESSING ONE NITRILASE ISOFORM IN SENSE OR ANTISENSE DIRECTION

The biosynthesis of IAA in Arabidopsis thaliana can proceed via differ ent pathways, which involve indole-3-acetonitrile (LAN) as an intermed iate. The enzyme nitrilase, which converts IAN to the auxin indole-3-a cetic acid (IAA), might be a key enzyme in this biosynthetic pathway. To elucidate the role of nitrilase during the development of the plant , we have investigated transgenic Arabidopsis plants, carrying one of the four nitrilase isoforms so far identified in Arabidopsis in either sense or antisense direction. The plants have been analyzed for their phenotype, auxin content, nitrilase transcript and protein amounts, a nd nitrilase activity. Analysis of free IAA content in seedlings did n ot reveal any prominent differences between the different lines, where as the total IAA content was slightly increased in the line overexpres sing NIT2 (sNIT2). In two of the antisense lines (aNIT1 and aNIT2) the total auxin content was about 75 % lower than in the wild type, while IAN was increased in these two antisense lines by about 2-fold. In sN IT2 plants the TAN content was slightly decreased compared with the wi ld type. When IAN was added to the medium, a strong increase in IAA co ntent was found in sNIT2 seedlings (ca. 22-fold), which was much lower in wild type and antisense plants. In mature plants levels of free IA A were decreased by about 25 % and 50 % in the antisense lines aNIT2 a nd aNIT1, respectively, whereas the IAA content in the other lines did not differ much. sNIT2/aNIT2 plants were further characterized becaus e it was shown by Northern and Western analysis as well as enzyme acti vity measurements that sNIT2 plants overexpress nitrilase constitutive ly. The results with these plants have shown that i) both nitrilase 1 and nitrilase 2 accept the same substrates, and ii) overexpression of nitrilase does not lead to a visible phenotype because in vivo IAN mig ht be the limiting factor in these plants. Application of IAN leads to an increase in total nitrilase protein as shown by Western blotting. Using transgenic lines with promoter-GUS fusions for each NIT gene, it was shown that i) Nln is highly expressed in seedlings and ii) NIT2 i s strongly induced (21-fold) after IAN application.